Application of real-time PCR for total airborne bacterial assessment: Comparison with epifluorescence microscopy and culture-dependent methods

NASA Astrophysics Data System (ADS)

Rinsoz, Thomas; Duquenne, Philippe; Greff-Mirguet, Guylaine; Oppliger, Anne

Traditional culture-dependent methods to quantify and identify airborne microorganisms are limited by factors such as short-duration sampling times and inability to count non-culturable or non-viable bacteria. Consequently, the quantitative assessment of bioaerosols is often underestimated. Use of the real-time quantitative polymerase chain reaction (Q-PCR) to quantify bacteria in environmental samples presents an alternative method, which should overcome this problem. The aim of this study was to evaluate the performance of a real-time Q-PCR assay as a simple and reliable way to quantify the airborne bacterial load within poultry houses and sewage treatment plants, in comparison with epifluorescence microscopy and culture-dependent methods. The estimates of bacterial load that we obtained from real-time PCR and epifluorescence methods, are comparable, however, our analysis of sewage treatment plants indicate these methods give values 270-290 fold greater than those obtained by the "impaction on nutrient agar" method. The culture-dependent method of air impaction on nutrient agar was also inadequate in poultry houses, as was the impinger-culture method, which gave a bacterial load estimate 32-fold lower than obtained by Q-PCR. Real-time quantitative PCR thus proves to be a reliable, discerning, and simple method that could be used to estimate airborne bacterial load in a broad variety of other environments expected to carry high numbers of airborne bacteria.

Real-time RT-PCR, a necessary tool to support the diagnosis and surveillance of rotavirus in Mexico.

PubMed

De La Cruz Hernández, Sergio Isaac; Anaya Molina, Yazmin; Gómez Santiago, Fabián; Terán Vega, Heidi Lizbeth; Monroy Leyva, Elda; Méndez Pérez, Héctor; García Lozano, Herlinda

2018-04-01

Rotavirus produces diarrhea in children under 5 years old. Most of those conventional methods such as polyacrylamide gel electrophoresis (PAGE) and reverse transcription-polymerase chain reaction (RT-PCR) have been used for rotavirus detection. However, these techniques need a multi-step process to get the results. In comparison with conventional methods, the real-time RT-PCR is a highly sensitive method, which allows getting the results in only one day. In this study a real-time RT-PCR assay was tested using a panel of 440 samples from patients with acute gastroenteritis, and characterized by PAGE and RT-PCR. The results show that the real-time RT-PCR detected rotavirus from 73% of rotavirus-negative samples analyzed by PAGE and RT-PCR; thus, the percentage of rotavirus-positive samples increased to 81%. The results indicate that this real-time RT-PCR should be part of a routine analysis, and as a support of the diagnosis of rotavirus in Mexico. Copyright © 2017 Elsevier Inc. All rights reserved.

[Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus].

PubMed

Yang, Yu; Bai, Lin; Hu, Kong-Xin; Yang, Zhi-Hong; Hu, Jian-Ping; Wang, Jing

2012-08-01

Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.

Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories

PubMed Central

2012-01-01

Background Ovine footrot is a contagious disease with worldwide occurrence in sheep. The main causative agent is the fastidious bacterium Dichelobacter nodosus. In Scandinavia, footrot was first diagnosed in Sweden in 2004 and later also in Norway and Denmark. Clinical examination of sheep feet is fundamental to diagnosis of footrot, but D. nodosus should also be detected to confirm the diagnosis. PCR-based detection using conventional PCR has been used at our institutes, but the method was laborious and there was a need for a faster, easier-to-interpret method. The aim of this study was to develop a TaqMan-based real-time PCR assay for detection of D. nodosus and to compare its performance with culturing and conventional PCR. Methods A D. nodosus-specific TaqMan based real-time PCR assay targeting the 16S rRNA gene was designed. The inclusivity and exclusivity (specificity) of the assay was tested using 55 bacterial and two fungal strains. To evaluate the sensitivity and harmonisation of results between different laboratories, aliquots of a single DNA preparation were analysed at three Scandinavian laboratories. The developed real-time PCR assay was compared to culturing by analysing 126 samples, and to a conventional PCR method by analysing 224 samples. A selection of PCR-products was cloned and sequenced in order to verify that they had been identified correctly. Results The developed assay had a detection limit of 3.9 fg of D. nodosus genomic DNA. This result was obtained at all three laboratories and corresponds to approximately three copies of the D. nodosus genome per reaction. The assay showed 100% inclusivity and 100% exclusivity for the strains tested. The real-time PCR assay found 54.8% more positive samples than by culturing and 8% more than conventional PCR. Conclusions The developed real-time PCR assay has good specificity and sensitivity for detection of D. nodosus, and the results are easy to interpret. The method is less time-consuming than either culturing or conventional PCR. PMID:22293440

Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize.

PubMed

Xu, Xiaoli; Peng, Cheng; Wang, Xiaofu; Chen, Xiaoyun; Wang, Qiang; Xu, Junfeng

2016-12-01

This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.

SMA Diagnosis: Detection of SMN1 Deletion with Real-Time mCOP-PCR System Using Fresh Blood DNA.

PubMed

Niba, Emma Tabe Eko; Ar Rochmah, Mawaddah; Harahap, Nur Imma Fatimah; Awano, Hiroyuki; Morioka, Ichiro; Iijima, Kazumoto; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Lai, Poh San; Bouike, Yoshihiro; Nishio, Hisahide; Shinohara, Masakazu

2017-12-18

Spinal muscular atrophy (SMA) is one of the most common autosomal recessive disorders. The symptoms are caused by defects of lower motor neurons in the spinal cord. More than 95% of SMA patients are homozygous for survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique using dried blood spot (DBS) on filter paper. This system is convenient for mass screening in the large population and/or first-tier diagnostic method of the patients in the remote areas. However, this system was still time-consuming and effort-taking, because it required pre-amplification procedure to avoid non-specific amplification and gel-electrophoresis to detect the presence or absence of SMN1 deletion. When the fresh blood samples are used instead of DBS, or when the gel-electrophoresis is replaced by real-time PCR, we may have a simpler and more rapid diagnostic method for SMA. To establish a simpler and more rapid diagnostic method of SMN1 deletion using fresh blood DNA. DNA samples extracted from fresh blood and stored at 4 ℃ for 1 month. The samples were assayed using a real-time mCOP-PCR system without pre-amplification procedures. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The DNA samples were directly subjected to the mCOP-PCR step. The amplification of mCOP-PCR was monitored in a real-time PCR apparatus. The genotyping results of the real-time mCOP-PCR system using fresh blood DNA were completely matched with those of PCR-RFLP. In this real-time mCOP-PCR system using fresh blood-DNA, it took only four hours from extraction of DNA to detection of the presence or absence of SMN1 deletion, while it took more than 12 hours in PCR-RFLP. Our real-time mCOP-PCR system using fresh blood DNA was rapid and accurate, suggesting it may be useful for the first-tier diagnostic method of SMA.

Agreement Rate of Rapid Urease Test, Conventional PCR, and Scorpion Real-Time PCR in Detecting Helicobacter Pylori from Tonsillar Samples of Patients with Chronic Tonsillitis

PubMed Central

Najafipour, Reza; Farivar, Taghi Naserpour; Pahlevan, Ali Akbar; Johari, Pouran; Safdarian, Farshid; Asefzadeh, Mina

2012-01-01

Background: Helicobacter pylori is capable of inducing systemic inflammatory reactions through immunological processes. There are several methods to identify the presence of H. pylori in clinical samples including rapid urease test (RUT), conventional polymerase chain reaction (PCR), and the Scorpion real-time PCR. Aim: The aim of the present study is to compare the agreement rate of these tests in identifying H. pylori in tonsillar biopsy specimens collected from patients with chronic tonsillitis. Materials and Methods: A total of 103 tonsil biopsy samples from patients with clinical signs of chronic tonsillitis were examined with RUT, PCR, and Scorpion real-time PCR. The degree of agreement between the three tests was later calculated. Results: There was a poor degree of agreement between RUT and PCR and also RUT and Scorpion real-time PCR (Kappa=0.269 and 0.249, respectively). In contrast with RUT, there was a strong degree of agreement between PCR and Scorpion real-time PCR (Kappa=0.970). Conclusion: The presence of a strong agreement between the Scorpion real-time PCR and PCR as well as its technical advantage over the conventional PCR assay, made the Scorpion real-time PCR an appropriate laboratory test to investigate the presence of H. pylori in tonsillar biopsy specimens in patients suffering from chronic tonsillitis. PMID:22754245

Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples ▿

PubMed Central

Létant, Sonia E.; Murphy, Gloria A.; Alfaro, Teneile M.; Avila, Julie R.; Kane, Staci R.; Raber, Ellen; Bunt, Thomas M.; Shah, Sanjiv R.

2011-01-01

In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples. PMID:21764960

Rapid detection of coliforms in drinking water of Arak city using multiplex PCR method in comparison with the standard method of culture (Most Probably Number)

PubMed Central

Fatemeh, Dehghan; Reza, Zolfaghari Mohammad; Mohammad, Arjomandzadegan; Salomeh, Kalantari; Reza, Ahmari Gholam; Hossein, Sarmadian; Maryam, Sadrnia; Azam, Ahmadi; Mana, Shojapoor; Negin, Najarian; Reza, Kasravi Alii; Saeed, Falahat

2014-01-01

Objective To analyse molecular detection of coliforms and shorten the time of PCR. Methods Rapid detection of coliforms by amplification of lacZ and uidA genes in a multiplex PCR reaction was designed and performed in comparison with most probably number (MPN) method for 16 artificial and 101 field samples. The molecular method was also conducted on isolated coliforms from positive MPN samples; standard sample for verification of microbial method certificated reference material; isolated strains from certificated reference material and standard bacteria. The PCR and electrophoresis parameters were changed for reducing the operation time. Results Results of PCR for lacZ and uidA genes were similar in all of standard, operational and artificial samples and showed the 876 bp and 147 bp bands of lacZ and uidA genes by multiplex PCR. PCR results were confirmed by MPN culture method by sensitivity 86% (95% CI: 0.71-0.93). Also the total execution time, with a successful change of factors, was reduced to less than two and a half hour. Conclusions Multiplex PCR method with shortened operation time was used for the simultaneous detection of total coliforms and Escherichia coli in distribution system of Arak city. It's recommended to be used at least as an initial screening test, and then the positive samples could be randomly tested by MPN. PMID:25182727

Evaluation of a real-time quantitative PCR method with propidium monazide treatment for analyses of viable fecal indicator bacteria in wastewater samples

EPA Science Inventory

The U.S. EPA is currently evaluating rapid, real-time quantitative PCR (qPCR) methods for determining recreational water quality based on measurements of fecal indicator bacteria DNA sequences. In order to potentially use qPCR for other Clean Water Act needs, such as updating cri...

Real-time PCR-based method for the rapid detection of extended RAS mutations using bridged nucleic acids in colorectal cancer.

PubMed

Iida, Takao; Mizuno, Yukie; Kaizaki, Yasuharu

2017-10-27

Mutations in RAS and BRAF are predictors of the efficacy of anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). Therefore, simple, rapid, cost-effective methods to detect these mutations in the clinical setting are greatly needed. In the present study, we evaluated BNA Real-time PCR Mutation Detection Kit Extended RAS (BNA Real-time PCR), a real-time PCR method that uses bridged nucleic acid clamping technology to rapidly detect mutations in RAS exons 2-4 and BRAF exon 15. Genomic DNA was extracted from 54 formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from mCRC patients. Among the 54 FFPE samples, BNA Real-time PCR detected 21 RAS mutations (38.9%) and 5 BRAF mutations (9.3%), and the reference assay (KRAS Mutation Detection Kit and MEBGEN™ RASKET KIT) detected 22 RAS mutations (40.7%). The concordance rate of detected RAS mutations between the BNA Real-time PCR assay and the reference assays was 98.2% (53/54). The BNA Real-time PCR assay proved to be a more simple, rapid, and cost-effective method for detecting KRAS and RAS mutations compared with existing assays. These findings suggest that BNA Real-time PCR is a valuable tool for predicting the efficacy of early anti-EGFR therapy in mCRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantification of mixed chimerism by real time PCR on whole blood-impregnated FTA cards.

PubMed

Pezzoli, N; Silvy, M; Woronko, A; Le Treut, T; Lévy-Mozziconacci, A; Reviron, D; Gabert, J; Picard, C

2007-09-01

This study has investigated quantification of chimerism in sex-mismatched transplantations by quantitative real time PCR (RQ-PCR) using FTA paper for blood sampling. First, we demonstrate that the quantification of DNA from EDTA-blood which has been deposit on FTA card is accurate and reproducible. Secondly, we show that fraction of recipient cells detected by RQ-PCR was concordant between the FTA and salting-out method, reference DNA extraction method. Furthermore, the sensitivity of detection of recipient cells is relatively similar with the two methods. Our results show that this innovative method can be used for MC assessment by RQ-PCR.

Real-Time PCR (qPCR) Primer Design Using Free Online Software

ERIC Educational Resources Information Center

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids.

PubMed

Yang, Litao; Liang, Wanqi; Jiang, Lingxi; Li, Wenquan; Cao, Wei; Wilson, Zoe A; Zhang, Dabing

2008-06-04

Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

High-throughput real-time quantitative reverse transcription PCR.

PubMed

Bookout, Angie L; Cummins, Carolyn L; Mangelsdorf, David J; Pesola, Jean M; Kramer, Martha F

2006-02-01

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Rectal swab sampling followed by an enrichment culture-based real-time PCR assay to detect Salmonella enterocolitis in children.

PubMed

Lin, L-H; Tsai, C-Y; Hung, M-H; Fang, Y-T; Ling, Q-D

2011-09-01

Although routine bacterial culture is the traditional reference standard method for the detection of Salmonella infection in children with diarrhoea, it is a time-consuming procedure that usually only gives results after 3-4 days. Some molecular detection methods can improve the turn-around time to within 24 h, but these methods are not applied directly from stool or rectal swab specimens as routine diagnostic methods for the detection of gastrointestinal pathogens. In this study, we tested the feasibility of a bacterial enrichment culture-based real-time PCR assay method for detecting and screening for diarrhoea in children caused by Salmonella. Our results showed that the minimum real-time PCR assay time required to detect enriched bacterial culture from a swab was 3 h. In all children with suspected Salmonella diarrhoea, the enrichment culture-based real-time PCR achieved 85.4% sensitivity and 98.1% specificity, as compared with the 53.7% sensitivity and 100% specificity of detection with the routine bacterial culture method. We suggest that rectal swab sampling followed by enrichment culture-based real-time PCR is suitable as a rapid method for detecting and screening for Salmonella in paediatric patients. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.

Targeting neuroendocrine differentiation for prostate cancer radiosensitization

DTIC Science & Technology

2017-12-01

Secondary HRP-conjugated antibodies were purchased fromGEHealthcare UK Ltd. (Buckinghamshire, UK). 2.5. RNA isolation and quantitative real- time PCR (qRT...gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T))Method,Methods 25 (2001) 402–408. [47] T.K. Kelly, T.B. Miranda, G...relative gene expression data using real- time quantitative PCR and the 2(−Delta Delta C(T)) method, Methods 25 (2001) 402–408. [37] J. Ren, L. Wen, X

Quantitative Real-Time PCR Analysis of Total Propidium Monazide -Resistant Fecal Indicator Bacteria in Wastewater

EPA Science Inventory

A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. Thes...

Detection and enumeration of Salmonella enteritidis in homemade ice cream associated with an outbreak: comparison of conventional and real-time PCR methods.

PubMed

Seo, K H; Valentin-Bon, I E; Brackett, R E

2006-03-01

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

Validation of PCR methods for quantitation of genetically modified plants in food.

PubMed

Hübner, P; Waiblinger, H U; Pietsch, K; Brodmann, P

2001-01-01

For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials.

Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution

EPA Science Inventory

There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method ...

Evaluation of Novel Broad-Range Real-Time PCR Assay for Rapid Detection of Human Pathogenic Fungi in Various Clinical Specimens▿

PubMed Central

Vollmer, Tanja; Störmer, Melanie; Kleesiek, Knut; Dreier, Jens

2008-01-01

In the present study, a novel broad-range real-time PCR was developed for the rapid detection of human pathogenic fungi. The assay targets a part of the 28S large-subunit ribosomal RNA (rDNA) gene. We investigated its application for the most important human pathogenic fungal genera, including Aspergillus, Candida, Cryptococcus, Mucor, Penicillium, Pichia, Microsporum, Trichophyton, and Scopulariopsis. Species were identified in PCR-positive reactions by direct DNA sequencing. A noncompetitive internal control was applied to prevent false-negative results due to PCR inhibition. The minimum detection limit for the PCR was determined to be one 28S rDNA copy per PCR, and the 95% detection limit was calculated to 15 copies per PCR. To assess the clinical applicability of the PCR method, intensive-care patients with artificial respiration and patients with infective endocarditis were investigated. For this purpose, 76 tracheal secretion samples and 70 heart valve tissues were analyzed in parallel by real-time PCR and cultivation. No discrepancies in results were observed between PCR analysis and cultivation methods. Furthermore, the application of the PCR method was investigated for other clinical specimens, including cervical swabs, nail and horny skin scrapings, and serum, blood, and urine samples. The combination of a broad-range real-time PCR and direct sequencing facilitates rapid screening for fungal infection in various clinical specimens. PMID:18385440

Evaluation of novel broad-range real-time PCR assay for rapid detection of human pathogenic fungi in various clinical specimens.

PubMed

Vollmer, Tanja; Störmer, Melanie; Kleesiek, Knut; Dreier, Jens

2008-06-01

In the present study, a novel broad-range real-time PCR was developed for the rapid detection of human pathogenic fungi. The assay targets a part of the 28S large-subunit ribosomal RNA (rDNA) gene. We investigated its application for the most important human pathogenic fungal genera, including Aspergillus, Candida, Cryptococcus, Mucor, Penicillium, Pichia, Microsporum, Trichophyton, and Scopulariopsis. Species were identified in PCR-positive reactions by direct DNA sequencing. A noncompetitive internal control was applied to prevent false-negative results due to PCR inhibition. The minimum detection limit for the PCR was determined to be one 28S rDNA copy per PCR, and the 95% detection limit was calculated to 15 copies per PCR. To assess the clinical applicability of the PCR method, intensive-care patients with artificial respiration and patients with infective endocarditis were investigated. For this purpose, 76 tracheal secretion samples and 70 heart valve tissues were analyzed in parallel by real-time PCR and cultivation. No discrepancies in results were observed between PCR analysis and cultivation methods. Furthermore, the application of the PCR method was investigated for other clinical specimens, including cervical swabs, nail and horny skin scrapings, and serum, blood, and urine samples. The combination of a broad-range real-time PCR and direct sequencing facilitates rapid screening for fungal infection in various clinical specimens.

Real-time PCR: Advanced technologies and applications

USDA-ARS?s Scientific Manuscript database

This book brings together contributions from 20 experts in the field of PCR, providing a broad perspective of the applications of quantitative real-time PCR (qPCR). The editors state in the preface that the aim is to provide detailed insight into underlying principles and methods of qPCR to provide ...

Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.

PubMed

Demeke, Tigst; Ratnayaka, Indira; Phan, Anh

2009-01-01

The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

Nested-PCR real time as alternative molecular tool for detection of Borrelia burgdorferi compared to the classical serological diagnosis of the blood.

PubMed

Sroka-Oleksiak, Agnieszka; Ufir, Krzysztof; Salamon, Dominika; Bulanda, Malgorzata; Gosiewski, Tomasz

Lyme disease, caused by Borrelia burgdorferi, is a multisystem disease that often makes difficulties to recognize caused by their genetic heterogenity. Currently, the gold standard for the detection of Lyme disease (LD) is serologic diagnostics based mainly on tests: ELISA and Western blot (WB). These methods, however, are subject to consider- able defect, especially in the initial phase of infection due to the occurrence of so-called serological window period and low specificity. For this reason, they might be replaced by molecular methods, for example polymerase chain reaction (PCR), which should be more sensitivity and specificity. In the present study we attempt to optimize the PCR reaction conditions and enhance existing test sensitivity by applying the equivalent of real time PCR - nested PCR for detection B. burgdorferi DNA in the patient's blood. The study involved 94 blood samples of patients with suspected LD. From each sample, 1.5 ml of blood was used for the isolation of bacterial DNA and PCR real time am- plification and its equivalent, in nested version. The remaining part earmarked for serologi- cal testing. Optimization of the reaction conditions made experimentally, using gradient of the temperature and gradient of the magnesium ions concentration for reaction real time in nested-PCR and PCR version. The results show that the nested-PCR real time, has a much higher sensitivity 45 (47.8%) of positive results for the detection of B. burgdorferi compared to the single- variety, without a preceding pre-amplification 2 (2.1%). Serological methods allowed the detection of infection in 41 (43.6%) samples. These results support of the nested PCR method as a better molecular tool for the detection of B. burgdorferi infection than classical PCR real time reaction. The nested-PCR real time method may be considered as a complement to ELISA and WB mainly in the early stages of infection, when in the blood circulating B. burgdorferi cells. By contrast, the results of serological and molecular tests should always be carried out tak- ing into account the patient's clinical status.

Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples.

PubMed

Kasturi, Kuppuswamy N; Drgon, Tomas

2017-07-15

The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA , group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non- Salmonella organisms. The invA - and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella -differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S Enteritidis isolates and 100% exclusivity for the 297 non-Enteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the V itek i mmuno d iagnostic a ssay s ystem (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method. IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples.

Real-Time PCR Method for Detection of Salmonella spp. in Environmental Samples

PubMed Central

Drgon, Tomas

2017-01-01

ABSTRACT The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA, group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non-Salmonella organisms. The invA- and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella-differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S. Enteritidis isolates and 100% exclusivity for the 297 non-Enteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the Vitek immunodiagnostic assay system (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method. IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples. PMID:28500041

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids

PubMed Central

Yang, Litao; Liang, Wanqi; Jiang, Lingxi; Li, Wenquan; Cao, Wei; Wilson, Zoe A; Zhang, Dabing

2008-01-01

Background Real-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule. Results We describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods. Conclusion The results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost. PMID:18522756

The diagnosis of microorganism involved in infective endocarditis (IE) by polymerase chain reaction (PCR) and real-time PCR: A systematic review.

PubMed

Faraji, Reza; Behjati-Ardakani, Mostafa; Moshtaghioun, Seyed Mohammad; Kalantar, Seyed Mehdi; Namayandeh, Seyedeh Mahdieh; Soltani, Mohammadhossien; Emami, Mahmood; Zandi, Hengameh; Firoozabadi, Ali Dehghani; Kazeminasab, Mahmood; Ahmadi, Nastaran; Sarebanhassanabadi, Mohammadtaghi

2018-02-01

Broad-range bacterial rDNA polymerase chain reaction (PCR) followed by sequencing may be identified as the etiology of infective endocarditis (IE) from surgically removed valve tissue; therefore, we reviewed the value of molecular testing in identifying organisms' DNA in the studies conducted until 2016. We searched Google Scholar, Scopus, ScienceDirect, Cochrane, PubMed, and Medline electronic databases without any time limitations up to December 2016 for English studies reporting microorganisms involved in infective endocarditis microbiology using PCR and real-time PCR. Most studies were prospective. Eleven out of 12 studies used valve tissue samples and blood cultures while only 1 study used whole blood. Also, 10 studies used the molecular method of PCR while 2 studies used real-time PCR. Most studies used 16S rDNA gene as the target gene. The bacteria were identified as the most common microorganisms involved in infective endocarditis. Streptococcus spp. and Staphylococcus spp. were, by far, the most predominant bacteria detected. In all studies, PCR and real-time PCR identified more pathogens than blood and tissue cultures; moreover, the sensitivity and specificity of PCR and real-time PCR were more than cultures in most of the studies. The highest sensitivity and specificity were 96% and 100%, respectively. The gram positive bacteria were the most frequent cause of infective endocarditis. The molecular methods enjoy a greater sensitivity compared to the conventional blood culture methods; yet, they are applicable only to the valve tissue of the patients undergoing cardiac valve surgery. Copyright © 2017. Published by Elsevier Taiwan.

Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution - Poster

EPA Science Inventory

There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method p...

EVALUATION OF QUANTITATIVE REAL TIME PCR FOR THE MEASUREMENT OF HELICOBATER PYLORI AT LOW CONCENTRATIONS IN DRINKING WATER

EPA Science Inventory

Aims: To determine the performance of a rapid, real time polymerase chain reaction (PCR) method for the detection and quantitative analysis Helicobacter pylori at low concentrations in drinking water.

Methods and Results: A rapid DNA extraction and quantitative PCR (QPCR)...

Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods.

PubMed

Edagawa, Akiko; Kimura, Akio; Kawabuchi-Kurata, Takako; Adachi, Shinichi; Furuhata, Katsunori; Miyamoto, Hiroshi

2015-10-19

We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples.

Investigation of Legionella Contamination in Bath Water Samples by Culture, Amoebic Co-Culture, and Real-Time Quantitative PCR Methods

PubMed Central

Edagawa, Akiko; Kimura, Akio; Kawabuchi-Kurata, Takako; Adachi, Shinichi; Furuhata, Katsunori; Miyamoto, Hiroshi

2015-01-01

We investigated Legionella contamination in bath water samples, collected from 68 bathing facilities in Japan, by culture, culture with amoebic co-culture, real-time quantitative PCR (qPCR), and real-time qPCR with amoebic co-culture. Using the conventional culture method, Legionella pneumophila was detected in 11 samples (11/68, 16.2%). Contrary to our expectation, the culture method with the amoebic co-culture technique did not increase the detection rate of Legionella (4/68, 5.9%). In contrast, a combination of the amoebic co-culture technique followed by qPCR successfully increased the detection rate (57/68, 83.8%) compared with real-time qPCR alone (46/68, 67.6%). Using real-time qPCR after culture with amoebic co-culture, more than 10-fold higher bacterial numbers were observed in 30 samples (30/68, 44.1%) compared with the same samples without co-culture. On the other hand, higher bacterial numbers were not observed after propagation by amoebae in 32 samples (32/68, 47.1%). Legionella was not detected in the remaining six samples (6/68, 8.8%), irrespective of the method. These results suggest that application of the amoebic co-culture technique prior to real-time qPCR may be useful for the sensitive detection of Legionella from bath water samples. Furthermore, a combination of amoebic co-culture and real-time qPCR might be useful to detect viable and virulent Legionella because their ability to invade and multiply within free-living amoebae is considered to correlate with their pathogenicity for humans. This is the first report evaluating the efficacy of the amoebic co-culture technique for detecting Legionella in bath water samples. PMID:26492259

Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples▿

PubMed Central

Delgado-Viscogliosi, Pilar; Solignac, Lydie; Delattre, Jean-Marie

2009-01-01

PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells. PMID:19363080

Evaluation of quantitative PCR measurement of bacterial colonization of epithelial cells.

PubMed

Schmidt, Marcin T; Olejnik-Schmidt, Agnieszka K; Myszka, Kamila; Borkowska, Monika; Grajek, Włodzimierz

2010-01-01

Microbial colonization is an important step in establishing pathogenic or probiotic relations to host cells and in biofilm formation on industrial or medical devices. The aim of this work was to verify the applicability of quantitative PCR (Real-Time PCR) to measure bacterial colonization of epithelial cells. Salmonella enterica and Caco-2 intestinal epithelial cell line was used as a model. To verify sensitivity of the assay a competition of the pathogen cells to probiotic microorganism was tested. The qPCR method was compared to plate count and radiolabel approach, which are well established techniques in this area of research. The three methods returned similar results. The best quantification accuracy had radiolabel method, followed by qPCR. The plate count results showed coefficient of variation two-times higher than this of qPCR. The quantitative PCR proved to be a reliable method for enumeration of microbes in colonization assay. It has several advantages that make it very useful in case of analyzing mixed populations, where several different species or even strains can be monitored at the same time.

Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

PubMed

Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

2014-09-01

The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

Evaluation of an Improved U.S. Food and Drug Administration Method for the Detection of Cyclospora cayetanensis in Produce Using Real-Time PCR.

PubMed

Murphy, Helen R; Lee, Seulgi; da Silva, Alexandre J

2017-07-01

Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease associated with the consumption of fresh produce or water contaminated with C. cayetanensis oocysts. In the United States, foodborne outbreaks of cyclosporiasis have been linked to various types of imported fresh produce, including cilantro and raspberries. An improved method was developed for identification of C. cayetanensis in produce at the U.S. Food and Drug Administration. The method relies on a 0.1% Alconox produce wash solution for efficient recovery of oocysts, a commercial kit for DNA template preparation, and an optimized TaqMan real-time PCR assay with an internal amplification control for molecular detection of the parasite. A single laboratory validation study was performed to assess the method's performance and compare the optimized TaqMan real-time PCR assay and a reference nested PCR assay by examining 128 samples. The samples consisted of 25 g of cilantro or 50 g of raspberries seeded with 0, 5, 10, or 200 C. cayetanensis oocysts. Detection rates for cilantro seeded with 5 and 10 oocysts were 50.0 and 87.5%, respectively, with the real-time PCR assay and 43.7 and 94.8%, respectively, with the nested PCR assay. Detection rates for raspberries seeded with 5 and 10 oocysts were 25.0 and 75.0%, respectively, with the real-time PCR assay and 18.8 and 68.8%, respectively, with the nested PCR assay. All unseeded samples were negative, and all samples seeded with 200 oocysts were positive. Detection rates using the two PCR methods were statistically similar, but the real-time PCR assay is less laborious and less prone to amplicon contamination and allows monitoring of amplification and analysis of results, making it more attractive to diagnostic testing laboratories. The improved sample preparation steps and the TaqMan real-time PCR assay provide a robust, streamlined, and rapid analytical procedure for surveillance, outbreak response, and regulatory testing of foods for detection of C. cayetanensis.

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

PubMed Central

Dinoop, K.P.; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R.P.; Narayanan, P.

2016-01-01

Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods. PMID:26997014

Simultaneous detection of papaya ringspot virus, papaya leaf distortion mosaic virus, and papaya mosaic virus by multiplex real-time reverse transcription PCR.

PubMed

Huo, P; Shen, W T; Yan, P; Tuo, D C; Li, X Y; Zhou, P

2015-12-01

Both the single infection of papaya ringspot virus (PRSV), papaya leaf distortion mosaic virus (PLDMV) or papaya mosaic virus (PapMV) and double infection of PRSV and PLDMV or PapMV which cause indistinguishable symptoms, threaten the papaya industry in Hainan Island, China. In this study, a multiplex real-time reverse transcription PCR (RT-PCR) was developed to detect simultaneously the three viruses based on their distinctive melting temperatures (Tms): 81.0±0.8°C for PRSV, 84.7±0.6°C for PLDMV, and 88.7±0.4°C for PapMV. The multiplex real-time RT-PCR method was specific and sensitive in detecting the three viruses, with a detection limit of 1.0×10(1), 1.0×10(2), and 1.0×10(2) copies for PRSV, PLDMV, and PapMV, respectively. Indeed, the reaction was 100 times more sensitive than the multiplex RT-PCR for PRSV, and 10 times more sensitive than multiplex RT-PCR for PLDMV. Field application of the multiplex real-time RT-PCR demonstrated that some non-symptomatic samples were positive for PLDMV by multiplex real-time RT-PCR but negative by multiplex RT-PCR, whereas some samples were positive for both PRSV and PLDMV by multiplex real-time RT-PCR assay but only positive for PLDMV by multiplex RT-PCR. Therefore, this multiplex real-time RT-PCR assay provides a more rapid, sensitive and reliable method for simultaneous detection of PRSV, PLDMV, PapMV and their mixed infections in papaya.

Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay.

PubMed

Wu, Qingqing; Xiang, Shengnan; Wang, Wenjun; Zhao, Jinyan; Xia, Jinhua; Zhen, Yueran; Liu, Bang

2018-05-01

Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209 bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R 2  > 0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤ 25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

Diagnosis of Cetacean morbillivirus: A sensitive one step real time RT fast-PCR method based on SYBR(®) Green.

PubMed

Sacristán, Carlos; Carballo, Matilde; Muñoz, María Jesús; Bellière, Edwige Nina; Neves, Elena; Nogal, Verónica; Esperón, Fernando

2015-12-15

Cetacean morbillivirus (CeMV) (family Paramyxoviridae, genus Morbillivirus) is considered the most pathogenic virus of cetaceans. It was first implicated in the bottlenose dolphin (Tursiops truncatus) mass stranding episode along the Northwestern Atlantic coast in the late 1980s, and in several more recent worldwide epizootics in different Odontoceti species. This study describes a new one step real-time reverse transcription fast polymerase chain reaction (real-time RT-fast PCR) method based on SYBR(®) Green to detect a fragment of the CeMV fusion protein gene. This primer set also works for conventional RT-PCR diagnosis. This method detected and identified all three well-characterized strains of CeMV: porpoise morbillivirus (PMV), dolphin morbillivirus (DMV) and pilot whale morbillivirus (PWMV). Relative sensitivity was measured by comparing the results obtained from 10-fold dilution series of PMV and DMV positive controls and a PWMV field sample, to those obtained by the previously described conventional phosphoprotein gene based RT-PCR method. Both the conventional and real-time RT-PCR methods involving the fusion protein gene were 100- to 1000-fold more sensitive than the previously described conventional RT-PCR method. Copyright © 2015 Elsevier B.V. All rights reserved.

Salmonella detection in poultry samples. Comparison of two commercial real-time PCR systems with culture methods for the detection of Salmonella spp. in environmental and fecal samples of poultry.

PubMed

Sommer, D; Enderlein, D; Antakli, A; Schönenbrücher, H; Slaghuis, J; Redmann, T; Lierz, M

2012-01-01

The efficiency of two commercial PCR methods based on real-time technology, the foodproof® Salmonella detection system and the BAX® PCR Assay Salmonella system was compared to standardized culture methods (EN ISO 6579:2002 - Annex D) for the detection of Salmonella spp. in poultry samples. Four sample matrices (feed, dust, boot swabs, feces) obtained directly from poultry flocks, as well as artificially spiked samples of the same matrices, were used. All samples were tested for Salmonella spp. using culture methods first as the gold standard. In addition samples spiked with Salmonella Enteridis were tested to evaluate the sensitivity of both PCR methods. Furthermore all methods were evaluated in an annual ring-trial of the National Salmonella Reference Laboratory of Germany. Salmonella detection in the matrices feed, dust and boot swabs were comparable in both PCR systems whereas the results from feces differed markedly. The quality, especially the freshness, of the fecal samples had an influence on the sensitivity of the real-time PCR and the results of the culture methods. In fresh fecal samples an initial spiking level of 100cfu/25g Salmonella Enteritidis was detected. Two-days-dried fecal samples allowed the detection of 14cfu/25g. Both real- time PCR protocols appear to be suitable for the detection of Salmonella spp. in all four matrices. The foodproof® system detected eight samples more to be positive compared to the BAX® system, but had a potential false positive result in one case. In 7-days-dried samples none of the methods was able to detect Salmonella likely through letal cell damage. In general the advantage of PCR analyses over the culture method is the reduction of working time from 4-5 days to only 2 days. However, especially for the analysis of fecal samples official validation should be conducted according to the requirement of EN ISO6579:2002 - Annex D.

Optimization and Verification of Droplet Digital PCR Even-Specific Methods for the Quantification of GM Maize DAS1507 and NK603.

PubMed

Grelewska-Nowotko, Katarzyna; Żurawska-Zajfert, Magdalena; Żmijewska, Ewelina; Sowa, Sławomir

2018-05-01

In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform. However, optimization of the PCR conditions might be necessary. In this study, we report the transfer of two validated qPCR methods for quantification of maize DAS1507 and NK603 events to the droplet dPCR (ddPCR) platform. After some optimization, both methods have been verified according to the guidance of the European Network of GMO Laboratories (ENGL) on analytical method verification (ENGL working group on "Method Verification." (2011) Verification of Analytical Methods for GMO Testing When Implementing Interlaboratory Validated Methods). Digital PCR methods performed equally or better than the qPCR methods. Optimized ddPCR methods confirm their suitability for GMO determination in food and feed.

Development of quantitative real-time PCR for detection and enumeration of Enterobacteriaceae.

PubMed

Takahashi, Hajime; Saito, Rumi; Miya, Satoko; Tanaka, Yuichiro; Miyamura, Natsumi; Kuda, Takashi; Kimura, Bon

2017-04-04

The family Enterobacteriaceae, members of which are widely distributed in the environment, includes many important human pathogens. In this study, a rapid real-time PCR method targeting rplP, coding for L16 protein, a component of the ribosome large subunit, was developed for enumerating Enterobacteriaceae strains, and its efficiency was evaluated using naturally contaminated food products. The rplP-targeted real-time PCR amplified Enterobacteriaceae species with Ct values of 14.0-22.8, whereas the Ct values for non-Enterobacteriaceae species were >30, indicating the specificity of this method for the Enterobacteriaceae. Using a calibration curve of Ct=-3.025 (log CFU/g)+37.35, which was calculated from individual plots of the cell numbers in different concentrations of 5 Enterobacteriaceae species, the rplP-targeted real-time PCR was applied to 51 food samples. A <1log difference between the real-time PCR and culture methods was obtained in a majority of the food samples (81.8%), with good correlation (r 2 =0.8285). This study demonstrated that the rplP-targeted real-time PCR method could detect and enumerate Enterobacteriaceae species in foods rapidly and accurately, and therefore, it can be used for the microbiological risk analysis of foods. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of sampling sites and laboratory diagnostic tests for S. equi subsp. equi in horses from confirmed strangles outbreaks.

PubMed

Lindahl, S; Båverud, V; Egenvall, A; Aspán, A; Pringle, J

2013-01-01

Strangles is a contagious equine-specific disease caused by Streptococcus equi subsp. equi. Unfortunately, detection of S. equi can fail in up to 40% of horses with strangles. Whereas recent molecular biologic methods and sampling techniques have improved recovery of S. equi optimal sampling methods and laboratory analyses remain ill-defined. To determine the yield of S. equi from horses with acute strangles in confirmed outbreaks by field-sampling methods subjected to culture and biochemical identification, and real-time PCR directly and after culture. Fifty-seven horses of varying breeds and ages from 8 strangles outbreaks. Prospective study. Culture with biochemical identification and real-time PCR directly, and from culture, were performed on nasal swabs, nasopharyngeal swabs, and nasopharyngeal lavages. Real-time PCR directly from samples identified the highest number of infected horses, with 45/57 nasal swabs, 41/57 nasopharyngeal swabs, and 48/57 nasopharyngeal lavages S. equi positive. Biochemical identification (highest positives 22/57) was inferior to real-time PCR for S. equi recovery regardless of sampling method. Real-time PCR of nasopharyngeal lavage directly and after culture yielded 52/57 positives whereas direct real-time PCR of nasopharyngeal lavage combined with either nasopharyngeal swabs or nasal swabs yielded 53/57 positives. Three horses were negative on all samples. Nasopharyngeal lavage analyzed by a combination of real-time PCR directly and after culture or, alternatively, real-time PCR directly on a nasopharyngeal lavage and a nasal/nasopharyngeal swab can identify S. equi in over 90% of acute strangles cases. Copyright © 2013 by the American College of Veterinary Internal Medicine.

The use of comparative duplex PCR in monitoring of patients with non-Hodgkin's lymphoma and chronic lymphocytic leukaemia.

PubMed

Slavícková, A; Forsterová, K; Ivánek, R; Cerný, J; Klener, P

2005-01-01

Various quantitative PCR approaches have been utilized during the last years to provide information about the treatment efficacy and the risk of recurrent disease in haematological malignancies. Apart from the frequently used real-time PCR, cost-saving modified standard PCR methods may be applied as well. This report evaluates the utility of the end-point comparative duplex PCR. We have used this method for monitoring of 35 patients with either NHL or CLL and observed a good correlation between quantitative molecular results and clinical outcome. There was also an agreement between comparative duplex PCR and real-time PCR in patients who were monitored by both methods. We therefore believe that use of this technique should be strongly considered instead of simple qualitative detection in monitoring of therapeutic outcome in NHL or CLL patients.

Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods.

PubMed

Camargo, Vinícius da Silva; Santana, Bruna Nicoleti; Ferrari, Elis Domingos; Nakamura, Alex Akira; Nagata, Walter Bertequini; Nardi, Ana Rita Moraes; Meireles, Marcelo Vasconcelos

2018-01-01

This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

High-Resolution Melting-Curve Analysis of Ligation-Mediated Real-Time PCR for Rapid Evaluation of an Epidemiological Outbreak of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli ▿

PubMed Central

Woksepp, Hanna; Jernberg, Cecilia; Tärnberg, Maria; Ryberg, Anna; Brolund, Alma; Nordvall, Michaela; Olsson-Liljequist, Barbro; Wisell, Karin Tegmark; Monstein, Hans-Jürg; Nilsson, Lennart E.; Schön, Thomas

2011-01-01

Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE. PMID:21956981

High-resolution melting-curve analysis of ligation-mediated real-time PCR for rapid evaluation of an epidemiological outbreak of extended-spectrum-beta-lactamase-producing Escherichia coli.

PubMed

Woksepp, Hanna; Jernberg, Cecilia; Tärnberg, Maria; Ryberg, Anna; Brolund, Alma; Nordvall, Michaela; Olsson-Liljequist, Barbro; Wisell, Karin Tegmark; Monstein, Hans-Jürg; Nilsson, Lennart E; Schön, Thomas

2011-12-01

Methods for the confirmation of nosocomial outbreaks of bacterial pathogens are complex, expensive, and time-consuming. Recently, a method based on ligation-mediated PCR (LM/PCR) using a low denaturation temperature which produces specific melting-profile patterns of DNA products has been described. Our objective was to further develop this method for real-time PCR and high-resolution melting analysis (HRM) in a single-tube system optimized in order to achieve results within 1 day. Following the optimization of LM/PCR for real-time PCR and HRM (LM/HRM), the method was applied for a nosocomial outbreak of extended-spectrum-beta-lactamase (ESBL)-producing and ST131-associated Escherichia coli isolates (n = 15) and control isolates (n = 29), including four previous clusters. The results from LM/HRM were compared to results from pulsed-field gel electrophoresis (PFGE), which served as the gold standard. All isolates from the nosocomial outbreak clustered by LM/HRM, which was confirmed by gel electrophoresis of the LM/PCR products and PFGE. Control isolates that clustered by LM/PCR (n = 4) but not by PFGE were resolved by confirmatory gel electrophoresis. We conclude that LM/HRM is a rapid method for the detection of nosocomial outbreaks of bacterial infections caused by ESBL-producing E. coli strains. It allows the analysis of isolates in a single-tube system within a day, and the discriminatory power is comparable to that of PFGE.

Efficacy of the detection of Legionella in hot and cold water samples by culture and PCR. I. Standardization of methods.

PubMed

Wójcik-Fatla, Angelina; Stojek, Nimfa Maria; Dutkiewicz, Jacek

2012-01-01

The aim of the present study was: - to compare methods for concentration and isolation of Legionella DNA from water; - to examine the efficacy of various modifications of PCR test (PCR, semi-nested PCR, and real-time PCR) for the detection of known numbers of Legionella pneumophila in water samples artificially contaminated with the strain of this bacterium and in randomly selected samples of environmental water, in parallel with examination by culture. It was found that filtration is much more effective than centrifugation for the concentration of DNA in water samples, and that the Qiamp DNA Mini-Kit is the most efficient for isolation of Legionella DNA from water. The semi-nested PCR and real-time PCR proved to be the most sensitive methods for detection of Legionella DNA in water samples. Both PCR modifications showed a high correlation with recovery of Legionella by culture (p<0.01), while no correlation occurred between the results of one-stage PCR and culture (p>0.1).

An insulated isothermal PCR method on a field-deployable device for rapid and sensitive detection of canine parvovirus type 2 at points of need.

PubMed

Wilkes, Rebecca P; Lee, Pei-Yu A; Tsai, Yun-Long; Tsai, Chuan-Fu; Chang, Hsiu-Hui; Chang, Hsiao-Fen G; Wang, Hwa-Tang T

2015-08-01

Canine parvovirus type 2 (CPV-2), including subtypes 2a, 2b and 2c, causes an acute enteric disease in both domestic and wild animals. Rapid and sensitive diagnosis aids effective disease management at points of need (PON). A commercially available, field-deployable and user-friendly system, designed with insulated isothermal PCR (iiPCR) technology, displays excellent sensitivity and specificity for nucleic acid detection. An iiPCR method was developed for on-site detection of all circulating CPV-2 strains. Limit of detection was determined using plasmid DNA. CPV-2a, 2b and 2c strains, a feline panleukopenia virus (FPV) strain, and nine canine pathogens were tested to evaluate assay specificity. Reaction sensitivity and performance were compared with an in-house real-time PCR using serial dilutions of a CPV-2b strain and 100 canine fecal clinical samples collected from 2010 to 2014, respectively. The 95% limit of detection of the iiPCR method was 13 copies of standard DNA and detection limits for CPV-2b DNA were equivalent for iiPCR and real-time PCR. The iiPCR reaction detected CPV-2a, 2b and 2c and FPV. Non-targeted pathogens were not detected. Test results of real-time PCR and iiPCR from 99 fecal samples agreed with each other, while one real-time PCR-positive sample tested negative by iiPCR. Therefore, excellent agreement (k = 0.98) with sensitivity of 98.41% and specificity of 100% in detecting CPV-2 in feces was found between the two methods. In conclusion, the iiPCR system has potential to serve as a useful tool for rapid and accurate PON, molecular detection of CPV-2. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

PubMed Central

DeShields, Joseph B.; Bomberger, Rachel A.; Woodhall, James W.; Wheeler, David L.; Moroz, Natalia; Johnson, Dennis A.; Tanaka, Kiwamu

2018-01-01

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis. PMID:29553557

Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence.

PubMed

Yang, Litao; Xu, Songci; Pan, Aihu; Yin, Changsong; Zhang, Kewei; Wang, Zhenying; Zhou, Zhigang; Zhang, Dabing

2005-11-30

Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.

A real-time PCR diagnostic method for detection of Naegleria fowleri.

PubMed

Madarová, Lucia; Trnková, Katarína; Feiková, Sona; Klement, Cyril; Obernauerová, Margita

2010-09-01

Naegleria fowleri is a free-living amoeba that can cause primary amoebic meningoencephalitis (PAM). While, traditional methods for diagnosing PAM still rely on culture, more current laboratory diagnoses exist based on conventional PCR methods; however, only a few real-time PCR processes have been described as yet. Here, we describe a real-time PCR-based diagnostic method using hybridization fluorescent labelled probes, with a LightCycler instrument and accompanying software (Roche), targeting the Naegleria fowleriMp2Cl5 gene sequence. Using this method, no cross reactivity with other tested epidemiologically relevant prokaryotic and eukaryotic organisms was found. The reaction detection limit was 1 copy of the Mp2Cl5 DNA sequence. This assay could become useful in the rapid laboratory diagnostic assessment of the presence or absence of Naegleria fowleri. Copyright 2009 Elsevier Inc. All rights reserved.

Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

PubMed

Taverniers, Isabel; Van Bockstaele, Erik; De Loose, Marc

2004-03-01

Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

PubMed

Gianfranceschi, Monica Virginia; Rodriguez-Lazaro, David; Hernandez, Marta; González-García, Patricia; Comin, Damiano; Gattuso, Antonietta; Delibato, Elisabetta; Sonnessa, Michele; Pasquali, Frederique; Prencipe, Vincenza; Sreter-Lancz, Zuzsanna; Saiz-Abajo, María-José; Pérez-De-Juan, Javier; Butrón, Javier; Kozačinski, Lidija; Tomic, Danijela Horvatek; Zdolec, Nevijo; Johannessen, Gro S; Jakočiūnė, Džiuginta; Olsen, John Elmerdahl; De Santis, Paola; Lovari, Sarah; Bertasi, Barbara; Pavoni, Enrico; Paiusco, Antonella; De Cesare, Alessandra; Manfreda, Gerardo; De Medici, Dario

2014-08-01

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of Mycoplasma pneumoniae by real-time PCR.

PubMed

Winchell, Jonas M; Mitchell, Stephanie L

2013-01-01

Mycoplasma pneumoniae is a significant cause of respiratory disease, accounting for approximately 20% of cases of community-acquired pneumonia. Although several diagnostic methods exist to detect M. pneumoniae in respiratory specimens, real-time PCR has emerged as a significant improvement for the rapid diagnosis of this pathogen. The method described herein details the procedure for the detection of M. pneumoniae by real-time PCR (qPCR). The qPCR assay described can be performed with three targets specific for M. pneumoniae (Mp181, Mp3, and Mp7) and one marker for the detection of the RNaseP gene found in human nucleic acid as an internal control reaction. Recent studies have demonstrated the ability of this procedure to reliably identify this agent and facilitate the timely recognition of an outbreak.

Principles and applications of polymerase chain reaction in medical diagnostic fields: a review

PubMed Central

Valones, Marcela Agne Alves; Guimarães, Rafael Lima; Brandão, Lucas André Cavalcanti; de Souza, Paulo Roberto Eleutério; de Albuquerque Tavares Carvalho, Alessandra; Crovela, Sergio

2009-01-01

Recent developments in molecular methods have revolutionized the detection and characterization of microorganisms in a broad range of medical diagnostic fields, including virology, mycology, parasitology, microbiology and dentistry. Among these methods, Polymerase Chain Reaction (PCR) has generated great benefits and allowed scientific advancements. PCR is an excellent technique for the rapid detection of pathogens, including those difficult to culture. Along with conventional PCR techniques, Real-Time PCR has emerged as a technological innovation and is playing an ever-increasing role in clinical diagnostics and research laboratories. Due to its capacity to generate both qualitative and quantitative results, Real-Time PCR is considered a fast and accurate platform. The aim of the present literature review is to explore the clinical usefulness and potential of both conventional PCR and Real-Time PCR assays in diverse medical fields, addressing its main uses and advances. PMID:24031310

Real-time quantitative PCR of Staphylococcus aureus and application in restaurant meals.

PubMed

Berrada, H; Soriano, J M; Mañes, J; Picó, Y

2006-01-01

Staphylococcus aureus is considered the second most common pathogen to cause outbreaks of food poisoning, exceeded only by Campylobacter. Consumption of foods containing this microorganism is often identified as the cause of illness. In this study, a rapid, reliable, and sensitive real-time quantitative PCR was developed and compared with conventional culture methods. Real-time quantitative PCR was carried out by purifying DNA extracts of S. aureus with a Staphylococcus sample preparation kit and quantifying it in the LightCycler system with hybridization probes. The assay was linear from a range of 10 to 10(6) S. aureus cells (r2 > 0.997). The PCR reaction presented an efficiency of >85%. Accuracy of the PCR-based assay, expressed as percent bias, was around 13%, and the precision, expressed as a percentage of the coefficient of variation, was 7 to 10%. Intraday and interday variability were studied at 10(2) CFU/g and was 12 and 14%, respectively. The proposed method was applied to the analysis of 77 samples of restaurant meals in Valencia (Spain). In 11.6% of samples S. aureus was detected by real-time quantitative PCR, as well as by the conventional microbiological method. An excellent correspondence between real-time quantitative PCR and microbiological numbers (CFU/g) was observed with deviations of < 28%.

Development of a fast PCR protocol enabling rapid generation of AmpFℓSTR® Identifiler® profiles for genotyping of human DNA

PubMed Central

2012-01-01

Background Traditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. The purpose of this study was to develop and validate a fast PCR protocol that enabled amplification of the 16 loci targeted by the AmpFℓSTR® Identifiler® primer set, allowing decreased cycling times. Methods Fast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR™ HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach. Results The total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA. Conclusions The fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations. PMID:22394458

Effect of Sequence Polymorphisms on Performance of Two Real-Time PCR Assays for Detection of Herpes Simplex Virus

PubMed Central

Stevenson, Jeffery; Hymas, Weston; Hillyard, David

2005-01-01

Herpes simplex virus (HSV) is the most common cause of acquired, sporadic encephalitis in the United States. PCR identification of HSV in spinal fluid has become the diagnostic gold standard due to its sensitivity and potential for speed, replacing other methods such as culture. We developed a real-time PCR assay to detect HSV, using a new type of hybridization probe, the Eclipse probe. In this study, we ran 323 samples (171 positives and 152 negatives) with the Eclipse real-time PCR assay and compared these results with another PCR assay using gel detection. The real-time assay agreed with our reference method for 319 out of the 323 samples tested (99%). Using two different real-time PCR platforms, we discovered that SNPs within the amplicon's probe binding region that are used to distinguish HSV-1 from HSV-2 can decrease assay sensitivity. This problem is potentially a general one for assays using fluorescent probes to detect target amplification in a real-time format. While real-time PCR can be a powerful tool in the field of infectious disease, careful sequence evaluation and clinical validation are essential in creating an effective assay. PMID:15872272

Comparison of viable plate count, turbidity measurement and real-time PCR for quantification of Porphyromonas gingivalis.

PubMed

Clais, S; Boulet, G; Van Kerckhoven, M; Lanckacker, E; Delputte, P; Maes, L; Cos, P

2015-01-01

The viable plate count (VPC) is considered as the reference method for bacterial enumeration in periodontal microbiology but shows some important limitations for anaerobic bacteria. As anaerobes such as Porphyromonas gingivalis are difficult to culture, VPC becomes time-consuming and less sensitive. Hence, efficient normalization of experimental data to bacterial cell count requires alternative rapid and reliable quantification methods. This study compared the performance of VPC with that of turbidity measurement and real-time PCR (qPCR) in an experimental context using highly concentrated bacterial suspensions. Our TaqMan-based qPCR assay for P. gingivalis 16S rRNA proved to be sensitive and specific. Turbidity measurements offer a fast method to assess P. gingivalis growth, but suffer from high variability and a limited dynamic range. VPC was very time-consuming and less repeatable than qPCR. Our study concludes that qPCR provides the most rapid and precise approach for P. gingivalis quantification. Although our data were gathered in a specific research context, we believe that our conclusions on the inferior performance of VPC and turbidity measurements in comparison to qPCR can be extended to other research and clinical settings and even to other difficult-to-culture micro-organisms. Various clinical and research settings require fast and reliable quantification of bacterial suspensions. The viable plate count method (VPC) is generally seen as 'the gold standard' for bacterial enumeration. However, VPC-based quantification of anaerobes such as Porphyromonas gingivalis is time-consuming due to their stringent growth requirements and shows poor repeatability. Comparison of VPC, turbidity measurement and TaqMan-based qPCR demonstrated that qPCR possesses important advantages regarding speed, accuracy and repeatability. © 2014 The Society for Applied Microbiology.

A new real-time PCR protocol for detection of avian haemosporidians.

PubMed

Bell, Jeffrey A; Weckstein, Jason D; Fecchio, Alan; Tkach, Vasyl V

2015-07-19

Birds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction. Using available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR). The real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ(2) = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples. Our real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research.

Development of real-time PCR technique for the estimation of population density of Pythium intermedium in forest soils.

PubMed

Li, Mingzhu; Senda, Masako; Komatsu, Tsutomu; Suga, Haruhisa; Kageyama, Koji

2010-10-20

Pythium intermedium is known to play an important role in the carbon cycling of cool-temperate forest soils. In this study, a fast, precise and effective real-time PCR technique for estimating the population densities of P. intermedium from soils was developed using species-specific primers. Specificity was confirmed both with conventional PCR and real-time PCR. The detection limit (sensitivity) was determined and amplification standard curves were generated using SYBR Green II fluorescent dye. A rapid and accurate assay for quantification of P. intermedium in Takayama forest soils of Japan was developed using a combination of a new DNA extraction method and PCR primers were developed for real-time PCR. And the distribution of P. intermedium in forest soil was investigated with both soil plating method and the developed real-time PCR technique. This new technique will be a useful tool and can be applied to practical use for studying the role of Pythium species in forest and agricultural ecosystems. Copyright © 2009 Elsevier GmbH. All rights reserved.

[Analytical performances of real-time PCR by Abbott RealTime CMV with m2000 for the detection of cytomegalovirus in urine].

PubMed

De Monte, Anne; Cannavo, Isabelle; Caramella, Anne; Ollier, Laurence; Giordanengo, Valérie

2016-01-01

Congenital cytomegalovirus (CMV) infection is the leading cause of sensoneurinal disability due to infectious congenital disease. The diagnosis of congenital CMV infection is based on the search of CMV in the urine within the first two weeks of life. Viral culture of urine is the gold standard. However, the PCR is highly sensitive and faster. It is becoming an alternative choice. The objective of this study is the validation of real-time PCR by Abbott RealTime CMV with m2000 for the detection of cytomegalovirus in urine. Repeatability, reproducibility, detection limit and inter-sample contamination were evaluated. Urine samples from patients (n=141) were collected and analyzed simultaneously in culture and PCR in order to assess the correlation of these two methods. The sensitivity and specificity of PCR were also calculated. The Abbott RealTime CMV PCR in urine is an automated and sensitive method (detection limit 200 UI/mL). Fidelity is very good (standard deviation of repeatability: 0.08 to 0.15 LogUI/mL and reproducibility 0.18 LogUI/mL). We can note a good correlation between culture and Abbott RealTime CMV PCR (kappa 96%). When considering rapid culture as reference, real-time PCR was highly sensitive (100%) and specific (98.2%). The real-time PCR by Abbott RealTime CMV with m2000 is optimal for CMV detection in urine.

A PCR primer bank for quantitative gene expression analysis.

PubMed

Wang, Xiaowei; Seed, Brian

2003-12-15

Although gene expression profiling by microarray analysis is a useful tool for assessing global levels of transcriptional activity, variability associated with the data sets usually requires that observed differences be validated by some other method, such as real-time quantitative polymerase chain reaction (real-time PCR). However, non-specific amplification of non-target genes is frequently observed in the latter, confounding the analysis in approximately 40% of real-time PCR attempts when primer-specific labels are not used. Here we present an experimentally validated algorithm for the identification of transcript-specific PCR primers on a genomic scale that can be applied to real-time PCR with sequence-independent detection methods. An online database, PrimerBank, has been created for researchers to retrieve primer information for their genes of interest. PrimerBank currently contains 147 404 primers encompassing most known human and mouse genes. The primer design algorithm has been tested by conventional and real-time PCR for a subset of 112 primer pairs with a success rate of 98.2%.

Real-time PCR detection chemistry.

PubMed

Navarro, E; Serrano-Heras, G; Castaño, M J; Solera, J

2015-01-15

Real-time PCR is the method of choice in many laboratories for diagnostic and food applications. This technology merges the polymerase chain reaction chemistry with the use of fluorescent reporter molecules in order to monitor the production of amplification products during each cycle of the PCR reaction. Thus, the combination of excellent sensitivity and specificity, reproducible data, low contamination risk and reduced hand-on time, which make it a post-PCR analysis unnecessary, has made real-time PCR technology an appealing alternative to conventional PCR. The present paper attempts to provide a rigorous overview of fluorescent-based methods for nucleic acid analysis in real-time PCR described in the literature so far. Herein, different real-time PCR chemistries have been classified into two main groups; the first group comprises double-stranded DNA intercalating molecules, such as SYBR Green I and EvaGreen, whereas the second includes fluorophore-labeled oligonucleotides. The latter, in turn, has been divided into three subgroups according to the type of fluorescent molecules used in the PCR reaction: (i) primer-probes (Scorpions, Amplifluor, LUX, Cyclicons, Angler); (ii) probes; hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon, MGB-Pleiades, MGB-Eclipse, ResonSense, Yin-Yang or displacing); and (iii) analogues of nucleic acids (PNA, LNA, ZNA, non-natural bases: Plexor primer, Tiny-Molecular Beacon). In addition, structures, mechanisms of action, advantages and applications of such real-time PCR probes and analogues are depicted in this review. Copyright © 2014 Elsevier B.V. All rights reserved.

Development and validation of a real-time PCR assay for the detection of Toxoplasma gondii DNA in animal and meat samples.

PubMed

Marino, Anna Maria Fausta; Percipalle, Maurizio; Giunta, Renato Paolo; Salvaggio, Antonio; Caracappa, Giulia; Alfonzetti, Tiziana; Aparo, Alessandra; Reale, Stefano

2017-03-01

We report a rapid and reliable method for the detection of Toxoplasma gondii in meat and animal tissues based on real-time polymerase chain reaction (PCR). Samples were collected from cattle, small ruminants, horses, and pigs raised or imported into Sicily, Italy. All DNA preparations were assayed by real-time PCR tests targeted to a 98-bp long fragment in the AF 529-bp repeat element and to the B1 gene using specific primers. Diagnostic sensitivity (100%), diagnostic specificity (100%), limit of detection (0.01 pg), efficiency (92-109%), and precision (mean coefficient of variation = 0.60%), repeatability (100%), reproducibility (100%), and robustness were evaluated using 240 DNA extracted samples (120 positives and 120 negative as per the OIE nested PCR method) from different matrices. Positive results were confirmed by the repetition of both real-time and nested PCR assays. Our study demonstrates the viability of a reliable, rapid, and specific real-time PCR on a large scale to monitor contamination with Toxoplasma cysts in meat and animal specimens. This validated method can be used for postmortem detection in domestic and wild animals and for food safety purposes.

TaqMan based real time PCR assay targeting EML4-ALK fusion transcripts in NSCLC.

PubMed

Robesova, Blanka; Bajerova, Monika; Liskova, Kvetoslava; Skrickova, Jana; Tomiskova, Marcela; Pospisilova, Sarka; Mayer, Jiri; Dvorakova, Dana

2014-07-01

Lung cancer with the ALK rearrangement constitutes only a small fraction of patients with non-small cell lung cancer (NSCLC). However, in the era of molecular-targeted therapy, efficient patient selection is crucial for successful treatment. In this context, an effective method for EML4-ALK detection is necessary. We developed a new highly sensitive variant specific TaqMan based real time PCR assay applicable to RNA from formalin-fixed paraffin-embedded tissue (FFPE). This assay was used to analyze the EML4-ALK gene in 96 non-selected NSCLC specimens and compared with two other methods (end-point PCR and break-apart FISH). EML4-ALK was detected in 33/96 (34%) specimens using variant specific real time PCR, whereas in only 23/96 (24%) using end-point PCR. All real time PCR positive samples were confirmed with direct sequencing. A total of 46 specimens were subsequently analyzed by all three detection methods. Using variant specific real time PCR we identified EML4-ALK transcript in 17/46 (37%) specimens, using end-point PCR in 13/46 (28%) specimens and positive ALK rearrangement by FISH was detected in 8/46 (17.4%) specimens. Moreover, using variant specific real time PCR, 5 specimens showed more than one EML4-ALK variant simultaneously (in 2 cases the variants 1+3a+3b, in 2 specimens the variants 1+3a and in 1 specimen the variant 1+3b). In one case of 96 EML4-ALK fusion gene and EGFR mutation were detected. All simultaneous genetic variants were confirmed using end-point PCR and direct sequencing. Our variant specific real time PCR assay is highly sensitive, fast, financially acceptable, applicable to FFPE and seems to be a valuable tool for the rapid prescreening of NSCLC patients in clinical practice, so, that most patients able to benefit from targeted therapy could be identified. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Direct PCR - A rapid method for multiplexed detection of different serotypes of Salmonella in enriched pork meat samples.

PubMed

Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Quyen, Than Linh; Engelsmann, Pia; Wolff, Anders; Bang, Dang Duong

2017-04-01

Salmonellosis, an infectious disease caused by Salmonella spp., is one of the most common foodborne diseases. Isolation and identification of Salmonella by conventional bacterial culture method is time consuming. In response to the demand for rapid on line or at site detection of pathogens, in this study, we developed a multiplex Direct PCR method for rapid detection of different Salmonella serotypes directly from pork meat samples without any DNA purification steps. An inhibitor-resistant Phusion Pfu DNA polymerase was used to overcome PCR inhibition. Four pairs of primers including a pair of newly designed primers targeting Salmonella spp. at subtype level were incorporated in the multiplex Direct PCR. To maximize the efficiency of the Direct PCR, the ratio between sample and dilution buffer was optimized. The sensitivity and specificity of the multiplex Direct PCR were tested using naturally contaminated pork meat samples for detecting and subtyping of Salmonella spp. Conventional bacterial culture methods were used as reference to evaluate the performance of the multiplex Direct PCR. Relative accuracy, sensitivity and specificity of 98.8%; 97.6% and 100%, respectively, were achieved by the method. Application of the multiplex Direct PCR to detect Salmonella in pork meat at slaughter reduces the time of detection from 5 to 6 days by conventional bacterial culture and serotyping methods to 14 h (including 12 h enrichment time). Furthermore, the method poses a possibility of miniaturization and integration into a point-of-need Lab-on-a-chip system for rapid online pathogen detection. Copyright © 2016 Elsevier Ltd. All rights reserved.

[A novel TaqMan® MGB probe for specifically detecting Streptococcus mutans].

PubMed

Zheng, Hui; Lin, Jiu-Xiang; DU, Ning; Chen, Feng

2013-10-18

To design a new TaqMan® MGB probe for improving the specificity of Streptococcus mutans's detection. We extracted six DNA samples from different streptococcal strains for PCR reaction. Conventional nested PCR and TaqMan® MGB real-time PCR were applied independently. The first round of nested PCR was carried out with the bacterial universal primers, while a second PCR was conducted by using primers specific for the 16S rRNA gene of Streptococcus mutans. The TaqMan® MGB probe for Streptococcus mutans was designed from sequence analyses, and the primers were the same as nested PCR. Streptococcus mutans DNA with 2.5 mg/L was sequentially diluted at 5-fold intervals to 0.16 μg/L. Standard DNA samples were used to generate standard curves by TaqMan® MGB real-time PCR. In the nested PCR, the primers specific for Streptococcus mutans also detected Streptococcus gordonii with visible band of 282 bp, giving false-positive results. In the TaqMan® MGB real-time PCR reaction, only Streptococcus mutans was detected. The detection limitation of TaqMan® MGB real-time PCR for Streptococcus mutans 16S rRNA gene was 20 μg/L. We designed a new TaqMan® MGB probe, and successfully set up a PCR based method for detecting oral Streptococcus mutans. TaqMan® MGB real-time PCR is a both specific and sensitive bacterial detection method.

Development and Evaluation of Event-Specific Quantitative PCR Method for Genetically Modified Soybean MON87701.

PubMed

Tsukahara, Keita; Takabatake, Reona; Masubuchi, Tomoko; Futo, Satoshi; Minegishi, Yasutaka; Noguchi, Akio; Kondo, Kazunari; Nishimaki-Mogami, Tomoko; Kurashima, Takeyo; Mano, Junichi; Kitta, Kazumi

2016-01-01

A real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event, MON87701. First, a standard plasmid for MON87701 quantification was constructed. The conversion factor (C f ) required to calculate the amount of genetically modified organism (GMO) was experimentally determined for a real-time PCR instrument. The determined C f for the real-time PCR instrument was 1.24. For the evaluation of the developed method, a blind test was carried out in an inter-laboratory trial. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr), respectively. The determined biases and the RSDr values were less than 30 and 13%, respectively, at all evaluated concentrations. The limit of quantitation of the method was 0.5%, and the developed method would thus be applicable for practical analyses for the detection and quantification of MON87701.

Development and validation of a SYBR Green I-based real-time polymerase chain reaction method for detection of haptoglobin gene deletion in clinical materials.

PubMed

Soejima, Mikiko; Tsuchiya, Yuji; Egashira, Kouichi; Kawano, Hiroyuki; Sagawa, Kimitaka; Koda, Yoshiro

2010-06-01

Anhaptoglobinemic patients run the risk of severe anaphylactic transfusion reaction because they produce serum haptoglobin (Hp) antibodies. Being homozygous for the Hp gene deletion (HP(del)) is the only known cause of congenital anhaptoglobinemia, and clinical diagnosis of HP(del) before transfusion is important to prevent anaphylactic shock. We recently developed a 5'-nuclease (TaqMan) real-time polymerase chain reaction (PCR) method. A SYBR Green I-based duplex real-time PCR assay using two forward primers and a common reverse primer followed by melting curve analysis was developed to determine HP(del) zygosity in a single tube. In addition, to obviate initial DNA extraction, we examined serially diluted blood samples as PCR templates. Allelic discrimination of HP(del) yielded optimal results at blood sample dilutions of 1:64 to 1:1024. The results from 2231 blood samples were fully concordant with those obtained by the TaqMan-based real-time PCR method. The detection rate of the HP(del) allele by the SYBR Green I-based method is comparable with that using the TaqMan-based method. This method is readily applicable due to its low initial cost and analyzability using economical real-time PCR machines and is suitable for high-throughput analysis as an alternative method for allelic discrimination of HP(del).

Evaluation of Legionella pneumophila contamination in Italian hotel water systems by quantitative real-time PCR and culture methods.

PubMed

Bonetta, Sa; Bonetta, Si; Ferretti, E; Balocco, F; Carraro, E

2010-05-01

This study was designed to define the extent of water contamination by Legionella pneumophila of certain Italian hotels and to compare quantitative real-time PCR with the conventional culture method. Nineteen Italian hotels of different sizes were investigated. In each hotel three hot water samples (boiler, room showers, recycling) and one cold water sample (inlet) were collected. Physico-chemical parameters were also analysed. Legionella pneumophila was detected in 42% and 74% of the hotels investigated by the culture method and by real-time PCR, respectively. In 21% of samples analysed by the culture method, a concentration of >10(4) CFU l(-1) was found, and Leg. pneumophila serogroup 1 was isolated from 10.5% of the hotels. The presence of Leg. pneumophila was significantly influenced by water sample temperature, while no association with water hardness or residual-free chlorine was found. This study showed a high percentage of buildings colonized by Leg. pneumophila. Moreover, real-time PCR proved to be sensitive enough to detect lower levels of contamination than the culture method. This study indicates that the Italian hotels represent a possible source of risk for Legionnaires' disease and confirms the sensitivity of the molecular method. To our knowledge, this is the first report to demonstrate Legionella contamination in Italian hotels using real-time PCR and culture methods.

Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

PubMed

Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

2015-05-05

Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

Evaluation of a real-time PCR assay for malaria diagnosis in patients from Vietnam and in returned travellers.

PubMed

Vo, Thi Kim Duy; Bigot, Patricia; Gazin, Pierre; Sinou, Veronique; De Pina, Jean Jacques; Huynh, Dinh Chien; Fumoux, Francis; Parzy, Daniel

2007-05-01

Real-time PCR diagnosis of malaria has advantages over traditional microscopic methods, especially when parasitaemia is low and when dealing with mixed infections. We have developed a new real-time PCR with specific genes in each Plasmodium species present only in one copy to identify the four pathogenic Plasmodium spp. for humans. The sensitivity was less than 25 parasites/microl. No cross-hybridisation was observed with human DNA or among the four Plasmodium spp. Using LightCycler PCR and conventional microscopy, we compared the diagnosis of malaria in patients from Vietnam and in returned European travellers with suspicion of malaria. In patients from Vietnam with suspicion of malaria, one mixed infection was observed by PCR only; the remaining data (54 of 55 patients) correlated with microscopy. In 79 patients without symptoms, low parasitaemia was detected in 7 samples by microscopy and in 16 samples by PCR. In returned travellers, PCR results were correlated with microscopy for all four species in 48 of 56 samples. The eight discrepant results were resolved in favour of real-time PCR diagnosis. This new real-time PCR is a rapid, accurate and efficient method for malaria diagnosis in returned travellers as well as for epidemiological studies or antimalarial efficiency trials in the field.

Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis.

PubMed

Choi, Yeonim; Jeon, Bo-Young; Shim, Tae Sun; Jin, Hyunwoo; Cho, Sang-Nae; Lee, Hyeyoung

2014-12-01

Rapid, accurate detection of Mycobacterium tuberculosis is crucial in the diagnosis of tuberculosis (TB), but conventional diagnostic methods have limited sensitivity and specificity or are time consuming. A new highly sensitive nucleic acid amplification test, combined nested and real-time polymerase chain reaction (PCR) in a single tube (one-tube nested real-time PCR), was developed for detecting M. tuberculosis, which takes advantage of two PCR techniques, i.e., nested PCR and real-time PCR. One-tube nested real-time PCR was designed to have two sequential reactions with two sets of primers and dual probes for the insertion sequence (IS) 6110 sequence of M. tuberculosis in a single closed tube. The minimum limits of detection of IS6110 real-time PCR and IS6110 one-tube nested real-time PCR were 100 fg/μL and 1 fg/μL of M. tuberculosis DNA, respectively. AdvanSure TB/non-tuberculous mycobacteria (NTM) real-time PCR, IS6110 real-time PCR, and two-tube nested real-time PCR showed 100% sensitivity and 100% specificity for clinical M. tuberculosis isolates and NTM isolates. In comparison, the sensitivities of AdvanSure TB/NTM real-time PCR, single IS6110 real-time PCR, and one-tube nested real-time PCR were 91% (152/167), 94.6% (158/167), and 100% (167/167) for sputum specimens, respectively. In conclusion, IS6110 one-tube nested real-time PCR is useful for detecting M. tuberculosis due to its high sensitivity and simple manipulation. Copyright © 2014 Elsevier Inc. All rights reserved.

DETECTION OF FECAL ENTEROCOCCI USING A REAL TIME PCR METHOD

EPA Science Inventory

In spite of their importance in public health, the detection of fecal enterococci is performed via culturing methods that are time consuming and that are subject to inaccuracies that relate to their culturable status. In order to address these problems, a real time PCR (TaqMan) ...

A rapid single-tube protocol for HAV detection by nested real-time PCR.

PubMed

Hu, Yuan; Arsov, Ivica

2014-09-01

Infections by food-borne viruses such as hepatitis A virus (HAV) and norovirus are significant public health concerns worldwide. Since food-borne viruses are rarely confirmed through direct isolation from contaminated samples, highly sensitive molecular techniques remain the methods of choice for the detection of viral genetic material. Our group has previously developed a specific nested real-time PCR (NRT-PCR) assay for HAV detection that improved overall sensitivity. Furthermore in this study, we have developed a single-tube NRT-PCR approach for HAV detection in food samples that reduces the likelihood of cross contamination between tubes during sample manipulation. HAV RNA was isolated from HAV-spiked food samples and HAV-infected cell cultures. All reactions following HAV RNA isolation, including conventional reverse transcriptase PCR, nested-PCR, and RT-PCR were performed in a single tube. Our results demonstrated that all the samples tested positive by RT-PCR and nested-PCR were also positive by a single-tube NRT-PCR. The detection limits observed for HAV-infected cell cultures and HAV-spiked green onions were 0.1 and 1 PFU, respectively. This novel method retained the specificity and robustness of the original NRT-PCR method, while greatly reducing sample manipulation, turnaround time, and the risk of carry-over contamination. Single-tube NRT-PCR thus represents a promising new tool that can potentially facilitate the detection of HAV in foods thereby improving food safety and public health.

A temperature control method for shortening thermal cycling time to achieve rapid polymerase chain reaction (PCR) in a disposable polymer microfluidic device

NASA Astrophysics Data System (ADS)

Bu, Minqiang; Perch-Nielsen, Ivan R.; Sørensen, Karen S.; Skov, Julia; Sun, Yi; Duong Bang, Dang; Pedersen, Michael E.; Hansen, Mikkel F.; Wolff, Anders

2013-07-01

We present a temperature control method capable of effectively shortening the thermal cycling time of polymerase chain reaction (PCR) in a disposable polymer microfluidic device with an external heater and a temperature sensor. The method employs optimized temperature overshooting and undershooting steps to achieve a rapid ramping between the temperature steps for DNA denaturation, annealing and extension. The temperature dynamics within the microfluidic PCR chamber was characterized and the overshooting and undershooting parameters were optimized using the temperature-dependent fluorescence signal from Rhodamine B. The method was validated with the PCR amplification of mecA gene (162 bp) from methicillin-resistant Staphylococcus aureus bacterium (MRSA), where the time for 30 cycles was reduced from 50 min (without over- and undershooting) to 20 min.

A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

PubMed Central

Coudray-Meunier, Coralie; Fraisse, Audrey; Martin-Latil, Sandra; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

2016-01-01

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log10. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log10. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log10 lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log10. Conversely, sensitivity was only 0.30 log10 better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health. PMID:26824897

Evaluation of loop-mediated isothermal amplification method (LAMP) for pathogenic Leptospira spp. detection with leptospires isolation and real-time PCR.

PubMed

Suwancharoen, Duangjai; Sittiwicheanwong, Busara; Wiratsudakul, Anuwat

2016-09-01

Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic Leptospira spp. Many molecular techniques have consecutively been developed to detect such pathogen including loop-mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold standard and to assess the agreement among these three tests using Cohen's kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5-99.8) and 97.0% (95% CI 94.9-98.2), respectively. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen's kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), respectively. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle.

A semi-nested real-time PCR method to detect low chimerism percentage in small quantity of hematopoietic stem cell transplant DNA samples.

PubMed

Aloisio, Michelangelo; Bortot, Barbara; Gandin, Ilaria; Severini, Giovanni Maria; Athanasakis, Emmanouil

2017-02-01

Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.

Tendency for interlaboratory precision in the GMO analysis method based on real-time PCR.

PubMed

Kodama, Takashi; Kurosawa, Yasunori; Kitta, Kazumi; Naito, Shigehiro

2010-01-01

The Horwitz curve estimates interlaboratory precision as a function only of concentration, and is frequently used as a method performance criterion in food analysis with chemical methods. The quantitative biochemical methods based on real-time PCR require an analogous criterion to progressively promote method validation. We analyzed the tendency of precision using a simplex real-time PCR technique in 53 collaborative studies of seven genetically modified (GM) crops. Reproducibility standard deviation (SR) and repeatability standard deviation (Sr) of the genetically modified organism (GMO) amount (%) was more or less independent of GM crops (i.e., maize, soybean, cotton, oilseed rape, potato, sugar beet, and rice) and evaluation procedure steps. Some studies evaluated whole steps consisting of DNA extraction and PCR quantitation, whereas others focused only on the PCR quantitation step by using DNA extraction solutions. Therefore, SR and Sr for GMO amount (%) are functions only of concentration similar to the Horwitz curve. We proposed S(R) = 0.1971C 0.8685 and S(r) = 0.1478C 0.8424, where C is the GMO amount (%). We also proposed a method performance index in GMO quantitative methods that is analogous to the Horwitz Ratio.

Human fecal source identification with real-time quantitative PCR

EPA Science Inventory

Waterborne diseases represent a significant public health risk worldwide, and can originate from contact with water contaminated with human fecal material. We describe a real-time quantitative PCR (qPCR) method that targets a Bacteroides dori human-associated genetic marker for...

[Comparative analysis between diatom nitric acid digestion method and plankton 16S rDNA PCR method].

PubMed

Han, Jun-ge; Wang, Cheng-bao; Li, Xing-biao; Fan, Yan-yan; Feng, Xiang-ping

2013-10-01

To compare and explore the application value of diatom nitric acid digestion method and plankton 16S rDNA PCR method for drowning identification. Forty drowning cases from 2010 to 2011 were collected from Department of Forensic Medicine of Wenzhou Medical University. Samples including lung, kidney, liver and field water from each case were tested with diatom nitric acid digestion method and plankton 16S rDNA PCR method, respectively. The Diatom nitric acid digestion method and plankton 16S rDNA PCR method required 20 g and 2 g of each organ, and 15 mL and 1.5 mL of field water, respectively. The inspection time and detection rate were compared between the two methods. Diatom nitric acid digestion method mainly detected two species of diatoms, Centriae and Pennatae, while plankton 16S rDNA PCR method amplified a length of 162 bp band. The average inspection time of each case of the Diatom nitric acid digestion method was (95.30 +/- 2.78) min less than (325.33 +/- 14.18) min of plankton 16S rDNA PCR method (P < 0.05). The detection rates of two methods for field water and lung were both 100%. For liver and kidney, the detection rate of plankton 16S rDNA PCR method was both 80%, higher than 40% and 30% of diatom nitric acid digestion method (P < 0.05), respectively. The laboratory testing method needs to be appropriately selected according to the specific circumstances in the forensic appraisal of drowning. Compared with diatom nitric acid digestion method, plankton 16S rDNA PCR method has practice values with such advantages as less quantity of samples, huge information and high specificity.

[Application of recombinase polymerase amplification in the detection of Pseudomonas aeruginosa].

PubMed

Jin, X J; Gong, Y L; Yang, L; Mo, B H; Peng, Y Z; He, P; Zhao, J N; Li, X L

2018-04-20

Objective: To establish an optimized method of recombinase polymerase amplification (RPA) to rapidly detect Pseudomonas aeruginosa in clinic. Methods: (1) The DNA templates of one standard Pseudomonas aeruginosa strain was extracted and detected by polymerase chain reaction (PCR), real-time fluorescence quantitative PCR and RPA. Time of sample loading, time of amplification, and time of detection of the three methods were recorded. (2) One standard Pseudomonas aeruginosa strain was diluted in 7 concentrations of 1×10(7,) 1×10(6,) 1×10(5,) 1×10(4,) 1×10(3,) 1×10(2,) and 1×10(1) colony forming unit (CFU)/mL after recovery and cultivation. The DNA templates of Pseudomonas aeruginosa and negative control strain Pseudomonas putida were extracted and detected by PCR, real-time fluorescence quantitative PCR, and RPA separately. The sensitivity of the three methods in detecting Pseudomonas aeruginosa was analyzed. (3) The DNA templates of one standard Pseudomonas aeruginosa strain and four negative control strains ( Staphylococcus aureus, Acinetobacter baumanii, Candida albicans, and Pseudomonas putida ) were extracted separately, and then they were detected by PCR, real-time fluorescence quantitative PCR, and RPA. The specificity of the three methods in detecting Pseudomonas aeruginosa was analyzed. (4) The DNA templates of 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin, 1 clinical strain of which was taken by cotton swab, and negative control strain Pseudomonas putida were extracted separately, and then they were detected by RPA. Positive amplification signals of the clinical strains were observed, and the detection rate was calculated. All experiments were repeated for 3 times. Sensitivity results were analyzed by GraphPad Prism 5.01 statistical software. Results: (1) The loading time of RPA, PCR, and real-time fluorescence quantitative PCR for detecting Pseudomonas aeruginosa were all 20 minutes. In PCR, time of amplification was 98 minutes, time of gel detection was 20 minutes, and the total time was 138 minutes. In real-time fluorescence quantitative PCR, amplification and detection could be completed simultaneously, which took 90 minutes, and the total time was 110 minutes. In RPA, amplification and detection could also be completed simultaneously, which took 15 minutes, and the total time was 35 minutes. (2) Pseudomonas putida did not show positive amplification signals or gel positive results in any of the three detection methods. The detection limit of Pseudomonas aeruginosa in real-time fluorescence quantitative PCR and PCR was 1×10(1) CFU/mL, and that of Pseudomonas aeruginosa in RPA was 1×10(2) CFU/mL. In RPA and real-time fluorescence quantitative PCR, the higher the concentration of Pseudomonas aeruginosa, the shorter threshold time and smaller the number of cycles, namely shorter time for detecting the positive amplified signal. In real-time fluorescence quantitative PCR, all positive amplification signal could be detected when the concentration of Pseudomonas aeruginosa was 1×10(1)-1×10(7) CFU/mL. In RPA, the detection rate of positive amplification signal was 0 when the concentration of Pseudomonas aeruginosa was 1×10(1) CFU/mL, while the detection rate of positive amplification signal was 67% when the concentration of Pseudomonas aeruginosa was 1×10(2) CFU/mL, and the detection rate of positive amplification signal was 100% when the concentration of Pseudomonas aeruginosa was 1×10(3)-1×10(7) CFU/mL. (3) In RPA, PCR, and real-time fluorescence quantitative PCR, Pseudomonas aeruginosa showed positive amplification signals and gel positive results, but there were no positive amplification signals or gel positive results in four negative control strains of Acinetobacter baumannii, Staphylococcus aureus, Candida albicans, and Pseudomonas putida . (4) In RPA, 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin and 1 clinical strain of Pseudomonas aeruginosa taken by cotton swab showed positive amplification signals, while Pseudomonas putida did not show positive amplification signal. The detection rate of positive amplification signal of 29 clinical strains of Pseudomonas aeruginosa in RPA was 100%. Conclusions: The established optimized RPA technology for fast detection of Pseudomonas aeruginosa requires shorter time, with high sensitivity and specificity. It was of great value in fast detection of Pseudomonas aeruginosa infection in clinic.

A novel quantitative real-time polymerase chain reaction method for detecting toxigenic Pasteurella multocida in nasal swabs from swine.

PubMed

Scherrer, Simone; Frei, Daniel; Wittenbrink, Max Michael

2016-12-01

Progressive atrophic rhinitis (PAR) in pigs is caused by toxigenic Pasteurella multocida. In Switzerland, PAR is monitored by selective culture of nasal swabs and subsequent polymerase chain reaction (PCR) screening of bacterial colonies for the P. multocida toxA gene. A panel of 203 nasal swabs from a recent PAR outbreak were used to evaluate a novel quantitative real-time PCR for toxigenic P. multocida in porcine nasal swabs. In comparison to the conventional PCR with a limit of detection of 100 genome equivalents per PCR reaction, the real-time PCR had a limit of detection of 10 genome equivalents. The real-time PCR detected toxA-positive P. multocida in 101 samples (49.8%), whereas the conventional PCR was less sensitive with 90 toxA-positive samples (44.3%). In comparison to the real-time PCR, 5.4% of the toxA-positive samples revealed unevaluable results by conventional PCR. The approach of culture-coupled toxA PCR for the monitoring of PAR in pigs is substantially improved by a novel quantitative real-time PCR.

Comparison of culture and qPCR methods in detection of mycobacteria from drinking waters.

PubMed

Räsänen, Noora H J; Rintala, Helena; Miettinen, Ilkka T; Torvinen, Eila

2013-04-01

Environmental mycobacteria are common bacteria in man-made water systems and may cause infections and hypersensitivity pneumonitis via exposure to water. We compared a generally used cultivation method and a quantitative polymerase chain reaction (qPCR) method to detect mycobacteria in 3 types of drinking waters: surface water, ozone-treated surface water, and groundwater. There was a correlation between the numbers of mycobacteria obtained by cultivation and qPCR methods, but the ratio of the counts obtained by the 2 methods varied among the types of water. The qPCR counts in the drinking waters produced from surface or groundwater were 5 to 34 times higher than culturable counts. In ozone-treated surface waters, both methods gave similar counts. The ozone-treated drinking waters had the highest concentration of assimilable organic carbon, which may explain the good culturability. In warm tap waters, qPCR gave 43 times higher counts than cultivation, but both qPCR counts and culturable counts were lower than those in the drinking waters collected from the same sites. The TaqMan qPCR method is a rapid and sensitive tool for total quantitation of mycobacteria in different types of clean waters. The raw water source and treatments affect both culturability and total numbers of mycobacteria in drinking waters.

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR.

PubMed

Aparecida de Oliveira, Maria; Abeid Ribeiro, Eliana Guimarães; Morato Bergamini, Alzira Maria; Pereira De Martinis, Elaine Cristina

2010-02-01

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE QPCR SYBR Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.

Distribution of Genetic Marker Concentrations for Fecal Indicator Bacteria in Sewage and Animal Feces

EPA Science Inventory

The application of quantitative real-time PCR (qPCR) methods for the identification of fecal microorganisms in surface waters has the potential to revolutionize water quality monitoring worldwide. Unlike traditional cultivation methods, qPCR estimates the concentration of gen...

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

PubMed

Abras, Alba; Ballart, Cristina; Llovet, Teresa; Roig, Carme; Gutiérrez, Cristina; Tebar, Silvia; Berenguer, Pere; Pinazo, María-Jesús; Posada, Elizabeth; Gascón, Joaquim; Schijman, Alejandro G; Gállego, Montserrat; Muñoz, Carmen

2018-01-01

Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.

Finding the joker among the maize endogenous reference genes for genetically modified organism (GMO) detection.

PubMed

Paternò, Annalisa; Marchesi, Ugo; Gatto, Francesco; Verginelli, Daniela; Quarchioni, Cinzia; Fusco, Cristiana; Zepparoni, Alessia; Amaddeo, Demetrio; Ciabatti, Ilaria

2009-12-09

The comparison of five real-time polymerase chain reaction (PCR) methods targeted at maize ( Zea mays ) endogenous sequences is reported. PCR targets were the alcohol dehydrogenase (adh) gene for three methods and high-mobility group (hmg) gene for the other two. The five real-time PCR methods have been checked under repeatability conditions at several dilution levels on both pooled DNA template from several genetically modified (GM) maize certified reference materials (CRMs) and single CRM DNA extracts. Slopes and R(2) coefficients of all of the curves obtained from the adopted regression model were compared within the same method and among all of the five methods, and the limit of detection and limit of quantitation were analyzed for each PCR system. Furthermore, method equivalency was evaluated on the basis of the ability to estimate the target haploid genome copy number at each concentration level. Results indicated that, among the five methods tested, one of the hmg-targeted PCR systems can be considered equivalent to the others but shows the best regression parameters and a higher repeteability along the dilution range. Thereby, it is proposed as a valid module to be coupled to different event-specific real-time PCR for maize genetically modified organism (GMO) quantitation. The resulting practicability improvement on the analytical control of GMOs is discussed.

Autoclave method for rapid preparation of bacterial PCR-template DNA.

PubMed

Simmon, Keith E; Steadman, Dewey D; Durkin, Sarah; Baldwin, Amy; Jeffrey, Wade H; Sheridan, Peter; Horton, Rene; Shields, Malcolm S

2004-02-01

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.

Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

PubMed Central

Alam, Mohammad J.; Tisdel, Naradah L.; Shah, Dhara N.; Yapar, Mehmet; Lasco, Todd M.; Garey, Kevin W.

2015-01-01

Background The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. Methods The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. Results A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. Conclusions The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run. PMID:25932438

Comparing real-time and conventional PCR to culture-based methods for detecting and quantifying Escherichia coli O157 in cattle feces.

PubMed

Jacob, M E; Bai, J; Renter, D G; Rogers, A T; Shi, X; Nagaraja, T G

2014-02-01

Detection of Escherichia coli O157 in cattle feces has traditionally used culture-based methods; PCR-based methods have been suggested as an alternative. We aimed to determine if multiplex real-time (mq) or conventional PCR methods could reliably detect cattle naturally shedding high (≥10(4) CFU/g of feces) and low (∼10(2) CFU/g of feces) concentrations of E. coli O157. Feces were collected from pens of feedlot cattle and evaluated for E. coli O157 by culture methods. Samples were categorized as (i) high shedders, (ii) immunomagnetic separation (IMS) positive after enrichment, or (iii) culture negative. DNA was extracted pre- and postenrichment from 100 fecal samples from each category (high shedder, IMS positive, culture negative) and subjected to mqPCR and conventional PCR assays based on detecting three genes, rfbE, stx1, and stx2. In feces from cattle determined to be E. coli O157 high shedders by culture, 37% were positive by mqPCR prior to enrichment; 85% of samples were positive after enrichment. In IMS-positive samples, 4% were positive by mqPCR prior to enrichment, while 43% were positive after enrichment. In culture-negative feces, 7% were positive by mqPCR prior to enrichment, and 40% were positive after enrichment. The proportion of high shedder-positive and culture-positive (high shedder and IMS) samples were significantly different from mqPCR-positive samples before and after enrichment (P < 0.01). Similar results were observed for conventional PCR. Our data suggest that mqPCR and conventional PCR are most useful in identifying high shedder animals and may not be an appropriate substitute to culture-based methods for detection of E. coli O157 in cattle feces.

Molecular detection of Toxoplasma gondii in water samples from Scotland and a comparison between the 529bp real-time PCR and ITS1 nested PCR.

PubMed

Wells, Beth; Shaw, Hannah; Innocent, Giles; Guido, Stefano; Hotchkiss, Emily; Parigi, Maria; Opsteegh, Marieke; Green, James; Gillespie, Simon; Innes, Elisabeth A; Katzer, Frank

2015-12-15

Waterborne transmission of Toxoplasma gondii is a potential public health risk and there are currently no agreed optimised methods for the recovery, processing and detection of T. gondii oocysts in water samples. In this study modified methods of T. gondii oocyst recovery and DNA extraction were applied to 1427 samples collected from 147 public water supplies throughout Scotland. T. gondii DNA was detected, using real time PCR (qPCR) targeting the 529bp repeat element, in 8.79% of interpretable samples (124 out of 1411 samples). The samples which were positive for T. gondii DNA originated from a third of the sampled water sources. The samples which were positive by qPCR and some of the negative samples were reanalysed using ITS1 nested PCR (nPCR) and results compared. The 529bp qPCR was the more sensitive technique and a full analysis of assay performance, by Bayesian analysis using a Markov Chain Monte Carlo method, was completed which demonstrated the efficacy of this method for the detection of T. gondii in water samples. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples.

PubMed

Mokhtari, W; Nsaibia, S; Gharbi, A; Aouni, M

2013-02-01

Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen. A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T(m) = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C(q)) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea. The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples. Copyright © 2012 Elsevier Ltd. All rights reserved.

Incidence of pulmonary aspergillosis and correlation of conventional diagnostic methods with nested PCR and real-time PCR assay using BAL fluid in intensive care unit patients.

PubMed

Zarrinfar, Hossein; Makimura, Koichi; Satoh, Kazuo; Khodadadi, Hossein; Mirhendi, Hossein

2013-05-01

Although the incidence of invasive aspergillosis in the intensive care unit (ICU) is scarce, it has emerged as major problems in critically ill patients. In this study, the incidence of pulmonary aspergillosis (PA) in ICU patients has evaluated and direct microscopy and culture has compared with nested polymerase chain reaction (PCR) and real-time PCR for detection of Aspergillus fumigatus and A. flavus in bronchoalveolar lavage (BAL) samples of the patients. Thirty BAL samples obtained from ICU patients during a 16-month period were subjected to direct examinations on 20% potassium hydroxide (KOH) and culture on two culture media. Nested PCR targeting internal transcribed spacer ribosomal DNA and TaqMan real-time PCR assay targeting β-tubulin gene were used for the detection of A. fumigatus and A. flavus. Of 30 patients, 60% were men and 40% were women. The diagnosis of invasive PA was probable in 1 (3%), possible in 11 (37%), and not IPA in 18 (60%). Nine samples were positive in nested PCR including seven samples by A. flavus and two by A. fumigatus specific primers. The lowest amount of DNA that TaqMan real-time PCR could detect was ≥40 copy numbers. Only one of the samples had a positive result of A. flavus real-time PCR with Ct value of 37.5. Although a significant number of specimens were positive in nested PCR, results of this study showed that establishment of a correlation between the conventional methods with nested PCR and real-time PCR needs more data confirmed by a prospective study with a larger sample group. © 2013 Wiley Periodicals, Inc.

A new diagnostic real-time PCR method for huanglongbing detection in citrus root tissue

USDA-ARS?s Scientific Manuscript database

Citrus fibrous root tissue was evaluated as an alternative source material for Huanglongbing (HLB) diagnosis by real-time PCR using primer-probe set TXCChlb, developed in the present study based on 16S rDNA of “Candidatus Liberibacter asiaticus” (CLas). Real-time PCR data obtained with DNA samples p...

Development of a real-time PCR assay with an internal amplification control for detection of Gram-negative histamine-producing bacteria in fish.

PubMed

Bjornsdottir-Butler, Kristin; Jones, Jessica L; Benner, Ronald; Burkhardt, William

2011-05-01

Prompt detection of bacteria that contribute to scombrotoxin (histamine) fish poisoning can aid in the detection of potentially toxic fish products and prevent the occurrence of illness. We report development of the first real-time PCR method for rapid detection of Gram-negative histamine-producing bacteria (HPB) in fish. The real-time PCR assay was 100% inclusive for detecting high-histamine producing isolates and did not detect any of the low- or non-histamine producing isolates. The efficiency of the assay with/without internal amplification control ranged from 96-104% and in the presence of background flora and inhibitory matrices was 92/100% and 73-96%, respectively. This assay was used to detect HPB from naturally contaminated yellowfin tuna, bluefish, and false albacore samples. Photobacterium damselae (8), Plesiomonas shigelloides (2), Shewanella sp. (1), and Morganella morganii (1) were subsequently isolated from the real-time PCR positive fish samples. These results indicate that the real-time PCR assay developed in this study is a rapid and sensitive method for detecting high-HPB. The assay may be adapted for quantification of HPB, either directly or with an MPN-PCR method. Copyright © 2010. Published by Elsevier Ltd.

EVALUATION OF RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

EPA Science Inventory

Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan (trademark)) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glab...

A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food

PubMed Central

Ren, Junan; Deng, Tingting; Huang, Wensheng; Chen, Ying; Ge, Yiqiang

2017-01-01

Meat adulteration is a worldwide concern. In this paper, a new droplet digital PCR (ddPCR) method was developed for the quantitative determination of the presence of chicken in sheep and goat meat products. Meanwhile, a constant (multiplication factor) was introduced to transform the ratio of copy numbers to the proportion of meats. The presented ddPCR method was also proved to be more accurate (showing bias of less than 9% in the range from 5% to 80%) than real-time PCR, which has been widely used in this determination. The method exhibited good repeatability and stability in different thermal treatments and at ultra-high pressure. The relative standard deviation (RSD) values of 5% chicken content was less than 5.4% for ultra-high pressure or heat treatment. Moreover, we confirmed that different parts of meat had no effect on quantification accuracy of the ddPCR method. In contrast to real-time PCR, we examined the performance of ddPCR as a more precise, sensitive and stable analytical strategy to overcome potential problems of discrepancies in amplification efficiency discrepancy and to obtain the copy numbers directly without standard curves. The method and strategy developed in this study can be applied to quantify the presence and to confirm the absence of adulterants not only to sheep but also to other kinds of meat and meat products. PMID:28319152

A noninvasive, direct real-time PCR method for sex determination in multiple avian species

USGS Publications Warehouse

Brubaker, Jessica L.; Karouna-Renier, Natalie K.; Chen, Yu; Jenko, Kathryn; Sprague, Daniel T.; Henry, Paula F.P.

2011-01-01

Polymerase chain reaction (PCR)-based methods to determine the sex of birds are well established and have seen few modifications since they were first introduced in the 1990s. Although these methods allowed for sex determination in species that were previously difficult to analyse, they were not conducive to high-throughput analysis because of the laboriousness of DNA extraction and gel electrophoresis. We developed a high-throughput real-time PCR-based method for analysis of sex in birds, which uses noninvasive sample collection and avoids DNA extraction and gel electrophoresis.

Methods for detection of GMOs in food and feed.

PubMed

Marmiroli, Nelson; Maestri, Elena; Gullì, Mariolina; Malcevschi, Alessio; Peano, Clelia; Bordoni, Roberta; De Bellis, Gianluca

2008-10-01

This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.

Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species.

PubMed

Trotta, Michele; Schönhuth, Susana; Pepe, Tiziana; Cortesi, M Luisa; Puyet, Antonio; Bautista, José M

2005-03-23

Mitochondrial 16S rRNA sequences from morphological validated grouper (Epinephelus aeneus, E. caninus, E. costae, and E. marginatus; Mycteroperca fusca and M. rubra), Nile perch (Lates niloticus), and wreck fish (Polyprion americanus) were used to develop an analytical system for group diagnosis based on two alternative Polymerase Chain Reaction (PCR) approaches. The first includes conventional multiplex PCR in which electrophoretic migration of different sizes of bands allowed identification of the fish species. The second approach, involving real-time PCR, produced a single amplicon from each species that showed different Tm values allowing the fish groups to be directly identified. Real-time PCR allows the quick differential diagnosis of the three groups of species and high-throughput screening of multiple samples. Neither PCR system cross-reacted with DNA samples from 41 common marketed fish species, thus conforming to standards for species validation. The use of these two PCR-based methods makes it now possible to discriminate grouper from substitute fish species.

Swab Protocol for Rapid Laboratory Diagnosis of Cutaneous Anthrax

PubMed Central

Marston, Chung K.; Bhullar, Vinod; Baker, Daniel; Rahman, Mahmudur; Hossain, M. Jahangir; Chakraborty, Apurba; Khan, Salah Uddin; Hoffmaster, Alex R.

2012-01-01

The clinical laboratory diagnosis of cutaneous anthrax is generally established by conventional microbiological methods, such as culture and directly straining smears of clinical specimens. However, these methods rely on recovery of viable Bacillus anthracis cells from swabs of cutaneous lesions and often yield negative results. This study developed a rapid protocol for detection of B. anthracis on clinical swabs. Three types of swabs, flocked-nylon, rayon, and polyester, were evaluated by 3 extraction methods, the swab extraction tube system (SETS), sonication, and vortex. Swabs were spiked with virulent B. anthracis cells, and the methods were compared for their efficiency over time by culture and real-time PCR. Viability testing indicated that the SETS yielded greater recovery of B. anthracis from 1-day-old swabs; however, reduced viability was consistent for the 3 extraction methods after 7 days and nonviability was consistent by 28 days. Real-time PCR analysis showed that the PCR amplification was not impacted by time for any swab extraction method and that the SETS method provided the lowest limit of detection. When evaluated using lesion swabs from cutaneous anthrax outbreaks, the SETS yielded culture-negative, PCR-positive results. This study demonstrated that swab extraction methods differ in their efficiency of recovery of viable B. anthracis cells. Furthermore, the results indicated that culture is not reliable for isolation of B. anthracis from swabs at ≥7 days. Thus, we recommend the use of the SETS method with subsequent testing by culture and real-time PCR for diagnosis of cutaneous anthrax from clinical swabs of cutaneous lesions. PMID:23035192

Direct identification of Streptococcus agalactiae and capsular type by real-time PCR in vaginal swabs from pregnant women.

PubMed

Morozumi, Miyuki; Chiba, Naoko; Igarashi, Yuko; Mitsuhashi, Naoki; Wajima, Takeaki; Iwata, Satoshi; Ubukata, Kimiko

2015-01-01

Most group B streptococcus (GBS) infections in newborns are with capsular type Ia, Ib, or III. To prevent these infections more effectively, we developed a real-time PCR method to simultaneously detect GBS species and identify these 3 capsular types in vaginal swab samples from women at 36-39 weeks of gestation. DNA to be detected included those of the dltS gene (encoding a histidine kinase specific to GBS) and cps genes encoding capsular types. PCR sensitivity was 10 CFU/well for a 33-35 threshold cycle. Results were obtained within 2 h. Direct PCR results were compared with results obtained from cultures. Samples numbering 1226 underwent PCR between September 2008 and August 2012. GBS positivity rates by direct PCR and after routine culture were 15.7% (n = 192) and 12.6% (n = 154), respectively. Sensitivity and specificity of direct PCR relative to culture were 96.1% and 95.9%. Of GBS positive samples identified by PCR, capsular types determined directly by real-time PCR were Ia (n = 24), Ib (n = 32), and III (n = 26). Real-time PCR using our designed cycling probe is a practical, highly sensitive method for identification of GBS in pregnant carriers, allowing use of prophylactic intrapartum antibiotics in time to cover the possibility of unexpected premature birth. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Real-time PCR for rapidly detecting aniline-degrading bacteria in activated sludge.

PubMed

Kayashima, Takakazu; Suzuki, Hisako; Maeda, Toshinari; Ogawa, Hiroaki I

2013-05-01

We developed a detection method that uses quantitative real-time PCR (qPCR) and the TaqMan system to easily and rapidly assess the population of aniline-degrading bacteria in activated sludge prior to conducting a biodegradability test on a chemical compound. A primer and probe set for qPCR was designed by a multiple alignment of conserved amino acid sequences encoding the large (α) subunit of aniline dioxygenase. PCR amplification tests showed that the designed primer and probe set targeted aniline-degrading strains such as Acidovorax sp., Gordonia sp., Rhodococcus sp., and Pseudomonas putida, thereby suggesting that the developed method can detect a wide variety of aniline-degrading bacteria. There was a strong correlation between the relative copy number of the α-aniline dioxygenase gene in activated sludge obtained with the developed qPCR method and the number of aniline-degrading bacteria measured by the Most Probable Number method, which is the conventional method, and a good correlation with the lag time of the BOD curve for aniline degradation produced by the biodegradability test in activated sludge samples collected from eight different wastewater treatment plants in Japan. The developed method will be valuable for the rapid and accurate evaluation of the activity of inocula prior to conducting a ready biodegradability test. Copyright © 2013 Elsevier Ltd. All rights reserved.

Real-time PCR detection of Brucella spp. DNA in lesions and viscera of bovine carcasses.

PubMed

Sola, Marília Cristina; da Veiga Jardim, Eurione A G; de Freitas, Marcius Ribeiro; de Mesquita, Albenones José

2014-09-01

This study reports a real-time PCR assay for the detection of Brucella spp. associated with the FTA® Elute method in lesions observed during sanitary inspections in beef slaughter. Of the total 276 samples, 78 (28.3%) tested positive and 198 (71.7%) negative for Brucella spp. The real-time PCR technique associated with the FTA® Elute method proved to be an important tool for the diagnosis, judgment about and disposal of carcasses and viscera of slaughtered animals. Copyright © 2014 Elsevier B.V. All rights reserved.

Intra-laboratory validation of chronic bee paralysis virus quantitation using an accredited standardised real-time quantitative RT-PCR method.

PubMed

Blanchard, Philippe; Regnault, Julie; Schurr, Frank; Dubois, Eric; Ribière, Magali

2012-03-01

Chronic bee paralysis virus (CBPV) is responsible for chronic bee paralysis, an infectious and contagious disease in adult honey bees (Apis mellifera L.). A real-time RT-PCR assay to quantitate the CBPV load is now available. To propose this assay as a reference method, it was characterised further in an intra-laboratory study during which the reliability and the repeatability of results and the performance of the assay were confirmed. The qPCR assay alone and the whole quantitation method (from sample RNA extraction to analysis) were both assessed following the ISO/IEC 17025 standard and the recent XP U47-600 standard issued by the French Standards Institute. The performance of the qPCR assay and of the overall CBPV quantitation method were validated over a 6 log range from 10(2) to 10(8) with a detection limit of 50 and 100 CBPV RNA copies, respectively, and the protocol of the real-time RT-qPCR assay for CBPV quantitation was approved by the French Accreditation Committee. Copyright © 2011 Elsevier B.V. All rights reserved.

Evaluation of a new commercial real-time PCR assay for diagnosis of Pneumocystis jirovecii pneumonia and identification of dihydropteroate synthase (DHPS) mutations.

PubMed

Montesinos, Isabel; Delforge, Marie-Luce; Ajjaham, Farida; Brancart, Françoise; Hites, Maya; Jacobs, Frederique; Denis, Olivier

2017-01-01

The PneumoGenius® real-time PCR assay is a new commercial multiplex real-time PCR method, which detects the Pneumocystis mitochondrial ribosomal large subunit (mtLSU) and two dihydropteroate synthase (DHPS) point mutations. To evaluate the clinical performance of this new real-time PCR assay we tested 120 extracted DNA samples from bronchoalveolar lavage specimens. These set of extracted DNA samples had already tested positive for Pneumocystis and patients had been classified in probable and unlikely PCP in a previous study. To evaluate de accuracy of the DHPS mutant's identification, an "in house" PCR and sequencing was performed. The sensitivity and specificity of PneumoGenius® PCR in discriminating between probable and unlikely Pneumocystis pneumonia (PCP) were 70% and 82% respectively. PneumoGenius® PCR was able to genotype more samples than "in house" DHPS PCR and sequencing. The same DHPS mutations were observed by both methods in four patients: two patients with a single mutation in position 171 (Pro57Ser) and two patients with a double mutation in position 165 (Thr55Ala) and in position 171 (Pro57Ser). A low rate of P. jirovecii (4.5%) harboring DHPS mutations was found, comparable to rates observed in other European countries. The PneumoGenius® real-time PCR is a suitable real-time PCR for PCP diagnosis and detection of DHPS mutants. The added value of DHPS mutation identification can assist in understanding the role of these mutations in prophylaxis failure or treatment outcome. Copyright © 2016 Elsevier Inc. All rights reserved.

Inter-laboratory Comparison of Real-time PCR Methods for Quantification of General Fecal Indicator Bacteria

EPA Science Inventory

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized prot...

Data Acceptance Criteria for Standardized Human-Associated Fecal Source Identification Quantitative Real-Time PCR Methods

EPA Science Inventory

There is a growing interest in the application of human-associated fecal sourceidentification quantitative real-time PCR (qPCR) technologies for water quality management. The transition from a research tool to a standardized protocol requires a high degree of confidence in data q...

EVALUATION OF A RAPID, QUANTITATIVE REAL-TIME PCR METHOD FOR ENUMERATION OF PATHOGENIC CANDIDA CELLS IN WATER

EPA Science Inventory

Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C....

Evaluation of High-Resolution Melting Curve Analysis of Ligation-Mediated Real-Time PCR, a Rapid Method for Epidemiological Typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) Pathogens

PubMed Central

Ryberg, Anna; Billström, Hanna; Hällgren, Anita; Nilsson, Lennart E.; Marklund, Britt-Inger; Olsson-Liljequist, Barbro; Schön, Thomas

2014-01-01

A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks. PMID:25232168

Real-time water quality monitoring at a Great Lakes National Park

USGS Publications Warehouse

Byappanahalli, Muruleedhara; Nevers, Meredith; Shively, Dawn; Spoljaric, Ashley; Otto, Christopher

2018-01-01

Quantitative polymerase chain reaction (qPCR) was used by the USEPA to establish new recreational water quality criteria in 2012 using the indicator bacteria enterococci. The application of this method has been limited, but resource managers are interested in more timely monitoring results. In this study, we evaluated the efficacy of qPCR as a rapid, alternative method to the time-consuming membrane filtration (MF) method for monitoring water at select beaches and rivers of Sleeping Bear Dunes National Lakeshore in Empire, MI. Water samples were collected from four locations (Esch Road Beach, Otter Creek, Platte Point Bay, and Platte River outlet) in 2014 and analyzed for culture-based (MF) and non-culture-based (i.e., qPCR) endpoints using Escherichia coli and enterococci bacteria. The MF and qPCR enterococci results were significantly, positively correlated overall (r = 0.686, p < 0.0001, n = 98) and at individual locations as well, except at the Platte River outlet location: Esch Road Beach (r = 0.441, p = 0.031, n = 24), Otter Creek (r = 0.592, p = 0.002, n = 24), and Platte Point Bay (r = 0.571, p = 0.004, n = 24). Similarly, E. coli MF and qPCR results were significantly, positively correlated (r = 0.469, p < 0.0001, n = 95), overall but not at individual locations. Water quality standard exceedances based on enterococci levels by qPCR were lower than by MF method: 3 and 16, respectively. Based on our findings, we conclude that qPCR may be a viable alternative to the culture-based method for monitoring water quality on public lands. Rapid, same-day results are achievable by the qPCR method, which greatly improves protection of the public from water-related illnesses.

Enhanced capillary electrophoretic screening of Alzheimer based on direct apolipoprotein E genotyping and one-step multiplex PCR.

PubMed

Woo, Nain; Kim, Su-Kang; Sun, Yucheng; Kang, Seong Ho

2018-01-01

Human apolipoprotein E (ApoE) is associated with high cholesterol levels, coronary artery disease, and especially Alzheimer's disease. In this study, we developed an ApoE genotyping and one-step multiplex polymerase chain reaction (PCR) based-capillary electrophoresis (CE) method for the enhanced diagnosis of Alzheimer's. The primer mixture of ApoE genes enabled the performance of direct one-step multiplex PCR from whole blood without DNA purification. The combination of direct ApoE genotyping and one-step multiplex PCR minimized the risk of DNA loss or contamination due to the process of DNA purification. All amplified PCR products with different DNA lengths (112-, 253-, 308-, 444-, and 514-bp DNA) of the ApoE genes were analyzed within 2min by an extended voltage programming (VP)-based CE under the optimal conditions. The extended VP-based CE method was at least 120-180 times faster than conventional slab gel electrophoresis methods In particular, all amplified DNA fragments were detected in less than 10 PCR cycles using a laser-induced fluorescence detector. The detection limits of the ApoE genes were 6.4-62.0pM, which were approximately 100-100,000 times more sensitive than previous Alzheimer's diagnosis methods In addition, the combined one-step multiplex PCR and extended VP-based CE method was also successfully applied to the analysis of ApoE genotypes in Alzheimer's patients and normal samples and confirmed the distribution probability of allele frequencies. This combination of direct one-step multiplex PCR and an extended VP-based CE method should increase the diagnostic reliability of Alzheimer's with high sensitivity and short analysis time even with direct use of whole blood. Copyright © 2017 Elsevier B.V. All rights reserved.

Prospective evaluation of the SeptiFAST multiplex real-time PCR assay for surveillance and diagnosis of infections in haematological patients after allogeneic stem cell transplantation compared to routine microbiological assays and an in-house real-time PCR method.

PubMed

Elges, Sandra; Arnold, Renate; Liesenfeld, Oliver; Kofla, Grzegorz; Mikolajewska, Agata; Schwartz, Stefan; Uharek, Lutz; Ruhnke, Markus

2017-12-01

We prospectively evaluated a multiplex real-time PCR assay (SeptiFast, SF) in a cohort of patients undergoing allo-BMT in comparison to an in-house PCR method (IH-PCR). Overall 847 blood samples (mean 8 samples/patient) from 104 patients with haematological malignancies were analysed. The majority of patients had acute leukaemia (62%) with a mean age of 52 years (54% female). Pathogens could be detected in 91 of 847 (11%) samples by SF compared to 38 of 205 (18.5%) samples by BC, and 57 of 847 (6.7%) samples by IH-PCR. Coagulase-negative staphylococci (n=41 in SF, n=29 in BC) were the most frequently detected bacteria followed by Escherichia coli (n=9 in SF, n=6 in BC). Candida albicans (n=17 in SF, n=0 in BC, n=24 in IH-PCR) was the most frequently detected fungal pathogen. SF gave positive results in 5% of samples during surveillance vs in 26% of samples during fever episodes. Overall, the majority of blood samples gave negative results in both PCR methods resulting in 93% overall agreement resulting in a negative predictive value of 0.96 (95% CI: 0.95-0.97), and a positive predictive value of 0.10 (95% CI: -0.01 to 0.21). SeptiFast appeared to be superior over BC and the IH-PCR method. © 2017 Blackwell Verlag GmbH.

Diagnosis of invasive fungal infections using real-time PCR assay in paediatric acute leukaemia induction.

PubMed

Mandhaniya, Sushil; Iqbal, Sobuhi; Sharawat, Surender Kumar; Xess, Immaculata; Bakhshi, Sameer

2012-07-01

Invasive fungal infections (IFI) lead to morbidity and mortality in neutropenic patients and in allogenic stem cell transplantation. Serum-based fungal detection assays have limitation of specificity or sensitivity. Studies on fungal DNA detection using real-time PCR in childhood leukaemia are lacking. The aim of this study was to develop sensitive and specific diagnostic tools for IFI in paediatric acute leukaemia patients using real-time PCR. Of 100 randomised paediatric acute leukaemia patients receiving antifungal prophylaxis with voriconazole/amphotericin B, single peripheral whole blood sample in EDTA was used for Pan-AC real-time PCR assay (detects nine Candida and six Aspergillus species) in patients who failed prophylaxis due to proven, probable, possible or suspected fungal infections. PCR results were retrospectively correlated with clinical profile. Real-time PCR test was positive in 18/29 (62%) patients who failed prophylaxis. The only patient with proven IFI (mucormycosis), real-time PCR assay was negative. Real-time PCR was positive in 2/4 (50%) patients with possible and 16/24 (66.6%) suspected IFI and 5/10 (50%) patients with pneumonia. By applying method A/B, sensitivity and positive predictive value could not be commented due to unproven Aspergillus or Candida infections; specificity and negative predictive values (NPV) were 41% and 100% respectively; by method C (included episodes of possible IFI as true positive), sensitivity, specificity, PPV and NPV were 50%, 36%, 11% and 81% respectively. In those with suspected IFI, 8/24 (33.3%) were PCR negative and unnecessarily received empirical antifungal therapy (EAFT). Real-time PCR is a practical, rapid, non-invasive screening test for excluding IFI in paediatric leukaemia. The high NPV makes real-time PCR a promising tool to use this prior to initiating EAFT in antibiotic-resistant febrile neutropenic patients; this would avoid toxicity, cost and hospitalisation for EAFT (ClinicalTrials.gov identifier:NCT00624143). © 2011 Blackwell Verlag GmbH.

Detection of Alicyclobacillus spp. in Fruit Juice by Combination of Immunomagnetic Separation and a SYBR Green I Real-Time PCR Assay

PubMed Central

Yuan, Yahong; Liu, Bin; Wang, Ling; Yue, Tianli

2015-01-01

An approach based on immunomagnetic separation (IMS) and SYBR Green I real-time PCR (real-time PCR) with species-specific primers and melting curve analysis was proposed as a rapid and effective method for detecting Alicyclobacillus spp. in fruit juices. Specific primers targeting the 16S rDNA sequences of Alicyclobacillus spp. were designed and then confirmed by the amplification of DNA extracted from standard strains and isolates. Spiked samples containing known amounts of target bacteria were used to obtain standard curves; the correlation coefficient was greater than 0.986 and the real-time PCR amplification efficiencies were 98.9%- 101.8%. The detection limit of the testing system was 2.8×101 CFU/mL. The coefficient of variation for intra-assay and inter-assay variability were all within the acceptable limit of 5%. Besides, the performance of the IMS-real-time PCR assay was further investigated by detecting naturally contaminated kiwi fruit juice; the sensitivity, specificity and accuracy were 91.7%, 95.9% and 95.3%, respectively. The established IMS-real-time PCR procedure provides a new method for identification and quantitative detection of Alicyclobacillus spp. in fruit juice. PMID:26488469

REAL TIME PCR ANALYSIS OF INDOOR MOLDS: PRINCIPLES, PROCEDURES AND APPLICATIONS

EPA Science Inventory

This presentation will endeavor to present an overview of the real time polymerase chain reaction method developed for indoor mold detection and quantification by the EPA. It will begin with a brief discussion of the PCR technology that provides the basis for this method and how ...

European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat.

PubMed

Delibato, Elisabetta; Rodriguez-Lazaro, David; Gianfranceschi, Monica; De Cesare, Alessandra; Comin, Damiano; Gattuso, Antonietta; Hernandez, Marta; Sonnessa, Michele; Pasquali, Frédérique; Sreter-Lancz, Zuzsanna; Saiz-Abajo, María-José; Pérez-De-Juan, Javier; Butrón, Javier; Prukner-Radovcic, Estella; Horvatek Tomic, Danijela; Johannessen, Gro S; Jakočiūnė, Džiuginta; Olsen, John E; Chemaly, Marianne; Le Gall, Francoise; González-García, Patricia; Lettini, Antonia Anna; Lukac, Maja; Quesne, Segolénè; Zampieron, Claudia; De Santis, Paola; Lovari, Sarah; Bertasi, Barbara; Pavoni, Enrico; Proroga, Yolande T R; Capuano, Federico; Manfreda, Gerardo; De Medici, Dario

2014-08-01

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

Novel and highly sensitive sybr® green real-time pcr for poxvirus detection in odontocete cetaceans.

PubMed

Sacristán, Carlos; Luiz Catão-Dias, José; Ewbank, Ana Carolina; Machado, Eduardo Ferreira; Neves, Elena; Santos-Neto, Elitieri Batista; Azevedo, Alexandre; Laison-Brito, José; De Castilho, Pedro Volkmer; Daura-Jorge, Fábio Gonçalves; Simões-Lopes, Paulo César; Carballo, Matilde; García-Párraga, Daniel; Manuel Sánchez-Vizcaíno, José; Esperón, Fernando

2018-06-08

Poxviruses are emerging pathogens in cetaceans, temporarily named 'Cetaceanpoxvirus' (CePV, family Poxviridae), classified into two main lineages: CePV-1 in odontocetes and CePV-2 in mysticetes. Only a few studies performed the molecular detection of CePVs, based on DNA-polymerase gene and/or DNA-topoisomerase I gene amplification. Herein we describe a new real-time PCR assay based on SYBR ® Green and a new primer set to detect a 150 bp fragment of CePV DNA-polymerase gene, also effective for conventional PCR detection. The novel real-time PCR was able to detect 5 up to 5 × 10 6 copies per reaction of a cloned positive control. Both novel PCR methods were 1000 to 100,000-fold more sensitive than those previously described in the literature. Samples of characteristic poxvirus skin lesions ('tattoo') from one Risso's dolphin (Grampus griseus), two striped dolphins (Stenella coeruleoalba) and two Guiana dolphins (Sotalia guianensis) were all positive to both our novel real time- and conventional PCR methods, even though three of these animals (a Risso's dolphin, a striped dolphin, and a Guiana dolphin) were previously negative to the conventional PCRs previously available. To our knowledge, this is the first real-time PCR detection method for Cetaceanpoxvirus, a much more sensitive tool for the detection of CePV-1 infections. Copyright © 2018 Elsevier B.V. All rights reserved.

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India.

PubMed

Dinoop, K P; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R P; Narayanan, P

2016-01-01

Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated ( P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods.

Calibration-free assays on standard real-time PCR devices

PubMed Central

Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr

2017-01-01

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration. PMID:28327545

Calibration-free assays on standard real-time PCR devices

NASA Astrophysics Data System (ADS)

Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr

2017-03-01

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.

Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp. bulgaricus.

PubMed

Shao, Yuyu; Wang, Zhaoxia; Bao, Qiuhua; Zhang, Heping

2016-12-01

In this study, a combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) was used to develop a method to determine the viability of cells of Lactobacillus delbrueckii ssp. bulgaricus ND02 (L. bulgaricus) that may have entered into a viable but nonculturable state. This can happen due to its susceptibility to cold shock during lyophilization and storage. Propidium monoazide concentration, PMA incubation time, and light exposure time were optimized to fully exploit the PMA-qPCR approach to accurately assess the total number of living L. bulgaricus ND02. Although PMA has little influence on living cells, when concentrations of PMA were higher than 30μg/mL the number of PCR-positive living bacteria decreased from 10 6 to 10 5 cfu/mL in comparison with qPCR enumeration. Mixtures of living and dead cells were used as method verification samples for enumeration by PMA-qPCR, demonstrating that this method was feasible and effective for distinguishing living cells of L. bulgaricus when mixed with a known number of dead cells. We suggest that several conditions need to be studied further before PMA-qPCR methods can be accurately used to distinguish living from dead cells for enumeration under more realistic sampling situations. However, this research provides a rapid way to enumerate living cells of L. bulgaricus and could be used to optimize selection of cryoprotectants in the lyophilization process and develop technologies for high cell density cultivation and optimal freeze-drying processes. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Rapid and sensitive detection of Zika virus by reverse transcription loop-mediated isothermal amplification.

PubMed

Wang, Xuan; Yin, Fenggui; Bi, Yuhai; Cheng, Gong; Li, Jing; Hou, Lidan; Li, Yunlong; Yang, Baozhi; Liu, Wenjun; Yang, Limin

2016-12-01

Zika virus (ZIKV) is an arbovirus that recently emerged and has expanded worldwide, causing a global threat and raising international concerns. Current molecular diagnostics, e.g., real-time PCR and reverse transcription PCR (RT-PCR), are time consuming, expensive, and can only be deployed in a laboratory instead of for field diagnostics. This study aimed to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) platform showing sensitivity, specificity, and more convenience than previous methods, being easily distributed and implemented. Specific primers were designed and screened to target the entire ZIKV genome. The analytical sensitivity and specificity of the assay were evaluated and compared with traditional PCR and quantitative real-time PCR. Three different simulated clinical sample quick preparation protocols were evaluated to establish a rapid and straightforward treatment procedure for clinical specimens in open field detection. The RT-LAMP assay for detection of ZIKV demonstrated superior specificity and sensitivity compared to traditional PCR at the optimum reaction temperature. For the ZIKV RNA standard, the limit of detection was 20 copies/test. For the simulated ZIKV clinical samples, the limit of detection was 0.02 pfu/test, which was one order of magnitude higher than RT-PCR and similar to real-time PCR. The detection limit of simulated ZIKV specimens prepared using a protease quick processing method was consistent with that of samples prepared using commercial nucleic acid extraction kits, indicating that our ZIKV detection method could be used in point-of-care testing. The RT-LAMP assay had excellent sensitivity and specificity for detecting ZIKV and can be deployed together with a rapid specimen processing method, offering the possibility for ZIKV diagnosis outside of the laboratory. Copyright © 2016 Elsevier B.V. All rights reserved.

Microfluidics-based digital quantitative PCR for single-cell small RNA quantification.

PubMed

Yu, Tian; Tang, Chong; Zhang, Ying; Zhang, Ruirui; Yan, Wei

2017-09-01

Quantitative analyses of small RNAs at the single-cell level have been challenging because of limited sensitivity and specificity of conventional real-time quantitative PCR methods. A digital quantitative PCR (dqPCR) method for miRNA quantification has been developed, but it requires the use of proprietary stem-loop primers and only applies to miRNA quantification. Here, we report a microfluidics-based dqPCR (mdqPCR) method, which takes advantage of the Fluidigm BioMark HD system for both template partition and the subsequent high-throughput dqPCR. Our mdqPCR method demonstrated excellent sensitivity and reproducibility suitable for quantitative analyses of not only miRNAs but also all other small RNA species at the single-cell level. Using this method, we discovered that each sperm has a unique miRNA profile. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A Nested PCR Assay to Avoid False Positive Detection of the Microsporidian Enterocytozoon hepatopenaei (EHP) in Environmental Samples in Shrimp Farms

PubMed Central

Jaroenlak, Pattana; Sanguanrut, Piyachat; Williams, Bryony A. P.; Stentiford, Grant D.; Flegel, Timothy W.; Sritunyalucksana, Kallaya

2016-01-01

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is an important disease of cultivated shrimp. Heavy infections may lead to retarded growth and unprofitable harvests. Existing PCR detection methods target the EHP small subunit ribosomal RNA (SSU rRNA) gene (SSU-PCR). However, we discovered that they can give false positive test results due to cross reactivity of the SSU-PCR primers with DNA from closely related microsporidia that infect other aquatic organisms. This is problematic for investigating and monitoring EHP infection pathways. To overcome this problem, a sensitive and specific nested PCR method was developed for detection of the spore wall protein (SWP) gene of EHP (SWP-PCR). The new SWP-PCR method did not produce false positive results from closely related microsporidia. The first PCR step of the SWP-PCR method was 100 times (104 plasmid copies per reaction vial) more sensitive than that of the existing SSU-PCR method (106 copies) but sensitivity was equal for both in the nested step (10 copies). Since the hepatopancreas of cultivated shrimp is not currently known to be infected with microsporidia other than EHP, the SSU-PCR methods are still valid for analyzing hepatopancreatic samples despite the lower sensitivity than the SWP-PCR method. However, due to its greater specificity and sensitivity, we recommend that the SWP-PCR method be used to screen for EHP in feces, feed and environmental samples for potential EHP carriers. PMID:27832178

A Nested PCR Assay to Avoid False Positive Detection of the Microsporidian Enterocytozoon hepatopenaei (EHP) in Environmental Samples in Shrimp Farms.

PubMed

Jaroenlak, Pattana; Sanguanrut, Piyachat; Williams, Bryony A P; Stentiford, Grant D; Flegel, Timothy W; Sritunyalucksana, Kallaya; Itsathitphaisarn, Ornchuma

2016-01-01

Hepatopancreatic microsporidiosis (HPM) caused by Enterocytozoon hepatopenaei (EHP) is an important disease of cultivated shrimp. Heavy infections may lead to retarded growth and unprofitable harvests. Existing PCR detection methods target the EHP small subunit ribosomal RNA (SSU rRNA) gene (SSU-PCR). However, we discovered that they can give false positive test results due to cross reactivity of the SSU-PCR primers with DNA from closely related microsporidia that infect other aquatic organisms. This is problematic for investigating and monitoring EHP infection pathways. To overcome this problem, a sensitive and specific nested PCR method was developed for detection of the spore wall protein (SWP) gene of EHP (SWP-PCR). The new SWP-PCR method did not produce false positive results from closely related microsporidia. The first PCR step of the SWP-PCR method was 100 times (104 plasmid copies per reaction vial) more sensitive than that of the existing SSU-PCR method (106 copies) but sensitivity was equal for both in the nested step (10 copies). Since the hepatopancreas of cultivated shrimp is not currently known to be infected with microsporidia other than EHP, the SSU-PCR methods are still valid for analyzing hepatopancreatic samples despite the lower sensitivity than the SWP-PCR method. However, due to its greater specificity and sensitivity, we recommend that the SWP-PCR method be used to screen for EHP in feces, feed and environmental samples for potential EHP carriers.

Analytical Validation of Quantitative Real-Time PCR Methods for Quantification of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

PubMed Central

Ramírez, Juan Carlos; Cura, Carolina Inés; Moreira, Otacilio da Cruz; Lages-Silva, Eliane; Juiz, Natalia; Velázquez, Elsa; Ramírez, Juan David; Alberti, Anahí; Pavia, Paula; Flores-Chávez, María Delmans; Muñoz-Calderón, Arturo; Pérez-Morales, Deyanira; Santalla, José; Guedes, Paulo Marcos da Matta; Peneau, Julie; Marcet, Paula; Padilla, Carlos; Cruz-Robles, David; Valencia, Edward; Crisante, Gladys Elena; Greif, Gonzalo; Zulantay, Inés; Costales, Jaime Alfredo; Alvarez-Martínez, Miriam; Martínez, Norma Edith; Villarroel, Rodrigo; Villarroel, Sandro; Sánchez, Zunilda; Bisio, Margarita; Parrado, Rudy; Galvão, Lúcia Maria da Cunha; da Câmara, Antonia Cláudia Jácome; Espinoza, Bertha; de Noya, Belkisyole Alarcón; Puerta, Concepción; Riarte, Adelina; Diosque, Patricio; Sosa-Estani, Sergio; Guhl, Felipe; Ribeiro, Isabela; Aznar, Christine; Britto, Constança; Yadón, Zaida Estela; Schijman, Alejandro G.

2015-01-01

An international study was performed by 26 experienced PCR laboratories from 14 countries to assess the performance of duplex quantitative real-time PCR (qPCR) strategies on the basis of TaqMan probes for detection and quantification of parasitic loads in peripheral blood samples from Chagas disease patients. Two methods were studied: Satellite DNA (SatDNA) qPCR and kinetoplastid DNA (kDNA) qPCR. Both methods included an internal amplification control. Reportable range, analytical sensitivity, limits of detection and quantification, and precision were estimated according to international guidelines. In addition, inclusivity and exclusivity were estimated with DNA from stocks representing the different Trypanosoma cruzi discrete typing units and Trypanosoma rangeli and Leishmania spp. Both methods were challenged against 156 blood samples provided by the participant laboratories, including samples from acute and chronic patients with varied clinical findings, infected by oral route or vectorial transmission. kDNA qPCR showed better analytical sensitivity than SatDNA qPCR with limits of detection of 0.23 and 0.70 parasite equivalents/mL, respectively. Analyses of clinical samples revealed a high concordance in terms of sensitivity and parasitic loads determined by both SatDNA and kDNA qPCRs. This effort is a major step toward international validation of qPCR methods for the quantification of T. cruzi DNA in human blood samples, aiming to provide an accurate surrogate biomarker for diagnosis and treatment monitoring for patients with Chagas disease. PMID:26320872

Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.

PubMed

Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James

2017-03-01

Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Quantification of measles, mumps and rubella viruses using real-time quantitative TaqMan-based RT-PCR assay.

PubMed

Ammour, Y; Faizuloev, E; Borisova, T; Nikonova, A; Dmitriev, G; Lobodanov, S; Zverev, V

2013-01-01

In this study, a rapid quantitative method using TaqMan-based real-time reverse transcription-polymerase chain reaction (qPCR-RT) has been developed for estimating the titers of measles, mumps and rubella (MMR) viruses in infected cell culture supernatants. The qPCR-RT assay was demonstrated to be a specific, sensitive, efficient and reproducible method. For MMR viral samples obtained during MMR viral propagations in Vero cells at a different multiplicity of infection, titers determined by the qPCR-RT assay have been compared with estimates of infectious virus obtained by a traditional commonly used method for MMR viruses - 50% cell culture infective dose (CCID(50)) assay, in paired samples. Pearson analysis evidenced a significant correlation between both methods for a certain period after viral inoculation. Furthermore, the established qPCR-RT assay was faster and less-laborious. The developed method could be used as an alternative method or a supplementary tool for the routine titer estimation during MMR vaccine production. Copyright © 2012 Elsevier B.V. All rights reserved.

Standardization of a two-step real-time polymerase chain reaction based method for species-specific detection of medically important Aspergillus species.

PubMed

Das, P; Pandey, P; Harishankar, A; Chandy, M; Bhattacharya, S; Chakrabarti, A

2017-01-01

Standardization of Aspergillus polymerase chain reaction (PCR) poses two technical challenges (a) standardization of DNA extraction, (b) optimization of PCR against various medically important Aspergillus species. Many cases of aspergillosis go undiagnosed because of relative insensitivity of conventional diagnostic methods such as microscopy, culture or antigen detection. The present study is an attempt to standardize real-time PCR assay for rapid sensitive and specific detection of Aspergillus DNA in EDTA whole blood. Three nucleic acid extraction protocols were compared and a two-step real-time PCR assay was developed and validated following the recommendations of the European Aspergillus PCR Initiative in our setup. In the first PCR step (pan-Aspergillus PCR), the target was 28S rDNA gene, whereas in the second step, species specific PCR the targets were beta-tubulin (for Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus), gene and calmodulin gene (for Aspergillus niger). Species specific identification of four medically important Aspergillus species, namely, A. fumigatus, A. flavus, A. niger and A. terreus were achieved by this PCR. Specificity of the PCR was tested against 34 different DNA source including bacteria, virus, yeast, other Aspergillus sp., other fungal species and for human DNA and had no false-positive reactions. The analytical sensitivity of the PCR was found to be 102 CFU/ml. The present protocol of two-step real-time PCR assays for genus- and species-specific identification for commonly isolated species in whole blood for diagnosis of invasive Aspergillus infections offers a rapid, sensitive and specific assay option and requires clinical validation at multiple centers.

Evaluation of a Rapid One-step Real-time PCR Method as a High-throughput Screening for Quantification of Hepatitis B Virus DNA in a Resource-limited Setting.

PubMed

Rashed-Ul Islam, S M; Jahan, Munira; Tabassum, Shahina

2015-01-01

Virological monitoring is the best predictor for the management of chronic hepatitis B virus (HBV) infections. Consequently, it is important to use the most efficient, rapid and cost-effective testing systems for HBV DNA quantification. The present study compared the performance characteristics of a one-step HBV polymerase chain reaction (PCR) vs the two-step HBV PCR method for quantification of HBV DNA from clinical samples. A total of 100 samples consisting of 85 randomly selected samples from patients with chronic hepatitis B (CHB) and 15 samples from apparently healthy individuals were enrolled in this study. Of the 85 CHB clinical samples tested, HBV DNA was detected from 81% samples by one-step PCR method with median HBV DNA viral load (VL) of 7.50 × 10 3 lU/ml. In contrast, 72% samples were detected by the two-step PCR system with median HBV DNA of 3.71 × 10 3 lU/ml. The one-step method showed strong linear correlation with two-step PCR method (r = 0.89; p < 0.0001). Both methods showed good agreement at Bland-Altman plot, with a mean difference of 0.61 log 10 IU/ml and limits of agreement of -1.82 to 3.03 log 10 IU/ml. The intra-assay and interassay coefficients of variation (CV%) of plasma samples (4-7 log 10 IU/ml) for the one-step PCR method ranged between 0.33 to 0.59 and 0.28 to 0.48 respectively, thus demonstrating a high level of concordance between the two methods. Moreover, elimination of the DNA extraction step in the one-step PCR kit allowed time-efficient and significant labor and cost savings for the quantification of HBV DNA in a resource limited setting. Rashed-Ul Islam SM, Jahan M, Tabassum S. Evaluation of a Rapid One-step Real-time PCR Method as a High-throughput Screening for Quantification of Hepatitis B Virus DNA in a Resource-limited Setting. Euroasian J Hepato-Gastroenterol 2015;5(1):11-15.

Evaluation of a Rapid One-step Real-time PCR Method as a High-throughput Screening for Quantification of Hepatitis B Virus DNA in a Resource-limited Setting

PubMed Central

Jahan, Munira; Tabassum, Shahina

2015-01-01

Virological monitoring is the best predictor for the management of chronic hepatitis B virus (HBV) infections. Consequently, it is important to use the most efficient, rapid and cost-effective testing systems for HBV DNA quantification. The present study compared the performance characteristics of a one-step HBV polymerase chain reaction (PCR) vs the two-step HBV PCR method for quantification of HBV DNA from clinical samples. A total of 100 samples consisting of 85 randomly selected samples from patients with chronic hepatitis B (CHB) and 15 samples from apparently healthy individuals were enrolled in this study. Of the 85 CHB clinical samples tested, HBV DNA was detected from 81% samples by one-step PCR method with median HBV DNA viral load (VL) of 7.50 × 103 lU/ml. In contrast, 72% samples were detected by the two-step PCR system with median HBV DNA of 3.71 × 103 lU/ml. The one-step method showed strong linear correlation with two-step PCR method (r = 0.89; p < 0.0001). Both methods showed good agreement at Bland-Altman plot, with a mean difference of 0.61 log10 IU/ml and limits of agreement of -1.82 to 3.03 log10 IU/ml. The intra-assay and interassay coefficients of variation (CV%) of plasma samples (4-7 log10 IU/ml) for the one-step PCR method ranged between 0.33 to 0.59 and 0.28 to 0.48 respectively, thus demonstrating a high level of concordance between the two methods. Moreover, elimination of the DNA extraction step in the one-step PCR kit allowed time-efficient and significant labor and cost savings for the quantification of HBV DNA in a resource limited setting. How to cite this article Rashed-Ul Islam SM, Jahan M, Tabassum S. Evaluation of a Rapid One-step Real-time PCR Method as a High-throughput Screening for Quantification of Hepatitis B Virus DNA in a Resource-limited Setting. Euroasian J Hepato-Gastroenterol 2015;5(1):11-15. PMID:29201678

A Human Fecal Contamination Score for Ranking Recreational Sites using the HF183/BacR287 Quantitative Real-Time PCR Method

EPA Science Inventory

Human fecal pollution of recreational waters remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for water quality research and manag...

The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumeratIng Cryptosporidium spp. oocysts in the environment

EPA Science Inventory

Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as ...

A human fecal contamination index for ranking impaired recreational watersusing the HF183 quantitative real-time PCR method

EPA Science Inventory

Human fecal pollution of surface water remains a public health concern worldwide. As a result, there is a growing interest in the application of human-associated fecal source identification quantitative real-time PCR (qPCR) technologies for recreational water quality risk managem...

Statistical tools for transgene copy number estimation based on real-time PCR.

PubMed

Yuan, Joshua S; Burris, Jason; Stewart, Nathan R; Mentewab, Ayalew; Stewart, C Neal

2007-11-01

As compared with traditional transgene copy number detection technologies such as Southern blot analysis, real-time PCR provides a fast, inexpensive and high-throughput alternative. However, the real-time PCR based transgene copy number estimation tends to be ambiguous and subjective stemming from the lack of proper statistical analysis and data quality control to render a reliable estimation of copy number with a prediction value. Despite the recent progresses in statistical analysis of real-time PCR, few publications have integrated these advancements in real-time PCR based transgene copy number determination. Three experimental designs and four data quality control integrated statistical models are presented. For the first method, external calibration curves are established for the transgene based on serially-diluted templates. The Ct number from a control transgenic event and putative transgenic event are compared to derive the transgene copy number or zygosity estimation. Simple linear regression and two group T-test procedures were combined to model the data from this design. For the second experimental design, standard curves were generated for both an internal reference gene and the transgene, and the copy number of transgene was compared with that of internal reference gene. Multiple regression models and ANOVA models can be employed to analyze the data and perform quality control for this approach. In the third experimental design, transgene copy number is compared with reference gene without a standard curve, but rather, is based directly on fluorescence data. Two different multiple regression models were proposed to analyze the data based on two different approaches of amplification efficiency integration. Our results highlight the importance of proper statistical treatment and quality control integration in real-time PCR-based transgene copy number determination. These statistical methods allow the real-time PCR-based transgene copy number estimation to be more reliable and precise with a proper statistical estimation. Proper confidence intervals are necessary for unambiguous prediction of trangene copy number. The four different statistical methods are compared for their advantages and disadvantages. Moreover, the statistical methods can also be applied for other real-time PCR-based quantification assays including transfection efficiency analysis and pathogen quantification.

Improvement in the detection rate of diarrhoeagenic bacteria in human stool specimens by a rapid real-time PCR assay.

PubMed

Iijima, Yoshio; Asako, Nahoko T; Aihara, Masanori; Hayashi, Kozaburo

2004-07-01

A rapid laboratory system has been developed and evaluated that can simultaneously identify major diarrhoeagenic bacteria, including Salmonella enterica, Vibrio parahaemolyticus, Campylobacter jejuni and Shiga toxin-producing Escherichia coli, in stool specimens by real-time PCR. Specific identification was achieved by using selective TaqMan probes, detecting two targets in each pathogen. A positive result was scored only when both targets of a pathogen were amplified and the difference between threshold cycles for detection was less than five. Diagnosis of enteric bacterial infections using this highly sensitive method, including DNA extraction and real-time PCR, requires only 3 h. Forty stool specimens related to suspected food poisoning outbreaks were analysed: 16 (40%) of these samples were found to be positive for diarrhoeagenic bacteria using a conventional culture method; 28 (70%) were positive using the real-time PCR assay. Of the 12 PCR-positive but culture-negative cases, 11 patients had consumed pathogen-contaminated or high-risk food. Analysis of faecal samples from 105 outpatients who complained of diarrhoea and/or abdominal pain identified 19 (18%) patients as being positive for diarrhoeagenic bacteria using the culture method. An additional six (6%) patients were found to be positive by PCR analysis.

Detection and identification of genetically modified EE-1 brinjal (Solanum melongena) by single, multiplex and SYBR® real-time PCR.

PubMed

Ballari, Rajashekhar V; Martin, Asha; Gowda, Lalitha R

2013-01-01

Brinjal is an important vegetable crop. Major crop loss of brinjal is due to insect attack. Insect-resistant EE-1 brinjal has been developed and is awaiting approval for commercial release. Consumer health concerns and implementation of international labelling legislation demand reliable analytical detection methods for genetically modified (GM) varieties. End-point and real-time polymerase chain reaction (PCR) methods were used to detect EE-1 brinjal. In end-point PCR, primer pairs specific to 35S CaMV promoter, NOS terminator and nptII gene common to other GM crops were used. Based on the revealed 3' transgene integration sequence, primers specific for the event EE-1 brinjal were designed. These primers were used for end-point single, multiplex and SYBR-based real-time PCR. End-point single PCR showed that the designed primers were highly specific to event EE-1 with a sensitivity of 20 pg of genomic DNA, corresponding to 20 copies of haploid EE-1 brinjal genomic DNA. The limits of detection and quantification for SYBR-based real-time PCR assay were 10 and 100 copies respectively. The prior development of detection methods for this important vegetable crop will facilitate compliance with any forthcoming labelling regulations. Copyright © 2012 Society of Chemical Industry.

Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

PubMed

Vela, J; Vitorica, J; Ruano, D

2001-12-01

We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

Real-time PCR assay for the detection and quantification of Legionella pneumophila in environmental water samples: utility for daily practice.

PubMed

Morio, Florent; Corvec, Stéphane; Caroff, Nathalie; Le Gallou, Florence; Drugeon, Henri; Reynaud, Alain

2008-07-01

We developed a quantitative real-time PCR assay targeting the mip gene of Legionella pneumophila for a prospective study from September 2004 to April 2005. It was compared with a standard culture method (French guideline AFNOR T90-431), analysing 120 water samples collected to monitor the risk related to Legionellae at Nantes hospital and to investigate a case of legionellosis acquired from hospital environment. Samples from six distinct water distribution systems were analysed by DNA extraction, amplification and detection with specific primers and FRET probes. The detection limit was 100 genomic units of L. pneumophila per liter (GU/l), the positivity threshold about 600 GU/l and the quantification limit 800 GU/l. PCR results were divided into three groups: negative (n=63), positive but non-quantifiable (n=22) or positive (n=35). PCR showed higher sensitivity than culture, whereas four culture-positive samples appeared negative by PCR (PCR inhibitor detected for two of them). Although no correlation was observed between both methods and real-time PCR cannot substitute for the reference method, it represents an interesting complement. Its sensitivity, reproducibility and rapidity appear particularly interesting in epidemic contexts in order to identify the source of contamination or to evaluate critical points of contamination in water distribution systems.

Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

PubMed

Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

2018-01-01

A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

Application of real-time PCR (qPCR) for characterization of microbial populations and type of milk in dairy food products.

PubMed

Agrimonti, Caterina; Bottari, Benedetta; Sardaro, Maria Luisa Savo; Marmiroli, Nelson

2017-09-08

Dairy foods represent an important sector of the food market for their nutritional qualities and their organoleptic characteristics, which are often linked to tradition and to region. These products are typically protected by labels such as PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication). Real-time PCR (qPCR) is a fundamental tool in "Food Genomics;" a discipline concerned with the residual DNA in food, which, alongside traditional physical and chemical methods, is frequently used to determine product safety, quality and authenticity. Compared to conventional or "end-point" PCR, qPCR incorporates continuous monitoring of reaction progress, thereby enabling quantification of target DNA. This review describes qPCR applications to the analysis of microbiota, and to the identification of the animal species source of milk from which dairy products have been made. These are important aspects for ensuring safety and authenticity. The various applications of qPCR are discussed, as well as advantages and disadvantages in comparison with other analytical methods.

Detection of Haemophilus influenzae in respiratory secretions from pneumonia patients by quantitative real-time polymerase chain reaction.

PubMed

Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

2009-08-01

A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

Development of a rapid method to quantify Salmonella Typhimurium using a combination of MPN with qPCR and a shortened time incubation.

PubMed

Kim, Sun Ae; Park, Si Hong; Lee, Sang In; Ricke, Steven C

2017-08-01

A novel method was developed for the specific quantification of S. Typhimurium using a most-probable-number (MPN) combined with qPCR and a shortened incubation time (MPN-qPCR-SIT). For S. Typhimurium enumeration, dilutions of samples were transferred into three wells on a microtiter plate and the plate was incubated for 4 h. The S. Typhimurium presence in the wells was identified using a qPCR and populations were determined based on an MPN calculation. The R 2 between the MPN-qPCR-SIT and conventional MPN exhibited a high level of correlation (0.9335-0.9752), suggesting that the MPN-qPCR-SIT offers a reliable alternative method for S. Typhimurium quantification. Although plating and qPCR were limited in their ability to detect low levels of S. Typhimurium (e.g. 0.18 log MPN/ml), these levels could be successfully detected with the MPN-qPCR-SIT. Chicken breast samples inoculated with S. Typhimurium were incubated at 0, 4, and 24 h and incubated samples were subjected to microbiome analysis. Levels of Salmonella and Enterobacteriaceae increased significantly with incubation time. The obvious benefits of the MPN-qPCR-SIT are: 1) a further confirmation step is not required, 2) the detection limit is as low as conventional MPN, but 3) is more rapid, requiring approximately 7 h to simultaneously complete quantification. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development and evaluation of the quantitative real-time PCR assay in detection and typing of herpes simplex virus in swab specimens from patients with genital herpes.

PubMed

Liu, Junlian; Yi, Yong; Chen, Wei; Si, Shaoyan; Yin, Mengmeng; Jin, Hua; Liu, Jianjun; Zhou, Jinlian; Zhang, Jianzhong

2015-01-01

Genital herpes (GH), which is caused mainly by herpes simplex virus (HSV)-2 and HSV-1, remains a worldwide problem. Laboratory confirmation of GH is important, particularly as there are other conditions which present similarly to GH, while atypical presentations of GH also occur. Currently, virus culture is the classical method for diagnosis of GH, but it is time consuming and with low sensitivity. A major advance for diagnosis of GH is to use Real-time polymerase chain reaction (PCR). In this study, to evaluate the significance of the real-time PCR method in diagnosis and typing of genital HSV, the primers and probes targeted at HSV-1 DNA polymerase gene and HSV-2 glycoprotein D gene fraction were designed and applied to amplify DNA from HSV-1 or HSV-2 by employing the real-time PCR technique. Then the PCR reaction system was optimized and evaluated. HSV in swab specimens from patients with genital herpes was detected by real-time PCR. The real-time PCR assay showed good specificity for detection and typing of HSV, with good linear range (5×10(2)~5×10(8) copies/ml, r=0.997), a sensitivity of 5×10(2) copies/ml, and good reproducibility (intra-assay coefficients of variation 2.29% and inter-assay coefficients of variation 4.76%). 186 swab specimens were tested for HSV by real-time PCR, and the positive rate was 23.7% (44/186). Among the 44 positive specimens, 8 (18.2%) were positive for HSV-1 with a viral load of 8.5546×10(6) copies/ml and 36 (81.2%) were positive for HSV-2 with a viral load of 1.9861×10(6) copies/ml. It is concluded that the real-time PCR is a specific, sensitive and rapid method for the detection and typing of HSV, which can be widely used in clinical diagnosis of GH.

High-throughput detection and screening of plants modified by gene editing using quantitative real-time polymerase chain reaction.

PubMed

Peng, Cheng; Wang, Hua; Xu, Xiaoli; Wang, Xiaofu; Chen, Xiaoyun; Wei, Wei; Lai, Yongmin; Liu, Guoquan; Godwin, Ian Douglas; Li, Jieqin; Zhang, Ling; Xu, Junfeng

2018-05-15

Gene editing techniques are becoming powerful tools for modifying target genes in organisms. Although several methods have been developed to detect gene-edited organisms, these techniques are time and labour intensive. Meanwhile, few studies have investigated high-throughput detection and screening strategies for plants modified by gene editing. In this study, we developed a simple, sensitive and high-throughput quantitative real-time (qPCR)-based method. The qPCR-based method exploits two differently labelled probes that are placed within one amplicon at the gene editing target site to simultaneously detect the wild-type and a gene-edited mutant. We showed that the qPCR-based method can accurately distinguish CRISPR/Cas9-induced mutants from the wild-type in several different plant species, such as Oryza sativa, Arabidopsis thaliana, Sorghum bicolor, and Zea mays. Moreover, the method can subsequently determine the mutation type by direct sequencing of the qPCR products of mutations due to gene editing. The qPCR-based method is also sufficiently sensitive to distinguish between heterozygous and homozygous mutations in T 0 transgenic plants. In a 384-well plate format, the method enabled the simultaneous analysis of up to 128 samples in three replicates without handling the post-polymerase chain reaction (PCR) products. Thus, we propose that our method is an ideal choice for screening plants modified by gene editing from many candidates in T 0 transgenic plants, which will be widely used in the area of plant gene editing. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction.

PubMed

Wilkes, Rebecca P; Tsai, Yun-Long; Lee, Pei-Yu; Lee, Fu-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

2014-09-09

Canine distemper virus (CDV) has been associated with outbreaks of canine infectious respiratory disease in shelters and boarding kennel environments. POCKITTM Nucleic Acid Analyzer is a field-deployable device capable of generating automatically interpreted insulated isothermal polymerase chain reaction (iiPCR) results from extracted nucleic acid within one hour. In this study, reverse transcription iiPCR (RT-iiPCR) was developed to facilitate point-of-need diagnosis of CDV infection. Analytical sensitivity (limit of detection 95%) of the established CDV RT-iiPCR was about 11 copies of in vitro transcribed RNA per reaction. CDV RT-iiPCR generated positive signals from CDV, but not Bordetella bronchiseptica, canine parvovirus, canine herpesvirus, canine adenovirus 2, canine influenza virus (subtype H3N8), canine parainfluenza virus, and canine respiratory coronavirus. To evaluate accuracy of the established reaction in canine distemper clinical diagnosis, 110 specimens from dogs, raccoons, and foxes suspected with CDV infection were tested simultaneously by CDV RT-iiPCR and real-time RT-PCR. CDV RT-iiPCR demonstrated excellent sensitivity (100%) and specificity (100%), compared to real-time RT-PCR. The results indicated an excellent correlation between RT-iiPCR and a reference real time RT-PCR method. Working in a lyophilized format, the established method has great potential to be used for point-of-care diagnosis of canine distemper in animals, especially in resource-limited facilities.

High-Throughput RT-PCR for small-molecule screening assays

PubMed Central

Bittker, Joshua A.

2012-01-01

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis. PMID:23487248

Methods for Real-Time PCR-Based Diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus Infections in an Opened Molecular Diagnostic Platform.

PubMed

Opota, Onya; Brouillet, René; Greub, Gilbert; Jaton, Katia

2017-01-01

The advances in molecular biology of the last decades have dramatically improved the field of diagnostic bacteriology. In particular, PCR-based technologies have impacted the diagnosis of infections caused by obligate intracellular bacteria such as pathogens from the Chlamydiacae family. Here, we describe a real-time PCR-based method using the Taqman technology for the diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus infection. The method presented here can be applied to various clinical samples and can be adapted on opened molecular diagnostic platforms.

Development of a fast PCR protocol enabling rapid generation of AmpFℓSTR® Identifiler® profiles for genotyping of human DNA.

PubMed

Foster, Amanda; Laurin, Nancy

2012-03-06

Traditional PCR methods for forensic STR genotyping require approximately 2.5 to 4 hours to complete, contributing a significant portion of the time required to process forensic DNA samples. The purpose of this study was to develop and validate a fast PCR protocol that enabled amplification of the 16 loci targeted by the AmpFℓSTR® Identifiler® primer set, allowing decreased cycling times. Fast PCR conditions were achieved by substituting the traditional Taq polymerase for SpeedSTAR™ HS DNA polymerase which is designed for fast PCR, by upgrading to a thermal cycler with faster temperature ramping rates and by modifying cycling parameters (less time at each temperature) and adopting a two-step PCR approach. The total time required for the optimized protocol is 26 min. A total of 147 forensically relevant DNA samples were amplified using the fast PCR protocol for Identifiler. Heterozygote peak height ratios were not affected by fast PCR conditions, and full profiles were generated for single-source DNA amounts between 0.125 ng and 2.0 ng. Individual loci in profiles produced with the fast PCR protocol exhibited average n-4 stutter percentages ranging from 2.5 ± 0.9% (THO1) to 9.9 ± 2.7% (D2S1338). No increase in non-adenylation or other amplification artefacts was observed. Minor contributor alleles in two-person DNA mixtures were reliably discerned. Low level cross-reactivity (monomorphic peaks) was observed with some domestic animal DNA. The fast PCR protocol presented offers a feasible alternative to current amplification methods and could aid in reducing the overall time in STR profile production or could be incorporated into a fast STR genotyping procedure for time-sensitive situations.

Evaluation of high-resolution melting curve analysis of ligation-mediated real-time PCR, a rapid method for epidemiological typing of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter Species) pathogens.

PubMed

Woksepp, Hanna; Ryberg, Anna; Billström, Hanna; Hällgren, Anita; Nilsson, Lennart E; Marklund, Britt-Inger; Olsson-Liljequist, Barbro; Schön, Thomas

2014-12-01

A single-tube method, ligation-mediated real-time PCR high-resolution melt analysis (LMqPCR HRMA), was modified for the rapid typing of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) pathogens. A 97% agreement (60/62 isolates) was achieved in comparison to pulsed-field gel electrophoresis (PFGE) results, which indicates that LMqPCR HRMA is a rapid and accurate screening tool for monitoring nosocomial outbreaks. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Duplex Real-Time PCR Method for the Differentiation of Cronobacter sakazakii and Cronobacter malonaticus.

PubMed

Li, Xiaofang; Cui, Jinghua; Du, Xiaoli; Cui, Zhigang; Huang, Yibing; Kan, Biao

2017-01-01

Cronobacter sakazakii and Cronobacter malonaticus are the most common species of Cronobacter , so it is necessary to detect the two species as soon as possible in surveillance programs. We developed a real-time PCR method for identifying C. sakazakii and C. malonaticus from the genus Cronobacter . In this study, the two pairs of primers and probes were designed, targeting 16S rRNA and fusA, respectively. The specificity of the real-time PCR assay was validated with 112 strains of Cronobacter , including 56 C. sakazakii , 32 C. malonaticus , 16 Cronobacter dublinensis , 6 Cronobacter turicensis , and 2 Cronobacter muytjensii . The results showed that C. sakazakii and C. malonaticus were all correctly identified, consistent with the results of another method by analyzing the clustering of the fusA sequence. The detection limit for pure culture was 10 2 CFU/ml and 10 3 CFU/g for artificially contaminated rehydrated powdered infant formula. Therefore, the developed real-time PCR was a rapid, sensitive, and reliable method for the identification of C. sakazakii and C. malonaticus .

Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman(®) real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay.

PubMed

Röder, Martin; Vieths, Stefan; Holzhauser, Thomas

2011-01-24

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg(-1) almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg(-1). We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman(®) probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg(-1) almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg(-1). Further, between 100 and 100,000 mg kg(-1) spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman(®) real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n=5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and potentially quantitative almond detection. This PCR method detects almond at a level where severe allergic reactions should not be expected for the majority of the almond allergic individuals. Copyright © 2010 Elsevier B.V. All rights reserved.

Culture and Real-Time PCR Based Maternal Screening and Antibiotic Susceptibility for Group B Streptococcus: An Iranian Experience.

PubMed

Goudarzi, Gholamreza; Ghafarzadeh, Masoumeh; Shakib, Pegah; Anbari, Khatereh

2015-04-19

Vertical Transmission of group B streptococcus (GBS) from a vagina colonized mother to her infant upon rupture of membranes (ROM) or after the onset of labor can cause life-threatening infections in newborn. Although intrapartum antibiotic prophylaxis (IAP) can significantly decrease neonatal GBS diseases, this issue has potentiated the emergence of antibiotic resistance strains. Our study examined the colonization rate of GBS using real-time PCR and culture methods, and trends in antibiotic resistance of GBS isolates obtained from pregnant women in Khorramabad, Iran. In this cross-sectional study, two vaginal-rectal swabs were collected and analyzed separately from 100 pregnant women at 35-37 weeks of gestation by convenience sampling method. The specimens were subjected to GBS detection using real-time PCR assay and standard culture. Susceptibility pattern of the GBS isolates was examined using the disk diffusion method. GBS carriage rate was 17% and 19% using culture and real-time PCR, respectively. In six samples, the culture was positive and the real-time PCR was negative. Sensitivity and specificity for real-time PCR were 72.7% and 96.1%, respectively using culture as the gold standard. Amongst twenty-two isolates examined, 100% resistance to erythromycin and clindamycin was observed. One isolate (4%) exhibited resistance to penicillin. Considering the relatively high GBS carriage rate in Khorramabad, routine antepartum screening for GBS is recommended. Penicillin can remain the antibiotic of choice for IAP; however, in penicillin-allergic mothers, vancomycin can be an alternative antibiotic.

[Rapid identification of meningitis due to bacterial pathogens].

PubMed

Ubukata, Kimiko

2013-01-01

We constructed a new real-time PCR method to detect causative pathogens in cerebrospinal fluid (CSF) from patient due to bacterial meningitis. The eight pathogens targeted in the PCR are Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus agalactiae, Staphylococcus aurues, Neisseria meningitides, Listeria monocytogenes, Esherichia coli, and Mycoplasma pneumoniae. The total time from DNA extraction from CSF to PCR analysis was 1.5 hour. The pathogens were detected in 72% of the CSF samples (n=115) by real-time PCR, but in only 48% by culture, although the microorganisms were completely concordant. The detection rate of pathogens with PCR was significantly better than that with cultures in patients with antibiotic administration.In conclusion, detection with real-time PCR is useful for rapidly identifying the causative pathogens of meningitis and for examining the clinical course of chemotherapy.

Evaluation of propidium monoazide real-time PCR for enumeration of probiotic lactobacilli microencapsulated in calcium alginate beads.

PubMed

Oketič, K; Matijašić, B Bogovič; Obermajer, T; Radulović, Z; Lević, S; Mirković, N; Nedović, V

2015-01-01

The aim of the study was to evaluate real-time PCR coupled with propidium monoazide (PMA) treatment for enumeration of microencapsulated probiotic lactobacilli microencapsulated in calcium alginate beads. Lactobacillus gasseri K7 (CCM 7710) and Lactobacillus delbrueckii subsp. bulgaricus (CCM 7712) were analysed by plate counting and PMA real-time PCR during storage at 4 °C for 90 days. PMA was effective in preventing PCR amplification of the target sequences of DNA released from heat-compromised bacteria. The values obtained by real-time PCR of non-treated samples were in general higher than those obtained by real-time PCR of PMA-treated samples or by plate counting, indicating the presence of sub-lethally injured cells. This study shows that plate count could not be completely replaced by culture independent method PMA real-time PCR for enumeration of probiotics, but may rather complement the well-established plate counting, providing useful information about the ratio of compromised bacteria in the samples.

Detection of foodborne pathogens using microarray technology

USDA-ARS?s Scientific Manuscript database

Assays based on the polymerase chain reaction (PCR) are now accepted methods for rapidly confirming the presence or absence of specific pathogens in foods and other types of samples. Conventional PCR requires the use of agarose gel electrophoresis to detect the PCR product; whereas, real-time PCR c...

Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.

PubMed

Sergueev, Kirill V; He, Yunxiu; Borschel, Richard H; Nikolich, Mikeljon P; Filippov, Andrey A

2010-06-28

Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.

Quantification of Wilms' tumor 1 mRNA by digital polymerase chain reaction.

PubMed

Koizumi, Yuki; Furuya, Daisuke; Endo, Teruo; Asanuma, Kouichi; Yanagihara, Nozomi; Takahashi, Satoshi

2018-02-01

Wilms' tumor 1 (WT1) is overexpressed in various hematopoietic tumors and widely used as a marker of minimal residual disease. WT1 mRNA has been analyzed using quantitative real-time polymerase chain reaction (real-time PCR). In the present study, we analyzed 40 peripheral blood and bone marrow samples obtained from cases of acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome at Sapporo Medical University Hospital from April 2012 to January 2015. We performed quantification of WT1 was performed using QuantStudio 3D Digital PCR System (Thermo Fisher Scientific‎) and compared the results between digital PCR and real-time PCR technology. The correlation between digital PCR and real-time PCR was very strong (R = 0.99), and the detection limits of the two methods were equivalent. Digital PCR was able to accurately detect lower WT levels compared with real-time PCR. Digital PCR technology can thus be utilized to predict WT1/ABL1 expression level accurately and should thus be useful for diagnosis or the evaluation of drug efficiency in patients with leukemia.

Targeted resequencing reveals ALK fusions in non-small cell lung carcinomas detected by FISH, immunohistochemistry, and real-time RT-PCR: a comparison of four methods.

PubMed

Tuononen, Katja; Sarhadi, Virinder Kaur; Wirtanen, Aino; Rönty, Mikko; Salmenkivi, Kaisa; Knuuttila, Aija; Remes, Satu; Telaranta-Keerie, Aino I; Bloor, Stuart; Ellonen, Pekka; Knuutila, Sakari

2013-01-01

Anaplastic lymphoma receptor tyrosine kinase (ALK) gene rearrangements occur in a subgroup of non-small cell lung carcinomas (NSCLCs). The identification of these rearrangements is important for guiding treatment decisions. The aim of our study was to screen ALK gene fusions in NSCLCs and to compare the results detected by targeted resequencing with results detected by commonly used methods, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and real-time reverse transcription-PCR (RT-PCR). Furthermore, we aimed to ascertain the potential of targeted resequencing in detection of ALK-rearranged lung carcinomas. We assessed ALK fusion status for 95 formalin-fixed paraffin-embedded tumor tissue specimens from 87 patients with NSCLC by FISH and real-time RT-PCR, for 57 specimens from 56 patients by targeted resequencing, and for 14 specimens from 14 patients by IHC. All methods were performed successfully on formalin-fixed paraffin-embedded tumor tissue material. We detected ALK fusion in 5.7% (5 out of 87) of patients examined. The results obtained from resequencing correlated significantly with those from FISH, real-time RT-PCR, and IHC. Targeted resequencing proved to be a promising method for ALK gene fusion detection in NSCLC. Means to reduce the material and turnaround time required for analysis are, however, needed.

Evaluation of Commercial Cell Preparations as Sources of Calibration Standards for Real-Time qPCR Analysis of Enterococci in Recreational Waters

EPA Science Inventory

In response to the Beach Act, the U.S. EPA has developed a quantitative PCR (qPCR) method for enterococci that meets requirements for rapid, risk-based water quality assessments of recreational waters. Widespread implementation of this method will require that different laborator...

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

PubMed Central

Ogrean, Christy; Jackson, Ben; Covino, James

2010-01-01

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

Comparison of real-time PCR with disk diffusion, agar screen and E-test methods for detection of methicillin-resistant Staphylococcus aureus.

PubMed

Shariati, Laleh; Validi, Majid; Tabatabaiefar, Mohammad Amin; Karimi, Ali; Nafisi, Mohammad Reza

2010-12-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the "gold standard" comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.

Nested PCR detection of malaria directly using blood filter paper samples from epidemiological surveys.

PubMed

Li, Peipei; Zhao, Zhenjun; Wang, Ying; Xing, Hua; Parker, Daniel M; Yang, Zhaoqing; Baum, Elizabeth; Li, Wenli; Sattabongkot, Jetsumon; Sirichaisinthop, Jeeraphat; Li, Shuying; Yan, Guiyun; Cui, Liwang; Fan, Qi

2014-05-08

Nested PCR is considered a sensitive and specific method for detecting malaria parasites and is especially useful in epidemiological surveys. However, the preparation of DNA templates for PCR is often time-consuming and costly. A simplified PCR method was developed to directly use a small blood filter paper square (2 × 2 mm) as the DNA template after treatment with saponin. This filter paper-based nested PCR method (FP-PCR) was compared to microscopy and standard nested PCR with DNA extracted by using a Qiagen DNA mini kit from filter paper blood spots of 204 febrile cases. The FP-PCR technique was further applied to evaluate malaria infections in 1,708 participants from cross-sectional epidemiological surveys conducted in Myanmar and Thailand. The FP-PCR method had a detection limit of ~0.2 parasites/μL blood, estimated using cultured Plasmodium falciparum parasites. With 204 field samples, the sensitivity of the FP-PCR method was comparable to that of the standard nested PCR method, which was significantly higher than that of microscopy. Application of the FP-PCR method in large cross-sectional studies conducted in Myanmar and Thailand detected 1.9% (12/638) and 6.2% (66/1,070) asymptomatic Plasmodium infections, respectively, as compared to the detection rates of 1.3% (8/638) and 0.04% (4/1,070) by microscopy. This FP-PCR method was much more sensitive than microscopy in detecting Plasmodium infections. It drastically increased the detection sensitivity of asymptomatic infections in cross-sectional surveys conducted in Thailand and Myanmar, suggesting that this FP-PCR method has a potential for future applications in malaria epidemiology studies.

Detection of Strongylus vulgaris in equine faecal samples by real-time PCR and larval culture - method comparison and occurrence assessment.

PubMed

Kaspar, A; Pfister, K; Nielsen, M K; Silaghi, C; Fink, H; Scheuerle, M C

2017-01-11

Strongylus vulgaris has become a rare parasite in Germany during the past 50 years due to the practice of frequent prophylactic anthelmintic therapy. To date, the emerging development of resistance in Cyathostominae and Parascaris spp. to numerous equine anthelmintics has changed deworming management and the frequency of anthelmintic usage. In this regard, reliable detection of parasitic infections, especially of the highly pathogenic S. vulgaris is essential. In the current study, two diagnostic methods for the detection of infections with S. vulgaris were compared and information on the occurrence of this parasite in German horses was gained. For this purpose, faecal samples of 501 horses were screened for S. vulgaris with real-time PCR and an additional larval culture was performed in samples of 278 horses. A subset of 26 horses underwent multiple follow-up examinations with both methods in order to evaluate both the persistence of S. vulgaris infections and the reproducibility of each diagnostic method. The real-time PCR revealed S. vulgaris-DNA in ten of 501 investigated equine samples (1.9%). The larval culture demonstrated larvae of S. vulgaris in three of the 278 samples (1.1%). A direct comparison of the two methods was possible in 321 samples including 43 follow-up examinations with the result of 11 S. vulgaris-positive samples by real-time PCR and 4 S. vulgaris-positive samples by larval culture. The McNemar's test (p-value = 0.016) revealed a significant difference and the kappa values (0.525) showed a moderate agreement between real-time PCR and larval culture. The real-time PCR detected a significantly higher proportion of positives of S. vulgaris compared to larval culture and should thus be considered as a routine diagnostic method for the detection of S. vulgaris in equine samples.

Assessment of Clinically Suspected Tubercular Lymphadenopathy by Real-Time PCR Compared to Non-Molecular Methods on Lymph Node Aspirates.

PubMed

Gupta, Vivek; Bhake, Arvind

2018-01-01

The diagnosis of tubercular lymphadenitis (TBLN) is challenging. This study assesses the role of diagnostic intervention with real-time PCR in clinically suspected tubercular lymphadenopathy in relation to cytology and microbiological methods. The cross-sectional study involved 214 patients, and PCR, cytology, and Ziehl-Neelsen (ZN) staining was performed on aspirates. The findings were compared with culture on Lowenstein-Jensen medium. The overall concordance of cytology and PCR, both individually and combined, was calculated. χ2 and Phi values were assessed between cytology, PCR, and culture. A cytological diagnosis of tuberculosis (TB), reactive lymphoid hyperplasia, and suppurative lymphadenitis was made in 71, 112, and 6 patients, respectively. PCR and culture were positive in 40% of the cases. Among the TBLN patients, PCR showed higher positivity in necrosis and culture showed higher positivity in necrotizing granuloma. Positive ZN staining was observed in 29.6% of the TBLN cases, with an overall positivity of 11%. PCR could additionally detect 82 cases missed by ZN staining. The overall concordance rate for either diagnostic modality, i.e., PCR or cytology, was highest (75%), and for PCR alone was 74%. Phi values were observed to be 0.47 between PCR and culture. Real-time PCR for Mycobacterium tuberculosis complex on aspirates offers a definitive and comparable diagnosis of TBLN. Including this approach as the primary investigation in the work-up of TBLN could reduce the burden of TB. © 2017 S. Karger AG, Basel.

Design and Assessment of a Real Time Reverse Transcription-PCR Method to Genotype Single-Stranded RNA Male-Specific Coliphages (Family Leviviridae).

EPA Science Inventory

A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assa...

Real-time PCR detection of Listeria monocytogenes in infant formula and lettuce following macrophage-based isolation and enrichment.

PubMed

Day, J B; Basavanna, U

2015-01-01

To develop a rapid detection procedure for Listeria monocytogenes in infant formula and lettuce using a macrophage-based enrichment protocol and real-time PCR. A macrophage cell culture system was employed for the isolation and enrichment of L. monocytogenes from infant formula and lettuce for subsequent identification using real-time PCR. Macrophage monolayers were exposed to infant formula and lettuce contaminated with a serial dilution series of L. monocytogenes. As few as approx. 10 CFU ml(-1) or g(-1) of L. monocytogenes were detected in infant formula and lettuce after 16 h postinfection by real-time PCR. Internal positive PCR controls were utilized to eliminate the possibility of false-negative results. Co-inoculation with Listeria innocua did not reduce the L. monocytogenes detection sensitivity. Intracellular L. monocytogenes could also be isolated on Listeria selective media from infected macrophage lysates for subsequent confirmation. The detection method is highly sensitive and specific for L. monocytogenes in infant formula and lettuce and establishes a rapid identification time of 20 and 48 h for presumptive and confirmatory identification, respectively. The method is a promising alternative to many currently used q-PCR detection methods which employ traditional selective media for enrichment of contaminated food samples. Macrophage enrichment of L. monocytogenes eliminates PCR inhibitory food elements and contaminating food microflora which produce cleaner samples that increase the rapidity and sensitivity of detection. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix.

PubMed

Kaufman, G E; Blackstone, G M; Vickery, M C L; Bej, A K; Bowers, J; Bowen, Michael D; Meyer, Richard F; DePaola, A

2004-11-01

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.

Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papaya L.).

PubMed

Nageswara-Rao, Madhugiri; Kwit, Charles; Agarwal, Sujata; Patton, Mariah T; Skeen, Jordan A; Yuan, Joshua S; Manshardt, Richard M; Stewart, C Neal

2013-09-01

Genetically engineered (GE) ringspot virus-resistant papaya cultivars 'Rainbow' and 'SunUp' have been grown in Hawai'i for over 10 years. In Hawai'i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities. We assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/μl of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%. This method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai'i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs.

Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya (Carica papaya L.)

PubMed Central

2013-01-01

Background Genetically engineered (GE) ringspot virus-resistant papaya cultivars ‘Rainbow’ and ‘SunUp’ have been grown in Hawai’i for over 10 years. In Hawai’i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities. Results We assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/μl of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%. Conclusions This method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai’i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs. PMID:24004548

Rapid detection of Porcine circovirus 2 by recombinase polymerase amplification.

PubMed

Wang, Jianchang; Wang, Jinfeng; Liu, Libing; Li, Ruiwen; Yuan, Wanzhe

2016-09-01

Porcine circovirus-associated disease, caused primarily by Porcine circovirus 2 (PCV-2), has become endemic in many pig-producing countries and has resulted in significant economic losses to the swine industry worldwide. Tests for PCV-2 infection include PCR, nested PCR, competitive PCR, and real-time PCR (rtPCR). Recombinase polymerase amplification (RPA) has emerged as an isothermal gene amplification technology for the molecular detection of infectious disease agents. RPA is performed at a constant temperature and therefore can be carried out in a water bath. In addition, RPA is completed in ~30 min, much faster than PCR, which usually takes >60 min. We developed a RPA-based method for the detection of PCV-2. The detection limit of RPA was 10(2) copies of PCV-2 genomic DNA. RPA showed the same sensitivity as rtPCR but was 10 times more sensitive than conventional PCR. Successful amplification of PCV-2 DNA, but not other viral templates, demonstrated high specificity of the RPA assay. This method was also validated using clinical samples. The results showed that the RPA assay had a diagnostic agreement rate of 93.7% with conventional PCR and 100% with rtPCR. These findings suggest that the RPA assay is a simple, rapid, and cost-effective method for PCV-2 detection, which could be potentially applied in clinical diagnosis and field surveillance of PCV-2 infection. © 2016 The Author(s).

A novel strategy to obtain quantitative data for modelling: combined enrichment and real-time PCR for enumeration of salmonellae from pig carcasses.

PubMed

Krämer, Nadine; Löfström, Charlotta; Vigre, Håkan; Hoorfar, Jeffrey; Bunge, Cornelia; Malorny, Burkhard

2011-03-01

Salmonella is a major zoonotic pathogen which causes outbreaks and sporadic cases of gastroenteritis in humans worldwide. The primary sources for Salmonella are food-producing animals such as pigs and poultry. For risk assessment and hazard analysis and critical control point (HACCP) concepts, it is essential to produce large amounts of quantitative data, which is currently not achievable with the standard cultural based methods for enumeration of Salmonella. This study presents the development of a novel strategy to enumerate low numbers of Salmonella in cork borer samples taken from pig carcasses as a first concept and proof of principle for a new sensitive and rapid quantification method based on combined enrichment and real-time PCR. The novelty of the approach is in the short pre-enrichment step, where for most bacteria, growth is in the log phase. The method consists of an 8h pre-enrichment of the cork borer sample diluted 1:10 in non-selective buffered peptone water, followed by DNA extraction, and Salmonella detection and quantification by real-time PCR. The limit of quantification was 1.4 colony forming units (CFU)/20 cm(2) (approximately 10 g) of artificially contaminated sample with 95% confidence interval of ± 0.7 log CFU/sample. The precision was similar to the standard reference most probable number (MPN) method. A screening of 200 potentially naturally contaminated cork borer samples obtained over seven weeks in a slaughterhouse resulted in 25 Salmonella-positive samples. The analysis of salmonellae within these samples showed that the PCR method had a higher sensitivity for samples with a low contamination level (<6.7 CFU/sample), where 15 of the samples negative with the MPN method was detected with the PCR method and 5 were found to be negative by both methods. For the samples with a higher contamination level (6.7-310 CFU/sample) a good agreement between the results obtained with the PCR and MPN methods was obtained. The quantitative real-time PCR method can easily be applied to other food and environmental matrices by adaptation of the pre-enrichment time and media. Copyright © 2010 Elsevier B.V. All rights reserved.

Protocol for the use of light upon extension real-time PCR for the determination of viral load in HBV infection.

PubMed

Li, Guimin; Li, Wangfeng; Liu, Lixia

2012-01-01

Real-time PCR has engendered wide acceptance for quantitation of hepatitis B virus (HBV) DNA in the blood due to its improved rapidity, sensitivity, reproducibility, and reduced contamination. Here we describe a cost-effective and highly sensitive HBV real-time quantitative assay based on the light upon extension real-time PCR platform and a simple and reliable HBV DNA preparation method using silica-coated magnetic beads.

dPCR: A Technology Review

PubMed Central

Quan, Phenix-Lan; Sauzade, Martin

2018-01-01

Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods. PMID:29677144

Rapid detection method for Bacillus anthracis using a combination of multiplexed real-time PCR and pyrosequencing and its application for food biodefense.

PubMed

Janzen, Timothy W; Thomas, Matthew C; Goji, Noriko; Shields, Michael J; Hahn, Kristen R; Amoako, Kingsley K

2015-02-01

Bacillus anthracis, the causative agent of anthrax, has the capacity to form highly resilient spores as part of its life cycle. The potential for the dissemination of these spores using food as a vehicle is a huge public health concern and, hence, requires the development of a foodborne bioterrorism response approach. In this work, we address a critical gap in food biodefense by presenting a novel, combined, sequential method involving the use of real-time PCR and pyrosequencing for the rapid, specific detection of B. anthracis spores in three food matrices: milk, apple juice, and bottled water. The food samples were experimentally inoculated with 40 CFU ml(-1), and DNA was extracted from the spores and analyzed after immunomagnetic separation. Applying the combination of multiplex real-time PCR and pyrosequencing, we successfully detected the presence of targets on both of the virulence plasmids and the chromosome. The results showed that DNA amplicons generated from a five-target multiplexed real-time PCR detection using biotin-labeled primers can be used for single-plex pyrosequencing detection. The combined use of multiplexed real-time PCR and pyrosequencing is a novel, rapid detection method for B. anthracis from food and provides a tool for accurate, quantitative identification with potential biodefense applications.

Recent developments in detection and enumeration of waterborne bacteria: a retrospective minireview.

PubMed

Deshmukh, Rehan A; Joshi, Kopal; Bhand, Sunil; Roy, Utpal

2016-12-01

Waterborne diseases have emerged as global health problems and their rapid and sensitive detection in environmental water samples is of great importance. Bacterial identification and enumeration in water samples is significant as it helps to maintain safe drinking water for public consumption. Culture-based methods are laborious, time-consuming, and yield false-positive results, whereas viable but nonculturable (VBNCs) microorganisms cannot be recovered. Hence, numerous methods have been developed for rapid detection and quantification of waterborne pathogenic bacteria in water. These rapid methods can be classified into nucleic acid-based, immunology-based, and biosensor-based detection methods. This review summarizes the principle and current state of rapid methods for the monitoring and detection of waterborne bacterial pathogens. Rapid methods outlined are polymerase chain reaction (PCR), digital droplet PCR, real-time PCR, multiplex PCR, DNA microarray, Next-generation sequencing (pyrosequencing, Illumina technology and genomics), and fluorescence in situ hybridization that are categorized as nucleic acid-based methods. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence are classified into immunology-based methods. Optical, electrochemical, and mass-based biosensors are grouped into biosensor-based methods. Overall, these methods are sensitive, specific, time-effective, and important in prevention and diagnosis of waterborne bacterial diseases. © 2016 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

FY*X real-time polymerase chain reaction with melting curve analysis associated with a complete one-step real-time FY genotyping.

PubMed

Ansart-Pirenne, H; Martin-Blanc, S; Le Pennec, P-Y; Rouger, P; Cartron, J-P; Tournamille, C

2007-02-01

The Duffy (FY) blood group system is controlled by four major alleles: FY*A and FY*B, the Caucasian common alleles, encoding Fy(a) and Fy(b) antigens; FY*X allele responsible for a poorly expressed Fy(b) antigen, and FY*Fy a silent predominant allele among Black population. Despite the recent development of a real-time fluorescent polymerase chain reaction (PCR) method for FY genotyping FY*X genotyping has not been described by this method. This study focused on the real-time FY*X genotyping development associated with a complete, one-step real-time FY genotyping, based on fluorescence resonance energy transfer (FRET) technology. Seventy-two blood samples from Fy(a+b-) Caucasian blood donors were studied by real-time PCR only. Forty-seven Caucasian and Black individual blood samples, referred to our laboratory, were studied by PCR-RFLP and real-time PCR. For each individual, the result of the genotype was compared to the known phenotype. The FY*X allele frequency calculated in an Fy(a+b-) Caucasian blood donors population was 0.014. With the Caucasian and Black patient samples we found a complete correlation between PCR-RFLP and the real-time PCR method whatever the alleles combination tested. When the known phenotype was not correlated to FY*X genotype, the presence of the Fy(b) antigen was always confirmed by adsorption-elution. The real-time technology method is rapid and accurate for FY genotyping. From now, we are able to detect the FY*X allele in all the alleles combinations studied. Regarding its significant frequency, the detection of the FY*X allele is useful for the correct typing of blood donors and recipients considering the therapeutic use of blood units and the preparation of test red blood cells for antibody screening.

An immunomagnetic separation-real-time PCR system for the detection of Alicyclobacillus acidoterrestris in fruit products.

PubMed

Wang, Zhouli; Cai, Rui; Yuan, Yahong; Niu, Chen; Hu, Zhongqiu; Yue, Tianli

2014-04-03

Alicyclobacillus acidoterrestris is the most important spoilage species within the Alicyclobacillus genus and has become a major issue in the pasteurized fruit juice industry. The aim of this study was to develop a method combining immunomagnetic separation (IMS) with real-time PCR system (IMS-PCR) for rapid and specific detection of A. acidoterrestris in fruit products. A real-time PCR with the TaqMan system was designed to target the 16S rDNA genes with specific primer and probe set. The specificity of the assay was confirmed using 9 A. acidoterrestris strains and 21 non-A. acidoterrestris strains. The results indicated that no combination of the designed primers and probe was found in any Alicyclobacillus genus except A. acidoterrestris. The detection limit of the established IMS-PCR was less than 10CFU/mL and the testing process was accomplished in 2-3h. For the three types of samples (sterile water, apple juice and kiwi juice), the correlation coefficient of standard curves was greater than 0.991, and the calculated PCR efficiencies were from 108% to 109%. As compared with the standard culture method performed concurrently on the same set of samples, the sensitivity, specificity and accuracy of IMS-PCR for 196 naturally contaminated fruit products were 90.0%, 98.3% and 97.5%, respectively. The results exhibited that the proposed IMS-PCR method was effective for the rapid detection of A. acidoterrestris in fruit products. Copyright © 2014. Published by Elsevier B.V.

Determining the prevalence of inv-positive and ail-positive Yersinia enterocolitica in pig tonsils using PCR and culture methods.

PubMed

Stachelska, Milena Alicja

2017-01-01

Yersiniosis is believed to be the third most common intestinal zoonosis in the European Union, after campylobacteriosis and salmonellosis. Yersinia enterocolitica is the most common species responsible for human infections. Pigs are regarded as the biggest reservoir of pathogenic Y. enterocolitica strains, which are mainly isolated from pig tonsils. The aim of this paper is to examine the prevalence of inv-positive and ail-positive Y. enterocolitica in pigs which were slaughtered in a Polish abattoir. Real-time PCR and culture methods were used to assess the prevalence of patho- genic Y. enterocolitica strains in pig tonsils. Real-time PCR was applied to detect inv-positive and ail-positive Y. enterocolitica. Y. enterocolitica was also isolated by applying direct plating, unselective (tryptic soy broth) and selective (irgasan-ticarcillin-potassium chlorate bouillon) enrichment. A total of 180 pigs were studied, of which 85% and 32% respectively were found to be infected with inv-positive and ail-positive Y. enterocolitica. The 92 inv-positive and ail-positive isolates, from 57 culture- positive tonsils, underwent bio- and serotyping. The most common was bioserotype 4/O:3, which was found in 53 (93%) out of 57 culture-positive tonsils. Strains of bioserotypes 2/O:5, 2/O:9 and 2/O:5.27 occurred in significantly lower numbers. The prevalence of inv-positive and ail-positive Y. enterocolitica was found to be high in the ton- sils of slaughtered pigs, using real-time PCR. The real-time PCR method for the detection and identification of pathogenic Y. enterocolitica is sensitive and specific, which has been verified by specificity and sensitivity tests using the pure cultures. Serotypes were distinguished from each other using PCR serotyping. The PCR method was essential in forming our conclusions.

Detection of 22 common leukemic fusion genes using a single-step multiplex qRT-PCR-based assay.

PubMed

Lyu, Xiaodong; Wang, Xianwei; Zhang, Lina; Chen, Zhenzhu; Zhao, Yu; Hu, Jieying; Fan, Ruihua; Song, Yongping

2017-07-25

Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management. We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization. Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL). We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an effective alternative to lengthy conventional diagnostic procedures requiring both cytogenetic analysis followed by targeted quantitative reverse transcription (qRT-PCR) methods, thus allowing timely patient management.

Development of PMA real-time PCR method to quantify viable cells of Pantoea agglomerans CPA-2, an antagonist to control the major postharvest diseases on oranges.

PubMed

Soto-Muñoz, Lourdes; Teixidó, Neus; Usall, Josep; Viñas, Inmaculada; Crespo-Sempere, Ana; Torres, Rosario

2014-06-16

Dilution plating is the quantification method commonly used to estimate the population level of postharvest biocontrol agents, but this method does not permit a distinction among introduced and indigenous strains. Recently, molecular techniques based on DNA amplification such as quantitative real-time PCR (qPCR) have been successfully applied for their high strain-specific detection level. However, the ability of qPCR to distinguish viable and nonviable cells is limited. A promising strategy to avoid this issue relies on the use of nucleic acid intercalating dyes, such as propidium monoazide (PMA), as a sample pretreatment prior to the qPCR. The objective of this study was to optimize a protocol based on PMA pre-treatment samples combined with qPCR to distinguish and quantify viable cells of the biocontrol agent P. agglomerans CPA-2 applied as a postharvest treatment on orange. The efficiency of PMA-qPCR method under the established conditions (30μM PMA for 20min of incubation followed by 30min of LED light exposure) was evaluated on an orange matrix. Results showed no difference in CFU or cells counts of viable cells between PMA-qPCR and dilution plating. Samples of orange matrix inoculated with a mixture of viable/dead cells showed 5.59log10 CFU/ml by dilution plating, 8.25log10 cells/ml by qPCR, and 5.93log10 cells/ml by PMA-qPCR. Furthermore, samples inoculated with heat-killed cells were not detected by dilution plating and PMA-qPCR, while by qPCR was of 8.16log10 cells/ml. The difference in quantification cycles (Cq) among qPCR and PMA-qPCR was approximately 16cycles, which means a reduction of 65,536 fold of the dead cells detected. In conclusion, PMA-qPCR method is a suitable tool for quantify viable CPA-2 cells, which could be useful to estimate the ability of this antagonist to colonize the orange surface. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of duplex real-time PCR for the detection of WSSV and PstDV1 in cultivated shrimp.

PubMed

Leal, Carlos A G; Carvalho, Alex F; Leite, Rômulo C; Figueiredo, Henrique C P

2014-07-05

The White spot syndrome virus (WSSV) and Penaeus stylirostris penstyldensovirus 1 (previously named Infectious hypodermal and hematopoietic necrosis virus-IHHNV) are two of the most important viral pathogens of penaeid shrimp. Different methods have been applied for diagnosis of these viruses, including Real-time PCR (qPCR) assays. A duplex qPCR method allows the simultaneous detection of two viruses in the same sample, which is more cost-effective than assaying for each virus separately. Currently, an assay for the simultaneous detection of the WSSV and the PstDV1 in shrimp is unavailable. The aim of this study was to develop and standardize a duplex qPCR assay for the simultaneous detection of the WSSV and the PstDV1 in clinical samples of diseased L. vannamei. In addition, to evaluate the performance of two qPCR master mixes with regard to the clinical sensitivity of the qPCR assay, as well as, different methods for qPCR results evaluation. The duplex qPCR assay for detecting WSSV and PstDV1 in clinical samples was successfully standardized. No difference in the amplification of the standard curves was observed between the duplex and singleplex assays. Specificities and sensitivities similar to those of the singleplex assays were obtained using the optimized duplex qPCR. The analytical sensitivities of duplex qPCR were two copies of WSSV control plasmid and 20 copies of PstDV1 control plasmid. The standardized duplex qPCR confirmed the presence of viral DNA in 28 from 43 samples tested. There was no difference for WSSV detection using the two kits and the distinct methods for qPCR results evaluation. High clinical sensitivity for PstDV1 was obtained with TaqMan Universal Master Mix associated with relative threshold evaluation. Three cases of simultaneous infection by the WSSV and the PstDV1 were identified with duplex qPCR. The standardized duplex qPCR was shown to be a robust, highly sensitive, and feasible diagnostic tool for the simultaneous detection of the WSSV and the PstDV1 in whiteleg shrimp. The use of the TaqMan Universal Master Mix and the relative threshold method of data analysis in our duplex qPCR method provided optimal levels of sensitivity and specificity.

Comparison of polymerase chain reaction methods and plating for analysis of enriched cultures of Listeria monocytogenes when using the ISO11290-1 method.

PubMed

Dalmasso, Marion; Bolocan, Andrei Sorin; Hernandez, Marta; Kapetanakou, Anastasia E; Kuchta, Tomáš; Manios, Stavros G; Melero, Beatriz; Minarovičová, Jana; Muhterem, Meryem; Nicolau, Anca Ioana; Rovira, Jordi; Skandamis, Panagiotis N; Stessl, Beatrix; Wagner, Martin; Jordan, Kieran; Rodríguez-Lázaro, David

2014-03-01

Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and subsequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to determine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the detection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid-phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR resulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was significantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is required, RTi-PCR could be an efficient way to get a preliminary result to be then confirmed by plating. Using DNA extraction from the second enrichment broth followed by RTi-PCR was reliable and a confirmed result could be obtained in three days, as against seven days by ISO11290-1. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid detection of Salmonella in pet food: design and evaluation of integrated methods based on real-time PCR detection.

PubMed

Balachandran, Priya; Friberg, Maria; Vanlandingham, V; Kozak, K; Manolis, Amanda; Brevnov, Maxim; Crowley, Erin; Bird, Patrick; Goins, David; Furtado, Manohar R; Petrauskene, Olga V; Tebbs, Robert S; Charbonneau, Duane

2012-02-01

Reducing the risk of Salmonella contamination in pet food is critical for both companion animals and humans, and its importance is reflected by the substantial increase in the demand for pathogen testing. Accurate and rapid detection of foodborne pathogens improves food safety, protects the public health, and benefits food producers by assuring product quality while facilitating product release in a timely manner. Traditional culture-based methods for Salmonella screening are laborious and can take 5 to 7 days to obtain definitive results. In this study, we developed two methods for the detection of low levels of Salmonella in pet food using real-time PCR: (i) detection of Salmonella in 25 g of dried pet food in less than 14 h with an automated magnetic bead-based nucleic acid extraction method and (ii) detection of Salmonella in 375 g of composite dry pet food matrix in less than 24 h with a manual centrifugation-based nucleic acid preparation method. Both methods included a preclarification step using a novel protocol that removes food matrix-associated debris and PCR inhibitors and improves the sensitivity of detection. Validation studies revealed no significant differences between the two real-time PCR methods and the standard U.S. Food and Drug Administration Bacteriological Analytical Manual (chapter 5) culture confirmation method.

Evaluation of two real time PCR assays for the detection of bacterial DNA in amniotic fluid.

PubMed

Girón de Velasco-Sada, Patricia; Falces-Romero, Iker; Quiles-Melero, Inmaculada; García-Perea, Adela; Mingorance, Jesús

2018-01-01

The aim of this study was to evaluate two non-commercial Real-Time PCR assays for the detection of microorganisms in amniotic fluid followed by identification by pyrosequencing. We collected 126 amniotic fluids from 2010 to 2015 for the evaluation of two Real-Time PCR assays for detection of bacterial DNA in amniotic fluid (16S Universal PCR and Ureaplasma spp. specific PCR). The method was developed in the Department of Microbiology of the University Hospital La Paz. Thirty-seven samples (29.3%) were positive by PCR/pyrosequencing and/or culture, 4 of them were mixed cultures with Ureaplasma urealyticum. The Universal 16S Real-Time PCR was compared with the standard culture (81.8% sensitivity, 97.4% specificity, 75% positive predictive value, 98% negative predictive value). The Ureaplasma spp. specific Real-Time PCR was compared with the Ureaplasma/Mycoplasma specific culture (92.3% sensitivity, 89.4% specificity, 50% positive predictive value, 99% negative predictive value) with statistically significant difference (p=0.005). Ureaplasma spp. PCR shows a rapid response time (5h from DNA extraction until pyrosequencing) when comparing with culture (48h). So, the response time of bacteriological diagnosis in suspected chorioamnionitis is reduced. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of Brucella spp. in milk from seronegative cows by real-time polymerase chain reaction in the region of Batna, Algeria

PubMed Central

Sabrina, Rabehi; Mossadak, Hamdi Taha; Bakir, Mamache; Asma, Meghezzi; Khaoula, Boushaba

2018-01-01

Aim: The aim of this study was to detect Brucella spp. DNA in milk samples collected from seronegative cows using the real-time polymerase chain reaction (PCR) assay for diagnosis of brucellosis in seronegative dairy cows to prevent transmission of disease to humans and to reduce economic losses in animal production. Materials and Methods: In this study, 65 milk samples were investigated for the detection of Brucella spp. The detection of the IS711 gene in all samples was done by real-time PCR assay by comparative cycle threshold method. Results: The results show that of the 65 DNA samples tested, 2 (3.08%) were positive for Brucella infection. The mean cyclic threshold values of IS711 real-time PCR test were 37.97 and 40.48, indicating a positive reaction. Conclusion: The results of the present study indicated that the real-time PCR appears to offer several advantages over serological tests. For this reason, the real-time PCR should be validated on representative numbers of Brucella-infected and free samples before being implemented in routine diagnosis in human and animal brucellosis for controlling this disease. PMID:29657430

Ultrafast, sensitive and large-volume on-chip real-time PCR for the molecular diagnosis of bacterial and viral infections.

PubMed

Houssin, Timothée; Cramer, Jérémy; Grojsman, Rébecca; Bellahsene, Lyes; Colas, Guillaume; Moulet, Hélène; Minnella, Walter; Pannetier, Christophe; Leberre, Maël; Plecis, Adrien; Chen, Yong

2016-04-21

To control future infectious disease outbreaks, like the 2014 Ebola epidemic, it is necessary to develop ultrafast molecular assays enabling rapid and sensitive diagnoses. To that end, several ultrafast real-time PCR systems have been previously developed, but they present issues that hinder their wide adoption, notably regarding their sensitivity and detection volume. An ultrafast, sensitive and large-volume real-time PCR system based on microfluidic thermalization is presented herein. The method is based on the circulation of pre-heated liquids in a microfluidic chip that thermalize the PCR chamber by diffusion and ultrafast flow switches. The system can achieve up to 30 real-time PCR cycles in around 2 minutes, which makes it the fastest PCR thermalization system for regular sample volume to the best of our knowledge. After biochemical optimization, anthrax and Ebola simulating agents could be respectively detected by a real-time PCR in 7 minutes and a reverse transcription real-time PCR in 7.5 minutes. These detections are respectively 6.4 and 7.2 times faster than with an off-the-shelf apparatus, while conserving real-time PCR sample volume, efficiency, selectivity and sensitivity. The high-speed thermalization also enabled us to perform sharp melting curve analyses in only 20 s and to discriminate amplicons of different lengths by rapid real-time PCR. This real-time PCR microfluidic thermalization system is cost-effective, versatile and can be then further developed for point-of-care, multiplexed, ultrafast and highly sensitive molecular diagnoses of bacterial and viral diseases.

Enhanced analysis of real-time PCR data by using a variable efficiency model: FPK-PCR

PubMed Central

Lievens, Antoon; Van Aelst, S.; Van den Bulcke, M.; Goetghebeur, E.

2012-01-01

Current methodology in real-time Polymerase chain reaction (PCR) analysis performs well provided PCR efficiency remains constant over reactions. Yet, small changes in efficiency can lead to large quantification errors. Particularly in biological samples, the possible presence of inhibitors forms a challenge. We present a new approach to single reaction efficiency calculation, called Full Process Kinetics-PCR (FPK-PCR). It combines a kinetically more realistic model with flexible adaptation to the full range of data. By reconstructing the entire chain of cycle efficiencies, rather than restricting the focus on a ‘window of application’, one extracts additional information and loses a level of arbitrariness. The maximal efficiency estimates returned by the model are comparable in accuracy and precision to both the golden standard of serial dilution and other single reaction efficiency methods. The cycle-to-cycle changes in efficiency, as described by the FPK-PCR procedure, stay considerably closer to the data than those from other S-shaped models. The assessment of individual cycle efficiencies returns more information than other single efficiency methods. It allows in-depth interpretation of real-time PCR data and reconstruction of the fluorescence data, providing quality control. Finally, by implementing a global efficiency model, reproducibility is improved as the selection of a window of application is avoided. PMID:22102586

Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall.

PubMed

Ooi, Delicia Shu Qin; Tan, Verena Ming Hui; Ong, Siong Gim; Chan, Yiong Huak; Heng, Chew Kiat; Lee, Yung Seng

2017-01-01

The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed.

[Quantitative PCR in the diagnosis of Leishmania].

PubMed

Mortarino, M; Franceschi, A; Mancianti, F; Bazzocchi, C; Genchi, C; Bandi, C

2004-06-01

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.

Application of quantitative real-time PCR compared to filtration methods for the enumeration of Escherichia coli in surface waters within Vietnam.

PubMed

Vital, Pierangeli G; Van Ha, Nguyen Thi; Tuyet, Le Thi Hong; Widmer, Kenneth W

2017-02-01

Surface water samples in Vietnam were collected from the Saigon River, rural and suburban canals, and urban runoff canals in Ho Chi Minh City, Vietnam, and were processed to enumerate Escherichia coli. Quantification was done through membrane filtration and quantitative real-time polymerase chain reaction (PCR). Mean log colony-forming unit (CFU)/100 ml E. coli counts in the dry season for river/suburban canals and urban canals were log 2.8 and 3.7, respectively, using a membrane filtration method, while using Taqman quantitative real-time PCR they were log 2.4 and 2.8 for river/suburban canals and urban canals, respectively. For the wet season, data determined by the membrane filtration method in river/suburban canals and urban canals samples had mean counts of log 3.7 and 4.1, respectively. While mean log CFU/100 ml counts in the wet season using quantitative PCR were log 3 and 2, respectively. Additionally, the urban canal samples were significantly lower than those determined by conventional culture methods for the wet season. These results show that while quantitative real-time PCR can be used to determine levels of fecal indicator bacteria in surface waters, there are some limitations to its application and it may be impacted by sources of runoff based on surveyed samples.

Micro-droplet Digital Polymerase Chain Reaction and Real-Time Quantitative Polymerase Chain Reaction Technologies Provide Highly Sensitive and Accurate Detection of Zika Virus.

PubMed

Hui, Yuan; Wu, Zhiming; Qin, Zhiran; Zhu, Li; Liang, Junhe; Li, Xujuan; Fu, Hanmin; Feng, Shiyu; Yu, Jianhai; He, Xiaoen; Lu, Weizhi; Xiao, Weiwei; Wu, Qinghua; Zhang, Bao; Zhao, Wei

2018-06-01

The establishment of highly sensitive diagnostic methods is critical in the early diagnosis and control of Zika virus (ZIKV) and in preventing serious neurological complications of ZIKV infection. In this study, we established micro-droplet digital polymerase chain reaction (ddPCR) and real-time quantitative PCR (RT-qPCR) protocols for the detection of ZIKV based on the amplification of the NS5 gene. For the ZIKV standard plasmid, the RT-qPCR results showed that the cycle threshold (Ct) value was linear from 10 1 to 10 8  copy/μL, with a standard curve R 2 of 0.999 and amplification efficiency of 92.203%; however, a concentration as low as 1 copy/μL could not be detected. In comparison with RT-qPCR, the ddPCR method resulted in a linear range of 10 1 -10 4  copy/μL and was able to detect concentrations as low as 1 copy/μL. Thus, for detecting ZIKV from clinical samples, RT-qPCR is a better choice for high-concentration samples (above 10 1  copy/μL), while ddPCR has excellent accuracy and sensitivity for low-concentration samples. These results indicate that the ddPCR method should be of considerable use in the early diagnosis, laboratory study, and monitoring of ZIKV.

Validating internal controls for quantitative plant gene expression studies.

PubMed

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-08-18

Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.

Rapid detection methods for viable Mycobacterium avium subspecies paratuberculosis in milk and cheese.

PubMed

Botsaris, George; Slana, Iva; Liapi, Maria; Dodd, Christine; Economides, Constantinos; Rees, Catherine; Pavlik, Ivo

2010-07-31

Mycobacterium avium subsp. paratuberculosis (MAP) may have a role in the development of Crohn's disease in humans via the consumption of contaminated milk and milk products. Detection of MAP from milk and dairy products has been reported from countries on the European continent, Argentina, the UK and Australia. In this study three different methods (quantitative real time PCR, combined phage IS900 PCR and conventional cultivation) were used to detect the presence of MAP in bulk tank milk (BTM) and cheese originating from sheep, goat and mixed milks from farms and products in Cyprus. During the first survey the presence of MAP was detected in 63 (28.6%) of cows' BTM samples by quantitative real time PCR. A second survey of BTM used a new combined phage IS900 PCR assay, and in this case MAP was detected in 50 (22.2%) samples showing a good level of agreement by both methods. None of the herds tested were known to be affected by Johne's disease and the presence of viable MAP was confirmed by conventional culture in only two cases of cows BTM. This suggests that either rapid method used is more sensitive than the conventional culture when testing raw milk samples for MAP. The two isolates recovered from BTM were identified by IS1311 PCR REA as cattle and sheep strains, respectively. In contrast when cheese samples were tested, MAP DNA was detected by quantitative real time PCR in seven (25.0%) samples (n=28). However no viable MAP was detected when either the combined phage IS900 PCR or conventional culture methods were used. Copyright 2010 Elsevier B.V. All rights reserved.

Detection of polyoma virus in brain tissue of patients with progressive multifocal leukoencephalopathy by real-time PCR and pyrosequencing.

PubMed

Beck, Rose C; Kohn, Debra J; Tuohy, Marion J; Prayson, Richard A; Yen-Lieberman, Belinda; Procop, Gary W

2004-03-01

We evaluated 2 methods, a LightCycler PCR assay and pyrosequencing for the detection of the JC polyoma virus (JCV) in fixed brain tissue of 10 patients with and 3 control patients without progressive multifocal leukoencephalopathy (PML). Nucleic acid extraction was performed after deparaffinization and proteinase K digestion. The LightCycler assay differentiates the BK virus (BKV), JCV, and SV40 using melt curve analysis. Conventional PCR was used with the same primers to generate products for pyrosequencing. Two sequencing primers were used that differentiate the polyoma viruses. Seven of 11 biopsies (1 patient had 2 biopsies) with PML were positive for JCV by real-time PCR and/or PCR/pyrosequencing. Three of 4 remaining biopsies were positive by real-time PCR but had melting points between JCV and SV40. The 4 specimens that were negative or atypical by LightCycler PCR were positive by traditional PCR, but 1 had an amplicon of lower molecular weight by gel electrophoresis. These were shown to represent JCV by at least 1 of the 2 pyrosequencing primers. The biopsies from patients without PML were PCR negative. Both the LightCycler and pyrosequencing assays are useful for confirming JCV in brain biopsies from patients with PML, but variant JCVs may require supplementary methods to confirm JCV infection.

Methods for Characterization of Alternative RNA Splicing.

PubMed

Harvey, Samuel E; Cheng, Chonghui

2016-01-01

Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semi-quantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

Species-specific diagnostic assays for Bonamia ostreae and B. exitiosa in European flat oyster Ostrea edulis: conventional, real-time and multiplex PCR.

PubMed

Ramilo, Andrea; Navas, J Ignacio; Villalba, Antonio; Abollo, Elvira

2013-05-27

Bonamia ostreae and B. exitiosa have caused mass mortalities of various oyster species around the world and co-occur in some European areas. The World Organisation for Animal Health (OIE) has included infections with both species in the list of notifiable diseases. However, official methods for species-specific diagnosis of either parasite have certain limitations. In this study, new species-specific conventional PCR (cPCR) and real-time PCR techniques were developed to diagnose each parasite species. Moreover, a multiplex PCR method was designed to detect both parasites in a single assay. The analytical sensitivity and specificity of each new method were evaluated. These new procedures were compared with 2 OIE-recommended methods, viz. standard histology and PCR-RFLP. The new procedures showed higher sensitivity than the OIE recommended ones for the diagnosis of both species. The sensitivity of tests with the new primers was higher using oyster gills and gonad tissue, rather than gills alone. The lack of a 'gold standard' prevented accurate estimation of sensitivity and specificity of the new methods. The implementation of statistical tools (maximum likelihood method) for the comparison of the diagnostic tests showed the possibility of false positives with the new procedures, although the absence of a gold standard precluded certainty. Nevertheless, all procedures showed negative results when used for the analysis of oysters from a Bonamia-free area.

Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes detection kit in combination with ShortPrep foodproof II Kit. Performance-Tested Method 070401.

PubMed

Junge, Benjamin; Berghof-Jäger, Kornelia

2006-01-01

A method was developed for the detection of L. monocytogenes in food based on real-time polymerase chain reaction (PCR). This advanced PCR method was designed to reduce the time needed to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the Roche/BIOTECON Diagnostics ShortPrep foodproof II Kit (formerly called Listeria ShortPrep Kit) designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is performed by using the Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes Detection Kit. This kit provides primers and hybridization probes for sequence-specific detection, convenient premixed reagents, and different controls for reliable interpretation of results. For repeatability studies, 20 different foods, covering the 15 food groups recommended from the AOAC Research Institute (AOAC RI) for L. monocytogenes detection were analyzed: raw meats, fresh produce/vegetables, processed meats, seafood, egg and egg products, dairy (cultured/noncultured), spices, dry foods, fruit/juices, uncooked pasta, nuts, confectionery, pet food, food dyes and colorings, and miscellaneous. From each food 20, samples were inoculated with a low level (1-10 colony-forming units (CFU)/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, 5 uninoculated samples were prepared from each food. The food samples were examined with the test kits and in correlation with the cultural methods according to U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) or U.S. Department of Agriculture (USDA)/Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook. After 48 h of incubation, the PCR method in all cases showed equal or better results than the reference cultural FDA/BAM or USDA/FSIS methods. Fifteen out of 20 tested food types gave exactly the same amount of positive samples for both methods in both inoculation levels. For 5 out of 20 foodstuffs, the PCR method resulted in more positives than the reference method after 48 h of incubation. Following AOAC RI definition, these were false positives because they were not confirmed by the reference method (false-positive rate for low inoculated foodstuffs: 5.4%; for high inoculated foodstuffs: 7.1%). Without calculating these unconfirmed positives, the PCR method showed equal sensitivity results compared to the alternative method. With the unconfirmed PCR-positives included into the calculations, the alternative PCR method showed a higher sensitivity than the microbiological methods (low inoculation level: 100 vs 98.0%; sensitivity rate: 1; high inoculation level: 99.7 vs 97.7%; sensitivity rate, 1). All in-house and independently tested uninoculated food samples were negative for L. monocytogenes. The ruggedness testing of both ShortPrep foodproof II Kit and Roche/BIOTECON LightCycler foodproof L. monocytogenes Detection Kit showed no noteworthy influences to any variation of the parameters component concentration, apparatus comparison, tester comparison, and sample volumes. In total, 102 L. monocytogenes isolates (cultures and pure DNA) were tested and detected for the inclusivity study, including all isolates claimed by the AOAC RI. The exclusivity study included 60 non-L. monocytogenes bacteria. None of the tested isolates gave a false-positive result; specificity was 100%. Three different lots were tested in the lot-to-lot study. All 3 lots gave equal results. The stability study was subdivided into 3 parts: long-term study, stress test, and freeze-defrost test. Three lots were tested in 4 time intervals within a period of 13 months. They all gave comparable results for all test intervals. For the stress test, LightCycler L. monocytogenes detection mixes were stored at different temperatures and tested at different time points during 1 month. Stable results were produced at all storage temperatures. The freeze-defrost analysis showed no noteworthy aggravation of test results. The independent validation study examined by Campden and Chorleywood Food Research Association Group (CCFRA) demonstrated again that the LightCycler L. monocytogenes detection system shows a comparable sensitivity to reference methods. With both the LightCycler PCR and BAM methods, 19 out of 20 inoculated food samples were detected. The 24 h PCR results generated by the LightCycler system corresponded directly with the FDA/BAM culture results. However, the 48 h PCR results did not relate exactly to the FDA/BAM results, as one sample found to be positive by the 48 h PCR could not be culturally confirmed and another sample which was negative by the 48 h PCR was culturally positive.

Real-time PCR assay for the diagnosis of pleural tuberculosis

PubMed Central

Cárdenas Bernal, Ana María; Giraldo-Cadavid, Luis Fernando; Prieto Diago, Enrique; Santander, Sandra Paola

2017-01-01

Abstract Introduction: The diagnosis of pleural tuberculosis requires an invasive and time-consuming reference method. Polymerase chain reaction (PCR) is rapid, but validation in pleural tuberculosis is still weak. Objective: To establish the operating characteristics of real-time polymerase chain reaction (RT-PCR) hybridization probes for the diagnosis of pleural tuberculosis. Methods: The validity of the RT-PCR hybridization probes was evaluated compared to a composite reference method by a cross-sectional study at the Hospital Universitario de la Samaritana. 40 adults with lymphocytic pleural effusion were included. Pleural tuberculosis was confirmed (in 9 patients) if the patient had at least one of three tests using the positive reference method: Ziehl-Neelsen or Mycobacterium tuberculosis culture in fluid or pleural tissue, or pleural biopsy with granulomas. Pleural tuberculosis was ruled out (in 31 patients) if all three tests were negative. The operating characteristics of the RT-PCR, using the Mid-P Exact Test, were determined using the OpenEpi 2.3 Software (2009). Results: The RT-PCR hybridization probes showed a sensitivity of 66.7% (95% CI: 33.2%-90.7%) and a specificity of 93.5% (95% CI: 80.3%-98.9%). The PPV was 75.0% (95% CI: 38.8%-95.6%) and a NPV of 90.6% (95% CI: 76.6%-97.6%). Two false positives were found for the test, one with pleural mesothelioma and the other with chronic pleuritis with mesothelial hyperplasia. Conclusions: The RT-PCR hybridization probes had good specificity and acceptable sensitivity, but a negative value cannot rule out pleural tuberculosis. PMID:29021638

Development of a real-time PCR protocol for the species origin confirmation of isolated animal particles detected by NIRM.

PubMed

Fumière, O; Marien, A; Fernández Pierna, J A; Baeten, V; Berben, G

2010-08-01

At present, European legislation prohibits totally the use of processed animal proteins in feed for all farmed animals (Commission Regulation (EC) No. 1234/2003-extended feed ban). A softening of the feed ban for non-ruminants would nevertheless be considered if alternative methods could be used to gain more information concerning the species origin of processed animal proteins than that which can be provided by classical optical microscopy. This would allow control provisions such as the ban of feeding animals with proteins from the same species or intra-species recycling (Regulation (EC) No. 1774/2002). Two promising alternative methods, near-infrared microscopy (NIRM) and real-time polymerase chain reaction (PCR), were combined to authenticate, at the species level, the presence of animal particles. The paper describes the improvements of the real-time PCR method made to the DNA extraction protocol, allowing five PCR analyses to be performed with the DNA extracted from a single particle.

Livers provide a reliable matrix for real-time PCR confirmation of avian botulism.

PubMed

Le Maréchal, Caroline; Ballan, Valentine; Rouxel, Sandra; Bayon-Auboyer, Marie-Hélène; Baudouard, Marie-Agnès; Morvan, Hervé; Houard, Emmanuelle; Poëzevara, Typhaine; Souillard, Rozenn; Woudstra, Cédric; Le Bouquin, Sophie; Fach, Patrick; Chemaly, Marianne

2016-04-01

Diagnosis of avian botulism is based on clinical symptoms, which are indicative but not specific. Laboratory investigations are therefore required to confirm clinical suspicions and establish a definitive diagnosis. Real-time PCR methods have recently been developed for the detection of Clostridium botulinum group III producing type C, D, C/D or D/C toxins. However, no study has been conducted to determine which types of matrices should be analyzed for laboratory confirmation using this approach. This study reports on the comparison of different matrices (pooled intestinal contents, livers, spleens and cloacal swabs) for PCR detection of C. botulinum. Between 2013 and 2015, 63 avian botulism suspicions were tested and 37 were confirmed as botulism. Analysis of livers using real-time PCR after enrichment led to the confirmation of 97% of the botulism outbreaks. Using the same method, spleens led to the confirmation of 90% of botulism outbreaks, cloacal swabs of 93% and pooled intestinal contents of 46%. Liver appears to be the most reliable type of matrix for laboratory confirmation using real-time PCR analysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

A multiplex PCR method for the identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in North America.

PubMed

Rasmussen Hellberg, Rosalee S; Morrissey, Michael T; Hanner, Robert H

2010-09-01

The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5' cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) "barcode" sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats. This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.

Validation and application of a quantitative real-time PCR assay to detect common wheat adulteration of durum wheat for pasta production.

PubMed

Carloni, Elisa; Amagliani, Giulia; Omiccioli, Enrica; Ceppetelli, Veronica; Del Mastro, Michele; Rotundo, Luca; Brandi, Giorgio; Magnani, Mauro

2017-06-01

Pasta is the Italian product par excellence and it is now popular worldwide. Pasta of a superior quality is made with pure durum wheat. In Italy, addition of Triticum aestivum (common wheat) during manufacturing is not allowed and, without adequate labeling, its presence is considered an adulteration. PCR-related techniques can be employed for the detection of common wheat contaminations. In this work, we demonstrated that a previously published method for the detection of T. aestivum, based on the gliadin gene, is inadequate. Moreover, a new molecular method, based on DNA extraction from semolina and real-time PCR determination of T. aestivum in Triticum spp., was validated. This multiplex real-time PCR, based on the dual-labeled probe strategy, guarantees target detection specificity and sensitivity in a short period of time. Moreover, the molecular analysis of common wheat contamination in commercial wheat and flours is described for the first time. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comparison of array comparative genomic hybridization and quantitative real-time PCR-based aneuploidy screening of blastocyst biopsies.

PubMed

Capalbo, Antonio; Treff, Nathan R; Cimadomo, Danilo; Tao, Xin; Upham, Kathleen; Ubaldi, Filippo Maria; Rienzi, Laura; Scott, Richard T

2015-07-01

Comprehensive chromosome screening (CCS) methods are being extensively used to select chromosomally normal embryos in human assisted reproduction. Some concerns related to the stage of analysis and which aneuploidy screening method to use still remain. In this study, the reliability of blastocyst-stage aneuploidy screening and the diagnostic performance of the two mostly used CCS methods (quantitative real-time PCR (qPCR) and array comparative genome hybridization (aCGH)) has been assessed. aCGH aneuploid blastocysts were rebiopsied, blinded, and evaluated by qPCR. Discordant cases were subsequently rebiopsied, blinded, and evaluated by single-nucleotide polymorphism (SNP) array-based CCS. Although 81.7% of embryos showed the same diagnosis when comparing aCGH and qPCR-based CCS, 18.3% (22/120) of embryos gave a discordant result for at least one chromosome. SNP array reanalysis showed that a discordance was reported in ten blastocysts for aCGH, mostly due to false positives, and in four cases for qPCR. The discordant aneuploidy call rate per chromosome was significantly higher for aCGH (5.7%) compared with qPCR (0.6%; P<0.01). To corroborate these findings, 39 embryos were simultaneously biopsied for aCGH and qPCR during blastocyst-stage aneuploidy screening cycles. 35 matched including all 21 euploid embryos. Blinded SNP analysis on rebiopsies of the four embryos matched qPCR. These findings demonstrate the high reliability of diagnosis performed at the blastocyst stage with the use of different CCS methods. However, the application of aCGH can be expected to result in a higher aneuploidy rate than other contemporary methods of CCS.

Factors affecting the relationship between quantitative polymerase chain reaction (qPCR) and culture-based enumeration of Enterococcus in environmental waters.

PubMed

Raith, M R; Ebentier, D L; Cao, Y; Griffith, J F; Weisberg, S B

2014-03-01

To determine the extent to which discrepancies between qPCR and culture-based results in beach water quality monitoring can be attributed to: (i) within-method variability, (ii) between-method difference within each method class (qPCR or culture) and (iii) between-class difference. We analysed 306 samples using two culture-based (EPA1600 and Enterolert) and two qPCR (Taqman and Scorpion) methods, each in duplicate. Both qPCR methods correlated with EPA1600, but regression analyses indicated approximately 0·8 log10 unit overestimation by qPCR compared to culture methods. Differences between methods within a class were less than half of this and were minimal for between-replicate within a method. Using the 104 Enterococcus per 100 ml management decision threshold, Taqman qPCR indicated the same decisions as EPA1600 for 87% of the samples, but indicated beach posting for unhealthful water when EPA1600 did not for 12% of the samples. After accounting for within-method and within-class variability, 8% of the samples exhibited true between-class discrepancy where both qPCR methods indicated beach posting while both culture methods did not. Measurement target difference (DNA vs growth) accounted for the majority of the qPCR-vs-culture discrepancy, but its influence on monitoring application is outweighed by frequent incorrect posting with culture methods due to incubation time delay. This is the first study to quantify the frequency with which culture-vs-qPCR discrepancies can be attributed to target difference - vs - method variability. © 2013 The Society for Applied Microbiology.

Diagnostics of Tree Diseases Caused by Phytophthora austrocedri Species.

PubMed

Mulholland, Vincent; Elliot, Matthew; Green, Sarah

2015-01-01

We present methods for the detection and quantification of four Phytophthora species which are pathogenic on trees; Phytophthora ramorum, Phytophthora kernoviae, Phytophthora lateralis, and Phytophthora austrocedri. Nucleic acid extraction methods are presented for phloem tissue from trees, soil, and pure cultures on agar plates. Real-time PCR methods are presented and include primer and probe sets for each species, general advice on real-time PCR setup and data analysis. A method for sequence-based identification, useful for pure cultures, is also included.

Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables.

PubMed

Vojkovska, H; Kubikova, I; Kralik, P

2015-03-01

Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false-negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice. In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model micro-organism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real-time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoil(™) Microbial DNA Isolation Kit, is suitable for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. Coupled with the triplex real-time qPCR assay, this DNA isolation kit is applicable to the samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. Several recent outbreaks of Listeria monocytogenes have been associated with the consumption of fruits and vegetables. Real-time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real-time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoil(™) Microbial DNA Isolation Kit can be used for the extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6 and 70.3%. This method is applicable to samples with bacterial loads of 10(3) bacterial cells per gram of L. monocytogenes. © 2014 The Society for Applied Microbiology.

A cost effective real-time PCR for the detection of adenovirus from viral swabs

PubMed Central

2013-01-01

Compared to traditional testing strategies, nucleic acid amplification tests such as real-time PCR offer many advantages for the detection of human adenoviruses. However, commercial assays are expensive and cost prohibitive for many clinical laboratories. To overcome fiscal challenges, a cost effective strategy was developed using a combination of homogenization and heat treatment with an “in-house” real-time PCR. In 196 swabs submitted for adenovirus detection, this crude extraction method showed performance characteristics equivalent to viral DNA obtained from a commercial nucleic acid extraction. In addition, the in-house real-time PCR outperformed traditional testing strategies using virus culture, with sensitivities of 100% and 69.2%, respectively. Overall, the combination of homogenization and heat treatment with a sensitive in-house real-time PCR provides accurate results at a cost comparable to viral culture. PMID:23758993

Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time.

PubMed

Sugita, Sunao; Ogawa, Manabu; Inoue, Shizu; Shimizu, Norio; Mochizuki, Manabu

2011-09-01

To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1 × 10(2)-2.1 × 10(6) copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

Application of RT-PCR in formalin-fixed and paraffin-embedded lung cancer tissues.

PubMed

Zhang, Fan; Wang, Zhuo-min; Liu, Hong-yu; Bai, Yun; Wei, Sen; Li, Ying; Wang, Min; Chen, Jun; Zhou, Qing-hua

2010-01-01

To analyze gene expression in formalin-fixed, paraffin-embedded lung cancer tissues using modified method. Total RNA from frozen tissues was extracted using TRIZOL reagent. RNA was extracted from formalin-fixed, paraffin-embedded tissues by digestion with proteinase K before the acid-phenol:chloroform extraction and carrier precipitation. We modified this method by using a higher concentration of proteinase K and a longer digestion time, optimized to 16 hours. RT-PCR and real-time RT-PCR were used to check reproducibility and the concordance between frozen and paraffin-embedded samples. The results showed that the RNA extracted from the paraffin-embedded lung tissues had high quality with the most fragment length between 28S and 18S bands (about 1000 to 2000 bases). The housekeeping gene GUSB exhibited low variation of expression in frozen and paraffin-embedded lung tissues, whereas PGK1 had the lowest variation in lymphoma tissues. Furthermore, real-time PCR analysis of the expression of known prognostic genes in non-small cell lung carcinoma (NSCLC) demonstrated an extremely high correlation (r>0.880) between the paired frozen and formalin-fixed, paraffin-embedded specimens. This improved method of RNA extraction is suitable for real-time quantitative RT-PCR, and may be used for global gene expression profiling of paraffin-embedded tissues.

Simultaneous Detection of Genetically Modified Organisms in a Mixture by Multiplex PCR-Chip Capillary Electrophoresis.

PubMed

Patwardhan, Supriya; Dasari, Srikanth; Bhagavatula, Krishna; Mueller, Steffen; Deepak, Saligrama Adavigowda; Ghosh, Sudip; Basak, Sanjay

2015-01-01

An efficient PCR-based method to trace genetically modified food and feed products is in demand due to regulatory requirements and contaminant issues in India. However, post-PCR detection with conventional methods has limited sensitivity in amplicon separation that is crucial in multiplexing. The study aimed to develop a sensitive post-PCR detection method by using PCR-chip capillary electrophoresis (PCR-CCE) to detect and identify specific genetically modified organisms in their genomic DNA mixture by targeting event-specific nucleotide sequences. Using the PCR-CCE approach, novel multiplex methods were developed to detect MON531 cotton, EH 92-527-1 potato, Bt176 maize, GT73 canola, or GA21 maize simultaneously when their genomic DNAs in mixtures were amplified using their primer mixture. The repeatability RSD (RSDr) of the peak migration time was 0.06 and 3.88% for the MON531 and Bt176, respectively. The RSD (RSDR) of the Cry1Ac peak ranged from 0.12 to 0.40% in multiplex methods. The method was sensitive in resolving amplicon of size difference up to 4 bp. The PCR-CCE method is suitable to detect multiple genetically modified events in a composite DNA sample by tagging their event specific sequences.

Using qPCR for Water Microbial Risk Assessments

EPA Science Inventory

Microbial risk assessment (MRA) has traditionally utilized microbiological data that was obtained by culture-based techniques that are expensive and time consuming. With the advent of PCR methods there is a realistic opportunity to conduct MRA studies economically, in less time,...

Ambient Stable Quantitative PCR Reagents for the Detection of Yersinia pestis

PubMed Central

Zhou, Lei; Guo, Zhaobiao; Zhou, Dongsheng; Zhai, Junhui; Yang, Ruifu

2010-01-01

Background Although assays for detecting Yersinia pestis using TaqMan probe-based real-time PCR have been developed for years, little is reported on room-temperature-stable PCR reagents, which will be invaluable for field epidemic surveillance, immediate response to public health emergencies, counter-bioterrorism investigation, etc. In this work, a set of real-time PCR reagents for rapid detection of Y. pestis was developed with extraordinary stability at 37°C. Methods/Principal Findings TaqMan-based real-time PCR assays were developed using the primers and probes targeting the 3a sequence in the chromosome and the F1 antigen gene caf1 in the plasmid pMT1of Y. pestis, respectively. Then, carbohydrate mixtures were added to the PCR reagents, which were later vacuum-dried for stability evaluation. The vacuum-dried reagents were stable at 37°C for at least 49 days for a lower concentration of template DNA (10 copies/µl), and up to 79 days for higher concentrations (≥102 copies/µl). The reagents were used subsequently to detect soil samples spiked with Y. pestis vaccine strain EV76, and 5×104 CFU per gram of soil could be detected by both 3a- and caf1-based PCR reagents. In addition, a simple and efficient method for soil sample processing is presented here. Conclusions/Significance The vacuum-dried reagents for real-time PCR maintain accuracy and reproducibility for at least 49 days at 37°C, indicating that they can be easily transported at room temperature for field application if the machine for performing real-time PCR is available. This dry reagent is of great significance for routine plague surveillance. PMID:20231881

Allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR), a powerful method for diagnosing loss of imprinting of the 11p15 region in Russell Silver and Beckwith Wiedemann syndromes.

PubMed

Azzi, Salah; Steunou, Virginie; Rousseau, Alexandra; Rossignol, Sylvie; Thibaud, Nathalie; Danton, Fabienne; Le Jule, Marilyne; Gicquel, Christine; Le Bouc, Yves; Netchine, Irène

2011-02-01

Many human syndromes involve a loss of imprinting (LOI) due to a loss (LOM) or a gain of DNA methylation (GOM). Most LOI occur as mosaics and can therefore be difficult to detect with conventional methods. The human imprinted 11p15 region is crucial for the control of fetal growth, and LOI at this locus is associated with two clinical disorders with opposite phenotypes: Beckwith-Wiedemann syndrome (BWS), characterized by fetal overgrowth and a high risk of tumors, and Russell-Silver syndrome (RSS), characterized by intrauterine and postnatal growth restriction. Until recently, we have been using Southern blotting for the diagnosis of RSS and BWS. We describe here a powerful quantitative technique, allele-specific methylated multiplex real-time quantitative PCR (ASMM RTQ-PCR), for the diagnosis of these two complex disorders. We first checked the specificity of the probes and primers used for ASMM RTQ-PCR. We then carried out statistical validation for this method, on both retrospective and prospective populations of patients. This analysis demonstrated that ASMM RTQ-PCR is more sensitive than Southern blotting for detecting low degree of LOI. Moreover, ASMM RTQ-PCR is a very rapid, reliable, simple, safe, and cost effective method. © 2011 Wiley-Liss, Inc.

Combined use of real-time PCR and nested sequence-based typing in survey of human Legionella infection.

PubMed

Qin, T; Zhou, H; Ren, H; Shi, W; Jin, H; Jiang, X; Xu, Y; Zhou, M; Li, J; Wang, J; Shao, Z; Xu, X

2016-07-01

Legionnaires' disease (LD) is a globally distributed systemic infectious disease. The burden of LD in many regions is still unclear, especially in Asian countries including China. A survey of Legionella infection using real-time PCR and nested sequence-based typing (SBT) was performed in two hospitals in Shanghai, China. A total of 265 bronchoalveolar lavage fluid (BALF) specimens were collected from hospital A between January 2012 and December 2013, and 359 sputum specimens were collected from hospital B throughout 2012. A total of 71 specimens were positive for Legionella according to real-time PCR focusing on the 5S rRNA gene. Seventy of these specimens were identified as Legionella pneumophila as a result of real-time PCR amplification of the dotA gene. Results of nested SBT revealed high genetic polymorphism in these L. pneumophila and ST1 was the predominant sequence type. These data revealed that the burden of LD in China is much greater than that recognized previously, and real-time PCR may be a suitable monitoring technology for LD in large sample surveys in regions lacking the economic and technical resources to perform other methods, such as urinary antigen tests and culture methods.

Quantitative detection of pork in commercial meat products by TaqMan® real-time PCR assay targeting the mitochondrial D-loop region.

PubMed

Kim, Miju; Yoo, Insuk; Lee, Shin-Young; Hong, Yeun; Kim, Hae-Yeong

2016-11-01

The TaqMan® real-time PCR assay using the mitochondrial D-loop region was developed for the quantitative detection of pork in processed meat products. The newly designed primers and probe specifically amplified pork without any cross-reactivity with non-target animal species. The limit of detection of the real-time PCR assay was 0.1pg of heat-treated pork meat and 0.1% (w/w) pork meat in beef and chicken meat mixtures. The quantitative real-time PCR assay was applied to analyze the pork meat content in 22 commercial processed meat products including jerkies, press hams, sausages, hamburger patties and steaks, grilled short rib patties, and nuggets. The developed real-time PCR method was able to detect pork meat in various types of processed meat products that declared the use of pork meat on their label. All processed meat products that declared no use of pork meat showed a negative result in the assay. The method developed in this study showed sensitivity and specificity in the quantification of pork meat in commercial processed meat products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development of a real-time PCR assay for monitoring anaerobic fungal and cellulolytic bacterial populations within the rumen.

PubMed

Denman, Stuart E; McSweeney, Christopher S

2006-12-01

Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.

[A new method of processing quantitative PCR data].

PubMed

Ke, Bing-Shen; Li, Guang-Yun; Chen, Shi-Min; Huang, Xiang-Yan; Chen, Ying-Jian; Xu, Jun

2003-05-01

Today standard PCR can't satisfy the need of biotechnique development and clinical research any more. After numerous dynamic research, PE company found there is a linear relation between initial template number and cycling time when the accumulating fluorescent product is detectable.Therefore,they developed a quantitative PCR technique to be used in PE7700 and PE5700. But the error of this technique is too great to satisfy the need of biotechnique development and clinical research. A better quantitative PCR technique is needed. The mathematical model submitted here is combined with the achievement of relative science,and based on the PCR principle and careful analysis of molecular relationship of main members in PCR reaction system. This model describes the function relation between product quantity or fluorescence intensity and initial template number and other reaction conditions, and can reflect the accumulating rule of PCR product molecule accurately. Accurate quantitative PCR analysis can be made use this function relation. Accumulated PCR product quantity can be obtained from initial template number. Using this model to do quantitative PCR analysis,result error is only related to the accuracy of fluorescence intensity or the instrument used. For an example, when the fluorescence intensity is accurate to 6 digits and the template size is between 100 to 1,000,000, the quantitative result accuracy will be more than 99%. The difference of result error is distinct using same condition,same instrument but different analysis method. Moreover,if the PCR quantitative analysis system is used to process data, it will get result 80 times of accuracy than using CT method.

Comparison of nested competitive RT-PCR and real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21) positive acute myelogenous leukemia.

PubMed

Wattjes, M P; Krauter, J; Nagel, S; Heidenreich, O; Ganser, A; Heil, G

2000-02-01

The chromosomal translocation t(8;21)(q22;q22) is one of the most frequent karyotypic aberrations in acute myeloid leukemia (AML) and results in a chimeric fusion transcript AML1/MTG8. Since AML1/MTG8 fusion transcripts remain detectable by RT-PCR in t(8;21) AML patients in long-term hematological remission, quantitative assessment of AML1/MTG8 transcripts is necessary for the monitoring of minimal residual disease (MRD) in these patients. Competitive RT-PCR and recently real-time RT-PCR are increasingly used for detection and quantification of leukemia specific fusion transcripts. For the direct comparison of both methods we cloned a 42 bp DNA fragment into the original AML1/MTG8 sequence. The resulting molecule was used as an internal competitor for our novel competitive nested RT-PCR for AML1/MTG8 and as an external standard for the generation of AML1/MTG8 standard curves in a real-time PCR assay. Using this standard molecule for both PCR techniques, we compared their sensitivity, linearity and reproducibility. Both methods were comparable with regard to all parameters tested irrespective of analyzing serial dilutions of plasmids, cell lines or samples from t(8;21) positive AML patients at different stages of the disease. Therefore, both techniques can be recommended for the monitoring of MRD in these particular AML patients. However, the automatization of the real-time PCR technique offers some technical advantages.

Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene

PubMed Central

Khan, Mehran; Li, Benjin; Jiang, Yue; Weng, Qiyong; Chen, Qinghe

2017-01-01

Late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases affecting potato and tomato worldwide. Early diagnosis of the P. infestans pathogen causing late blight should be the top priority for addressing disease epidemics and management. In this study, we performed a loop-mediated isothermal amplification (LAMP) assay, conventional polymerase chain reaction (PCR), nested PCR, and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1 (Ras-related protein) gene of P. infestans. In comparison with the PCR-based assays, the LAMP technique led to higher specificity and sensitivity, using uncomplicated equipment with an equivalent time frame. All 43 P. infestans isolates, yielded positive detection results using LAMP assay showing no cross reaction with other Phytophthora spp., oomycetes or fungal pathogens. The LAMP assay yielded the lowest detectable DNA concentration (1.28 × 10-4 ng μL-1), being 10 times more sensitive than nested PCR (1.28 × 10-3 ng μL-1), 100 times more sensitive than real-time PCR (1.28 × 10-2 ng μL-1) and 103 times more sensitive than the conventional PCR assay (1.28 × 10-1 ng μL-1). In the field experiment, the LAMP assay outperformed the other tests by amplifying only diseased tissues (leaf and stem), and showing no positive reaction in healthy tissues. Overall, the LAMP assay developed in this study provides a specific, sensitive, simple, and effective visual method for detection of the P. infestans pathogen, and is therefore suitable for application in early prediction of the disease to reduce the risk of epidemics. PMID:29051751

Evaluation of Different PCR-Based Assays and LAMP Method for Rapid Detection of Phytophthora infestans by Targeting the Ypt1 Gene.

PubMed

Khan, Mehran; Li, Benjin; Jiang, Yue; Weng, Qiyong; Chen, Qinghe

2017-01-01

Late blight, caused by the oomycete Phytophthora infestans , is one of the most devastating diseases affecting potato and tomato worldwide. Early diagnosis of the P. infestans pathogen causing late blight should be the top priority for addressing disease epidemics and management. In this study, we performed a loop-mediated isothermal amplification (LAMP) assay, conventional polymerase chain reaction (PCR), nested PCR, and real-time PCR to verify and compare the sensitivity and specificity of the reaction based on the Ypt1 (Ras-related protein) gene of P. infestans. In comparison with the PCR-based assays, the LAMP technique led to higher specificity and sensitivity, using uncomplicated equipment with an equivalent time frame. All 43 P. infestans isolates, yielded positive detection results using LAMP assay showing no cross reaction with other Phytophthora spp., oomycetes or fungal pathogens. The LAMP assay yielded the lowest detectable DNA concentration (1.28 × 10 -4 ng μL -1 ), being 10 times more sensitive than nested PCR (1.28 × 10 -3 ng μL -1 ), 100 times more sensitive than real-time PCR (1.28 × 10 -2 ng μL -1 ) and 10 3 times more sensitive than the conventional PCR assay (1.28 × 10 -1 ng μL -1 ). In the field experiment, the LAMP assay outperformed the other tests by amplifying only diseased tissues (leaf and stem), and showing no positive reaction in healthy tissues. Overall, the LAMP assay developed in this study provides a specific, sensitive, simple, and effective visual method for detection of the P. infestans pathogen, and is therefore suitable for application in early prediction of the disease to reduce the risk of epidemics.

Application of the Modular Approach to an In-House Validation Study of Real-Time PCR Methods for the Detection and Serogroup Determination of Verocytotoxigenic Escherichia coli ▿ †

PubMed Central

Kagkli, Dafni-Maria; Weber, Thomas P.; Van den Bulcke, Marc; Folloni, Silvia; Tozzoli, Rosangela; Morabito, Stefano; Ermolli, Monica; Gribaldo, Laura; Van den Eede, Guy

2011-01-01

European Commission regulation 2073/2005 on the microbiological criteria for food requires that Escherichia coli is monitored as an indicator of hygienic conditions. Since verocytotoxigenic E. coli (VTEC) strains often cause food-borne infections by the consumption of raw food, the Biological Hazards (BIOHAZ) panel of the European Food Safety Authority (EFSA) recommended their monitoring in food as well. In particular, VTEC strains belonging to serogroups such as O26, O103, O111, O145, and O157 are known causative agents of several human outbreaks. Eight real-time PCR methods for the detection of E. coli toxin genes and their variants (stx1, stx2), the intimin gene (eae), and five serogroup-specific genes have been proposed by the European Reference Laboratory for VTEC (EURL-VTEC) as a technical specification to the European Normalization Committee (CEN TC275/WG6). Here we applied a “modular approach” to the in-house validation of these PCR methods. The modular approach subdivides an analytical process into separate parts called “modules,” which are independently validated based on method performance criteria for a limited set of critical parameters. For the VTEC real-time PCR module, the following parameters are being assessed: specificity, dynamic range, PCR efficiency, and limit of detection (LOD). This study describes the modular approach for the validation of PCR methods to be used in food microbiology, using single-target plasmids as positive controls and showing their applicability with food matrices. PMID:21856838

Sample pooling for real-time PCR detection and virulence determination of the footrot pathogen Dichelobacter nodosus.

PubMed

Frosth, Sara; König, Ulrika; Nyman, Ann-Kristin; Aspán, Anna

2017-09-01

Dichelobacter nodosus is the principal cause of ovine footrot and strain virulence is an important factor in disease severity. Therefore, detection and virulence determination of D. nodosus is important for proper diagnosis of the disease. Today this is possible by real-time PCR analysis. Analysis of large numbers of samples is costly and laborious; therefore, pooling of individual samples is common in surveillance programs. However, pooling can reduce the sensitivity of the method. The aim of this study was to develop a pooling method for real-time PCR analysis that would allow sensitive detection and simultaneous virulence determination of D. nodosus. A total of 225 sheep from 17 flocks were sampled using ESwabs within the Swedish Footrot Control Program in 2014. Samples were first analysed individually and then in pools of five by real-time PCR assays targeting the 16S rRNA and aprV2/B2 genes of D. nodosus. Each pool consisted of four negative and one positive D. nodosus samples with varying amounts of the bacterium. In the individual analysis, 61 (27.1%) samples were positive in the 16S rRNA and the aprV2/B2 PCR assays and 164 (72.9%) samples were negative. All samples positive in the aprV2/B2 PCR-assay were of aprB2 variant. The pooled analysis showed that all 41 pools were also positive for D. nodosus 16S rRNA and the aprB2 variant. The diagnostic sensitivity for pooled and individual samples was therefore similar. Our method includes concentration of the bacteria before DNA-extraction. This may account for the maintenance of diagnostic sensitivity. Diagnostic sensitivity in the real-time PCR assays of the pooled samples were comparable to the sensitivity obtained for individually analysed samples. Even sub-clinical infections were able to be detected in the pooled PCR samples which is important for control of the disease. This method may therefore be implemented in footrot control programs where it can replace analysis of individual samples.

Real-time PCR for type-specific identification of herpes simplex in clinical samples: evaluation of type-specific results in the context of CNS diseases.

PubMed

Meylan, Sylvain; Robert, Daniel; Estrade, Christine; Grimbuehler, Valérie; Péter, Olivier; Meylan, Pascal R; Sahli, Roland

2008-02-01

HSV-1 and HSV-2 cause CNS infections of dissimilar clinico-pathological characteristics with prognostic and therapeutic implications. To validate a type-specific real-time PCR that uses MGB/LNA Taqman probes and to review the virologico-clinical data of 25 eligible patients with non-neonatal CNS infections. This real-time PCR was evaluated against conventional PCR (26 CSF and 20 quality controls), and LightCycler assay (51 mucocutaneous, 8 CSF and 32 quality controls) and culture/immunofluorescence (75 mucocutaneous) to assess typing with independent methods. Taqman real-time PCR detected 240 HSV genomes per ml CSF, a level appropriate for the management of patients, and provided unambiguous typing for the 104 positive (62 HSV-1 and 42 HSV-2) out the 160 independent clinical samples tested. HSV type diagnosed by Taqman real-time PCR predicted final diagnosis (meningitis versus encephalitis/meningoencephalitis, p<0.001) in 24/25 patients at time of presentation, in contrast to clinical evaluation. Our real-time PCR, as a sensitive and specific means for type-specific HSV diagnosis, provided rapid prognostic information for patient management.

Molecular diagnostics and ITS-based phylogenic analysis of Streptococcus suis serotype 2 in central Vietnam.

PubMed

Nguyen, Bach Hoang; Phan, Dieu Hong Nu; Nguyen, Hien Xuan; Le, An Van; Alberti, Alberto

2015-07-04

Streptococcus suis (S. suis) serotype 2 has recently become the most prevalent cause of meningitis in adults in many areas of Vietnam. This study provides data on S. suis molecular diagnosis in central Vietnam using a real-time polymerase chain reaction (PCR) assay targeting the S. suis serotype 2 cps2J gene. Additionally, 16S-23S rDNA intragenic spacer (ITS)-based phylogenic analysis of strains isolated from cerebrospinal fluid (CSF) in Thua Thien Hue Province, Vietnam, is presented and discussed. Pathogenic bacteria were isolated from 40 CSF samples, and 18 were identified as S. suis by culture-dependent methods. Capsular serotyping was assessed by real-time PCR. ITS sequences were obtained after traditional PCR and were used in phylogenic analyses. Pathogenic bacteria were isolated from 36 out of 40 CSF samples. A total of 18 S. suis strains were isolated and assigned to serotype 2 by real-time PCR. One CSF sample, negative when tested by culture-dependent methods, was positive to S. suis serotype 2 by real-time PCR. Pairwise alignments of the 18 ITS sequences did not reveal any variable nucleotide position, and resulted in a single sequence type. Sequences were similar to S. suis serotype 2 reference ITS sequences (> 98.1%), and there was no lack of an ITS spacer region in the isolates. S. suis serotype 2 is the most prevalent serotype in central Vietnam. Real-time PCR assay proved to be a reliable diagnostic method for early detection of S. suis 2 in CSF samples.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

PubMed Central

Cura, Carolina I.; Duffy, Tomas; Lucero, Raúl H.; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J.; Valencia Ayala, Edward; Kjos, Sonia A.; Santalla, José; Mahaney, Susan M.; Cayo, Nelly M.; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S.; Acosta Viana, Karla Y.; Brutus, Laurent; Ocampo, Susana B.; Aznar, Christine; Cuba Cuba, Cesar A.; Gürtler, Ricardo E.; Ramsey, Janine M.; Ribeiro, Isabela; VandeBerg, John L.; Yadon, Zaida E.; Osuna, Antonio; Schijman, Alejandro G.

2015-01-01

Background Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). Methods/Principal Findings The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Conclusions/Significance Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production. PMID:25993316

Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia

PubMed Central

Church, Deirdre L; Ambasta, Anshula; Wilmer, Amanda; Williscroft, Holly; Ritchie, Gordon; Pillai, Dylan R; Champagne, Sylvie; Gregson, Daniel G

2015-01-01

BACKGROUND: Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP. METHODS: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ−]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP. RESULTS: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%. CONCLUSION: PJ real-time PCR improved detection of PJ in immunocompromised patients. PMID:26600815

Evaluation of Simplexa Group A Strep Direct Kit Compared to Hologic Group A Streptococcal Direct Assay for Detection of Group A Streptococcus in Throat Swabs.

PubMed

Church, Deirdre L; Lloyd, Tracie; Larios, Oscar; Gregson, Daniel B

2018-03-01

Diagnosis of bacterial pharyngitis is confirmed by detection of group A Streptococcus (GAS) in patient throat samples. Testing of throat samples has historically relied on culture, but new molecular methods allow much faster test turnaround time (i.e., same day versus 48 to 72 h for culture). Our laboratory uses the Hologic GAS Direct (GASD) assay for screening more than 125,000 throat samples per year. Simplexa GAS Direct is a new real-time quantitative PCR (qPCR) assay that does not require initial DNA extraction. Performance of Simplexa qPCR was compared to GASD. A total of 289 throat swabs were collected from patients attending ambulatory clinics in Calgary, Alberta, Canada. A total of 60 (20.8%) of the samples were initially GAS positive by either method: 54 by both methods, 4 by Simplex qPCR alone, and 2 by GASD alone. An in-house PCR using a unique GAS primer set was used to resolve the 6 discrepant results. Overall, GASD compared to Simplexa qPCR had a sensitivity, specificity, positive predictive value, and negative predictive value of 93.1% versus 100%, 100% versus 100%, 100% versus 100%, and 98.31% versus 100%, respectively. Implementation of Simplexa qPCR in our laboratory setting would cost more but allow the high sample volume to be reported in half the time and save 0.62 medical laboratory technician (MLT) full-time equivalent (FTE). In comparison to culture, the implementation of Simplexa qPCR would save 2.79 medical laboratory assistant (MLA) FTE plus 0.94 MLT FTE. Simplexa qPCR has improved performance and diagnostic efficiency in a high-volume laboratory compared to GASD for GAS detection in throat swabs. Copyright © 2018 American Society for Microbiology.

Rapid screening of toxigenic vibrio cholerae O1 strains from south Iran by PCR-ELISA.

PubMed

Mousavi, Seyed Latif; Nazarian, Shahram; Amani, Jafar; Rahgerdi, Ahmad Karimi

2008-01-01

The ability to sensitively detect Vibrio cholera with PCR-ELISA method represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen. The aim of this research is to evaluate the suitability of a PCR-enzyme-linked immunosorbent assay for sensitive and rapid detection of V. cholera O1. The 398-bp sequence of a gene that codes for the cholera toxin B subunit was amplified by PCR. The digoxigenin-labeled amplified products were coated on microplates and detected by ELISA. The PCR product was also hybridized with biotin labelled probe and detected by ELISA using streptavidin. The specificity of the PCR was determined using 10 bacterial strains and 50 samples from south Iran. The detection limit was 0.5 pg of the genomic DNA and five bacterial cells. Adaptation of PCR into PCR-ELISA assay format facilitates specific and sensitive detection and diagnosis of human cholera disease. We conclude that this PCR-ELISA is a diagnostic method that specifically detects toxin genes in V. cholera O1 strains. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.

Development of a real-time microchip PCR system for portable plant disease diagnosis.

PubMed

Koo, Chiwan; Malapi-Wight, Martha; Kim, Hyun Soo; Cifci, Osman S; Vaughn-Diaz, Vanessa L; Ma, Bo; Kim, Sungman; Abdel-Raziq, Haron; Ong, Kevin; Jo, Young-Ki; Gross, Dennis C; Shim, Won-Bo; Han, Arum

2013-01-01

Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25 × 16 × 8 cm(3) in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample.

Development of a Real-Time Microchip PCR System for Portable Plant Disease Diagnosis

PubMed Central

Kim, Hyun Soo; Cifci, Osman S.; Vaughn-Diaz, Vanessa L.; Ma, Bo; Kim, Sungman; Abdel-Raziq, Haron; Ong, Kevin; Jo, Young-Ki; Gross, Dennis C.; Shim, Won-Bo; Han, Arum

2013-01-01

Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25×16×8 cm3 in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample. PMID:24349341

Quantitative assessment of hematopoietic chimerism by quantitative real-time polymerase chain reaction of sequence polymorphism systems after hematopoietic stem cell transplantation.

PubMed

Qin, Xiao-ying; Li, Guo-xuan; Qin, Ya-zhen; Wang, Yu; Wang, Feng-rong; Liu, Dai-hong; Xu, Lan-ping; Chen, Huan; Han, Wei; Wang, Jing-zhi; Zhang, Xiao-hui; Li, Jin-lan; Li, Ling-di; Liu, Kai-yan; Huang, Xiao-jun

2011-08-01

Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT. A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally. Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation <1.85%), which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method. This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.

Development and evaluation of a Quadruplex Taq Man real-time PCR assay for simultaneous detection of clinical isolates of Enterococcus faecalis, Enterococcus faecium and their vanA and vanB genotypes.

PubMed

Naserpour Farivar, Taghi; Najafipour, Reza; Johari, Pouran; Aslanimehr, Masoumeh; Peymani, Amir; Jahani Hashemi, Hoasan; Mirzaui, Baman

2014-10-01

We developed and evaluated the utility of a quadruplex Taqman real-time PCR assay that allows simultaneous identification of vancomycin-resistant genotypes and clinically relevant enterococci. The specificity of the assay was tested using reference strains of vancomycin-resistant and susceptible enterococci. In total, 193 clinical isolates were identified and subsequently genotyped using a Quadruplex Taqman real-time PCR assay and melting curve analysis. Representative Quadruplex Taqman real-time PCR amplification curve were obtained for Enterococcus faecium, Enterococcus faecalis, vanA-containing E. faecium, vanB-containing E. faecalis. Phenotypic and genotypic analysis of the isolates gave same results for 82 enterococcal isolates, while in 5 isolates, they were inconsistent. We had three mixed strains, which were detected by the TaqMan real-time PCR assay and could not be identified correctly using phenotypic methods. Vancomycin resistant enterococci (VRE) genotyping and identification of clinically relevant enterococci were rapidly and correctly performed using TaqMan real-time multiplex real-time PCR assay.

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods.

PubMed

Yang, Litao; Pan, Aihu; Zhang, Kewei; Guo, Jinchao; Yin, Changsong; Chen, Jianxiu; Huang, Cheng; Zhang, Dabing

2005-08-10

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

PubMed

Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

2014-12-01

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Comparison of the genexpert enterovirus assay (GXEA) with real-time one step RT-PCR for the detection of enteroviral RNA in the cerebrospinal fluid of patients with meningitis.

PubMed

Hong, JiYoung; Kim, Ahyoun; Hwang, Seoyeon; Cheon, Doo-Sung; Kim, Jong-Hyen; Lee, June-Woo; Park, Jae-Hak; Kang, Byunghak

2015-02-13

Enteroviruses (EVs) are the leading cause of aseptic meningitis worldwide. Detection of enteroviral RNA in clinical specimens has been demonstrated to improve the management of patient care, especially that of neonates and young children. To establish a sensitive and reliable assay for routine laboratory diagnosis, we compared the sensitivity and specificity of the GeneXpert Enterovirus Assay (GXEA) with that of the reverse transcription polymerase chain reaction (RT-PCR) based assay referred to as real-time one step RT-PCR (RTo-PCR). The sensitivity/specificity produced by GXEA and RTo-PCR were 100%/100% and 65%/100%, respectively. Both methods evaluated in this article can be used for detection of enterovirus in clinical specimens and these nucleic acid amplification methods are useful assays for the diagnosis of enteroviral infection.

Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR.

PubMed

Liu, Ya-Mei; Qiu, Liang; Sheng, An-Zhi; Wan, Xiao-Yuan; Cheng, Dong-Yuan; Huang, Jie

2018-01-01

A TaqMan probe and a pair of specific primers were selected from the small subunit ribosomal DNA (SSU rDNA) sequence of Enterocytozoon hepatopenaei (EHP); this real-time PCR assay was developed and optimized. It showed a good linearity in detecting standards of EHP SSU rDNA fragments from 4 × 10 2 to 4 × 10 8 copies/reaction using the established method. The detection limit of the qPCR method was as low as 4 × 10 1 copies per reaction, which was higher than the conventional PCR and SYBR Green I-based EHP qPCR reported. Using the qPCR assay, EHP was detected in four batches of slow-growing Penaeus vannamei specimens collected from Tianjin and Zhejiang Province in China was detected using qPCR. The results showed that all the hepatopancreas from the slow-growing P. vannamei specimens were detected as EHP-positive. EHP copies of hepatopancreas in some batches had a negative correlation with the body mass index (BMI) of shrimps; however, not all batches of specimens had this negative correlation between EHP copies of hepatopancreas and BMI. This qPCR technique is sensitive, specific and easy to perform (96 tests in <3 h), which provides technical support for the detection and prevention of EHP. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification and quantification of genetically modified Moonshade carnation lines using conventional and TaqMan real-time polymerase chain reaction methods.

PubMed

Li, Peng; Jia, Junwei; Bai, Lan; Pan, Aihu; Tang, Xueming

2013-07-01

Genetically modified carnation (Dianthus caryophyllus L.) Moonshade was approved for planting and commercialization in several countries from 2004. Developing methods for analyzing Moonshade is necessary for implementing genetically modified organism labeling regulations. In this study, the 5'-transgene integration sequence was isolated using thermal asymmetric interlaced (TAIL)-PCR. Based upon the 5'-transgene integration sequence, conventional and TaqMan real-time PCR assays were established. The relative limit of detection for the conventional PCR assay was 0.05 % for Moonshade using 100 ng total carnation genomic DNA, corresponding to approximately 79 copies of the carnation haploid genome, and the limits of detection and quantification of the TaqMan real-time PCR assay were estimated to be 51 and 254 copies of haploid carnation genomic DNA, respectively. These results are useful for identifying and quantifying Moonshade and its derivatives.

Diagnosis and identification of Leishmania spp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran.

PubMed

Khademvatan, S; Neisi, N; Maraghi, S; Saki, J

2011-12-01

The aim of present study was describing a real-time PCR assay for the diagnosis and direct identification of Leishmania species on Giemsa-stained slides in south-west of Iran. Altogether, 102 Giemsa-stained slides were collected from different part of south-west of Iran between 2008 and 2011. All the Giemsa-stained slides were examined under light microscope. After DNA extraction, real-time PCR amplification and detection were conducted with fluorescent SYBR Green I. For identification, PCR products were analysed with melting curve analysis. One hundred and two archived slides from suspected lesion examined by microscopy and real-time PCR. The sensitivity of the real-time PCR on Giemsa-stained slid was 98% (96/102). The melting curve analysis (T(m)) were 88·3±0·2°C for L. tropica (MHOM/IR/02/Mash10), 86·5±0·2°C for L. major (MHOM/IR/75/ER) and 89·4±0·3°C for L. infantum (MCAN/IR/97/LON 49), respectively. This study is first report in use of real-time PCR for diagnosis and identification of Leishmania spp. in Iran. Up to now, in Iran, the majority of identification of Leishmania species is restriction fragment length polymorphism (PCR-RFLP) of ITS1 and kinetoplast DNA. Our data showed that Giemsa-stained slides that were stored more than 3 years, can be use for Leishmania DNA extraction and amplification by real-time PCR. Compared to conventional PCR-based methods, the real-time PCR is extremely rapid with results and more samples can be processed at one time.

[Experimental studies of using real-time fluorescence quantitative PCR and RT-PCR to detect E6 and E7 genes of human papillomavirus type 16 in cervical carcinoma cell lines].

PubMed

Chen, Yue-yue; Peng, Zhi-lan; Liu, Shan-ling; He, Bing; Hu, Min

2007-06-01

To establish a method of using real-time fluorescence quantitative PCR and RT-PCR to detect the E6 and E7 genes of human papillomavirus type 16 (HPV-16). Plasmids containing HPV-16 E6 or E7 were used to generate absolute standard curves. Three cervical carcinoma cell lines CaSki, SiHa and HeLa were tested by real-time fluorescence quantitative PCR and RT-PCR analyses for the expressions of HPV-16 E6 and E7. The correlation coefficients of standard curves were larger than 0. 99, and the PCR efficiency was more than 90%. The relative levels of HPV-16 E6 and E7 DNA and RNA were CaSki>SiHa>HeLa cell. HPV-16 E6 and E7 quantum by real-time fluorescence quantitative PCR and RT-PCR analyses may serve as a reliable and sensitive tool. This study provides the possibility of further researches on the relationship between HPV-16 E6 or E7 copy number and cervical carcinoma.

A new visually improved and sensitive loop mediated isothermal amplification (LAMP) for diagnosis of symptomatic falciparum malaria.

PubMed

Mohon, Abu Naser; Elahi, Rubayet; Khan, Wasif A; Haque, Rashidul; Sullivan, David J; Alam, Mohammad Shafiul

2014-06-01

Molecular diagnosis of malaria by nucleotide amplification requires sophisticated and expensive instruments, typically found only in well-established laboratories. Loop-mediated isothermal amplification (LAMP) has provided a new platform for an easily adaptable molecular technique for molecular diagnosis of malaria without the use of expensive instruments. A new primer set has been designed targeting the 18S rRNA gene for the detection of Plasmodium falciparum in whole blood samples. The efficacy of LAMP using the new primer set was assessed in this study in comparison to that of a previously described set of LAMP primers as well as with microscopy and real-time PCR as reference methods for detecting P. falciparum. Pre-addition of hydroxy napthol blue (HNB) in the LAMP reaction caused a distinct color change, thereby improving the visual detection system. The new LAMP assay was found to be 99.1% sensitive compared to microscopy and 98.1% when compared to real-time PCR. Meanwhile, its specificity was 99% and 100% in contrast to microscopy and real-time PCR, respectively. Moreover, the LAMP method was in very good agreement with microscopy and real-time PCR (0.94 and 0.98, respectively). This new LAMP method can detect at least 5parasites/μL of infected blood within 35min, while the other LAMP method tested in this study, could detect a minimum of 100parasites/μL of human blood after 60min of amplification. Thus, the new method is sensitive and specific, can be carried out in a very short time, and can substitute PCR in healthcare clinics and standard laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

Rapid detection of Listeria monocytogenes in raw milk and soft cheese by a redox potential measurement based method combined with real-time PCR.

PubMed

Erdősi, Orsolya; Szakmár, Katalin; Reichart, Olivér; Szili, Zsuzsanna; László, Noémi; Székely Körmöczy, Péter; Laczay, Péter

2014-09-01

The incidence of outbreaks of foodborne listeriosis has indicated the need for a reliable and rapid detection of the microbe in different foodstuffs. A method combining redox potential measurement and real-time polymerase chain reaction (PCR) was developed to detect Listeria monocytogenes in artificially contaminated raw milk and soft cheese. Food samples of 25 g or 25 ml were homogenised in 225 ml of Listeria Enrichment Broth (LEB) with Oxford supplement, and the redox potential measurement technique was applied. For Listeria species the measuring time was maximum 34 h. The absence of L. monocytogenes could reliably be proven by the redox potential measurement method, but Listeria innocua and Bacillus subtilis could not be differentiated from L. monocytogenes on the basis of the redox curves. The presence of L. monocytogenes had to be confirmed by real-time PCR. The combination of these two methods proved to detect < 10 cfu/g of L. monocytogenes in a cost- and time-effective manner. This method can potentially be used as an alternative to the standard nutrient method for the rapid detection of L. monocytogenes in food.

Development of a screening method for genetically modified soybean by plasmid-based quantitative competitive polymerase chain reaction.

PubMed

Shimizu, Eri; Kato, Hisashi; Nakagawa, Yuki; Kodama, Takashi; Futo, Satoshi; Minegishi, Yasutaka; Watanabe, Takahiro; Akiyama, Hiroshi; Teshima, Reiko; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

2008-07-23

A novel type of quantitative competitive polymerase chain reaction (QC-PCR) system for the detection and quantification of the Roundup Ready soybean (RRS) was developed. This system was designed based on the advantage of a fully validated real-time PCR method used for the quantification of RRS in Japan. A plasmid was constructed as a competitor plasmid for the detection and quantification of genetically modified soy, RRS. The plasmid contained the construct-specific sequence of RRS and the taxon-specific sequence of lectin1 (Le1), and both had 21 bp oligonucleotide insertion in the sequences. The plasmid DNA was used as a reference molecule instead of ground seeds, which enabled us to precisely and stably adjust the copy number of targets. The present study demonstrated that the novel plasmid-based QC-PCR method could be a simple and feasible alternative to the real-time PCR method used for the quantification of genetically modified organism contents.

Comparison of methods for in-house screening of HLA-B*57:01 to prevent abacavir hypersensitivity in HIV-1 care.

PubMed

De Spiegelaere, Ward; Philippé, Jan; Vervisch, Karen; Verhofstede, Chris; Malatinkova, Eva; Kiselinova, Maja; Trypsteen, Wim; Bonczkowski, Pawel; Vogelaers, Dirk; Callens, Steven; Ruelle, Jean; Kabeya, Kabamba; De Wit, Stephane; Van Acker, Petra; Van Sandt, Vicky; Emonds, Marie-Paule; Coucke, Paul; Sermijn, Erica; Vandekerckhove, Linos

2015-01-01

Abacavir is a nucleoside reverse transcriptase inhibitor used as part of combination antiretroviral therapy in HIV-1-infected patients. Because this drug can cause a hypersensitivity reaction that is correlated with the presence of the HLA-B*57:01 allotype, screening for the presence of HLA-B*57:01 is recommended before abacavir initiation. Different genetic assays have been developed for HLA-B*57:01 screening, each with specific sensitivity, turnaround time and assay costs. Here, a new real-time PCR (qPCR) based analysis is described and compared to sequence specific primer PCR with capillary electrophoresis (SSP PCR CE) on 149 patient-derived samples, using sequence specific oligonucleotide hybridization combined with high resolution SSP PCR as gold standard. In addition to these PCR based methods, a complementary approach was developed using flow cytometry with an HLA-B17 specific monoclonal antibody as a pre-screening assay to diminish the number of samples for genetic testing. All three assays had a maximum sensitivity of >99. However, differences in specificity were recorded, i.e. 84.3%, 97.2% and >99% for flow cytometry, qPCR and SSP PCR CE respectively. Our data indicate that the most specific and sensitive of the compared methods is the SSP PCR CE. Flow cytometry pre-screening can substantially decrease the number of genetic tests for HLA-B*57:01 typing in a clinical setting.

Inactivation conditions for human Norovirus measured by an in situ capture-qRT-PCR Method

USDA-ARS?s Scientific Manuscript database

Human noroviruses (HuNoVs) are the major cause of epidemic non-bacterial gastroenteritis. Due to the inability to cultivate HuNoVs, it has been a challenge to determine their infectivity. Quantitative real-time RT-PCR (qRT-PCR) is widely used in detecting HuNoVs. However, qRT-PCR only detects the...

Detection of five potentially periodontal pathogenic bacteria in peri-implant disease: A comparison of PCR and real-time PCR.

PubMed

Schmalz, Gerhard; Tsigaras, Sandra; Rinke, Sven; Kottmann, Tanja; Haak, Rainer; Ziebolz, Dirk

2016-07-01

The aim of this study was to compare the microbial analysis methods of polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in terms of detection of five selected potentially periodontal pathogenic bacteria in peri-implant disease. Therefore 45 samples of healthy, mucositis and peri-implantitis (n = 15 each) were assessed according to presence of the following bacteria using PCR (DNA-strip technology) and RT-PCR (fluorescent dye SYBR green-system): Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tanerella forsythia (Tf), and Fusobacterium nucleatum (Fn). There were no significant correlations between the bacterial and disease patterns, so the benefit of using microbiological tests for the diagnosis of peri-implant diseases is questionable. Correlations between the methods were highest for Tf (Kendall's Tau: 0.65, Spearman: 0.78), Fn (0.49, 0.61) and Td (0.49, 0.59). For Aa (0.38, 0.42) and Pg (0.04, 0.04), lower correlation values were detected. Accordingly, conventional semi-quantitative PCR seems to be sufficient for analyzing potentially periodontal pathogenic bacterial species. Copyright © 2016 Elsevier Inc. All rights reserved.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

Validating internal controls for quantitative plant gene expression studies

PubMed Central

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-01-01

Background Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Results Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Conclusion Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments. PMID:15317655

Detection of the tetM resistance determinant among phenotypically sensitive Ureaplasma species by a novel real-time PCR method.

PubMed

Kotrotsiou, Tzimoula; Tzimoula, Kotrotsiou; Exindari, Maria; Maria, Exindari; Diza, Eudoxia; Eudoxia, Diza; Gioula, Georgia; Georgia, Gioula; Melidou, Angeliki; Angeliki, Melidou; Malisiovas, Nikolaos; Nikolaos, Malisiovas

2015-02-01

The study aimed to identify the proportion of tetM-positive Ureaplasma spp. isolates phenotypically susceptible to tetracycline by real-time PCR. Ureaplasma spp. strains of urogenital origin were isolated from 100 female or male adults on A7 agar plates. The presence of Ureaplasma was confirmed by the presence of urease gene by a novel real-time PCR method. Genotyping and sensitivity to tetracyclines were examined using commercial methods. The tetM gene was detected by a novel real-time PCR method especially designed for this study. Ureaplasma parvum was isolated from 87 of the specimens; Ureaplasma urealyticum, from 12; and both species were isolated from a single specimen. All isolates were phenotypically susceptible to tetracyclines. Thirty-five strains were tetM carriers; 29 (82.9%), U. parvum; 5 (14.3%), U. urealyticum; and 1 (2.9%), U. parvum/U. urealyticum. No statistically significant difference was observed between the 3 groups. Four (40%) tetM carriers were isolated from 10 symptomatic men; 11 (32.4%), from 34 symptomatic women; and 20 (35.7%), from 56 asymptomatic women. No statistically significant difference was observed between the 3 groups. The tetM determinant is detected in 35% of phenotypically susceptible to tetracycline Ureaplasma spp. Greek isolates. The use of a real-time PCR technique is particularly helpful, as it makes its detection easy; cost-effective; rapid; and, therefore, more convenient for the surveillance of the dissemination of the tetM resistance gene. Copyright © 2015 Elsevier Inc. All rights reserved.

[Selection of reference genes of Siraitia grosvenorii by real-time PCR].

PubMed

Tu, Dong-ping; Mo, Chang-ming; Ma, Xiao-jun; Zhao, Huan; Tang, Qi; Huang, Jie; Pan, Li-mei; Wei, Rong-chang

2015-01-01

Siraitia grosvenorii is a traditional Chinese medicine also as edible food. This study selected six candidate reference genes by real-time quantitative PCR, the expression stability of the candidate reference genes in the different samples was analyzed by using the software and methods of geNorm, NormFinder, BestKeeper, Delta CT method and RefFinder, reference genes for S. grosvenorii were selected for the first time. The results showed that 18SrRNA expressed most stable in all samples, was the best reference gene in the genetic analysis. The study has a guiding role for the analysis of gene expression using qRT-PCR methods, providing a suitable reference genes to ensure the results in the study on differential expressed gene in synthesis and biological pathways, also other genes of S. grosvenorii.

The use of quantitative real-time reverse transcriptase PCR for 5' and 3' portions of ALK transcripts to detect ALK rearrangements in lung cancers.

PubMed

Wang, Rui; Pan, Yunjian; Li, Chenguang; Hu, Haichuan; Zhang, Yang; Li, Hang; Luo, Xiaoyang; Zhang, Jie; Fang, Zhaoyuan; Li, Yuan; Shen, Lei; Ji, Hongbin; Garfield, David; Sun, Yihua; Chen, Haiquan

2012-09-01

Approximately 3% to 7% of non-small cell lung cancers (NSCLC) harbor an ALK fusion gene, thus defining a tumor group that may be responsive to targeted therapy. The breakpoint in ALK consistently occurs at exon 20 and EML4 or other fusion partners, thus driving a strong expression of ALK kinase domain and resulting in an unbalanced expression in 5' and 3' portions of ALK transcripts. We have developed a rapid and accurate method by simultaneously detecting the expression in 5' and 3' portions of ALK mRNA. Quantitative real-time reverse transcriptase PCR (qRT-PCR) was used to examine expression levels of the 5' and 3' portions of ALK transcripts in177 NSCLCs, in which EGFR, KRAS, HER2, and BRAF mutations were absent. If unbalanced ALK mRNA expression was seen, ALK rearrangement was assumed to exist. ALK FISH was used to confirm the accuracy of qRT-PCR. RT-PCR and 5' RACE coupling sequencing identified the fusion variants. Real-time RT-PCR showed excellent sensitivity and specificity (100% and 100%, respectively) for detection of ALK rearrangements in resected specimens. In addition, six novel ALK fusion variants were identified, including one KIF5B-ALK (E17;A20) and five EML4-ALK variants (E6a;A19, E6a/b ins 18;A20, E17b ins 39;A20, E10a/b, E13;A20, and E17 ins 65;A20). Real-time RT-PCR is a rapid and accurate method for diagnosing ALK-rearranged lung cancers. Coupling of 5' RACE to this method should further facilitate rapid identification of novel ALK fusion genes. ©2012 AACR.

Real-time PCR (qPCR) primer design using free online software.

PubMed

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

Application of real-time PCR and melting curve analysis in rapid Diego blood group genotyping.

PubMed

Novaretti, M C Z; Ruiz, A S; Dorlhiac-Llacer, P E; Chamone, D A F

2010-01-01

The paucity of appropriate reagents for serologic typing of the Diego blood group antigens has prompted the development of a real-time PCR and melting curve analysis for Diego blood group genotyping. In this study, we phenotyped 4326 donor blood samples for Di(a) using semiautomated equipment. All 157 Di(a+) samples were then genotyped by PCR using sequence-specific primers (PCR-SSP) for DI*02 because of anti-Di(b) scarcity. Of the 4326 samples, we simultaneously tested 160 samples for Di(a) and Di(b) serology, and DI*01 and DI*02 by PCR-SSP and by real-time PCR. We used the same primers for Diego genotyping by real-time PCR and PCR-SSP. Melting curve profiles obtained using the dissociation software of the real-time PCR apparatus enabled the discrimination of Diego alleles. Of the total samples tested, 4169 blood donors, 96.4 percent (95% confidence interval [CI], 95.8-96.9%), were homozygous for DI*02 and 157, 3.6 percent (95% CI, 3.1%-4.2%), were heterozygous DI*01/02. No blood donor was found to be homozygous for DI*01 in this study. The calculated DI*01 and DI*02 allele frequencies were 0.0181 (95% CI, 0.0173-0.0189) and 0.9819 (95% CI, 0.9791-0.9847), respectively, showing a good fit for the Hardy-Weinberg equilibrium. There was full concordance among Diego phenotype results by PCR-SSP and real-time PCR. DI*01 and DI*02 allele determination with SYBR Green I and thermal cycler technology are useful methods for Diego determination. The real-time PCR with SYBR Green I melting temperature protocol can be used as a rapid screening tool for DI*01 and DI*02 blood group genotyping.

Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

PubMed Central

Paudel, Damodar; Jarman, Richard; Limkittikul, Kriengsak; Klungthong, Chonticha; Chamnanchanunt, Supat; Nisalak, Ananda; Gibbons, Robert; Chokejindachai, Watcharee

2011-01-01

Background: Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti). Materials and Methods: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR® Green assay, real time Taqman® assay to compare the sensitivity and specificity. Results: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR® green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay. Conclusion: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus. PMID:22363089

Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

PubMed Central

Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

2006-01-01

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages. PMID:16957227

Rapid quantitative detection of Lactobacillus sakei in meat and fermented sausages by real-time PCR.

PubMed

Martín, Belén; Jofré, Anna; Garriga, Margarita; Pla, Maria; Aymerich, Teresa

2006-09-01

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages.

Detection of Streptococcus pneumoniae and Haemophilus influenzae Type B by Real-Time PCR from Dried Blood Spot Samples among Children with Pneumonia: A Useful Approach for Developing Countries

PubMed Central

Selva, Laura; Benmessaoud, Rachid; Lanaspa, Miguel; Jroundi, Imane; Moraleda, Cinta; Acacio, Sozinho; Iñigo, Melania; Bastiani, Alien; Monsonis, Manuel; Pallares, Roman; Bassat, Quique; Muñoz-Almagro, Carmen

2013-01-01

Background Dried blood spot (DBS) is a reliable blood collection method for storing samples at room temperature and easily transporting them. We have previously validated a Real-Time PCR for detection of Streptococcus pneumoniae in DBS. The objective of this study was to apply this methodology for the diagnosis of S. pneumoniae and Haemophilus influenzae b (Hib) in DBS samples of children with pneumonia admitted to two hospitals in Mozambique and Morocco. Methods Ply and wzg genes of S. pneumoniae and bexA gene of Hib, were used as targets of Real-Time PCR. 329 DBS samples of children hospitalized with clinical diagnosis of pneumonia were tested. Results Real-Time PCR in DBS allowed for a significant increase in microbiological diagnosis of S. pneumoniae and Hib. When performing blood bacterial culture, only ten isolates of S. pneumoniae and none of Hib were detected (3·0% positivity rate, IC95% 1·4-5·5%). Real-Time PCR from DBS samples increased the detection yield by 4x fold, as 30 S. pneumoniae and 11 Hib cases were detected (12·4% positivity rate, IC95% 9·0-16·5%; P<0·001). Conclusion Real-Time PCR applied in DBS may be a valuable tool for improving diagnosis and surveillance of pneumonia caused by S. pneumoniae or Hib in developing countries. PMID:24116190

Evaluation of RIDA®GENE norovirus GI/GII real time RT-PCR using stool specimens collected from children and adults with acute gastroenteritis.

PubMed

Kanwar, N; Hassan, F; Barclay, L; Langley, C; Vinjé, J; Bryant, P W; George, K St; Mosher, L; Matthews-Greer, J M; Rocha, M A; Beenhouwer, D O; Harrison, C J; Moffatt, M; Shastri, N; Selvarangan, R

2018-04-10

Norovirus is the leading cause of epidemic and sporadic acute gastroenteritis (AGE) in the United States. Widespread prevalence necessitates implementation of accurate norovirus detection assays in clinical diagnostic laboratories. To evaluate RIDA ® GENE norovirus GI/GII real-time RT-PCR assay (RGN RT-PCR) using stool samples from patients with sporadic AGE. Patients between 14 days to 101 years of age with symptoms of AGE were enrolled prospectively at four sites across the United States during 2014-2015. Stool specimens were screened for the presence of norovirus RNA by the RGN RT-PCR assay. Results were compared with a reference method that included conventional RT-PCR and sequencing of a partial region of the 5'end of the norovirus ORF2 gene. A total of 259 (36.0%) of 719 specimens tested positive for norovirus by the reference method. The RGN RT-PCR assay detected norovirus in 244 (94%) of these 259 norovirus positive specimens. The sensitivity and specificity (95% confidence interval) of the RGN RT-PCR assay for detecting norovirus genogroup (G) I was 82.8% (63.5-93.5) and 99.1% (98.0-99.6) and for GII was 94.8% (90.8-97.2) and 98.6% (96.9-99.4), respectively. Seven specimens tested positive by the RGN-RT PCR that were negative by the reference method. The fifteen false negative samples were typed as GII.4 Sydney, GII.13, GI.3, GI.5, GI.2, GII.1, and GII.3 in the reference method. The RGN RT-PCR assay had a high sensitivity and specificity for the detection of norovirus in stool specimens from patients with sporadic AGE. Copyright © 2018. Published by Elsevier B.V.

Rapid and Specific Method for Evaluating Streptomyces Competitive Dynamics in Complex Soil Communities

USDA-ARS?s Scientific Manuscript database

Quantifying target microbial populations in complex communities remains a barrier to studying species interactions in soil environments. Quantitative real-time PCR (qPCR) offers a rapid and specific means to assess populations of target microorganisms. SYBR Green and TaqMan-based qPCR assays were de...

Detection of newly produced T and B lymphocytes by digital PCR in blood stored dry on nylon flocked swabs.

PubMed

Tessitore, Marion Vaglio; Sottini, Alessandra; Roccaro, Aldo M; Ghidini, Claudia; Bernardi, Simona; Martellosio, Giovanni; Serana, Federico; Imberti, Luisa

2017-04-05

A normal number of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) is considered a biomarker for adequate new T- and B-cell production. In newborns, detection of TRECs and KRECs by real time PCR from dried blood spotted on filter paper is used for the screening of severe immunodeficiency. In adults, elderly and during diseases, where the number of TRECs is lower than in newborns and children, a large amount of DNA and a sensitive method of amplification are necessary to identify newly produced lymphocytes. DNA was prepared from blood of 203 healthy adults (range: 18-91 years old) absorbed for 10 s on flocked swabs and let to dry, or from peripheral blood mononuclear cells. DNA was subjected to digital PCR and to well established conventional real time PCR-based method using TREC- and KREC-specific primers and probes. The number of TRECs and KRECs was expressed per mL of blood. Statistical analysis was performed by nested ANOVA, Pearson coefficient of determination, and by linear regression tests. The novel method for the storage of dried blood on nylon flocked swabs and the use of digital PCR allow quantification of TRECs and KRECs with high degree of sensitivity, specificity, accuracy, and precision. TRECs and KRECs were amplified by digital PCR in all tested blood samples, including those obtained from elderly individuals (>70 years old) and that were negative by real time PCR. Furthermore, values of TRECs and KRECs obtained by digital PCR were in the range of those acquired by real time PCR. Our findings demonstrate that DNA isolation from dried blood on flocked swabs followed by digital PCR-based analysis represents a useful tool for studying new lymphocyte production in adults and elderly individuals. This suggests the potential use of the methodology when monitoring of clinical variables is limited by the number of molecules that can be amplified and detected, such as in patients with immunodeficiency or under immunosuppressive therapies.

Quantitative analysis of periodontal pathogens by ELISA and real-time polymerase chain reaction.

PubMed

Hamlet, Stephen M

2010-01-01

The development of analytical methods enabling the accurate identification and enumeration of bacterial species colonizing the oral cavity has led to the identification of a small number of bacterial pathogens that are major factors in the etiology of periodontal disease. Further, these methods also underpin more recent epidemiological analyses of the impact of periodontal disease on general health. Given the complex milieu of over 700 species of microorganisms known to exist within the complex biofilms found in the oral cavity, the identification and enumeration of oral periodontopathogens has not been an easy task. In recent years however, some of the intrinsic limitations of the more traditional microbiological analyses previously used have been overcome with the advent of immunological and molecular analytical methods. Of the plethora of methodologies reported in the literature, the enzyme-linked immunosorbent assay (ELISA), which combines the specificity of antibody with the sensitivity of simple enzyme assays and the polymerase chain reaction (PCR), has been widely utilized in both laboratory and clinical applications. Although conventional PCR does not allow quantitation of the target organism, real-time PCR (rtPCR) has the ability to detect amplicons as they accumulate in "real time" allowing subsequent quantitation. These methods enable the accurate quantitation of as few as 10(2) (using rtPCR) to 10(4) (using ELISA) periodontopathogens in dental plaque samples.

Real-Time PCR with an Internal Control for Detection of All Known Human Adenovirus Serotypes▿

PubMed Central

Damen, Marjolein; Minnaar, René; Glasius, Patricia; van der Ham, Alwin; Koen, Gerrit; Wertheim, Pauline; Beld, Marcel

2008-01-01

The “gold standard” for the diagnosis of adenovirus (AV) infection is virus culture, which is rather time-consuming. Especially for immunocompromised patients, in whom severe infections with AV have been described, rapid diagnosis is important. Therefore, an internally controlled AV real-time PCR assay detecting all known human AV serotypes was developed. Primers were chosen from the hexon region, which is the most conserved region, and in order to cover all known serotypes, degenerate primers were used. The internal control (IC) DNA contained the same primer binding sites as the AV DNA control but had a shuffled probe region compared to the conserved 24-nucleotide consensus AV hexon probe region (the target). The IC DNA was added to the clinical sample in order to monitor extraction and PCR efficiency. The sensitivity and the linearity of the AV PCR were determined. For testing the specificity of this PCR assay for human AVs, a selection of 51 AV prototype strains and 66 patient samples positive for other DNA viruses were tested. Moreover, a comparison of the AV PCR method described herein with culture and antigen (Ag) detection was performed with a selection of 151 clinical samples. All 51 AV serotypes were detected in the selection of AV prototype strains. Concordant results from culture or Ag detection and PCR were found for 139 (92.1%) of 151 samples. In 12 cases (7.9%), PCR was positive while the culture was negative. In conclusion, a sensitive, internally controlled nonnested AV real-time PCR assay which is able to detect all known AV serotypes with higher sensitivity than a culture or Ag detection method was developed. PMID:18923006

Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR.

PubMed

Gentilini, Fabio; Turba, Maria E

2014-01-01

A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR. In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5°C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations. Copyright © 2014 Elsevier B.V. All rights reserved.

Risk assessment of false-positive quantitative real-time PCR results in food, due to detection of DNA originating from dead cells.

PubMed

Wolffs, Petra; Norling, Börje; Rådström, Peter

2005-03-01

Real-time PCR technology is increasingly used for detection and quantification of pathogens in food samples. A main disadvantage of nucleic acid detection is the inability to distinguish between signals originating from viable cells and DNA released from dead cells. In order to gain knowledge concerning risks of false-positive results due to detection of DNA originating from dead cells, quantitative PCR (qPCR) was used to investigate the degradation kinetics of free DNA in four types of meat samples. Results showed that the fastest degradation rate was observed (1 log unit per 0.5 h) in chicken homogenate, whereas the slowest rate was observed in pork rinse (1 log unit per 120.5 h). Overall results indicated that degradation occurred faster in chicken samples than in pork samples and faster at higher temperatures. Based on these results, it was concluded that, especially in pork samples, there is a risk of false-positive PCR results. This was confirmed in a quantitative study on cell death and signal persistence over a period of 28 days, employing three different methods, i.e. viable counts, direct qPCR, and finally floatation, a recently developed discontinuous density centrifugation method, followed by qPCR. Results showed that direct qPCR resulted in an overestimation of up to 10 times of the amount of cells in the samples compared to viable counts, due to detection of DNA from dead cells. However, after using floatation prior to qPCR, results resembled the viable count data. This indicates that by using of floatation as a sample treatment step prior to qPCR, the risk of false-positive PCR results due to detection of dead cells, can be minimized.

Establishment of a multiplex real-time RT-PCR assay for rapid identification of H6 subtype avian influenza viruses.

PubMed

Yang, Fan; Wu, Haibo; Liu, Fumin; Lu, Xiangyun; Peng, Xiuming; Wu, Nanping

2018-06-01

The H6 subtype avian influenza viruses (AIVs) possess the capacity for zoonotic transmission from avian species to humans. Establishment of a specific, rapid and sensitive method to screen H6 AIVs is necessary. Based on the conserved domain of the matrix and H6 AIV hemagglutinin genes, two TaqMan minor-groove-binder probes and multiplex real-time RT-PCR primers were designed in this study. The multiplex real-time RT-PCR assay developed in this study had high specificity and repeatability and a detection limit of 30 copies per reaction. This rapid diagnostic method will be useful for clinical detection and surveillance of H6 AIVs in China.

Identification of Pseudallescheria and Scedosporium species by three molecular methods.

PubMed

Lu, Qiaoyun; Gerrits van den Ende, A H G; Bakkers, J M J E; Sun, Jiufeng; Lackner, M; Najafzadeh, M J; Melchers, W J G; Li, Ruoyu; de Hoog, G S

2011-03-01

The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial β-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 10(3), and 5 × 10(2) cells/μl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species.

Rapid Screening for Deleted Form of β-thalassemia by Real-Time Quantitative PCR.

PubMed

Ke, Liang-Yin; Chang, Jan-Gowth; Chang, Chao-Sung; Hsieh, Li-Ling; Liu, Ta-Chih

2017-01-01

Thalassemia is the most common single gene disease in human beings. The prevalence rate of β-thalassemia in Taiwan is approximately 1-3%. Previously methods to reveal and diagnose severe deleted form of α- or β-thalassemia were insufficient and inappropriate for prenatal diagnosis. A real-time quantitative PCR method was set up for rapid screening of the deleted form of β-thalassemia. Our results show that ΔΔCt between deleted form of β-thalassemia and normal individuals were 1.0674 ± 0.0713. On the contrary, mutation form β-thalassemia showed no difference with normal healthy control. The HBB/CCR5 ratio for deleted form of β-thalassemia patients was 0.48, whether normal individuals and mutation form of β-thalassemia was 1.0. This RQ-PCR technique is an alternative rapid screening assay for deleted form of β-thalassemia. In addition, it could also identify undefined type. Our technique by using RQ-PCR to quantify gene copies is a reliable and time-saving method that can screen deleted form of β-thalassemia. © 2016 Wiley Periodicals, Inc.

Development of SYBR Green I Based Real-Time RT-PCR Assay for Specific Detection of Watermelon silver mottle Virus.

PubMed

Rao, Xueqin; Sun, Jie

2015-09-01

Watermelon silver mottle virus (WSMoV), which belongs to the genus Tospovirus , causes significant loss in Cucurbitaceae plants. Development of a highly sensitive and reliable detection method for WSMoV. Recombinant plasmids for targeting the sequence of nucleocapsid protein gene of WSMoV were constructed. SYBR Green I real-time PCR was established and evaluated with standard recombinant plasmids and 27 watermelon samples showing WSMoV infection symptoms. The recombinant plasmid was used as template for SYBR Green I real-time PCR to generate standard and melting curves. Melting curve analysis indicated no primer-dimers and non-specific products in the assay. No cross-reaction was observed with Capsicum chlorosis virus (genus Tospovirus ) and Cucumber mosaic virus (genus Cucumovirus). Repeatability tests indicated that inter-assay variability of the Ct values was 1.6%. A highly sensitive, reliable and rapid detection method of SYBR Green I real-time PCR for timely detection of WSMoV plants and vector thrips was established, which will facilitate disease forecast and control.

Application of the SYBR Green real-time HRM PCR technique in the differentiation of the Babesia canis canis protozoa isolated in the areas of eastern Poland.

PubMed

Adaszek, Lukasz; Winiarczyk, Stanisław

2010-04-01

The aim of this study was to determine the usefulness of the real-time polymerised chain reaction (PCR) high-resolution melting (HRM) method in the differentiation of the Babesia canis canis protozoa isolated from dogs in the areas of eastern Poland. The studies involved 20 isolates of B. canis canis qualified depending on the analysis of the 18S RNA gene sequence to group A (EU 622792) and 20 isolates qualified to group B (EU 622793). It was proven with the real-time PCR technique that the melting temperature (Tm) of the obtained products of amplification was 78 degrees C for the representatives of group A and 81 degrees C for the representatives of group B, which proves that the real-time SYBR Green HRM PCR method is a technique allowing for the differentiation of the B. canis isolates which are slightly different with respect to the genetic structure, without the necessity to carry out time-consuming studies, i.e., sequencing and restriction fragment length polymorphism.

Simplified Pan-species Real-time PCR-based Detection of Plasmodium Spp. in Blood Smear

PubMed Central

HASSANPOUR, Gholamreza; MIRHENDI, Hossein; MOHEBALI, Mehdi; RAEISI, Ahmad; ZERAATI, Hojjat; KESHAVARZ, Hossein

2016-01-01

Background: We aimed to quicken and simplify the detection of Plasmodium in blood samples by developing and testing a pan-Plasmodium real-time PCR for accurate screening of individuals suspected of malaria. Methods: A single primer/probe set for pan-species Plasmodium-specific real time PCR targeting a conserved region of the small subunit 18S ribosomal DNA was designed and evaluated for rapid diagnosis and screening of malaria infections using dried blood smears. FTA cards were used for rapid and simple DNA extraction. Results: The primers and probes showed a positive response with the DNA extracted from bloods infected with P. falciparum and P. vivax but not with DNA extracted from various smears from uninfected blood samples. Seven positive cases positive by both microscopy and nested PCR were found among 280 blood samples taken from in South and Southeast Iran. Five samples were identified as positive for P. vivax and two as positive for P. falciparum. All positive samples were positive by real-time PCR. Furthermore, all 38-blood samples positive by microscopy were positive by real-time PCR. No microscopy-negative samples were positive by real-time PCR. Conclusion: By using a simple FTA card for DNA extraction and by application of the real-time PCR developed in this study, sensitivity similar to nested-PCR and microscopy was achieved. This format simplifies the detection of Plasmodium in large numbers of samples. PMID:28127357

An Improved Quantitative Real-Time PCR Assay for the Enumeration of Heterosigma akashiwo (Raphidophyceae) Cysts Using a DNA Debris Removal Method and a Cyst-Based Standard Curve.

PubMed

Kim, Joo-Hwan; Kim, Jin Ho; Wang, Pengbin; Park, Bum Soo; Han, Myung-Soo

2016-01-01

The identification and quantification of Heterosigma akashiwo cysts in sediments by light microscopy can be difficult due to the small size and morphology of the cysts, which are often indistinguishable from those of other types of algae. Quantitative real-time PCR (qPCR) based assays represent a potentially efficient method for quantifying the abundance of H. akashiwo cysts, although standard curves must be based on cyst DNA rather than on vegetative cell DNA due to differences in gene copy number and DNA extraction yield between these two cell types. Furthermore, qPCR on sediment samples can be complicated by the presence of extracellular DNA debris. To solve these problems, we constructed a cyst-based standard curve and developed a simple method for removing DNA debris from sediment samples. This cyst-based standard curve was compared with a standard curve based on vegetative cells, as vegetative cells may have twice the gene copy number of cysts. To remove DNA debris from the sediment, we developed a simple method involving dilution with distilled water and heating at 75°C. A total of 18 sediment samples were used to evaluate this method. Cyst abundance determined using the qPCR assay without DNA debris removal yielded results up to 51-fold greater than with direct counting. By contrast, a highly significant correlation was observed between cyst abundance determined by direct counting and the qPCR assay in conjunction with DNA debris removal (r2 = 0.72, slope = 1.07, p < 0.001). Therefore, this improved qPCR method should be a powerful tool for the accurate quantification of H. akashiwo cysts in sediment samples.

Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

PubMed Central

Wang, Hye-young; Kim, Sunghyun; Kim, Jungho; Park, Soon-Deok

2014-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 103 CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene. PMID:24648566

Quantification Bias Caused by Plasmid DNA Conformation in Quantitative Real-Time PCR Assay

PubMed Central

Lin, Chih-Hui; Chen, Yu-Chieh; Pan, Tzu-Ming

2011-01-01

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997

Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR

PubMed Central

Chemidlin Prévost-Bouré, Nicolas; Christen, Richard; Dequiedt, Samuel; Mougel, Christophe; Lelièvre, Mélanie; Jolivet, Claudy; Shahbazkia, Hamid Reza; Guillou, Laure; Arrouays, Dominique; Ranjard, Lionel

2011-01-01

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1 / FF390. This in silico analysis of the specificity of FR1 / FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1 / FF390 for Fungi was validated in vitro by cloning - sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils. PMID:21931659

Operational Evaluation of the Rapid Viability PCR Method for ...

EPA Pesticide Factsheets

Journal Article This research work has a significant impact on the use of the RV-PCR method to analyze post-decontamination environmental samples during an anthrax event. The method has shown 98% agreement with the traditional culture based method. With such a success, this method, upon validation, will significantly increase the laboratory throughput/capacity to analyze a large number of anthrax event samples in a relatively short time.

Multiparametric comparison of chromogenic-based culture methods used to assess the microbiological quality of drinking water and the mFC method combined with a molecular confirmation procedure.

PubMed

Maheux, Andrée F; Dion-Dupont, Vanessa; Bisson, Marc-Antoine; Bouchard, Sébastien; Jubinville, Éric; Nkuranga, Martine; Rodrigue, Lynda; Bergeron, Michel G; Rodriguez, Manuel J

2015-03-01

MI agar and Colilert(®), as well as mFC agar combined with an Escherichia coli-specific molecular assay (mFC + E. coli rtPCR), were compared in terms of their sensitivity, ease of use, time to result and affordability. The three methods yielded a positive E. coli signal for 11.5, 10.8, and 11.5% of the 968 well water samples tested, respectively. One hundred and thirty-six (136) samples gave blue colonies on mFC agar and required confirmation. E. coli-specific rtPCR showed false-positive results in 23.5% (32/136) of cases. In terms of ease of use, Colilert was the simplest method to use while the MI method provided ease of use comparable to all membrane filtration methods. However, the mFC + E. coli rtPCR assay required highly trained employees for confirmation purposes. In terms of affordability, and considering contamination rate of well water samples tested, the Colilert method and the mFC + E. coli rtPCR assay were at least five times more costly than the MI agar method. Overall, compared with the other two methods tested, the MI agar method offers the most advantages to assess drinking water quality.

Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood

PubMed Central

Boyd, MA; Tennant, SM; Melendez, JH; Toema, D; Galen, JE; Geddes, CD; Levine, MM

2015-01-01

Aims Isolation of Salmonella Typhi from blood culture is the standard diagnostic for confirming typhoid fever but it is unavailable in many developing countries. We previously described a Microwave Accelerated Metal Enhanced Fluorescence (MAMEF)-based assay to detect Salmonella in medium. Attempts to detect Salmonella in blood were unsuccessful, presumably due to the interference of erythrocytes. The objective of this study was to evaluate various blood treatment methods that could be used prior to PCR, real-time PCR or MAMEF to increase sensitivity of detection of Salmonella. Methods and Results We tested ammonium chloride and erythrocyte lysis buffer, water, Lymphocyte Separation Medium, BD Vacutainer® CPT™ Tubes and dextran. Erythrocyte lysis buffer was the best isolation method as it is fast, inexpensive and works with either fresh or stored blood. The sensitivity of PCR- and real-time PCR detection of Salmonella in spiked blood was improved when whole blood was first lysed using erythrocyte lysis buffer prior to DNA extraction. Removal of erythrocytes and clotting factors also enabled reproducible lysis of Salmonella and fragmentation of DNA, which are necessary for MAMEF sensing. Conclusions Use of the erythrocyte lysis procedure prior to DNA extraction has enabled improved sensitivity of Salmonella detection by PCR and real-time PCR and has allowed lysis and fragmentation of Salmonella using microwave radiation (for future detection by MAMEF). Significance and Impact of the Study Adaptation of the blood lysis method represents a fundamental breakthrough that improves the sensitivity of DNA-based detection of Salmonella in blood. PMID:25630831

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources

EPA Science Inventory

The state of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusio...

Real-Time Polymerase Chain Reaction Detection of Angiostrongylus cantonensis DNA in Cerebrospinal Fluid from Patients with Eosinophilic Meningitis

PubMed Central

Qvarnstrom, Yvonne; Xayavong, Maniphet; da Silva, Ana Cristina Aramburu; Park, Sarah Y.; Whelen, A. Christian; Calimlim, Precilia S.; Sciulli, Rebecca H.; Honda, Stacey A. A.; Higa, Karen; Kitsutani, Paul; Chea, Nora; Heng, Seng; Johnson, Stuart; Graeff-Teixeira, Carlos; Fox, LeAnne M.; da Silva, Alexandre J.

2016-01-01

Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis. Timely diagnosis of these infections is difficult, partly because reliable laboratory diagnostic methods are unavailable. The aim of this study was to evaluate the usefulness of a real-time polymerase chain reaction (PCR) assay for the detection of A. cantonensis DNA in human cerebrospinal fluid (CSF) specimens. A total of 49 CSF specimens from 33 patients with eosinophilic meningitis were included: A. cantonensis DNA was detected in 32 CSF specimens, from 22 patients. Four patients had intermittently positive and negative real-time PCR results on subsequent samples, indicating that the level of A. cantonensis DNA present in CSF may fluctuate during the course of the illness. Immunodiagnosis and/or supplemental PCR testing supported the real-time PCR findings for 30 patients. On the basis of these observations, this real-time PCR assay can be useful to detect A. cantonensis in the CSF from patients with eosinophilic meningitis. PMID:26526920

Assessment of SCAR markers to design real-time PCR primers for rhizosphere quantification of Azospirillum brasilense phytostimulatory inoculants of maize.

PubMed

Couillerot, O; Poirier, M-A; Prigent-Combaret, C; Mavingui, P; Caballero-Mellado, J; Moënne-Loccoz, Y

2010-08-01

To assess the applicability of sequence characterized amplified region (SCAR) markers obtained from BOX, ERIC and RAPD fragments to design primers for real-time PCR quantification of the phytostimulatory maize inoculants Azospirillum brasilense UAP-154 and CFN-535 in the rhizosphere. Primers were designed based on strain-specific SCAR markers and were screened for successful amplification of target strain and absence of cross-reaction with other Azospirillum strains. The specificity of primers thus selected was verified under real-time PCR conditions using genomic DNA from strain collection and DNA from rhizosphere samples. The detection limit was 60 fg DNA with pure cultures and 4 x 10(3) (for UAP-154) and 4 x 10(4) CFU g(-1) (for CFN-535) in the maize rhizosphere. Inoculant quantification was effective from 10(4) to 10(8) CFU g(-1) soil. BOX-based SCAR markers were useful to find primers for strain-specific real-time PCR quantification of each A. brasilense inoculant in the maize rhizosphere. Effective root colonization is a prerequisite for successful Azospirillum phytostimulation, but cultivation-independent monitoring methods were lacking. The real-time PCR methods developed here will help understand the effect of environmental conditions on root colonization and phytostimulation by A. brasilense UAP-154 and CFN-535.

Rapid, cost-effective, sensitive and quantitative detection of Acinetobacter baumannii from pneumonia patients

PubMed Central

Nomanpour, B; Ghodousi, A; Babaei, A; Abtahi, HR; Tabrizi, M; Feizabadi, MM

2011-01-01

Background and Objectives Pneumonia with Acinetobacter baumannii has a major therapeutic problem in health care settings. Decision to initiate correct antibiotic therapy requires rapid identification and quantification of organism. The aim of this study was to develop a rapid and sensitive method for direct detection of A. baumannii from respiratory specimens. Materials and Methods A Taqman real time PCR based on the sequence of bla oxa-51 was designed and used for direct detection of A. baumannii from 361 respiratory specimens of patients with pneumonia. All specimens were checked by conventional bacteriology in parallel. Results The new real time PCR could detect less than 200 cfu per ml of bacteria in specimens. There was agreement between the results of real time PCR and culture (Kappa value 1.0, p value<0.001). The sensitivity, specificity and predictive values of real time PCR were 100%. The prevalence of A. baumannii in pneumonia patients was 10.53 % (n=38). Poly-microbial infections were detected in 65.71% of specimens. Conclusion Acinetobacter baumannii is the third causative agent in nosocomial pneumonia after Pseudomonas aeroginosa (16%) and Staphylococcus aureus (13%) at Tehran hospitals. We recommend that 104 CFU be the threshold for definition of infection with A. baumannii using real time PCR. PMID:22530083

Clinical utility of an optimised multiplex real-time PCR assay for the identification of pathogens causing sepsis in Vietnamese patients.

PubMed

Tat Trung, Ngo; Van Tong, Hoang; Lien, Tran Thi; Van Son, Trinh; Thanh Huyen, Tran Thi; Quyen, Dao Thanh; Hoan, Phan Quoc; Meyer, Christian G; Song, Le Huu

2018-02-01

For the identification of bacterial pathogens, blood culture is still the gold standard diagnostic method. However, several disadvantages apply to blood cultures, such as time and rather large volumes of blood sample required. We have previously established an optimised multiplex real-time PCR method in order to diagnose bloodstream infections. In the present study, we evaluated the diagnostic performance of this optimised multiplex RT-PCR in blood samples collected from 110 septicaemia patients enrolled at the 108 Military Central Hospital, Hanoi, Vietnam. Positive results were obtained by blood culture, the Light Cylcler-based SeptiFast ® assay and our multiplex RT-PCR in 35 (32%), 31 (28%), and 31 (28%) samples, respectively. Combined use of the three methods confirmed 50 (45.5%) positive cases of bloodstream infection, a rate significantly higher compared to the exclusive use of one of the three methods (P=0.052, 0.012 and 0.012, respectively). The sensitivity, specificity and area under the curve (AUC) of our assay were higher compared to that of the SeptiFast ® assay (77.4%, 86.1% and 0.8 vs. 67.7%, 82.3% and 0.73, respectively). Combined use of blood culture and multiplex RT-PCR assay showed a superior diagnostic performance, as the sensitivity, specificity, and AUC reached 83.3%, 100%, and 0.95, respectively. The concordance between blood culture and the multiplex RT-PCR assay was highest for Klebsiella pneumonia (100%), followed by Streptococcus spp. (77.8%), Escherichia coli (66.7%), Staphylococcus spp. (50%) and Salmonella spp. (50%). In addition, the use of the newly established multiplex RT-PCR assay increased the spectrum of identifiable agents (Acintobacter baumannii, 1/32; Proteus mirabilis, 1/32). The combination of culture and the multiplex RT-PCR assay provided an excellent diagnostic accomplishment and significantly supported the identification of causative pathogens in clinical samples obtained from septic patients. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Development and evaluation of event-specific quantitative PCR method for genetically modified soybean A2704-12.

PubMed

Takabatake, Reona; Akiyama, Hiroshi; Sakata, Kozue; Onishi, Mari; Koiwa, Tomohiro; Futo, Satoshi; Minegishi, Yasutaka; Teshima, Reiko; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

2011-01-01

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (C(f)), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined C(f) values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.

Accuracy of real-time PCR, Gram stain and culture for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae meningitis diagnosis.

PubMed

Wu, Henry M; Cordeiro, Soraia M; Harcourt, Brian H; Carvalho, Mariadaglorias; Azevedo, Jailton; Oliveira, Tainara Q; Leite, Mariela C; Salgado, Katia; Reis, Mitermayer G; Plikaytis, Brian D; Clark, Thomas A; Mayer, Leonard W; Ko, Albert I; Martin, Stacey W; Reis, Joice N

2013-01-22

Although cerebrospinal fluid (CSF) culture is the diagnostic reference standard for bacterial meningitis, its sensitivity is limited, particularly when antibiotics were previously administered. CSF Gram staining and real-time PCR are theoretically less affected by antibiotics; however, it is difficult to evaluate these tests with an imperfect reference standard. CSF from patients with suspected meningitis from Salvador, Brazil were tested with culture, Gram stain, and real-time PCR using S. pneumoniae, N. meningitidis, and H. influenzae specific primers and probes. An antibiotic detection disk bioassay was used to test for the presence of antibiotic activity in CSF. The diagnostic accuracy of tests were evaluated using multiple methods, including direct evaluation of Gram stain and real-time PCR against CSF culture, evaluation of real-time PCR against a composite reference standard, and latent class analysis modeling to evaluate all three tests simultaneously. Among 451 CSF specimens, 80 (17.7%) had culture isolation of one of the three pathogens (40 S. pneumoniae, 36 N. meningitidis, and 4 H. influenzae), and 113 (25.1%) were real-time PCR positive (51 S. pneumoniae, 57 N. meningitidis, and 5 H. influenzae). Compared to culture, real-time PCR sensitivity and specificity were 95.0% and 90.0%, respectively. In a latent class analysis model, the sensitivity and specificity estimates were: culture, 81.3% and 99.7%; Gram stain, 98.2% and 98.7%; and real-time PCR, 95.7% and 94.3%, respectively. Gram stain and real-time PCR sensitivity did not change significantly when there was antibiotic activity in the CSF. Real-time PCR and Gram stain were highly accurate in diagnosing meningitis caused by S. pneumoniae, N. meningitidis, and H. influenzae, though there were few cases of H. influenzae. Furthermore, real-time PCR and Gram staining were less affected by antibiotic presence and might be useful when antibiotics were previously administered. Gram staining, which is inexpensive and commonly available, should be encouraged in all clinical settings.

A BAYESIAN METHOD FOR CALCULATING REAL-TIME QUANTITATIVE PCR CALIBRATION CURVES USING ABSOLUTE PLASMID DNA STANDARDS

EPA Science Inventory

In real-time quantitative PCR studies using absolute plasmid DNA standards, a calibration curve is developed to estimate an unknown DNA concentration. However, potential differences in the amplification performance of plasmid DNA compared to genomic DNA standards are often ignore...

Direct Fluorescence Detection of Allele-Specific PCR Products Using Novel Energy-Transfer Labeled Primers.

PubMed

Winn-Deen

1998-12-01

Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the PCR product liberated during the detection step. The author has combined the PCR amplification and detection steps into a single procedure suitable for closed-tube analysis. Methods and Results: Allele-specific PCR primers were designed as Sunrise energy-transfer primers and contained a 3' terminal mismatch to distinguish between normal and mutant DNA. Cloned normal (W64) and mutant (R64) templates of the beta3-adrenergic receptor gene were tested to verify amplification specificity and yield. A no-target negative control was also run with each reaction. After PCR, each reaction was tested for fluorescence yield by measuring fluorescence on a spectrofluorimeter or fluorescent microtitreplate reader. The cloned controls and 24 patient samples were tested for the W64R mutation by two methods. The direct fluorescence results with the Sunrise allele-specific PCR method gave comparable genotypes to those obtained with the PCR/ restriction digest/gel electrophoresis control method. No PCR artifacts were observed in the negative controls or in the PCR reactions run with the mismatched target. Conclusions: The results of this pilot study indicate good PCR product and fluorescence yield from allele-specific energy-transfer labeled primers, and the capability of distinguishing between normal and mutant alleles based on fluorescence alone, without the need for restriction digestion, gel electrophoresis, or hybridization with an allele-specific probe.

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

PubMed Central

Ghosh, Prakash; Khan, Md. Anik Ashfaq; Duthie, Malcolm S.; Vallur, Aarthy C.; Picone, Alessandro; Howard, Randall F.; Reed, Steven G.

2017-01-01

Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L. donovani parasites in VL, PKDL and RVL patients, as well as being capable of assessing response to treatment. As such, this real time PCR assay represents an important contribution in efforts to eliminate VL. PMID:28957391

Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples.

PubMed

Stark, D; Al-Qassab, S E; Barratt, J L N; Stanley, K; Roberts, T; Marriott, D; Harkness, J; Ellis, J T

2011-01-01

The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.

Technical note: a novel method for routine genotyping of horse coat color gene polymorphisms.

PubMed

Royo, L J; Fernández, I; Azor, P J; Alvarez, I; Pérez-Pardal, L; Goyache, F

2008-06-01

The aim of this note is to describe a reliable, fast, and cost-effective real-time PCR method for routine genotyping of mutations responsible for most coat color variation in horses. The melanocortin-1 receptor, Agouti-signaling peptide, and membrane-associated transporter protein alleles were simultaneously determined using 2 PCR protocols. The assay described here is an alternative method for routine genotyping of a defined number of polymorphisms. Allelic variants are detected in real time and no post-PCR manipulations are required, therefore limiting costs and possible carryover contamination. Data can be copied to a Microsoft Excel spreadsheet for semiautomatic determination of the genotype using a macro freely available at http://www.igijon.com/personales/fgoyache/software_i.htm (last accessed February 26, 2007). The performance of the method is demonstrated on 156 Spanish Purebred horses.

The applicability of real-time PCR in the diagnostic of cutaneous leishmaniasis and parasite quantification for clinical management: Current status and perspectives.

PubMed

Moreira, Otacilio C; Yadon, Zaida E; Cupolillo, Elisa

2017-09-29

Cutaneous leishmaniasis (CL) is spread worldwide and is the most common manifestation of leishmaniasis. Diagnosis is performed by combining clinical and epidemiological features, and through the detection of Leishmania parasites (or DNA) in tissue specimens or trough parasite isolation in culture medium. Diagnosis of CL is challenging, reflecting the pleomorphic clinical manifestations of this disease. Skin lesions vary in severity, clinical appearance, and duration, and in some cases, they can be indistinguishable from lesions related to other diseases. Over the past few decades, PCR-based methods, including real-time PCR assays, have been developed for Leishmania detection, quantification and species identification, improving the molecular diagnosis of CL. This review provides an overview of many real-time PCR methods reported for the diagnostic evaluation of CL and some recommendations for the application of these methods for quantification purposes for clinical management and epidemiological studies. Furthermore, the use of real-time PCR for Leishmania species identification is also presented. The advantages of real-time PCR protocols are numerous, including increased sensitivity and specificity and simpler standardization of diagnostic procedures. However, despite the numerous assays described, there is still no consensus regarding the methods employed. Furthermore, the analytical and clinical validation of CL molecular diagnosis has not followed international guidelines so far. A consensus methodology comprising a DNA extraction protocol with an exogenous quality control and an internal reference to normalize parasite load is still needed. In addition, the analytical and clinical performance of any consensus methodology must be accurately assessed. This review shows that a standardization initiative is essential to guide researchers and clinical laboratories towards the achievement of a robust and reproducible methodology, which will permit further evaluation of parasite load as a surrogate marker of prognosis and monitoring of aetiological treatment, particularly in multi-centric observational studies and clinical trials. Copyright © 2017 Elsevier B.V. All rights reserved.

Complementary techniques: validation of gene expression data by quantitative real time PCR.

PubMed

Provenzano, Maurizio; Mocellin, Simone

2007-01-01

Microarray technology can be considered the most powerful tool for screening gene expression profiles of biological samples. After data mining, results need to be validated with highly reliable biotechniques allowing for precise quantitation of transcriptional abundance of identified genes. Quantitative real time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Currently, qrt-PCR is considered by most experts the most appropriate method to confirm or confute microarray-generated data. The knowledge of the biochemical principles underlying qrt-PCR as well as some related technical issues must be beard in mind when using this biotechnology.

Real-Time PCR Assay for Detection and Enumeration of Dekkera bruxellensis in Wine

PubMed Central

Phister, Trevor G.; Mills, David A.

2003-01-01

Traditional methods to detect the spoilage yeast Dekkera bruxellensis from wine involve lengthy enrichments. To overcome this difficulty, we developed a quantitative real-time PCR method to directly detect and enumerate D. bruxellensis in wine. Specific PCR primers to D. bruxellensis were designed to the 26S rRNA gene, and nontarget yeast and bacteria common to the winery environment were not amplified. The assay was linear over a range of cell concentrations (6 log units) and could detect as little as 1 cell per ml in wine. The addition of large amounts of nontarget yeasts did not impact the efficiency of the assay. This method will be helpful to identify possible routes of D. bruxellensis infection in winery environments. Moreover, the time involved in performing the assay (3 h) should enable winemakers to more quickly make wine processing decisions in order to reduce the threat of spoilage by D. bruxellensis. PMID:14660395

Protocol for the use of a rapid real-time PCR method for the detection of HIV-1 proviral DNA using double-stranded primer.

PubMed

Pau, Chou-Pong; Wells, Susan K; Granade, Timothy C

2012-01-01

This chapter describes a real-time PCR method for the detection of HIV-1 proviral DNA in whole blood samples using a novel double-stranded primer system. The assay utilizes a simple commercially available DNA extraction method and a rapid and easy-to-perform real-time PCR protocol to consistently detect a minimum of four copies of HIV-1 group M proviral DNA in as little as 90 min after sample (whole blood) collection. Co-amplification of the human RNase P gene serves as an internal control to monitor the efficiency of both the DNA extraction and amplification. Once the assay is validated properly, it may be suitable as an alternative confirmation test for HIV-1 infections in a variety of HIV testing venues including the mother-to-child transmission testing sites, clinics, and diagnostic testing centers.

Direct urine polymerase chain reaction for chlamydia and gonorrhoea: a simple means of bringing high-throughput rapid testing to remote settings?

PubMed

Rahimi, Frashta; Goire, Namraj; Guy, Rebecca; Kaldor, John M; Ward, James; Nissen, Michael D; Sloots, Theo P; Whiley, David M

2013-08-01

Background Rapid point-of-care tests (POCTs) for chlamydia (Chlamydia trachomatis) and gonorrhoea (Neisseria gonorrhoeae) have the potential to confer health benefits in certain populations even at moderate sensitivities; however, suitable POCTs for these organisms are currently lacking. In this study, we investigated the use of direct urine polymerase chain reaction (PCR), with the view of implementing a simplified PCR strategy for high-throughput chlamydia and gonorrhoea screening in remote settings. Briefly, a simple dilution of the urine was performed before adding it directly to a real-time PCR reaction. The method was evaluated using 134 stored urine specimens that had been submitted for chlamydia and gonorrhoea testing and had been tested using a commercial C. trachomatis and N. gonorrhoeae PCR method. These included samples that were PCR-positive for chlamydia (n=87), gonorrhoea (n=16) or both (n=2). Direct urine testing was conducted using previously described in-house real-time PCR methods for C. trachomatis and N. gonorrhoeae as well as for recognised N.gonorrhoeae antimicrobial resistance mechanisms. The overall sensitivities and specificities of the direct urine PCR were 78% and 100% for chlamydia, and 83% and 100% for gonorrhoea. N.gonorrhoeae penicillin and quinolone resistance mechanisms were characterised in 14 of the 18 N. gonorrhoeae-positive samples. The results of this study show that the simplified PCR strategy may be a feasible approach for rapid screening and improving chlamydia and gonorrhoea treatment in remote settings.

Overestimation of the Legionella spp. load in environmental samples by quantitative real-time PCR: pretreatment with propidium monoazide as a tool for the assessment of an association between Legionella concentration and sanitary risk.

PubMed

Ditommaso, Savina; Ricciardi, Elisa; Giacomuzzi, Monica; Arauco Rivera, Susan R; Ceccarelli, Adriano; Zotti, Carla M

2014-12-01

Quantitative polymerase chain reaction (qPCR) offers rapid, sensitive, and specific detection of Legionella in environmental water samples. In this study, qPCR and qPCR combined with propidium monoazide (PMA-qPCR) were both applied to hot-water system samples and compared to traditional culture techniques. In addition, we evaluated the ability of PMA-qPCR to monitor the efficacy of different disinfection strategies. Comparison between the quantification obtained by culture and by qPCR or PMA-qPCR on environmental water samples confirms that the concentration of Legionella estimated by GU/L is generally higher than that estimated in CFU/L. Our results on 57 hot-water-system samples collected from 3 different sites show that: i) qPCR results were on average 178-fold higher than the culture results (Δ log10=2.25), ii) PMA-qPCR results were on average 27-fold higher than the culture results (Δ log10=1.43), iii) propidium monoazide-induced signal reduction in qPCR were nearly 10-fold (Δ log10=0.95), and that iv) different degrees of correlations between the 3 methods might be explained by different matrix properties, but also by different disinfection methods affecting cultivability of Legionella. In our study, we calculated the logarithmic differences between the results obtained by PMA-qPCR and those obtained by culture, and we suggested an algorithm for the interpretation of PMA-qPCR results for the routine monitoring of healthcare water systems using a commercial qPCR system (iQ-check real-time PCR kit; Bio-Rad, Marnes-la-Coquette, France). Copyright © 2014 Elsevier Inc. All rights reserved.

Error minimization algorithm for comparative quantitative PCR analysis: Q-Anal.

PubMed

OConnor, William; Runquist, Elizabeth A

2008-07-01

Current methods for comparative quantitative polymerase chain reaction (qPCR) analysis, the threshold and extrapolation methods, either make assumptions about PCR efficiency that require an arbitrary threshold selection process or extrapolate to estimate relative levels of messenger RNA (mRNA) transcripts. Here we describe an algorithm, Q-Anal, that blends elements from current methods to by-pass assumptions regarding PCR efficiency and improve the threshold selection process to minimize error in comparative qPCR analysis. This algorithm uses iterative linear regression to identify the exponential phase for both target and reference amplicons and then selects, by minimizing linear regression error, a fluorescence threshold where efficiencies for both amplicons have been defined. From this defined fluorescence threshold, cycle time (Ct) and the error for both amplicons are calculated and used to determine the expression ratio. Ratios in complementary DNA (cDNA) dilution assays from qPCR data were analyzed by the Q-Anal method and compared with the threshold method and an extrapolation method. Dilution ratios determined by the Q-Anal and threshold methods were 86 to 118% of the expected cDNA ratios, but relative errors for the Q-Anal method were 4 to 10% in comparison with 4 to 34% for the threshold method. In contrast, ratios determined by an extrapolation method were 32 to 242% of the expected cDNA ratios, with relative errors of 67 to 193%. Q-Anal will be a valuable and quick method for minimizing error in comparative qPCR analysis.

Identification and genotyping of molluscum contagiosum virus from genital swab samples by real-time PCR and Pyrosequencing.

PubMed

Trama, Jason P; Adelson, Martin E; Mordechai, Eli

2007-12-01

Laboratory diagnosis of molluscum contagiosum virus (MCV) is important as lesions can be confused with those caused by Cryptococcus neoformans, herpes simplex virus, human papillomavirus, and varicella-zoster virus. To develop a rapid method for identifying patients infected with MCV via swab sampling. Two dual-labeled probe real-time PCR assays, one homologous to the p43K gene and one to the MC080R gene, were designed. The p43K PCR was designed to be used in conjunction with Pyrosequencing for confirmation of PCR products and discrimination between MCV1 and MCV2. Both PCR assays were optimized with respect to reaction components, thermocycling parameters, and primer and probe concentrations. The specificities of both PCR assays were confirmed by non-amplification of 38 known human pathogens. Sensitivity assays demonstrated detection of as few as 10 copies per reaction. Testing 703 swabs, concordance between the two real-time PCR assays was 99.9%. Under the developed conditions, Pyrosequencing of the p43K PCR product was capable of providing enough nucleotide sequence to definitively differentiate MCV1 and MCV2. These real-time PCR assays can be used for the rapid, sensitive, and specific detection of MCV and, when combined with Pyrosequencing, can further discriminate between MCV1 and MCV2.

A novel rapid genotyping technique for Collie eye anomaly: SYBR Green-based real-time polymerase chain reaction method applicable to blood and saliva specimens on Flinders Technology Associates filter paper.

PubMed

Chang, Hye-Sook; Mizukami, Keijiro; Yabuki, Akira; Hossain, Mohammad A; Rahman, Mohammad M; Uddin, Mohammad M; Arai, Toshiro; Yamato, Osamu

2010-09-01

Collie eye anomaly (CEA) is a canine inherited ocular disease that shows a wide variety of manifestations and severity of clinical lesions. Recently, a CEA-associated mutation was reported, and a DNA test that uses conventional polymerase chain reaction (PCR) has now become available. The objective of the current study was to develop a novel rapid genotyping technique by using SYBR Green-based real-time PCR for future large-scale surveys as a key part in the strategy to eradicate CEA by selective breeding. First, a SYBR Green-based real-time PCR assay for genotyping of CEA was developed and evaluated by using purified DNA samples from normal, carrier, and affected Border Collies in which genotypes had previously been determined by conventional PCR. This real-time PCR assay demonstrated appropriate amplifications in all genotypes, and the results were consistent with those of conventional PCR. Second, the availability of Flinders Technology Associates filter paper (FTA card) as DNA templates for the real-time PCR assay was evaluated by using blood and saliva specimens to determine suitability for CEA screening. DNA-containing solution prepared from a disc of blood- or saliva-spotted FTA cards was available directly as templates for the real-time PCR assay when the volume of solution was 2.5% of the PCR mixture. In conclusion, SYBR Green-based real-time PCR combined with FTA cards is a rapid genotyping technique for CEA that can markedly shorten the overall time required for genotyping as well as simplify the sample preparation. Therefore, this newly developed technique suits large-scale screening in breeding populations of Collie-related breeds.

Development of a panel of seven duplex real-time PCR assays for detecting 13 streptococcal superantigens.

PubMed

Yang, Peng; Peng, Xiaomin; Cui, Shujuan; Shao, Junbin; Zhu, Xuping; Zhang, Daitao; Liang, Huijie; Wang, Quanyi

2013-07-30

Streptococcal superantigens (SAgs) are the major virulence factors of infection in humans for group A Streptococcus (GAS) bacteria. A panel consisting of seven duplex real-time PCR assays was developed to simultaneously detect 13 streptococcal SAgs and one internal control which may be important in the control of GAS-mediated diseases. Primer and probe sequences were selected based on the highly conserved region from an alignment of nucleotide sequences of the 13 streptococcal SAgs. The reaction conditions of the duplex real-time PCR were optimized and the specificity of the duplex assays was evaluated using SAg positive strains. The limit of detection of the duplex assays was determined by using 10-fold serial dilutions of the DNA of 13 streptococcal SAgs and compared to a conventional polymerase chain reaction (PCR) method for evaluating the duplex assays sensitivity. Using the duplex assays, we were able to differentiate between 13 SAgs from Streptococcus strains and other non-Streptococcus bacteria without cross-reaction. On the other hand, the limit of detection of the duplex assays was at least one or two log dilutions lower than that of the conventional PCR. The panel was highly specific (100%) and the limit of detection of these duplex groups was at least ten times lower than that obtained by using a conventional PCR method.

Quantification of intestinal bacterial populations by real-time PCR with a universal primer set and minor groove binder probes: a global approach to the enteric flora.

PubMed

Ott, Stephan J; Musfeldt, Meike; Ullmann, Uwe; Hampe, Jochen; Schreiber, Stefan

2004-06-01

The composition of the human intestinal flora is important for the health status of the host. The global composition and the presence of specific pathogens are relevant to the effects of the flora. Therefore, accurate quantification of all major bacterial populations of the enteric flora is needed. A TaqMan real-time PCR-based method for the quantification of 20 dominant bacterial species and groups of the intestinal flora has been established on the basis of 16S ribosomal DNA taxonomy. A PCR with conserved primers was used for all reactions. In each real-time PCR, a universal probe for quantification of total bacteria and a specific probe for the species in question were included. PCR with conserved primers and the universal probe for total bacteria allowed relative and absolute quantification. Minor groove binder probes increased the sensitivity of the assays 10- to 100-fold. The method was evaluated by cross-reaction experiments and quantification of bacteria in complex clinical samples from healthy patients. A sensitivity of 10(1) to 10(3) bacterial cells per sample was achieved. No significant cross-reaction was observed. The real-time PCR assays presented may facilitate understanding of the intestinal bacterial flora through a normalized global estimation of the major contributing species.

[Comparative analysis of real-time quantitative PCR-Sanger sequencing method and TaqMan probe method for detection of KRAS/BRAF mutation in colorectal carcinomas].

PubMed

Zhang, Xun; Wang, Yuehua; Gao, Ning; Wang, Jinfen

2014-02-01

To compare the application values of real-time quantitative PCR-Sanger sequencing and TaqMan probe method in the detection of KRAS and BRAF mutations, and to correlate KRAS/BRAF mutations with the clinicopathological characteristics in colorectal carcinomas. Genomic DNA of the tumor cells was extracted from formalin fixed paraffin embedded (FFPE) tissue samples of 344 colorectal carcinomas by microdissection. Real-time quantitative PCR-Sanger sequencing and TaqMan probe method were performed to detect the KRAS/BRAF mutations. The frequency and types of KRAS/BRAF mutations, clinicopathological characteristics and survival time were analyzed. KRAS mutations were detected in 39.8% (137/344) and 38.7% (133/344) of 344 colorectal carcinomas by using real-time quantitative PCR-Sanger sequencing and TaqMan probe method, respectively. BRAF mutation was detected in 4.7% (16/344) and 4.1% (14/344), respectively. There was no significant correlation between the two methods. The frequency of the KRAS mutation in female was higher than that in male (P < 0.05). The frequency of the BRAF mutation in colon was higher than that in rectum. The frequency of the BRAF mutation in stage III-IV cases was higher than that in stageI-II cases. The frequency of the BRAF mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma and nonspecific adenocarcinoma had the lowest mutation rate. The frequency of the BRAF mutation in grade III cases was higher than that in grade II cases (P < 0.05). The overall concordance for the two methods of KRAS/BRAF mutation detection was 98.8% (kappa = 0.976). There was statistic significance between BRAF and KRAS mutations for the survival time of colorectal carcinomas (P = 0.039). There were no statistic significance between BRAF mutation type and BRAF/KRAS wild type (P = 0.058). (1) Compared with real-time quantitative PCR-Sanger sequencing, TaqMan probe method is better with regard to handling time, efficiency, repeatability, cost and equipment. (2) The frequency of the KRAS mutation is correlated with gender. BRAF mutation is correlated with primary tumor site, TNM stage, histological types and histological grades.(3) BRAF gene mutation is an independent prognostic marker for colorectal carcinomas.

Measuring and mitigating inhibition during real-time, quantitative PCR analysis of viral nucleic acid extracts from large-volume environmental water samples

USDA-ARS?s Scientific Manuscript database

Naturally-occurring inhibitory compounds are a major concern during qPCR and RT-qPCR analysis of environmental samples, particularly large volume water samples. Here, a standardized method for measuring and mitigating sample inhibition in environmental water concentrates is described. Specifically, ...

Improved Specificity for Detection of Mycobacterium bovis in Fresh Tissues Using IS6110 Real-time PCR

USDA-ARS?s Scientific Manuscript database

Background: Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the US. Detection of M. bovis by PCR in tissue homogenates may provide a simple, rapid method to complement diagnostic culture. A significant impediment to PCR based assays on tissue...

Proficiency program for real-time PCR diagnosis of Bordetella pertussis infections in French hospital laboratories and at the French National Reference Center for Whooping Cough and other Bordetelloses.

PubMed

Caro, Valérie; Guiso, Nicole; Alberti, Corinne; Liguori, Sandrine; Burucoa, Christophe; Couetdic, Gérard; Doucet-Populaire, Florence; Ferroni, Agnès; Papin-Gibaud, Sophie; Grattard, Florence; Réglier-Poupet, Hélène; Raymond, Josette; Soler, Catherine; Bouchet, Sylvie; Charreau, Sandrine; Couzon, Brigitte; Leymarie, Isabelle; Tavares, Nicole; Choux, Mathilde; Bingen, Edouard; Bonacorsi, Stéphane

2009-10-01

With the support of a ministerial program for innovative and expensive technologies, dedicated to the economic evaluation of laboratory diagnosis of pertussis by real-time PCR, external quality assessment for real-time IS481 PCR was carried out. Coordinated by the National Centre of Reference of Pertussis and other Bordetelloses (NCR), this study aimed to harmonize and to assess the performances of eight participating microbiology hospital laboratories throughout the French territory. Between January 2006 and February 2007, 10 proficiency panels were sent by the NCR (ascending proficiency program), representing a total of 49 samples and including eight panels to analyze and evaluate the global sensitivity and specificity of real-time PCR, one to assess the limit of detection, and one to evaluate nucleic acid extraction methods. As part of the descending proficiency program, extracted DNA from clinical samples was sent by the eight participating laboratories in different panels and analyzed by the NCR. In the ascending proficiency analysis, the sensitivity and specificity of the real-time PCR methods were 92.2% and 94.3%, respectively. The limit of detection of the different methods ranged between 0.1 and 1 fg/microl (0.2 to 2 CFU/microl). The nucleic acid extraction methods showed similar performances. During the descending proficiency analysis, performed with 126 samples, the result of the NCR for 15 samples (11.9%) was discordant with the result obtained by the source laboratory. Despite several initial differences, harmonization was easy and performances were homogeneous. However, the risk of false-positive results remains quite high, and we strongly recommend establishment of uniform quality control procedures performed regularly.

A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

PubMed

Marin, M S; Quintana, S; Leunda, M R; Recavarren, M; Pagnuco, I; Späth, E; Pérez, S; Odeón, A

2016-01-01

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle. Copyright © 2015 Elsevier B.V. All rights reserved.

Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture.

PubMed

Hiitiö, Heidi; Riva, Rauna; Autio, Tiina; Pohjanvirta, Tarja; Holopainen, Jani; Pyörälä, Satu; Pelkonen, Sinikka

2015-05-01

Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85.7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83.0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97.0 and 95.8% compared with BC. For Staphylococcus spp., the corresponding figures were 86.7 and 75.4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37.0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.

[Clinical significance of monitoring BK polyomavirus in patients after hematopoietic stem cell transplantation].

PubMed

Yin, Chang-Xin; Jiang, Qian-Li; He, Han; Yu, Guo-Pan; Xu, Yue; Meng, Fan-Yi; Yang, Mo

2012-02-01

This study was aimed to establish a method for rapid detecting BK polyomavirus (BKV) and to investigate the feasibility and value used in leukemia patients undergoing hematopoietic stem cell transplantation. Primers were designed according to BKV gene sequence; the quantitative standards for BKV and a real-time fluorescent quantitative PCR for BKV were established. The BKV level in urine samples from 36 patients after hematopoietic stem cell transplantation were detected by established method. The results showed that the standard of reconstructed plasmid and real time fluorescent quantitative PCR method were successfully established, its good specificity, sensitivity and stability were confirmed by experiments. BKV was found in 55.56% of urine samples, and the BKV load in urine was 2.46 × 10(4) - 7.8 × 10(9) copy/ml. It is concluded that the establishment of real-time fluorescent quantitative PCR for BKV detection provides a method for early diagnosis of the patients with hemorrhagic cystitis after hematopoietic stem cell transplantation.

Comparison of PCR and quantitative real-time PCR methods for the characterization of ruminant and cattle fecal pollution sources.

PubMed

Raith, Meredith R; Kelty, Catherine A; Griffith, John F; Schriewer, Alexander; Wuertz, Stefan; Mieszkin, Sophie; Gourmelon, Michele; Reischer, Georg H; Farnleitner, Andreas H; Ervin, Jared S; Holden, Patricia A; Ebentier, Darcy L; Jay, Jennifer A; Wang, Dan; Boehm, Alexandria B; Aw, Tiong Gim; Rose, Joan B; Balleste, E; Meijer, W G; Sivaganesan, Mano; Shanks, Orin C

2013-11-15

The State of California has mandated the preparation of a guidance document on the application of fecal source identification methods for recreational water quality management. California contains the fifth highest population of cattle in the United States, making the inclusion of cow-associated methods a logical choice. Because the performance of these methods has been shown to change based on geography and/or local animal feeding practices, laboratory comparisons are needed to determine which assays are best suited for implementation. We describe the performance characterization of two end-point PCR assays (CF128 and CF193) and five real-time quantitative PCR (qPCR) assays (Rum2Bac, BacR, BacCow, CowM2, and CowM3) reported to be associated with either ruminant or cattle feces. Each assay was tested against a blinded set of 38 reference challenge filters (19 duplicate samples) containing fecal pollution from 12 different sources suspected to impact water quality. The abundance of each host-associated genetic marker was measured for qPCR-based assays in both target and non-target animals and compared to quantities of total DNA mass, wet mass of fecal material, as well as Bacteroidales, and enterococci determined by 16S rRNA qPCR and culture-based approaches (enterococci only). Ruminant- and cow-associated genetic markers were detected in all filters containing a cattle fecal source. However, some assays cross-reacted with non-target pollution sources. A large amount of variability was evident across laboratories when protocols were not fixed suggesting that protocol standardization will be necessary for widespread implementation. Finally, performance metrics indicate that the cattle-associated CowM2 qPCR method combined with either the BacR or Rum2Bac ruminant-associated methods are most suitable for implementation. Published by Elsevier Ltd.

Development and clinical validation of a multiplex real-time PCR assay for herpes simplex and varicella zoster virus.

PubMed

Tan, Thean Yen; Zou, Hao; Ong, Danny Chee Tiong; Ker, Khor Jia; Chio, Martin Tze Wei; Teo, Rachael Yu Lin; Koh, Mark Jean Aan

2013-12-01

Herpes simplex virus (HSV) and varicella zoster virus (VZV) are related members of the Herpesviridae family and are well-documented human pathogens causing a spectrum of diseases, from mucocutaneous disease to infections of the central nervous system. This study was carried out to evaluate and validate the performance of a multiplex real-time polymerase chain reaction (PCR) assay in detecting and differentiating HSV1, HSV2, and VZV from clinical samples. Consensus PCR primers for HSV were designed from the UL30 component of the DNA polymerase gene of HSV, with 2 separate hydrolysis probes designed to differentiate HSV1 and HSV2. Separate primers and a probe were also designed against the DNA polymerase gene of VZV. A total of 104 clinical samples were available for testing by real-time PCR, conventional PCR, and viral culture. The sensitivity and specificity of the real-time assay was calculated by comparing the multiplex PCR result with that of a combined standard of virus culture and conventional PCR. The sensitivity of the real-time assay was 100%, with specificity ranging from 98% to 100% depending on the target gene. Both PCR methods detected more positive samples for HSV or VZV, compared with conventional virus culture. This multiplex PCR assay provides accurate and rapid diagnostic capabilities for the diagnosis and differentiation of HSV1, HSV2, and VZV infections, with the presence of an internal control to monitor for inhibition of the PCR reaction.

Utility of Real-Time PCR for Detection of Exserohilum rostratum in Body and Tissue Fluids during the Multistate Outbreak of Fungal Meningitis and Other Infections

PubMed Central

Gade, Lalitha; Grgurich, Dale E.; Kerkering, Thomas M.; Brandt, Mary E.

2014-01-01

Exserohilum rostratum was the major cause of the multistate outbreak of fungal meningitis linked to contaminated injections of methylprednisolone acetate produced by the New England Compounding Center. Previously, we developed a fungal DNA extraction procedure and broad-range and E. rostratum-specific PCR assays and confirmed the presence of fungal DNA in 28% of the case patients. Here, we report the development and validation of a TaqMan real-time PCR assay for the detection of E. rostratum in body fluids, which we used to confirm infections in 57 additional case patients, bringing the total number of case patients with PCR results positive for E. rostratum to 171 (37% of the 461 case patients with available specimens). Compared to fungal culture and the previous PCR assays, this real-time PCR assay was more sensitive. Of the 139 identical specimens from case patients tested by all three methods, 19 (14%) were positive by culture, 41 (29%) were positive by the conventional PCR assay, and 65 (47%) were positive by the real-time PCR assay. We also compared the utility of the real-time PCR assay with that of the previously described beta-d-glucan (BDG) detection assay for monitoring response to treatment in case patients with serially collected CSF. Only the incident CSF specimens from most of the case patients were positive by real-time PCR, while most of the subsequently collected specimens were negative, confirming our previous observations that the BDG assay was more appropriate than the real-time PCR assay for monitoring the response to treatment. Our results also demonstrate that the real-time PCR assay is extremely susceptible to contamination and its results should be used only in conjunction with clinical and epidemiological data. PMID:25520443

Comparison of bacteriological culture and PCR for detection of bacteria in ovine milk--sheep are not small cows.

PubMed

Zadoks, Ruth N; Tassi, Riccardo; Martin, Elena; Holopainen, Jani; McCallum, Sarah; Gibbons, James; Ballingall, Keith T

2014-10-01

Mastitis, inflammation of the mammary gland, is an important cause of disease, mortality, and production losses in dairy and meat sheep. Mastitis is commonly caused by intramammary infection with bacteria, which can be detected by bacterial culture or PCR. PathoProof (Thermo Fisher Scientific Ltd., Vantaa, Finland) is a commercially available real-time PCR system for the detection of bovine mastitis pathogens. Sheep differ from cattle in the bacterial species or bacterial strains that cause mastitis, as well as in the composition of their milk. The aim of this study was to evaluate whether the PathoProof system was suitable for detection of mastitis pathogens in sheep milk. Milk samples were collected aseptically from 219 udder halves of 113 clinically healthy ewes in a single flock. Aliquots were used for bacteriological culture and real-time PCR-based detection of bacteria. For species identified by culture, the diagnosis was confirmed by species-specific conventional PCR or by sequencing of a housekeeping gene. The majority of samples were negative by culture (74.4% of 219 samples) and real-time PCR (82.3% of 192 samples). Agreement was observed for 138 of 192 samples. Thirty-four samples were positive by culture only, mostly due to presence of species that are not covered by primers in the PCR system (e.g., Mannheimia spp.). Two samples were positive for Streptococcus uberis by culture but not by PCR directly from the milk samples. This was not due to inability of the PCR primers to amplify ovine Streptococcus uberis, as diluted DNA extracts from the same samples and DNA extracts from the bacterial isolates were positive by real-time PCR. For samples containing Staphylococcus spp., 11 samples were positive by culture and PCR, 9 by culture only, and 20 by PCR only. Samples that were negative by either method had lower bacterial load than samples that were positive for both methods, whereas no clear relation with species identity was observed. This study provides proof of principle that real-time PCR can be used for detection of mastitis pathogens in ovine milk. Routine use in sheep may require inclusion of primer sets for sheep-specific mastitis pathogens. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Immunohistochemistry and Polymerase Chain Reaction for Detection Human Papilloma Virus in Warts: A Comparative Study

PubMed Central

Lee, Hong Sun; Lee, Ji Hyun; Choo, Ji Yoon; Byun, Hee Jin; Jun, Jin Hyun

2016-01-01

Background Immunohistochemistry and polymerase chain reaction (PCR) are the most widely used methods for the detection of viruses. PCR is known to be a more sensitive and specific method than the immunohistochemical method at this time, but PCR has the disadvantages of high cost and skilled work to use widely. With the progress of technology, the immunohistochemical methods used in these days has come to be highly sensitive and actively used in the diagnostic fields. Objective To evaluate and compare the usefulness of immunohistochemistry and PCR for detection human papilloma virus (HPV) in wart lesions. Methods Nine biopsy samples of verruca vulgaris and 10 of condyloma accuminatum were examined. Immunohistochemical staining using monoclonal antibody to HPV L1 capsid protein and PCR were done for the samples. DNA sequencing of the PCR products and HPV genotyping were also done. Results HPV detection rate was 78.9% (88.9% in verruca vulgaris, 70.0% in condyloma accuminatum) on immunohistochemistry and 100.0% for PCR. HPV-6 genotype showed a lower positivity rate on immunohistochemistry (50.0%) as compared to that of the other HPV genotypes. Conclusion Immunohistochemistry for HPV L1 capsid protein showed comparable sensitivity for detection HPV. Considering the high cost and great effort needed for the PCR methods, we can use immunohistochemistry for HPV L1 capsid protein with the advantage of lower cost and simple methods for HPV detection. PMID:27489431

Interlaboratory validation of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

PubMed

Takabatake, Reona; Koiwa, Tomohiro; Kasahara, Masaki; Takashima, Kaori; Futo, Satoshi; Minegishi, Yasutaka; Akiyama, Hiroshi; Teshima, Reiko; Oguchi, Taichi; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

2011-01-01

To reduce the cost and time required to routinely perform the genetically modified organism (GMO) test, we developed a duplex quantitative real-time PCR method for a screening analysis simultaneously targeting an event-specific segment for GA21 and Cauliflower Mosaic Virus 35S promoter (P35S) segment [Oguchi et al., J. Food Hyg. Soc. Japan, 50, 117-125 (2009)]. To confirm the validity of the method, an interlaboratory collaborative study was conducted. In the collaborative study, conversion factors (Cfs), which are required to calculate the GMO amount (%), were first determined for two real-time PCR instruments, the ABI PRISM 7900HT and the ABI PRISM 7500. A blind test was then conducted. The limit of quantitation for both GA21 and P35S was estimated to be 0.5% or less. The trueness and precision were evaluated as the bias and reproducibility of the relative standard deviation (RSD(R)). The determined bias and RSD(R) were each less than 25%. We believe the developed method would be useful for the practical screening analysis of GM maize.

Review of Detection of Brucella sp. by Polymerase Chain Reaction

PubMed Central

Yu, Wei Ling; Nielsen, Klaus

2010-01-01

Here we present a review of most of the currently used polymerase chain reaction (PCR)-based methods for identification of Brucella bacteria in biological samples. We focused in particular on methods using single-pair primers, multiplex primers, real-time PCRs, PCRs for marine Brucella, and PCRs for molecular biotyping. These methods are becoming very important tools for the identification of Brucella, at the species level and recently also at the biovar level. These techniques require minimum biological containment and can provide results in a very short time. In addition, genetic fingerprinting of isolates aid in epidemiological studies of the disease and its control. PCR-based methods are more useful and practical than conventional methods used to identify Brucella spp., and new methods for Brucella spp identification and typing are still being developed. However, the sensitivity, specificity, and issues of quality control and quality assurance using these methods must be fully validated on clinical samples before PCR can be used in routine laboratory testing for brucellosis. PMID:20718083

Real-time PCR machine system modeling and a systematic approach for the robust design of a real-time PCR-on-a-chip system.

PubMed

Lee, Da-Sheng

2010-01-01

Chip-based DNA quantification systems are widespread, and used in many point-of-care applications. However, instruments for such applications may not be maintained or calibrated regularly. Since machine reliability is a key issue for normal operation, this study presents a system model of the real-time Polymerase Chain Reaction (PCR) machine to analyze the instrument design through numerical experiments. Based on model analysis, a systematic approach was developed to lower the variation of DNA quantification and achieve a robust design for a real-time PCR-on-a-chip system. Accelerated lift testing was adopted to evaluate the reliability of the chip prototype. According to the life test plan, this proposed real-time PCR-on-a-chip system was simulated to work continuously for over three years with similar reproducibility in DNA quantification. This not only shows the robustness of the lab-on-a-chip system, but also verifies the effectiveness of our systematic method for achieving a robust design.

A Droplet Digital PCR Method for Severe Combined Immunodeficiency Newborn Screening.

PubMed

Vidal-Folch, Noemi; Milosevic, Dragana; Majumdar, Ramanath; Gavrilov, Dimitar; Matern, Dietrich; Raymond, Kimiyo; Rinaldo, Piero; Tortorelli, Silvia; Abraham, Roshini S; Oglesbee, Devin

2017-09-01

Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch. TREC and RPP30 levels were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. DBSs from 610 presumed-normal, 29 lymphocyte-profiled, and 10 clinically diagnosed infants (1 X-linked SCID, 1 RAG1 Omenn syndrome, and other conditions) were tested. Control infants showed 14 to 474 TREC copies/μL blood. SCID infants, and other TCL conditions, had ≤15 TREC copies/μL. The ddPCR lower limit of quantitation was 14 TREC copies/μL, and the limit of detection was 4 TREC copies/μL. Intra-assay and interassay imprecision was <20% CV for DBSs at 54 to 60 TREC copies/μL. Testing 29 infants with known lymphocyte profiles resulted in a sensitivity of 88.9% and a specificity of 100% at TRECs <20 copies/μL. We developed a multiplex ddPCR method for the absolute quantitation of DBS TRECs that can detect SCID and other TCL conditions associated with absent or low TRECs and validated this method for NBS. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Detection of bacterial pathogens in Mongolia meningitis surveillance with a new real-time PCR assay to detect Haemophilus influenzae.

PubMed

Wang, Xin; Mair, Raydel; Hatcher, Cynthia; Theodore, M Jordan; Edmond, Karen; Wu, Henry M; Harcourt, Brian H; Carvalho, Maria da Gloria S; Pimenta, Fabiana; Nymadawa, Pagbajab; Altantsetseg, Dorjpurev; Kirsch, Mariah; Satola, Sarah W; Cohn, Amanda; Messonnier, Nancy E; Mayer, Leonard W

2011-04-01

Since the implementation of Haemophilus influenzae (Hi) serotype b vaccine, other serotypes and non-typeable strains have taken on greater importance as a cause of Hi diseases. A rapid and accurate method is needed to detect all Hi regardless of the encapsulation status. We developed 2 real-time PCR (rt-PCR) assays to detect specific regions of the protein D gene (hpd). Both hpd assays are very specific and sensitive for detection of Hi. Of the 63 non-Hi isolates representing 21 bacterial species, none was detected by the hpd #1 assay, and only one of 2 H. aphrophilus isolates was detected by the hpd #3 assay. The hpd #1 and #3 assays detected 97% (229/237) and 99% (234/237) of Hi isolates, respectively, and were superior for detection of both typeable and non-typeable Hi isolates, as compared to previously developed rt-PCR targeting ompP2 or bexA. The diagnostic sensitivity and specificity of these rt-PCR assays were assessed on cerebrospinal fluid specimens collected as part of meningitis surveillance in Ulaanbaatar, Mongolia. The etiology (Neisseria meningitidis, Hi, and Streptococcus pneumoniae) of 111 suspected meningitis cases was determined by conventional methods (culture and latex agglutination), previously developed rt-PCR assays, and the new hpd assays. The rt-PCR assays were more sensitive for detection of meningitis pathogens than other classical methods and improved detection from 50% (56/111) to 75% (83/111). The hpd #3 assay identified a non-b Hi that was missed by the bexA assay and other methods. A sensitive rt-PCR assay to detect both typeable and non-typeable Hi is a useful tool for improving Hi disease surveillance especially after Hib vaccine introduction. Published by Elsevier GmbH.

Comparison between culture and a multiplex quantitative real-time polymerase chain reaction assay detecting Ureaplasma urealyticum and U. parvum.

PubMed

Frølund, Maria; Björnelius, Eva; Lidbrink, Peter; Ahrens, Peter; Jensen, Jørgen Skov

2014-01-01

A novel multiplex quantitative real-time polymerase chain reaction (qPCR) for simultaneous detection of U. urealyticum and U. parvum was developed and compared with quantitative culture in Shepard's 10 C medium for ureaplasmas in urethral swabs from 129 men and 66 women, and cervical swabs from 61 women. Using culture as the gold standard, the sensitivity of the qPCR was 96% and 95% for female urethral and cervical swabs, respectively. In male urethral swabs the sensitivity was 89%. The corresponding specificities were 100%, 87% and 99%. The qPCR showed a linear increasing DNA copy number with increasing colour-changing units. Although slightly less sensitive than culture, this multiplex qPCR assay detecting U. urealyticum and U. parvum constitutes a simple and fast alternative to the traditional methods for identification of ureaplasmas and allows simultaneous species differentiation and quantitation in clinical samples. Furthermore, specimens overgrown by other bacteria using the culture method can be evaluated in the qPCR.

Serotype 3 is a common serotype causing invasive pneumococcal disease in children less than 5 years old, as identified by real-time PCR.

PubMed

Selva, L; Ciruela, P; Esteva, C; de Sevilla, M F; Codina, G; Hernandez, S; Moraga, F; García-García, J J; Planes, A; Coll, F; Jordan, I; Cardeñosa, N; Batalla, J; Salleras, L; Dominguez, A; Muñoz-Almagro, C

2012-07-01

Serotype 3 is one of the most often detected pneumococcal serotypes in adults and it is associated with serious disease. In contrast, the isolation of serotype 3 by bacterial culture is unusual in children with invasive pneumococcal disease (IPD). The purpose of this study was to learn the serotype distribution of IPD, including culture-negative episodes, by using molecular methods in normal sterile samples. We studied all children<5 years of age with IPD admitted to two paediatric hospitals in Catalonia, Spain, from 2007 to 2009. A sequential real-time polymerase chain reaction (PCR) approach was added to routine methods for the detection and serotyping of pneumococcal infection. Among 257 episodes (219 pneumonia, 27 meningitis, six bacteraemia and five others), 33.5% were identified by culture and the rest, 66.5%, were detected exclusively by real-time PCR. The most common serotypes detected by culture were serotypes 1 (26.7%) and 19A (25.6%), and by real-time PCR, serotypes 1 (19.8%) and 3 (18.1%). Theoretical coverage rates by the PCV7, PCV10 and PCV13 vaccines were 10.5, 52.3 and 87.2%, respectively, for those episodes identified by culture, compared to 5.3, 31.6 and 60.2% for those identified only by real-time PCR. Multiplex real-time PCR has been shown to be useful for surveillance studies of IPD. Serotype 3 is underdiagnosed by culture and is important in paediatric IPD.

Design and performance testing of a real-time PCR assay for sensitive and reliable direct quantification of Brettanomyces in wine.

PubMed

Tessonnière, H; Vidal, S; Barnavon, L; Alexandre, H; Remize, F

2009-02-28

Because the yeast Brettanomyces produces volatile phenols and acetic acid, it is responsible for wine spoilage. The uncontrolled accumulation of these molecules in wine leads to sensorial defects that compromise wine quality. The need for a rapid, specific, sensitive and reliable method to detect this spoilage yeast has increased over the last decade. All these requirements are met by real-time PCR. We here propose improvements of existing methods to enhance the robustness of the assay. Six different protocols to isolate DNA from a wine and three PCR mix compositions were tested, and the best method was selected. Insoluble PVPP addition during DNA extraction by a classical phenol:chloroform protocol succeeded in the relief of PCR inhibitors from wine. We developed an internal control which was efficient to avoid false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The method was evaluated by an intra-laboratory study for its specificity, linearity, repeatability and reproducibility. A standard curve was established from 14 different wines artificially inoculated. The quantification limit was 31 cfu/mL.

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

PubMed

Böttcher, S; Ritgen, M; Pott, C; Brüggemann, M; Raff, T; Stilgenbauer, S; Döhner, H; Dreger, P; Kneba, M

2004-10-01

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

Detection of adulterated murine components in meat products by TaqMan© real-time PCR.

PubMed

Fang, Xin; Zhang, Chi

2016-02-01

Using murine meat to substitute mutton has been identified as a new type of meat fraud in China, yet no detection method for murine species has been reported. Here, three kinds of rodent were used as target species to establish a murine-specific real-time PCR method of detection. The mitochondrial cytochrome b gene (cytb) of each target was sequenced and a TaqMan probe was designed based on the cytb. Simultaneously, an internal positive control (IPC) plasmid along with its respective probe were designed to monitor the PCR reaction. As a result, the duplex real-time PCR system was verified to be specific. The limit of detection (LOD) was lower than 1 pg of DNA per reaction and 0.1% murine contamination in meat mixtures. Standard curves were generated for a quantitative analysis. Thus, this study provided a new tool to control the quality of meat products for official and third-party laboratories. Copyright © 2015. Published by Elsevier Ltd.

Detection of Histoplasma capsulatum from clinical specimens by cycling probe-based real-time PCR and nested real-time PCR.

PubMed

Muraosa, Yasunori; Toyotome, Takahito; Yahiro, Maki; Watanabe, Akira; Shikanai-Yasuda, Maria Aparecida; Kamei, Katsuhiko

2016-05-01

We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples

EPA Science Inventory

Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protectio...

Salmonella detection from chicken rinsate with surface enhanced Raman spectroscopy and RT-PCR validation

USDA-ARS?s Scientific Manuscript database

Optical detection of bacteria has been approached in recent years as a bacteria detection method that can counter time restraints of traditional plating or the high reoccurring cost of real-time polymerase chain reaction (RT-PCR). The goal of optical detection is to identify bacteria with spectral s...

Validation of a HLA-A2 tetramer flow cytometric method, IFNgamma real time RT-PCR, and IFNgamma ELISPOT for detection of immunologic response to gp100 and MelanA/MART-1 in melanoma patients

PubMed Central

Xu, Yuanxin; Theobald, Valerie; Sung, Crystal; DePalma, Kathleen; Atwater, Laura; Seiger, Keirsten; Perricone, Michael A; Richards, Susan M

2008-01-01

Background HLA-A2 tetramer flow cytometry, IFNγ real time RT-PCR and IFNγ ELISPOT assays are commonly used as surrogate immunological endpoints for cancer immunotherapy. While these are often used as research assays to assess patient's immunologic response, assay validation is necessary to ensure reliable and reproducible results and enable more accurate data interpretation. Here we describe a rigorous validation approach for each of these assays prior to their use for clinical sample analysis. Methods Standard operating procedures for each assay were established. HLA-A2 (A*0201) tetramer assay specific for gp100209(210M) and MART-126–35(27L), IFNγ real time RT-PCR and ELISPOT methods were validated using tumor infiltrating lymphocyte cell lines (TIL) isolated from HLA-A2 melanoma patients. TIL cells, specific for gp100 (TIL 1520) or MART-1 (TIL 1143 and TIL1235), were used alone or spiked into cryopreserved HLA-A2 PBMC from healthy subjects. TIL/PBMC were stimulated with peptides (gp100209, gp100pool, MART-127–35, or influenza-M1 and negative control peptide HIV) to further assess assay performance characteristics for real time RT-PCR and ELISPOT methods. Validation parameters included specificity, accuracy, precision, linearity of dilution, limit of detection (LOD) and limit of quantification (LOQ). In addition, distribution was established in normal HLA-A2 PBMC samples. Reference ranges for assay controls were established. Results The validation process demonstrated that the HLA-A2 tetramer, IFNγ real time RT-PCR, and IFNγ ELISPOT were highly specific for each antigen, with minimal cross-reactivity between gp100 and MelanA/MART-1. The assays were sensitive; detection could be achieved at as few as 1/4545–1/6667 cells by tetramer analysis, 1/50,000 cells by real time RT-PCR, and 1/10,000–1/20,000 by ELISPOT. The assays met criteria for precision with %CV < 20% (except ELISPOT using high PBMC numbers with %CV < 25%) although flow cytometric assays and cell based functional assays are known to have high assay variability. Most importantly, assays were demonstrated to be effective for their intended use. A positive IFNγ response (by RT-PCR and ELISPOT) to gp100 was demonstrated in PBMC from 3 melanoma patients. Another patient showed a positive MART-1 response measured by all 3 validated methods. Conclusion Our results demonstrated the tetramer flow cytometry assay, IFNγ real-time RT-PCR, and INFγ ELISPOT met validation criteria. Validation approaches provide a guide for others in the field to validate these and other similar assays for assessment of patient T cell response. These methods can be applied not only to cancer vaccines but to other therapeutic proteins as part of immunogenicity and safety analyses. PMID:18945350

Rapid diagnosis of sepsis with TaqMan-Based multiplex real-time PCR.

PubMed

Liu, Chang-Feng; Shi, Xin-Ping; Chen, Yun; Jin, Ye; Zhang, Bing

2018-02-01

The survival rate of septic patients mainly depends on a rapid and reliable diagnosis. A rapid, broad range, specific and sensitive quantitative diagnostic test is the urgent need. Thus, we developed a TaqMan-Based Multiplex real-time PCR assays to identify bloodstream pathogens within a few hours. Primers and TaqMan probes were designed to be complementary to conserved regions in the 16S rDNA gene of different kinds of bacteria. To evaluate accurately, sensitively, and specifically, the known bacteria samples (Standard strains, whole blood samples) are determined by TaqMan-Based Multiplex real-time PCR. In addition, 30 blood samples taken from patients with clinical symptoms of sepsis were tested by TaqMan-Based Multiplex real-time PCR and blood culture. The mean frequency of positive for Multiplex real-time PCR was 96% at a concentration of 100 CFU/mL, and it was 100% at a concentration greater than 1000 CFU/mL. All the known blood samples and Standard strains were detected positively by TaqMan-Based Multiplex PCR, no PCR products were detected when DNAs from other bacterium were used in the multiplex assay. Among the 30 patients with clinical symptoms of sepsis, 18 patients were confirmed positive by Multiplex real-time PCR and seven patients were confirmed positive by blood culture. TaqMan-Based Multiplex real-time PCR assay with highly sensitivity, specificity and broad detection range, is a rapid and accurate method in the detection of bacterial pathogens of sepsis and should have a promising usage in the diagnosis of sepsis. © 2017 Wiley Periodicals, Inc.

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.

PubMed

Devonshire, Alison S; O'Sullivan, Denise M; Honeyborne, Isobella; Jones, Gerwyn; Karczmarczyk, Maria; Pavšič, Jernej; Gutteridge, Alice; Milavec, Mojca; Mendoza, Pablo; Schimmel, Heinz; Van Heuverswyn, Fran; Gorton, Rebecca; Cirillo, Daniela Maria; Borroni, Emanuele; Harris, Kathryn; Barnard, Marinus; Heydenrych, Anthenette; Ndusilo, Norah; Wallis, Carole L; Pillay, Keshree; Barry, Thomas; Reddington, Kate; Richter, Elvira; Mozioğlu, Erkan; Akyürek, Sema; Yalçınkaya, Burhanettin; Akgoz, Muslum; Žel, Jana; Foy, Carole A; McHugh, Timothy D; Huggett, Jim F

2016-08-03

Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.

Development and validation of duplex, triplex, and pentaplex real-time PCR screening assays for the detection of genetically modified organisms in food and feed.

PubMed

Huber, Ingrid; Block, Annette; Sebah, Daniela; Debode, Frédéric; Morisset, Dany; Grohmann, Lutz; Berben, Gilbert; Stebih, Dejan; Milavec, Mojca; Zel, Jana; Busch, Ulrich

2013-10-30

Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

Quantification of M13 and T7 bacteriophages by TaqMan and SYBR green qPCR.

PubMed

Peng, Xiujuan; Nguyen, Alex; Ghosh, Debadyuti

2018-02-01

TaqMan and SYBR Green quantitative PCR (qPCR) methods were developed as DNA-based approaches to reproducibly enumerate M13 and T7 phages from phage display selection experiments individually and simultaneously. The genome copies of M13 and T7 phages were quantified by TaqMan or SYBR Green qPCR referenced against M13 and T7 DNA standard curves of known concentrations. TaqMan qPCR was capable of quantifying M13 and T7 phage DNA simultaneously with a detection range of 2.75*10 1 -2.75*10 8 genome copies(gc)/μL and 2.66*10 1 -2.66*10 8 genome copies(gc)/μL respectively. TaqMan qPCR demonstrated an efficient amplification efficiency (E s ) of 0.97 and 0.90 for M13 and T7 phage DNA, respectively. SYBR Green qPCR was ten-fold more sensitive than TaqMan qPCR, able to quantify 2.75-2.75*10 7 gc/μL and 2.66*10 1 -2.66*10 7 gc/μL of M13 and T7 phage DNA, with an amplification efficiency E s of 1.06 and 0.78, respectively. Due to its superior sensitivity, SYBR Green qPCR was used to enumerate M13 and T7 phage display clones selected against a cell line, and quantified titers demonstrated accuracy comparable to titers from traditional double-layer plaque assay. Compared to enzyme linked immunosorbent assay, both qPCR methods exhibited increased detection sensitivity and reproducibility. These qPCR methods are reproducible, sensitive, and time-saving to determine their titers and to quantify a large number of phage samples individually or simultaneously, thus avoiding the need for time-intensive double-layer plaque assay. These findings highlight the attractiveness of qPCR for phage enumeration for applications ranging from selection to next-generation sequencing (NGS). Copyright © 2017 Elsevier B.V. All rights reserved.

An interlaboratory study on efficient detection of Shiga toxin-producing Escherichia coli O26, O103, O111, O121, O145, and O157 in food using real-time PCR assay and chromogenic agar.

PubMed

Hara-Kudo, Yukiko; Konishi, Noriko; Ohtsuka, Kayoko; Iwabuchi, Kaori; Kikuchi, Rie; Isobe, Junko; Yamazaki, Takumiko; Suzuki, Fumie; Nagai, Yuhki; Yamada, Hiroko; Tanouchi, Atsuko; Mori, Tetsuya; Nakagawa, Hiroshi; Ueda, Yasufumi; Terajima, Jun

2016-08-02

To establish an efficient detection method for Shiga toxin (Stx)-producing Escherichia coli (STEC) O26, O103, O111, O121, O145, and O157 in food, an interlaboratory study using all the serogroups of detection targets was firstly conducted. We employed a series of tests including enrichment, real-time PCR assays, and concentration by immunomagnetic separation, followed by plating onto selective agar media (IMS-plating methods). This study was particularly focused on the efficiencies of real-time PCR assays in detecting stx and O-antigen genes of the six serogroups and of IMS-plating methods onto selective agar media including chromogenic agar. Ground beef and radish sprouts samples were inoculated with the six STEC serogroups either at 4-6CFU/25g (low levels) or at 22-29CFU/25g (high levels). The sensitivity of stx detection in ground beef at both levels of inoculation with all six STEC serogroups was 100%. The sensitivity of stx detection was also 100% in radish sprouts at high levels of inoculation with all six STEC serogroups, and 66.7%-91.7% at low levels of inoculation. The sensitivity of detection of O-antigen genes was 100% in both ground beef and radish sprouts at high inoculation levels, while at low inoculation levels, it was 95.8%-100% in ground beef and 66.7%-91.7% in radish sprouts. The sensitivity of detection with IMS-plating was either the same or lower than those of the real-time PCR assays targeting stx and O-antigen genes. The relationship between the results of IMS-plating methods and Ct values of real-time PCR assays were firstly analyzed in detail. Ct values in most samples that tested negative in the IMS-plating method were higher than the maximum Ct values in samples that tested positive in the IMS-plating method. This study indicates that all six STEC serogroups in food contaminated with more than 29CFU/25g were detected by real-time PCR assays targeting stx and O-antigen genes and IMS-plating onto selective agar media. Therefore, screening of stx and O-antigen genes followed by isolation of STECs by IMS-plating methods may be an efficient method to detect the six STEC serogroups. Copyright © 2016 Elsevier B.V. All rights reserved.

Cross-linking gold nanoparticles aggregation method based on localised surface plasmon resonance for quantitative detection of miR-155.

PubMed

Esmaeili-Bandboni, Aghil; Amini, Seyed Mohammad; Faridi-Majidi, Reza; Bagheri, Jamshid; Mohammadnejad, Javad; Sadroddiny, Esmaeil

2018-06-01

MiR-155 plays a critical role in the formation of cancers and other diseases. In this study, the authors aimed to design and fabricate a biosensor based on cross-linking gold nanoparticles (AuNPs) aggregation for the detection and quantification of miR-155. Also, they intended to compare this method with SYBR Green real-time polymerase chain reaction (PCR). Primers for real-time PCR, and two thiolated capture probes for biosensor, complementary with miR-155, were designed. Citrate capped AuNPs (18.7 ± 3.6 nm) were synthesised and thiolated capture probes immobilised to AuNPs. The various concentrations of synthetic miR-155 were measured by this biosensor and real-time PCR method. Colorimetric changes were studied, and the calibration curves were plotted. Results showed the detection limit of 10 nM for the fabricated biosensor and real-time PCR. Also, eye detection using colour showed the weaker detection limit (1 µM), for this biosensor. MiR-133b as the non-complementary target could not cause a change in both colour and UV-visible spectrum. The increase in hydrodynamic diameter and negative zeta potential of AuNPs after the addition of probes verified the biosensor accurately fabricated. This fabricated biosensor could detect miR-155 simpler and faster than previous methods.

Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations

PubMed Central

Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han

2015-01-01

The incidence of foodborne diseases has increased over the years and resulted in major public health problem globally. Foodborne pathogens can be found in various foods and it is important to detect foodborne pathogens to provide safe food supply and to prevent foodborne diseases. The conventional methods used to detect foodborne pathogen are time consuming and laborious. Hence, a variety of methods have been developed for rapid detection of foodborne pathogens as it is required in many food analyses. Rapid detection methods can be categorized into nucleic acid-based, biosensor-based and immunological-based methods. This review emphasizes on the principles and application of recent rapid methods for the detection of foodborne bacterial pathogens. Detection methods included are simple polymerase chain reaction (PCR), multiplex PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP) and oligonucleotide DNA microarray which classified as nucleic acid-based methods; optical, electrochemical and mass-based biosensors which classified as biosensor-based methods; enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay which classified as immunological-based methods. In general, rapid detection methods are generally time-efficient, sensitive, specific and labor-saving. The developments of rapid detection methods are vital in prevention and treatment of foodborne diseases. PMID:25628612

Rapid and simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes by magnetic capture hybridization and multiplex real-time PCR.

PubMed

Carloni, Elisa; Rotundo, Luca; Brandi, Giorgio; Amagliani, Giulia

2018-05-25

The application of rapid, specific, and sensitive methods for pathogen detection and quantification is very advantageous in diagnosis of human pathogens in several applications, including food analysis. The aim of this study was the evaluation of a method for the multiplexed detection and quantification of three significant foodborne pathogenic species (Escherichia coli O157, Salmonella spp., and Listeria monocytogenes). The assay combines specific DNA extraction by multiplex magnetic capture hybridization (mMCH) with multiplex real-time PCR. The amplification assay showed linearity in the range 10 6 -10 genomic units (GU)/PCR for each co-amplified species. The sensitivity corresponded to 1 GU/PCR for E. coli O157 and L. monocytogenes, and 10 GU/PCR for Salmonella spp. The immobilization process and the hybrid capture of the MCH showed good efficiency and reproducibility for all targets, allowing the combination in equal amounts of the different nanoparticle types in mMCH. MCH and mMCH efficiencies were similar. The detection limit of the method was 10 CFU in samples with individual pathogens and 10 2  CFU in samples with combination of the three pathogens in unequal amounts (amount's differences of 2 or 3 log). In conclusion, this multiplex molecular platform can be applied to determine the presence of target species in food samples after culture enrichment. In this way, this method could be a time-saving and sensitive tool to be used in routine diagnosis.

Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis Sterne using Propidium Monoazide in the Presence of White Powders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hess, Becky M.; Kaiser, Brooke LD; Sydor, Michael A.

ABSTRACT Aims To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Methods and Results PMA selectively enters nonviable cells and binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for B. anthracis Sterne and Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 formore » both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Conclusions The developed assay enables simultaneous identification and viability assessment for B. anthracis Sterne and Y. pestis pgm- under laboratory conditions, even in the presence of white powders. Eliminating the DNA extraction step that is typically used reduces total assay time and labor requirements for sample analysis. Significance and Impact of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a biothreat event or the safety of food. Keywords Bacillus anthracis, Yersinia pestis, Propidium Monoazide, qPCR, White Powders, Rapid Viability Detection« less

Optimization of PMA-PCR Protocol for Viability Detection of Pathogens

NASA Technical Reports Server (NTRS)

Mikkelson, Brian J.; Lee, Christine M.; Ponce, Adrian

2011-01-01

This presented study demonstrates the need that PMA-PCR can be used to capture the loss of viability of a sample that is much more specific and time-efficient than alternative methods. This protocol is particularly useful in scenarios in which sterilization treatments may inactivate organisms but not degrade their DNA. The use of a PCR-based method of pathogen detection without first inactivating the DNA of nonviable cells will potentially lead to false positives. The loss of culturability, by heat-killing, did not prevent amplified PCR products, which supports the use of PMA to prevent amplification and differentiate between viable and dead cells. PMA was shown to inhibit the amplification of DNA by PCR in vegetative cells that had been heat-killed.

International Study to Evaluate PCR Methods for Detection of Trypanosoma cruzi DNA in Blood Samples from Chagas Disease Patients

PubMed Central

Schijman, Alejandro G.; Bisio, Margarita; Orellana, Liliana; Sued, Mariela; Duffy, Tomás; Mejia Jaramillo, Ana M.; Cura, Carolina; Auter, Frederic; Veron, Vincent; Qvarnstrom, Yvonne; Deborggraeve, Stijn; Hijar, Gisely; Zulantay, Inés; Lucero, Raúl Horacio; Velazquez, Elsa; Tellez, Tatiana; Sanchez Leon, Zunilda; Galvão, Lucia; Nolder, Debbie; Monje Rumi, María; Levi, José E.; Ramirez, Juan D.; Zorrilla, Pilar; Flores, María; Jercic, Maria I.; Crisante, Gladys; Añez, Néstor; De Castro, Ana M.; Gonzalez, Clara I.; Acosta Viana, Karla; Yachelini, Pedro; Torrico, Faustino; Robello, Carlos; Diosque, Patricio; Triana Chavez, Omar; Aznar, Christine; Russomando, Graciela; Büscher, Philippe; Assal, Azzedine; Guhl, Felipe; Sosa Estani, Sergio; DaSilva, Alexandre; Britto, Constança; Luquetti, Alejandro; Ladzins, Janis

2011-01-01

Background A century after its discovery, Chagas disease still represents a major neglected tropical threat. Accurate diagnostics tools as well as surrogate markers of parasitological response to treatment are research priorities in the field. The purpose of this study was to evaluate the performance of PCR methods in detection of Trypanosoma cruzi DNA by an external quality evaluation. Methodology/Findings An international collaborative study was launched by expert PCR laboratories from 16 countries. Currently used strategies were challenged against serial dilutions of purified DNA from stocks representing T. cruzi discrete typing units (DTU) I, IV and VI (set A), human blood spiked with parasite cells (set B) and Guanidine Hidrochloride-EDTA blood samples from 32 seropositive and 10 seronegative patients from Southern Cone countries (set C). Forty eight PCR tests were reported for set A and 44 for sets B and C; 28 targeted minicircle DNA (kDNA), 13 satellite DNA (Sat-DNA) and the remainder low copy number sequences. In set A, commercial master mixes and Sat-DNA Real Time PCR showed better specificity, but kDNA-PCR was more sensitive to detect DTU I DNA. In set B, commercial DNA extraction kits presented better specificity than solvent extraction protocols. Sat-DNA PCR tests had higher specificity, with sensitivities of 0.05–0.5 parasites/mL whereas specific kDNA tests detected 5.10−3 par/mL. Sixteen specific and coherent methods had a Good Performance in both sets A and B (10 fg/µl of DNA from all stocks, 5 par/mL spiked blood). The median values of sensitivities, specificities and accuracies obtained in testing the Set C samples with the 16 tests determined to be good performing by analyzing Sets A and B samples varied considerably. Out of them, four methods depicted the best performing parameters in all three sets of samples, detecting at least 10 fg/µl for each DNA stock, 0.5 par/mL and a sensitivity between 83.3–94.4%, specificity of 85–95%, accuracy of 86.8–89.5% and kappa index of 0.7–0.8 compared to consensus PCR reports of the 16 good performing tests and 63–69%, 100%, 71.4–76.2% and 0.4–0.5, respectively compared to serodiagnosis. Method LbD2 used solvent extraction followed by Sybr-Green based Real time PCR targeted to Sat-DNA; method LbD3 used solvent DNA extraction followed by conventional PCR targeted to Sat-DNA. The third method (LbF1) used glass fiber column based DNA extraction followed by TaqMan Real Time PCR targeted to Sat-DNA (cruzi 1/cruzi 2 and cruzi 3 TaqMan probe) and the fourth method (LbQ) used solvent DNA extraction followed by conventional hot-start PCR targeted to kDNA (primer pairs 121/122). These four methods were further evaluated at the coordinating laboratory in a subset of human blood samples, confirming the performance obtained by the participating laboratories. Conclusion/Significance This study represents a first crucial step towards international validation of PCR procedures for detection of T. cruzi in human blood samples. PMID:21264349

Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques

PubMed Central

Baldi, Paolo; La Porta, Nicola

2017-01-01

In the never ending struggle against plant pathogenic bacteria, a major goal is the early identification and classification of infecting microorganisms. Xylella fastidiosa, a Gram-negative bacterium belonging to the family Xanthmonadaceae, is no exception as this pathogen showed a broad range of vectors and host plants, many of which may carry the pathogen for a long time without showing any symptom. Till the last years, most of the diseases caused by X. fastidiosa have been reported from North and South America, but recently a widespread infection of olive quick decline syndrome caused by this fastidious pathogen appeared in Apulia (south-eastern Italy), and several cases of X. fastidiosa infection have been reported in other European Countries. At least five different subspecies of X. fastidiosa have been reported and classified: fastidiosa, multiplex, pauca, sandyi, and tashke. A sixth subspecies (morus) has been recently proposed. Therefore, it is vital to develop fast and reliable methods that allow the pathogen detection during the very early stages of infection, in order to prevent further spreading of this dangerous bacterium. To this purpose, the classical immunological methods such as ELISA and immunofluorescence are not always sensitive enough. However, PCR-based methods exploiting specific primers for the amplification of target regions of genomic DNA have been developed and are becoming a powerful tool for the detection and identification of many species of bacteria. The aim of this review is to illustrate the application of the most commonly used PCR approaches to X. fastidiosa study, ranging from classical PCR, to several PCR-based detection methods: random amplified polymorphic DNA (RAPD), quantitative real-time PCR (qRT-PCR), nested-PCR (N-PCR), immunocapture PCR (IC-PCR), short sequence repeats (SSRs, also called VNTR), single nucleotide polymorphisms (SNPs) and multilocus sequence typing (MLST). Amplification and sequence analysis of specific targets is also mentioned. The fast progresses achieved during the last years in the DNA-based classification of this pathogen are described and discussed and specific primers designed for the different methods are listed, in order to provide a concise and useful tool to all the researchers working in the field. PMID:28642764

Development of a One-Step Duplex RT-PCR Method for the Simultaneous Detection of VP3/VP1 and VP1/P2B Regions of the Hepatitis A Virus.

PubMed

Kim, Mi-Ju; Lee, Shin-Young; Kim, Hyun-Joong; Lee, Jeong Su; Joo, In Sun; Kwak, Hyo Sun; Kim, Hae-Yeong

2016-08-28

The simultaneous detection and accurate identification of hepatitis A virus (HAV) is critical in food safety and epidemiological studies to prevent the spread of HAV outbreaks. Towards this goal, a one-step duplex reverse-transcription (RT)-PCR method was developed targeting the VP1/P2B and VP3/VP1 regions of the HAV genome for the qualitative detection of HAV. An HAV RT-qPCR standard curve was produced for the quantification of HAV RNA. The detection limit of the duplex RT-PCR method was 2.8 × 10(1) copies of HAV. The PCR products enabled HAV genotyping analysis through DNA sequencing, which can be applied for epidemiological investigations. The ability of this duplex RT-PCR method to detect HAV was evaluated with HAV-spiked samples of fresh lettuce, frozen strawberries, and oysters. The limit of detection of the one-step duplex RT-PCR for each food model was 9.4 × 10(2) copies/20 g fresh lettuce, 9.7 × 10(3) copies/20 g frozen strawberries, and 4.1 × 10(3) copies/1.5 g oysters. Use of a one-step duplex RT-PCR method has advantages such as shorter time, decreased cost, and decreased labor owing to the single amplification reaction instead of four amplifications necessary for nested RT-PCR.

Quantitative analysis of diet structure by real-time PCR, reveals different feeding patterns by two dominant grasshopper species

PubMed Central

Huang, Xunbing; Wu, Huihui; McNeill, Mark Richard; Qin, Xinghu; Ma, Jingchuan; Tu, Xiongbing; Cao, Guangchun; Wang, Guangjun; Nong, Xiangqun; Zhang, Zehua

2016-01-01

Studies on grasshopper diets have historically employed a range of methodologies, each with certain advantages and disadvantages. For example, some methodologies are qualitative instead of quantitative. Others require long experimental periods or examine population-level effects, only. In this study, we used real-time PCR to examine diets of individual grasshoppers. The method has the advantage of being both fast and quantitative. Using two grasshopper species, Oedaleus asiaticus and Dasyhippus barbipes, we designed ITS primer sequences for their three main host plants, Stipa krylovii, Leymus chinensis and Cleistogenes squarrosa and used real-time PCR method to test diet structure both qualitatively and quantitatively. The lowest detection efficiency of the three grass species was ~80% with a strong correlation between actual and PCR-measured food intake. We found that Oedaleus asiaticus maintained an unchanged diet structure across grasslands with different grass communities. By comparison, Dasyhippus barbipes changed its diet structure. These results revealed why O. asiaticus distribution is mainly confined to Stipa-dominated grassland, and D. barbipes is more widely distributed across Inner Mongolia. Overall, real-time PCR was shown to be a useful tool for investigating grasshopper diets, which in turn offers some insight into grasshopper distributions and improved pest management. PMID:27562455

Quantifying spontaneous metastasis in a syngeneic mouse melanoma model using real time PCR.

PubMed

Deng, Wentao; McLaughlin, Sarah L; Klinke, David J

2017-08-07

Modeling metastasis in vivo with animals is a priority for both revealing mechanisms of tumor dissemination and developing therapeutic methods. While conventional intravenous injection of tumor cells provides an efficient and consistent system for studying tumor cell extravasation and colonization, studying spontaneous metastasis derived from orthotopic tumor sites has the advantage of modeling more aspects of the metastatic cascade, but is challenging as it is difficult to detect small numbers of metastatic cells. In this work, we developed an approach for quantifying spontaneous metastasis in the syngeneic mouse B16 system using real time PCR. We first transduced B16 cells with lentivirus expressing firefly luciferase Luc2 gene for bioluminescence imaging. Next, we developed a real time quantitative PCR (qPCR) method for the detection of luciferase-expressing, metastatic tumor cells in mouse lungs and other organs. To illustrate the approach, we quantified lung metastasis in both spontaneous and experimental scenarios using B16F0 and B16F10 cells in C57BL/6Ncrl and NOD-Scid Gamma (NSG) mice. We tracked B16 melanoma metastasis with both bioluminescence imaging and qPCR, which were found to be self-consistent. Using this assay, we can quantitatively detect one Luc2 positive tumor cell out of 10 4 tissue cells, which corresponds to a metastatic burden of 1.8 × 10 4 metastatic cells per whole mouse lung. More importantly, the qPCR method was at least a factor of 10 more sensitive in detecting metastatic cell dissemination and should be combined with bioluminescence imaging as a high-resolution, end-point method for final metastatic cell quantitation. Given the rapid growth of primary tumors in many mouse models, assays with improved sensitivity can provide better insight into biological mechanisms that underpin tumor metastasis.

Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.

PubMed

Jia, Xianbo; Lin, Xinjian; Chen, Jichen

2017-11-02

Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.

Efficiency of peracetic acid in inactivating bacteria, viruses, and spores in water determined with ATP bioluminescence, quantitative PCR, and culture-based methods.

PubMed

Park, Eunyoung; Lee, Cheonghoon; Bisesi, Michael; Lee, Jiyoung

2014-03-01

The disinfection efficiency of peracetic acid (PAA) was investigated on three microbial types using three different methods (filtration-based ATP (adenosine-triphosphate) bioluminescence, quantitative polymerase chain reaction (qPCR), culture-based method). Fecal indicator bacteria (Enterococcus faecium), virus indicator (male-specific (F(+)) coliphages (coliphages)), and protozoa disinfection surrogate (Bacillus subtilis spores (spores)) were tested. The mode of action for spore disinfection was visualized using scanning electron microscopy. The results indicated that PAA concentrations of 5 ppm (contact time: 5 min), 50 ppm (10 min), and 3,000 ppm (5 min) were needed to achieve 3-log reduction of E. faecium, coliphages, and spores, respectively. Scanning electron microscopy observation showed that PAA targets the external layers of spores. The lower reduction rates of tested microbes measured with qPCR suggest that qPCR may overestimate the surviving microbes. Collectively, PAA showed broad disinfection efficiency (susceptibility: E. faecium > coliphages > spores). For E. faecium and spores, ATP bioluminescence was substantially faster (∼5 min) than culture-based method (>24 h) and qPCR (2-3 h). This study suggests PAA as an effective alternative to inactivate broad types of microbial contaminants in water. Together with the use of rapid detection methods, this approach can be useful for urgent situations when timely response is needed for ensuring water quality.

Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey.

PubMed

Döskaya, Mert; Caner, Ayse; Degirmenci, Aysu; Wengenack, Nancy L; Yolasigmaz, Aysegül; Turgay, Nevin; Ozensoy Töz, Seray; Gürüz, Yüksel

2011-07-01

Routine laboratory diagnosis of Pneumocystis jirovecii is currently achieved by PCR in almost all laboratories with sufficient equipment due to its high sensitivity and specificity compared to staining methods. A current issue that limits the reliability and sensitivity of PCR is the degree of inhibition caused by inhibitory substances in respiratory samples. The present study aimed to analyse the degree and frequency of inhibition in real-time PCR detecting P. jirovecii in respiratory specimens submitted to a Pneumocystis pneumonia (PcP) diagnosis laboratory in Ege University Medical School, Turkey. Between July 2009 and December 2010, 76 respiratory specimens [63 bronchoalveolar lavage (BAL) fluid, 10 sputum samples, two tracheal aspiration fluid and one thoracentesis fluid] obtained from 69 PcP-suspected patients were investigated for the presence of P. jirovecii using real-time PCR targeting the cdc2 gene. Of these samples, 42 of the specimens were stained and examined by microscopy according to the request of the clinicians. PCR was positive in 15 specimens in the initial run. Of the remaining 61 samples, 41 of them were negative with positive internal inhibition controls (i.e. true-negative group). The frequency of inhibition in the initial run was 26.31 % (20/76) as determined by spiked negative controls. All of the inhibited samples were resolved after 1 : 2, 1 : 5, 1 : 10 and 1 : 20 dilutions. P. jirovecii was detected by PCR in two inhibited specimens after retesting with diluted samples which were also positive by microscopy. The incidence of P. jirovecii in respiratory specimens was 22.36 % (17/76) as determined by real-time PCR and 7.14 % (3/42) by microscopy. Overall, the incidence of P. jirovecii in respiratory samples was 23.68 % (18/76) as detected by both methods. In conclusion, inclusion of spiked positive controls in each sample and retesting with diluted samples to resolve inhibition increased the reliability of the real-time PCR assay in terms of determining false-negative results and influencing the treatment of the patient. Furthermore, results of the present study determined for the first time the frequency and degree of inhibition in a real-time PCR detecting P. jirovecii in respiratory specimens during routine diagnosis of PcP.

Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems.

PubMed

Touron-Bodilis, A; Pougnard, C; Frenkiel-Lebossé, H; Hallier-Soulier, S

2011-08-01

This study was designed to evaluate the usefulness of quantification by real-time PCR as a management tool to monitor concentrations of Legionella spp. and Legionella pneumophila in industrial cooling systems and its ability to anticipate culture trends by the French standard method (AFNOR T90-431). Quantifications of Legionella bacteria were achieved by both methods on samples from nine cooling systems with different water qualities. Proportion of positive samples for L. pneumophila quantified by PCR was clearly lower in deionized or river waters submitted to a biocide treatment than in raw river waters, while positive samples for Legionella spp. were quantified for almost all the samples. For some samples containing PCR inhibitors, high quantification limits (up to 4·80 × 10(5) GU l(-1) ) did not allow us to quantify L. pneumophila, when they were quantified by culture. Finally, the monitoring of concentrations of L. pneumophila by both methods showed similar trends for 57-100% of the samples. These results suggest that, if some methodological steps designed to reduce inhibitory problems and thus decrease the quantification limits, could be developed to quantify Legionella in complex waters, the real-time PCR could be a valuable complementary tool to monitor the evolution of L. pneumophila concentrations. This study shows the possibility of using real-time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology. No claim to French Government works.

Buccal DNA collection: comparison of buccal swabs with FTA cards.

PubMed

Milne, Elizabeth; van Bockxmeer, Frank M; Robertson, Laila; Brisbane, Joanna M; Ashton, Lesley J; Scott, Rodney J; Armstrong, Bruce K

2006-04-01

Collection and analysis of DNA, most commonly from blood or buccal cells, is becoming more common in epidemiologic studies. Buccal samples, which are painless to take and relatively easily collected, are often the preferred source. There are several buccal cell collection methods: swabs, brushes, mouthwash, and treated cards, such as FTA or IsoCode cards. Few studies have systematically compared methods of buccal cell collection with respect to DNA yield and amplification success under similar conditions. We compared buccal DNA collection and amplification using buccal swabs and FTA cards in 122 control subjects from our Australian case-control study of childhood acute lymphoblastic leukaemia. Buccal DNA was quantified using a real-time PCR for beta-actin and genotyped at the loci of three polymorphisms (MTHFR 677C>T, ACE I/D, and XPD 1012G>A). PCR was successful with DNA from buccal swabs for 62% to 89% of subjects and from FTA cards for 83% to 100% of subjects, depending on the locus. The matched pair odds ratios (95% confidence interval) comparing success of FTA cards with buccal swabs are as follows: MTHFR 677C>T using PCR-RFLP, 12.5 (11.6-13.5) and using real-time PCR, 130.0 (113.1-152.8); ACE I/D using PCR-amplified fragment length polymorphism, 3.36 (3.2-3.5); XPD 1012G>A using real-time PCR, 150.0 (132.7-172.3). FTA cards are a robust DNA collection method and generally produce DNA suitable for PCR more reliably than buccal swabs. There are, however, technical challenges in handling discs punched from FTA cards that intending users should be aware of.

Simultaneous detection of Fusarium culmorum and F. graminearum in plant material by duplex PCR with melting curve analysis.

PubMed

Brandfass, Christoph; Karlovsky, Petr

2006-01-23

Fusarium head blight (FHB) is a disease of cereal crops, which has a severe impact on wheat and barley production worldwide. Apart from reducing the yield and impairing grain quality, FHB leads to contamination of grain with toxic secondary metabolites (mycotoxins), which pose a health risk to humans and livestock. The Fusarium species primarily involved in FHB are F. graminearum and F. culmorum. A key prerequisite for a reduction in the incidence of FHB is an understanding of its epidemiology. We describe a duplex-PCR-based method for the simultaneous detection of F. culmorum and F. graminearum in plant material. Species-specific PCR products are identified by melting curve analysis performed in a real-time thermocycler in the presence of the fluorescent dye SYBR Green I. In contrast to multiplex real-time PCR assays, the method does not use doubly labeled hybridization probes. PCR with product differentiation by melting curve analysis offers a cost-effective means of qualitative analysis for the presence of F. culmorum and F. graminearum in plant material. This method is particularly suitable for epidemiological studies involving a large number of samples.

Development of real-time PCR tests for the detection of Tenebrio molitor in food and feed.

PubMed

Debode, Frédéric; Marien, Aline; Gérard, Amaury; Francis, Frédéric; Fumière, Olivier; Berben, Gilbert

2017-08-01

Insects are rich in proteins and could be an alternative source of proteins to feed animals and humans. Numerous companies have started the production of insects for feed purposes. In Europe, these processed animal proteins are not yet authorised by legislation as many questions still need to be answered concerning this 'novel food'. Authorisations will be possible when methods of authentication of the products are available. In this study we propose real-time PCR methods for the specific detection of the mealworm (Tenebriomolitor), one of the most widely used insects for food and feed production. Two PCR assays are proposed: the first based on the wingless gene and the second based on the cadherin gene. The PCR tests amplify fragments of 87 bp. These qualitative methods were tested according to several performance criteria. The specificity was tested on 34 insect species' DNA, but also on non-insect species including crustacean, mammals, birds and plants. The limit of detection was determined and was below 20 copies for the two PCR tests. The applicability of the tests was demonstrated by the analysis of real-life processed samples containing T. molitor.

A multiplex real-time PCR assay, based on invA and pagC genes, for the detection and quantification of Salmonella enterica from cattle lymph nodes.

PubMed

Bai, Jianfa; Trinetta, Valentina; Shi, Xiaorong; Noll, Lance W; Magossi, Gabriela; Zheng, Wanglong; Porter, Elizabeth P; Cernicchiaro, Natalia; Renter, David G; Nagaraja, Tiruvoor G

2018-05-01

Cattle lymph nodes can harbor Salmonella and potentially contaminate beef products. We have developed and validated a new real-time PCR (qPCR) assay for the detection and quantification of Salmonella enterica in cattle lymph nodes. The assay targets both the invA and pagC genes, the most conserved molecular targets in Salmonella enterica. An 18S rRNA gene assay that amplifies from cattle and other animal species was also included as an internal control. Available DNA sequences for invA, pagC and 18S rRNA genes were used for primer and probe selections. Three Salmonella serotypes, S. Typhimurium, S. Anatum, and S. Montevideo, were used to assess the assay's analytical sensitivity. Correlation coefficients of standard curves generated for each target and for all three serotypes were >99% and qPCR amplification efficiencies were between 93% and 110%. Assay sensitivity was also determined using standard curve data generated from Salmonella-negative cattle lymph nodes spiked with 10-fold dilutions of the three Salmonella serotypes. Assay specificity was determined using Salmonella culture method, and qPCR testing on 36 Salmonella strains representing 33 serotypes, 38 Salmonella strains of unknown serotypes, 252 E. coli strains representing 40 serogroups, and 31 other bacterial strains representing 18 different species. A collection of 647 cattle lymph node samples from steers procured from the Midwest region of the US were tested by the qPCR, and compared to culture-method of detection. Salmonella prevalence by qPCR for pre-enriched and enriched lymph nodes was 19.8% (128/647) and 94.9% (614/647), respectively. A majority of qPCR positive pre-enriched samples (105/128) were at concentrations between 10 4 and 10 5  CFU/mL. Culture method detected Salmonella in 7.7% (50/647) and 80.7% (522/647) of pre- and post-enriched samples, respectively; 96.0% (48/50) of pre-enriched and 99.4% (519/522) of post-enriched culture-positive samples were also positive by qPCR. More samples tested positive by qPCR than by culture method, indicating that the real-time PCR assay was more sensitive. Our data indicate that this triplex qPCR can be used to accurately detect and quantify Salmonella enterica strains from cattle lymph node samples. The assay may serve as a useful tool to monitor the prevalence of Salmonella in beef production systems. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of an in-house fast real-time PCR method for detection of fish allergen in foods and comparison with a commercial kit.

PubMed

Herrero, Beatriz; Vieites, Juan M; Espiñeira, Montserrat

2014-05-15

Food allergy is recognised as an important human health problem. Fish represent one of the most important causes of food hypersensitivity reaction. Small amounts of the allergen can cause severe reactions in sensitive individuals, so correct labelling is essential to ensure the protection of consumers. The objective of the present work was to develop a reliable, sensitive and specific real-time PCR method for the detection of fish and traces of fish in all kind of products included those that have undergone aggressive treatments such as high temperature or pressure. This methodology was validated simulating products likely to contain this allergen and spiking them with fish cooking water. In addition, a comparison between the performance of in-house methodology and a commercial kit, both of them based on real-time PCR, was carried out. This work is relevant because it is the first, rapid real-time PCR method developed to date for the detection of fish in processed food products. The results obtained confirm the present assay is a useful tool in detecting fish and, therefore, minimising exposure and reducing incidences of allergic reaction to fish in contaminated products. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

PubMed

Noguchi, Akio; Nakamura, Kosuke; Sakata, Kozue; Sato-Fukuda, Nozomi; Ishigaki, Takumi; Mano, Junichi; Takabatake, Reona; Kitta, Kazumi; Teshima, Reiko; Kondo, Kazunari; Nishimaki-Mogami, Tomoko

2016-04-19

A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

Molecular identification and real-time quantitative PCR (qPCR) for rapid detection of Thelohanellus kitauei, a Myxozoan parasite causing intestinal giant cystic disease in the Israel carp.

PubMed

Seo, Jung Soo; Jeon, Eun Ji; Kim, Moo Sang; Woo, Sung Ho; Kim, Jin Do; Jung, Sung Hee; Park, Myoung Ae; Jee, Bo Young; Kim, Jin Woo; Kim, Yi-Cheong; Lee, Eun Hye

2012-06-01

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

The Next-Generation PCR-Based Quantification Method for Ambient Waters: Digital PCR.

PubMed

Cao, Yiping; Griffith, John F; Weisberg, Stephen B

2016-01-01

Real-time quantitative PCR (qPCR) is increasingly being used for ambient water monitoring, but development of digital polymerase chain reaction (digital PCR) has the potential to further advance the use of molecular techniques in such applications. Digital PCR refines qPCR by partitioning the sample into thousands to millions of miniature reactions that are examined individually for binary endpoint results, with DNA density calculated from the fraction of positives using Poisson statistics. This direct quantification removes the need for standard curves, eliminating the labor and materials associated with creating and running standards with each batch, and removing biases associated with standard variability and mismatching amplification efficiency between standards and samples. Confining reactions and binary endpoint measurements to small partitions also leads to other performance advantages, including reduced susceptibility to inhibition, increased repeatability and reproducibility, and increased capacity to measure multiple targets in one analysis. As such, digital PCR is well suited for ambient water monitoring applications and is particularly advantageous as molecular methods move toward autonomous field application.

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

PubMed Central

Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

2006-01-01

Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. Conclusion The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR. PMID:16907967

Optimized Pan-species and Speciation Duplex Real-time PCR Assays for Plasmodium Parasites Detection in Malaria Vectors

PubMed Central

Sandeu, Maurice Marcel; Moussiliou, Azizath; Moiroux, Nicolas; Padonou, Gilles G.; Massougbodji, Achille; Corbel, Vincent; Tuikue Ndam, Nicaise

2012-01-01

Background An accurate method for detecting malaria parasites in the mosquito’s vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus. Methods Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin. Results The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was “excellent” (κ = 0.8, P<0.05). The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P = 0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed. Conclusion This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the utility of employing an accurate molecular diagnostic tool for detecting malaria parasites in field mosquito populations. PMID:23285168

Detection of Giardia intestinalis in water samples collected from natural water reservoirs and wells in northern and north-eastern Poland using LAMP, real-time PCR and nested PCR.

PubMed

Lass, Anna; Szostakowska, Beata; Korzeniewski, Krzysztof; Karanis, Panagiotis

2017-10-01

Giardia intestinalis is a protozoan parasite, transmitted to humans and animals by the faecal-oral route, mainly through contaminated water and food. Knowledge about the distribution of this parasite in surface water in Poland is fragmentary and incomplete. Accordingly, 36 environmental water samples taken from surface water reservoirs and wells were collected in Pomerania and Warmia-Masuria provinces, Poland. The 50 L samples were filtered and subsequently analysed with three molecular detection methods: loop-mediated isothermal amplification (LAMP), real-time polymerase chain reaction (real-time PCR) and nested PCR. Of the samples examined, Giardia DNA was found in 15 (42%) samples with the use of LAMP; in 12 (33%) of these samples, Giardia DNA from this parasite was also detected using real-time PCR; and in 9 (25%) using nested PCR. Sequencing of selected positive samples confirmed that the PCR products were fragments of the Giardia intestinalis small subunit rRNA gene. Genotyping using multiplex real-time PCR indicated the presence of assemblages A and B, with the latter predominating. The results indicate that surface water in Poland, as well as water taken from surface wells, may be a source of Giardia strains which are potentially pathogenic for humans. It was also demonstrated that LAMP assay is more sensitive than the other two molecular assays.

A method of multiplex PCR for detection of field released Beauveria bassiana, a fungal entomopathogen applied for pest management in jute (Corchorus olitorius).

PubMed

Biswas, Chinmay; Dey, Piyali; Gotyal, B S; Satpathy, Subrata

2015-04-01

The fungal entomopathogen Beauveria bassiana is a promising biocontrol agent for many pests. Some B. bassiana strains have been found effective against jute pests. To monitor the survival of field released B. bassiana a rapid and efficient detection technique is essential. Conventional methods such as plating method or direct culture method which are based on cultivation on selective media followed by microscopy are time consuming and not so sensitive. PCR based methods are rapid, sensitive and reliable. A single primer PCR may fail to amplify some of the strains. However, multiplex PCR increases the possibility of detection as it uses multiple primers. Therefore, in the present investigation a multiplex PCR protocol was developed by multiplexing three primers SCA 14, SCA 15 and SCB 9 to detect field released B. bassiana strains from soil as well as foliage of jute field. Using our multiplex PCR protocol all the five B. bassiana strains could be detected from soil and three strains viz., ITCC 6063, ITCC 4563 and ITCC 4796 could be detected even from the crop foliage after 45 days of spray.

Detection of respiratory viruses and bacteria in children using a twenty-two target reverse-transcription real-time PCR (RT-qPCR) panel.

PubMed

Ellis, Chelsey; Misir, Amita; Hui, Charles; Jabbour, Mona; Barrowman, Nicholas; Langill, Jonathan; Bowes, Jennifer; Slinger, Robert

2016-05-01

Rapid detection of the wide range of viruses and bacteria that cause respiratory infection in children is important for patient care and antibiotic stewardship. We therefore designed and evaluated a ready-to-use 22 target respiratory infection reverse-transcription real-time polymerase chain reaction (RT-qPCR) panel to determine if this would improve detection of these agents at our pediatric hospital. RT-qPCR assays for twenty-two target organisms were dried-down in individual wells of 96 well plates and saved at room temperature. Targets included 18 respiratory viruses and 4 bacteria. After automated nucleic acid extraction of nasopharyngeal aspirate (NPA) samples, rapid qPCR was performed. RT-qPCR results were compared with those obtained by the testing methods used at our hospital laboratories. One hundred fifty-nine pediatric NPA samples were tested with the RT-qPCR panel. One or more respiratory pathogens were detected in 132/159 (83%) samples. This was significantly higher than the detection rate of standard methods (94/159, 59%) (P<0.001). This difference was mainly due to improved RT-qPCR detection of rhinoviruses, parainfluenza viruses, bocavirus, and coronaviruses. The panel internal control assay performance remained stable at room temperature storage over a two-month testing period. The RT-qPCR panel was able to identify pathogens in a high proportion of respiratory samples. The panel detected more positive specimens than the methods in use at our hospital. The pre-made panel format was easy to use and rapid, with results available in approximately 90 minutes. We now plan to determine if use of this panel improves patient care and antibiotic stewardship.

Development of a high-speed real-time PCR system for rapid and precise nucleotide recognition

NASA Astrophysics Data System (ADS)

Terazono, Hideyuki; Takei, Hiroyuki; Hattori, Akihiro; Yasuda, Kenji

2010-04-01

Polymerase chain reaction (PCR) is a common method used to create copies of a specific target region of a DNA sequence and to produce large quantities of DNA. A few DNA molecules, which act as templates, are rapidly amplified by PCR into many billions of copies. PCR is a key technology in genome-based biological analysis, revolutionizing many life science fields such as medical diagnostics, food safety monitoring, and countermeasures against bioterrorism. Thus, many applications have been developed with the thermal cycling. For these PCR applications, one of the most important key factors is reduction in the data acquisition time. To reduce the acquisition time, it is necessary to decrease the temperature transition time between the high and low ends as much as possible. We have developed a novel rapid real-time PCR system based on rapid exchange of media maintained at different temperatures. This system consists of two thermal reservoirs and a reaction chamber for PCR observation. The temperature transition was achieved within 0.3 sec, and good thermal stability was achieved during thermal cycling with rapid exchange of circulating media. This system allows rigorous optimization of the temperatures required for each stage of the PCR processes. Resulting amplicons were confirmed by electrophoresis. Using the system, rapid DNA amplification was accomplished within 3.5 min, including initial heating and complete 50 PCR cycles. It clearly shows that the device could allow us faster temperature switching than the conventional conduction-based heating systems based on Peltier heating/cooling.

Comparison of microscopy, ELISA, and real-time PCR for detection of Giardia intestinalis in human stool specimens

PubMed

Beyhan, Yunus Emre; Taş Cengiz, Zeynep

2017-08-23

Background/aim: This study included patients who had digestive system complaints between August 2015 and October 2015. The research was designed to compare conventional microscopy with an antigen detection ELISA kit and the TaqMan-based real-time PCR (RT-PCR) technique for detection of Giardia intestinalis in human stool specimens. Materials and methods: Samples were concentrated by formalin-ether sedimentation technique and microscopic examinations were carried out on wet mount slides. A commercially available ELISA kit (Giardia CELISA, Cellabs, Brookvale, Australia) was used for immunoassay. DNA was extracted from fecal samples of about 200 mg using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) and the LightCycler Nano system (Roche Diagnostics, Mannheim, Germany) was used for the TaqMan-based RT-PCR assay. Results: A total of 94 stool samples, 38 of them diagnosed positive (40.4%) and 56 of them diagnosed negative by microscopy, were selected for evaluation by antigen detection and molecular assays. The prevalence of G. intestinalis infection was found as 46.8% (n: 44) and 79.8% (n: 75) by ELISA and RT-PCR, respectively. RT-PCR revealed by far the highest positivity rate compared to the other two methods. The difference between these methods was found to be statistically significant (P < 0.05). In comparison to PCR, the sensitivity and specificity of microscopy and ELISA were 50.7% and 100% and 53.3% and 79%, respectively. Conclusion: RT-PCR seems to be much more sensitive and beneficial for rapid and accurate diagnosis of G. intestinalis in human stools.

An Alu-based, MGB Eclipse real-time PCR method for quantitation of human DNA in forensic samples.

PubMed

Nicklas, Janice A; Buel, Eric

2005-09-01

The forensic community needs quick, reliable methods to quantitate human DNA in crime scene samples to replace the laborious and imprecise slot blot method. A real-time PCR based method has the possibility of allowing development of a faster and more quantitative assay. Alu sequences are primate-specific and are found in many copies in the human genome, making these sequences an excellent target or marker for human DNA. This paper describes the development of a real-time Alu sequence-based assay using MGB Eclipse primers and probes. The advantages of this assay are simplicity, speed, less hands-on-time and automated quantitation, as well as a large dynamic range (128 ng/microL to 0.5 pg/microL).

A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi.

PubMed

Komaki-Yasuda, Kanako; Vincent, Jeanne Perpétue; Nakatsu, Masami; Kato, Yasuyuki; Ohmagari, Norio; Kano, Shigeyuki

2018-01-01

A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient's blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a "fast PCR enzyme". In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the "fast PCR enzyme", with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses.

A novel PCR-based system for the detection of four species of human malaria parasites and Plasmodium knowlesi

PubMed Central

Komaki-Yasuda, Kanako; Vincent, Jeanne Perpétue; Nakatsu, Masami; Kato, Yasuyuki; Ohmagari, Norio

2018-01-01

A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient’s blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a “fast PCR enzyme”. In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the “fast PCR enzyme”, with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses. PMID:29370297

Real-Time Polymerase Chain Reaction Detection of Angiostrongylus cantonensis DNA in Cerebrospinal Fluid from Patients with Eosinophilic Meningitis.

PubMed

Qvarnstrom, Yvonne; Xayavong, Maniphet; da Silva, Ana Cristina Aramburu; Park, Sarah Y; Whelen, A Christian; Calimlim, Precilia S; Sciulli, Rebecca H; Honda, Stacey A A; Higa, Karen; Kitsutani, Paul; Chea, Nora; Heng, Seng; Johnson, Stuart; Graeff-Teixeira, Carlos; Fox, LeAnne M; da Silva, Alexandre J

2016-01-01

Angiostrongylus cantonensis is the most common infectious cause of eosinophilic meningitis. Timely diagnosis of these infections is difficult, partly because reliable laboratory diagnostic methods are unavailable. The aim of this study was to evaluate the usefulness of a real-time polymerase chain reaction (PCR) assay for the detection of A. cantonensis DNA in human cerebrospinal fluid (CSF) specimens. A total of 49 CSF specimens from 33 patients with eosinophilic meningitis were included: A. cantonensis DNA was detected in 32 CSF specimens, from 22 patients. Four patients had intermittently positive and negative real-time PCR results on subsequent samples, indicating that the level of A. cantonensis DNA present in CSF may fluctuate during the course of the illness. Immunodiagnosis and/or supplemental PCR testing supported the real-time PCR findings for 30 patients. On the basis of these observations, this real-time PCR assay can be useful to detect A. cantonensis in the CSF from patients with eosinophilic meningitis. © The American Society of Tropical Medicine and Hygiene.

Identification of Pseudallescheria and Scedosporium Species by Three Molecular Methods▿

PubMed Central

Lu, Qiaoyun; Gerrits van den Ende, A. H. G.; Bakkers, J. M. J. E.; Sun, Jiufeng; Lackner, M.; Najafzadeh, M. J.; Melchers, W. J. G.; Li, Ruoyu; de Hoog, G. S.

2011-01-01

The major clinically relevant species in Scedosporium (teleomorph Pseudallescheria) are Pseudallescheria boydii, Scedosporium aurantiacum, Scedosporium apiospermum, and Scedosporium prolificans, while Pseudallescheria minutispora, Petriellopsis desertorum, and Scedosporium dehoogii are exceptional agents of disease. Three molecular methods targeting the partial β-tubulin gene were developed and evaluated to identify six closely related species of the S. apiospermum complex using quantitative real-time PCR (qPCR), PCR-based reverse line blot (PCR-RLB), and loop-mediated isothermal amplification (LAMP). qPCR was not specific enough for the identification of all species but had the highest sensitivity. The PCR-RLB assay was efficient for the identification of five species. LAMP distinguished all six species unambiguously. The analytical sensitivities of qPCR, PCR-RLB, and LAMP combined with MagNAPure, CTAB (cetyltrimethylammonium bromide), and FTA filter (Whatman) extraction were 50, 5 × 103, and 5 × 102 cells/μl, respectively. When LAMP was combined with a simplified DNA extraction method using an FTA filter, identification to the species level was achieved within 2 h, including DNA extraction. The FTA-LAMP assay is therefore recommended as a cost-effective, simple, and rapid method for the identification of Scedosporium species. PMID:21177887

Comparison of four PCR methods for efficient detection of Trypanosoma cruzi in routine diagnostics.

PubMed

Seiringer, Peter; Pritsch, Michael; Flores-Chavez, María; Marchisio, Edoardo; Helfrich, Kerstin; Mengele, Carolin; Hohnerlein, Stefan; Bretzel, Gisela; Löscher, Thomas; Hoelscher, Michael; Berens-Riha, Nicole

2017-07-01

Due to increased migration, Chagas disease has become an international health problem. Reliable diagnosis of chronically infected people is crucial for prevention of non-vectorial transmission as well as treatment. This study compared four distinct PCR methods for detection of Trypanosoma cruzi DNA for the use in well-equipped routine diagnostic laboratories. DNA was extracted of T. cruzi-positive and negative patients' blood samples and cultured T. cruzi, T. rangeli as well as Leishmania spp. One conventional and two real-time PCR methods targeting a repetitive Sat-DNA sequence as well as one conventional PCR method targeting the variable region of the kDNA minicircle were compared for sensitivity, intra- and interassay precision, limit of detection, specificity and cross-reactivity. Considering the performance, costs and ease of use, an algorithm for PCR-diagnosis of patients with a positive serology for T. cruzi antibodies was developed. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Probe-based real-time PCR method for multilocus melt typing of Xylella fastidiosa strains.

PubMed

Brady, Jeff A; Faske, Jennifer B; Ator, Rebecca A; Castañeda-Gill, Jessica M; Mitchell, Forrest L

2012-04-01

Epidemiological studies of Pierce's disease (PD) can be confounded by a lack of taxonomic detail on the bacterial causative agent, Xylella fastidiosa (Xf). PD in grape is caused by strains of Xylella fastidiosa subsp. fastidiosa, but is not caused by other subspecies of Xf that typically colonize plants other than grape. Detection assays using ELISA and qPCR are effective at detecting and quantifying Xf presence or absence, but offer no information on Xf subspecies or strain identity. Surveying insects or host plants for Xf by current ELISA or qPCR methods provides only presence/absence and quantity information for any and all Xf subspecies, potentially leading to false assessments of disease threat. This study uses a series of adjacent-hybridizing DNA melt analysis probes that are capable of efficiently discriminating Xf subspecies and strain relationships in rapid real-time PCR reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of PMS-PCR technology for detection of Mycobacterium avium subsp. paratuberculosis directly from bovine fecal specimens.

PubMed

Salgado, M; Steuer, P; Troncoso, E; Collins, M T

2013-12-27

Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis, or Johne's disease, in animals. Diagnosis of MAP infection is challenging because of the pathogen's fastidious in vitro growth requirements and low-level intermittent shedding in feces during the preclinical phase of the infection. Detection of these "low-shedders" is important for effective control of paratuberculosis as these animals serve as sources of infection for susceptible calves. Magnetic separation technology, used in combination with culture or molecular methods for the isolation and detection of pathogenic bacteria, enhances the analytical sensitivity and specificity of detection methods. The aim of the present study was to evaluate peptide-mediated magnetic separation (PMS) capture technology coupled with IS900 PCR using the Roche real-time PCR system (PMS-PCR), in comparison with fecal culture using BACTEC-MGIT 960 system, for detection of MAP in bovine fecal samples. Among the 351 fecal samples 74.9% (263/351) were PMS-PCR positive while only 12.3% (43/351) were MGIT culture-positive (p=0.0001). All 43 MGIT culture-positive samples were also positive by PMS-PCR. Mean PMS-PCR crossing-point (Cp) values for the 13 fecal samples with the highest number of MAP, based on time to detection, (26.3) were significantly lower than for the 17 fecal samples with <100 MAP per 2g feces (30.06) (p<0.05). PMS-PCR technology provided results in a shorter time and yielded a higher number of positive results than MGIT culture. Earlier and faster detection of animals shedding MAP by PMS-PCR should significantly strengthen control efforts for MAP-infected cattle herds by helping to limit infection transmission at earlier stages of the infection. Copyright © 2013 Elsevier B.V. All rights reserved.

The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus

PubMed Central

Dung, Tran Thi Ngoc; Phat, Voong Vinh; Nga, Tran Vu Thieu; My, Phan Vu Tra; Duy, Pham Thanh; Campbell, James I.; Thuy, Cao Thu; Hoang, Nguyen Van Minh; Van Minh, Pham; Le Phuc, Hoang; Tuyet, Pham Thi Ngoc; Vinh, Ha; Kien, Duong Thi Hue; Huy, Huynh Le Anh; Vinh, Nguyen Thanh; Nga, Tran Thi Thu; Hau, Nguyen Thi Thu; Chinh, Nguyen Tran; Thuong, Tang Chi; Tuan, Ha Manh; Simmons, Cameron; Farrar, Jeremy J.; Baker, Stephen

2013-01-01

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits. PMID:23046990

Establishment and application of event-specific polymerase chain reaction methods for two genetically modified soybean events, A2704-12 and A5547-127.

PubMed

Li, Xiang; Pan, Liangwen; Li, Junyi; Zhang, Qigang; Zhang, Shuya; Lv, Rong; Yang, Litao

2011-12-28

For implementation of the issued regulations and labeling policies for genetically modified organism (GMO) supervision, the polymerase chain reaction (PCR) method has been widely used due to its high specificity and sensitivity. In particular, use of the event-specific PCR method based on the flanking sequence of transgenes has become the primary trend. In this study, both qualitative and quantitative PCR methods were established on the basis of the 5' flanking sequence of transgenic soybean A2704-12 and the 3' flanking sequence of transgenic soybean A5547-127, respectively. In qualitative PCR assays, the limits of detection (LODs) were 10 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127. In quantitative real-time PCR assays, the LODs were 5 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127, and the limits of quantification (LOQs) were 10 copies for both. Low bias and acceptable SD and RSD values were also achieved in quantification of four blind samples using the developed real-time PCR assays. In addition, the developed PCR assays for the two transgenic soybean events were used for routine analysis of soybean samples imported to Shanghai in a 6 month period from October 2010 to March 2011. A total of 27 lots of soybean from the United States and Argentina were analyzed: 8 lots from the Unites States were found to have the GM soybean A2704-12 event, and the GM contents were <1.5% in all eight analyzed lots. On the contrary, no GM soybean A5547-127 content was found in any of the eight lots. These results demonstrated that the established event-specific qualitative and quantitative PCR methods could be used effectively in routine identification and quantification of GM soybeans A2704-12 and A5547-127 and their derived products.

Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

PubMed Central

Villarreal, Martha Lissete Morales; Padilha, Marina; Vieira, Antonio Diogo Silva; Franco, Bernadette Dora Gombossy de Melo; Martinez, Rafael Chacon Ruiz; Saad, Susana Marta Isay

2013-01-01

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions. PMID:24358142

Quantification of viable bacterial starter cultures of Virgibacillus sp. and Tetragenococcus halophilus in fish sauce fermentation by real-time quantitative PCR.

PubMed

Udomsil, Natteewan; Chen, Shu; Rodtong, Sureelak; Yongsawatdigul, Jirawat

2016-08-01

Real-time quantitative polymerase chain reaction (qPCR) methods were developed for the quantification of Virgibacillus sp. SK37 and Tetragenococcus halophilus MS33, which were added as starter cultures in fish sauce fermentation. The PCR assays were coupled with propidium monoazide (PMA) treatment of samples to selectively quantify viable cells and integrated with exogenous recombinant Escherichia coli cells to control variabilities in analysis procedures. The qPCR methods showed species-specificity for both Virgibacillus halodenitrificans and T. halophilus as evaluated using 6 reference strains and 28 strains of bacteria isolated from fish sauce fermentation. The qPCR efficiencies were 101.1% for V. halodenitrificans and 90.2% for T. halophilus. The quantification limits of the assays were 10(3) CFU/mL and 10(2) CFU/mL in fish sauce samples with linear correlations over 4 Logs for V. halodenitrificans and T. halophilus, respectively. The matrix effect was not observed when evaluated using fish sauce samples fermented for 1-6 months. The developed PMA-qPCR methods were successfully applied to monitor changes of Virgibacillus sp. SK37 and T. halophilus MS33 in a mackerel fish sauce fermentation model where culture-dependent techniques failed to quantify the starter cultures. The results demonstrated the usability of the methods as practical tools for monitoring the starter cultures in fish sauce fermentation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of repetitive-PCR and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid strain typing of Bacillus coagulans

PubMed Central

Nakayama, Motokazu; Tomita, Ayumi; Sonoda, Takumi; Hasumi, Motomitsu; Miyamoto, Takahisa

2017-01-01

In order to establish rapid and accurate typing method for Bacillus coagulans strains which is important for controlling in some canned foods and tea-based beverages manufacturing because of the high-heat resistance of the spores and high tolerance of the vegetative cells to catechins and chemicals, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and repetitive-PCR (rep-PCR) were evaluated. For this purpose, 28 strains of B. coagulans obtained from various culture collections were tested. DNA sequence analyses of the genes encoding 16S rRNA and DNA gyrase classified the test strains into two and three groups, respectively, regardless of their phenotypes. Both MALDI-TOF MS and rep-PCR methods classified the test strains in great detail. Strains classified in each group showed similar phenotypes, such as carbohydrate utilization determined using API 50CH. In particular, the respective two pairs of strains which showed the same metabolic characteristic were classified into the same group by both MALDI-TOF MS and rep-PCR methods separating from the other strains. On the other hand, the other strains which have the different profiles of carbohydrate utilization were separated into different groups by these methods. These results suggested that the combination of MALDI-TOF MS and rep-PCR analyses was advantageous for the rapid and detailed typing of bacterial strains in respect to both phenotype and genotype. PMID:29020109

Evaluation of repetitive-PCR and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid strain typing of Bacillus coagulans.

PubMed

Sato, Jun; Nakayama, Motokazu; Tomita, Ayumi; Sonoda, Takumi; Hasumi, Motomitsu; Miyamoto, Takahisa

2017-01-01

In order to establish rapid and accurate typing method for Bacillus coagulans strains which is important for controlling in some canned foods and tea-based beverages manufacturing because of the high-heat resistance of the spores and high tolerance of the vegetative cells to catechins and chemicals, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and repetitive-PCR (rep-PCR) were evaluated. For this purpose, 28 strains of B. coagulans obtained from various culture collections were tested. DNA sequence analyses of the genes encoding 16S rRNA and DNA gyrase classified the test strains into two and three groups, respectively, regardless of their phenotypes. Both MALDI-TOF MS and rep-PCR methods classified the test strains in great detail. Strains classified in each group showed similar phenotypes, such as carbohydrate utilization determined using API 50CH. In particular, the respective two pairs of strains which showed the same metabolic characteristic were classified into the same group by both MALDI-TOF MS and rep-PCR methods separating from the other strains. On the other hand, the other strains which have the different profiles of carbohydrate utilization were separated into different groups by these methods. These results suggested that the combination of MALDI-TOF MS and rep-PCR analyses was advantageous for the rapid and detailed typing of bacterial strains in respect to both phenotype and genotype.

Diagnosis of bacteremia in pediatric oncologic patients by in-house real-time PCR.

PubMed

Quiles, Milene Gonçalves; Menezes, Liana Carballo; Bauab, Karen de Castro; Gumpl, Elke Kreuscher; Rocchetti, Talita Trevizani; Palomo, Flavia Silva; Carlesse, Fabianne; Pignatari, Antonio Carlos Campos

2015-07-23

Infections are the major cause of morbidity and mortality in children with cancer. Gaining a favorable prognosis for these patients depends on selecting the appropriate therapy, which in turn depends on rapid and accurate microbiological diagnosis. This study employed real-time PCR (qPCR) to identify the main pathogens causing bloodstream infection (BSI) in patients treated at the Pediatric Oncology Institute IOP-GRAACC-UNIFESP-Brazil. Antimicrobial resistance genes were also investigated using this methodology. A total of 248 samples from BACTEC® blood culture bottles and 99 whole-blood samples collected in tubes containing EDTA K2 Gel were isolated from 137 patients. All samples were screened by specific Gram probes for multiplex qPCR. Seventeen sequences were evaluated using gender-specific TaqMan probes and the resistance genes bla SHV, bla TEM, bla CTX, bla KPC, bla IMP, bla SPM, bla VIM, vanA, vanB and mecA were detected using the SYBR Green method. Positive qPCR results were obtained in 112 of the blood culture bottles (112/124), and 90 % agreement was observed between phenotypic and molecular microbial detection methods. For bacterial and fungal identification, the performance test showed: sensitivity 87 %; specificity 91 %; NPV 90 %; PPV 89 % and accuracy of 89 % when compared with the phenotypic method. The mecA gene was detected in 37 samples, extended-spectrum β-lactamases were detected in six samples and metallo-β-lactamase coding genes in four samples, with 60 % concordance between the two methods. The qPCR on whole blood detected eight samples possessing the mecA gene and one sample harboring the vanB gene. The bla KPC, bla VIM, bla IMP and bla SHV genes were not detected in this study. Real-time PCR is a useful tool in the early identification of pathogens and antimicrobial resistance genes from bloodstream infections of pediatric oncologic patients.

Direct PCR Offers a Fast and Reliable Alternative to Conventional DNA Isolation Methods for Gut Microbiomes.

PubMed

Videvall, Elin; Strandh, Maria; Engelbrecht, Anel; Cloete, Schalk; Cornwallis, Charlie K

2017-01-01

The gut microbiome of animals is emerging as an important factor influencing ecological and evolutionary processes. A major bottleneck in obtaining microbiome data from large numbers of samples is the time-consuming laboratory procedures required, specifically the isolation of DNA and generation of amplicon libraries. Recently, direct PCR kits have been developed that circumvent conventional DNA extraction steps, thereby streamlining the laboratory process by reducing preparation time and costs. However, the reliability and efficacy of direct PCR for measuring host microbiomes have not yet been investigated other than in humans with 454 sequencing. Here, we conduct a comprehensive evaluation of the microbial communities obtained with direct PCR and the widely used Mo Bio PowerSoil DNA extraction kit in five distinct gut sample types (ileum, cecum, colon, feces, and cloaca) from 20 juvenile ostriches, using 16S rRNA Illumina MiSeq sequencing. We found that direct PCR was highly comparable over a range of measures to the DNA extraction method in cecal, colon, and fecal samples. However, the two methods significantly differed in samples with comparably low bacterial biomass: cloacal and especially ileal samples. We also sequenced 100 replicate sample pairs to evaluate repeatability during both extraction and PCR stages and found that both methods were highly consistent for cecal, colon, and fecal samples ( r s > 0.7) but had low repeatability for cloacal ( r s = 0.39) and ileal ( r s = -0.24) samples. This study indicates that direct PCR provides a fast, cheap, and reliable alternative to conventional DNA extraction methods for retrieving 16S rRNA data, which can aid future gut microbiome studies. IMPORTANCE The microbial communities of animals can have large impacts on their hosts, and the number of studies using high-throughput sequencing to measure gut microbiomes is rapidly increasing. However, the library preparation procedure in microbiome research is both costly and time-consuming, especially for large numbers of samples. We investigated a cheaper and faster direct PCR method designed to bypass the DNA isolation steps during 16S rRNA library preparation and compared it with a standard DNA extraction method. We used both techniques on five different gut sample types collected from 20 juvenile ostriches and sequenced samples with Illumina MiSeq. The methods were highly comparable and highly repeatable in three sample types with high microbial biomass (cecum, colon, and feces), but larger differences and low repeatability were found in the microbiomes obtained from the ileum and cloaca. These results will help microbiome researchers assess library preparation procedures and plan their studies accordingly.

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection

PubMed Central

Schunk, Mirjam; Kebede Mekonnen, Seleshi; Wondafrash, Beyene; Mengele, Carolin; Fleischmann, Erna; Herbinger, Karl-Heinz; Verweij, Jaco J.; Geldmacher, Christof; Bretzel, Gisela; Löscher, Thomas; Zeynudin, Ahmed

2015-01-01

Background In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study. Methodology Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months. Principal Findings Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic “gold standard”, the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100). Conclusions The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments PMID:26360049

Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection.

PubMed

Schunk, Mirjam; Kebede Mekonnen, Seleshi; Wondafrash, Beyene; Mengele, Carolin; Fleischmann, Erna; Herbinger, Karl-Heinz; Verweij, Jaco J; Geldmacher, Christof; Bretzel, Gisela; Löscher, Thomas; Zeynudin, Ahmed

2015-01-01

In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study. Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months. Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic "gold standard", the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100). The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments.

A novel, multiplex, real-time PCR-based approach for the detection of the commonly occurring pathogenic fungi and bacteria.

PubMed

Horváth, Ádám; Pető, Zoltán; Urbán, Edit; Vágvölgyi, Csaba; Somogyvári, Ferenc

2013-12-23

Polymerase chain reaction (PCR)-based techniques are widely used to identify fungal and bacterial infections. There have been numerous reports of different, new, real-time PCR-based pathogen identification methods although the clinical practicability of such techniques is not yet fully clarified.The present study focuses on a novel, multiplex, real-time PCR-based pathogen identification system developed for rapid differentiation of the commonly occurring bacterial and fungal causative pathogens of bloodstream infections. A multiplex, real-time PCR approach is introduced for the detection and differentiation of fungi, Gram-positive (G+) and Gram-negative (G-) bacteria. The Gram classification is performed with the specific fluorescence resonance energy transfer (FRET) probes recommended for LightCycler capillary real-time PCR. The novelty of our system is the use of a non-specific SYBR Green dye instead of labelled anchor probes or primers, to excite the acceptor dyes on the FRET probes. In conjunction with this, the use of an intercalating dye allows the detection of fungal amplicons.With the novel pathogen detection system, fungi, G + and G- bacteria in the same reaction tube can be differentiated within an hour after the DNA preparation via the melting temperatures of the amplicons and probes in the same tube. This modified FRET technique is specific and more rapid than the gold-standard culture-based methods. The fact that fungi, G + and G- bacteria were successfully identified in the same tube within an hour after the DNA preparation permits rapid and early evidence-based management of bloodstream infections in clinical practice.

Validation of the Applied Biosystems 7500 Fast Instrument for Detection of Listeria Species with the SureTect Listeria Species PCR Assay.

PubMed

Cloke, Jonathan; Arizanova, Julia; Crabtree, David; Simpson, Helen; Evans, Katharine; Vaahtoranta, Laura; Palomäki, Jukka-Pekka; Artimo, Paulus; Huang, Feng; Liikanen, Maria; Koskela, Suvi; Chen, Yi

2016-01-01

The Thermo Scientific™ SureTect™ Listeria species Real-Time PCR Assay was certified during 2013 by the AOAC Research Institute (RI) Performance Tested Methods(SM) program as a rapid method for the detection of Listeria species from a wide range of food matrixes and surface samples. A method modification study was conducted in 2015 to extend the matrix claims of the product to a wider range of food matrixes. This report details the method modification study undertaken to extend the use of this PCR kit to the Applied Biosystems™ 7500 Fast PCR Instrument and Applied Biosystems RapidFinder™ Express 2.0 software allowing use of the assay on a 96-well format PCR cycler in addition to the current workflow, using the 24-well Thermo Scientific PikoReal™ PCR Instrument and Thermo Scientific SureTect software. The method modification study presented in this report was assessed by the AOAC-RI as being a level 2 method modification study, necessitating a method developer study on a representative range of food matrixes covering raw ground turkey, 2% fat pasteurized milk, and bagged lettuce as well as stainless steel surface samples. All testing was conducted in comparison to the reference method detailed in International Organization for Standardization (ISO) 6579:2002. No significant difference by probability of detection statistical analysis was found between the SureTect Listeria species PCR Assay or the ISO reference method methods for any of the three food matrixes and the surface samples analyzed during the study.

Rapid Identification and Quantification of Aureococcus anophagefferens by qPCR Method (Taqman) in the Qinhuangdao Coastal Area: A Region for Recurrent Brown Tide Breakout in China.

PubMed

Wang, Li-Ping; Lei, Kun

2016-12-01

Since 2009, Aureococcus anophagefferens has caused brown tide to occur recurrently in Qinhuangdao coastal area, China. Because the algal cells of A. anophagefferens are so tiny (~3 µm) that it is very hard to identify exactly under a microscope for natural water samples, it is very urgent to develop a method for efficient and continuous monitoring. Here specific primers and Taqman probe are designed to develop a real-time quantitative PCR (qPCR) method for identification and quantification continually. The algal community and cell abundance of A. anophagefferens in the study area (E 119°20'-119°50' and N 39°30'-39°50') from April to October in 2013 are detected by pyrosequencing, and are used to validate the specification and precision of qPCR method for natural samples. Both pyrosequencing and qPCR shows that the targeted cells are present only in May, June and July, and the cell abundance are July > June > May. Although there are various algal species including dinoflagellata, diatom, Cryptomonadales, Chrysophyceae and Chlorophyta living in the natural seawater simultaneously, no disturbance happens to qPCR method. This qPCR method could detect as few as 10 targeted cells, indicating it is able to detect the algal cells at pre-bloom levels. Therefore, qPCR with Taqman probe provides a powerful and sensitive method to monitor the brown tide continually in Qinhuangdao coastal area, China. The results provide a necessary technology support for forecasting the brown tide initiation, in China.

Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet "Beta vulgaris L.": GMO application.

PubMed

Chaouachi, Maher; Alaya, Akram; Ali, Imen Ben Haj; Hafsa, Ahmed Ben; Nabi, Nesrine; Bérard, Aurélie; Romaniuk, Marcel; Skhiri, Fethia; Saïd, Khaled

2013-01-01

KEY MESSAGE : Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis. The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R (2) > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.

New PCR diagnostic systems for the detection and quantification of porcine cytomegalovirus (PCMV).

PubMed

Morozov, Vladimir A; Morozov, Alexey V; Denner, Joachim

2016-05-01

Pigs are frequently infected with porcine cytomegalovirus (PCMV). Infected adult animals may not present with symptoms of disease, and the virus remains latent. However, the virus may be transmitted to human recipients receiving pig transplants. Recently, it was shown that pig-to-non-human-primate xenotransplantations showed 2 to 3 times lower transplant survival when the donor pig was infected with PCMV. Therefore, highly sensitive methods are required to select virus-free pigs and to examine xenotransplants. Seven previously established PCR detection systems targeting the DNA polymerase gene of PCMV were examined by comparison of thermodynamic parameters of oligonucleotides, and new diagnostic nested PCR and real-time PCR systems with improved parameters and high sensitivity were established. The detection limit of conventional PCR was estimated to be 15 copies, and that of the nested PCR was 5 copies. The sensitivity of the real-time PCR with a TaqMan probe was two copies. An equal efficiency of the newly established detection systems was shown by parallel testing of DNA from sera and blood of six pigs, identifying the same animals as PCMV infected. These new diagnostic PCR systems will improve the detection of PCMV and therefore increase the safety of porcine xenotransplants.

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples.

PubMed

Cura, Carolina I; Duffy, Tomas; Lucero, Raúl H; Bisio, Margarita; Péneau, Julie; Jimenez-Coello, Matilde; Calabuig, Eva; Gimenez, María J; Valencia Ayala, Edward; Kjos, Sonia A; Santalla, José; Mahaney, Susan M; Cayo, Nelly M; Nagel, Claudia; Barcán, Laura; Málaga Machaca, Edith S; Acosta Viana, Karla Y; Brutus, Laurent; Ocampo, Susana B; Aznar, Christine; Cuba Cuba, Cesar A; Gürtler, Ricardo E; Ramsey, Janine M; Ribeiro, Isabela; VandeBerg, John L; Yadon, Zaida E; Osuna, Antonio; Schijman, Alejandro G

2015-05-01

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI-TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR). The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm. Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.

Faster and economical screening for vancomycin-resistant enterococci by sequential use of chromogenic agar and real-time polymerase chain reaction.

PubMed

Tan, Thean Yen; Jiang, Boran; Ng, Lily Siew Yong

2017-08-01

Screening for vancomycin-resistant enterococci (VRE) by culture takes days to generate results, while polymerase chain reaction (PCR) testing directly from clinical specimens lacks specificity. The aims of this study were to develop a real-time PCR to detect and identify Enterococcus faecium, Enterococcus faecalis, and vanA and vanB genes, and to evaluate the impact of this PCR on test-reporting times when performing it directly from suspect VRE isolates present on screening chromogenic media. The tetraplex PCR primers were designed to amplify E. faecium, E. faecalis, and vanA and vanB genes, with melt-curve analysis of PCR products. Following analytical and clinical validation of the molecular assay, PCR testing was performed for target colonies present on VRE chromogenic media. PCR results were evaluated against conventional phenotypic identification and susceptibility testing, with the time to result being monitored for both modalities. A total of 519 colonies from clinical specimens were tested concurrently by real-time PCR and phenotypic methods. In all, 223 isolates were identified with phenotypic vancomycin resistance (vanA, n = 108; vanB, n = 105; non-vanA/vanB = 10), with complete agreement between PCR and phenotypic testing for vancomycin-resistant E. faecium and E. faecalis. The majority (88.6%) of PCR results were reported, on average, 24.8 hours earlier than those of phenotypic testing, with 68% reduction in total costs. The use of culture on selective media, followed by direct colony PCR confirmation allows faster and economical VRE screening. Copyright © 2015. Published by Elsevier B.V.

Detection of malaria infection in blood transfusion: a comparative study among real-time PCR, rapid diagnostic test and microscopy: sensitivity of Malaria detection methods in blood transfusion.

PubMed

Hassanpour, Gholamreza; Mohebali, Mehdi; Raeisi, Ahmad; Abolghasemi, Hassan; Zeraati, Hojjat; Alipour, Mohsen; Azizi, Ebrahim; Keshavarz, Hossein

2011-06-01

The transmission of malaria by blood transfusion was one of the first transfusion-transmitted infections recorded in the world. Transfusion-transmitted malaria may lead to serious problems because infection with Plasmodium falciparum may cause rapidly fatal death. This study aimed to compare real-time polymerase chain reaction (real-time PCR) with rapid diagnostic test (RDT) and light microscopy for the detection of Plasmodium spp. in blood transfusion, both in endemic and non-endemic areas of malaria disease in Iran. Two sets of 50 blood samples were randomly collected. One set was taken from blood samples donated in blood bank of Bandar Abbas, a city located in a malarious-endemic area, and the other set from Tehran, a non-endemic one. Light microscopic examination on both thin and thick smears, RDTs, and real-time PCR were performed on the blood samples and the results were compared. Thin and thick light microscopic examinations of all samples as well as RDT results were negative for Plasmodium spp. Two blood samples from endemic area were positive only with real-time PCR. It seems that real-time PCR as a highly sensitive method can be helpful for the confirmation of malaria infection in different units of blood transfusion organization especially in malaria-endemic areas where the majority of donors may be potentially infected with malaria parasites.

Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

PubMed Central

2013-01-01

Background A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. Methods A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. Results This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. Conclusions The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples. PMID:23433252

A TaqMan real-time PCR method based on alternative oxidase genes for detection of plant species in animal feed samples.

PubMed

Campos, Maria Doroteia; Valadas, Vera; Campos, Catarina; Morello, Laura; Braglia, Luca; Breviario, Diego; Cardoso, Hélia G

2018-01-01

Traceability of processed food and feed products has been gaining importance due to the impact that those products can have on human/animal health and to the associated economic and legal concerns, often related to adulterations and frauds as it can be the case for meat and milk. Despite mandatory traceability requirements for the analysis of feed composition, few reliable and accurate methods are presently available to enforce the legislative frame and allow the authentication of animal feeds. In this study, nine sensitive and species-specific real-time PCR TaqMan MGB assays are described for plant species detection in animal feed samples. The method is based on selective real-time qPCR (RT-qPCR) amplification of target genes belonging to the alternative oxidase (AOX) gene family. The plant species selected for detection in feed samples were wheat, maize, barley, soybean, rice and sunflower as common components of feeds, and cotton, flax and peanut as possible undesirable contaminants. The obtained results were compared with end-point PCR methodology. The applicability of the AOX TaqMan assays was evaluated through the screening of commercial feed samples, and by the analysis of plant mixtures with known composition. The RT-qPCR methodology allowed the detection of the most abundant species in feeds but also the identification of contaminant species present in lower amounts, down to 1% w/w. AOX-based methodology provides a suitable molecular marker approach to ascertain plant species composition of animal feed samples, thus supporting feed control and enforcement of the feed sector and animal production.

Detection of central nervous system leukemia in children with acute lymphoblastic leukemia by real-time polymerase chain reaction.

PubMed

Pine, Sharon R; Yin, Changhong; Matloub, Yousif H; Sabaawy, Hatem E; Sandoval, Claudio; Levendoglu-Tugal, Oya; Ozkaynak, M Fevzi; Jayabose, Somasundaram

2005-02-01

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification.

Detection of Central Nervous System Leukemia in Children with Acute Lymphoblastic Leukemia by Real-Time Polymerase Chain Reaction

PubMed Central

Pine, Sharon R.; Yin, Changhong; Matloub, Yousif H.; Sabaawy, Hatem E.; Sandoval, Claudio; Levendoglu-Tugal, Oya; Ozkaynak, M. Fevzi; Jayabose, Somasundaram

2005-01-01

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification. PMID:15681484

Detection of SEA-type α-thalassemia in embryo biopsies by digital PCR.

PubMed

Lee, Ta-Hsien; Hsu, Ya-Chiung; Chang, Chia Lin

2017-08-01

Accurate and efficient pre-implantation genetic diagnosis (PGD) based on the analysis of single or oligo-cells is needed for timely identification of embryos that are affected by deleterious genetic traits in in vitro fertilization (IVF) clinics. Polymerase chain reaction (PCR) is the backbone of modern genetic diagnoses, and a spectrum of PCR-based techniques have been used to detect various thalassemia mutations in prenatal diagnosis (PND) and PGD. Among thalassemias, SEA-type α-thalassemia is the most common variety found in Asia, and can lead to Bart's hydrops fetalis and serious maternal complications. To formulate an efficient digital PCR for clinical diagnosis of SEA-type α-thalassemia in cultured embryos, we conducted a pilot study to detect the α-globin and SEA-type deletion alleles in blastomere biopsies with a highly sensitive microfluidics-based digital PCR method. Genomic DNA from embryo biopsy samples were extracted, and crude DNA extracts were first amplified by a conventional PCR procedure followed by a nested PCR reaction with primers and probes that are designed for digital PCR amplification. Analysis of microfluidics-based PCR reactions showed that robust signals for normal α-globin and SEA-type deletion alleles, together with an internal control gene, can be routinely generated using crude embryo biopsies after a 10 6 -fold dilution of primary PCR products. The SEA-type deletion in cultured embryos can be sensitively diagnosed with the digital PCR procedure in clinics. The adoption of this robust PGD method could prevent the implantation of IVF embryos that are destined to develop Bart's hydrops fetalis in a timely manner. The results also help inform future development of a standard digital PCR procedure for cost-effective PGD of α-thalassemia in a standard IVF clinic. Copyright © 2017. Published by Elsevier B.V.

A simple and rapid technique for the authentication of the ginseng cultivar, Yunpoong, using an SNP marker in a large sample of ginseng leaves.

PubMed

Sun, Hua; Wang, Hong-Tao; Kwon, Woo-Saeng; Kim, Yeon-Ju; In, Jun-Gyo; Yang, Deok-Chun

2011-11-01

Yunpoong is an important Korean ginseng (Panax ginseng C. A. Meyer) cultivar, but no molecular marker has been available to identify Yunpoong from other cultivars. In this study, we developed a single nucleotide polymorphism (SNP) marker for Yunpoong based on analysis of expressed sequence tags (ESTs) in an exon region of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. This SNP marker had high specificity to authenticate Yunpoong in twelve different main ginseng cultivars. For application of the molecular marker, a rapid identification method was established based on the NaOH-Tris method and real-time polymerase chain reaction (PCR) in order to ensure more efficiency in the cultivar selection. The biggest feature of the NaOH-Tris method was that it made the extraction of DNA very simple and rapid in young leaf tissues. We only spent 1 min to extract DNA and directly used it to do PCR. In this report, the conventional DNA extraction method was used to develop molecular marker process, and the NaOH-Tris method was applied in screening large numbers of cultivars. Moreover, the greatest advantage of the real-time PCR compared with traditional PCR, is time saving and high efficiency. Thus, this strategy provides a rapid and reliable method for the specific identification of Yunpoong in a large number of samples. Copyright © 2011 Elsevier B.V. All rights reserved.

Quantitative phenotyping of X-disease resistance in chokecherry using real-time PCR.

PubMed

Huang, Danqiong; Walla, James A; Dai, Wenhao

2014-03-01

A quantitative real-time SYBR Green PCR (qPCR) assay has been developed to detect and quantify X-disease phytoplasmas in chokecherry. An X-disease phytoplasma-specific and high sensitivity primer pair was designed based on the 16S rRNA gene sequence of X-disease phytoplasmas. This primer pair was specific to the 16SrIII group (X-disease) phytoplasmas. The qPCR method can quantify phytoplasmas from a DNA mix (a mix of both chokecherry and X-disease phytoplasma DNA) at as low as 0.001 ng, 10-fold lower than conventional PCR using the same primer pair. A significant correlation between the copy number of phytoplasmas and visual phenotypic rating scores of X-disease resistance in chokecherry plants was observed. Disease resistant chokecherries had a significantly lower titer of X-disease phytoplasmas than susceptible plants. This suggests that the qPCR assay provides a more objective tool to phenotype phytoplasma disease severity, particularly for early evaluation of host resistance; therefore, this method will facilitate quantitative phenotyping of disease resistance and has great potential in enhancing plant breeding. Copyright © 2013 Elsevier B.V. All rights reserved.

Development and validation of an event-specific quantitative PCR method for genetically modified maize MIR162.

PubMed

Takabatake, Reona; Masubuchi, Tomoko; Futo, Satoshi; Minegishi, Yasutaka; Noguchi, Akio; Kondo, Kazunari; Teshima, Reiko; Kurashima, Takeyo; Mano, Junichi; Kitta, Kazumi

2014-01-01

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) maize event, MIR162. We first prepared a standard plasmid for MIR162 quantification. The conversion factor (Cf) required to calculate the genetically modified organism (GMO) amount was empirically determined for two real-time PCR instruments, the Applied Biosystems 7900HT (ABI7900) and the Applied Biosystems 7500 (ABI7500) for which the determined Cf values were 0.697 and 0.635, respectively. To validate the developed method, a blind test was carried out in an interlaboratory study. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr). The determined biases were less than 25% and the RSDr values were less than 20% at all evaluated concentrations. These results suggested that the limit of quantitation of the method was 0.5%, and that the developed method would thus be suitable for practical analyses for the detection and quantification of MIR162.

Enhanced Reverse Transcription-PCR Assay for Detection of Norovirus Genogroup I

PubMed Central

Dreier, Jens; Störmer, Melanie; Mäde, Dietrich; Burkhardt, Sabine; Kleesiek, Knut

2006-01-01

We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities. PMID:16891482

Evaluation of automated repetitive-sequence-based PCR (DiversiLab) compared to PCR ribotyping for rapid molecular typing of community- and nosocomial-acquired Clostridium difficile.

PubMed

Church, Deirdre L; Chow, Barbara L; Lloyd, Tracie; Gregson, Daniel B

2011-06-01

Automated repetitive PCR (rep-PCR; DiversiLab) was compared to PCR ribotyping of the 16S-23S RNA intergenic spacer of Clostridium difficile (CD) as the "gold standard" method for CD typing. PCR products were separated on DiversiLab LabChips (bioMérieux, St. Laurent, Quebec, Canada) utilizing a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) operating the DiversiLab v1.4 assay. Bioanalyzer data were exported to a secure DiversiLab website and analyzed with DiversiLab v3.4 software. Replicability of each method was verified by confirming that the 5 CD reference strains (RS) formed distinct clusters (CD4, CD6, VL0047, VL0013 [ribotype 027], VL0018 [ribotype 001]) by both typing methods. Ninety randomly selected clinical isolates (CS) were analyzed by both methods: 49 from community-acquired and 41 from hospital-acquired cases. A similarity index (SI) of ≥90% was used to define clusters when comparing the known RS cluster to the PCR ribotyping and rep-PCR patterns of CS. Fourteen different PCR-ribotype clusters were identified, but most CS formed 4 major clusters (i.e., CD4 [15/90; 17%], CD6 [17%], 027 [12%], and 001 [9%]). A total of 7 rep-PCR types were identified, but most CS formed 2 major rep-PCR clusters (i.e., CD4 [29/90; 32%] and CD6 [23%]); several PCR ribotypes occurred within a single rep-PCR cluster. Rep-PCR did not distinguish 027 or 001 isolates; i) 027 RS strain did not cluster, ii) eleven 027 CS strains clustered as CD4, iii) no 027 CS strains clustered with the 027 RS, and iv) only 2 001 CS clustered with the RS. Agreement between the PCR-ribotype and rep-PCR clusters only occurred for 35/90 (39%) of the CS using a rep-PCR SI of ≥90%. Rep-PCR time to results was similar, but the annual costs of routinely using this method are 32% higher than PCR ribotyping. Routine use of rep-PCR for CD typing is limited by its lack of definitive separation of the hypertoxigenic 027 or 001 outbreak CD strains. Copyright © 2011 Elsevier Inc. All rights reserved.

Development of duplex real-time RT-PCR based on Taqman technology for detecting simultaneously the genome of pan-enterovirus and enterovirus 71.

PubMed

Hwang, Seoyeon; Kang, Byunghak; Hong, Jiyoung; Kim, Ahyoun; Kim, Hyejin; Kim, Kisang; Cheon, Doo-Sung

2013-07-01

Human enterovirus (EV) 71 is the main etiological agent of hand, foot, and mouth disease (HFMD). It is associated with neurological complications, and caused fatalities during recent outbreaks in the Asia-Pacific region. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. In this study, a duplex real-time RT-PCR assay was developed in order to simultaneously detect pan-EV and EV71. EV71-specific primers and probes were designed based on the highly conserved VP1 region of EV71. Five EV71 strains were detected as positive, and no positive fluorescence signal was observed in the duplex real-time RT-PCR for other viral RNA, which showed 100% specificity for the selected panel, and no cross-reactions were observed in this duplex real-time RT-PCR. The EV71-specific duplex real-time RT-PCR was more sensitive than conventional RT-PCR, and detected viral titers that were 10-fold lower than those measured by the latter. Of the 381 HFMD clinical specimens, 196 (51.4%) cases were pan-EV-positive, of which 170 (86.7%) were EV71-positive when tested by pan-EV and EV71-specific duplex real-time RT-PCR. EV71-specific duplex real-time RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks. Copyright © 2013 Wiley Periodicals, Inc.

Comparison of real-time PCR methods for the detection of Naegleria fowleri in surface water and sediment.

PubMed

Streby, Ashleigh; Mull, Bonnie J; Levy, Karen; Hill, Vincent R

2015-05-01

Naegleria fowleri is a thermophilic free-living ameba found in freshwater environments worldwide. It is the cause of a rare but potentially fatal disease in humans known as primary amebic meningoencephalitis. Established N. fowleri detection methods rely on conventional culture techniques and morphological examination followed by molecular testing. Multiple alternative real-time PCR assays have been published for rapid detection of Naegleria spp. and N. fowleri. Foursuch assays were evaluated for the detection of N. fowleri from surface water and sediment. The assays were compared for thermodynamic stability, analytical sensitivity and specificity, detection limits, humic acid inhibition effects, and performance with seeded environmental matrices. Twenty-one ameba isolates were included in the DNA panel used for analytical sensitivity and specificity analyses. N. fowleri genotypes I and III were used for method performance testing. Two of the real-time PCR assays were determined to yield similar performance data for specificity and sensitivity for detecting N. fowleri in environmental matrices.

Comparison of real-time PCR methods for the detection of Naegleria fowleri in surface water and sediment

PubMed Central

Streby, Ashleigh; Mull, Bonnie J.; Levy, Karen

2015-01-01

Naegleria fowleri is a thermophilic free-living ameba found in freshwater environments worldwide. It is the cause of a rare but potentially fatal disease in humans known as primary amebic meningoencephalitis. Established N. fowleri detection methods rely on conventional culture techniques and morphological examination followed by molecular testing. Multiple alternative real-time PCR assays have been published for rapid detection of Naegleria spp. and N. fowleri. Four such assays were evaluated for the detection of N. fowleri from surface water and sediment. The assays were compared for thermodynamic stability, analytical sensitivity and specificity, detection limits, humic acid inhibition effects, and performance with seeded environmental matrices. Twenty-one ameba isolates were included in the DNA panel used for analytical sensitivity and specificity analyses. N. fowleri genotypes I and III were used for method performance testing. Two of the real-time PCR assays were determined to yield similar performance data for specificity and sensitivity for detecting N. fowleri in environmental matrices. PMID:25855343

Development, optimization, and single laboratory validation of an event-specific real-time PCR method for the detection and quantification of Golden Rice 2 using a novel taxon-specific assay.

PubMed

Jacchia, Sara; Nardini, Elena; Savini, Christian; Petrillo, Mauro; Angers-Loustau, Alexandre; Shim, Jung-Hyun; Trijatmiko, Kurniawan; Kreysa, Joachim; Mazzara, Marco

2015-02-18

In this study, we developed, optimized, and in-house validated a real-time PCR method for the event-specific detection and quantification of Golden Rice 2, a genetically modified rice with provitamin A in the grain. We optimized and evaluated the performance of the taxon (targeting rice Phospholipase D α2 gene)- and event (targeting the 3' insert-to-plant DNA junction)-specific assays that compose the method as independent modules, using haploid genome equivalents as unit of measurement. We verified the specificity of the two real-time PCR assays and determined their dynamic range, limit of quantification, limit of detection, and robustness. We also confirmed that the taxon-specific DNA sequence is present in single copy in the rice genome and verified its stability of amplification across 132 rice varieties. A relative quantification experiment evidenced the correct performance of the two assays when used in combination.

A multiplex real-time PCR assay for the identification and differentiation of Salmonella enterica serovar Typhimurium and monophasic serovar 4,[5],12:i:-.

PubMed

Prendergast, Deirdre M; Hand, Darren; Nί Ghallchóir, Eadaoin; McCabe, Evonne; Fanning, Seamus; Griffin, Margaret; Egan, John; Gutierrez, Montserrat

2013-08-16

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is considered to be a monophasic variant of Salmonella Typhimurium and is increasingly associated with human infections. The use of PCR for the unequivocal identification of strains identified by conventional serotyping as 4,[5],12:i:- has been recommended by the European Food Safety Authority (EFSA), in particular the conventional multiplex PCR developed by Tennant et al. (2010). An alternative protocol for the identification and differentiation of S. Typhimurium and S. Typhimurium-like strains, including its monophasic variants, based on a multiplex real-time PCR assay was developed in our laboratory. A panel of 206 Salmonella strains was used to validate our multiplex real-time PCR against the conventional multiplex PCR recommended by EFSA, i.e. 43 Salmonella strains of serovars other than Typhimurium and 163 routine isolates determined by slide agglutination serotyping to have an incomplete antigenic formula compatible with the S. Typhimurium formula 4,[5],12:i:1,2. Both methods correctly identified the 43 Salmonella strains as non S. Typhimurium. Among the 163 isolates of undetermined serovar by conventional serotyping, both PCR protocols identified 54 isolates as S. Typhimurium, 101 as monophasic S. Typhimurium and 8 as non-S. Typhimurium. Twenty isolates phenotypically lacking the phase-2 H antigen were positive for the fljB.1,2 gene. These strains have been recently described in the literature by other workers and have been referred to as "inconsistent" variants of S. Typhimurium. Antimicrobial resistance and phage typing were also performed on the S. Typhimurium isolates, including monophasic variants, and approximately half of the isolates identified as monophasic S. Typhimurium by our multiplex real-time PCR protocol were DT193 with the resistance pattern ASSuT. There was 100% concordance between the conventional PCR and the multiplex real-time PCR method developed in this study which proved that our protocol is equivalent to the one recommended by EFSA. In comparison to the conventional PCR, this new protocol is faster and is currently being applied routinely in our laboratory to all isolates that could potentially be S. Typhimurium. Copyright © 2013 Elsevier B.V. All rights reserved.

Improved Methods for Capture, Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

PubMed Central

Turner, Cameron R.; Miller, Derryl J.; Coyne, Kathryn J.; Corush, Joel

2014-01-01

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species. PMID:25474207

Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp.).

PubMed

Turner, Cameron R; Miller, Derryl J; Coyne, Kathryn J; Corush, Joel

2014-01-01

Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

Increased efficacy for in-house validation of real-time PCR GMO detection methods.

PubMed

Scholtens, I M J; Kok, E J; Hougs, L; Molenaar, B; Thissen, J T N M; van der Voet, H

2010-03-01

To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision. Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML (residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility (RSD(r) and RSD(R)) were calculated. The results showed that not only the PCR reaction but also the factors 'DNA isolation' and 'PCR day' are important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methods.

Evaluation by latent class analysis of a magnetic capture based DNA extraction followed by real-time qPCR as a new diagnostic method for detection of Echinococcus multilocularis in definitive hosts.

PubMed

Maas, Miriam; van Roon, Annika; Dam-Deisz, Cecile; Opsteegh, Marieke; Massolo, Alessandro; Deksne, Gunita; Teunis, Peter; van der Giessen, Joke

2016-10-30

A new method, based on a magnetic capture based DNA extraction followed by qPCR, was developed for the detection of the zoonotic parasite Echinococcus multilocularis in definitive hosts. Latent class analysis was used to compare this new method with the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. In total, 60 red foxes and coyotes from three different locations were tested with both molecular methods and the sedimentation and counting technique (SCT) or intestinal scraping technique (IST). Though based on a limited number of samples, it could be established that the magnetic capture based DNA extraction followed by qPCR showed similar sensitivity and specificity as the currently used phenol-chloroform DNA extraction followed by single tube nested PCR. All methods have a high specificity as shown by Bayesian latent class analysis. Both molecular assays have higher sensitivities than the combined SCT and IST, though the uncertainties in sensitivity estimates were wide for all assays tested. The magnetic capture based DNA extraction followed by qPCR has the advantage of not requiring hazardous chemicals like the phenol-chloroform DNA extraction followed by single tube nested PCR. This supports the replacement of the phenol-chloroform DNA extraction followed by single tube nested PCR by the magnetic capture based DNA extraction followed by qPCR for molecular detection of E. multilocularis in definitive hosts. Copyright © 2016 Elsevier B.V. All rights reserved.

Real-time PCR Machine System Modeling and a Systematic Approach for the Robust Design of a Real-time PCR-on-a-Chip System

PubMed Central

Lee, Da-Sheng

2010-01-01

Chip-based DNA quantification systems are widespread, and used in many point-of-care applications. However, instruments for such applications may not be maintained or calibrated regularly. Since machine reliability is a key issue for normal operation, this study presents a system model of the real-time Polymerase Chain Reaction (PCR) machine to analyze the instrument design through numerical experiments. Based on model analysis, a systematic approach was developed to lower the variation of DNA quantification and achieve a robust design for a real-time PCR-on-a-chip system. Accelerated lift testing was adopted to evaluate the reliability of the chip prototype. According to the life test plan, this proposed real-time PCR-on-a-chip system was simulated to work continuously for over three years with similar reproducibility in DNA quantification. This not only shows the robustness of the lab-on-a-chip system, but also verifies the effectiveness of our systematic method for achieving a robust design. PMID:22315563

Application of clone library analysis and real-time PCR for comparison of microbial communities in a low-grade copper sulfide ore bioheap leachate.

PubMed

Bowei, Chen; Xingyu, Liu; Wenyan, Liu; Jiankang, Wen

2009-11-01

The microbial communities of leachate from a bioleaching heap located in China were analyzed using the 16S rRNA gene clone library and real-time quantitative PCR. Both methods showed that Leptospirillum spp. were the dominant bacteria, and Ferroplasma acidiphilum were the only archaea detected in the leachate. Clone library results indicated that nine operational taxonomic units (OTUs) were obtained, which fell into four divisions, the Nitrospirae (74%), the gamma-Proteobacteria (14%), the Actinobacteria (6%) and the Euryarchaeota (6%). The results obtained by real-time PCR in some ways were the same as clone library analysis. Furthermore, Sulfobacillus spp., detected only by real-time PCR, suggests that real-time PCR was a reliable technology to study the microbial communities in bioleaching environments. It is a useful tool to assist clone library analysis, to further understand microbial consortia and to have comprehensive and exact microbiological information about bioleaching environments. Finally, the interactions among the microorganisms detected in the leachate were summarized according to the characteristics of these species.

Development of real-time PCR for detection and quantitation of Streptococcus parauberis.

PubMed

Nguyen, T L; Lim, Y J; Kim, D-H; Austin, B

2016-01-01

Streptococcus parauberis is an increasing threat to aquaculture of olive flounder, Paralichthys olivaceus Temminck & Schlegel, in South Korea. We developed a real-time polymerase chain reaction (PCR) method using the TaqMan probe assay to detect and quantify S. parauberis by targeting the gyrB gene sequences, which are effective for molecular analysis of the genus Streptococcus. Our real-time PCR assay is capable of detecting 10 fg of genomic DNA per reaction. The intra- and interassay coefficient of variation (CV) values ranged from 0.42-1.95%, demonstrating that the assay has good reproducibility. There was not any cross-reactivity to Streptococcus iniae or to other streptococcal/lactococcal fish pathogens, such as S. agalactiae and Lactococcus garvieae, indicating that the assay is highly specific to S. parauberis. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. parauberis from inoculated tissue homogenates (r = 0.957; P < 0.05). Hence, this sensitive and specific real-time PCR is a valuable tool for diagnostic quantitation of S. parauberis in clinical samples. © 2014 John Wiley & Sons Ltd.

Rapid extraction from and direct identification in clinical samples of methicillin-resistant staphylococci using the PCR.

PubMed

Jaffe, R I; Lane, J D; Albury, S V; Niemeyer, D M

2000-09-01

Methicillin-resistant staphylococci (MRS) are one of the most common causes of nosocomial infections and bacteremia. Standard bacterial identification and susceptibility testing frequently require as long as 72 h to report results, and there may be difficulty in rapidly and accurately identifying methicillin resistance. The use of the PCR is a rapid and simple process for the amplification of target DNA sequences, which can be used to identify and test bacteria for antimicrobial resistance. However, many sample preparation methods are unsuitable for PCR utilization in the clinical laboratory because they either are not cost-effective, take too long to perform, or do not provide a satisfactory DNA template for PCR. Our goal was to provide same-day results to facilitate rapid diagnosis and therapy. In this report, we describe a rapid method for extraction of bacterial DNA directly from blood culture bottles that gave quality DNA for PCR in as little as 20 min. We compared this extraction method to the standard QIAGEN method for turnaround time (TAT), cost, purity, and use of template in PCR. Specific identification of MRS was determined using intragenic primer sets for bacterial and Staphylococcus 16S rRNA and mecA gene sequences. The PCR primer sets were validated with 416 isolates of staphylococci, including methicillin-resistant Staphylococcus aureus (n = 106), methicillin-sensitive S. aureus (n = 134), and coagulase-negative Staphylococcus (n = 176). The total supply cost of our extraction method and PCR was $2.15 per sample with a result TAT of less than 4 h. The methods described herein represent a rapid and accurate DNA extraction and PCR-based identification system, which makes the system an ideal candidate for use under austere field conditions and one that may have utility in the clinical laboratory.

Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer.

PubMed

Bièche, I; Olivi, M; Champème, M H; Vidaud, D; Lidereau, R; Vidaud, M

1998-11-23

Gene amplification is a common event in the progression of human cancers, and amplified oncogenes have been shown to have diagnostic, prognostic and therapeutic relevance. A kinetic quantitative polymerase-chain-reaction (PCR) method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real-time, was used to quantify gene amplification in tumor DNA. Reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components is limited during the exponential phase, meaning that values are highly reproducible in reactions starting with the same copy number. This greatly improves the precision of DNA quantification. Moreover, real-time PCR does not require post-PCR sample handling, thereby preventing potential PCR-product carry-over contamination; it possesses a wide dynamic range of quantification and results in much faster and higher sample throughput. The real-time PCR method, was used to develop and validate a simple and rapid assay for the detection and quantification of the 3 most frequently amplified genes (myc, ccndl and erbB2) in breast tumors. Extra copies of myc, ccndl and erbB2 were observed in 10, 23 and 15%, respectively, of 108 breast-tumor DNA; the largest observed numbers of gene copies were 4.6, 18.6 and 15.1, respectively. These results correlated well with those of Southern blotting. The use of this new semi-automated technique will make molecular analysis of human cancers simpler and more reliable, and should find broad applications in clinical and research settings.

A molecular approach for the rapid, selective and sensitive detection of Exophiala jeanselmei in environmental samples: development and performance assessment of a real-time PCR assay.

PubMed

Libert, X; Chasseur, C; Packeu, A; Bureau, F; Roosens, N H; De Keersmaecker, S J C

2016-02-01

Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR®green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR®green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days.

Shell-vial culture, coupled with real-time PCR, applied to Rickettsia conorii and Rickettsia massiliae-Bar29 detection, improving the diagnosis of the Mediterranean spotted fever.

PubMed

Segura, Ferran; Pons, Immaculada; Sanfeliu, Isabel; Nogueras, María-Mercedes

2016-04-01

Rickettsia conorii and Rickettsia massiliae-Bar29 are related to Mediterranean spotted fever (MSF). They are intracellular microorganisms. The Shell-vial culture assay (SV) improved Rickettsia culture but it still has some limitations: blood usually contains low amount of microorganisms and the samples that contain the highest amount of them are non-sterile. The objectives of this study were to optimize SV culture conditions and monitoring methods and to establish antibiotic concentrations useful for non-sterile samples. 12 SVs were inoculated with each microorganism, incubated at different temperatures and monitored by classical methods and real-time PCR. R. conorii was detected by all methods at all temperatures since 7th day of incubation. R. massiliae-Bar29 was firstly observed at 28°C. Real-time PCR allowed to detected it 2-7 days earlier (depend on temperature) than classical methods. Antibiotics concentration needed for the isolation of these Rickettsia species from non-sterile samples was determined inoculating SV with R. conorii, R. massiliae-Bar29, biopsy or tick, incubating them with different dilutions of antibiotics and monitoring them weekly. To sum up, if a MSF diagnosis is suspected, SV should be incubated at both 28°C and 32°C for 1-3 weeks and monitored by a sensitive real-time PCR. If the sample is non-sterile the panel of antibiotics tested can be added. Copyright © 2016 Elsevier GmbH. All rights reserved.

Development and validation of a SYBR Green real-time PCR assay for rapid and quantitative detection of goose interferons and proinflammatory cytokines.

PubMed

Zhou, Hao; Chen, Shun; Qi, Yulin; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

2015-10-01

Real time quantitative polymerase chain reaction (RT-qPCR) based on SYBR-Green I binding is a quick, reliable, and easy method for analyzing small amounts of mRNA. Viral pathogens are recognized at the time of infection by pattern recognition receptors; thus, the inflammatory cytokines (IL1β, IL6, and IL18) and antiviral cytokines (IFNα, IFNγ) are secreted by innate immune cells and induced to respond to the pathogens. The objective of this study was to develop an effective and sensitive RT-qPCR assay for the rapid and accurate quantification of goose cytokines: IFNα, IFNγ, IL1β, IL6, and IL18. Subsequently, the established methods were employed to detect the immune response in agonist-stimulated goose spleen cells in vitro. These data indicated that the established RT-qPCR is a reliable method for determining relative gene expression. The results revealed that Imiquimod led to the significant upregulation of goose IFNα (P < 0.01), IFNγ (P < 0.01), IL1β (P < 0.01), IL6 (P < 0.01), and IL18 (P < 0.05). The established methods are important for scientific research and clinical applications, which require rapid and accurate results in a short period of time. The technique can potentially be used in the further research of goose molecular immunology, which will help us understand the interactions between hosts and pathogens. © 2015 Poultry Science Association Inc.

Rapid diagnosis of common deletional α-thalassemia in the Chinese population by qPCR based on identical primer homologous fragments.

PubMed

Long, Ju

2016-05-01

In China, -(SEA), -α(3.7) and -α(4.2) are common deletional α-thalassemia alleles. Gap-PCR is the currently used detection method for these alleles, whose disadvantages include time-consuming procedure and increased potential for PCR product contamination. Therefore, this detection method needs to be improved. Based on identical-primer homologous fragments, a qPCR system was developed for deletional α-thalassemia genotyping, which was composed of a group of quantitatively-related primers and their corresponding probes plus two groups of qualitatively-related primers and their corresponding probes. In order to verify the accuracy of the qPCR system, known genotype samples and random samples are employed. The standard curve result demonstrated that designed primers and probes all yielded good amplification efficiency. In the tests of known genotype samples and random samples, sample detection results were consistent with verification results. In detecting αα, -(SEA), -α(3.7) and -α(4.2) alleles, deletional α-thalassemia alleles are accurately detected by this method. In addition, this method is provided with a wider detection range, greater speed and reduced PCR product contamination risk when compared with current common gap-PCR detection reagents. Copyright © 2016 Elsevier B.V. All rights reserved.

MRPrimer: a MapReduce-based method for the thorough design of valid and ranked primers for PCR

PubMed Central

Kim, Hyerin; Kang, NaNa; Chon, Kang-Wook; Kim, Seonho; Lee, NaHye; Koo, JaeHyung; Kim, Min-Soo

2015-01-01

Primer design is a fundamental technique that is widely used for polymerase chain reaction (PCR). Although many methods have been proposed for primer design, they require a great deal of manual effort to generate feasible and valid primers, including homology tests on off-target sequences using BLAST-like tools. That approach is inconvenient for many target sequences of quantitative PCR (qPCR) due to considering the same stringent and allele-invariant constraints. To address this issue, we propose an entirely new method called MRPrimer that can design all feasible and valid primer pairs existing in a DNA database at once, while simultaneously checking a multitude of filtering constraints and validating primer specificity. Furthermore, MRPrimer suggests the best primer pair for each target sequence, based on a ranking method. Through qPCR analysis using 343 primer pairs and the corresponding sequencing and comparative analyses, we showed that the primer pairs designed by MRPrimer are very stable and effective for qPCR. In addition, MRPrimer is computationally efficient and scalable and therefore useful for quickly constructing an entire collection of feasible and valid primers for frequently updated databases like RefSeq. Furthermore, we suggest that MRPrimer can be utilized conveniently for experiments requiring primer design, especially real-time qPCR. PMID:26109350

Development of a novel hexa-plex PCR method for identification and serotyping of Salmonella species.

PubMed

Li, Ruichao; Wang, Yang; Shen, Jianzhong; Wu, Congming

2014-01-01

Salmonella is one of the most important foodborne pathogens, which causes a huge economic burden worldwide. To detect Salmonella rapidly is very meaningful in preventing salmonellosis and decreasing economic losses. Currently, isolation of Salmonella is confirmed by biochemical and serobased serotyping methods, which are time consuming, labor intensive, and complicated. To solve this problem, a hexa-plex polymerase chain reaction (PCR) method was developed using comparative genomics analysis and multiplex PCR technology to detect Salmonella and Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Agona, Salmonella Choleraesuis, and Salmonella Pullorum simultaneously. The accuracy of this method was tested by a collection of 142 Salmonella. Furthermore, the strategy described in this article to mine serovar-specific fragments for Salmonella could be used to find specific fragments for other Salmonella serotypes and bacteria. The combination of this strategy and multiplex PCR is promising in the rapid identification of foodborne pathogens.

Simultaneous digital quantification and fluorescence-based size characterization of massively parallel sequencing libraries.

PubMed

Laurie, Matthew T; Bertout, Jessica A; Taylor, Sean D; Burton, Joshua N; Shendure, Jay A; Bielas, Jason H

2013-08-01

Due to the high cost of failed runs and suboptimal data yields, quantification and determination of fragment size range are crucial steps in the library preparation process for massively parallel sequencing (or next-generation sequencing). Current library quality control methods commonly involve quantification using real-time quantitative PCR and size determination using gel or capillary electrophoresis. These methods are laborious and subject to a number of significant limitations that can make library calibration unreliable. Herein, we propose and test an alternative method for quality control of sequencing libraries using droplet digital PCR (ddPCR). By exploiting a correlation we have discovered between droplet fluorescence and amplicon size, we achieve the joint quantification and size determination of target DNA with a single ddPCR assay. We demonstrate the accuracy and precision of applying this method to the preparation of sequencing libraries.

Rapid identification and quantification of Campylobacter coli and Campylobacter jejuni by real-time PCR in pure cultures and in complex samples

PubMed Central

2011-01-01

Background Campylobacter spp., especially Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying Campylobacter pose an important risk for human contamination. Pigs are known to be frequently colonized with Campylobacter, especially C. coli, and to excrete high numbers of this pathogen in their faeces. Molecular tools, notably real-time PCR, provide an effective, rapid, and sensitive alternative to culture-based methods for the detection of C. coli and C. jejuni in various substrates. In order to serve as a diagnostic tool supporting Campylobacter epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of C. coli and C. jejuni directly in faecal, feed, and environmental samples. Results With a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the C. coli and C. jejuni real-time PCR assays allowed a precise quantification of purified DNA from C. coli and C. jejuni. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 102 CFU/g of faeces, 1.3 × 102 CFU/g of feed, and 1.0 × 103 CFU/m2 for the environmental samples. Compared to the results obtained by culture, both C. coli and C. jejuni real-time PCR assays exhibited a specificity of 96.2% with a kappa of 0.94 and 0.89 respectively. For faecal samples of experimentally infected pigs, the coefficients of correlation between the C. coli or C. jejuni real-time PCR assay and culture enumeration were R2 = 0.90 and R2 = 0.93 respectively. Conclusion The C. coli and C. jejuni real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying C. coli and C. jejuni in faeces, feed, and environmental samples. These assays represent a new diagnostic tool for studying the epidemiology of Campylobacter by, for instance, investigating the carriage and excretion of C. coli and C. jejuni by pigs from conventional herds. PMID:21600037

Signal Amplification by Glyco-qPCR for Ultrasensitive Detection of Carbohydrates: Applications in Glycobiology**

PubMed Central

Kwon, Seok Joon; Lee, Kyung Bok; Solakyildirim, Kemal; Masuko, Sayaka; Ly, Mellisa; Zhang, Fuming; Li, Lingyun; Dordick, Jonathan S.; Linhardt, Robert J.

2012-01-01

Tiny amounts of carbohydrates (ca. 1 zmol) can be detected quantitatively by a real-time method based on the conjugation of carbohydrates with DNA markers (see picture). The proposed method (glyco-qPCR) provides uniform, ultrasensitive detection of carbohydrates, which can be applied to glycobiology, as well as carbohydrate-based drug discovery. PMID:23073897

Digital PCR: A Sensitive and Precise Method for KIT D816V Quantification in Mastocytosis.

PubMed

Greiner, Georg; Gurbisz, Michael; Ratzinger, Franz; Witzeneder, Nadine; Simonitsch-Klupp, Ingrid; Mitterbauer-Hohendanner, Gerlinde; Mayerhofer, Matthias; Müllauer, Leonhard; Sperr, Wolfgang R; Valent, Peter; Hoermann, Gregor

2018-03-01

The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR ( P < 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival ( P < 0.001). dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis. © 2017 American Association for Clinical Chemistry.

Development and validation of a multiplex real-time PCR method to simultaneously detect 47 targets for the identification of genetically modified organisms.

PubMed

Cottenet, Geoffrey; Blancpain, Carine; Sonnard, Véronique; Chuah, Poh Fong

2013-08-01

Considering the increase of the total cultivated land area dedicated to genetically modified organisms (GMO), the consumers' perception toward GMO and the need to comply with various local GMO legislations, efficient and accurate analytical methods are needed for their detection and identification. Considered as the gold standard for GMO analysis, the real-time polymerase chain reaction (RTi-PCR) technology was optimised to produce a high-throughput GMO screening method. Based on simultaneous 24 multiplex RTi-PCR running on a ready-to-use 384-well plate, this new procedure allows the detection and identification of 47 targets on seven samples in duplicate. To comply with GMO analytical quality requirements, a negative and a positive control were analysed in parallel. In addition, an internal positive control was also included in each reaction well for the detection of potential PCR inhibition. Tested on non-GM materials, on different GM events and on proficiency test samples, the method offered high specificity and sensitivity with an absolute limit of detection between 1 and 16 copies depending on the target. Easy to use, fast and cost efficient, this multiplex approach fits the purpose of GMO testing laboratories.

Simultaneous quantification of alternatively spliced transcripts in a single droplet digital PCR reaction.

PubMed

Sun, Bing; Tao, Lian; Zheng, Yun-Ling

2014-06-01

Human telomerase reverse transcriptase (hTERT) is an essential component required for telomerase activity and telomere maintenance. Several alternatively spliced forms of hTERT mRNA have been reported in human primary and tumor cells. Currently, however, there is no sensitive and accurate method for the simultaneous quantification of multiple alternatively spliced RNA transcripts, such as in the case of hTERT. Here we show droplet digital PCR (ddPCR) provides sensitive, simultaneous digital quantification in a single reaction of two alternatively spliced single deletion hTERT transcripts (α-/β+ and α+/β-) as well as the opportunity to manually quantify non-deletion (α+/β+) and double deletion (α-/β-) transcripts. Our ddPCR method enables direct comparison among four alternatively spliced mRNAs without the need for internal standards or multiple primer pairs specific for each variant as real-time PCR (qPCR) requires, thus eliminating potential variation due to differences in PCR amplification efficiency.

Development of loop-mediated isothermal amplification and SYBR green real-time PCR methods for the detection of Citrus yellow mosaic badnavirus in citrus species.

PubMed

Anthony Johnson, A M; Dasgupta, I; Sai Gopal, D V R

2014-07-01

Citrus yellow mosaic badnavirus (CMBV) is an important pathogen in southern India spread by infected citrus propagules. One of the measures to arrest the spread of CMBV is to develop methods to screen and certify citrus propagules as CMBV-free. The methods loop-mediated isothermal amplification (LAMP) and SYBR green real-time PCR (SGRTPCR) have been developed for the efficient detection of CMBV in citrus propagules. This paper compares the sensitivities of LAMP and SGRTPCR with polymerase chain reaction (PCR) for the detection of CMBV. Whereas PCR and LAMP were able to detect CMBV from a minimum of 10 ng of total DNA of infected leaf samples, SGRTPCR could detect the same from 1 ng of total DNA. Using SGRTPCR, the viral titres were estimated to be the highest in rough lemon and lowest in Nagpur Mandarin of the five naturally infected citrus species tested. The results will help in designing suitable strategies for the sensitive detection of CMBV from citrus propagules. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of Nested and Real-Time PCR for the Detection of Ceratocystis fagacearum in the Sapwood of Diseased Oak Species in Minnesota

Treesearch

A. Yang; J. Juzwik

2017-01-01

Oak wilt caused by Ceratocystis fagacearum is a significant disease of Quercus spp. in the eastern United States. Early and accurate detection of the pathogen is particularly important when disease control is planned. Nested and real-time polymerase chain reaction (PCR) methods utilizing fungal DNA extracted from sapwood drill...

Legionella detection by culture and qPCR: Comparing apples and oranges.

PubMed

Whiley, Harriet; Taylor, Michael

2016-01-01

Legionella spp. are the causative agent of Legionnaire's disease and an opportunistic pathogen of significant public health concern. Identification and quantification from environmental sources is crucial for identifying outbreak origins and providing sufficient information for risk assessment and disease prevention. Currently there are a range of methods for Legionella spp. quantification from environmental sources, but the two most widely used and accepted are culture and real-time polymerase chain reaction (qPCR). This paper provides a review of these two methods and outlines their advantages and limitations. Studies from the last 10 years which have concurrently used culture and qPCR to quantify Legionella spp. from environmental sources have been compiled. 26/28 studies detected Legionella at a higher rate using qPCR compared to culture, whilst only one study detected equivalent levels of Legionella spp. using both qPCR and culture. Aggregating the environmental samples from all 28 studies, 2856/3967 (72%) tested positive for the presence of Legionella spp. using qPCR and 1331/3967 (34%) using culture. The lack of correlation between methods highlights the need to develop an acceptable standardized method for quantification that is sufficient for risk assessment and management of this human pathogen.

Comparison between nasopharyngeal swab and nasal wash, using culture and PCR, in the detection of potential respiratory pathogens.

PubMed

Gritzfeld, Jenna F; Roberts, Paul; Roche, Lorna; El Batrawy, Sherouk; Gordon, Stephen B

2011-04-13

Nasopharyngeal carriage of potential pathogens is important as it is both the major source of transmission and the prerequisite of invasive disease. New methods for detecting carriage could improve comfort, accuracy and laboratory utility. The aims of this study were to compare the sensitivities of a nasopharyngeal swab (NPS) and a nasal wash (NW) in detecting potential respiratory pathogens in healthy adults using microbiological culture and PCR. Healthy volunteers attended for nasal washing and brushing of the posterior nasopharynx. Conventional and real-time PCR were used to detect pneumococcus and meningococcus. Statistical differences between the two nasal sampling methods were determined using a nonparametric Mann-Whitney U test; differences between culture and PCR methods were determined using the McNemar test.Nasal washing was more comfortable for volunteers than swabbing (n = 24). In detection by culture, the NW was significantly more likely to detect pathogens than the NPS (p < 0.00001). Overall, there was a low carriage rate of pathogens in this sample; no significant difference was seen in the detection of bacteria between culture and PCR methods. Nasal washing and PCR may provide effective alternatives to nasopharyngeal swabbing and classical microbiology, respectively.

Evaluation of a Commercial Multiplex PCR for Rapid Detection of Multi Drug Resistant Gram Negative Infections

PubMed Central

Chavada, Ruchir; Maley, Michael

2015-01-01

Introduction: Community and healthcare associated infections caused by multi-drug resistant gram negative organisms (MDR GN) represent a worldwide threat. Nucleic Acid Detection tests are becoming more common for their detection; however they can be expensive requiring specialised equipment and local expertise. This study was done to evaluate the utility of a commercial multiplex tandem (MT) PCR for detection of MDR GN. Methods: The study was done on stored laboratory MDR GN isolates from sterile and non-sterile specimens (n=126, out of stored 567 organisms). Laboratory validation of the MT PCR was done to evaluate sensitivity, specificity and agreement with the current phenotypic methods used in the laboratory. Amplicon sequencing was also done on selected isolates for assessing performance characteristics. Workflow and cost implications of the MT PCR were evaluated. Results: The sensitivity and specificity of the MT PCR were calculated to be 95% and 96.7% respectively. Agreement with the phenotypic methods was 80%. Major lack of agreement was seen in detection of AmpC beta lactamase in enterobacteriaceae and carbapenemase in non-fermenters. Agreement of the MT PCR with another multiplex PCR was found to be 87%. Amplicon sequencing confirmed the genotype detected by MT PCR in 94.2 % of cases tested. Time to result was faster for the MT PCR but cost per test was higher. Conclusion: This study shows that with carefully chosen targets for detection of resistance genes in MDR GN, rapid and efficient identification is possible. MT PCR was sensitive and specific and likely more accurate than phenotypic methods. PMID:26464612

A novel COLD-PCR/FMCA assay enhances the detection of low-abundance IDH1 mutations in gliomas.

PubMed

Pang, Brendan; Durso, Mary B; Hamilton, Ronald L; Nikiforova, Marina N

2013-03-01

Point mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in many gliomas. The detection of IDH1 mutations becomes challenging on suboptimal glioma biopsies when a limited number of tumor cells is available for analysis. Coamplification at lower denaturing-polymerase chain reaction (COLD-PCR) is a PCR technique that deliberately lowers the denaturing cycle temperature to selectively favor amplification of mutant alleles, allowing for the sensitive detection of low-abundance mutations. We developed a novel COLD-PCR assay on the LightCycler platform (Roche, Applied Science, Indianapolis, IN), using post-PCR fluorescent melting curve analysis (FMCA) for the detection of mutant IDH1 with a detection limit of 1%. Thirty-five WHO grade I to IV gliomas and 9 non-neoplastic brain and spinal cord biopsies were analyzed with this technique and the results were compared with the conventional real-time PCR and the Sanger sequencing analysis. COLD-PCR/FMCA was able to detect the most common IDH1 R132H mutation and rare mutation types including R132H, R132C, R132L, R132S, and R132G mutations. Twenty-five glioma cases were positive for IDH1 mutations by COLD-PCR/FMCA, and 23 gliomas were positive by the conventional real-time PCR and Sanger sequencing. A pilocytic astrocytoma (PA I) and a glioblastoma multiforme (GBM IV) showed low-abundance IDH1 mutations detected by COLD-PCR/FMCA. The remaining 10 glioma and 9 non-neoplastic samples were negative by all the 3 methods. In summary, we report a novel COLD-PCR/FMCA method that provides rapid and sensitive detection of IDH1 mutations in formalin-fixed paraffin-embedded tissue and can be used in the clinical setting to assess the small brain biopsies.

Design of primers and probes for quantitative real-time PCR methods.

PubMed

Rodríguez, Alicia; Rodríguez, Mar; Córdoba, Juan J; Andrade, María J

2015-01-01

Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and reliable quantification depends on using efficient primers and probes. Design of primers and probes should meet several criteria to find potential primers and probes for specific qPCR assays. The formation of primer-dimers and other non-specific products should be avoided or reduced. This factor is especially important when designing primers for SYBR(®) Green protocols but also in designing probes to ensure specificity of the developed qPCR protocol. To design primers and probes for qPCR, multiple software programs and websites are available being numerous of them free. These tools often consider the default requirements for primers and probes, although new research advances in primer and probe design should be progressively added to different algorithm programs. After a proper design, a precise validation of the primers and probes is necessary. Specific consideration should be taken into account when designing primers and probes for multiplex qPCR and reverse transcription qPCR (RT-qPCR). This chapter provides guidelines for the design of suitable primers and probes and their subsequent validation through the development of singlex qPCR, multiplex qPCR, and RT-qPCR protocols.

Relative sensitivity of conventional and real-time PCR assays for detection of SFG Rickettsia in blood and tissue samples from laboratory animals.

PubMed

Zemtsova, Galina E; Montgomery, Merrill; Levin, Michael L

2015-01-01

Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays.

Relative Sensitivity of Conventional and Real-Time PCR Assays for Detection of SFG Rickettsia in Blood and Tissue Samples from Laboratory Animals

PubMed Central

Zemtsova, Galina E.; Montgomery, Merrill; Levin, Michael L.

2015-01-01

Studies on the natural transmission cycles of zoonotic pathogens and the reservoir competence of vertebrate hosts require methods for reliable diagnosis of infection in wild and laboratory animals. Several PCR-based applications have been developed for detection of infections caused by Spotted Fever group Rickettsia spp. in a variety of animal tissues. These assays are being widely used by researchers, but they differ in their sensitivity and reliability. We compared the sensitivity of five previously published conventional PCR assays and one SYBR green-based real-time PCR assay for the detection of rickettsial DNA in blood and tissue samples from Rickettsia- infected laboratory animals (n = 87). The real-time PCR, which detected rickettsial DNA in 37.9% of samples, was the most sensitive. The next best were the semi-nested ompA assay and rpoB conventional PCR, which detected as positive 18.4% and 14.9% samples respectively. Conventional assays targeting ompB, gltA and hrtA genes have been the least sensitive. Therefore, we recommend the SYBR green-based real-time PCR as a tool for the detection of rickettsial DNA in animal samples due to its higher sensitivity when compared to more traditional assays. PMID:25607846

Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs.

PubMed

Sehata, Go; Sato, Hiroaki; Ito, Toshihiro; Imaizumi, Yoshitaka; Noro, Taichi; Oishi, Eiji

2015-07-01

We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication.

Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs

PubMed Central

SEHATA, Go; SATO, Hiroaki; ITO, Toshihiro; IMAIZUMI, Yoshitaka; NORO, Taichi; OISHI, Eiji

2015-01-01

We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication. PMID:25728411

Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene.

PubMed

Chae, H S; Park, G N; Kim, S H; Jo, H J; Kim, J T; Jeoung, H Y; An, D J; Kim, N H; Shin, B W; Kang, Y I; Chang, K S

2012-08-01

Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) μg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.

Validation of a quantitative Eimeria spp. PCR for fresh droppings of broiler chickens.

PubMed

Peek, H W; Ter Veen, C; Dijkman, R; Landman, W J M

2017-12-01

A quantitative Polymerase Chain Reaction (qPCR) for the seven chicken Eimeria spp. was modified and validated for direct use on fresh droppings. The analytical specificity of the qPCR on droppings was 100%. Its analytical sensitivity (non-sporulated oocysts/g droppings) was 41 for E. acervulina, ≤2900 for E. brunetti, 710 for E. praecox, 1500 for E. necatrix, 190 for E. tenella, 640 for E. maxima, and 1100 for E. mitis. Field validation of the qPCR was done using droppings with non-sporulated oocysts from 19 broiler flocks. To reduce the number of qPCR tests five grams of each pooled sample (consisting of ten fresh droppings) per time point were blended into one mixed sample. Comparison of the oocysts per gram (OPG)-counting method with the qPCR using pooled samples (n = 1180) yielded a Pearson's correlation coefficient of 0.78 (95% CI: 0.76-0.80) and a Pearson's correlation coefficient of 0.76 (95% CI: 0.70-0.81) using mixed samples (n = 236). Comparison of the average of the OPG-counts of the five pooled samples with the mixed sample per time point (n = 236) showed a Pearson's correlation coefficient (R) of 0.94 (95% CI: 0.92-0.95) for the OPG-counting method and 0.87 (95% CI: 0.84-0.90) for the qPCR. This indicates that mixed samples are practically equivalent to the mean of five pooled samples. The good correlation between the OPG-counting method and the qPCR was further confirmed by the visual agreement between the total oocyst/g shedding patterns measured with both techniques in the 19 broiler flocks using the mixed samples.

Detection of shigella in lettuce by the use of a rapid molecular assay with increased sensitivity

PubMed Central

Jiménez, Kenia Barrantes; McCoy², Clyde B.; Achí, Rosario

2010-01-01

A Multiplex Polymerase Chain Reaction (PCR) assay to be used as an alternative to the conventional culture method in detecting Shigella and enteroinvasive Escherichia coli (EIEC) virulence genes ipaH and ial in lettuce was developed. Efficacy and rapidity of the molecular method were determined as compared to the conventional culture. Lettuce samples were inoculated with different Shigella flexneri concentrations (from 10 CFU/ml to 107 CFU/ml). DNA was extracted directly from lettuce after inoculation (direct-PCR) and after an enrichment step (enrichment PCR). Multiplex PCR detection limit was 104CFU/ml, diagnostic sensitivity and specificity were 100% accurate. An internal amplification control (IAC) of 100 bp was used in order to avoid false negative results. This method produced results in 1 to 2 days while the conventional culture method required 5 to 6 days. Also, the culture method detection limit was 106 CFU/ml, diagnostic sensitivity was 53% and diagnostic specificity was 100%. In this study a Multiplex PCR method for detection of virulence genes in Shigella and EIEC was shown to be effective in terms of diagnostic sensitivity, detection limit and amount of time as compared to Shigella conventional culture. PMID:24031579

Real-time PCR for detection and quantification of the biocontrol agent Trichoderma atroviride strain SC1 in soil.

PubMed

Savazzini, Federica; Longa, Claudia Maria Oliveira; Pertot, Ilaria; Gessler, Cesare

2008-05-01

Trichoderma (Hypocreales, Ascomycota) is a widespread genus in nature and several Trichoderma species are used in industrial processes and as biocontrol agents against crop diseases. It is very important that the persistence and spread of microorganisms released on purpose into the environment are accurately monitored. Real-time PCR methods for genus/species/strain identification of microorganisms are currently being developed to overcome the difficulties of classical microbiological and enzymatic methods for monitoring these populations. The aim of the present study was to develop and validate a specific real-time PCR-based method for detecting Trichoderma atroviride SC1 in soil. We developed a primer and TaqMan probe set constructed on base mutations in an endochitinase gene. This tool is highly specific for the detection and quantification of the SC1 strain. The limits of detection and quantification calculated from the relative standard deviation were 6000 and 20,000 haploid genome copies per gram of soil. Together with the low throughput time associated with this procedure, which allows the evaluation of many soil samples within a short time period, these results suggest that this method could be successfully used to trace the fate of T. atroviride SC1 applied as an open-field biocontrol agent.

Introduction of a dermatophyte polymerase chain reaction assay to the diagnostic mycology service in Scotland.

PubMed

Alexander, C L; Shankland, G S; Carman, W; Williams, C

2011-05-01

Dermatophytes are the major cause of superficial mycoses in samples submitted to Clinical Mycology, Glasgow. The most prevalent species is Trichophyton rubrum as identified classically by microscopy and culture. Recent advances in polymerase chain reaction (PCR) technology were examined for the feasibility of introducing a T. rubrum real-time PCR assay into a routine diagnostic service. To improve the diagnostic mycology service by the introduction of a real-time PCR test for T. rubrum. The DNA from 4972 nail and skin samples was obtained using the Qiagen QIAsymphony automated extractor. This DNA was subjected to real-time PCR using T. rubrum-specific primers and a probe. During phase 1 of the study, 862 samples were analysed; 446 of 470 specimens that grew T. rubrum were detected by PCR. Out of 4110 samples analysed during phase 2, 753 T. rubrum infections were diagnosed and reported within 72 h. A total of 3357 samples were negative for a fungal infection by PCR and microscopy; these were also reported within 72 h. A vast reduction in the turnaround times can be achieved using this technique as opposed to classical methods. Samples which are PCR negative but microscopy positive are still subjected to culture. Screening samples for their suitability for PCR prior to processing eliminates the application of PCR for T. rubrum on inappropriate samples such those from the scalp or pityriasis versicolor. © 2011 The Authors. BJD © 2011 British Association of Dermatologists.

Real-time PCR evaluation of Strongylus vulgaris in horses on farms in Denmark and Central Kentucky.

PubMed

Nielsen, M K; Olsen, S N; Lyons, E T; Monrad, J; Thamsborg, S M

2012-12-21

Strongyle parasites are ubiquitous in grazing horses, and the large strongyle Strongylus vulgaris is considered the most pathogenic helminth parasite of horses. Recent investigations have suggested an association between occurrence of this parasite and usage of selective therapy based on regular fecal egg counts. The established diagnostic method for S. vulgaris involves larval culture and subsequent morphological identification of third stage larvae under the microscope. Recently, a real-time PCR assay was developed and validated for the detection and semi-quantification of S. vulgaris eggs in equine fecal samples. The purposes of the present study were (a) to determine the presence of S. vulgaris by real-time PCR in Danish and American horses on farms using vastly different anthelmintic treatment regimens and (b) to evaluate the association between larval culture results and the PCR. A total of 991 horses representing 53 different horse farms in Denmark and Central Kentucky were studied. Fresh fecal samples were collected from all horses, and strongyle eggs retrieved for DNA extraction and subsequent real-time PCR analysis. Individual larval cultures were performed on the Danish part of the data set (663 horses on 42 farms). On the Danish farms, the S. vulgaris PCR prevalence was found to be 9.2% on farms not basing parasite control on fecal egg counts, and 14.1% on farms using selective therapy. No horses were PCR positive in the American part of the study (328 horses on 11 farms). Kappa-values indicated a moderate agreement between PCR and larval culture results, while McNemar tests revealed no statistical difference between the paired proportions. Significant associations were found between PCR cycle of threshold (Ct) value groups and larval culture counts. Results indicate that both diagnostic methods can be useful for determining the occurrence of S. vulgaris on horse farms, but that they both are affected by potential sources of error. The PCR results confirmed previous findings suggesting that S. vulgaris can reemerge under selective therapy regimens. Copyright © 2012 Elsevier B.V. All rights reserved.

Detection and quantification of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting.

PubMed

Atkins, S D; Clark, I M; Sosnowska, D; Hirsch, P R; Kerry, B R

2003-08-01

Potato cyst nematodes (PCN) are serious pests in commercial potato production, causing yield losses valued at approximately $300 million in the European Community. The nematophagous fungus Plectosphaerella cucumerina has demonstrated its potential as a biological control agent against PCN populations by reducing field populations by up to 60% in trials. The use of biological control agents in the field requires the development of specific techniques to monitor the release, population size, spread or decline, and pathogenicity against its host. A range of methods have therefore been developed to monitor P. cucumerina. A species-specific PCR primer set (PcCF1-PcCR1) was designed that was able to detect the presence of P. cucumerina in soil, root, and nematode samples. PCR was combined with a bait method to identify P. cucumerina from infected nematode eggs, confirming the parasitic ability of the fungus. A selective medium was adapted to isolate the fungus from root and soil samples and was used to quantify the fungus from field sites. A second P. cucumerina-specific primer set (PcRTF1-PcRTR1) and a Taqman probe (PcRTP1) were designed for real-time PCR quantification of the fungus and provided a very sensitive means of detecting the fungus from soil. PCR, bait, and culture methods were combined to investigate the presence and abundance of P. cucumerina from two field sites in the United Kingdom where PCN populations were naturally declining. All methods enabled differences in the activity of P. cucumerina to be detected, and the results demonstrated the importance of using a combination of methods to investigate population size and activity of fungi.

An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

PubMed Central

Chang, Shy-Shin; Hsu, Hsung-Ling; Cheng, Ju-Chien; Tseng, Ching-Ping

2011-01-01

Background Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. Methodology/Principal Findings We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3′-end complementary to the template bacterial sequence and a 5′-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10–100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. Conclusions/Significance Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories. PMID:21637859

Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus.

PubMed

Lakshmi, I Karthika; Putty, Kalyani; Raut, Satya Samparna; Patil, Sunil R; Rao, P P; Bhagyalakshmi, B; Jyothi, Y Krishna; Susmitha, B; Reddy, Y Vishnuvardhan; Kasulanati, Sowmya; Jyothi, J Shiva; Reddy, Y N

2018-04-01

The present study was designed to standardize real-time polymerase chain reaction (PCR) for detecting the bluetongue virus from blood samples of sheep collected during outbreaks of bluetongue disease in the year 2014 in Andhra Pradesh and Telangana states of India. A 10-fold serial dilution of Plasmid PUC59 with bluetongue virus (BTV) NS3 insert was used to plot the standard curve. BHK-21 and KC cells were used for in vitro propagation of virus BTV-9 at a TCID50/ml of 10 5 ml and RNA was isolated by the Trizol method. Both reverse transcription-PCR and real-time PCR using TaqMan probe were carried out with RNA extracted from virus-spiked culture medium and blood to compare the sensitivity by means of finding out the limit of detection (LoD). The results were verified by inoculating the detected and undetected dilutions onto cell cultures with further cytological (cytopathic effect) and molecular confirmation (by BTV-NS1 group-specific PCR). The standardized technique was then applied to field samples (blood) for detecting BTV. The slope of the standard curve obtained was -3.23, and the efficiency was 103%. The LoD with RT-PCR was 8.269E×10 3 number of copies of plasmid, whereas it was 13 with real-time PCR for plasmid dilutions. Similarly, LoD was determined for virus-spiked culture medium, and blood with both the types of PCR and the values were 10 3 TCID 50/ml and 10 4 TCID 50/ml with RT-PCR and 10° TCID 50/ml and 10 2 TCID 50/ml with real-time PCR, respectively. The standardized technique was applied to blood samples collected from BTV suspected animals; 10 among 20 samples were found positive with Cq values ranging from 27 to 39. The Cq value exhibiting samples were further processed in cell cultures and were confirmed to be BT positive. Likewise, Cq undetected samples on processing in cell cultures turned out to be BTV negative. Real-time PCR was found to be a very sensitive as well as reliable method to detect BTV present in different types of samples, including blood samples collected from BTV-infected sheep, compared to RT-PCR. The LoD of BTV is likely influenced by sample type, possibly by the interference by the other components present in the sample.

Comparative evaluation of in-house manual, and commercial semi-automated and automated DNA extraction platforms in the sample preparation of human stool specimens for a Salmonella enterica 5'-nuclease assay.

PubMed

Schuurman, Tim; de Boer, Richard; Patty, Rachèl; Kooistra-Smid, Mirjam; van Zwet, Anton

2007-12-01

In the present study, three methods (NucliSens miniMAG [bioMérieux], MagNA Pure DNA Isolation Kit III Bacteria/Fungi [Roche], and a silica-guanidiniumthiocyanate {Si-GuSCN-F} procedure for extracting DNA from stool specimens were compared with regard to analytical performance (relative DNA recovery and down stream real-time PCR amplification of Salmonella enterica DNA), stability of the extracted DNA, hands-on time (HOT), total processing time (TPT), and costs. The Si-GuSCN-F procedure showed the highest analytical performance (relative recovery of 99%, S. enterica real-time PCR sensitivity of 91%) at the lowest associated costs per extraction (euro 4.28). However, this method did required the longest HOT (144 min) and subsequent TPT (176 min) when processing 24 extractions. Both miniMAG and MagNA Pure extraction showed similar performances at first (relative recoveries of 57% and 52%, S. enterica real-time PCR sensitivity of 85%). However, when difference in the observed Ct values after real-time PCR were taken into account, MagNA Pure resulted in a significant increase in Ct value compared to both miniMAG and Si-GuSCN-F (with on average +1.26 and +1.43 cycles). With regard to inhibition all methods showed relatively low inhibition rates (< 4%), with miniMAG providing the lowest rate (0.7%). Extracted DNA was stable for at least 1 year for all methods. HOT was lowest for MagNA Pure (60 min) and TPT was shortest for miniMAG (121 min). Costs, finally, were euro 4.28 for Si-GuSCN, euro 6.69 for MagNA Pure and euro 9.57 for miniMAG.

A comparative study of digital PCR and real-time qPCR for the detection and quantification of HPV mRNA in sentinel lymph nodes of cervical cancer patients.

PubMed

Carow, Katrin; Read, Christina; Häfner, Norman; Runnebaum, Ingo B; Corner, Adam; Dürst, Matthias

2017-10-30

Qualitative analyses showed that the presence of HPV mRNA in sentinel lymph nodes of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival. To further address the clinical potential of the strategy and to define prognostic threshold levels it is necessary to use a quantitative assay. Here, we compare two methods of quantification: digital PCR and standard quantitative PCR. Serial dilutions of 5 ng-5 pg RNA (≙ 500-0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 sentinel lymph nodes with varying HPV transcript levels. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analyzed by qPCR and dPCR. Digital PCR was run in the RainDrop ® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template. For the analysis of both, clinical samples and serial dilution samples, dPCR and qPCR showed comparable sensitivity. With regard to reproducibility, both methods differed considerably, especially for low template samples. Here, we found with qPCR a mean variation coefficient of 126% whereas dPCR enabled a significantly lower mean variation coefficient of 40% (p = 0.01). Generally, we saw with dPCR a substantial reduction of subsampling errors, which most likely reflects the large cDNA amounts available for analysis. Compared to real-time PCR, dPCR shows higher reliability. Thus, our HPV mRNA dPCR assay holds promise for the clinical evaluation of occult tumor cells in histologically tumor-free lymph nodes in future studies.

TaqMan real-time RT-PCR detection of infectious salmon anaemia virus (ISAV) from formalin-fixed paraffin-embedded Atlantic salmon Salmo salar tissues.

PubMed

Godoy, M G; Kibenge, F S; Kibenge, M J; Olmos, P; Ovalle, L; Yañez, A J; Avendaño-Herrera, R

2010-05-18

The objective of this study was to evaluate the application of a TaqMan real-time reverse transcriptase PCR (RT-PCR) assay for the detection of infectious salmon anaemia virus (ISAV) in formalin-fixed paraffin-embedded (FFPE) fish tissues from Atlantic salmon Salmo salar with and without clinical signs of infection, and to compare it with histological and immunohistochemical (IHC) techniques. Sixteen fish samples obtained in 2007 and 2008 from 4 different farms in Chile were examined. The real-time RT-PCR allowed the detection of ISAV in FFPE samples from 9 of 16 fish, regardless of the organs analyzed, whereas 4 of the real-time RT-PCR negative fish were positive as indicated by histological examination and 3 of the real-time RT-PCR positive fish were negative as indicated by immunohistochemistry evaluation. The presence of ISAV in RT-PCR positive samples was confirmed by amplicon sequencing. This work constitutes the first report on the use of real-time RT-PCR for the detection of ISAV in FFPE sections. The assay is very useful for the examination of archival wax-embedded tissues, and allows for both prospective and retrospective evaluation of tissue samples for the presence of ISAV. However, the method only confirms the presence of the pathogen and should be used in combination with histopathology, which is a more precise tool. The combination of both techniques would be invaluable for confirmatory diagnosis of infectious salmon anaemia (ISA), which is essential for solving salmon farm problems.

[REAL TIME POLYMERASE CHAIN REACTION IN TULAREMIA LABORATORY DIAGNOSTICS].

PubMed

Kormilitsyna, M I; Mescheryakova, I S; Mikhailova, T V; Dobrovolsky, A A

2015-01-01

Enhancement of tularemia laboratory diagnostics by F. tularensis DNA determination in blood sera of patients using real time polymerase chain reaction (RT-PCR). 39 blood sera of patients obtained during transmissive epidemic outbreak of tularemia in Khanty-Mansiysk in 2013 were studied in agglutination reaction, passive hemagglutination, RT-PCR. Specific primers and fluorescent probes were used: ISFTu2F/R+ISFTu2P, Tu14GF/R+tul4-PR2. Advantages of using RT-PCR for early diagnostics of tularemia, when specific antibodies are not detected using traditional immunologic methods, were established. Use of a combination of primers and ISFTu2F/R+ISFTu2P probe allowed to detect F. tularensis DNA in 100% of sera, whereas Tul4G F/R+tul4-PR2 combination--92% of sera. The data were obtained when DNA was isolated from sera using "Proba Rapid" express method. Clinical-epidemiologic diagnosis oftularemia was confirmed by both immune-serologic and RT-PCR methods when sera were studied 3-4 weeks after the onset of the disease. RT-PCR with ISFTu2F/R primers and fluorescent probe ISFTu2P, having high sensitivity and specificity, allows to determine F. tularensis DNA in blood sera of patients at both the early stage and 3-4 weeks after the onset of the disease.

Comparative analysis of gene expression level by quantitative real-time PCR has limited application in objects with different morphology.

PubMed

Demidenko, Natalia V; Penin, Aleksey A

2012-01-01

qRT-PCR is a generally acknowledged method for gene expression analysis due to its precision and reproducibility. However, it is well known that the accuracy of qRT-PCR data varies greatly depending on the experimental design and data analysis. Recently, a set of guidelines has been proposed that aims to improve the reliability of qRT-PCR. However, there are additional factors that have not been taken into consideration in these guidelines that can seriously affect the data obtained using this method. In this study, we report the influence that object morphology can have on qRT-PCR data. We have used a number of Arabidopsis thaliana mutants with altered floral morphology as models for this study. These mutants have been well characterised (including in terms of gene expression levels and patterns) by other techniques. This allows us to compare the results from the qRT-PCR with the results inferred from other methods. We demonstrate that the comparison of gene expression levels in objects that differ greatly in their morphology can lead to erroneous results.

Detection of MPLW515L/K Mutations and Determination of Allele Frequencies with a Single-Tube PCR Assay

PubMed Central

Takei, Hiraku; Morishita, Soji; Araki, Marito; Edahiro, Yoko; Sunami, Yoshitaka; Hironaka, Yumi; Noda, Naohiro; Sekiguchi, Yuji; Tsuneda, Satoshi; Ohsaka, Akimichi; Komatsu, Norio

2014-01-01

A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner. PMID:25144224

Detection of MPLW515L/K mutations and determination of allele frequencies with a single-tube PCR assay.

PubMed

Takei, Hiraku; Morishita, Soji; Araki, Marito; Edahiro, Yoko; Sunami, Yoshitaka; Hironaka, Yumi; Noda, Naohiro; Sekiguchi, Yuji; Tsuneda, Satoshi; Ohsaka, Akimichi; Komatsu, Norio

2014-01-01

A gain-of-function mutation in the myeloproliferative leukemia virus (MPL) gene, which encodes the thrombopoietin receptor, has been identified in patients with essential thrombocythemia and primary myelofibrosis, subgroups of classic myeloproliferative neoplasms (MPNs). The presence of MPL gene mutations is a critical diagnostic criterion for these diseases. Here, we developed a rapid, simple, and cost-effective method of detecting two major MPL mutations, MPLW515L/K, in a single PCR assay; we termed this method DARMS (dual amplification refractory mutation system)-PCR. DARMS-PCR is designed to produce three different PCR products corresponding to MPLW515L, MPLW515K, and all MPL alleles. The amplicons are later detected and quantified using a capillary sequencer to determine the relative frequencies of the mutant and wild-type alleles. Applying DARMS-PCR to human specimens, we successfully identified MPL mutations in MPN patients, with the exception of patients bearing mutant allele frequencies below the detection limit (5%) of this method. The MPL mutant allele frequencies determined using DARMS-PCR correlated strongly with the values determined using deep sequencing. Thus, we demonstrated the potential of DARMS-PCR to detect MPL mutations and determine the allele frequencies in a timely and cost-effective manner.

Molecular differentiation of Russian wild ginseng using mitochondrial nad7 intron 3 region.

PubMed

Li, Guisheng; Cui, Yan; Wang, Hongtao; Kwon, Woo-Saeng; Yang, Deok-Chun

2017-07-01

Cultivated ginseng is often introduced as a substitute and adulterant of Russian wild ginseng due to its lower cost or misidentification caused by similarity in appearance with wild ginseng. The aim of this study is to develop a simple and reliable method to differentiate Russian wild ginseng from cultivated ginseng. The mitochondrial NADH dehydrogenase subunit 7 ( nad 7) intron 3 regions of Russian wild ginseng and Chinese cultivated ginseng were analyzed. Based on the multiple sequence alignment result, a specific primer for Russian wild ginseng was designed by introducing additional mismatch and allele-specific polymerase chain reaction (PCR) was performed for identification of wild ginseng. Real-time allele-specific PCR with endpoint analysis was used for validation of the developed Russian wild ginseng single nucleotide polymorphism (SNP) marker. An SNP site specific to Russian wild ginseng was exploited by multiple alignments of mitochondrial nad 7 intron 3 regions of different ginseng samples. With the SNP-based specific primer, Russian wild ginseng was successfully discriminated from Chinese and Korean cultivated ginseng samples by allele-specific PCR. The reliability and specificity of the SNP marker was validated by checking 20 individuals of Russian wild ginseng samples with real-time allele-specific PCR assay. An effective DNA method for molecular discrimination of Russian wild ginseng from Chinese and Korean cultivated ginseng was developed. The established real-time allele-specific PCR was simple and reliable, and the present method should be a crucial complement of chemical analysis for authentication of Russian wild ginseng.

Development and Validation of a P-35S, T-nos, T-35S and P-FMV Tetraplex Real-time PCR Screening Method to Detect Regulatory Genes of Genetically Modified Organisms in Food.

PubMed

Eugster, Albert; Murmann, Petra; Kaenzig, Andre; Breitenmoser, Alda

2014-10-01

In routine analysis screening methods based on real-time PCR (polymerase chain reaction) are most commonly used for the detection of genetically modified (GM) plant material in food and feed. Screening tests are based on sequences frequently used for GM development, allowing the detection of a large number of GMOs (genetically modified organisms). Here, we describe the development and validation of a tetraplex real-time PCR screening assay comprising detection systems for the regulatory genes Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens nos terminator, Cauliflower Mosaic Virus 35S terminator and Figwort Mosaic Virus 34S promoter. Three of the four primer and probe combinations have already been published elsewhere, whereas primers and probe for the 35S terminator have been developed in-house. Adjustment of primer and probe concentrations revealed a high PCR sensitivity with insignificant physical cross-talk between the four detection channels. The sensitivity of each PCR-system is sufficient to detect a GMO concentration as low as 0.05% of the containing respective element. The specificity of the described tetraplex is high when tested on DNA from GM maize, soy, rapeseed and tomato. We also demonstrate the robustness of the system by inter-laboratory tests. In conclusion, this method provides a sensitive and reliable screening procedure for the detection of the most frequently used regulatory elements present in GM crops either authorised or unauthorised for food.

Effects of topical anaesthetics and fluorescein on the real‐time PCR used for the diagnosis of Herpesviruses and Acanthamoeba keratitis

PubMed Central

Goldschmidt, P; Rostane, H; Saint‐Jean, C; Batellier, L; Alouch, C; Zito, E; Bourcier, T; Laroche, L; Chaumeil, C

2006-01-01

Background The early microbiological diagnosis of corneal infections may prevent the condition from worsening. Aim To study the potential interferences of oxybuprocain and fluorescein solutions used by ophthalmologists on the performances of the real‐time polymerase chain reaction (PCR) carried out as routine test for diagnosis of keratitis. Methods Quantified suspensions of Herpes simplex virus (HSV1), Varicella zoster virus (VZV), Cytomegalovirus (CMV) and Acanthamoeba with and without oxybuprocain or fluorescein added before DNA extraction were tested by real‐time PCR. Results The capacities of the real‐time PCR to detect HSV, VZV, CMV and Acanthamoeba were reduced by oxybuprocain and fluorescein. Both products diluted to 1/16 reduced the PCR detection capacities for more than 2 logs (DNA copies/sample). Conclusions The simultaneous introduction of fluorescein or topical anaesthetics into the tubes containing the specimens to be tested by PCR may lead to false negative results. Because corneal specimens for microbiological diagnosis of keratitis are obtained after topical administration of anaesthetics and corneal staining with fluorescein, ophthalmologists should be aware to rinse the eye surface intensively with appropriate eye solutions to minimise the risks of misdiagnosis. PMID:16899529

Molecular Analysis of Spinal Muscular Atrophy: A genotyping protocol based on TaqMan(®) real-time PCR.

PubMed

de Souza Godinho, Fernanda Marques; Bock, Hugo; Gheno, Tailise Conte; Saraiva-Pereira, Maria Luiza

2012-12-01

Spinal muscular atrophy (SMA) is an autosomal recessive inherited disorder caused by alterations in the survival motor neuron I (SMN1) gene. SMA patients are classified as type I-IV based on severity of symptoms and age of onset. About 95% of SMA cases are caused by the homozygous absence of SMN1 due to gene deletion or conversion into SMN2. PCR-based methods have been widely used in genetic testing for SMA. In this work, we introduce a new approach based on TaqMan(®)real-time PCR for research and diagnostic settings. DNA samples from 100 individuals with clinical signs and symptoms suggestive of SMA were analyzed. Mutant DNA samples as well as controls were confirmed by DNA sequencing. We detected 58 SMA cases (58.0%) by showing deletion of SMN1 exon 7. Considering clinical information available from 56 of them, the patient distribution was 26 (46.4%) SMA type I, 16 (28.6%) SMA type II and 14 (25.0%) SMA type III. Results generated by the new method was confirmed by PCR-RFLP and by DNA sequencing when required. In conclusion, a protocol based on real-time PCR was shown to be effective and specific for molecular analysis of SMA patients.

Use of extremely short Förster resonance energy transfer probes in real-time polymerase chain reaction

PubMed Central

Kutyavin, Igor V.

2013-01-01

Described in the article is a new approach for the sequence-specific detection of nucleic acids in real-time polymerase chain reaction (PCR) using fluorescently labeled oligonucleotide probes. The method is based on the production of PCR amplicons, which fold into dumbbell-like secondary structures carrying a specially designed ‘probe-luring’ sequence at their 5′ ends. Hybridization of this sequence to a complementary ‘anchoring’ tail introduced at the 3′ end of a fluorescent probe enables the probe to bind to its target during PCR, and the subsequent probe cleavage results in the florescence signal. As it has been shown in the study, this amplicon-endorsed and guided formation of the probe-target duplex allows the use of extremely short oligonucleotide probes, up to tetranucleotides in length. In particular, the short length of the fluorescent probes makes possible the development of a ‘universal’ probe inventory that is relatively small in size but represents all possible sequence variations. The unparalleled cost-effectiveness of the inventory approach is discussed. Despite the short length of the probes, this new method, named Angler real-time PCR, remains highly sequence specific, and the results of the study indicate that it can be effectively used for quantitative PCR and the detection of polymorphic variations. PMID:24013564

Application of EMA-qPCR as a complementary tool for the detection and monitoring of Legionella in different water systems.

PubMed

Qin, Tian; Tian, Zhengan; Ren, Hongyu; Hu, Guangchun; Zhou, Haijian; Lu, Jinxing; Luo, Chengwang; Liu, Zunyu; Shao, Zhujun

2012-05-01

Legionella are prevalent in human-made water systems and cause legionellosis in humans. Conventional culturing and polymerase chain reaction (PCR) techniques are not sufficiently accurate for the quantitative analysis of live Legionella bacteria in water samples because of the presence of viable but nonculturable cells and dead cells. Here, we report a rapid detection method for viable Legionella that combines ethidium monoazide (EMA) with quantitative real-time PCR (qPCR) and apply this method to detect Legionella in a large number of water samples from different sources. Results yielded that samples treated with 5 μg/ml EMA for 10 min and subsequently exposed to light irradiation for 5 min were optimal for detecting Legionella. EMA treatment before qPCR could block the signal from approximately 4 log(10) of dead cells. When investigating environmental water samples, the percent-positive rate obtained by EMA-qPCR was significantly higher than conventional PCR and culture methods, and slightly lower than qPCR. The bacterial count of Legionella determined by EMA-qPCR were mostly greater than those determined by culture assays and lower than those determined by qPCR. Acceptable correlations were found between the EMA-qPCR and qPCR results for cooling towers, piped water and hot spring water samples (r = 0.849, P < 0.001) and also found between the EMA-qPCR and culture results for hot spring water samples (r = 0.698, P < 0.001). The results indicate that EMA-qPCR could be used as a complementary tool for the detection and monitoring of Legionella in water systems, especially in hot spring water samples.

Evaluation of a Campylobacter fetus subspecies venerealis real-time quantitative polymerase chain reaction for direct analysis of bovine preputial samples

PubMed Central

Chaban, Bonnie; Chu, Shirley; Hendrick, Steven; Waldner, Cheryl; Hill, Janet E.

2012-01-01

The detection and subspeciation of Campylobacter fetus subsp. venerealis (CFV) from veterinary samples is important for both clinical and economic reasons. Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis, a venereal disease that can lead to serious reproductive problems in cattle, and strict international regulations require animals and animal products to be CFV-free for trade. This study evaluated methods reported in the literature for CFV detection and reports the translation of an extensively tested CFV-specific polymerase chain reaction (PCR) primer set; including the VenSF/VenSR primers and a real-time, quantitative PCR (qPCR) platform using SYBR Green chemistry. Three methods of preputial sample preparation for direct qPCR were evaluated and a heat lysis DNA extraction method was shown to allow for CFV detection at the level of approximately one cell equivalent per reaction (or 1.0 × 103 CFU/mL) from prepuce. The optimized sample preparation and qPCR protocols were then used to evaluate 3 western Canadian bull cohorts, which included 377 bulls, for CFV. The qPCR assay detected 11 positive bulls for the CFV-specific parA gene target. DNA sequence data confirmed the identity of the amplified product and revealed that positive samples were comprised of 2 sequence types; one identical to previously reported CFV parA gene sequences and one with a 9% sequence divergence. These results add valuable information towards our understanding of an important CFV subspeciation target and offer a significantly improved format for an internationally recognized PCR test. PMID:23277694

Detection and Quantification of Human Fecal Pollution with Real-Time PCR

EPA Science Inventory

ABSTRACT Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for enumeration of two recently described ...

Comparative evaluation of three automated systems for DNA extraction in conjunction with three commercially available real-time PCR assays for quantitation of plasma Cytomegalovirus DNAemia in allogeneic stem cell transplant recipients.

PubMed

Bravo, Dayana; Clari, María Ángeles; Costa, Elisa; Muñoz-Cobo, Beatriz; Solano, Carlos; José Remigia, María; Navarro, David

2011-08-01

Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting.

Next day Salmonella spp. detection method based on real-time PCR for meat, dairy and vegetable food products.

PubMed

Rodriguez-Lazaro, David; Gonzalez-García, Patricia; Delibato, Elisabetta; De Medici, Dario; García-Gimeno, Rosa Maria; Valero, Antonio; Hernandez, Marta

2014-08-01

The microbiological standard for detection of Salmonella relies on several cultural steps and requires more than 5 days for final confirmation, and as consequence there is a need for an alternative rapid methodology for its detection. The aim of this study was to compare different detection strategies based on real-time PCR for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, ready to eat lettuce salad and raw sheep milk cured cheese. Three main parameters were evaluated to reduce the time and cost for final results: the initial sample size (25 and 50 g), the incubation times (6, 10 and 18 h) and the bacterial DNA extraction (simple boiling of the culture after washing the bacterial pellet, the use of the Chelex resin, and a commercial silica column). The results obtained demonstrate that a combination of an incubation in buffered peptone water for 18 h of a 25 g-sample coupled to a DNA extraction by boiling and a real-time PCR assay detected down to 2-4 Salmonella spp.CFU per sample in less than 21 h in different types of food products. This RTi-PCR-based method is fully compatible with the ISO standard, providing results more rapidly and cost-effectively. The results were confirmed in a large number of naturally contaminated food samples with at least the same analytical performance as the reference method. Copyright © 2014 Elsevier B.V. All rights reserved.

Validated method for quantification of genetically modified organisms in samples of maize flour.

PubMed

Kunert, Renate; Gach, Johannes S; Vorauer-Uhl, Karola; Engel, Edwin; Katinger, Hermann

2006-02-08

Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR) amplification has easier sample preparation and detection limits are lower. Of the different methods of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green detection method showed that the method is highly sensitive to sample matrices and genomic DNA content influencing the interpretation of results. Therefore, this paper describes a real-time DNA quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with detection limits of lower than 18 copies per sample applicable and comparable to highly purified plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard deviation.

Species level identification of coagulase negative Staphylococcus spp. from buffalo using matrix-assisted laser desorption ionization-time of flight mass spectrometry and cydB real-time quantitative PCR.

PubMed

Pizauro, Lucas J L; de Almeida, Camila C; Soltes, Glenn A; Slavic, Durda; Rossi-Junior, Oswaldo D; de Ávila, Fernando A; Zafalon, Luiz F; MacInnes, Janet I

2017-05-01

Incorrect identification of Staphylococcus spp. can have serious clinical and zoonotic repercussions. Accordingly, the aim of this study was to determine if matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or cydB real- time quantitative PCR (qPCR) could be used to accurately identify coagulase negative Staphylococcus spp. (CoNS) obtained from buffalo milk and milking environment samples. Seventy-five of 84 CoNS isolates could be identified to the species level (score value >1.99) using MALDI-TOF MS. However, as determined by cytochrome d ubiquinol oxidase subunit II (cydB) qPCR and by 16S RNA and cydB gene sequencing, 10S. agnetis strains were wrongly identified as S. hyicus by MALDI-TOF MS. In addition, 9 isolates identified by MALDI-TOF only to the genus level (score values between 1.70 and 1.99) could be identified to species by cydB qPCR. Our findings suggest that MALDI-TOF MS is a reliable method for rapid identification of S. chromogenes and S. epidermidis (species of interest both in human and veterinary medicine) and may be able to correctly identify other Staphylococcus spp. However, at present not all Staphylococcus spp. found in buffalo milk can be accurately identified by MALDI-TOF MS and for these organisms, the cydB qPCR developed in the current study may provide a reliable alternative method for rapid identification of CoNS species. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jothikumar, N., E-mail: jin2@cdc.gov; Hill, Vincent R.

Highlights: •Uses a single-labeled fluorescent primer for real-time PCR. •The detection sensitivity of PET PCR was comparable to TaqMan PCR. •Melt curve analysis can be performed to confirm target amplicon production. •Conventional PCR primers can be converted to PET PCR primers. -- Abstract: We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3′-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5′-end forms a hairpin structure. A fluorescent dye is attached to 5′-end of either the forwardmore » or reverse primer. The presence of guanosine residues at the first and second position of the 3′ dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000–0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource-limited environments.« less

Development of a test for bovine tuberculosis in cattle based on measurement of gamma interferon mRNA by real-time PCR.

PubMed

Gan, W; Zhou, X; Yang, H; Chen, H; Qiao, J; Khan, S H; Yang, L; Yin, X; Zhao, D

2013-08-03

The infection status of cattle for bovine tuberculosis (bTB) was determined by real-time PCR, comparing the levels of IFN-γ mRNA in blood cultures stimulated with either bovine or avian tuberculin with non-stimulated control (phosphate buffer saline, PBS) blood culture. Totally, 137 cattle were tested to validate the assay, in which 54 were IFN-γ real-time quantitative PCR (RT-qPCR) positive, while the remaining 83 were found negative. Meanwhile, the IFN-γ ELISA test was carried out using the Bovigam IFN-γ detection ELISA kit and these results were used as a standard. The results of the single intradermal tuberculin tests (SIDT) and IFN-γ RT-qPCR tests were compared and revealed that the RT-qPCR correlated better with the ELISA and its accuracy was higher than SIDT. This indicates the RT-qPCR is a useful diagnostic method for bTB in cattle. However, several limitations remain for our approach, such as lack of a TB lesions or postmortem test results as a gold standard. Further improvements should be made in the future to increase accuracy of diagnosis of bTB in cattle.

Gene expression analysis of immunostained endothelial cells isolated from formaldehyde-fixated paraffin embedded tumors using laser capture microdissection--a technical report.

PubMed

Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E

2009-12-01

Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by reverse transcription-PCR (RT-PCR) and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4 degrees C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. Glyceraldehyde-3-phosphate dehydrogenase and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM.

Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA

PubMed Central

Jahan, Munira; Lutful Moben, Ahmed; Tabassum, Shahina

2014-01-01

ABSTRACT Background Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. Materials and methods A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Results Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log10 copies/ml and 6.95 ± 1.08 log10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. Conclusion HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35. PMID:29264316

Validation of a high-throughput real-time polymerase chain reaction assay for the detection of capripoxviral DNA.

PubMed

Stubbs, Samuel; Oura, Chris A L; Henstock, Mark; Bowden, Timothy R; King, Donald P; Tuppurainen, Eeva S M

2012-02-01

Capripoxviruses, which are endemic in much of Africa and Asia, are the aetiological agents of economically devastating poxviral diseases in cattle, sheep and goats. The aim of this study was to validate a high-throughput real-time PCR assay for routine diagnostic use in a capripoxvirus reference laboratory. The performance of two previously published real-time PCR methods were compared using commercially available reagents including the amplification kits recommended in the original publication. Furthermore, both manual and robotic extraction methods used to prepare template nucleic acid were evaluated using samples collected from experimentally infected animals. The optimised assay had an analytical sensitivity of at least 63 target DNA copies per reaction, displayed a greater diagnostic sensitivity compared to conventional gel-based PCR, detected capripoxviruses isolated from outbreaks around the world and did not amplify DNA from related viruses in the genera Orthopoxvirus or Parapoxvirus. The high-throughput robotic DNA extraction procedure did not adversely affect the sensitivity of the assay compared to manual preparation of PCR templates. This laboratory-based assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis.

PubMed

Chang, Chia-Hao; Mau-Hsu, Daxen; Chen, Ke-Cheng; Wei, Cheng-Wey; Chiu, Chiung-Ying; Young, Tai-Horng

2018-02-21

In a single-cell study, isolating and identifying single cells are essential, but these processes often require a large investment of time or money. The aim of this study was to isolate and analyse single cells using a novel platform, the PanelChip™ Analysis System, which includes 2500 microwells chip and a digital real-time polymerase chain reaction (dqPCR) assay, in comparison with a standard PCR (qPCR) assay. Through the serial dilution of a known concentration standard, namely pUC19, the accuracy and sensitivity levels of two methodologies were compared. The two systems were tested on the basis of expression levels of the genetic markers vimentin, E-cadherin, N-cadherin and GAPDH in A549 lung carcinoma cells at two known concentrations. Furthermore, the influence of a known PCR inhibitor commonly found in blood samples, heparin, was evaluated in both methodologies. Finally, mathematical models were proposed and separation method of single cells was verified; moreover, gene expression levels during epithelial-mesenchymal transition in single cells under TGFβ1 treatment were measured. The drawn conclusion is that dqPCR performed using PanelChip™ is superior to the standard qPCR in terms of sensitivity, precision, and heparin tolerance. The dqPCR assay is a potential tool for clinical diagnosis and single-cell applications.

Real-time PCR and NASBA for rapid and sensitive detection of Vibrio cholerae in ballast water.

PubMed

Fykse, Else M; Nilsen, Trine; Nielsen, Agnete Dessen; Tryland, Ingun; Delacroix, Stephanie; Blatny, Janet M

2012-02-01

Transport of ballast water is one major factor in the transmission of aquatic organisms, including pathogenic bacteria. The IMO-guidelines of the Convention for the Control and Management of Ships' Ballast Water and Sediments, states that ships are to discharge <1 CFU per 100 ml ballast water of toxigenic Vibrio cholerae, emphasizing the need to establish test methods. To our knowledge, there are no methods sensitive and rapid enough available for cholera surveillance of ballast water. In this study real-time PCR and NASBA methods have been evaluated to specifically detect 1 CFU/100ml of V. cholerae in ballast water. Ballast water samples spiked with V. cholerae cells were filtered and enriched in alkaline peptone water before PCR or NASBA detection. The entire method, including sample preparation and analysis was performed within 7 h, and has the potential to be used for analysis of ballast water for inspection and enforcement control. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of a multiplex probe combination-based one-step real-time reverse transcription-PCR for NA subtype typing of avian influenza virus.

PubMed

Sun, Zhihao; Qin, Tao; Meng, Feifei; Chen, Sujuan; Peng, Daxin; Liu, Xiufan

2017-10-18

Nine influenza virus neuraminidase (NA) subtypes have been identified in poultry and wild birds. Few methods are available for rapid and simple NA subtyping. Here we developed a multiplex probe combination-based one-step real-time reverse transcriptase PCR (rRT-PCR) to detect nine avian influenza virus NA subtypes. Nine primer-probe pairs were assigned to three groups based on the different fluorescent dyes of the probes (FAM, HEX, or Texas Red). Each probe detected only one NA subtype, without cross reactivity. The detection limit was less than 100 EID 50 or 100 copies of cDNA per reaction. Data obtained using this method with allantoic fluid samples isolated from live bird markets and H9N2-infected chickens correlated well with data obtained using virus isolation and sequencing, but was more sensitive. This new method provides a specific and sensitive alternative to conventional NA-subtyping methods.

RUCS: rapid identification of PCR primers for unique core sequences.

PubMed

Thomsen, Martin Christen Frølund; Hasman, Henrik; Westh, Henrik; Kaya, Hülya; Lund, Ole

2017-12-15

Designing PCR primers to target a specific selection of whole genome sequenced strains can be a long, arduous and sometimes impractical task. Such tasks would benefit greatly from an automated tool to both identify unique targets, and to validate the vast number of potential primer pairs for the targets in silico. Here we present RUCS, a program that will find PCR primer pairs and probes for the unique core sequences of a positive genome dataset complement to a negative genome dataset. The resulting primer pairs and probes are in addition to simple selection also validated through a complex in silico PCR simulation. We compared our method, which identifies the unique core sequences, against an existing tool called ssGeneFinder, and found that our method was 6.5-20 times more sensitive. We used RUCS to design primer pairs that would target a set of genomes known to contain the mcr-1 colistin resistance gene. Three of the predicted pairs were chosen for experimental validation using PCR and gel electrophoresis. All three pairs successfully produced an amplicon with the target length for the samples containing mcr-1 and no amplification products were produced for the negative samples. The novel methods presented in this manuscript can reduce the time needed to identify target sequences, and provide a quick virtual PCR validation to eliminate time wasted on ambiguously binding primers. Source code is freely available on https://bitbucket.org/genomicepidemiology/rucs. Web service is freely available on https://cge.cbs.dtu.dk/services/RUCS. mcft@cbs.dtu.dk. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Sizing up arthropod genomes: an evaluation of the impact of environmental variation on genome size estimates by flow cytometry and the use of qPCR as a method of estimation.

PubMed

Gregory, T Ryan; Nathwani, Paula; Bonnett, Tiffany R; Huber, Dezene P W

2013-09-01

A study was undertaken to evaluate both a pre-existing method and a newly proposed approach for the estimation of nuclear genome sizes in arthropods. First, concerns regarding the reliability of the well-established method of flow cytometry relating to impacts of rearing conditions on genome size estimates were examined. Contrary to previous reports, a more carefully controlled test found negligible environmental effects on genome size estimates in the fly Drosophila melanogaster. Second, a more recently touted method based on quantitative real-time PCR (qPCR) was examined in terms of ease of use, efficiency, and (most importantly) accuracy using four test species: the flies Drosophila melanogaster and Musca domestica and the beetles Tribolium castaneum and Dendroctonus ponderosa. The results of this analysis demonstrated that qPCR has the tendency to produce substantially different genome size estimates from other established techniques while also being far less efficient than existing methods.

Evaluation of Polymerase Chain Reaction for Detecting Coliform Bacteria in Drinking Water Sources.

PubMed

Isfahani, Bahram Nasr; Fazeli, Hossein; Babaie, Zeinab; Poursina, Farkhondeh; Moghim, Sharareh; Rouzbahani, Meisam

2017-01-01

Coliform bacteria are used as indicator organisms for detecting fecal pollution in water. Traditional methods including microbial culture tests in lactose-containing media and enzyme-based tests for the detection of β-galactosidase; however, these methods are time-consuming and less specific. The aim of this study was to evaluate polymerase chain reaction (PCR) for detecting coliform. Totally, 100 of water samples from Isfahan drinking water source were collected. Coliform bacteria and Escherichia coli were detected in drinking water using LacZ and LamB genes in PCR method performed in comparison with biochemical tests for all samples. Using phenotyping, 80 coliform isolates were found. The results of the biochemical tests illustrated 78.7% coliform bacteria and 21.2% E. coli . PCR results for LacZ and LamB genes were 67.5% and 17.5%, respectively. The PCR method was shown to be an effective, sensitive, and rapid method for detecting coliform and E. coli in drinking water from the Isfahan drinking water sources.