Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers

PubMed Central

Schneider, Uffe Vest; Mikkelsen, Nikolaj Dam; Lindqvist, Anja; Okkels, Limei Meng; Jøhnk, Nina; Lisby, Gorm

2012-01-01

We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5′-end. In qPCR, the 5′-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5′-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5′-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5′-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5′-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5′-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions. PMID:22701644

Apparent polyploidization after gamma irradiation: pitfalls in the use of quantitative polymerase chain reaction (qPCR) for the estimation of mitochondrial and nuclear DNA gene copy numbers.

PubMed

Kam, Winnie W Y; Lake, Vanessa; Banos, Connie; Davies, Justin; Banati, Richard

2013-05-30

Quantitative polymerase chain reaction (qPCR) has been widely used to quantify changes in gene copy numbers after radiation exposure. Here, we show that gamma irradiation ranging from 10 to 100 Gy of cells and cell-free DNA samples significantly affects the measured qPCR yield, due to radiation-induced fragmentation of the DNA template and, therefore, introduces errors into the estimation of gene copy numbers. The radiation-induced DNA fragmentation and, thus, measured qPCR yield varies with temperature not only in living cells, but also in isolated DNA irradiated under cell-free conditions. In summary, the variability in measured qPCR yield from irradiated samples introduces a significant error into the estimation of both mitochondrial and nuclear gene copy numbers and may give spurious evidence for polyploidization.

Longitudinal monitoring of bottlenose dolphins leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Longitudinal monitoring of bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review.

PubMed

Irshad, Mohammad; Gupta, Priyanka; Mankotia, Dhananjay Singh; Ansari, Mohammad Ahmad

2016-05-28

The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process.

Multiplex qPCR for serodetection and serotyping of hepatitis viruses: A brief review

PubMed Central

Irshad, Mohammad; Gupta, Priyanka; Mankotia, Dhananjay Singh; Ansari, Mohammad Ahmad

2016-01-01

The present review describes the current status of multiplex quantitative real time polymerase chain reaction (qPCR) assays developed and used globally for detection and subtyping of hepatitis viruses in body fluids. Several studies have reported the use of multiplex qPCR for the detection of hepatitis viruses, including hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). In addition, multiplex qPCR has also been developed for genotyping HBV, HCV, and HEV subtypes. Although a single step multiplex qPCR assay for all six hepatitis viruses, i.e., A to G viruses, is not yet reported, it may be available in the near future as the technologies continue to advance. All studies use a conserved region of the viral genome as the basis of amplification and hydrolysis probes as the preferred chemistries for improved detection. Based on a standard plot prepared using varying concentrations of template and the observed threshold cycle value, it is possible to determine the linear dynamic range and to calculate an exact copy number of virus in the specimen. Advantages of multiplex qPCR assay over singleplex or other molecular techniques in samples from patients with co-infection include fast results, low cost, and a single step investigation process. PMID:27239109

Detection and quantification limits of the EPA Enterococcus qPCR method

EPA Science Inventory

The U.S. EPA will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality in 2013 and has published preliminary proposed water quality criteria guidelines for the method. An im...

Interpreting dual ELISA and qPCR data for bacterial kidney disease of salmonids.

PubMed

Nance, Shelly L; Riederer, Michael; Zubkowski, Tyler; Trudel, Marc; Rhodes, Linda D

2010-09-02

Although there are a variety of methods available for the detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmon and trout, the enzyme-linked immunosorbent assay (ELISA) is probably the most widely used method. However, ELISA measures bacterial antigen, which does not necessarily reflect the number of cells present. We hypothesized that dual analysis of kidney tissue by ELISA and a quantitative real-time polymerase chain reaction assay (qPCR) would provide complementary information about antigen level and the number of bacterial genomes. We found that DNA extracted from the insoluble fraction of the ELISA tissue preparation produced the same qPCR result as DNA extracted directly from frozen tissue, permitting true dual analysis of the same tissue sample. We examined kidney tissue in this manner from individual free-ranging juvenile Chinook salmon and antibiotic-treated captive subadult Chinook salmon and observed 3 different patterns of results. Among the majority of fish, there was a strong correlation between the ELISA value and the qPCR value. However, subsets of fish exhibited either low ELISA values with elevated qPCR values or higher ELISA values with very low qPCR values. These observations suggest a conceptual model that allows inferences about the state of infection of individual fish based on dual ELISA/qPCR results. Although this model requires further assessment through experimental infections and treatments, it may have utility in broodstock selection programs that currently apply egg-culling practices based on ELISA alone.

Contribution of the qPCR for the Diagnosis of Pneumocystosis

PubMed Central

Issa, Nahema; Gabriel, Frederic; Baulier, Gildas; Accoceberry, Isabelle; Mourissoux, Gaelle; Guisset, Olivier; Camou, Fabrice

2017-01-01

Abstract Background Pneumocystis jirovecii pneumonia (PCP) is an opportunistic fungal respiratory infection. The incidence of PCP has decreased among HIV patients, however among non HIV-negative patients on immunosuppressive drugs; an increase in incidence is noted. In this population, the diagnosis of PCP is difficult because the clinical presentation is atypical and the direct examination (DE) of the respiratory secretions is often negative. In this context, detection of Pneumocystis jirovecii DNA in respiratory secretions by real-time quantitative chain reaction (qPCR) should be usefull. Methods In order to evaluate the usefulness of qPCR, all patients hospitalized in medicine or intensive care unit (ICU) in a university hospital and having a positive qPCR in respiratory secretions were included in a retrospective study conducted between 2013 and 2016. Based on clinical data, respiratory secretions, imaging and treatment, patients were classified into three groups: certain PCP, possible, or colonization, irrespective of the value of qPCR. Results One hundred and fifty patients, including 38 infected with HIV, were included: 75 in medicine and 75 in intensive care. Ninety patients (60%) had bronchoalveolar lavage. The diagnosis of PCP was considered certain or possible for 52 and 77 patients respectively and rejected (colonization) for 21 patients. DE was negative for 78% of non-HIV patients and 29% of HIV patients. Among the 129 patients with PCP, the hospital mortality was 35.9% in ICU and 21.5% in medicine. The median value of qPCR was 76,650 copies/mL among patients with PCP and 3,220 copies/mL among colonized patients (P < 0.001) with no significant difference in type of respiratory specimen or place of hospitalization. The optimal threshold value of qPCR determined from the ROC curve was 10,100 copies/mL with a sensitivity of 76.6% and a specificity of 86%. Specificity was 100% at the threshold of 59,250 copies/mL. Conclusion If qPCR alone is imperfect

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.

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.

Optimization of Diamond Nucleic Acid Dye for quantitative PCR.

PubMed

Haines, Alicia M; Tobe, Shanan S; Linacre, Adrian

2016-10-01

Here, we evaluate Diamond Nucleic Acid Dye (DD) for use in quantitative PCR (qPCR) applications. Although DD is a commercially available stain for detection of DNA separated by gel electrophoresis, its use as a detection dye in qPCR has yet to be described. To determine if DD can be used in qPCR, we investigated its inhibitory effects on qPCR at concentrations ranging 0.1-2.5×. Serial dilution of DNA was used to determine the efficiency, sensitivity, and linearity of DD-generated qPCR data in comparison to other commonly used fluorescent dyes such as SYBR Green (SG), EvaGreen (EG), and BRYT Green (BG). DD was found to be comparable with other dyes for qPCR applications, with an R2 value >0.9 and an efficiency of 0.83. Mitochondrial DNA (mtDNA) target signals were successfully produced by DD over a DNA dilution range of ~28 ng- 0.28 pg, demonstrating comparable sensitivity to the other dyes investigated. Cq values obtained using DD were lower than those using EG by almost 7 cycles. We conclude that Diamond Nucleic Acid Dye is a cheaper, less toxic alternative for qPCR applications.

Improving qPCR telomere length assays: Controlling for well position effects increases statistical power.

PubMed

Eisenberg, Dan T A; Kuzawa, Christopher W; Hayes, M Geoffrey

2015-01-01

Telomere length (TL) is commonly measured using quantitative PCR (qPCR). Although, easier than the southern blot of terminal restriction fragments (TRF) TL measurement method, one drawback of qPCR is that it introduces greater measurement error and thus reduces the statistical power of analyses. To address a potential source of measurement error, we consider the effect of well position on qPCR TL measurements. qPCR TL data from 3,638 people run on a Bio-Rad iCycler iQ are reanalyzed here. To evaluate measurement validity, correspondence with TRF, age, and between mother and offspring are examined. First, we present evidence for systematic variation in qPCR TL measurements in relation to thermocycler well position. Controlling for these well-position effects consistently improves measurement validity and yields estimated improvements in statistical power equivalent to increasing sample sizes by 16%. We additionally evaluated the linearity of the relationships between telomere and single copy gene control amplicons and between qPCR and TRF measures. We find that, unlike some previous reports, our data exhibit linear relationships. We introduce the standard error in percent, a superior method for quantifying measurement error as compared to the commonly used coefficient of variation. Using this measure, we find that excluding samples with high measurement error does not improve measurement validity in our study. Future studies using block-based thermocyclers should consider well position effects. Since additional information can be gleaned from well position corrections, rerunning analyses of previous results with well position correction could serve as an independent test of the validity of these results. © 2015 Wiley Periodicals, Inc.

Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp.

PubMed

Criado-Fornelio, A; Buling, A; Cunha-Filho, N A; Ruas, J L; Farias, N A R; Rey-Valeiron, C; Pingret, J L; Etievant, M; Barba-Carretero, J C

2007-12-25

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I((R)). The method, consisting of amplification of a 235bp fragment of the 18S rRNA gene, is able to detect at least 0.1fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1ng-0.1fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).

Detection of mandarin in orange juice by single-nucleotide polymorphism qPCR assay.

PubMed

Aldeguer, Miriam; López-Andreo, María; Gabaldón, José A; Puyet, Antonio

2014-02-15

A dual-probe real time PCR (qPCR) DNA-based analysis was devised for the identification of mandarin in orange juice. A single nucleotide polymorphism at the trnL-trnF intergenic region of the chloroplast chromosome was confirmed in nine orange (Citrus sinensis) and thirteen commercial varieties of mandarin, including Citrus reticulata and Citrus unshiu species and a mandarin × tangelo hybrid. Two short minor-groove binding fluorescent probes targeting the polymorphic sequence were used in the dual-probe qPCR, which allowed the detection of both species in single-tube reactions. The similarity of PCR efficiencies allowed a simple estimation of the ratio mandarin/orange in the juice samples, which correlated to the measured difference of threshold cycle values for both probes. The limit of detection of the assay was 5% of mandarin in orange juice, both when the juice was freshly prepared (not from concentrate) or reconstituted from concentrate, which would allow the detection of fraudulently added mandarin juice. The possible use of the dual-probe system for quantitative measurements was also tested on fruit juice mixtures. qPCR data obtained from samples containing equal amounts of mandarin and orange juice revealed that the mandarin target copy number was approximately 2.6-fold higher than in orange juice. The use of a matrix-adapted control as calibrator to compensate the resulting C(T) bias allowed accurate quantitative measurements to be obtained. Copyright © 2013 Elsevier Ltd. All rights reserved.

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

A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments.

PubMed

Ganger, Michael T; Dietz, Geoffrey D; Ewing, Sarah J

2017-12-01

qPCR has established itself as the technique of choice for the quantification of gene expression. Procedures for conducting qPCR have received significant attention; however, more rigorous approaches to the statistical analysis of qPCR data are needed. Here we develop a mathematical model, termed the Common Base Method, for analysis of qPCR data based on threshold cycle values (C q ) and efficiencies of reactions (E). The Common Base Method keeps all calculations in the logscale as long as possible by working with log 10 (E) ∙ C q , which we call the efficiency-weighted C q value; subsequent statistical analyses are then applied in the logscale. We show how efficiency-weighted C q values may be analyzed using a simple paired or unpaired experimental design and develop blocking methods to help reduce unexplained variation. The Common Base Method has several advantages. It allows for the incorporation of well-specific efficiencies and multiple reference genes. The method does not necessitate the pairing of samples that must be performed using traditional analysis methods in order to calculate relative expression ratios. Our method is also simple enough to be implemented in any spreadsheet or statistical software without additional scripts or proprietary components.

A ready-to-use duplex qPCR to detect Leishmania infantum DNA in naturally infected dogs.

PubMed

Rampazzo, Rita de Cássia Pontello; Solcà, Manuela da Silva; Santos, Liliane Celestino Sales; Pereira, Lais de Novaes; Guedes, José Carlos Oliveira; Veras, Patrícia Sampaio Tavares; Fraga, Deborah Bittencourt Mothé; Krieger, Marco Aurélio; Costa, Alexandre Dias Tavares

2017-11-15

Canine visceral leishmaniasis (CVL) is a systemic disease caused by Leishmania infantum. A precise CVL diagnosis would allow for a faster and more specific treatment. Quantitative PCR (qPCR) is a sensitive and specific technique that can diagnose CVL and also monitor parasite load in the animal during the course of the infection or treatment. The aim of this study was to develop a ready-to-use (gelified and freezer-free) duplex qPCR for the identification of infected animals. We combined a new qPCR protocol that detects the canine 18S rRNA gene with an existing protocol for L. infantum kDNA detection, creating a duplex qPCR. This duplex method was then developed into a ready-to-use format. The performance of the duplex and singleplex reactions were compared in the traditional format (liquid and freezer-stored). Furthermore, the duplex qPCR performance was compared between the ready-to-use and traditional formats. The singleplex and new duplex qPCR exhibited the same detection limit in the traditional format (0.1 parasites/reaction). The ready-to-use format showed a detection limit of 1 parasite/reaction without affecting the reaction efficiency. The performance of the new qPCR protocol in the two formats was assessed using canine tissue samples from 82 dogs in an endemic CVL area that were previously characterized by standard serological and parasitological protocols. Splenic aspirates provided a higher rate of positivity (92.9%) followed by skin (50%) and blood (35.7%). The reported detection limits were observed for all tissues studied. Our results show that the amplification of L. infantum kDNA and canine DNA in a single tube, using either the traditional or ready-to-use format, exhibited the same diagnostic performance as amplification of the parasite kDNA alone. The detection of the host gene strengthens the qPCR results by confirming the presence and quality of DNA in the samples and the absence of polymerase inhibitors. The ready-to-use duplex qPCR format

Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR

PubMed Central

Morisset, Dany; Štebih, Dejan; Milavec, Mojca; Gruden, Kristina; Žel, Jana

2013-01-01

In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed. PMID:23658750

qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms.

PubMed

Azevedo, Michelle de Campos Soriani; Ramuno, Natália Mortari; Fachin, Luciana Raquel Vincenzi; Tassa, Mônica; Rosa, Patrícia Sammarco; Belone, Andrea de Faria Fernandes; Diório, Suzana Madeira; Soares, Cleverson Teixeira; Garlet, Gustavo Pompermaier; Trombone, Ana Paula Favaro

Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events In Vivo.

PubMed

Falabella, Micol; Sun, Linqing; Barr, Justin; Pena, Andressa Z; Kershaw, Erin E; Gingras, Sebastien; Goncharova, Elena A; Kaufman, Brett A

2017-10-05

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by nonhomologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR editing in founder mice. Using this DNA sample collection, we validated specific quantitative and digital PCR methods (qPCR and dPCR, respectively) for measuring the frequency of on-target editing in founder mice. We found that locked nucleic acid (LNA) probes combined with an internal reference probe (Drop-Off Assay) provide accurate measurements of editing rates. The Drop-Off LNA Assay also detected on-target CRISPR-Cas9 gene editing in blastocysts with a sensitivity comparable to PCR-clone sequencing. Lastly, we demonstrate that the allele-specific LNA probes used in qPCR competitor assays can accurately detect recombinant mutations in founder mice. In summary, we show that LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo , testing RNA guides, and detecting recombinant mutations. Copyright © 2017 Falabella et al.

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

PubMed

Smith, Cindy J; Osborn, A Mark

2009-01-01

Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

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.

Relationship and variation of qPCR and culturable enterococci estimates in ambient surface waters are predictable

USGS Publications Warehouse

Whitman, Richard L.; Ge, Zhongfu; Nevers, Meredith B.; Boehm, Alexandria B.; Chern, Eunice C.; Haugland, Richard A.; Lukasik, Ashley M.; Molina, Marirosa; Przybyla-Kelly, Kasia; Shively, Dawn A.; White, Emily M.; Zepp, Richard G.; Byappanahalli, Muruleedhara N.

2010-01-01

The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based methods, the U.S. Environmental Protection Agency is currently considering its use as a basis for revised ambient water quality criteria. In anticipation of this possibility, we sought to examine the relationship between qPCR-based and culture-based estimates of enterococci in surface waters. Using data from several research groups, we compared enterococci estimates by the two methods in water samples collected from 37 sites across the United States. A consistent linear pattern in the relationship between cell equivalents (CCE), based on the qPCR method, and colony-forming units (CFU), based on the traditional culturable method, was significant (P 10CFU > 2.0/100 mL) while uncertainty increases at lower CFU values. It was further noted that the relative error in replicated qPCR estimates was generally higher than that in replicated culture counts even at relatively high target levels, suggesting a greater need for replicated analyses in the qPCR method to reduce relative error. Further studies evaluating the relationship between culture and qPCR should take into account analytical uncertainty as well as potential differences in results of these methods that may arise from sample variability, different sources of pollution, and environmental factors.

Reference genes for normalization of qPCR assays in sugarcane plants under water deficit.

PubMed

de Andrade, Larissa Mara; Dos Santos Brito, Michael; Fávero Peixoto Junior, Rafael; Marchiori, Paulo Eduardo Ribeiro; Nóbile, Paula Macedo; Martins, Alexandre Palma Boer; Ribeiro, Rafael Vasconcelos; Creste, Silvana

2017-01-01

Sugarcane ( Saccharum spp.) is the main raw material for sugar and ethanol production. Among the abiotic stress, drought is the main one that negatively impact sugarcane yield. Although gene expression analysis through quantitative PCR (qPCR) has increased our knowledge about biological processes related to drought, gene network that mediates sugarcane responses to water deficit remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving qPCR. In this study, candidate genes were tested for their suitable as reference genes for qPCR analyses in two sugarcane cultivars with varying drought tolerance. Eight candidate reference genes were evaluated in leaves sampled in plants subjected to water deficit in both field and greenhouse conditions. In addition, five genes were evaluated in shoot roots of plants subjected to water deficit by adding PEG8000 to the nutrient solution. NormFinder and RefFinder algorithms were used to identify the most stable gene(s) among genotypes and under different experimental conditions. Both algorithms revealed that in leaf samples, UBQ1 and GAPDH genes were more suitable as reference genes, whereas GAPDH was the best reference one in shoot roots. Reference genes suitable for sugarcane under water deficit were identified, which would lead to a more accurate and reliable analysis of qPCR. Thus, results obtained in this study may guide future research on gene expression in sugarcane under varying water conditions.

Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

PubMed

Rojas, Alicia; Segev, Gilad; Markovics, Alex; Aroch, Itamar; Baneth, Gad

2017-09-19

Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The performance of these molecular assays in feces was compared to fecal flotation and to the previously described cox1 gene semi-nested PCR in 18 fecal samples from dogs with clinical oesophageal spirocercosis diagnosed by oesophagoscopy. The HRM qPCR for ITS1 and 18S were both able to detect 0.2 S. lupi eggs per gram (epg), while the HRM qPCR for the cytb and the semi-nested PCR for the cox1 detected 6 epg and 526 epg, respectively. Spirocerca lupi was detected in 61.1%, 44.4%, 27.8%, 11.1% and 5.6% of the fecal samples of dogs diagnosed with spirocercosis by using the ITS1 and 18S HRM qPCR assays, fecal flotation, cytb HRM qPCR and cox1 semi-nested PCR, respectively. All dogs positive by fecal flotation were also positive by ITS1 and 18S HRM qPCRs. Quantification of S. lupi eggs was successfully achieved in the HRM qPCRs and compared to the fecal flotation with no significant difference in the calculated concentrations between the HRM qPCRs that detected the 18S and ITS1 loci and the fecal flotation. The HRM qPCR for the 18S cross-amplified DNA from Toxocara canis and Toxascaris leonina. In contrast, the HRM qPCR for ITS1 did not cross-amplify DNA from other canine gastrointestinal parasites. This study presents two new molecular assays with significantly increased sensitivity for confirming and quantifying fecal S. lupi eggs. Of these, the HRM qPCR for ITS1 showed the best performance in terms of the limit of detection and absence of cross-amplification with other parasites. These assays will be

Effect of Environmental Parameters on the qPCR Signal of Enterococci in Tropical Waters

EPA Science Inventory

Fecal contamination is the major source of pathogens in recreational waters. The need for quick public notifications has expanded the interest in the use of a rapid, quantitative polymerase chain reaction method (qPCR) to determine enterococci density. However, very little info...

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.

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.

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.

Kinetics of Poliovirus Shedding following Oral Vaccination as Measured by Quantitative Reverse Transcription-PCR versus Culture

PubMed Central

Begum, Sharmin; Uddin, Md Jashim; Platts-Mills, James A.; Liu, Jie; Kirkpatrick, Beth D.; Chowdhury, Anwarul H.; Jamil, Khondoker M.; Haque, Rashidul; Petri, William A.; Houpt, Eric R.

2014-01-01

Amid polio eradication efforts, detection of oral polio vaccine (OPV) virus in stool samples can provide information about rates of mucosal immunity and allow estimation of the poliovirus reservoir. We developed a multiplex one-step quantitative reverse transcription-PCR (qRT-PCR) assay for detection of OPV Sabin strains 1, 2, and 3 directly in stool samples with an external control to normalize samples for viral quantity and compared its performance with that of viral culture. We applied the assay to samples from infants in Dhaka, Bangladesh, after the administration of trivalent OPV (tOPV) at weeks 14 and 52 of life (on days 0 [pre-OPV], +4, +11, +18, and +25 relative to vaccination). When 1,350 stool samples were tested, the sensitivity and specificity of the quantitative PCR (qPCR) assay were 89 and 91% compared with culture. A quantitative relationship between culture+/qPCR+ and culture−/qPCR+ stool samples was observed. The kinetics of shedding revealed by qPCR and culture were similar. qPCR quantitative cutoffs based on the day +11 or +18 stool samples could be used to identify the culture-positive shedders, as well as the long-duration or high-frequency shedders. Interestingly, qPCR revealed that a small minority (7%) of infants contributed the vast majority (93 to 100%) of the total estimated viral excretion across all subtypes at each time point. This qPCR assay for OPV can simply and quantitatively detect all three Sabin strains directly in stool samples to approximate shedding both qualitatively and quantitatively. PMID:25378579

Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples

USGS Publications Warehouse

Stelzer, Erin A.; Strickler, Kriston M.; Schill, William B.

2012-01-01

During summer and early fall 2010, 15 river samples and 6 fecal-source samples were collected in West Virginia. These samples were analyzed by three laboratories for three microbial source tracking (MST) markers: AllBac, a general fecal indicator; BacHum, a human-associated fecal indicator; and BoBac, a ruminant-associated fecal indicator. MST markers were analyzed by means of the quantitative polymerase chain reaction (qPCR) method. The aim was to assess interlaboratory precision when the three laboratories used the same MST marker and shared deoxyribonucleic acid (DNA) extracts of the samples, but different equipment, reagents, and analyst experience levels. The term assay refers to both the markers and the procedure differences listed above. Interlaboratory precision was best for all three MST assays when using the geometric mean absolute relative percent difference (ARPD) and Friedman's statistical test as a measure of interlaboratory precision. Adjustment factors (one for each MST assay) were calculated using results from fecal-source samples analyzed by all three laboratories and applied retrospectively to sample concentrations to account for differences in qPCR results among labs using different standards and procedures. Following the application of adjustment factors to qPCR results, ARPDs were lower; however, statistically significant differences between labs were still observed for the BacHum and BoBac assays. This was a small study and two of the MST assays had 52 percent of samples with concentrations at or below the limit of accurate quantification; hence, more testing could be done to determine if the adjustment factors would work better if the majority of sample concentrations were above the quantification limit.

Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR.

PubMed

Zhong, Qun; Bhattacharya, Smiti; Kotsopoulos, Steven; Olson, Jeff; Taly, Valérie; Griffiths, Andrew D; Link, Darren R; Larson, Jonathan W

2011-07-07

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented. This journal is © The Royal Society of Chemistry 2011

Quantification of hookworm ova from wastewater matrices using quantitative PCR.

PubMed

Gyawali, Pradip; Ahmed, Warish; Sidhu, Jatinder P; Jagals, Paul; Toze, Simon

2017-07-01

A quantitative PCR (qPCR) assay was used to quantify Ancylostoma caninum ova in wastewater and sludge samples. We estimated the average gene copy numbers for a single ovum using a mixed population of ova. The average gene copy numbers derived from the mixed population were used to estimate numbers of hookworm ova in A. caninum seeded and unseeded wastewater and sludge samples. The newly developed qPCR assay estimated an average of 3.7×10 3 gene copies per ovum, which was then validated by seeding known numbers of hookworm ova into treated wastewater. The qPCR estimated an average of (1.1±0.1), (8.6±2.9) and (67.3±10.4) ova for treated wastewater that was seeded with (1±0), (10±2) and (100±21) ova, respectively. The further application of the qPCR assay for the quantification of A. caninum ova was determined by seeding a known numbers of ova into the wastewater matrices. The qPCR results indicated that 50%, 90% and 67% of treated wastewater (1L), raw wastewater (1L) and sludge (~4g) samples had variable numbers of A. caninum gene copies. After conversion of the qPCR estimated gene copy numbers to ova for treated wastewater, raw wastewater, and sludge samples, had an average of 0.02, 1.24 and 67 ova, respectively. The result of this study indicated that qPCR can be used for the quantification of hookworm ova from wastewater and sludge samples; however, caution is advised in interpreting qPCR generated data for health risk assessment. Copyright © 2017. Published by Elsevier B.V.

Comparison of traditional culture and molecular qPCR for detection of simultaneous carriage of multiple pneumococcal serotypes in African children.

PubMed

Olwagen, Courtney P; Adrian, Peter V; Madhi, Shabir A

2017-07-05

S. pneumoniae is a common colonizer of the human nasopharynx in high income and low-middle income countries. Due to limitations of standard culture methods, the prevalence of concurrent colonization with multiple serotypes is unclear. We evaluated the use of multiplex quantitative PCR (qPCR) to detect multiple pneumococcal serotypes/group colonization in archived nasopharyngeal swabs of pneumococcal conjugate vaccine naive children who had previously been investigated by traditional culture methods. Overall the detection of pneumococcal colonization was higher by qPCR (82%) compared to standard culture (71%; p < 0.001), with a high concordance (kappa = 0.73) of serotypes/groups identified by culture also being identified by qPCR. Also, qPCR was more sensitive in detecting multiple serotype/groups among colonized cases (28.7%) compared to culture (4.5%; p < 0.001). Of the additional serotypes detected only by qPCR, the majority were of lower density (<10 4 CFU/ml) than the dominant colonizing serotype, with serotype/group 6A/B, 19B/F and 23F being the highest density colonizers, followed by serotype 5 and serogroup 9A/L/N/V being the most common second and third colonizers respectively. The ability of qPCR to detect multiple pneumococcal serotypes at a low carriage density might provide better insight into underlying mechanism for changes in serotype colonization in PCV vaccinated children.

Molecular Quantification of Zooplankton Gut Content: The Case For qPCR

NASA Astrophysics Data System (ADS)

Frischer, M. E.; Walters, T. L.; Gibson, D. M.; Nejstgaard, J. C.; Troedsson, C.

2016-02-01

The ability to obtain information about feeding selectivity and rates in situ for zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, directly estimating feeding selection and rates of zooplankton, without bias, associated with culturing conditions has been notoriously difficult. A potential approach for addressing this problem is to target prey-specific DNA as a marker for prey ingestion and selection. In this study we report the development of a differential length amplification quantitative PCR (dla-qPCR) assay targeting the 18S rRNA gene to validate the use of a DNA-based approach to quantify consumption of specific plankton prey by the pelagic tunicate (doliolid) Dolioletta gegenbauri. Compared to copepods and other marine animals, the digestion of prey genomic DNA inside the gut of doliolids is low. This method minimizes potential underestimations, and therefore allows prey DNA to be used as an effective indicator of prey consumption. We also present an initial application of a qPCR-assay to estimate consumption of specific prey species on the southeastern continental shelf of the U.S., where doliolids stochastically bloom in response to upwelling events. Estimated feeding rates, based on qPCR, were in the same range as those estimated from clearance rates in laboratory feeding studies. In the field, consumption of specific prey, including the centric diatom Thalassiosira spp. was detected in the gut of wild caught D. gegenbauri at the levels consistent with their abundance in the water column at the time of collection. Thus, both experimental and field investigations support the hypothesis that a qPCR approach will be useful for the quantitative investigation of the in situ diet of D. gegenbauri without introduced bias' associated with cultivation.

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.

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...

Detection of group a streptococcal pharyngitis by quantitative PCR.

PubMed

Dunne, Eileen M; Marshall, Julia L; Baker, Ciara A; Manning, Jayne; Gonis, Gena; Danchin, Margaret H; Smeesters, Pierre R; Satzke, Catherine; Steer, Andrew C

2013-07-11

Group A streptococcus (GAS) is the most common bacterial cause of sore throat. School-age children bear the highest burden of GAS pharyngitis. Accurate diagnosis is difficult: the majority of sore throats are viral in origin, culture-based identification of GAS requires 24-48 hours, and up to 15% of children are asymptomatic throat carriers of GAS. The aim of this study was to develop a quantitative polymerase chain reaction (qPCR) assay for detecting GAS pharyngitis and assess its suitability for clinical diagnosis. Pharyngeal swabs were collected from children aged 3-18 years (n = 91) and adults (n = 36) located in the Melbourne area who presented with sore throat. Six candidate PCR assays were screened using a panel of reference isolates, and two of these assays, targeting speB and spy1258, were developed into qPCR assays. The qPCR assays were compared to standard culture-based methods for their ability to detect GAS pharyngitis. GAS isolates from culture positive swabs underwent emm-typing. Clinical data were used to calculate McIsaac scores as an indicator of disease severity. Twenty-four of the 127 samples (18.9%) were culture-positive for GAS, and all were in children (26%). The speB qPCR had 100% sensitivity and 100% specificity compared with gold-standard culture, whereas the spy1258 qPCR had 87% sensitivity and 100% specificity. Nine different emm types were found, of which emm 89, 3, and 28 were most common. Bacterial load as measured by qPCR correlated with culture load. There were no associations between symptom severity as indicated by McIsaac scores and GAS bacterial load. The speB qPCR displayed high sensitivity and specificity and may be a useful tool for GAS pharyngitis diagnosis and research.

Rapid quantification of plant-powdery mildew interactions by qPCR and conidiospore counts.

PubMed

Weßling, Ralf; Panstruga, Ralph

2012-08-31

The powdery mildew disease represents a valuable patho-system to study the interaction between plant hosts and obligate biotrophic fungal pathogens. Numerous discoveries have been made on the basis of the quantitative evaluation of plant-powdery mildew interactions, especially in the context of hyper-susceptible and/or resistant plant mutants. However, the presently available methods to score the pathogenic success of powdery mildew fungi are laborious and thus not well suited for medium- to high-throughput analysis. Here we present two new protocols that allow the rapid quantitative assessment of powdery mildew disease development. One procedure depends on quantitative polymerase chain reaction (qPCR)-based evaluation of fungal biomass, while the other relies on the quantification of fungal conidiospores. We validated both techniques using the powdery mildew pathogen Golovinomyces orontii on a set of hyper-susceptible and resistant Arabidopsis thaliana mutants and found that both cover a wide dynamic range of one to two (qPCR) and four to five (quantification of conidia) orders of magnitude, respectively. The two approaches yield reproducible results and are easy to perform without specialized equipment. The qPCR and spore count assays rapidly and reproducibly quantify powdery mildew pathogenesis. Our methods are performed at later stages of infection and discern mutant phenotypes accurately. The assays therefore complement currently used procedures of powdery mildew quantification and can overcome some of their limitations. In addition, they can easily be adapted to other plant-powdery mildew patho-systems.

Sample-ready multiplex qPCR assay for detection of malaria.

PubMed

Kamau, Edwin; Alemayehu, Saba; Feghali, Karla C; Juma, Dennis W; Blackstone, George M; Marion, William R; Obare, Peter; Ogutu, Bernhards; Ockenhouse, Christian F

2014-04-25

Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, "wet" assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance. The limit of detection for the MMSR assay was 0.244 parasites/μL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to "wet" assay which was 0.39 and 3.13 parasites/μL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the "wet" assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the "wet" assay. The MMSR

Is qPCR a Reliable Indicator of Cyanotoxin Risk in Freshwater?

PubMed Central

Pacheco, Ana Beatriz F.; Guedes, Iame A.; Azevedo, Sandra M.F.O.

2016-01-01

The wide distribution of cyanobacteria in aquatic environments leads to the risk of water contamination by cyanotoxins, which generate environmental and public health issues. Measurements of cell densities or pigment contents allow both the early detection of cellular growth and bloom monitoring, but these methods are not sufficiently accurate to predict actual cyanobacterial risk. To quantify cyanotoxins, analytical methods are considered the gold standards, but they are laborious, expensive, time-consuming and available in a limited number of laboratories. In cyanobacterial species with toxic potential, cyanotoxin production is restricted to some strains, and blooms can contain varying proportions of both toxic and non-toxic cells, which are morphologically indistinguishable. The sequencing of cyanobacterial genomes led to the description of gene clusters responsible for cyanotoxin production, which paved the way for the use of these genes as targets for PCR and then quantitative PCR (qPCR). Thus, the quantification of cyanotoxin genes appeared as a new method for estimating the potential toxicity of blooms. This raises a question concerning whether qPCR-based methods would be a reliable indicator of toxin concentration in the environment. Here, we review studies that report the parallel detection of microcystin genes and microcystin concentrations in natural populations and also a smaller number of studies dedicated to cylindrospermopsin and saxitoxin. We discuss the possible issues associated with the contradictory findings reported to date, present methodological limitations and consider the use of qPCR as an indicator of cyanotoxin risk. PMID:27338471

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

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.

PubMed

Joshi, Molishree; Keith Pittman, H; Haisch, Carl; Verbanac, Kathryn

2008-09-01

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

EPA Science Inventory

Quantitative PCR (qPCR) techniques have been widely used to measure Dehalococcoides (Dhc) DNA in the groundwater at field sites for several years. Interpretation of these data may be complicated when different laboratories using alternate methods conduct the analysis. An...

Relationship and Variation of qPCR and Culturable Enterococci Estimates in Ambient Surface Waters Are Predictable

EPA Science Inventory

The quantitative polymerase chain reaction (qPCR) method provides rapid estimates of fecal indicator bacteria densities that have been indicated to be useful in the assessment of water quality. Primarily because this method provides faster results than standard culture-based meth...

The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE).

PubMed

Taylor, Sean C; Mrkusich, Eli M

2014-01-01

In the past decade, the techniques of quantitative PCR (qPCR) and reverse transcription (RT)-qPCR have become accessible to virtually all research labs, producing valuable data for peer-reviewed publications and supporting exciting research conclusions. However, the experimental design and validation processes applied to the associated projects are the result of historical biases adopted by individual labs that have evolved and changed since the inception of the techniques and associated technologies. This has resulted in wide variability in the quality, reproducibility and interpretability of published data as a direct result of how each lab has designed their RT-qPCR experiments. The 'minimum information for the publication of quantitative real-time PCR experiments' (MIQE) was published to provide the scientific community with a consistent workflow and key considerations to perform qPCR experiments. We use specific examples to highlight the serious negative ramifications for data quality when the MIQE guidelines are not applied and include a summary of good and poor practices for RT-qPCR. © 2013 S. Karger AG, Basel.

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.

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

PubMed Central

Te, Shu Harn; Chen, Enid Yingru

2015-01-01

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

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.

Sample-ready multiplex qPCR assay for detection of malaria

PubMed Central

2014-01-01

Background Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. Methods A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, “wet” assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance. Results The limit of detection for the MMSR assay was 0.244 parasites/μL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to “wet” assay which was 0.39 and 3.13 parasites/μL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the “wet” assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity

Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

PubMed

Arvia, Rosaria; Sollai, Mauro; Pierucci, Federica; Urso, Carmelo; Massi, Daniela; Zakrzewska, Krystyna

2017-08-01

Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥10 5 copies per reaction, while the upper limit of ddPCR was 10 4 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome. Copyright © 2017 Elsevier B.V. All rights reserved.

QUANTITATIVE PCR ANALYSIS OF HOUSE DUST CAN REVEAL ABNORMAL MOLD CONDITIONS

EPA Science Inventory

Indoor mold populations were measured in the dust of homes in Cleveland and Cincinnati, OH, by quantitative PCR (QPCR) and, in Cincinnati, also by culturing. QPCR assays for 82 species (or groups of species) were used to identify and quantify indoor mold populations in moldy home...

Design and optimization of reverse-transcription quantitative PCR experiments.

PubMed

Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael

2009-10-01

Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

EPA Science Inventory

Purified oocysts of Cryptosporidium parvum were used to evaluate applicability of two quantitative PCR (qPCR) viability detection methods in raw surface water and disinfection treated water. Propidium monoazide-qPCR targeting hsp70 gene was compared to reverse transcription (RT)-...

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

PubMed

Te, Shu Harn; Chen, Enid Yingru; Gin, Karina Yew-Hoong

2015-08-01

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

PubMed Central

Staggemeier, Rodrigo; Pilger, Diogo André; Spilki, Fernando Rosado; Cantarelli, Vlademir Vicente

2014-01-01

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species. PMID:24626408

Multiplex SYBR® green-real time PCR (qPCR) assay for the detection and differentiation of Bartonella henselae and Bartonella clarridgeiae in cats.

PubMed

Staggemeier, Rodrigo; Pilger, Diogo André; Spilki, Fernando Rosado; Cantarelli, Vlademir Vicente

2014-01-01

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR.

PubMed

D'Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

2016-01-01

Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new "Free-ITR" qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

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.

Reference Gene Selection for qPCR Normalization of Kosteletzkya virginica under Salt Stress

PubMed Central

Tang, Xiaoli; Wang, Hongyan; Shao, Chuyang; Shao, Hongbo

2015-01-01

Kosteletzkya virginica (L.) is a newly introduced perennial halophytic plant. Presently, reverse transcription quantitative real-time PCR (qPCR) is regarded as the best choice for analyzing gene expression and its accuracy mainly depends on the reference genes which are used for gene expression normalization. In this study, we employed qPCR to select the most stable reference gene in K. virginica which showed stable expression profiles under our experimental conditions. The candidate reference genes were 18S ribosomal RNA (18SrRNA), β-actin (ACT), α-tubulin (TUA), and elongation factor (EF). We tracked the gene expression profiles of the candidate genes and analyzed their stabilities through BestKeeper, geNorm, and NormFinder software programs. The results of the three programs were identical and 18SrRNA was assessed to be the most stable reference gene in this study. However, TUA was identified to be the most unstable. Our study proved again that the traditional reference genes indeed displayed a certain degree of variations under given experimental conditions. Importantly, our research also provides guidance for selecting most suitable reference genes and lays the foundation for further studies in K. virginica. PMID:26581422

Synthetic internal control sequences to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi

USDA-ARS?s Scientific Manuscript database

Quantitative PCR (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5’-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi, the soybean rust pathogen. Because of the extreme sensitivity of Q-PCR, the DNA of a single u...

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 approaches for microbial community studies in wastewater treatment systems: applications and considerations.

PubMed

Kim, Jaai; Lim, Juntaek; Lee, Changsoo

2013-12-01

Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results. Copyright © 2013 Elsevier Inc. All rights reserved.

Quantitative PCR and Digital PCR for Detection of Ascaris lumbricoides Eggs in Reclaimed Water

PubMed Central

Santísima-Trinidad, Ana Belén; Bornay-Llinares, Fernando Jorge; Martín González, Marcos; Pascual Valero, José Antonio; Ros Muñoz, Margarita

2017-01-01

The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control. Ascaris lumbricoides is one of the soil-transmitted helminths (STH) with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO) recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR) and digital PCR (dPCR) were able to detect low amounts of A. lumbricoides eggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as one A. lumbricoides egg equivalent, while dPCR can detect DNA concentrations as low as five A. lumbricoides egg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20 A. lumbricoides eggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks. PMID:28377928

Quantitative polymerase chain reaction (PCR) for detection of aquatic animal pathogens in a diagnostic laboratory setting

USGS Publications Warehouse

Purcell, Maureen K.; Getchell, Rodman G.; McClure, Carol A.; Weber, S.E.; Garver, Kyle A.

2011-01-01

Real-time, or quantitative, polymerase chain reaction (qPCR) is quickly supplanting other molecular methods for detecting the nucleic acids of human and other animal pathogens owing to the speed and robustness of the technology. As the aquatic animal health community moves toward implementing national diagnostic testing schemes, it will need to evaluate how qPCR technology should be employed. This review outlines the basic principles of qPCR technology, considerations for assay development, standards and controls, assay performance, diagnostic validation, implementation in the diagnostic laboratory, and quality assurance and control measures. These factors are fundamental for ensuring the validity of qPCR assay results obtained in the diagnostic laboratory setting.

Development of a Dry-Reagent-Based qPCR to Facilitate the Diagnosis of Mycobacterium ulcerans Infection in Endemic Countries

PubMed Central

Babonneau, Jérémie; Bernard, Christian; Marion, Estelle; Chauty, Annick; Kempf, Marie; Robert, Raymond; Marsollier, Laurent

2015-01-01

Background Buruli ulcer is a neglected tropical disease caused by Mycobacterium ulcerans. This skin disease is the third most common mycobacterial disease and its rapid diagnosis and treatment are necessary. Polymerase chain reaction (PCR) is considered to be the most sensitive method for the laboratory confirmation of Buruli ulcer. However, PCR remains expensive and involves reagents unsuitable for use in tropical countries with poor storage conditions, hindering the development of reliable quantitative PCR (qPCR) diagnosis. We aimed to overcome this problem by developing a ready-to-use dry qPCR mix for the diagnosis of M. ulcerans infection. Methodology/Principal Findings We compared the efficiency of three different dry qPCR mixes, lyophilized with various concentrations of cryoprotectants, with that of a freshly prepared mixture, for the detection of a standard range of M. ulcerans DNA concentrations. We evaluated the heat resistance of the dry mixes, comparing them with the fresh mix after heating. We also evaluated one of the dry mixes in field conditions, by analyzing 93 specimens from patients with suspected Buruli ulcers. The dry mix was (i) highly resistant to heat; (ii) of similar sensitivity and efficiency to the fresh mix and (iii) easier to use than the fresh mix. Conclusions Dry qPCR mixes are suitable for use in the diagnosis of M. ulcerans infection in endemic countries. The user-friendly format of this mix makes it possible for untrained staff to perform diagnostic tests with a limited risk of contamination. The possibility of using this mix in either vial or strip form provides considerable flexibility for the management of small or large amounts of sample. Thus, dry-mix qPCR could be used as a reliable tool for the diagnosis of Buruli ulcer in the field. PMID:25830546

A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum.

PubMed

Marques, Lucas M; Amorim, Aline T; Martins, Hellen Braga; Rezende, Izadora Souza; Barbosa, Maysa Santos; Lobão, Tassia Neves; Campos, Guilherme B; Timenetsky, Jorge

2013-12-27

Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples. Copyright © 2013. Published by Elsevier B.V.

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.

An international trial of quantitative PCR for monitoring Legionella in artificial water systems.

PubMed

Lee, J V; Lai, S; Exner, M; Lenz, J; Gaia, V; Casati, S; Hartemann, P; Lück, C; Pangon, B; Ricci, M L; Scaturro, M; Fontana, S; Sabria, M; Sánchez, I; Assaf, S; Surman-Lee, S

2011-04-01

  To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae.   Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ≥10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l(-1) ) and culture (CFU l(-1) ) for Legionella pneumophila (0·71) and for Legionella spp. (2·03). In hot and cold water (506 samples), the differences were less, 0·62 for Leg. pneumophila and 1·05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l(-1) always exceeded CFU l(-1) , and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ≥50°C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring.   Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required.   This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

The use of ELISA, nPCR and qPCR for diagnosis of ocular toxoplasmosis in experimentally infected pigs.

PubMed

Garcia, João Luis; Burrells, Alison; Bartley, Paul M; Bartley, Kathryn; Innes, Elisabeth A; Katzer, Frank

2017-12-01

In the present study we experimentally infected pigs with T. gondii tachyzoites, bradyzoites and oocysts in order to evaluate IgG-ELISA, nested-PCR, and qPCR for diagnosis of ocular infection. Eighteen pigs were divided into four groups: G1 (infected with 10 3 tissue cysts of the M4 strain (type II) at day 28, n=5), G2 (infected with 10 3 oocysts of the M4 strain at day 28, n=5), G3 (infected with tachyzoites of S48 strain (type 1) at day 0, n=5), and G4 (uninfected unchallenged, control group n=3). At day 70 of the experiment all animals were culled, and serum, aqueous humor (AH) and vitreous humor (VH) samples were collected to perform indirect ELISA, and PCR (nPCR, and qPCR). By ELISA nine pigs (60%) out of 15 were positive in VH samples, and seven out of 15 (46%) were positive in AH samples. Both molecular techniques used here, nPCR and qPCR, were able to detect <50fg of T. gondii tachyzoite DNA. The nPCR and qPCR detected six (7/15, 47%) and two (2/15, 13.3%) positive animals respectively. Antibody responses were detected in serum and in AH and VH from the eye, suggesting that pigs may be an animal that could be used as a model to further our understanding of diagnosis of human ocular infection with T. gondii. Copyright © 2017 Elsevier Ltd. All rights reserved.

O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis.

PubMed

Mekonnen, Solomon A; Beissner, Marcus; Saar, Malkin; Ali, Solomon; Zeynudin, Ahmed; Tesfaye, Kassahun; Adbaru, Mulatu G; Battke, Florian; Poppert, Sven; Hoelscher, Michael; Löscher, Thomas; Bretzel, Gisela; Herbinger, Karl-Heinz

2017-10-02

Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR methods

EPA Science Inventory

Quantitative polymerase chain reaction (qPCR) is increasingly being used for the quantitative detection of fecal indicator bacteria in beach water. QPCR allows for same-day health warnings, and its application is being considered as an optionn for recreational water quality testi...

Comparison of Enterococcus qPCR analysis results from fresh and marine water samples on two real-time instruments -

EPA Science Inventory

EPA is currently considering a quantitative polymerase chain reaction (qPCR) method, targeting Enterococcus spp., for beach monitoring. Improvements in the method’s cost-effectiveness may be realized by the use of newer instrumentation such as the Applied Biosystems StepOneTM a...

Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters.

PubMed

Riedel, Timothy E; Zimmer-Faust, Amity G; Thulsiraj, Vanessa; Madi, Tania; Hanley, Kaitlyn T; Ebentier, Darcy L; Byappanahalli, Muruleedhara; Layton, Blythe; Raith, Meredith; Boehm, Alexandria B; Griffith, John F; Holden, Patricia A; Shanks, Orin C; Weisberg, Stephen B; Jay, Jennifer A

2014-04-01

Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample. Copyright © 2014 Elsevier Ltd. All rights reserved.

Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters

USGS Publications Warehouse

Riedel, Timothy E.; Zimmer-Faust, Amity G.; Thulsiraj, Vanessa; Madi, Tania; Hanley, Kaitlyn T.; Ebentier, Darcy L.; Byappanahalli, Muruleedhara N.; Layton, Blythe; Raith, Meredith; Boehm, Alexandria B.; Griffith, John F.; Holden, Patricia A.; Shanks, Orin C.; Weisberg, Stephen B.; Jay, Jennifer A.

2014-01-01

Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample.

Detection and quantitation of HPV in genital and oral tissues and fluids by real time PCR

PubMed Central

2010-01-01

Background Human papillomaviruses (HPVs) remain a serious world health problem due to their association with anogenital/oral cancers and warts. While over 100 HPV types have been identified, a subset is associated with malignancy. HPV16 and 18 are the most prevalent oncogenic types, while HPV6 and 11 are most commonly responsible for anogenital warts. While other quantitative PCR (qPCR) assays detect oncogenic HPV, there is no single tube assay distinguishing the most frequent oncogenic types and the most common types found in warts. Results A Sybr Green-based qPCR assay was developed utilizing degenerate primers to the highly conserved HPV E1 theoretically detecting any HPV type. A single tube multiplex qPCR assay was also developed using type-specific primer pairs and TaqMan probes that allowed for detection and quantitation of HPV6,11,16,18. Each HPV type was detected over a range from 2 × 101 to 2 × 106copies/reaction providing a reliable method of quantitating type-specific HPV in 140 anogenital/cutaneous/oral benign and malignant specimens. 35 oncogenic and low risk alpha genus HPV types were detected. Concordance was detected in previously typed specimens. Comparisons to the gold standard detected an overall sensitivity of 89% (95% CI: 77% - 96%) and specificity of 90% (95%CI: 52% - 98%). Conclusion There was good agreement between the ability of the qPCR assays described here to identify HPV types in malignancies previously typed using standard methods. These novel qPCR assays will allow rapid detection and quantitation of HPVs to assess their role in viral pathogenesis. PMID:20723234

Laser micro-dissection and qPCR for identifying specific HPV types responsible for malignancy in penile lesions.

PubMed

Lebelo, Ramokone L; Thys, Sofie; Benoy, Ina; Depuydt, Christophe E; Bogers, John-Paul; Bida, Meshack N; Mphahlele, M Jeffrey

2015-10-01

The aim of the study was to identify specific human papillomavirus (HPV) type responsible for malignancy in penile tissue samples using laser micro-dissection and TaqMan quantitative real-time PCR (qPCR). The study was based on two pre-malignant and seven malignant penile tissue samples and laser micro-dissection was performed on all. Genotyping was performed on whole tissue sections and laser micro-dissection samples using qPCR. Two whole tissue section samples were HPV negative while seven were HPV positive. In four samples that were single HPV infections with whole tissue section PCR, identical HPV types were confirmed with laser micro-dissection PCR. Clearly confirming that the single HPV type detected is responsible for malignancy. In two samples that had multiple HPV infections with whole tissue section PCR, only one HPV type with the highest viral load was detected with laser micro-dissection PCR, suggesting that the HPV type with the highest viral load is most likely the cause of that particular lesion. HPV 11 and/or HPV 16 were the only types detected with laser micro-dissection PCR in these cases, compared to multiple HPV types (HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, and HPV 39) initially detected with whole tissue section PCR. HPV 11 was associated with verrucous lesions while HPV 16 was associated with squamous cell carcinoma and PIN 3 lesions. This study confirms that laser micro-dissection and qPCR are essential tools in identifying the HPV types responsible for malignancy in penile lesions, particularly in samples with multiple infections. © 2015 Wiley Periodicals, Inc.

Telomere content measurement in human hematopoietic cells: Comparative analysis of qPCR and Flow-FISH techniques.

PubMed

Wand, Taylor; Fang, Mike; Chen, Christina; Hardy, Nathan; McCoy, J Philip; Dumitriu, Bogdan; Young, Neal S; Biancotto, Angélique

2016-10-01

Abnormal telomere lengths have been linked to cancer and other hematologic disorders. Determination of mean telomere content (MTC) is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Here, we compared a quantitative Polymerase Chain Reaction approach (qPCR) and a flow cytometric approach, fluorescence in situ hybridization (Flow-FISH), to evaluate telomere content distribution in total patient peripheral blood mononuclear cells or specific cell populations. Flow-FISH is based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA) 3 probe and DNA staining with propidium iodide. We showed that both qPCR and Flow-FISH provide a robust measurement, with Flow-FISH measuring a relative content longer than qPCR at a single cell approach and that TRF2 fluorescence intensity did not correlate with MTC. Both methods showed comparable telomere content reduction with age, and the rate of relative telomere loss was similar. Published 2016 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America. Published 2016 Wiley Periodicals Inc. This article is a US government work and, as such, is in the public domain in the United States of America.

Comparison of Enterococcus qPCR analysis results from fresh and marine water samples on two real-time instruments - poster

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for widespread implementation of this or ...

Detection of Legionella species in environmental water by the quantitative PCR method in combination with ethidium monoazide treatment.

PubMed

Inoue, Hiroaki; Takama, Tomoko; Yoshizaki, Miwa; Agata, Kunio

2015-01-01

We detected Legionella species in 111 bath water samples and 95 cooling tower water samples by using a combination of conventional plate culture, quantitative polymerase chain reaction (qPCR) and qPCR combined with ethidium monoazide treatment (EMA-qPCR) methods. In the case of bath water samples, Legionella spp. were detected in 30 samples by plate culture, in 85 samples by qPCR, and in 49 samples by EMA-qPCR. Of 81 samples determined to be Legionella-negative by plate culture, 56 and 23 samples were positive by qPCR and EMA-qPCR, respectively. Therefore, EMA treatment decreased the number of Legionella-positive bath water samples detected by qPCR. In contrast, EMA treatment had no effect on cooling tower water samples. We therefore expect that EMA-qPCR is a useful method for the rapid detection of viable Legionella spp. from bath water samples.

Evaluation and utilization of preassembled frozen commercial fast real-time qPCR master mixes for detection of cytomegalovirus and BK virus.

PubMed

Glover, William A; Atienza, Ederlyn E; Nesbitt, Shannon; Kim, Woo J; Castor, Jared; Cook, Linda; Jerome, Keith R

2016-01-01

Quantitative DNA detection of cytomegalovirus (CMV) and BK virus (BKV) is critical in the management of transplant patients. Quantitative laboratory-developed procedures for CMV and BKV have been described in which much of the processing is automated, resulting in rapid, reproducible, and high-throughput testing of transplant patients. To increase the efficiency of such assays, the performance and stability of four commercial preassembled frozen fast qPCR master mixes (Roche FastStart Universal Probe Master Mix with Rox, Bio-Rad SsoFast Probes Supermix with Rox, Life Technologies TaqMan FastAdvanced Master Mix, and Life Technologies Fast Universal PCR Master Mix), in combination with in-house designed primers and probes, was evaluated using controls and standards from standard CMV and BK assays. A subsequent parallel evaluation using patient samples was performed comparing the performance of freshly prepared assay mixes versus aliquoted frozen master mixes made with two of the fast qPCR mixes (Life Technologies TaqMan FastAdvanced Master Mix, and Bio-Rad SsoFast Probes Supermix with Rox), chosen based on their performance and compatibility with existing PCR cycling conditions. The data demonstrate that the frozen master mixes retain excellent performance over a period of at least 10 weeks. During the parallel testing using clinical specimens, no difference in quantitative results was observed between the preassembled frozen master mixes and freshly prepared master mixes. Preassembled fast real-time qPCR frozen master mixes perform well and represent an additional strategy laboratories can implement to reduce assay preparation times, and to minimize technical errors and effort necessary to perform clinical PCR. © 2015 Wiley Periodicals, Inc.

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.

Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.

PubMed

Whale, Alexandra S; Huggett, Jim F; Cowen, Simon; Speirs, Valerie; Shaw, Jacqui; Ellison, Stephen; Foy, Carole A; Scott, Daniel J

2012-06-01

One of the benefits of Digital PCR (dPCR) is the potential for unparalleled precision enabling smaller fold change measurements. An example of an assessment that could benefit from such improved precision is the measurement of tumour-associated copy number variation (CNV) in the cell free DNA (cfDNA) fraction of patient blood plasma. To investigate the potential precision of dPCR and compare it with the established technique of quantitative PCR (qPCR), we used breast cancer cell lines to investigate HER2 gene amplification and modelled a range of different CNVs. We showed that, with equal experimental replication, dPCR could measure a smaller CNV than qPCR. As dPCR precision is directly dependent upon both the number of replicate measurements and the template concentration, we also developed a method to assist the design of dPCR experiments for measuring CNV. Using an existing model (based on Poisson and binomial distributions) to derive an expression for the variance inherent in dPCR, we produced a power calculation to define the experimental size required to reliably detect a given fold change at a given template concentration. This work will facilitate any future translation of dPCR to key diagnostic applications, such as cancer diagnostics and analysis of cfDNA.

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

Development of qPCR systems to quantify shoot infections by canker-causing pathogens in stone fruits and nut crops.

PubMed

Luo, Y; Gu, S; Felts, D; Puckett, R D; Morgan, D P; Michailides, T J

2017-02-01

To develop real-time PCR assays for quantification of shoot infection levels of canker disease of stone fruits and nut crops caused by six fungal pathogen groups. This study focused on six major canker-causing fungal pathogen groups: Phomopsis sp., Botryosphaeria dothidea, Lasiodiplodia sp., Cytospora sp., Neofusicoccum sp. and Diplodia sp., occurring in stone fruits and nut crops in California. DNA primers were designed to specifically target each of the six pathogen groups after the specificity tests using canker-causing and non-canker-causing pathogens and by using DNA sequences of other species from GenBank using blast. The quantitative real-time PCR (qPCR) systems were developed and used to quantify the infection levels of inoculated dried plum shoots. For Neofusicoccum sp. and Phomopsis sp., which were used in inoculation of walnut shoots, the values of the molecular severity ranged from 5·60 to 6·94 during the 16 days of latent infection period. The qPCR assays were more efficient, accurate and precise to quantify latent infections caused by canker-causing pathogens as compared to the traditional plating methods. This study demonstrated the potential of using the developed qPCR systems for epidemiological studies on canker diseases of woody plants. © 2016 The Society for Applied Microbiology.

Human-associated fecal qPCR measurements and predicted risk of gastrointestinal illness in recreational waters contaminated with raw sewage

EPA Science Inventory

We used quantitative microbial risk assessment (QMRA) to estimate the risk of gastrointestinal (GI) illness associated with swimming in recreational waters containing different concentrations of human-associated fecal qPCR markers from raw sewage– HF183 and HumM2. The volume/volu...

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.

QUANTITATIVE PCR OF SELECTED ASPERGILLUS, PENICILLIUM AND PAECILOMYCES SPECIES

EPA Science Inventory

A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan®) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of Aspergillus, Penicillium and ...

Evaluation of quantitative PCR for early diagnosis of Pseudomonas aeruginosa infection in cystic fibrosis: a prospective cohort study.

PubMed

Héry-Arnaud, G; Nowak, E; Caillon, J; David, V; Dirou, A; Revert, K; Munck, M-R; Frachon, I; Haloun, A; Horeau-Langlard, D; Le Bihan, J; Danner-Boucher, I; Ramel, S; Pelletier, M-P; Rosec, S; Gouriou, S; Poulhazan, E; Payan, C; Férec, C; Rault, G; Le Gal, G; Le Berre, R

2017-03-01

Early detection of Pseudomonas aeruginosa lung positivity is a key element in cystic fibrosis (CF) management. PCR has increased the accuracy of detection of many microorganisms. Clinical relevance of P. aeruginosa quantitative PCR (qPCR) in this context is unclear. Our aim was to determine P. aeruginosa qPCR sensitivity and specificity, and to assess the possible time saved by qPCR in comparison with standard practice (culture). A multicentre cohort study was conducted over a 3-year period in 96 patients with CF without chronic P. aeruginosa colonization. Sputum samples were collected at each visit. Conventional culture and two-step qPCR (oprL qPCR and gyrB/ecfX qPCR) were performed for 707 samples. The positivity criteria were based on the qPCR results, defined in a previous study as follow: oprL qPCR positivity alone if bacterial density was <730 CFU/mL or oprL qPCR combined with gyrB/ecfX qPCR if bacterial density was ≥730 CFU/mL. During follow up, 36 of the 96 patients with CF were diagnosed on culture as colonized with P. aeruginosa. This two-step qPCR displayed a sensitivity of 94.3% (95% CI 79.7%-98.6%), and a specificity of 86.3% (95% CI 83.4%-88.7%). It enabled P. aeruginosa acquisition to be diagnosed earlier in 20 patients, providing a median detection time gain of 8 months (interquartile range 3.7-17.6) for them. Implementing oprL and gyrB/ecfX qPCR in the management of patients with CF allowed earlier detection of first P. aeruginosa lung positivity than culture alone. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia.

PubMed

León, Cielo M; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R; Ramírez, Juan D

2017-01-01

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania . Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 10 1 and 1 × 10 -1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

PubMed Central

León, Cielo M.; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S.; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R.; Ramírez, Juan D.

2017-01-01

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. PMID:29046670

Development of a real-time quantitative PCR assay to enumerate Yersinia pestis in fleas.

PubMed

Gabitzsch, Elizabeth S; Vera-Tudela, Rommelle; Eisen, Rebecca J; Bearden, Scott W; Gage, Kenneth L; Zeidner, Nordin S

2008-07-01

A real-time quantitative polymerase chain reaction (qPCR) assay was developed for Yersina pestis. The qPCR assay was developed utilizing a conserved region of the Y. pestis ferric iron uptake regulator gene (fur) to design primers and a fluorescent (FAM-labeled) TaqMan probe. The assay was optimized using cultured Y. pestis (UG05-0454) and was confirmed to work with strains from 3 Y. pestis biovars. The optimized assay was capable of detecting a single organism of cultured Y. pestis and as little as 300 bacteria in infected flea triturates. This qPCR assay enables rapid enumeration of Y. pestis bacterium in laboratory-infected fleas when compared with conventional serial dilution plating.

MeltMan: Optimization, Evaluation, and Universal Application of a qPCR System Integrating the TaqMan qPCR and Melting Analysis into a Single Assay

PubMed Central

Nagy, Alexander; Černíková, Lenka; Vitásková, Eliška; Křivda, Vlastimil; Dán, Ádám; Dirbáková, Zuzana; Jiřincová, Helena; Procházka, Bohumír; Sedlák, Kamil; Havlíčková, Martina

2016-01-01

In the present work, we optimised and evaluated a qPCR system integrating 6-FAM (6-carboxyfluorescein)-labelled TaqMan probes and melting analysis using the SYTO 82 (S82) DNA binding dye in a single reaction. We investigated the influence of the S82 on various TaqMan and melting analysis parameters and defined its optimal concentration. In the next step, the method was evaluated in 36 different TaqMan assays with a total of 729 paired reactions using various DNA and RNA templates, including field specimens. In addition, the melting profiles of interest were correlated with the electrophoretic patterns. We proved that the S82 is fully compatible with the FAM-TaqMan system. Further, the advantages of this approach in routine diagnostic TaqMan qPCR were illustrated with practical examples. These included solving problems with flat or other atypical amplification curves or even false negativity as a result of probe binding failure. Our data clearly show that the integration of the TaqMan qPCR and melting analysis into a single assay provides an additional control option as well as the opportunity to perform more complex analyses, get more data from the reactions, and obtain analysis results with higher confidence. PMID:27031831

Legionella in water samples: how can you interpret the results obtained by quantitative PCR?

PubMed

Ditommaso, Savina; Ricciardi, Elisa; Giacomuzzi, Monica; Arauco Rivera, Susan R; Zotti, Carla M

2015-02-01

Evaluation of the potential risk associated with Legionella has traditionally been determined from culture-based methods. Quantitative polymerase chain reaction (qPCR) is an alternative tool that offers rapid, sensitive and specific detection of Legionella in environmental water samples. In this study we compare the results obtained by conventional qPCR (iQ-Check™ Quanti Legionella spp.; Bio-Rad) and by culture method on artificial samples prepared in Page's saline by addiction of Legionella pneumophila serogroup 1 (ATCC 33152) and we analyse the selective quantification of viable Legionella cells by the qPCR-PMA method. The amount of Legionella DNA (GU) determined by qPCR was 28-fold higher than the load detected by culture (CFU). Applying the qPCR combined with PMA treatment we obtained a reduction of 98.5% of the qPCR signal from dead cells. We observed a dissimilarity in the ability of PMA to suppress the PCR signal in samples with different amounts of bacteria: the effective elimination of detection signals by PMA depended on the concentration of GU and increasing amounts of cells resulted in higher values of reduction. Using the results from this study we created an algorithm to facilitate the interpretation of viable cell level estimation with qPCR-PMA. Copyright © 2014 Elsevier Ltd. All rights reserved.

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.

Comparative evaluation of a laboratory developed real-time PCR assay and the RealStar® HHV-6 PCR Kit for quantitative detection of human herpesvirus 6.

PubMed

Yip, Cyril C Y; Sridhar, Siddharth; Cheng, Andrew K W; Fung, Ami M Y; Cheng, Vincent C C; Chan, Kwok-Hung; Yuen, Kwok-Yung

2017-08-01

HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar ® HHV-6 PCR Kit. The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar ® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log 10 copies/ml and a coefficient of determination (R 2 ) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log 10 and 2 log 10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar ® HHV-6 PCR Kit (R 2 =0.926; P<0.0001), with an average bias of -0.24 log 10 copies/ml. The in-house developed HHV-6 qPCR method is a sensitive and reliable assay with lower cost for the detection and quantification of HHV-6 DNA when compared to the RealStar ® HHV-6 PCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of stable reference genes for quantitative PCR in cells derived from chicken lymphoid organs.

PubMed

Borowska, D; Rothwell, L; Bailey, R A; Watson, K; Kaiser, P

2016-02-01

Quantitative polymerase chain reaction (qPCR) is a powerful technique for quantification of gene expression, especially genes involved in immune responses. Although qPCR is a very efficient and sensitive tool, variations in the enzymatic efficiency, quality of RNA and the presence of inhibitors can lead to errors. Therefore, qPCR needs to be normalised to obtain reliable results and allow comparison. The most common approach is to use reference genes as internal controls in qPCR analyses. In this study, expression of seven genes, including β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-glucuronidase (GUSB), TATA box binding protein (TBP), α-tubulin (TUBAT) and 28S ribosomal RNA (r28S), was determined in cells isolated from chicken lymphoid tissues and stimulated with three different mitogens. The stability of the genes was measured using geNorm, NormFinder and BestKeeper software. The results from both geNorm and NormFinder were that the three most stably expressed genes in this panel were TBP, GAPDH and r28S. BestKeeper did not generate clear answers because of the highly heterogeneous sample set. Based on these data we will include TBP in future qPCR normalisation. The study shows the importance of appropriate reference gene normalisation in other tissues before qPCR analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of commercial RNA extraction kits and qPCR master mixes for studying gene expression in small biopsy tissue samples from the equine gastric epithelium.

PubMed

Tesena, Parichart; Korchunjit, Wasamon; Taylor, Jane; Wongtawan, Tuempong

2017-01-01

Gastric tissue biopsy and gene expression analysis are important tools for disease diagnosis and study of the physiology of the equine stomach. However, RNA extraction from gastric biopsy samples is a complex procedure because the samples contain low quantities of RNA and are contaminated with mucous protein and bacterial flora. The objectives of these studies were to compare the performance of RNA extraction methods and to investigate the sensitivity of commercial qPCR master mixes for gene expression analysis of gastric biopsy samples. Three commercial RNA extraction methods (TRIzol ™ , GENEzol ™ and MiniPrep ™ ) and four qPCR master mixes with SYBR ® green (qPCRBIO, KAPA, QuantiNova, and PerfeCTa) were compared. RNA qualification and quantitation were compared. Real-time PCR was used to compare qPCR master mixes. The results revealed that TRIzol and GENEzol obtained significantly higher yield of RNA (P<0.01) but that TRIzol had the highest contamination of protein and DNA (P<0.05). Conversely, MiniPrep resulting in a significantly higher purification of RNA (P<0.05) but provided the lowest yield of RNA (P<0.01). For PCR master mixes, KAPA was significantly (P<0.05) more sensitive than other qPCR kits for all amounts of DNA template, particularly at the lowest amount of cDNA. In conclusion, GENEzol is the best method to obtain a high RNA yield and purification and it is more cost-effective than the others as well. Regarding the qPCR master mixes, KAPA SYBR qPCR Master Mix (2x) Universal is superior to the other tested master mixes for studying gene expression in equine gastric biopsies.

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

PubMed Central

Bliem, Rupert; Schauer, Sonja; Plicka, Helga; Obwaller, Adelheid; Sommer, Regina; Steinrigl, Adolf; Alam, Munirul; Reischer, Georg H.; Farnleitner, Andreas H.

2015-01-01

Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 × 102 to 2.3 × 104 cell equivalents liter−1, whereas GR-corrected abundances ranged from 4.7 × 103 to 1.6 × 106 cell equivalents liter−1. GR-corrected qPCR results were in good agreement with an independent cell-based direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs. PMID:25724966

Characterization of vaginal Lactobacillus species by rplK -based multiplex qPCR in Russian women.

PubMed

Demkin, Vladimir V; Koshechkin, Stanislav I

2017-10-01

We describe a multiplex qPCR assay for identification and quantitative assessment of a set of vaginal Lactobacillus species, including L. acidophilus, L. crispatus, L. gasseri, L. helveticus, L. iners, and L. jensenii. The assay extends the previously developed qPCR method for Lactobacillus detection and total quantification based on targeting the rplK gene. Both assays use only single pair of primers and a set of probes combined in three reactions, comprising a vaginal Lactobacillus diagnostic assay panel. The utility of the diagnostic panel was evaluated by analyzing of vaginal swab specimens from 145 patients with different status of vaginal health. Most frequently, only one Lactobacillus species was dominant (68,9%), mostly L. crispatus (18,6%) or L. iners (33,1%), but two or three Lactobacillus species were also being simultaneously detected (24,9%). The diagnostic panel will facilitate investigations of the role of Lactobacillus species in the health of the female reproductive system and promote studies of variability of the vaginal microbiota. Copyright © 2017 Elsevier Ltd. All rights reserved.

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...

Development of a quantitative PCR (qPCR) for Giardia and analysis of the prevalence, cyst shedding and genotypes of Giardia present in sheep across four states in Australia.

PubMed

Yang, Rongchang; Jacobson, Caroline; Gardner, Graham; Carmichael, Ian; Campbell, Angus J D; Ryan, Una

2014-02-01

A novel quantitative PCR (qPCR) for Giardia at the glutamate dehydrogenase (gdh) locus was developed and validated. The qPCR was used to screen a total of 3412 lamb faecal samples collected from approximately 1189 lambs at three sampling periods (weaning, post-weaning and pre-slaughter) from eight farms across South Australia (SA), New South Wales (NSW), Victoria (Vic) and Western Australia (WA). The overall prevalence was 20.2% (95% CI 18.9-21.6) and of the 690 positives, 473 were successfully typed. In general, the prevalence of Giardia varied widely across the different farms with the highest prevalence in one WA farm (42.1%) at pre-slaughter sampling and the lowest prevalence in one Victorian farm (7.2%) at weaning. The range of cyst shedding at weaning, post-weaning and pre-slaughter overall across all states was 63-1.3×10(9) cysts g(-1) (median=1.7×10(4)), 63-1.1×10(9) cysts g(-1) (median=9.6×10(3)), 63-4.7×10(9) cysts g(-1) (median=8.1×10(4)) respectively. Assemblage specific primers at the triose phosphate isomerase (tpi) locus identified assemblage A in 22.4% (106/473) of positive samples typed, assemblage E in 75.9% (359/473) and mixed A and E assemblages in 1.7% (8/473) of samples. A subset of representative samples from the 8 farms (n=32) were typed at both the gdh and beta-giardin loci and confirmed these results and identified sub-assemblage AII in 16 representative assemblage A isolates across the 8 farms. This demonstrates a prevalence of Giardia previously not recognised in Australian sheep, highlighting a need for further research to quantify the production impacts of this protozoan parasite. Copyright © 2013 Elsevier Inc. All rights reserved.

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.

Use of real-time qPCR to quantify members of the unculturable heterotrophic bacterial community in a deep sea marine sponge, Vetulina sp.

PubMed

Cassler, M; Peterson, C L; Ledger, A; Pomponi, S A; Wright, A E; Winegar, R; McCarthy, P J; Lopez, J V

2008-04-01

In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

PubMed

Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

2017-04-01

Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

Duplex Quantitative PCR Assay for Detection of Haemophilus influenzae That Distinguishes Fucose- and Protein D-Negative Strains

PubMed Central

de Gier, Camilla; Pickering, Janessa L.; Richmond, Peter C.; Thornton, Ruth B.

2016-01-01

We have developed a specific Haemophilus influenzae quantitative PCR (qPCR) that also identifies fucose-negative and protein D-negative strains. Analysis of 100 H. influenzae isolates, 28 Haemophilus haemolyticus isolates, and 14 other bacterial species revealed 100% sensitivity (95% confidence interval [CI], 96% to 100%) and 100% specificity (95% CI, 92% to 100%) for this assay. The evaluation of 80 clinical specimens demonstrated a strong correlation between semiquantitative culture and the qPCR (P < 0.001). PMID:27335148

Monitoring the dynamics of syntrophic β-oxidizing bacteria during anaerobic degradation of oleic acid by quantitative PCR.

PubMed

Ziels, Ryan M; Beck, David A C; Martí, Magalí; Gough, Heidi L; Stensel, H David; Svensson, Bo H

2015-04-01

The ecophysiology of long-chain fatty acid-degrading syntrophic β-oxidizing bacteria has been poorly understood due to a lack of quantitative abundance data. Here, TaqMan quantitative PCR (qPCR) assays targeting the 16S rRNA gene of the known mesophilic syntrophic β-oxidizing bacterial genera Syntrophomonas and Syntrophus were developed and validated. Microbial community dynamics were followed using qPCR and Illumina-based high-throughput amplicon sequencing in triplicate methanogenic bioreactors subjected to five consecutive batch feedings of oleic acid. With repeated oleic acid feeding, the initial specific methane production rate significantly increased along with the relative abundances of Syntrophomonas and methanogenic archaea in the bioreactor communities. The novel qPCR assays showed that Syntrophomonas increased from 7 to 31% of the bacterial community 16S rRNA gene concentration, whereas that of Syntrophus decreased from 0.02 to less than 0.005%. High-throughput amplicon sequencing also revealed that Syntrophomonas became the dominant genus within the bioreactor microbiomes. These results suggest that increased specific mineralization rates of oleic acid were attributed to quantitative shifts within the microbial communities toward higher abundances of syntrophic β-oxidizing bacteria and methanogenic archaea. The novel qPCR assays targeting syntrophic β-oxidizing bacteria may thus serve as monitoring tools to indicate the fatty acid β-oxidization potential of anaerobic digester communities. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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...

Probabilistic PCA of censored data: accounting for uncertainties in the visualization of high-throughput single-cell qPCR data.

PubMed

Buettner, Florian; Moignard, Victoria; Göttgens, Berthold; Theis, Fabian J

2014-07-01

High-throughput single-cell quantitative real-time polymerase chain reaction (qPCR) is a promising technique allowing for new insights in complex cellular processes. However, the PCR reaction can be detected only up to a certain detection limit, whereas failed reactions could be due to low or absent expression, and the true expression level is unknown. Because this censoring can occur for high proportions of the data, it is one of the main challenges when dealing with single-cell qPCR data. Principal component analysis (PCA) is an important tool for visualizing the structure of high-dimensional data as well as for identifying subpopulations of cells. However, to date it is not clear how to perform a PCA of censored data. We present a probabilistic approach that accounts for the censoring and evaluate it for two typical datasets containing single-cell qPCR data. We use the Gaussian process latent variable model framework to account for censoring by introducing an appropriate noise model and allowing a different kernel for each dimension. We evaluate this new approach for two typical qPCR datasets (of mouse embryonic stem cells and blood stem/progenitor cells, respectively) by performing linear and non-linear probabilistic PCA. Taking the censoring into account results in a 2D representation of the data, which better reflects its known structure: in both datasets, our new approach results in a better separation of known cell types and is able to reveal subpopulations in one dataset that could not be resolved using standard PCA. The implementation was based on the existing Gaussian process latent variable model toolbox (https://github.com/SheffieldML/GPmat); extensions for noise models and kernels accounting for censoring are available at http://icb.helmholtz-muenchen.de/censgplvm. © The Author 2014. Published by Oxford University Press. All rights reserved.

Probabilistic PCA of censored data: accounting for uncertainties in the visualization of high-throughput single-cell qPCR data

PubMed Central

Buettner, Florian; Moignard, Victoria; Göttgens, Berthold; Theis, Fabian J.

2014-01-01

Motivation: High-throughput single-cell quantitative real-time polymerase chain reaction (qPCR) is a promising technique allowing for new insights in complex cellular processes. However, the PCR reaction can be detected only up to a certain detection limit, whereas failed reactions could be due to low or absent expression, and the true expression level is unknown. Because this censoring can occur for high proportions of the data, it is one of the main challenges when dealing with single-cell qPCR data. Principal component analysis (PCA) is an important tool for visualizing the structure of high-dimensional data as well as for identifying subpopulations of cells. However, to date it is not clear how to perform a PCA of censored data. We present a probabilistic approach that accounts for the censoring and evaluate it for two typical datasets containing single-cell qPCR data. Results: We use the Gaussian process latent variable model framework to account for censoring by introducing an appropriate noise model and allowing a different kernel for each dimension. We evaluate this new approach for two typical qPCR datasets (of mouse embryonic stem cells and blood stem/progenitor cells, respectively) by performing linear and non-linear probabilistic PCA. Taking the censoring into account results in a 2D representation of the data, which better reflects its known structure: in both datasets, our new approach results in a better separation of known cell types and is able to reveal subpopulations in one dataset that could not be resolved using standard PCA. Availability and implementation: The implementation was based on the existing Gaussian process latent variable model toolbox (https://github.com/SheffieldML/GPmat); extensions for noise models and kernels accounting for censoring are available at http://icb.helmholtz-muenchen.de/censgplvm. Contact: fbuettner.phys@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24618470

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)...

Miltenberger blood group typing by real-time polymerase chain reaction (qPCR) melting curve analysis in Thai population.

PubMed

Vongsakulyanon, A; Kitpoka, P; Kunakorn, M; Srikhirin, T

2015-12-01

To develop reliable and convenient methods for Miltenberger (Mi(a) ) blood group typing. To apply real-time polymerase chain reaction (qPCR) melting curve analysis to Mi(a) blood group typing. The Mi(a) blood group is the collective set of glycophorin hybrids in the MNS blood group system. Mi(a+) blood is common among East Asians and is also found in the Thai population. Incompatible Mi(a) blood transfusions pose the risk of life-threatening haemolysis; therefore, Mi(a) blood group typing is necessary in ethnicities where the Mi(a) blood group is prevalent. One hundred and forty-three blood samples from Thai blood donors were used in the study. The samples included 50 Mi(a+) samples and 93 Mi(a-) samples, which were defined by serology. The samples were typed by Mi(a) typing qPCR, and 50 Mi(a+) samples were sequenced to identify the Mi(a) subtypes. Mi(a) subtyping qPCR was performed to define GP.Mur. Both Mi(a) typing and Mi(a) subtyping were tested on a conventional PCR platform. The results of Mi(a) typing qPCR were all concordant with serology. Sequencing of the 50 Mi(a+) samples revealed 47 GP.Mur samples and 3 GP.Hop or Bun samples. Mi(a) subtyping qPCR was the supplementary test used to further define GP.Mur from other Mi(a) subtypes. Both Mi(a) typing and Mi(a) subtyping performed well using a conventional PCR platform. Mi(a) typing qPCR correctly identified Mi(a) blood groups in a Thai population with the feasibility of Mi(a) subtype discrimination, and Mi(a) subtyping qPCR was able to further define GP.Mur from other Mi(a) subtypes. © 2015 British Blood Transfusion Society.

Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR.

PubMed

Pessoa-E-Silva, Rômulo; Mendonça Trajano-Silva, Lays Adrianne; Lopes da Silva, Maria Almerice; da Cunha Gonçalves-de-Albuquerque, Suênia; de Goes, Tayná Correia; Silva de Morais, Rayana Carla; Lopes de Melo, Fábio; de Paiva-Cavalcanti, Milena

2016-12-01

The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per μL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

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...

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

EPA Science Inventory

There are numerous quantitative real-time PCR (qPCR) assays available to detect and enumerate fecal pollution in ambient waters. Each assay employs distinct primers and probes that target different rRNA genes and microorganisms leading to potential variations in concentration es...

Pneumocystis jirovecii (Pj) quantitative PCR to differentiate Pj pneumonia from Pj colonization in immunocompromised patients.

PubMed

Maillet, M; Maubon, D; Brion, J P; François, P; Molina, L; Stahl, J P; Epaulard, O; Bosseray, A; Pavese, P

2014-03-01

Conventional polymerase chain reaction (PCR) in respiratory samples does not differentiate between Pneumocystis pneumonia (PCP) and Pneumocystis jirovecii (Pj) colonization. We used Pj real-time quantitative PCR (qPCR) with the objective to discriminate PCP from Pj colonization in immunocompromised patients. All positive Pj qPCR [targeting the major surface glycoprotein (MSG) gene] obtained in respiratory samples from immunocompromised patients presenting pneumonia at the Grenoble University Hospital, France, were collected between August 2009 and April 2011. Diagnoses were retrospectively determined by a multidisciplinary group of experts blinded to the Pj qPCR results. Thirty-one bronchoalveolar lavages and four broncho aspirations positive for the Pj qPCR were obtained from 35 immunocompromised patients. Diagnoses of definite, probable, and possible PCP, and pneumonia from another etiology were retrospectively made for 7, 4, 5, and 19 patients, respectively. Copy numbers were significantly higher in the "definite group" (median 465,000 copies/ml) than in the "probable group" (median 38,600 copies/ml), the "possible group" (median 1,032 copies/ml), and the "other diagnosis group" (median 390 copies/ml). With the value of 3,160 copies/ml, the sensitivity and specificity of qPCR for the diagnosis of PCP were 100 % and 70 %, respectively. With the value of 31,600 copies/ml, the sensitivity and specificity were 80 % and 100 %, respectively. The positive predictive value was 100 % for results with more than 31,600 copies/ml and the negative predictive value was 100 % for results with fewer than 3,160 copies/ml. qPCR targeting the MSG gene can be helpful to discriminate PCP from Pj colonization in immunocompromised patients, using two cut-off values, with a gray zone between them.

Viability qPCR, a new tool for Legionella risk management.

PubMed

Lizana, X; López, A; Benito, S; Agustí, G; Ríos, M; Piqué, N; Marqués, A M; Codony, F

2017-11-01

Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups. The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy. Copyright © 2017 Elsevier GmbH. All rights reserved.

A quantitative PCR assay for aerobic, vinyl chloride- and ethene-assimilating microorganisms in groundwater.

PubMed

Jin, Yang Oh; Mattes, Timothy E

2010-12-01

Vinyl chloride (VC) is a known human carcinogen that is primarily formed in groundwater via incomplete anaerobic dechlorination of chloroethenes. Aerobic, ethene-degrading bacteria (etheneotrophs), which are capable of both fortuitous and growth-linked VC oxidation, could be important in natural attenuation of VC plumes that escape anaerobic treatment. In this work, we developed a quantitative, real-time PCR (qPCR) assay for etheneotrophs in groundwater. We designed and tested degenerate qPCR primers for two functional genes involved in aerobic, growth-coupled VC- and ethene-oxidation (etnC and etnE). Primer specificity to these target genes was tested by comparison to nucleotide sequence databases, PCR analysis of template DNA extracted from isolates and environmental samples, and sequencing of qPCR products obtained from VC-contaminated groundwater. The assay was made quantitative by constructing standard curves (threshold cycle vs log gene copy number) with DNA amplified from Mycobacterium strain JS60, an etheneotrophic isolate. Analysis of groundwater samples from three different VC-contaminated sites revealed that etnC abundance ranged from 1.6 × 10(3) - 1.0 × 10(5) copies/L groundwater while etnE abundance ranged from 4.3 × 10(3) - 6.3 × 10(5) copies/L groundwater. Our data suggest this novel environmental measurement method will be useful for supporting VC bioremediation strategies, assisting in site closure, and conducting microbial ecology studies involving etheneotrophs.

Characterization and Comparison of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR Assay for Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid from Experimental Invasive Pulmonary Aspergillosis

PubMed Central

Francesconi, Andrea; Kasai, Miki; Petraitiene, Ruta; Petraitis, Vidmantas; Kelaher, Amy M.; Schaufele, Robert; Hope, William W.; Shea, Yvonne R.; Bacher, John; Walsh, Thomas J.

2006-01-01

Bronchoalveolar lavage (BAL) is widely used for evaluation of patients with suspected invasive pulmonary aspergillosis (IPA). However, the diagnostic yield of BAL for detection of IPA by culture and direct examination is limited. Earlier diagnosis may be facilitated by assays that can detect Aspergillus galactomannan antigen or DNA in BAL fluid. We therefore characterized and compared the diagnostic yields of a galactomannan enzyme immunoassay (GM EIA), quantitative real-time PCR (qPCR), and quantitative cultures in experiments using BAL fluid from neutropenic rabbits with experimentally induced IPA defined as microbiologically and histologically evident invasion. The qPCR assay targeted the rRNA gene complex of Aspergillus fumigatus. The GM EIA and qPCR assay were characterized by receiver operator curve analysis. With an optimal cutoff of 0.75, the GM EIA had a sensitivity and specificity of 100% in untreated controls. A decline in sensitivity (92%) was observed when antifungal therapy (AFT) was administered. The optimal cutoff for qPCR was a crossover of 36 cycles, with sensitivity and specificity of 80% and 100%, respectively. The sensitivity of qPCR also decreased with AFT to 50%. Quantitative culture of BAL had a sensitivity of 46% and a specificity of 100%. The sensitivity of quantitative culture decreased with AFT to 16%. The GM EIA and qPCR assay had greater sensitivity than culture in detection of A. fumigatus in BAL fluid in experimentally induced IPA (P ± 0.04). Use of the GM EIA and qPCR assay in conjunction with culture-based diagnostic methods applied to BAL fluid could facilitate accurate diagnosis and more-timely initiation of specific therapy. PMID:16825367

Development of strain-specific PCR primers for quantitative detection of Bacillus mesentericus strain TO-A in human feces.

PubMed

Sato, Naoki; Seo, Genichiro; Benno, Yoshimi

2014-01-01

Strain-specific polymerase chain reaction (PCR) primers for detection of Bacillus mesentericus strain TO-A (BM TO-A) were developed. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. A 991-bp RAPD marker found to be strain-specific was sequenced, and two primer pairs specific to BM TO-A were constructed based on this sequence. In addition, we explored a more specific DNA region using inverse PCR, and designed a strain-specific primer set for use in real-time quantitative PCR (qPCR). These primer pairs were tested against 25 Bacillus subtilis strains and were found to be strain-specific. After examination of the detection limit and linearity of detection of BM TO-A in feces, the qPCR method and strain-specific primers were used to quantify BM TO-A in the feces of healthy volunteers who had ingested 3×10(8) colony forming unit (CFU) of BM TO-A per day in tablets. During the administration period, BM TO-A was detected in the feces of all 24 subjects, and the average number of BM TO-A detected using the culture method and qPCR was about 10(4.8) and 10(5.8) cells per gram of feces, respectively. Using the qPCR method, BM TO-A was detected in the feces of half of the subjects 3 d after withdrawal, and was detected in the feces of only one subject 1 week after withdrawal. These results suggest that the qPCR method using BM TO-A strain-specific primers is useful for the quantitative detection of this strain in feces.

Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

PubMed Central

Harshman, Dustin K.; Rao, Brianna M.; McLain, Jean E.; Watts, George S.; Yoon, Jeong-Yeol

2015-01-01

Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections. PMID:26601245

Comparative analysis of techniques for detection of quiescent Botrytis cinerea in grapes by quantitative PCR

USDA-ARS?s Scientific Manuscript database

Quantitative PCR (qPCR) can be used to detect and monitor pathogen colonization, but early attempts to apply the technology to quiescent Botrytis cinerea infections of grape berries identified some specific limitations. In this study, four DNA extraction methods, two tissue-grinding methods, two gra...

Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

EPA Science Inventory

Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

Genome-Wide Identification and Evaluation of Reference Genes for Quantitative RT-PCR Analysis during Tomato Fruit Development.

PubMed

Cheng, Yuan; Bian, Wuying; Pang, Xin; Yu, Jiahong; Ahammed, Golam J; Zhou, Guozhi; Wang, Rongqing; Ruan, Meiying; Li, Zhimiao; Ye, Qingjing; Yao, Zhuping; Yang, Yuejian; Wan, Hongjian

2017-01-01

Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR) is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs). Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified 38 most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red) using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170), SlFRG12 (Solyc04g009770), SlFRG16 (Solyc10g081190), SlFRG27 (Solyc06g007510), and SlFRG37 (Solyc11g005330) were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070) and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species.

Genome-Wide Identification and Evaluation of Reference Genes for Quantitative RT-PCR Analysis during Tomato Fruit Development

PubMed Central

Cheng, Yuan; Bian, Wuying; Pang, Xin; Yu, Jiahong; Ahammed, Golam J.; Zhou, Guozhi; Wang, Rongqing; Ruan, Meiying; Li, Zhimiao; Ye, Qingjing; Yao, Zhuping; Yang, Yuejian; Wan, Hongjian

2017-01-01

Gene expression analysis in tomato fruit has drawn increasing attention nowadays. Quantitative real-time PCR (qPCR) is a routine technique for gene expression analysis. In qPCR operation, reliability of results largely depends on the choice of appropriate reference genes (RGs). Although tomato is a model for fruit biology study, few RGs for qPCR analysis in tomato fruit had yet been developed. In this study, we initially identified 38 most stably expressed genes based on tomato transcriptome data set, and their expression stabilities were further determined in a set of tomato fruit samples of four different fruit developmental stages (Immature, mature green, breaker, mature red) using qPCR analysis. Two statistical algorithms, geNorm and Normfinder, concordantly determined the superiority of these identified putative RGs. Notably, SlFRG05 (Solyc01g104170), SlFRG12 (Solyc04g009770), SlFRG16 (Solyc10g081190), SlFRG27 (Solyc06g007510), and SlFRG37 (Solyc11g005330) were proved to be suitable RGs for tomato fruit development study. Further analysis using geNorm indicate that the combined use of SlFRG03 (Solyc02g063070) and SlFRG27 would provide more reliable normalization results in qPCR experiments. The identified RGs in this study will be beneficial for future qPCR analysis of tomato fruit developmental study, as well as for the potential identification of optimal normalization controls in other plant species. PMID:28900431

Influences of sample interference and interference controls on quantification of enterococci fecal indicator bacteria in surface water samples by the qPCR method

EPA Science Inventory

A quantitative polymerase chain reaction (qPCR) method for the detection of entercocci fecal indicator bacteria has been shown to be generally applicable for the analysis of temperate fresh (Great Lakes) and marine coastal waters and for providing risk-based determinations of wat...

A theoretical introduction to "combinatory SYBRGreen qPCR screening", a matrix-based approach for the detection of materials derived from genetically modified plants.

PubMed

Van den Bulcke, Marc; Lievens, Antoon; Barbau-Piednoir, Elodie; MbongoloMbella, Guillaume; Roosens, Nancy; Sneyers, Myriam; Casi, Amaya Leunda

2010-03-01

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. "Combinatory qPCR SYBRGreen screening" (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBRGREEN qPCR analysis based on four values: the C(t)- and T(m) values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, "Prime number tracing", matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBRGREEN qPCR methods and through application of a newly developed "prime number"-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product.

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 ...

Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

PubMed

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

Q-PCR based bioburden assessment of drinking water throughout treatment and delivery to the International Space Station

NASA Technical Reports Server (NTRS)

Newcombe, David; Stuecker, Tara; La Duc, Myron; Venkateswaran, Kasthuri

2005-01-01

Previous studies indicated evidence of opportunistic pathogens samples obtained during missions to the International Space Station (ISS). This study utilized TaqMan quantitative PCR to determine specific gene abundance in potable and non-potable ISS waters. Probe and primer sets specific to the small subunit rRNA genes were used to elucidate overall bacterial rRNA gene numbers. while those specific for Burkholderia cepacia and Stenotrophomonas maltophilia were optimized and used to probe for the presence of these two opportunistic pathogens. This research builds upon previous microbial diversity studies of ISS water and demonstrates the utility of Q-PCR tool to examine water quality.

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.

Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

PubMed Central

Lucero, David E.; Ribera, Wilma; Pizarro, Juan Carlos; Plaza, Carlos; Gordon, Levi W.; Peña, Reynaldo; Morrissey, Leslie A.; Rizzo, Donna M.; Stevens, Lori

2014-01-01

Background In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia. Methodology/Principal Findings We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors). Conclusions/Significance We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors. PMID:25474154

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.

Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches

PubMed Central

Mellerup, Anders; Ståhl, Marie

2015-01-01

The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces. PMID:26114765

Decay Of Bacterial Pathogens, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure-Amended Soils

EPA Science Inventory

This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria (FIB), and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manure-amended agricultural soils. Known concentrations of transformed green...

Decay Of Bacterial Pathogen, Fecal Indicators, And Real-Time Quantitative PCR Genetic Markers In Manure Amended Soils

EPA Science Inventory

This study examined persistence and decay of bacterial pathogens, fecal indicator bacteria, and emerging real-time quantitative PCR (qPCR) genetic markers for rapid detection of fecal pollution in manre-amended agricultural soils. Known concentrations of transformed green fluore...

DNA Extraction Method Affects the Detection of a Fungal Pathogen in Formalin-Fixed Specimens Using qPCR.

PubMed

Adams, Andrea J; LaBonte, John P; Ball, Morgan L; Richards-Hrdlicka, Kathryn L; Toothman, Mary H; Briggs, Cheryl J

2015-01-01

Museum collections provide indispensable repositories for obtaining information about the historical presence of disease in wildlife populations. The pathogenic amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has played a significant role in global amphibian declines, and examining preserved specimens for Bd can improve our understanding of its emergence and spread. Quantitative PCR (qPCR) enables Bd detection with minimal disturbance to amphibian skin and is significantly more sensitive to detecting Bd than histology; therefore, developing effective qPCR methodologies for detecting Bd DNA in formalin-fixed specimens can provide an efficient and effective approach to examining historical Bd emergence and prevalence. Techniques for detecting Bd in museum specimens have not been evaluated for their effectiveness in control specimens that mimic the conditions of animals most likely to be encountered in museums, including those with low pathogen loads. We used American bullfrogs (Lithobates catesbeianus) of known infection status to evaluate the success of qPCR to detect Bd in formalin-fixed specimens after three years of ethanol storage. Our objectives were to compare the most commonly used DNA extraction method for Bd (PrepMan, PM) to Macherey-Nagel DNA FFPE (MN), test optimizations for Bd detection with PM, and provide recommendations for maximizing Bd detection. We found that successful detection is relatively high (80-90%) when Bd loads before formalin fixation are high, regardless of the extraction method used; however, at lower infection levels, detection probabilities were significantly reduced. The MN DNA extraction method increased Bd detection by as much as 50% at moderate infection levels. Our results indicate that, for animals characterized by lower pathogen loads (i.e., those most commonly encountered in museum collections), current methods may underestimate the proportion of Bd-infected amphibians. Those extracting DNA from archived museum

Determination of viable Salmonellae from potable and source water through PMA assisted qPCR.

PubMed

Singh, Gulshan; Vajpayee, Poornima; Bhatti, Saurabh; Ronnie, Nirmala; Shah, Nimish; McClure, Peter; Shanker, Rishi

2013-07-01

Resource constrained countries identified as endemic zones for pathogenicity of Salmonella bear an economic burden due to recurring expenditure on medical treatment. qPCR used for Salmonella detection could not discriminate between viable and nonviable cells. Propidium monoazide (PMA) that selectively penetrates nonviable cells to cross-link their DNA, was coupled with ttr gene specific qPCR for quantifying viable salmonellae in source/potable waters collected from a north Indian city. Source water (raw water for urban potable water supply) and urban potable water exhibited viable salmonellae in the range of 2.1×10(4)-2.6×10(6) and 2-7160CFU/100mL, respectively. Potable water at water works exhibited DNA from dead cells but no viable cells were detected. PMA assisted qPCR could specifically detect low numbers of live salmonellae in Source and potable waters. This strategy can be used in surveillance of urban potable water distribution networks to map contamination points for better microbial risk management. Copyright © 2013 Elsevier Inc. All rights reserved.

Species identification of Cannabis sativa using real-time quantitative PCR (qPCR).

PubMed

Johnson, Christopher E; Premasuthan, Amritha; Satkoski Trask, Jessica; Kanthaswamy, Sree

2013-03-01

Most narcotics-related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3' exon-trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real-time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa. © 2013 American Academy of Forensic Sciences.

Development and optimization of an efficient qPCR system for olive authentication in edible oils.

PubMed

Alonso-Rebollo, Alba; Ramos-Gómez, Sonia; Busto, María D; Ortega, Natividad

2017-10-01

The applicability of qPCR in olive-oil authentication depends on the DNA obtained from the oils and the amplification primers. Therefore, four olive-specific amplification systems based on the trnL gene were designed (A-, B-, C- and D-trnL systems). The qPCR conditions, primer concentration and annealing temperature, were optimized. The systems were tested for efficiency and sensitivity to select the most suitable for olive oil authentication. The selected system (D-trnL) demonstrated specificity toward olive in contrast to other oleaginous species (canola, soybean, sunflower, maize, peanut and coconut) and showed high sensitivity in a broad linear dynamic range (LOD and LOQ: 500ng - 0.0625pg). This qPCR system enabled detection, with high sensitivity and specificity, of olive DNA isolated from oils processed in different ways, establishing it as an efficient method for the authentication of olive oil regardless of its category. Copyright © 2017 Elsevier Ltd. All rights reserved.

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...

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.

A 10-Year Retrospective Comparison of Two Target Sequences, REP-529 and B1, for Toxoplasma gondii Detection by Quantitative PCR

PubMed Central

Belaz, Sorya; Gangneux, Jean-Pierre; Dupretz, Peggy; Guiguen, Claude

2015-01-01

This study aimed to evaluate the repeated sequence REP-529 compared to that of the B1 gene in the molecular diagnosis of toxoplasmosis by quantitative PCR (qPCR) in routine diagnosis. Over a 10-year period (2003 to 2013), all patients prospectively diagnosed with a positive REP-529 qPCR result for toxoplasmosis were included. All DNA samples (76 samples from 56 patients) were simultaneously tested using the two qPCR methods (REP-529 and B1). The mean cycle threshold (CT) obtained with the B1 qPCR was significantly higher (+4.71 cycles) than that obtained with REP-529 qPCR (P < 0.0001). Thirty-one out of 69 extracts (45.6%) positive with REP-529 qPCR were not amplified with the B1 qPCR (relative sensitivity of 54.4% compared to that with REP-529), yielding false-negative results with 15/28 placenta, 5 cord blood, 2 amniotic fluid, 4 cerebrospinal fluid, 1 aqueous humor, 2 lymph node puncture, and 1 abortion product sample. This defect in sensitivity would have left 20/56 patients undiagnosed, distributed as follows: 12/40 congenital toxoplasmosis, 4/5 cerebral toxoplasmosis, 2/8 patients with retinochoroiditis, and 2 patients with chronic lymphadenopathy. This poor performance of B1 qPCR might be related to low parasite loads, since the mean Toxoplasma quantification in extracts with B1 false-negative results was 0.4 parasite/reaction. These results clearly show the superiority of the REP-529 sequence in the diagnosis of toxoplasmosis by PCR and suggest that this target should be adopted as part of the standardization of the PCR assay. PMID:25653416

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.

Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods.

PubMed

Olsen, Morten Tange; Bérubé, Martine; Robbins, Jooke; Palsbøll, Per J

2012-09-06

Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments.

Validation of Reference Genes for Real-Time Quantitative PCR (qPCR) Analysis of Avibacterium paragallinarum.

PubMed

Wen, Shuxiang; Chen, Xiaoling; Xu, Fuzhou; Sun, Huiling

2016-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels.

mcrA-Targeted Real-Time Quantitative PCR Method To Examine Methanogen Communities▿

PubMed Central

Steinberg, Lisa M.; Regan, John M.

2009-01-01

Methanogens are of great importance in carbon cycling and alternative energy production, but quantitation with culture-based methods is time-consuming and biased against methanogen groups that are difficult to cultivate in a laboratory. For these reasons, methanogens are typically studied through culture-independent molecular techniques. We developed a SYBR green I quantitative PCR (qPCR) assay to quantify total numbers of methyl coenzyme M reductase α-subunit (mcrA) genes. TaqMan probes were also designed to target nine different phylogenetic groups of methanogens in qPCR assays. Total mcrA and mcrA levels of different methanogen phylogenetic groups were determined from six samples: four samples from anaerobic digesters used to treat either primarily cow or pig manure and two aliquots from an acidic peat sample stored at 4°C or 20°C. Only members of the Methanosaetaceae, Methanosarcina, Methanobacteriaceae, and Methanocorpusculaceae and Fen cluster were detected in the environmental samples. The three samples obtained from cow manure digesters were dominated by members of the genus Methanosarcina, whereas the sample from the pig manure digester contained detectable levels of only members of the Methanobacteriaceae. The acidic peat samples were dominated by both Methanosarcina spp. and members of the Fen cluster. In two of the manure digester samples only one methanogen group was detected, but in both of the acidic peat samples and two of the manure digester samples, multiple methanogen groups were detected. The TaqMan qPCR assays were successfully able to determine the environmental abundance of different phylogenetic groups of methanogens, including several groups with few or no cultivated members. PMID:19447957

Quantitative PCR: an appropriate tool to detect viable but not culturable Brettanomyces bruxellensis in wine.

PubMed

Willenburg, Elize; Divol, Benoit

2012-11-15

Quantitative PCR as a tool has been used to detect Brettanomyces bruxellensis directly from wine samples. Accurate and timely detection of this yeast is important to prevent unwanted spoilage of wines and beverages. The aim of this study was to distinguish differences between DNA and mRNA as template for the detection of this yeast. The study was also used to determine if it is possible to accurately detect cells in the viable but not culturable (VBNC) state of B. bruxellensis by qPCR. Several methods including traditional plating, epifluorescence counts and qPCR were used to amplify DNA and mRNA. It was observed that mRNA was a better template for the detection in terms of standard curve analysis and qPCR efficiencies. Various primers previously published were tested for their specificity, qPCR efficiency and accuracy of enumeration. A single primer set was selected which amplified a region of the actin-encoding gene. The detection limit for this assay was 10cellsmL(-1). B. bruxellensis could also be quantified in naturally contaminated wines with this assay. The mRNA gave a better indication of the viability of the cells which compared favourably to fluorescent microscopy and traditional cell counts. The ability of the assay to accurately estimate the number of cells in the VBNC state was also demonstrated. Copyright © 2012 Elsevier B.V. All rights reserved.

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

Evaluation of Suitable Reference Genes for Normalization of qPCR Gene Expression Studies in Brinjal (Solanum melongena L.) During Fruit Developmental Stages.

PubMed

Kanakachari, Mogilicherla; Solanke, Amolkumar U; Prabhakaran, Narayanasamy; Ahmad, Israr; Dhandapani, Gurusamy; Jayabalan, Narayanasamy; Kumar, Polumetla Ananda

2016-02-01

Brinjal/eggplant/aubergine is one of the major solanaceous vegetable crops. Recent availability of genome information greatly facilitates the fundamental research on brinjal. Gene expression patterns during different stages of fruit development can provide clues towards the understanding of its biological functions. Quantitative real-time PCR (qPCR) has become one of the most widely used methods for rapid and accurate quantification of gene expression. However, its success depends on the use of a suitable reference gene for data normalization. For qPCR analysis, a single reference gene is not universally suitable for all experiments. Therefore, reference gene validation is a crucial step. Suitable reference genes for qPCR analysis of brinjal fruit development have not been investigated so far. In this study, we have selected 21 candidate reference genes from the Brinjal (Solanum melongena) Plant Gene Indices database (compbio.dfci.harvard.edu/tgi/plant.html) and studied their expression profiles by qPCR during six different fruit developmental stages (0, 5, 10, 20, 30, and 50 days post anthesis) along with leaf samples of the Pusa Purple Long (PPL) variety. To evaluate the stability of gene expression, geNorm and NormFinder analytical softwares were used. geNorm identified SAND (SAND family protein) and TBP (TATA binding protein) as the best pairs of reference genes in brinjal fruit development. The results showed that for brinjal fruit development, individual or a combination of reference genes should be selected for data normalization. NormFinder identified Expressed gene (expressed sequence) as the best single reference gene in brinjal fruit development. In this study, we have identified and validated for the first time reference genes to provide accurate transcript normalization and quantification at various fruit developmental stages of brinjal which can also be useful for gene expression studies in other Solanaceae plant species.

Latent class analysis of real time qPCR and bacteriological culturing for the diagnosis of Streptococcus agalactiae in cow composite milk samples.

PubMed

Holmøy, Ingrid H; Toft, Nils; Jørgensen, Hannah J; Mørk, Tormod; Sølverød, Liv; Nødtvedt, Ane

2018-06-01

Streptococcus agalactiae (S. agalactiae) has re-emerged as a mastitis pathogen among Norwegian dairy cows. The Norwegian cattle health services recommend that infected herds implement measures to eradicate S. agalactiae, this includes a screening of milk samples from all lactating cows. The performance of the qPCR-test currently in use for this purpose has not been evaluated under field conditions. The objective of this study was to estimate the sensitivity and specificity of the real-time qPCR assay in use in Norway (Mastitis 4 qPCR, DNA Diagnostics A/S, Risskov, Denmark) and compare it to conventional bacteriological culturing for detection of S. agalactiae in milk samples. Because none of these tests are considered a perfect reference test, the evaluation was performed using latent class models in a Bayesian analysis. Aseptically collected cow-composite milk samples from 578 cows belonging to 6 herds were cultured and tested by qPCR. While 37 (6.4%) samples were positive for S. agalactiae by bacteriological culture, 66 (11.4%) samples were positive by qPCR. The within-herd prevalence in the six herds, as estimated by the latent class models ranged from 7.7 to 50.8%. At the recommended cut-off (cycle threshold 37), the sensitivity of the qPCR was significantly higher at 95.3 (95% posterior probability interval [PPI] [84.2; 99.6]) than that of bacteriological culture at 58.2 (95% PPI [43.8; 74.4]). However, bacterial culture had a higher specificity of 99.7 (95% PPI [98.5; 100.0]) compared to the qPCR at 98.5 (95% PPI [94.6; 99.9]). The median estimated negative predictive values of qPCR was consistently higher than those of the BC at all estimated prevalences, and the superiority of the qPCR increased with increasing within-herd prevalence. The median positive predictive values of BC was in general higher than the estimates for the qPCR, however, at the highest prevalence the predictive ability of both tests were similar. Copyright © 2018 Elsevier B.V. All

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

Spatiotemporal Dynamics of Vibrio cholerae in Turbid Alkaline Lakes as Determined by Quantitative PCR.

PubMed

Bliem, Rupert; Reischer, Georg; Linke, Rita; Farnleitner, Andreas; Kirschner, Alexander

2018-06-01

In recent years, global warming has led to a growing number of Vibrio cholerae infections in bathing water users in regions formerly unaffected by this pathogen. It is therefore of high importance to monitor V. cholerae in aquatic environments and to elucidate the main factors governing its prevalence and abundance. For this purpose, rapid and standardizable methods that can be performed by routine water laboratories are prerequisite. In this study, we applied a recently developed multiplex quantitative PCR (qPCR) strategy (i) to monitor the spatiotemporal variability of V. cholerae abundance in two small soda pools and a large lake that is intensively used for recreation and (ii) to elucidate the main factors driving V. cholerae dynamics in these environments. V. cholerae was detected with qPCR at high concentrations of up to 970,000 genomic units 100 ml -1 during the warm season, up to 2 orders of magnitude higher than values obtained by cultivation. An independent cytometric approach led to results comparable to qPCR data but with significantly more positive samples due to problems with DNA recovery for qPCR. Not a single sample was positive for toxigenic V. cholerae , indicating that only nontoxigenic V. cholerae (NTVC) was present. Temperature was the main predictor of NTVC abundance, but the quality and quantity of dissolved organic matter were also important environmental correlates. Based on this study, we recommend using the developed qPCR strategy for quantification of toxigenic and nontoxigenic V. cholerae in bathing waters with the need for improvements in DNA recovery. IMPORTANCE There is a definitive need for rapid and standardizable methods to quantify waterborne bacterial pathogens. Such methods have to be thoroughly tested for their applicability to environmental samples. In this study, we critically tested a recently developed multiplex qPCR strategy for its applicability to determine the spatiotemporal variability of V. cholerae abundance in

A 10-year retrospective comparison of two target sequences, REP-529 and B1, for Toxoplasma gondii detection by quantitative PCR.

PubMed

Belaz, Sorya; Gangneux, Jean-Pierre; Dupretz, Peggy; Guiguen, Claude; Robert-Gangneux, Florence

2015-04-01

This study aimed to evaluate the repeated sequence REP-529 compared to that of the B1 gene in the molecular diagnosis of toxoplasmosis by quantitative PCR (qPCR) in routine diagnosis. Over a 10-year period (2003 to 2013), all patients prospectively diagnosed with a positive REP-529 qPCR result for toxoplasmosis were included. All DNA samples (76 samples from 56 patients) were simultaneously tested using the two qPCR methods (REP-529 and B1). The mean cycle threshold (CT) obtained with the B1 qPCR was significantly higher (+4.71 cycles) than that obtained with REP-529 qPCR (P<0.0001). Thirty-one out of 69 extracts (45.6%) positive with REP-529 qPCR were not amplified with the B1 qPCR (relative sensitivity of 54.4% compared to that with REP-529), yielding false-negative results with 15/28 placenta, 5 cord blood, 2 amniotic fluid, 4 cerebrospinal fluid, 1 aqueous humor, 2 lymph node puncture, and 1 abortion product sample. This defect in sensitivity would have left 20/56 patients undiagnosed, distributed as follows: 12/40 congenital toxoplasmosis, 4/5 cerebral toxoplasmosis, 2/8 patients with retinochoroiditis, and 2 patients with chronic lymphadenopathy. This poor performance of B1 qPCR might be related to low parasite loads, since the mean Toxoplasma quantification in extracts with B1 false-negative results was 0.4 parasite/reaction. These results clearly show the superiority of the REP-529 sequence in the diagnosis of toxoplasmosis by PCR and suggest that this target should be adopted as part of the standardization of the PCR assay. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Quantitative PCR estimates Angiostrongylus cantonensis (rat lungworm) infection levels in semi-slugs (Parmarion martensi)

PubMed Central

Jarvi, Susan I.; Farias, Margaret E.M.; Howe, Kay; Jacquier, Steven; Hollingsworth, Robert; Pitt, William

2013-01-01

The life cycle of the nematode Angiostrongylus cantonensis involves rats as the definitive host and slugs and snails as intermediate hosts. Humans can become infected upon ingestion of intermediate or paratenic (passive carrier) hosts containing stage L3 A. cantonensis larvae. Here, we report a quantitative PCR (qPCR) assay that provides a reliable, relative measure of parasite load in intermediate hosts. Quantification of the levels of infection of intermediate hosts is critical for determining A. cantonensis intensity on the Island of Hawaii. The identification of high intensity infection ‘hotspots’ will allow for more effective targeted rat and slug control measures. qPCR appears more efficient and sensitive than microscopy and provides a new tool for quantification of larvae from intermediate hosts, and potentially from other sources as well. PMID:22902292

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...

Analysis of Enterococci and Bacteriodales Fecal Indicator Bacteria in a Lake Michigan Tributary by Real-Time Quantitative PCR

EPA Science Inventory

The Salt Creek watershed in northwest Indiana drains into Lake Michigan near several heavily used recreational beaches. This study aimed to investigate the levels of fecal indicator bacteria, enterococci and Bacteroidales, in Salt Creek using real-time quantitative PCR (qPCR) an...

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR.

PubMed

Yáñez, M Adela; Nocker, Andreas; Soria-Soria, Elena; Múrtula, Raquel; Martínez, Lorena; Catalán, Vicente

2011-05-01

One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells. Copyright © 2011 Elsevier B.V. All rights reserved.

Detection and discrimination of five E. coli pathotypes using a combinatory SYBR® Green qPCR screening system.

PubMed

Barbau-Piednoir, Elodie; Denayer, Sarah; Botteldoorn, Nadine; Dierick, Katelijne; De Keersmaecker, Sigrid C J; Roosens, Nancy H

2018-04-01

A detection and discrimination system for five Escherichia coli pathotypes, based on a combination of 13 SYBR® Green qPCR, has been developed, i.e., combinatory SYBR® Green qPCR screening system for pathogenic E. coli (CoSYPS Path E. coli). It allows the discrimination on isolates and the screening of potential presence in food of the following pathotypes of E. coli: shigatoxigenic (STEC) (including enterohemorrhagic (EHEC)), enteropathogenic (EPEC), enteroaggregative (EAggEC), enteroaggregative shigatoxigenic (EAggSTEC), and enteroinvasive (EIEC) E. coli. The SYBR® Green qPCR assays target the uidA, ipaH, eae, aggR, aaiC, stx1, and stx2 genes. uidA controls for E. coli presence and all the other genes are specific targets of E. coli pathotypes. For each gene, two primer pairs have been designed to guarantee a sufficient detection even in case of deletion or polymorphisms in the target gene. Moreover, all the qPCR have been designed to be run together in a single analytical PCR plate. This study includes the primer pairs' design, in silico and in situ selectivity, sensitivity, repeatability, and reproducibility evaluation of the 13 SYBR® Green qPCR assays. Each target displayed a selectivity of 100%. The limit of detection of the 13 assays is between 1 and 10 genomic copies. Their repeatability and reproducibility comply with the European requirements. As a preliminary feasibility study on food, the CoSYPS Path E. coli system was subsequently evaluated on four food matrices artificially contaminated with pathogenic E. coli. It allowed the detection of an initial contamination level as low as 2 to 7 cfu of STEC/25 g of food matrix after 24 h of enrichment.

Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata

NASA Astrophysics Data System (ADS)

Pu, Fei; Yang, Bingye; Ke, Caihuan

2015-07-01

Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 ( ACT-2), elongation factor 1 alpha ( EF-1α), elongation factor 1 beta ( EF-1β), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH), ubiquitin ( UBQ), β-tubulin ( β-TUB), and 18S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene ( Gβ) across tissue types was also examined and normalized to the expression of each or both of UBQ and β-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further functional genomics studies in this economically valuable marine bivalve.

Assessment of nitrification in groundwater filters for drinking water production by qPCR and activity measurement.

PubMed

de Vet, W W J M; Kleerebezem, R; van der Wielen, P W J J; Rietveld, L C; van Loosdrecht, M C M

2011-07-01

In groundwater treatment for drinking water production, the causes of nitrification problems and the effectiveness of process optimization in rapid sand filters are often not clear. To assess both issues, the performance of a full-scale groundwater filter with nitrification problems and another filter with complete nitrification and pretreatment by subsurface aeration was monitored over nine months. Quantitative real-time polymerase chain reaction (qPCR) targeting the amoA gene of bacteria and archaea and activity measurements of ammonia oxidation were used to regularly evaluate water and filter sand samples. Results demonstrated that subsurface aeration stimulated the growth of ammonia-oxidizing prokaryotes (AOP) in the aquifer. Cell balances, using qPCR counts of AOP for each filter, showed that the inoculated AOP numbers from the aquifer were marginal compared with AOP numbers detected in the filter. Excessive washout of AOP was not observed and did not cause the nitrification problems. Ammonia-oxidizing archaea grew in both filters, but only in low numbers compared to bacteria. The cell-specific nitrification rate in the sand and backwash water samples was high for the subsurface aerated filter, but systematically much lower for the filter with nitrification problems. From this, we conclude that incomplete nitrification was caused by nutrient limitation. Copyright © 2011 Elsevier Ltd. All rights reserved.

A human fecal contamination score for ranking recreational sites using the HF183/BacR287 quantitative real-time PCR method.

PubMed

Cao, Yiping; Sivaganesan, Mano; Kelty, Catherine A; Wang, Dan; Boehm, Alexandria B; Griffith, John F; Weisberg, Stephen B; Shanks, Orin C

2018-01-01

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 management. However, there are currently no standardized approaches for field implementation and interpretation of qPCR data. In this study, a standardized HF183/BacR287 qPCR method was combined with a water sampling strategy and a novel Bayesian weighted average approach to establish a human fecal contamination score (HFS) that can be used to prioritize sampling sites for remediation based on measured human waste levels. The HFS was then used to investigate 975 study design scenarios utilizing different combinations of sites with varying sampling intensities (daily to once per week) and number of qPCR replicates per sample (2-14 replicates). Findings demonstrate that site prioritization with HFS is feasible and that both sampling intensity and number of qPCR replicates influence reliability of HFS estimates. The novel data analysis strategy presented here provides a prescribed approach for the implementation and interpretation of human-associated HF183/BacR287 qPCR data with the goal of site prioritization based on human fecal pollution levels. In addition, information is provided for future users to customize study designs for optimal HFS performance. Published by Elsevier Ltd.

Multi-laboratory survey of qPCR enterococci analysis method performance

EPA Pesticide Factsheets

Quantitative polymerase chain reaction (qPCR) has become a frequently used technique for quantifying enterococci in recreational surface waters, but there are several methodological options. Here we evaluated how three method permutations, type of mastermix, sample extract dilution and use of controls in results calculation, affect method reliability among multiple laboratories with respect to sample interference. Multiple samples from each of 22 sites representing an array of habitat types were analyzed using EPA Method 1611 and 1609 reagents with full strength and five-fold diluted extracts. The presence of interference was assessed three ways: using sample processing and PCR amplifications controls; consistency of results across extract dilutions; and relative recovery of target genes from spiked enterococci in water sample compared to control matrices with acceptable recovery defined as 50 to 200%. Method 1609, which is based on an environmental mastermix, was found to be superior to Method 1611, which is based on a universal mastermix. Method 1611 had over a 40% control assay failure rate with undiluted extracts and a 6% failure rate with diluted extracts. Method 1609 failed in only 11% and 3% of undiluted and diluted extracts analyses. Use of sample processing control assay results in the delta-delta Ct method for calculating relative target gene recoveries increased the number of acceptable recovery results. Delta-delta tended to bias recoveries fr

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 ...

Evaluation of Reference Genes for Quantitative Real-Time PCR in Songbirds

PubMed Central

Zinzow-Kramer, Wendy M.; Horton, Brent M.; Maney, Donna L.

2014-01-01

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbird: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology. PMID:24780145

Empirical evaluation of humpback whale telomere length estimates; quality control and factors causing variability in the singleplex and multiplex qPCR methods

PubMed Central

2012-01-01

Background Telomeres, the protective cap of chromosomes, have emerged as powerful markers of biological age and life history in model and non-model species. The qPCR method for telomere length estimation is one of the most common methods for telomere length estimation, but has received recent critique for being too error-prone and yielding unreliable results. This critique coincides with an increasing awareness of the potentials and limitations of the qPCR technique in general and the proposal of a general set of guidelines (MIQE) for standardization of experimental, analytical, and reporting steps of qPCR. In order to evaluate the utility of the qPCR method for telomere length estimation in non-model species, we carried out four different qPCR assays directed at humpback whale telomeres, and subsequently performed a rigorous quality control to evaluate the performance of each assay. Results Performance differed substantially among assays and only one assay was found useful for telomere length estimation in humpback whales. The most notable factors causing these inter-assay differences were primer design and choice of using singleplex or multiplex assays. Inferred amplification efficiencies differed by up to 40% depending on assay and quantification method, however this variation only affected telomere length estimates in the worst performing assays. Conclusion Our results suggest that seemingly well performing qPCR assays may contain biases that will only be detected by extensive quality control. Moreover, we show that the qPCR method for telomere length estimation can be highly precise and accurate, and thus suitable for telomere measurement in non-model species, if effort is devoted to optimization at all experimental and analytical steps. We conclude by highlighting a set of quality controls which may serve for further standardization of the qPCR method for telomere length estimation, and discuss some of the factors that may cause variation in qPCR experiments

Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil.

PubMed

Fornazari, Felipe; da Silva, Rodrigo Costa; Richini-Pereira, Virginia Bodelão; Beserra, Hugo Enrique Orsini; Luvizotto, Maria Cecília Rui; Langoni, Helio

2012-09-01

Leptospirosis is an infectious disease of worldwide importance. The development of diagnostic techniques allows sick animals to be identified, reservoirs to be eliminated and the disease prevented and controlled. The present study aimed to compare different techniques for diagnosing leptospirosis in sheep. Samples of kidney, liver and blood were collected from 465 animals that originated from a slaughterhouse. The sera were analyzed by the Microscopic Agglutination Test (MAT), and kidney and liver samples of seropositive animals were analyzed using four techniques: bacteriological culture, the Warthin Starry (WS) technique, conventional PCR (cPCR), and quantitative PCR (qPCR). With the MAT, 21 animals were positive (4.5%) to serovars Hardjo (n=12), Hebdomadis (n=5), Sentot (n=2), Wolfii (n=1) and Shermani (n=1). Titers were 100 (n=10), 200 (n=2), 400 (n=6) and 1600 (n=3). No animal was positive by bacteriological culture; four animals were positive by the WS technique in kidney samples; six animals were positive by cPCR in kidney samples; and 11 animals were positive by qPCR, eight of which in kidney samples and three in liver. The bacterial quantification revealed a median of 4.3 bacteria/μL in liver samples and 36.6 bacteria/μL in kidney samples. qPCR presented the highest sensitivity among the techniques, followed by cPCR, the WS technique and bacteriological culture. These results indicate that sheep can carry leptospires of the Sejroe serogroup, and demonstrate the efficiency of quantitative PCR to detect Leptospira spp. in tissue samples. Published by Elsevier B.V.

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

Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome.

PubMed

Badri, Amine; Stefani, Franck O P; Lachance, Geneviève; Roy-Arcand, Line; Beaudet, Denis; Vialle, Agathe; Hijri, Mohamed

2016-10-01

Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172 ± 304 SE (n = 6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.

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

Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples.

PubMed

Pulford, D J; Gias, E; Bueno, I M; McFadden, Amj

2016-01-01

To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per

Quantitative analysis of pork and chicken products by droplet digital PCR.

PubMed

Cai, Yicun; Li, Xiang; Lv, Rong; Yang, Jielin; Li, Jian; He, Yuping; Pan, Liangwen

2014-01-01

In this project, a highly precise quantitative method based on the digital polymerase chain reaction (dPCR) technique was developed to determine the weight of pork and chicken in meat products. Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of species-specific DNAs in meat products. However, it is limited in amplification efficiency and relies on standard curves based Ct values, detecting and quantifying low copy number target DNA, as in some complex mixture meat products. By using the dPCR method, we find the relationships between the raw meat weight and DNA weight and between the DNA weight and DNA copy number were both close to linear. This enabled us to establish formulae to calculate the raw meat weight based on the DNA copy number. The accuracy and applicability of this method were tested and verified using samples of pork and chicken powder mixed in known proportions. Quantitative analysis indicated that dPCR is highly precise in quantifying pork and chicken in meat products and therefore has the potential to be used in routine analysis by government regulators and quality control departments of commercial food and feed enterprises.

Multi-laboratory comparison of quantitative PCR assays for detection and quantification of Fusarium virguliforme from soybean roots and soil

USDA-ARS?s Scientific Manuscript database

Accurate identification and quantification of Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, within root tissue and soil are important tasks. Several quantitative PCR (qPCR) assays have been developed but there are no reports comparing their use in sensitive and specific...

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence

PubMed Central

2017-01-01

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies. PMID:28591185

Enhanced Detection of Surface-Associated Bacteria in Indoor Environments by Quantitative PCR

PubMed Central

Buttner, Mark P.; Cruz-Perez, Patricia; Stetzenbach, Linda D.

2001-01-01

Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitate Bacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling

MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples

EPA Science Inventory

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from ...

Determining Fungi rRNA Copy Number by PCR

EPA Science Inventory

The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus".

PubMed

Selvaraj, Vijayanandraj; Maheshwari, Yogita; Hajeri, Subhas; Chen, Jianchi; McCollum, Thomas Greg; Yokomi, Raymond

2018-01-01

Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer.

Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus"

PubMed Central

Hajeri, Subhas; Chen, Jianchi; McCollum, Thomas Greg

2018-01-01

Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium “Candidatus Liberibacter asiaticus” (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer. PMID:29772016

OPPORTUNISTIC ASPERGILLUS PATHOGENS MEASURED IN HOME AND HOSPITAL TAP WATER BY MOLD SPECIFIC QUANTITATIVE PCR (MSQPCR)

EPA Science Inventory

Opportunistic fungal pathogens are a concern because of the increasing number of immunocompromised patients. The goal of this research was to test a simple extraction method and rapid quantitative PCR (QPCR) measurement of the occurrence of potential pathogens, Aspergillus fumiga...

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.

Volume dependency for culture of fungi from respiratory secretions and increased sensitivity of Aspergillus quantitative PCR.

PubMed

Fraczek, Marcin G; Kirwan, Marie B; Moore, Caroline B; Morris, Julie; Denning, David W; Richardson, Malcolm D

2014-02-01

Diagnosis of aspergillosis is often difficult. We compared fungal yields from respiratory specimens using the Health Protection Agency standard culture method (BSOP57), a higher volume undiluted culture method Mycology Reference Centre Manchester (MRCM) and Aspergillus quantitative real time polymerase chain reaction (qPCR). Sputum, bronchial aspirate and bronchoalveolar lavage (BAL) samples (total 23) were collected from aspergillosis patients. One fraction of all samples was cultured using the MRCM method, one BSOP57 and one was used for qPCR. The recovery rate for fungi was significantly higher by MRCM (87%) than by BSOP57 (8.7%) from all 23 specimens. Sputum samples were 44% positive by MRCM compared to no fungi isolated (0%) by BSOP57. Bronchial aspirates were 75% positive by MRCM and 0% by BSOP57. BAL samples were positive in 20% by MRCM and 10% by BSOP57. qPCR was always more sensitive than culture (95.6%) from all samples. In general, over 100 mould colonies (81 Aspergillus fumigatus) were grown using the MRCM method compared with only one colony from BSOP57. This study provides a reference point for standardisation of respiratory sample processing in diagnostic laboratories. Culture from higher volume undiluted respiratory specimens has a much higher yield for Aspergillus than BSOP57. qPCR is much more sensitive than culture and the current UK method requires revision. © 2013 Blackwell Verlag GmbH.

Standardization of Quantitative PCR for Human T-Cell Leukemia Virus Type 1 in Japan: a Collaborative Study

PubMed Central

Okuma, Kazu; Yamochi, Tadanori; Sato, Tomoo; Sasaki, Daisuke; Hasegawa, Hiroo; Umeki, Kazumi; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Naruse, Isao; Yamagishi, Makoto; Nakashima, Makoto; Momose, Haruka; Araki, Kumiko; Mizukami, Takuo; Mizusawa, Saeko; Okada, Yoshiaki; Ochiai, Masaki; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Nosaka, Kisato; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Satake, Masahiro; Okayama, Akihiko; Mochizuki, Manabu; Izumo, Shuji; Saito, Shigeru; Itabashi, Kazuo; Kamihira, Shimeru; Yamaguchi, Kazunari; Watanabe, Toshiki

2015-01-01

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory. PMID:26292315

Standardization of Quantitative PCR for Human T-Cell Leukemia Virus Type 1 in Japan: a Collaborative Study.

PubMed

Kuramitsu, Madoka; Okuma, Kazu; Yamochi, Tadanori; Sato, Tomoo; Sasaki, Daisuke; Hasegawa, Hiroo; Umeki, Kazumi; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Naruse, Isao; Yamagishi, Makoto; Nakashima, Makoto; Momose, Haruka; Araki, Kumiko; Mizukami, Takuo; Mizusawa, Saeko; Okada, Yoshiaki; Ochiai, Masaki; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Nosaka, Kisato; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Satake, Masahiro; Okayama, Akihiko; Mochizuki, Manabu; Izumo, Shuji; Saito, Shigeru; Itabashi, Kazuo; Kamihira, Shimeru; Yamaguchi, Kazunari; Watanabe, Toshiki; Hamaguchi, Isao

2015-11-01

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evaluation of Reference Genes for RT qPCR Analyses of Structure-Specific and Hormone Regulated Gene Expression in Physcomitrella patens Gametophytes

PubMed Central

Le Bail, Aude; Scholz, Sebastian; Kost, Benedikt

2013-01-01

The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions. PMID:23951063

Simultaneous detection of ricin and abrin DNA by real-time PCR (qPCR).

PubMed

Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wölfel, Roman

2012-09-01

Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5'-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

Validation of miRNA genes suitable as reference genes in qPCR analyses of miRNA gene expression in Atlantic salmon (Salmo salar).

PubMed

Johansen, Ilona; Andreassen, Rune

2014-12-23

MicroRNAs (miRNAs) are an abundant class of endogenous small RNA molecules that downregulate gene expression at the post-transcriptional level. They play important roles by regulating genes that control multiple biological processes, and recent years there has been an increased interest in studying miRNA genes and miRNA gene expression. The most common method applied to study gene expression of single genes is quantitative PCR (qPCR). However, before expression of mature miRNAs can be studied robust qPCR methods (miRNA-qPCR) must be developed. This includes identification and validation of suitable reference genes. We are particularly interested in Atlantic salmon (Salmo salar). This is an economically important aquaculture species, but no reference genes dedicated for use in miRNA-qPCR methods has been validated for this species. Our aim was, therefore, to identify suitable reference genes for miRNA-qPCR methods in Salmo salar. We used a systematic approach where we utilized similar studies in other species, some biological criteria, results from deep sequencing of small RNAs and, finally, experimental validation of candidate reference genes by qPCR to identify the most suitable reference genes. Ssa-miR-25-3p was identified as most suitable single reference gene. The best combinations of two reference genes were ssa-miR-25-3p and ssa-miR-455-5p. These two genes were constitutively and stably expressed across many different tissues. Furthermore, infectious salmon anaemia did not seem to affect their expression levels. These genes were amplified with high specificity, good efficiency and the qPCR assays showed a good linearity when applying a simple cybergreen miRNA-PCR method using miRNA gene specific forward primers. We have identified suitable reference genes for miRNA-qPCR in Atlantic salmon. These results will greatly facilitate further studies on miRNA genes in this species. The reference genes identified are conserved genes that are identical in their mature

Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus.

PubMed

Ciulli, Sara; Pinheiro, Ana Cristina de Aguiar Saldana; Volpe, Enrico; Moscato, Michele; Jung, Tae Sung; Galeotti, Marco; Stellino, Sabrina; Farneti, Riccardo; Prosperi, Santino

2015-03-01

Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/μl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains. Copyright © 2014 Elsevier B.V. All rights reserved.

Detection of Mycobacterium avium subsp. paratuberculosis in Drinking Water and Biofilms by Quantitative PCR ▿ †

PubMed Central

Beumer, Amy; King, Dawn; Donohue, Maura; Mistry, Jatin; Covert, Terry; Pfaller, Stacy

2010-01-01

It has been suggested that Mycobacterium avium subspecies paratuberculosis has a role in Crohn's disease. The organism may be acquired but is difficult to culture from the environment. We describe a quantitative PCR (qPCR) method to detect M. avium subsp. paratuberculosis in drinking water and the results of its application to drinking water and faucet biofilm samples collected in the United States. PMID:20817803

Effectiveness of qPCR permutations, internal controls and dilution as means for minimizing the impact of inhibition while measuring Enterococcus in environmental waters.

PubMed

Cao, Y; Griffith, J F; Dorevitch, S; Weisberg, S B

2012-07-01

  Draft criteria for the optional use of qPCR for recreational water quality monitoring have been published in the United States. One concern is that inhibition of the qPCR assay can lead to false-negative results and potentially inadequate public health protection. We evaluate the effectiveness of strategies for minimizing the impact of inhibition.   Five qPCR method permutations for measuring Enterococcus were challenged with 133 potentially inhibitory fresh and marine water samples. Serial dilutions were conducted to assess Enterococcus target assay inhibition, to which inhibition identified using four internal controls (IC) was compared. The frequency and magnitude of inhibition varied considerably among qPCR methods, with the permutation using an environmental master mix performing substantially better. Fivefold dilution was also effective at reducing inhibition in most samples (>78%). ICs were variable and somewhat ineffective, with 54-85% agreement between ICs and serial dilution.   The current IC methods appear to not accurately predict Enterococcus inhibition and should be used with caution; fivefold dilution and the use of reagents designed for environmental sample analysis (i.e. more robust qPCR chemistry) may be preferable.   Suitable approaches for defining, detecting and reducing inhibition will improve implementation of qPCR for water monitoring. © 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

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...

A Java program for LRE-based real-time qPCR that enables large-scale absolute quantification.

PubMed

Rutledge, Robert G

2011-03-02

Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples.

Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei.

PubMed

Janse, Ingmar; Hamidjaja, Raditijo A; Hendriks, Amber C A; van Rotterdam, Bart J

2013-02-14

Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification.

One time quantitative PCR detection of Pseudomonas aeruginosa to discriminate intermittent from chronic infection in cystic fibrosis.

PubMed

Boutin, Sébastien; Weitnauer, Michael; Hassel, Selina; Graeber, Simon Y; Stahl, Mirjam; Dittrich, A Susanne; Mall, Marcus A; Dalpke, Alexander H

2018-05-01

Chronic airway infection with Pseudomonas aeruginosa is a major risk factor of progression of lung disease in patients with cystic fibrosis (CF). Chronic P. aeruginosa infection evolves from intermittent infection that is amenable to antibiotic eradication, whereas chronically adapted P. aeruginosa becomes resistant to antibiotic therapy. Discrimination of intermittent versus chronic infection is therefore of high therapeutic relevance, yet the available diagnostic methods are only partly satisfactory. The aim of the present study was, therefore, to evaluate the usage of quantitative PCR (qPCR) to measure pathogen abundance and to discriminate between intermittent and chronic Pseudomonas infection in patients with CF. Using an established qPCR protocol, we analyzed the abundance of P. aeruginosa in 141 throats swabs and 238 sputa from CF patients with intermittent or chronic infection with P. aeruginosa, as determined by standard culture based diagnostics. We observed a large increase of abundance of P. aeruginosa in throat swabs and sputum samples from patients with chronic compared to intermittent infections with P. aeruginosa. The data show that abundance of P. aeruginosa as measured by qPCR is a valuable tool to discriminate intermittent from chronic infection. Of note, P. aeruginosa burden seems more sensitive than mucoidity phenotype to discriminate chronic from intermittent strains. Furthermore we observed that molecular detection in throat swabs was linked to a viable culture in the sputum when sputum was available. This result is of special interest in young patients with cystic fibrosis that often cannot expectorate sputum. We also observed that qPCR in comparison to culture detected the infection earlier. The results suggest that qPCR detection and quantification of P. aeruginosa is a precious tool to be added to the diagnostic toolbox in cystic fibrosis. Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Enumeration of verocytotoxigenic Escherichia coli (VTEC) O157 and O26 in milk by quantitative PCR.

PubMed

Mancusi, Rocco; Trevisani, Marcello

2014-08-01

Quantitative real-time polymerase chain reaction (qPCR) can be a convenient alternative to the Most Probable Number (MPN) methods to count VTEC in milk. The number of VTEC is normally very low in milk; therefore with the aim of increasing the method sensitivity a qPCR protocol that relies on preliminary enrichment was developed. The growth pattern of six VTEC strains (serogroups O157 and O26) was studied using enrichment in Buffered Peptone Water (BPW) with or without acriflavine for 4-24h. Milk samples were inoculated with these strains over a five Log concentration range between 0.24-0.50 and 4.24-4.50 Log CFU/ml. DNA was extracted from the enriched samples in duplicate and each extract was analysed in duplicate by qPCR using pairs of primers specific for the serogroups O157 and O26. When samples were pre-enriched in BPW at 37°C for 8h, the relationship between threshold cycles (CT values) and VTEC Log numbers was linear over a five Log concentration range. The regression of PCR threshold cycle numbers on VTEC Log CFU/ml had a slope coefficient equal to -3.10 (R(2)=0.96) which is indicative of a 10-fold difference of the gene copy numbers between samples (with a 100 ± 10% PCR efficiency). The same 10-fold proportion used for inoculating the milk samples with VTEC was observed, therefore, also in the enriched samples at 8h. A comparison of the CT values of milk samples and controls revealed that the strains inoculated in milk grew with 3 Log increments in the 8h enrichment period. Regression lines that fitted the qPCR and MPN data revealed that the error of the qPCR estimates is lower than the error of the estimated MPN (r=0.982, R(2)=0.965 vs. r=0.967, R(2)=0.935). The growth rates of VTEC strains isolated from milk should be comparatively assessed before qPCR estimates based on the regression model are considered valid. Comparative assessment of the growth rates can be done using spectrophotometric measurements of standardized cultures of isolates and

Fluorescence acquisition during hybridization phase in quantitative real-time PCR improves specificity and signal-to-noise ratio.

PubMed

Mehndiratta, Mohit; Palanichamy, Jayanth Kumar; Ramalingam, Pradeep; Pal, Arnab; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

2008-12-01

Quantitative real-time PCR (qPCR) is a standard method used for quantification of specific gene expression. This utilizes either dsDNA binding dyes or probe based chemistry. While dsDNA binding dyes have the advantage of low cost and flexibility, fluorescence due to primer dimers also interferes with the fluorescence of the specific product. Sometimes it is difficult, if not impossible, to standardize conditions and redesign primers in such a way that only specific fluorescence of the products of test and reference genes are acquired. Normally, the fluorescence acquisition in qPCR using dsDNA binding dyes is done during the melting phase of the PCR at a temperature between the melting points of primer dimers and the specific product. We have modified the protocol to acquire fluorescence during the hybridization phase. This significantly increased the signal-to-noise ratio and enabled the use of dsDNA binding dyes for mRNA quantification in situations where it was not possible when measurement was done in the melting phase. We have demonstrated it for three mRNAs, E6, E7, and DNMT1 with beta-actin as the reference gene, and for two miRNAs. This modification broadens the scope of qPCR using dsDNA binding dyes.

Detection of Toxoplasma gondii DNA by qPCR in the feces of a cat that recently ingested infected prey does not necessarily imply oocyst shedding.

PubMed

Poulle, Marie-Lazarine; Forin-Wiart, Marie-Amélie; Josse-Dupuis, Émilie; Villena, Isabelle; Aubert, Dominique

2016-01-01

Detection of Toxoplasma gondii DNA in cat feces is considered indicative of the presence of T. gondii oocysts. This study aims to demonstrate that the high sensitivity of qPCR can lead to T. gondii DNA detection in cat feces in the absence of oocysts. A cat immune to toxoplasmosis was fed with a mouse experimentally infected with T. gondii. Detection of DNA of this parasite was performed by qPCR on feces passed: (i) on the day the cat ingested the infected prey; (ii) during the three previous days; and (iii) during the three following days. The kinetics of qPCR results are clearly not linked to oocyst shedding and this result demonstrates that qPCR can detect T. gondii DNA related to bradyzoites from an infected prey, in the absence of oocysts. Caution is thus recommended when interpreting T. gondii qPCR results for samples of cat feces. © M.-L. Poulle et al., published by EDP Sciences, 2016.

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

PubMed

Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

2016-07-01

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea. © 2016 The Author(s).

Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

USGS Publications Warehouse

Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

2016-01-01

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control.

PubMed

Das, Amaresh; Deng, Ming Y; Babiuk, Shawn; McIntosh, Michael T

2017-05-01

Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

USDA-ARS?s Scientific Manuscript database

The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay rather than the entire sample process. Our objective was to develop a method to determine the 95% LOD (lowest co...

Evaluation of qPCR curve analysis methods for reliable biomarker discovery: bias, resolution, precision, and implications.

PubMed

Ruijter, Jan M; Pfaffl, Michael W; Zhao, Sheng; Spiess, Andrej N; Boggy, Gregory; Blom, Jochen; Rutledge, Robert G; Sisti, Davide; Lievens, Antoon; De Preter, Katleen; Derveaux, Stefaan; Hellemans, Jan; Vandesompele, Jo

2013-01-01

RNA transcripts such as mRNA or microRNA are frequently used as biomarkers to determine disease state or response to therapy. Reverse transcription (RT) in combination with quantitative PCR (qPCR) has become the method of choice to quantify small amounts of such RNA molecules. In parallel with the democratization of RT-qPCR and its increasing use in biomedical research or biomarker discovery, we witnessed a growth in the number of gene expression data analysis methods. Most of these methods are based on the principle that the position of the amplification curve with respect to the cycle-axis is a measure for the initial target quantity: the later the curve, the lower the target quantity. However, most methods differ in the mathematical algorithms used to determine this position, as well as in the way the efficiency of the PCR reaction (the fold increase of product per cycle) is determined and applied in the calculations. Moreover, there is dispute about whether the PCR efficiency is constant or continuously decreasing. Together this has lead to the development of different methods to analyze amplification curves. In published comparisons of these methods, available algorithms were typically applied in a restricted or outdated way, which does not do them justice. Therefore, we aimed at development of a framework for robust and unbiased assessment of curve analysis performance whereby various publicly available curve analysis methods were thoroughly compared using a previously published large clinical data set (Vermeulen et al., 2009) [11]. The original developers of these methods applied their algorithms and are co-author on this study. We assessed the curve analysis methods' impact on transcriptional biomarker identification in terms of expression level, statistical significance, and patient-classification accuracy. The concentration series per gene, together with data sets from unpublished technical performance experiments, were analyzed in order to assess the

Performance of two quantitative PCR methods for microbial source tracking of human sewage and implications for microbial risk assessment in recreational waters

EPA Science Inventory

Before new, rapid quantitative PCR (qPCR) methods for recreational water quality assessment and microbial source tracking (MST) can be useful in a regulatory context, an understanding of the ability of the method to detect a DNA target (marker) when the contaminant soure has been...

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.

Enhanced analytical sensitivity of a quantitative PCR for CMV using a modified nucleic-acid extraction procedure.

PubMed

Ferreira-Gonzalez, A; Yanovich, S; Langley, M R; Weymouth, L A; Wilkinson, D S; Garrett, C T

2000-01-01

Accurate and rapid diagnosis of CMV disease in immunocompromised individuals remains a challenge. Quantitative polymerase chain reaction (QPCR) methods for detection of CMV in peripheral blood mononuclear cells (PBMC) have improved the positive and negative predictive value of PCR for diagnosis of CMV disease. However, detection of CMV in plasma has demonstrated a lower negative predictive value for plasma as compared with PBMC. To enhance the sensitivity of the QPCR assay for plasma specimens, plasma samples were centrifuged before nucleic-acid extraction and the extracted DNA resolubilized in reduced volume. Optimization of the nucleic-acid extraction focused on decreasing or eliminating the presence of inhibitors in the pelleted plasma. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) with the same primer sequences as CMV. PCR products were detected by hybridization in a 96-well microtiter plate coated with a CMV or IS specific probe. The precision of the QPCR assay for samples prepared from untreated and from pelleted plasma was then assessed. The coefficient of variation for both types of samples was almost identical and the magnitude of the coefficient of variations was reduced by a factor of ten if the data were log transformed. Linearity of the QPCR assay extended over a 3.3-log range for both types of samples but the range of linearity for pelleted plasma was 20 to 40,000 viral copies/ml (vc/ml) in contrast to 300 to 400,000 vc/ml for plasma. Thus, centrifugation of plasma before nucleic-acid extraction and resuspension of extracted CMV DNA in reduced volume enhanced the analytical sensitivity approximately tenfold over the dynamic range of the assay. Copyright 2000 Wiley-Liss, Inc.

A Java Program for LRE-Based Real-Time qPCR that Enables Large-Scale Absolute Quantification

PubMed Central

Rutledge, Robert G.

2011-01-01

Background Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Findings Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. Conclusions The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples. PMID:21407812

A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina.

PubMed

Buling, A; Criado-Fornelio, A; Asenzo, G; Benitez, D; Barba-Carretero, J C; Florin-Christensen, M

2007-06-20

The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome b gene, this method shows both high sensitivity and specificity, and allows quantification of parasite DNA. In tests, reproducible quantitative results were obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curve analysis differentiated between B. bovis and B. bigemina. To assess the performance of the new qPCR procedure it was used to screen for babesiosis in 40 cows and 80 horses. B. bigemina was detected in five cows (three of these were also found to be positive by standard PCR techniques targeting the 18S rRNA gene). In addition, B. bovis was detected in one horse and B. bigemina in two horses using the proposed method, while none was found positive by ribosomal standard PCR. The sequences of the B. bigemina cytochrome b and 18S rRNA genes were completely conserved in isolates from Spain and Argentina, while those of B. bovis showed moderate polymorphism.

Aspergillus Galactomannan Enzyme Immunoassay and Quantitative PCR for Diagnosis of Invasive Aspergillosis with Bronchoalveolar Lavage Fluid

PubMed Central

Musher, Benjamin; Fredricks, David; Leisenring, Wendy; Balajee, S. Arunmozhi; Smith, Caitlin; Marr, Kieren A.

2004-01-01

Invasive pulmonary aspergillosis (IPA) is frequent and often fatal in hematopoietic stem cell transplant patients. Diagnosis requires microbiological or histopathologic demonstration of the organism in tissues; however, cultivation of Aspergillus species from respiratory secretions has low diagnostic sensitivity. Assays to detect Aspergillus antigen or DNA in bronchoalveolar lavage (BAL) fluid could facilitate earlier diagnosis, thereby guiding optimal therapy and obviating the need for additional costly and potentially morbid diagnostic evaluation. We evaluated the performance of a galactomannan enzyme immunoassay (GM EIA; Bio-Rad) by using a range of index cutoffs to define positivity and a quantitative PCR (qPCR) assay for the detection of Aspergillus species from BAL samples of patients with proven and probable IPA (case patients; n = 49) and without IPA (control patients; n = 50). The sensitivity of the GM EIA was 61% with an index cutoff of 1.0 and 76% with an index cutoff of 0.5; the corresponding specificities were 98 and 94%, respectively. The sensitivity and specificity of qPCR assay were 67 and 100%, respectively. The sensitivity with 22 culture-negative BAL specimens from patients with IPA was 41% for GM EIA with an index cutoff of 1.0, 59% for GM EIA with an index cutoff of 0.5, and 36% for qPCR assay. GM EIA indices and DNA quantities corresponded to BAL fungal burdens, with culture-positive samples having larger amounts of antigen and DNA compared to culture-negative samples. GM EIA and qPCR assay add to the sensitivity of BAL for diagnosing IPA in high-risk patients, with excellent specificity. Adjunctive use of these tests may reduce dependence on invasive diagnostic procedures. PMID:15583275

Identification of Reference Genes for Real-Time Quantitative PCR Experiments in the Liverwort Marchantia polymorpha

PubMed Central

Dolan, Liam; Langdale, Jane A.

2015-01-01

Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data. PMID:25798897

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.

Quantitative PCR Profiling of Escherichia coli in Livestock Feces Reveals Increased Population Resilience Relative to Culturable Counts under Temperature Extremes.

PubMed

Oliver, David M; Bird, Clare; Burd, Emmy; Wyman, Michael

2016-09-06

The relationship between culturable counts (CFU) and quantitative PCR (qPCR) cell equivalent counts of Escherichia coli in dairy feces exposed to different environmental conditions and temperature extremes was investigated. Fecal samples were collected in summer and winter from dairy cowpats held under two treatments: field-exposed versus polytunnel-protected. A significant correlation in quantified E. coli was recorded between the qPCR and culture-based methods (r = 0.82). Evaluation of the persistence profiles of E. coli over time revealed no significant difference in the E. coli numbers determined as either CFU or gene copies during the summer for the field-exposed cowpats, whereas significantly higher counts were observed by qPCR for the polytunnel-protected cowpats, which were exposed to higher ambient temperatures. In winter, the qPCR returned significantly higher counts of E. coli for the field-exposed cowpats, thus representing a reversal of the findings from the summer sampling campaign. Results from this study suggest that with increasing time post-defecation and with the onset of challenging environmental conditions, such as extremes in temperature, culture-based counts begin to underestimate the true resilience of viable E. coli populations in livestock feces. This is important not only in the long term as the Earth changes in response to climate-change drivers but also in the short term during spells of extremely cold or hot weather.

Coupling spore traps and quantitative PCR assays for detection of the downy mildew pathogens of spinach (Peronospora effusa) and beet (Peronospora schachtii)

USDA-ARS?s Scientific Manuscript database

Downy mildew of spinach (Spinacia oleracea L.), caused by Peronospora effusa, is a disease constraint on production worldwide, including in California where the majority of United States spinach is grown. The aim of this study was to develop a real-time quantitative PCR (qPCR) assay for detection o...

Combination of Competitive Quantitative PCR and Constant-Denaturant Capillary Electrophoresis for High-Resolution Detection and Enumeration of Microbial Cells

PubMed Central

Lim, Eelin L.; Tomita, Aoy V.; Thilly, William G.; Polz, Martin F.

2001-01-01

A novel quantitative PCR (QPCR) approach, which combines competitive PCR with constant-denaturant capillary electrophoresis (CDCE), was adapted for enumerating microbial cells in environmental samples using the marine nanoflagellate Cafeteria roenbergensis as a model organism. Competitive PCR has been used successfully for quantification of DNA in environmental samples. However, this technique is labor intensive, and its accuracy is dependent on an internal competitor, which must possess the same amplification efficiency as the target yet can be easily discriminated from the target DNA. The use of CDCE circumvented these problems, as its high resolution permitted the use of an internal competitor which differed from the target DNA fragment by a single base and thus ensured that both sequences could be amplified with equal efficiency. The sensitivity of CDCE also enabled specific and precise detection of sequences over a broad range of concentrations. The combined competitive QPCR and CDCE approach accurately enumerated C. roenbergensis cells in eutrophic, coastal seawater at abundances ranging from approximately 10 to 104 cells ml−1. The QPCR cell estimates were confirmed by fluorescent in situ hybridization counts, but estimates of samples with <50 cells ml−1 by QPCR were less variable. This novel approach extends the usefulness of competitive QPCR by demonstrating its ability to reliably enumerate microorganisms at a range of environmentally relevant cell concentrations in complex aquatic samples. PMID:11525983

Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR

PubMed Central

Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2012-01-01

Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant–pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1α, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under

Isolation and Identification of Lactic Acid Bacteria from Traditional Dairy Products in Baotou and Bayannur of Midwestern Inner Mongolia and q-PCR Analysis of Predominant Species

PubMed Central

2016-01-01

In this study, traditional culture method and 16S rRNA gene analysis were applied to reveal the composition and diversity of lactic acid bacteria (LAB) of fermented cow milk, huruud and urum from Baotou and Bayannur of midwestern Inner Mongolia. Also, the quantitative results of dominant LAB species in three different types of dairy products from Baotou and Bayannur were gained by quantitative polymerase chain reaction (q-PCR) technology. Two hundred and two LAB strains isolated from sixty-six samples were identified and classified into four genera, namely Enterococcus, Lactococcus, Lactobacillus, Leuconostoc, and twenty-one species and subspecies. From these isolates, Lactococcus lactis subsp. lactis (32.18%), Lactobacillus plantarum (12.38%) and Leuconosto mesenteroides (11.39%) were considered as the dominated LAB species under the condition of cultivating in MRS and M17 medium. And the q-PCR results revealed that the number of dominant species varied from samples to samples and from region to region. This study clearly shows the composition and diversity of LAB existing in fermented cow milk, huruud and urum, which could be considered as valuable resources for LAB isolation and further probiotic selection. PMID:27621691

qPCR standard operating procedure for measuring microorganisms in dust from dwellings in large cohort studies.

PubMed

Scherer, Emeline; Rocchi, Steffi; Reboux, Gabriel; Vandentorren, Stéphanie; Roussel, Sandrine; Vacheyrou, Mallory; Raherison, Chantal; Millon, Laurence

2014-01-01

The aim of the present study was to assess performance, feasibility and relevance of a Standard Operational Procedure (SOP) for large-scale use in the microbial analysis of children's indoor environments. We analyzed dust settled on Electrostatic Dust Fall Collectors (EDCs) by using qPCR which targeted 6 molds, 3 bacteria and 1 mite, chosen for their involvement in allergic or inflammatory processes. Six types of commercialized electrostatic wipes were tested for their releasing capacity of fungal DNA from fungal spores captured by the wipes. Specificity, repeatability and detection limits of the qPCR procedure were tested using calibrated microbial suspensions. The feasibility and relevance of this sampling and analysis method were assessed in a 75-home pilot study. Our result showed that one specific make of wipe was more effective than the others in releasing fungal DNA. qPCR procedure showed good repeatability. The quantification limit was about 5 fg DNA/μL for all species except Penicillium chrysogenum (0.5 fg DNA/μL) and Dermatophagoïdes pteronyssinus (10 fg DNA/μL). No cross-reactivity was observed. DNA concentrations in the 53/75 homes participating in the pilot study were between 0 and 24 625, 0 and 69 738 equivalent cells per cm(2) for the fungi and bacteria, and between 0 and 1 equivalent mites per cm(2) for D. pteronyssinus. Using the SOP described, we were able to classify the 53 dwellings from the least to the most contaminated according to the quantity of DNA measured for each species. Our SOP measured fungi, bacteria and mites using a cost-efficient, discreet and well-accepted sampling method with just one qPCR tool. The whole procedure can be used for microbial analysis in large cohort studies such as the ELFE study ("Etude Longitudinale Française depuis l'Enfance") and could help improve our understanding of the interactions between the environment, allergic diseases and child development. © 2013.

METHODS TO CLASSIFY ENVIRONMENTAL SAMPLES BASED ON MOLD ANALYSES BY QPCR

EPA Science Inventory

Quantitative PCR (QPCR) analysis of molds in indoor environmental samples produces highly accurate speciation and enumeration data. In a number of studies, eighty of the most common or potentially problematic indoor molds were identified and quantified in dust samples from homes...

High-throughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunodeficiency in population-based newborn screening.

PubMed

Gerstel-Thompson, Jacalyn L; Wilkey, Jonathan F; Baptiste, Jennifer C; Navas, Jennifer S; Pai, Sung-Yun; Pass, Kenneth A; Eaton, Roger B; Comeau, Anne Marie

2010-09-01

Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample. We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded. The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR. Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

Development and Use of Fluorescent Antibody and qPCR Protocols for the Electrostatic Spore Trap

USDA-ARS?s Scientific Manuscript database

Fluorescent antibody (FA) and qPCR protocols were evaluated for the newly developed aerobiological sampler (Ionic Spore Trap), which depends upon electrostatic deposition of particulates onto a 25 mm aluminum disk (stub). This device was originally designed for assessment of captured particulates by...

A new whole mitochondrial genome qPCR (WMG-qPCR) with SYBR Green® to identify phlebotomine sand fly blood meals.

PubMed

Rodrigues, Ana Caroline Moura; Magalhães, Rafaela Damasceno; Romcy, Kalil Andrade Mubarac; Freitas, Jeferson Lucas Sousa; Melo, Ana Carolina Fonseca Lindoso; Rodon, Fernanda Cristina Macedo; Bevilaqua, Claudia Maria Leal; Melo, Luciana Magalhães

2017-04-30

Phlebotomine sand flies are blood-feeding insects of marked medical and veterinary significance. Investigations on the biology of these insects hold great importance for both ecological and epidemiological purposes. The present work describes a new approach for real-time PCR (qPCR) with SYBR Green ® , named WMG-qPCR, to identify phlebotomine blood meals. The novelty of the assay was to design primers based on the Whole Mitochondrial Genome (WMG) of the potential hosts (human, dog, cat, brown rat and chicken) aiming to amplify through qPCR the regions of mitochondrial DNA (mtDNA) which are less conserved among all species. Initially, the best method for mtDNA extraction to be applied in WMG-qPCR was determined. Afterwards, amplification specificities were accessed by cross-reaction assays with mtDNA samples from all animal species, besides phlebotomine DNA. Finally, the selected primers were also tested for their limit of DNA detection through standard curves constructed by serial dilution of blood DNA obtained for each target animal species. The WMG-qPCR was able to detect as low as 10pL of blood, equivalent to 26, 84, 130, and 320fg DNA of cat, human, dog and rat, respectively. The assay was also capable to amplify as low as 5pL of chicken blood (5pg DNA). In conclusion, WMG-qPCR seems to be a promising tool to identify phlebotomine blood meals, with high species-specificity and sensitivity. Furthermore, as no supplementary techniques are required, this new approach presents minimized costs and simplified technical-training requirements for execution. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods.

PubMed

Rodríguez, Alicia; Werning, María L; Rodríguez, Mar; Bermúdez, Elena; Córdoba, Juan J

2012-12-01

A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1-4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain. Copyright © 2012 Elsevier Ltd. All rights reserved.

Development, application, and results of routine monitoring of Marek's disease virus in broiler house dust using real-time quantitative PCR.

PubMed

Walkden-Brown, Stephen W; Islam, A F Aminul; Groves, Peter J; Rubite, Ambrosio; Sharpe, Sue M; Burgess, Susan K

2013-06-01

Results are presented from four studies between 2002 and 2011 into the feasibility of routinely monitoring Marek's disease virus serotype 1 (MDV-1) in broiler house dust using real-time quantitative PCR (qPCR) measurement. Study 1 on two farms showed that detection of MDV-1 occurred earlier on average in dust samples tested using qPCR than standard PCR and in spleen samples from five birds per shed assayed for MDV-1 by qPCR or standard PCR. DNA quality following extraction from dust had no effect on detection of MDV-1. Study 2 demonstrated that herpesvirus of turkeys (HVT) and MDV serotype 2 (MDV-2) in addition to MDV-1 could be readily amplified from commercial farm dust samples, often in mixtures. MDV-2 was detected in 11 of 20 samples despite the absence of vaccination with this serotype. Study 3 investigated the reproducibility and sensitivity of the qPCR test and the presence of inhibitors in the samples. Samples extracted and amplified in triplicate showed a high level of reproducibility except at very low levels of virus near the limit of detection. Mixing of samples prior to extraction provided results consistent with the proportions in the mixture. Tests for inhibition showed that if the template contained DNA in the range 0.5-20 ng/microl no inhibition of the reaction was detectable. The sensitivity of the tests in terms of viral copy number (VCN) per milligram of dust was calculated to be in the range 24-600 VCN/mg for MDV-1, 48-1200 VCN/mg for MDV-2, and 182-4560 VCN/mg for HVT. In study 4 the results of 1976 commercial tests carried out for one company were analyzed. Overall 23.1% of samples were positive for MDV-1, 26.1% in unvaccinated and 16.4% in vaccinated chickens. There was marked regional and temporal variation in the proportion of positive samples and the MDV-1 load. The tests were useful in formulating Marek's disease vaccination strategies. The number of samples submitted has increased recently, as has the incidence of positive samples

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.

A nested real-time PCR assay for the quantification of Plasmodium falciparum DNA extracted from dried blood spots.

PubMed

Tran, Tuan M; Aghili, Amirali; Li, Shanping; Ongoiba, Aissata; Kayentao, Kassoum; Doumbo, Safiatou; Traore, Boubacar; Crompton, Peter D

2014-10-04

As public health efforts seek to eradicate malaria, there has been an emphasis on eliminating low-density parasite reservoirs in asymptomatic carriers. As such, diagnosing submicroscopic Plasmodium infections using PCR-based techniques has become important not only in clinical trials of malaria vaccines and therapeutics, but also in active malaria surveillance campaigns. However, PCR-based quantitative assays that rely on nucleic acid extracted from dried blood spots (DBS) have demonstrated lower sensitivity than assays that use cryopreserved whole blood as source material. The density of Plasmodium falciparum asexual parasites was quantified using genomic DNA extracted from dried blood spots (DBS) and the sensitivity of two approaches was compared: quantitative real-time PCR (qPCR) targeting the P. falciparum 18S ribosomal RNA gene, either with an initial conventional PCR amplification prior to qPCR (nested qPCR), or without an initial amplification (qPCR only). Parasite densities determined by nested qPCR, qPCR only, and light microscopy were compared. Nested qPCR results in 10-fold higher sensitivity (0.5 parasites/μl) when compared to qPCR only (five parasites/ul). Among microscopy-positive samples, parasite densities calculated by nested qPCR correlated strongly with microscopy for both asymptomatic (Pearson's r=0.58, P<0.001) and symptomatic (Pearson's r=0.70, P<0.0001) P. falciparum infections. Nested qPCR improves the sensitivity for the detection of P. falciparum blood-stage infection from clinical DBS samples. This approach may be useful for active malaria surveillance in areas where submicroscopic asymptomatic infections are prevalent.

Development of a rapid HRM qPCR for the diagnosis of the four most prevalent Plasmodium lineages in New Zealand.

PubMed

Schoener, E R; Hunter, S; Howe, L

2017-07-01

Although wildlife rehabilitation and translocations are important tools in wildlife conservation in New Zealand, disease screening of birds has not been standardized. Additionally, the results of the screening programmes are often difficult to interpret due to missing disease data in resident or translocating avian populations. Molecular methods have become the most widespread method for diagnosing avian malaria (Plasmodium spp.) infections. However, these methods can be time-consuming, expensive and are less specific in diagnosing mixed infections. Thus, this study developed a new real-time PCR (qPCR) method that was able to detect and specifically identify infections of the three most common lineages of avian malaria in New Zealand (Plasmodium (Novyella) sp. SYAT05, Plasmodium elongatum GRW6 and Plasmodium spp. LINN1) as well as a less common, pathogenic Plasmodium relictum GRW4 lineage. The assay was also able to discern combinations of these parasites in the same sample and had a detection limit of five parasites per microlitre. Due to concerns relating to the presence of the potentially highly pathogenic P. relictum GRW4 lineage in avian populations, an additional confirmatory high resolution (HRM) qPCR was developed to distinguish between commonly identified P. elongatum GRW6 from P. relictum GRW4. The new qPCR assays were tested using tissue samples containing Plasmodium schizonts from three naturally infected dead birds resulting in the identified infection of P. elongatum GRW6. Thus, these rapid qPCR assays have shown to be cost-effective and rapid screening tools for the detection of Plasmodium infection in New Zealand native birds.

A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl

USGS Publications Warehouse

Smith, Matthew M.; Schmutz, Joel A.; Apelgren, Chloe; Ramey, Andy M.

2015-01-01

Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n = 105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R2 = 0.694, P = 0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species.

Identification of TL-Om1, an Adult T-Cell Leukemia (ATL) Cell Line, as Reference Material for Quantitative PCR for Human T-Lymphotropic Virus 1

PubMed Central

Okuma, Kazu; Yamagishi, Makoto; Yamochi, Tadanori; Firouzi, Sanaz; Momose, Haruka; Mizukami, Takuo; Takizawa, Kazuya; Araki, Kumiko; Sugamura, Kazuo; Yamaguchi, Kazunari; Watanabe, Toshiki

2014-01-01

Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TL-Om1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR. PMID:25502533

Analysis of the bacterial community in aged and aging pit mud of Chinese Luzhou-flavour liquor by combined PCR-DGGE and quantitative PCR assay.

PubMed

Liang, Huipeng; Li, Wenfang; Luo, Qingchun; Liu, Chaolan; Wu, Zhengyun; Zhang, Wenxue

2015-10-01

The community structure of bacteria in aged and aging pit mud, which was judged according to their sensory and physicochemical characteristics, was analysed using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real-time PCR (qPCR). The phyla Firmicutes, Actinobacteria, Proteobacteria, Synergistetes and Unclassified Bacteria were detected and the fermentative Firmicutes was predominant in both types of pit mud in the PCR-DGGE analysis. Among Firmicutes, Clostridiales was dominant in aged pit mud while Bacillales and Lactobacillales were dominant in aging pit mud. The diversity of bacterial communities in aged pit mud was higher than that in aging pit mud. In the qPCR analysis the abundance of Clostridium IV in aged pit mud was higher than that in aging pit mud and there were significant differences in the quantity of Clostridium IV between aged and aging pit mud of the same cellar (P < 0.05). There were some significant differences in the microbial community structure between aged and aging pit mud. The differences in the quantity of Clostridium IV might be involved in the distinction that the aged pit mud has a strong aroma while the aging pit mud does not. © 2014 Society of Chemical Industry.

Underwater Application of Quantitative PCR on an Ocean Mooring

PubMed Central

Preston, Christina M.; Harris, Adeline; Ryan, John P.; Roman, Brent; Marin, Roman; Jensen, Scott; Everlove, Cheri; Birch, James; Dzenitis, John M.; Pargett, Douglas; Adachi, Masao; Turk, Kendra; Zehr, Jonathon P.; Scholin, Christopher A.

2011-01-01

The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions. PMID:21829630

miPrimer: an empirical-based qPCR primer design method for small noncoding microRNA

PubMed Central

Kang, Shih-Ting; Hsieh, Yi-Shan; Feng, Chi-Ting; Chen, Yu-Ting; Yang, Pok Eric; Chen, Wei-Ming

2018-01-01

MicroRNAs (miRNAs) are 18–25 nucleotides (nt) of highly conserved, noncoding RNAs involved in gene regulation. Because of miRNAs’ short length, the design of miRNA primers for PCR amplification remains a significant challenge. Adding to the challenge are miRNAs similar in sequence and miRNA family members that often only differ in sequences by 1 nt. Here, we describe a novel empirical-based method, miPrimer, which greatly reduces primer dimerization and increases primer specificity by factoring various intrinsic primer properties and employing four primer design strategies. The resulting primer pairs displayed an acceptable qPCR efficiency of between 90% and 110%. When tested on miRNA families, miPrimer-designed primers are capable of discriminating among members of miRNA families, as validated by qPCR assays using Quark Biosciences’ platform. Of the 120 miRNA primer pairs tested, 95.6% and 93.3% were successful in amplifying specifically non-family and family miRNA members, respectively, after only one design trial. In summary, miPrimer provides a cost-effective and valuable tool for designing miRNA primers. PMID:29208706

Evaluation of 16S rRNA qPCR for detection of Mycobacterium leprae DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases.

PubMed

Marques, Lívia Érika Carlos; Frota, Cristiane Cunha; Quetz, Josiane da Silva; Bindá, Alexandre Havt; Mota, Rosa Maria Salane; Pontes, Maria Araci de Andrade; Gonçalves, Heitor de Sá; Kendall, Carl; Kerr, Ligia Regina Franco Sansigolo

2017-12-26

Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (T m  = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 10 3 -8.02 × 10 5 , and in SB samples from MB patients were 1.87 × 10 3 -1.50 × 10 6 . Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

PubMed

Kimura, Yasumasa; Soma, Takahiro; Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J L; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

2016-01-01

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.

Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays

PubMed Central

Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J. L.; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

2016-01-01

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download. PMID:26863543

MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples.

PubMed

Sivaganesan, Mano; Siefring, Shawn; Varma, Manju; Haugland, Richard A

2011-12-01

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for

An event-specific method for the detection and quantification of ML01, a genetically modified Saccharomyces cerevisiae wine strain, using quantitative PCR.

PubMed

Vaudano, Enrico; Costantini, Antonella; Garcia-Moruno, Emilia

2016-10-03

The availability of genetically modified (GM) yeasts for winemaking and, in particular, transgenic strains based on the integration of genetic constructs deriving from other organisms into the genome of Saccharomyces cerevisiae, has been a reality for several years. Despite this, their use is only authorized in a few countries and limited to two strains: ML01, able to convert malic acid into lactic acid during alcoholic fermentation, and ECMo01 suitable for reducing the risk of carbamate production. In this work we propose a quali-quantitative culture-independent method for the detection of GM yeast ML01 in commercial preparations of ADY (Active Dry Yeast) consisting of efficient extraction of DNA and qPCR (quantitative PCR) analysis based on event-specific assay targeting MLC (malolactic cassette), and a taxon-specific S. cerevisiae assay detecting the MRP2 gene. The ADY DNA extraction methodology has been shown to provide good purity DNA suitable for subsequent qPCR. The MLC and MRP2 qPCR assay showed characteristics of specificity, dynamic range, limit of quantification (LOQ) limit of detection (LOD), precision and trueness, which were fully compliant with international reference guidelines. The method has been shown to reliably detect 0.005% (mass/mass) of GM ML01 S. cerevisiae in commercial preparations of ADY. Copyright © 2016 Elsevier B.V. All rights reserved.

Validation of qPCR Methods for the Detection of Mycobacterium in New World Animal Reservoirs.

PubMed

Housman, Genevieve; Malukiewicz, Joanna; Boere, Vanner; Grativol, Adriana D; Pereira, Luiz Cezar M; Silva, Ita de Oliveira; Ruiz-Miranda, Carlos R; Truman, Richard; Stone, Anne C

2015-11-01

Zoonotic pathogens that cause leprosy (Mycobacterium leprae) and tuberculosis (Mycobacterium tuberculosis complex, MTBC) continue to impact modern human populations. Therefore, methods able to survey mycobacterial infection in potential animal hosts are necessary for proper evaluation of human exposure threats. Here we tested for mycobacterial-specific single- and multi-copy loci using qPCR. In a trial study in which armadillos were artificially infected with M. leprae, these techniques were specific and sensitive to pathogen detection, while more traditional ELISAs were only specific. These assays were then employed in a case study to detect M. leprae as well as MTBC in wild marmosets. All marmosets were negative for M. leprae DNA, but 14 were positive for the mycobacterial rpoB gene assay. Targeted capture and sequencing of rpoB and other MTBC genes validated the presence of mycobacterial DNA in these samples and revealed that qPCR is useful for identifying mycobacterial-infected animal hosts.

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.

A Quantitative PCR Protocol for Detection of Oxyspirura petrowi in Northern Bobwhites (Colinus virginianus).

PubMed

Kistler, Whitney M; Parlos, Julie A; Peper, Steven T; Dunham, Nicholas R; Kendall, Ronald J

2016-01-01

Oxyspirura petrowi is a parasitic nematode that infects wild birds. This parasite has a broad host range, but has recently been reported in high prevalences from native Galliformes species in the United States. In order to better understand the impact O. petrowi has on wild bird populations, we developed a quantitative PCR protocol to detect infections in wild northern bobwhites (Colinus virginianus). We used paired fecal and cloacal swab samples from wild caught and experimentally infected northern bobwhites and matching fecal float data from experimentally infected birds to validate our assay. Overall we detected more positive birds from fecal samples than the paired cloacal swabs and there was strong agreement between the qPCR results from fecal samples and from fecal flotation (84%; κ = 0.69 [0.53-0.84 95% CI]). We also detected O. petrowi DNA in ten replicates of samples spiked with one O. petrowi egg. This qPCR assay is an effective assay to detect O. petrowi infections in wild birds. Our results suggest that fecal samples are the most appropriate sample for detecting infections; although, cloacal swabs can be useful for determining if O. petrowi is circulating in a population.

A Quantitative PCR Protocol for Detection of Oxyspirura petrowi in Northern Bobwhites (Colinus virginianus)

PubMed Central

Kistler, Whitney M.; Parlos, Julie A.; Peper, Steven T.; Dunham, Nicholas R.; Kendall, Ronald J.

2016-01-01

Oxyspirura petrowi is a parasitic nematode that infects wild birds. This parasite has a broad host range, but has recently been reported in high prevalences from native Galliformes species in the United States. In order to better understand the impact O. petrowi has on wild bird populations, we developed a quantitative PCR protocol to detect infections in wild northern bobwhites (Colinus virginianus). We used paired fecal and cloacal swab samples from wild caught and experimentally infected northern bobwhites and matching fecal float data from experimentally infected birds to validate our assay. Overall we detected more positive birds from fecal samples than the paired cloacal swabs and there was strong agreement between the qPCR results from fecal samples and from fecal flotation (84%; κ = 0.69 [0.53–0.84 95% CI]). We also detected O. petrowi DNA in ten replicates of samples spiked with one O. petrowi egg. This qPCR assay is an effective assay to detect O. petrowi infections in wild birds. Our results suggest that fecal samples are the most appropriate sample for detecting infections; although, cloacal swabs can be useful for determining if O. petrowi is circulating in a population. PMID:27893772

Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples.

PubMed

Holmes, Amy S; Houston, Rachel; Elwick, Kyleen; Gangitano, David; Hughes-Stamm, Sheree

2018-05-01

DNA quantification is a vital step in forensic DNA analysis to determine the optimal input amount for DNA typing. A quantitative real-time polymerase chain reaction (qPCR) assay that can predict DNA degradation or inhibitors present in the sample prior to DNA amplification could aid forensic laboratories in creating a more streamlined and efficient workflow. This study compares the results from four commercial qPCR kits: (1) Investigator® Quantiplex® Pro Kit, (2) Quantifiler® Trio DNA Quantification Kit, (3) PowerQuant® System, and (4) InnoQuant® HY with high molecular weight DNA, low template samples, degraded samples, and DNA spiked with various inhibitors.The results of this study indicate that all kits were comparable in accurately predicting quantities of high quality DNA down to the sub-picogram level. However, the InnoQuant(R) HY kit showed the highest precision across the DNA concentration range tested in this study. In addition, all kits performed similarly with low concentrations of forensically relevant PCR inhibitors. However, in general, the Investigator® Quantiplex® Pro Kit was the most tolerant kit to inhibitors and provided the most accurate quantification results with higher concentrations of inhibitors (except with salt). PowerQuant® and InnoQuant® HY were the most sensitive to inhibitors, but they did indicate significant levels of PCR inhibition. When quantifying degraded samples, each kit provided different degradation indices (DI), with Investigator® Quantiplex® Pro indicating the largest DI and Quantifiler® Trio indicating the smallest DI. When the qPCR kits were paired with their respective STR kit to genotype highly degraded samples, the Investigator® 24plex QS and GlobalFiler® kits generated more complete profiles when the small target concentrations were used for calculating input amount.

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR.

PubMed

Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

2016-01-01

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR.

RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR

PubMed Central

Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

2016-01-01

Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR. PMID:27304673

Development of Quantitative Real-Time PCR Assays for Detection and Quantification of Surrogate Biological Warfare Agents in Building Debris and Leachate▿

PubMed Central

Saikaly, Pascal E.; Barlaz, Morton A.; de los Reyes, Francis L.

2007-01-01

Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus anthracis (anthrax) and Yersinia pestis (plague), respectively. The targets for SYBR green Q-PCR assays were the 16S-23S rRNA intergenic transcribed spacer (ITS) region and recA gene for B. atrophaeus and the gyrB, wzm, and recA genes for S. marcescens. All assays showed high specificity when tested against 5 ng of closely related Bacillus and Serratia nontarget DNA from 21 organisms. Several spore lysis methods that include a combination of one or more of freeze-thaw cycles, chemical lysis, hot detergent treatment, bead beat homogenization, and sonication were evaluated. All methods tested showed similar threshold cycle values. The limit of detection of the developed Q-PCR assays was determined using DNA extracted from a pure bacterial culture and DNA extracted from sterile water, leachate, and SBD samples spiked with increasing quantities of surrogates. The limit of detection for B. atrophaeus genomic DNA using the ITS and B. atrophaeus recA Q-PCR assays was 7.5 fg per PCR. The limits of detection of S. marcescens genomic DNA using the gyrB, wzm, and S. marcescens recA Q-PCR assays were 7.5 fg, 75 fg, and 7.5 fg per PCR, respectively. Quantification of B. atrophaeus vegetative cells and spores was linear (R2 > 0.98) over a 7-log-unit dynamic range down to 101 B. atrophaeus cells or spores. Quantification of S. marcescens (R2 > 0.98) was linear over a 6-log-unit dynamic range down to 102 S. marcescens cells. The developed Q-PCR assays are highly specific and sensitive and can

The combination of quantitative PCR and western blot detecting CP4-EPSPS component in Roundup Ready soy plant tissues and commercial soy-related foodstuffs.

PubMed

Xiao, Xiao; Wu, Honghong; Zhou, Xinghu; Xu, Sheng; He, Jian; Shen, Wenbiao; Zhou, Guanghong; Huang, Ming

2012-06-01

With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods

Quantification of Plasma miRNAs by Digital PCR for Cancer Diagnosis

PubMed Central

Ma, Jie; Li, Ning; Guarnera, Maria; Jiang, Feng

2013-01-01

Analysis of plasma microRNAs (miRNAs) by quantitative polymerase chain reaction (qPCR) provides a potential approach for cancer diagnosis. However, absolutely quantifying low abundant plasma miRNAs is challenging with qPCR. Digital PCR offers a unique means for assessment of nucleic acids presenting at low levels in plasma. This study aimed to evaluate the efficacy of digital PCR for quantification of plasma miRNAs and the potential utility of this technique for cancer diagnosis. We used digital PCR to quantify the copy number of plasma microRNA-21-5p (miR-21–5p) and microRNA-335–3p (miR-335–3p) in 36 lung cancer patients and 38 controls. Digital PCR showed a high degree of linearity and quantitative correlation with miRNAs in a dynamic range from 1 to 10,000 copies/μL of input, with high reproducibility. qPCR exhibited a dynamic range from 100 to 1×107 copies/μL of input. Digital PCR had a higher sensitivity to detect copy number of the miRNAs compared with qPCR. In plasma, digital PCR could detect copy number of both miR-21–5p and miR-335–3p, whereas qPCR was only able to assess miR-21–5p. Quantification of the plasma miRNAs by digital PCR provided 71.8% sensitivity and 80.6% specificity in distinguishing lung cancer patients from cancer-free subjects. PMID:24277982

The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment

PubMed Central

Staggs, Sarah E.; Beckman, Erin M.; Keely, Scott P.; Mackwan, Reena; Ware, Michael W.; Moyer, Alan P.; Ferretti, James A.; Sayed, Abu; Xiao, Lihua; Villegas, Eric N.

2013-01-01

Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources. PMID:23805235

Integrating quantitative PCR and Bayesian statistics in quantifying human adenoviruses in small volumes of source water.

PubMed

Wu, Jianyong; Gronewold, Andrew D; Rodriguez, Roberto A; Stewart, Jill R; Sobsey, Mark D

2014-02-01

Rapid quantification of viral pathogens in drinking and recreational water can help reduce waterborne disease risks. For this purpose, samples in small volume (e.g. 1L) are favored because of the convenience of collection, transportation and processing. However, the results of viral analysis are often subject to uncertainty. To overcome this limitation, we propose an approach that integrates Bayesian statistics, efficient concentration methods, and quantitative PCR (qPCR) to quantify viral pathogens in water. Using this approach, we quantified human adenoviruses (HAdVs) in eighteen samples of source water collected from six drinking water treatment plants. HAdVs were found in seven samples. In the other eleven samples, HAdVs were not detected by qPCR, but might have existed based on Bayesian inference. Our integrated approach that quantifies uncertainty provides a better understanding than conventional assessments of potential risks to public health, particularly in cases when pathogens may present a threat but cannot be detected by traditional methods. © 2013 Elsevier B.V. All rights reserved.

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.

A real-time, quantitative PCR protocol for assessing the relative parasitemia of Leucocytozoon in waterfowl.

PubMed

Smith, Matthew M; Schmutz, Joel; Apelgren, Chloe; Ramey, Andrew M

2015-04-01

Microscopic examination of blood smears can be effective at diagnosing and quantifying hematozoa infections. However, this method requires highly trained observers, is time consuming, and may be inaccurate for detection of infections at low levels of parasitemia. To develop a molecular methodology for identifying and quantifying Leucocytozoon parasite infection in wild waterfowl (Anseriformes), we designed a real-time, quantitative PCR protocol to amplify Leucocytozoon mitochondrial DNA using TaqMan fluorogenic probes and validated our methodology using blood samples collected from waterfowl in interior Alaska during late summer and autumn (n=105). By comparing our qPCR results to those derived from a widely used nested PCR protocol, we determined that our assay showed high levels of sensitivity (91%) and specificity (100%) in detecting Leucocytozoon DNA from host blood samples. Additionally, results of a linear regression revealed significant correlation between the raw measure of parasitemia produced by our qPCR assay (Ct values) and numbers of parasites observed on blood smears (R(2)=0.694, P=0.003), indicating that our assay can reliably determine the relative parasitemia levels among samples. This methodology provides a powerful new tool for studies assessing effects of haemosporidian infection in wild avian species. Published by Elsevier B.V.

Combining real-time PCR and next-generation DNA sequencing to provide quantitative comparisons of fungal aerosol populations

NASA Astrophysics Data System (ADS)

Dannemiller, Karen C.; Lang-Yona, Naama; Yamamoto, Naomichi; Rudich, Yinon; Peccia, Jordan

2014-02-01

We examined fungal communities associated with the PM10 mass of Rehovot, Israel outdoor air samples collected in the spring and fall seasons. Fungal communities were described by 454 pyrosequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal RNA encoding gene. To allow for a more quantitative comparison of fungal exposure in humans, the relative abundance values of specific taxa were transformed to absolute concentrations through multiplying these values by the sample's total fungal spore concentration (derived from universal fungal qPCR). Next, the sequencing-based absolute concentrations for Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, and Penicillium/Aspergillus spp. were compared to taxon-specific qPCR concentrations for A. alternata, C. cladosporioides, E. nigrum, and Penicillium/Aspergillus spp. derived from the same spring and fall aerosol samples. Results of these comparisons showed that the absolute concentration values generated from pyrosequencing were strongly associated with the concentration values derived from taxon-specific qPCR (for all four species, p < 0.005, all R > 0.70). The correlation coefficients were greater for species present in higher concentrations. Our microbial aerosol population analyses demonstrated that fungal diversity (number of fungal operational taxonomic units) was higher in the spring compared to the fall (p = 0.02), and principal coordinate analysis showed distinct seasonal differences in taxa distribution (ANOSIM p = 0.004). Among genera containing allergenic and/or pathogenic species, the absolute concentrations of Alternaria, Aspergillus, Fusarium, and Cladosporium were greater in the fall, while Cryptococcus, Penicillium, and Ulocladium concentrations were greater in the spring. The transformation of pyrosequencing fungal population relative abundance data to absolute concentrations can improve next-generation DNA sequencing-based quantitative aerosol exposure

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.

Developing noninvasive diagnosis for single-gene disorders: the role of digital PCR.

PubMed

Barrett, Angela N; Chitty, Lyn S

2014-01-01

Cell-free fetal DNA constitutes approximately 10 % of the cell-free DNA found in maternal plasma and can be used as a reliable source of fetal genetic material for noninvasive prenatal diagnosis (NIPD) from early pregnancy. The relatively high levels of maternal background can make detection of paternally inherited point mutations challenging. Diagnosis of inheritance of autosomal recessive disorders using qPCR is even more challenging due to the high background of mutant maternal allele. Digital PCR is a very sensitive modified method of quantitative real-time PCR (qPCR), allowing absolute quantitation and rare allele detection without the need for standards or normalization. Samples are diluted and then partitioned into a large number of small qPCR reactions, some of which contain the target molecule and some which do not; the proportion of positive reactions can be used to calculate the concentration of targets in the initial sample. Here we discuss the use of digital PCR as an accurate approach to NIPD for single-gene disorders.

Creation of a bovine herpes virus 1 (BoHV-1) quantitative particle standard by transmission electron microscopy and comparison with established standards for use in real-time PCR.

PubMed

Hoferer, Marc; Braun, Anne; Sting, Reinhard

2017-07-01

Standards are pivotal for pathogen quantification by real-time PCR (qPCR); however, the creation of a complete and universally applicable virus particle standard is challenging. In the present study a procedure based on purification of bovine herpes virus type 1 (BoHV-1) and subsequent quantification by transmission electron microscopy (TEM) is described. Accompanying quantitative quality controls of the TEM preparation procedure using qPCR yielded recovery rates of more than 95% of the BoHV-1 virus particles on the grid used for virus counting, which was attributed to pre-treatment of the grid with 5% bovine albumin. To compare the value of the new virus particle standard for use in qPCR, virus counter based quantification and established pure DNA standards represented by a plasmid and an oligonucleotide were included. It could be shown that the numbers of virus particles, plasmid and oligonucleotide equivalents were within one log10 range determined on the basis of standard curves indicating that different approaches provide comparable quantitative values. However, only virus particles represent a complete, universally applicable quantitative virus standard that meets the high requirements of an RNA and DNA virus gold standard. In contrast, standards based on pure DNA have to be considered as sub-standard due to limited applications. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

A TaqMan-based multiplex qPCR assay and DNA extraction method for phylotype IIB sequevars 1&2 (select agent) strains of Ralstonia solanacearum

DOE PAGES

Stulberg, Michael J.; Huang, Qi

2015-10-01

Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regionsmore » of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Moreover, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum.« less

A TaqMan-Based Multiplex qPCR Assay and DNA Extraction Method for Phylotype IIB Sequevars 1&2 (Select Agent) Strains of Ralstonia solanacearum

PubMed Central

Stulberg, Michael J.; Huang, Qi

2015-01-01

Ralstonia solanacearum race 3 biovar 2 strains belonging to phylotype IIB, sequevars 1 and 2 (IIB-1&2) cause brown rot of potato in temperate climates, and are quarantined pathogens in Canada and Europe. Since these strains are not established in the U.S. and because of their potential risk to the potato industry, the U.S. government has listed them as select agents. Cultivated geraniums are also a host and have the potential to spread the pathogen through trade, and its extracts strongly inhibits DNA-based detection methods. We designed four primer and probe sets for an improved qPCR method that targets stable regions of DNA. RsSA1 and RsSA2 recognize IIB-1&2 strains, RsII recognizes the current phylotype II (the newly proposed R. solanacearum species) strains (and a non-plant associated R. mannitolilytica), and Cox1 recognizes eight plant species including major hosts of R. solanacearum such as potato, tomato and cultivated geranium as an internal plant control. We multiplexed the RsSA2 with the RsII and Cox1 sets to provide two layers of detection of a positive IIB-1&2 sample, and to validate plant extracts and qPCR reactions. The TaqMan-based uniplex and multiplex qPCR assays correctly identified 34 IIB-1&2 and 52 phylotype II strains out of 90 R. solanacearum species complex strains. Additionally, the multiplex qPCR assay was validated successfully using 169 artificially inoculated symptomatic and asymptomatic plant samples from multiple plant hosts including geranium. Furthermore, we developed an extraction buffer that allowed for a quick and easy DNA extraction from infected plants including geranium for detection of R. solanacearum by qPCR. Our multiplex qPCR assay, especially when coupled with the quick extraction buffer method, allows for quick, easy and reliable detection and differentiation of the IIB-1&2 strains of R. solanacearum. PMID:26426354

Comparison of Enterococcus qPCR analysis results from fresh and marine waters on two real-tme instruments

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) will be recommending a quantitativ e polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for widespread implementation of this o...

Multiplex real-time PCR using temperature sensitive primer-supplying hydrogel particles and its application for malaria species identification

PubMed Central

Byoun, Mun Sub; Yoo, Changhoon; Sim, Sang Jun; Lim, Chae Seung; Kim, Sung Woo

2018-01-01

Real-time PCR, also called quantitative PCR (qPCR), has been powerful analytical tool for detection of nucleic acids since it developed. Not only for biological research but also for diagnostic needs, qPCR technique requires capacity to detect multiple genes in recent years. Solid phase PCR (SP-PCR) where one or two directional primers are immobilized on solid substrates could analyze multiplex genetic targets. However, conventional SP-PCR was subjected to restriction of application for lack of PCR efficiency and quantitative resolution. Here we introduce an advanced qPCR with primer-incorporated network (PIN). One directional primers are immobilized in the porous hydrogel particle by covalent bond and the other direction of primers are temporarily immobilized at so-called 'Supplimers'. Supplimers released the primers to aqueous phase in the hydrogel at the thermal cycling of PCR. It induced the high PCR efficiency over 92% with high reliability. It reduced the formation of primer dimers and improved the selectivity of qPCR thanks to the strategy of 'right primers supplied to right place only'. By conducting a six-plex qPCR of 30 minutes, we analyzed DNA samples originated from malaria patients and successfully identified malaria species in a single reaction. PMID:29293604

Development of a quantitative PCR assay for measurement of trichechid herpesvirus 1 load in the Florida manatee ( Trichechus manatus latirostris).

PubMed

Ferrante, Jason A; Cortés-Hinojosa, Galaxia; Archer, Linda L; Wellehan, James F X

2017-07-01

Trichechid herpesvirus 1 (TrHV-1) is currently the only known herpesvirus in any sirenian. We hypothesized that stress may lead to recrudescence of TrHV-1 in manatees, thus making TrHV-1 a potential biomarker of stress. We optimized and validated a TrHV-1 real-time quantitative probe hybridization PCR (qPCR) assay that was used to quantify TrHV-1 in manatee peripheral blood mononuclear cells (PBMCs). Average baseline TrHV-1 loads in a clinically healthy wild Florida manatee ( Trichechus manatus latirostris) population ( n = 42) were 40.9 ± SD 21.2 copies/100 ng DNA; 19 of 42 manatees were positive. TrHV-1 loads were significantly different between the 2 field seasons ( p < 0.025). This optimized and validated qPCR assay may be used as a tool for further research into TrHV-1 in Florida manatees.

A multiplexed droplet digital PCR assay performs better than qPCR on inhibition prone samples.

PubMed

Sedlak, Ruth Hall; Kuypers, Jane; Jerome, Keith R

2014-12-01

We demonstrate the development of a multiplex droplet digital PCR assay for human cytomegalovirus (CMV), human adenovirus species F, and an internal plasmid control that may be useful for PCR inhibition-prone clinical samples. This assay performs better on inhibition-prone stool samples than a quantitative PCR assay for CMV and is the first published clinical virology droplet digital PCR assay to incorporate an internal control. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of a rapid, quantitative real-time PCR method for enumeration of pathogenic Candida cells in water

USGS Publications Warehouse

Brinkman, Nichole E.; Haugland, Richard A.; Wymer, Larry J.; Byappanahalli, Muruleedhara N.; Whitman, Richard L.; Vesper, Stephen J.

2003-01-01

Quantitative PCR (QPCR) technology, incorporating fluorigenic 5′ nuclease (TaqMan) chemistry, was utilized for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaniae) in water. Known numbers of target cells were added to distilled and tap water samples, filtered, and disrupted directly on the membranes for recovery of DNA for QPCR analysis. The assay's sensitivities were between one and three cells per filter. The accuracy of the cell estimates was between 50 and 200% of their true value (95% confidence level). In similar tests with surface water samples, the presence of PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts, with resultant reductions in sensitivity but generally not in quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QPCR results and colony counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus organisms. With a combined sample processing and analysis time of less than 4 h, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking and recreational waters and may have applications in the detection of fecal pollution.

Use of Serial Quantitative PCR of the vapA Gene of Rhodococcus equi in Feces for Early Detection of R. equi Pneumonia in Foals.

PubMed

Madrigal, R G; Shaw, S D; Witkowski, L A; Sisson, B E; Blodgett, G P; Chaffin, M K; Cohen, N D

2016-01-01

Current screening tests for Rhodococcus equi pneumonia in foals lack adequate accuracy for clinical use. Real-time, quantitative PCR (qPCR) for virulent R. equi in feces has not been systematically evaluated as a screening test. The objective of this study was to evaluate the accuracy of qPCR for vapA in serially collected fecal samples as a screening test for R. equi pneumonia in foals. One hundred and twenty-five foals born in 2011 at a ranch in Texas. Fecal samples were collected concurrently with thoracic ultrasonography (TUS) screening examinations at ages 3, 5, and 7 weeks. Affected (pneumonic) foals (n = 25) were matched by age and date-of-birth to unaffected (n = 25) and subclinical (ie, having thoracic TUS lesions but no clinical signs of pneumonia) foals (n = 75). DNA was extracted from feces using commercial kits and concentration of virulent R. equi in feces was determined by qPCR. Subsequently affected foals had significantly greater concentrations of vapA in feces than foals that did not develop pneumonia (unaffected and subclinical foals) at 5 and 7 weeks of age. Accuracy of fecal qPCR, however, was poor as a screening test to differentiate foals that would develop clinical signs of pneumonia from those that would remain free of clinical signs (including foals with subclinical pulmonary lesions attributed to R. equi) using receiver operating characteristic (ROC) methods. In the population studied, serial qPCR on feces lacked adequate accuracy as a screening test for clinical R. equi foal pneumonia. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Reconstruction of the original mycoflora in pelleted feed by PCR-SSCP and qPCR.

PubMed

Dorn-In, Samart; Fahn, Carmen; Hölzel, Christina S; Wenz, Sebastian; Hartwig, Isabella; Schwaiger, Karin; Bauer, Johann

2014-10-01

Ground feeds for pigs were investigated for fungal contamination before and after pelleting (subsamples in total n = 24) by cultural and molecular biological methods. A fungal-specific primer pair ITS1/ITS5.8R was used to amplify fungal DNA; PCR products were processed for the PCR-SSCP method. In the resulting acrylamide gel, more than 85% of DNA bands of ground feeds were preserved after pelleting. Twenty-two DNA bands were sequenced; all represented fungal DNA. The level of fungal DNA in ground feed samples was equivalent to 4.77-5.69 log10  CFU g(-1) , calculated by qPCR using a standard curve of Aspergillus flavus. In pelleted feed, the level of fungal DNA was in average ± 0.07 log10 different from ground feed. Quantified by cultural methods, the fresh ground feeds contained up to 4.51 log10  CFU g(-1) culturable fungi, while there was < 2.83 log10  CFU g(-1) detected in pelleted feeds. This result shows that, while the process of pelleting reduced the amount of living fungi dramatically, it did not affect the total fungal DNA in feed. Thus, the described methodology was able to reconstruct the fungal microbiota in feeds and reflected a considerable fungal contamination of raw materials such as grains. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Whole Genome Sequencing and Multiplex qPCR Methods to Identify Campylobacter jejuni Encoding cst-II or cst-III Sialyltransferase

PubMed Central

Neal-McKinney, Jason M.; Liu, Kun C.; Jinneman, Karen C.; Wu, Wen-Hsin; Rice, Daniel H.

2018-01-01

Campylobacter jejuni causes more than 2 million cases of gastroenteritis annually in the United States, and is also linked to the autoimmune sequelae Guillan–Barre syndrome (GBS). GBS often results in flaccid paralysis, as the myelin sheaths of nerve cells are degraded by the adaptive immune response. Certain strains of C. jejuni modify their lipooligosaccharide (LOS) with the addition of neuraminic acid, resulting in LOS moieties that are structurally similar to gangliosides present on nerve cells. This can trigger GBS in a susceptible host, as antibodies generated against C. jejuni can cross-react with gangliosides, leading to demyelination of nerves and a loss of signal transduction. The goal of this study was to develop a quantitative PCR (qPCR) method and use whole genome sequencing data to detect the Campylobacter sialyltransferase (cst) genes responsible for the addition of neuraminic acid to LOS. The qPCR method was used to screen a library of 89 C. jejuni field samples collected by the Food and Drug Administration Pacific Northwest Lab (PNL) as well as clinical isolates transferred to PNL. In silico analysis was used to screen 827 C. jejuni genomes in the FDA GenomeTrakr SRA database. The results indicate that a majority of C. jejuni strains could produce LOS with ganglioside mimicry, as 43.8% of PNL isolates and 46.9% of the GenomeTrakr isolates lacked the cst genes. The methods described in this study can be used by public health laboratories to rapidly determine whether a C. jejuni isolate has the potential to induce GBS. Based on these results, a majority of C. jejuni in the PNL collection and submitted to GenomeTrakr have the potential to produce LOS that mimics human gangliosides. PMID:29615986

Whole Genome Sequencing and Multiplex qPCR Methods to Identify Campylobacter jejuni Encoding cst-II or cst-III Sialyltransferase.

PubMed

Neal-McKinney, Jason M; Liu, Kun C; Jinneman, Karen C; Wu, Wen-Hsin; Rice, Daniel H

2018-01-01

Campylobacter jejuni causes more than 2 million cases of gastroenteritis annually in the United States, and is also linked to the autoimmune sequelae Guillan-Barre syndrome (GBS). GBS often results in flaccid paralysis, as the myelin sheaths of nerve cells are degraded by the adaptive immune response. Certain strains of C. jejuni modify their lipooligosaccharide (LOS) with the addition of neuraminic acid, resulting in LOS moieties that are structurally similar to gangliosides present on nerve cells. This can trigger GBS in a susceptible host, as antibodies generated against C. jejuni can cross-react with gangliosides, leading to demyelination of nerves and a loss of signal transduction. The goal of this study was to develop a quantitative PCR (qPCR) method and use whole genome sequencing data to detect the Campylobacter sialyltransferase ( cst ) genes responsible for the addition of neuraminic acid to LOS. The qPCR method was used to screen a library of 89 C. jejuni field samples collected by the Food and Drug Administration Pacific Northwest Lab (PNL) as well as clinical isolates transferred to PNL. In silico analysis was used to screen 827 C. jejuni genomes in the FDA GenomeTrakr SRA database. The results indicate that a majority of C. jejuni strains could produce LOS with ganglioside mimicry, as 43.8% of PNL isolates and 46.9% of the GenomeTrakr isolates lacked the cst genes. The methods described in this study can be used by public health laboratories to rapidly determine whether a C. jejuni isolate has the potential to induce GBS. Based on these results, a majority of C. jejuni in the PNL collection and submitted to GenomeTrakr have the potential to produce LOS that mimics human gangliosides.

Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea▿

PubMed Central

Andree, Karl B.; Fernández-Tejedor, Margarita; Elandaloussi, Laurence M.; Quijano-Scheggia, Sonia; Sampedro, Nagore; Garcés, Esther; Camp, Jordi; Diogène, Jorge

2011-01-01

The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy. PMID:21193668

Selection of reference genes for gene expression studies in virus-infected monocots using quantitative real-time PCR.

PubMed

Zhang, Kun; Niu, Shaofang; Di, Dianping; Shi, Lindan; Liu, Deshui; Cao, Xiuling; Miao, Hongqin; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2013-10-10

Both genome-wide transcriptomic surveys of the mRNA expression profiles and virus-induced gene silencing-based molecular studies of target gene during virus-plant interaction involve the precise estimation of the transcript abundance. Quantitative real-time PCR (qPCR) is the most widely adopted technique for mRNA quantification. In order to obtain reliable quantification of transcripts, identification of the best reference genes forms the basis of the preliminary work. Nevertheless, the stability of internal controls in virus-infected monocots needs to be fully explored. In this work, the suitability of ten housekeeping genes (ACT, EF1α, FBOX, GAPDH, GTPB, PP2A, SAND, TUBβ, UBC18 and UK) for potential use as reference genes in qPCR were investigated in five different monocot plants (Brachypodium, barley, sorghum, wheat and maize) under infection with different viruses including Barley stripe mosaic virus (BSMV), Brome mosaic virus (BMV), Rice black-streaked dwarf virus (RBSDV) and Sugarcane mosaic virus (SCMV). By using three different algorithms, the most appropriate reference genes or their combinations were identified for different experimental sets and their effectiveness for the normalisation of expression studies were further validated by quantitative analysis of a well-studied PR-1 gene. These results facilitate the selection of desirable reference genes for more accurate gene expression studies in virus-infected monocots. Copyright © 2013 Elsevier B.V. All rights reserved.

Real-time quantitative PCR detection of circulating tumor cells using tag DNA mediated signal amplification strategy.

PubMed

Mei, Ting; Lu, Xuewen; Sun, Ning; Li, Xiaomei; Chen, Jitao; Liang, Min; Zhou, Xinke; Fang, Zhiyuan

2018-06-05

The level of circulating tumor cell (CTCs) is a reliable marker for tumor burden and malignant progression. Quantification of CTCs remains technically challenging due to the rarity of these cells in peripheral blood. In the present study, we established a real-time quantitative PCR (Q-PCR) based method for sensitive detection of CTCs without DNA extraction. Blood sample was first turned to erythrocyte lyses and then incubated with two antibodies, tag-DNA modified CK-19 antibody and magnetic beads conjugated EpCAM antibody. Tumor cells were further enriched by magnetic separation. Tag-DNA that immobilized on tumor cells through CK-19 antibodies were also retrieved, which was further quantified by Q-PCR. This assay was able to detect single tumor cell in a 5 mL blood sample. The detection rate of clinical tumor blood sample was 92.3%. Furthermore, CTC count in patient was correlated with tumor stage and tumor status. The signal amplification was based on tag DNA rather than tumor gene, which was independent of nucleic acid extraction. With high sensitivity and convenience, this method can be a good alternative for the determination of cancer progress. Copyright © 2018 Elsevier B.V. All rights reserved.

Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples.

PubMed

Durant, Jean-Francois; Irenge, Leonid M; Fogt-Wyrwas, Renata; Dumont, Catherine; Doucet, Jean-Pierre; Mignon, Bernard; Losson, Bertrand; Gala, Jean-Luc

2012-12-07

Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis) and/or Toxocara cati (T. cati), two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum) and Parascaris equorum (P. equorum). The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

Studies of plant colonisation by closely related Bacillus amyloliquefaciens biocontrol agents using strain specific quantitative PCR assays.

PubMed

Johansson, Anna H; Bejai, Sarosh; Niazi, Adnan; Manzoor, Shahid; Bongcam-Rudloff, Erik; Meijer, Johan

2014-12-01

Certain strains of Bacillus amyloliquefaciens can colonize plants and improve growth and stress management. In order to study these effects, bacterial growth dynamics on plants and in the rhizosphere are of interest calling for specific analytical tools. For that purpose, quantitative real-time PCR (qPCR) assays were developed in order to differentiate among three closely related B. amyloliquefaciens subsp. plantarum strains (UCMB5033, UCMB5036, UCMB5113) and to determine their levels with high accuracy. Oligonucleotide primers were designed for strain unique gene sequences and used for SYBR green based qPCR analysis. Standard curves covered a wide linear range (10(6)) of DNA amounts with the lowest detection level at 50 fg. Post-reaction melting curve analysis showed only a single product. Accurate threshold cycles were obtained, even in the presence of high excess of related Bacillus strains and total bacterial DNA from soil. Analysis of Bacillus colonisation after seed treatment of two oilseed rape cultivars (Oase and Ritz) grown on agar support showed a time dependent effect but that the bacteria mostly were found on root tissues and little on green tissues. The colonisation on plants grown in soil varied among the Bacillus strains where Oase seemed to house more bacteria than Ritz. Applied as a mixture, all three Bacillus strains co-existed on the roots of plants grown in soil. The qPCR assay in combination with other techniques will be a powerful tool to study plant interactions of these B. amyloliquefaciens biocontrol agents to further understand the requirements for successful interactions and improvement of plant properties.

Comparison of Enterococcus quantitative polymerase chain reaction analysis results from midwest U.S. river samples using EPA Method 1611 and Method 1609 PCR reagents

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) has provided recommended beach advisory values in its 2012 recreational water quality criteria (RWQC) for states wishing to use quantitative polymerase chain reaction (qPCR) for the monitoring of Enterococcus fecal indicator bacteria...

A tool for design of primers for microRNA-specific quantitative RT-qPCR.

PubMed

Busk, Peter K

2014-01-28

MicroRNAs are small but biologically important RNA molecules. Although different methods can be used for quantification of microRNAs, quantitative PCR is regarded as the reference that is used to validate other methods. Several commercial qPCR assays are available but they often come at a high price and the sequences of the primers are not disclosed. An alternative to commercial assays is to manually design primers but this work is tedious and, hence, not practical for the design of primers for a larger number of targets. I have developed the software miRprimer for automatic design of primers for the method miR-specific RT-qPCR, which is one of the best performing microRNA qPCR methods available. The algorithm is based on an implementation of the previously published rules for manual design of miR-specific primers with the additional feature of evaluating the propensity of formation of secondary structures and primer dimers. Testing of the primers showed that 76 out of 79 primers (96%) worked for quantification of microRNAs by miR-specific RT-qPCR of mammalian RNA samples. This success rate corresponds to the success rate of manual primer design. Furthermore, primers designed by this method have been distributed to several labs and used successfully in published studies. The software miRprimer is an automatic and easy method for design of functional primers for miR-specific RT-qPCR. The application is available as stand-alone software that will work on the MS Windows platform and in a developer version written in the Ruby programming language.

Digital PCR for detection of citrus pathogens

USDA-ARS?s Scientific Manuscript database

Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

Quantitative PCR-based parasite burden estimation of Babesia gibsoni in the vector tick, Haemaphysalis longicornis (Acari: Ixodidae), fed on an experimentally infected dog.

PubMed

Hatta, Takeshi; Matsubayashi, Makoto; Miyoshi, Takeharu; Islam, Khyrul; Alim, M Abdul; Anisuzzaman; Yamaji, Kayoko; Fujisaki, Kozo; Tsuji, Naotoshi

2013-01-31

Most causative agents of babesiosis, Babesia parasites, are transmitted transovarially in ixodid ticks. In this study, B. gibsoni, the causative agent of canine babesiosis which has transovarial transmission, was detected in tissues of the vector tick, Haemaphysalis longicornis using a modified quantitative PCR assay. Conventional PCR results showed that the newly designed primer set, which amplifies a 143-bp fragment of rhoptry-associated protein-1 (BgRAP-1) gene in B. gibsoni, was 100 times more sensitive than primers targeting P18 gene encoding 18 kDa protein of B. gibsoni, which was recently renamed as thrombospondin related adhesive protein (BgTRAP) gene, in an artificially generated sample solution containing metagenomic DNA (B. gibsoni DNA extracted from infected dog blood mixed with tick DNA). The TaqMan probe-based quantitative PCR (qPCR) for BgRAP-1 could also detect infected RBCs (iRBCs) at levels of 3.5 × 10(5) to 3.5 × 10(1)/μl, a range that is broader than that of a past SYBR Green-based qPCR method for P18/BgTRAP, which had a detection limit of 3.5 × 10(3) iRBCs/μl. Using this qPCR assay, we attempted to quantify the B. gibsoni burden in tick ovaries and embryonated eggs. Levels of infection were normalized to the copy number of tick's genomic DNA fragment of ribosomal DNA internal transcribed spacer region 2 (ITS2) for the standardization. According to this, low levels of parasite burden were quantified in ovaries and eggs. This detection system is sensitive and is recommended as a tool for elucidating the biological interactions between the vector tick H. longicornis and the parasite, B. gibsoni.

Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies.

PubMed

de Vega-Bartol, José J; Santos, Raquen Raissa; Simões, Marta; Miguel, Célia M

2013-05-01

Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species. Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

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.

Development, validation and field evaluation of a quantitative real-time PCR able to differentiate between field Mycoplasma synoviae and the MS-H-live vaccine strain.

PubMed

Dijkman, R; Feberwee, A; Landman, W J M

2017-08-01

A quantitative PCR (qPCR) able to differentiate between field Mycoplasma synoviae and MS-H vaccine strain was developed, validated and evaluated. It was developed using nucleotide differences in the obg gene. Analytical specificity and sensitivity assessed using DNA from 194 M. synoviae field samples, three different batches of MS-H vaccine and from 43 samples representing four other avian Mycoplasma species proved to be 100%. The detection limit for field M. synoviae and MS-H vaccine strain was 10 2-3 and 10 2 colony-forming units PCR equivalents/g trachea mucus, respectively. The qPCR was able to detect both, field M. synoviae and MS-H vaccine strain in ratios of 1:100 determined both using spiked and field samples. One hundred and twenty samples from M. synoviae-infected non-vaccinated birds, 110 samples from M. synoviae-vaccinated birds from a bird experiment and 224 samples from M. synoviae negative (serology and PCR) birds were used to determine the relative sensitivity and specificity using a previously described M. synoviae PCR as reference. The relative sensitivity and specificity for field M. synoviae were 95.0% and 99.6%, respectively, and 94.6% and 100% for the MS-H-live vaccine, respectively. Field validation and confirmation by multi locus sequence typing revealed that the qPCR correctly distinguished between MS-H and field M. synoviae. Evaluation of the differentiating M. synoviae qPCR in three commercial flocks suggested transmission of MS-H-live vaccine from vaccinated to non-vaccinated flocks at the same farm. Furthermore, it showed evidence for the colonization with field M. synoviae in MS-H-vaccinated flocks.

Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in low-income countries.

PubMed

Lindsay, Brianna; Ochieng, John B; Ikumapayi, Usman N; Toure, Aliou; Ahmed, Dilruba; Li, Shan; Panchalingam, Sandra; Levine, Myron M; Kotloff, Karen; Rasko, David A; Morris, Carolyn R; Juma, Jane; Fields, Barry S; Dione, Michel; Malle, Dramane; Becker, Stephen M; Houpt, Eric R; Nataro, James P; Sommerfelt, Halvor; Pop, Mihai; Oundo, Joe; Antonio, Martin; Hossain, Anowar; Tamboura, Boubou; Stine, O Colin

2013-06-01

Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 10(4) ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10(4) ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥ 2.9 × 10(4) ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 10(4) ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 10(4) ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.

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.

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.

Development and validation of a quantitative PCR for rapid and specific detection of California sea lion adenovirus 1 and prevalence in wild and managed populations.

PubMed

Cortés-Hinojosa, Galaxia; Gulland, Frances M D; Goldstein, Tracey; Venn-Watson, Stephanie; Rivera, Rebecca; Archer, Linda L; Waltzek, Thomas B; Gray, Gregory C; Wellehan, James F X

2017-03-01

California sea lion adenovirus 1 (CSLAdV-1) has been associated with hepatitis and enteritis in several wild and captive populations of diverse pinniped species. Currently available tests have been limited to pan-adenoviral polymerase chain reaction (PCR) followed by sequencing. We present the development of a quantitative probe-hybridization PCR (qPCR) assay for rapid, sensitive, and specific detection of this virus in California sea lions ( Zalophus californianus) and other pinnipeds. This assay did not amplify other mammalian adenoviruses and is able to detect consistently down to 10 viral copies per well. Compared with the gold standard conventional pan-adenovirus PCR/sequencing assay, diagnostic sensitivity and specificity of 100% and 88.2% were found, respectively. The lower diagnostic specificity of this qPCR assay may be the result of the lower limit of detection of this assay compared with the gold standard rather than the result of detection of true false-positives.

Effectiveness of onsite wastewater reuse system in reducing bacterial contaminants measured with human-specific IMS/ATP and qPCR.

PubMed

Agidi, Senyo; Vedachalam, Sridhar; Mancl, Karen; Lee, Jiyoung

2013-01-30

Water shortages and the drive to recycle is increasing interest in reuse of reclaimed wastewater. Timely and cost-effective ways to detect fecal pollutants prior to reuse increases confidence of residents and neighbors concerned about reuse of reclaimed wastewater. The on-site wastewater treatment and reuse systems (OWTRS) used in this study include a septic tank, peat bioreactor, ClO(2) disinfection and land spray irrigation system. Bacteroides fragilis, Escherichia coli and Enterococcus spp., were tested with immunomagnetic separation/ATP bioluminescence (IMS/ATP), qPCR and culture-based methods. The results displayed a 2-log reduction in fecal bacteria in the peat bioreactor and a 5-log reduction following chloride dioxide disinfection. The fecal bacteria levels measured by IMS/ATP correlated with qPCR results: HuBac 16S (R(2) = 0.903), Bf-group 16S (R(2) = 0.956), gyrB (R(2) = 0.673), and Ent 23S (R(2) = 0.724). This is the first study in which the newly developed human-specific IMS/ATP and previously developed IMS/ATP were applied for determining OWTRS efficiency. Results of the study revealed that IMS/ATP is a timely and cost-effective way to detect fecal contaminants, and results were validated with qPCR and culture based methods. The new IMS/ATP can also be applied broadly in the detection of human-originated fecal contamination. Copyright © 2012 Elsevier Ltd. All rights reserved.

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors

PubMed Central

Ikten, Cengiz; Ustun, Rustem; Catal, Mursel; Yol, Engin; Uzun, Bulent

2016-01-01

Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 102 and 1.6 x 102 DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease. PMID:27195795

Stepwise kinetic equilibrium models of quantitative polymerase chain reaction.

PubMed

Cobbs, Gary

2012-08-16

Numerous models for use in interpreting quantitative PCR (qPCR) data are present in recent literature. The most commonly used models assume the amplification in qPCR is exponential and fit an exponential model with a constant rate of increase to a select part of the curve. Kinetic theory may be used to model the annealing phase and does not assume constant efficiency of amplification. Mechanistic models describing the annealing phase with kinetic theory offer the most potential for accurate interpretation of qPCR data. Even so, they have not been thoroughly investigated and are rarely used for interpretation of qPCR data. New results for kinetic modeling of qPCR are presented. Two models are presented in which the efficiency of amplification is based on equilibrium solutions for the annealing phase of the qPCR process. Model 1 assumes annealing of complementary targets strands and annealing of target and primers are both reversible reactions and reach a dynamic equilibrium. Model 2 assumes all annealing reactions are nonreversible and equilibrium is static. Both models include the effect of primer concentration during the annealing phase. Analytic formulae are given for the equilibrium values of all single and double stranded molecules at the end of the annealing step. The equilibrium values are then used in a stepwise method to describe the whole qPCR process. Rate constants of kinetic models are the same for solutions that are identical except for possibly having different initial target concentrations. Analysis of qPCR curves from such solutions are thus analyzed by simultaneous non-linear curve fitting with the same rate constant values applying to all curves and each curve having a unique value for initial target concentration. The models were fit to two data sets for which the true initial target concentrations are known. Both models give better fit to observed qPCR data than other kinetic models present in the literature. They also give better estimates of

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.

Incorporating expert judgments in utility evaluation of bacteroidales qPCR assays for microbial source tracking in a drinking water source.

PubMed

Åström, Johan; Pettersson, Thomas J R; Reischer, Georg H; Norberg, Tommy; Hermansson, Malte

2015-02-03

Several assays for the detection of host-specific genetic markers of the order Bacteroidales have been developed and used for microbial source tracking (MST) in environmental waters. It is recognized that the source-sensitivity and source-specificity are unknown and variable when introducing these assays in new geographic regions, which reduces their reliability and use. A Bayesian approach was developed to incorporate expert judgments with regional assay sensitivity and specificity assessments in a utility evaluation of a human and a ruminant-specific qPCR assay for MST in a drinking water source. Water samples from Lake Rådasjön were analyzed for E. coli, intestinal enterococci and somatic coliphages through cultivation and for human (BacH) and ruminant-specific (BacR) markers through qPCR assays. Expert judgments were collected regarding the probability of human and ruminant fecal contamination based on fecal indicator organism data and subjective information. Using Bayes formula, the conditional probability of a true human or ruminant fecal contamination given the presence of BacH or BacR was determined stochastically from expert judgments and regional qPCR assay performance, using Beta distributions to represent uncertainties. A web-based computational tool was developed for the procedure, which provides a measure of confidence to findings of host-specific markers and demonstrates the information value from these assays.

Incorporating Expert Judgments in Utility Evaluation of Bacteroidales qPCR Assays for Microbial Source Tracking in a Drinking Water Source

PubMed Central

Åström, Johan; Pettersson, Thomas J. R.; Reischer, Georg H.; Norberg, Tommy; Hermansson, Malte

2017-01-01

Several assays for the detection of host-specific genetic markers of the order Bacteroidales have been developed and used for microbial source tracking (MST) in environmental waters. It is recognized that the source-sensitivity and source-specificity are unknown and variable when introducing these assays in new geographic regions, which reduces their reliability and use. A Bayesian approach was developed to incorporate expert judgments with regional assay sensitivity and specificity assessments in a utility evaluation of a human and a ruminant-specific qPCR assay for MST in a drinking water source. Water samples from Lake Rådasjön were analyzed for E. coli, intestinal enterococci and somatic coliphages through cultivation and for human (BacH) and ruminant-specific (BacR) markers through qPCR assays. Expert judgments were collected regarding the probability of human and ruminant fecal contamination based on fecal indicator organism data and subjective information. Using Bayes formula, the conditional probability of a true human or ruminant fecal contamination given the presence of BacH or BacR was determined stochastically from expert judgments and regional qPCR assay performance, using Beta distributions to represent uncertainties. A web-based computational tool was developed for the procedure, which provides a measure of confidence to findings of host-specific markers and demonstrates the information value from these assays. PMID:25545113

Development of Conventional and Real-Time Quantitative PCR Assays for Diagnosis and Monitoring of Scabies

PubMed Central

Wong, Samson S. Y.; Poon, Rosana W. S.; Chau, Sandy; Wong, Sally C. Y.; To, Kelvin K. W.; Cheng, Vincent C. C.; Fung, Kitty S. C.

2015-01-01

Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration of Sarcoptes scabiei mites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of S. scabiei. The cox1 gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. The S. scabiei DNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (median S. scabiei DNA copy number, 3.604 versus 2.457 log10 copies per reaction; P = 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated. PMID:25903566

Development of Conventional and Real-Time Quantitative PCR Assays for Diagnosis and Monitoring of Scabies.

PubMed

Wong, Samson S Y; Poon, Rosana W S; Chau, Sandy; Wong, Sally C Y; To, Kelvin K W; Cheng, Vincent C C; Fung, Kitty S C; Yuen, K Y

2015-07-01

Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration of Sarcoptes scabiei mites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of S. scabiei. The cox1 gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. The S. scabiei DNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (median S. scabiei DNA copy number, 3.604 versus 2.457 log10 copies per reaction; P = 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots.

PubMed

Horn, Ivo R; van Rijn, Menno; Zwetsloot, Tom J J; Basmagi, Said; Dirks-Mulder, Anita; van Leeuwen, Willem B; Ravensberg, Willem J; Gravendeel, Barbara

2016-02-01

The fungal species Trichoderma harzianum is widely used as a biological agent in crop protection. To verify the continued presence of this fungus on plant roots manually inoculated with T. harzianum strain T22, a Q-PCR was designed using specific probes for this particular strain. To develop these molecular diagnostic tools, genome mining was first carried out to retrieve putative new regions by which different strains of T. harzianum could be distinguished. Subsequently, Sanger sequencing of the L-aminoacid oxidase gene (aox1) in T. harzianum was applied to determine the mutations differing between various strains isolated from the Trichoderma collection of Koppert Biological Systems. Based on the sequence information obtained, a set of hydrolysis probes was subsequently developed which discriminated T. harzianum T22 strains varying in only a single nucleotide. Probes designed for two strains uniquely recognized the respective strains in Q-PCR with a detection limit of 12,5ng DNA. Titration assays in which T. harzianum DNA from distinct strains was varied further underscored the specificity of the probes. Lastly, fungal DNA extracted from roots of greenhouse cultured tomato plants was analyzed using the probe-based assay. DNA from T. harzianum strain T22 could readily be identified on roots of greenhouse reared tomato plants inoculated with varying concentrations up to one week after treatment with a detection limit of 3e6 colony forming units of T. harzianum T22. We conclude that the Q-PCR method is a reliable and robust method for assessing the presence and quantity of T. harzianum strain T22 in manually inoculated plant material. Our method provides scope for the development of DNA based strain specific identification of additional strains of Trichoderma and other fungal biological control agents. Copyright © 2015 Elsevier B.V. All rights reserved.

A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus Hormoconis resinae.

PubMed

Martin-Sanchez, Pedro M; Gorbushina, Anna A; Kunte, Hans-Jörg; Toepel, Jörg

2016-07-01

A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus Hormoconis resinae, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify H. resinae in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of H. resinae in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms.

Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital PCR assay.

PubMed

Kelley, Kashonda; Cosman, Angela; Belgrader, Phillip; Chapman, Brenda; Sullivan, Donna C

2013-07-01

Health care-associated infections with methicillin-resistant Staphylococcus aureus (MRSA) contribute to significant hospitalization costs. We report here a droplet digital PCR (ddPCR) assay, which is a next-generation emulsion-based endpoint PCR assay for high-precision MRSA analysis. Reference cultures of MRSA, methicillin-susceptible S. aureus (MSSA), and confounders were included as controls. Copan swabs were used to sample cultures and collect specimens for analysis from patients at a large teaching hospital. Swab extraction and cell lysis were accomplished using magnetic-driven agitation of silica beads. Quantitative PCR (qPCR) (Roche Light Cycler 480) and ddPCR (Bio-Rad QX100 droplet digital PCR system) assays were used to detect genes for the staphylococcal protein SA0140 (SA) and the methicillin resistance (mecA) gene employing standard TaqMan chemistries. Both qPCR and ddPCR assays correctly identified culture controls for MRSA (76), MSSA (12), and confounder organisms (36) with 100% sensitivity and specificity. Analysis of the clinical samples (211 negative and 186 positive) collected during a study of MRSA nasal carriage allowed direct comparison of the qPCR and ddPCR assays to the Cepheid MRSA GeneXpert assay. A total of 397 clinical samples were examined in this study. Cepheid MRSA GeneXpert values were used to define negative and positive samples. Both the qPCR and ddPCR assays were in good agreement with the reference assay. The sensitivities for the qPCR and ddPCR assays were 96.8% (95% confidence interval [CI], 93.1 to 98.5%) and 96.8% (95% CI, 93.1 to 98.5%), respectively. Both the qPCR and ddPCR assays had specificities of 91.9% (95% CI, 87.5 to 94.9%) for qPCR and 91.0% (95% CI, 86.4 to 94.2%) for ddPCR technology.

Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples

PubMed Central

2012-01-01

Background Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis) and/or Toxocara cati (T. cati), two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. Methods A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum) and Parascaris equorum (P. equorum). The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. Results 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. Conclusion The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits. PMID:23216873

Identification and quantification of virulence factors of enterotoxigenic Escherichia coli by high-resolution melting curve quantitative PCR.

PubMed

Wang, Weilan; Zijlstra, Ruurd T; Gänzle, Michael G

2017-05-15

Diagnosis of enterotoxigenic E. coli (ETEC) associated diarrhea is complicated by the diversity of E.coli virulence factors. This study developed a multiplex quantitative PCR assay based on high-resolution melting curves analysis (HRM-qPCR) to identify and quantify genes encoding five ETEC fimbriae related to diarrhea in swine, i.e. K99, F41, F18, F6 and K88. Five fimbriae expressed by ETEC were amplified in multiple HRM-qPCR reactions to allow simultaneous identification and quantification of five target genes. The assay was calibrated to allow quantification of the most abundant target gene, and validated by analysis of 30 samples obtained from piglets with diarrhea and healthy controls, and comparison to standard qPCR detection. The five amplicons with melting temperatures (Tm) ranging from 74.7 ± 0.06 to 80.5 ± 0.15 °C were well-separated by HRM-qPCR. The area of amplicons under the melting peak correlated linearly to the proportion of the template in the calibration mixture if the proportion exceeded 4.8% (K88) or <1% (all other amplicons). The suitability of the method was evaluated using 30 samples from weaned pigs aged 6-7 weeks; 14 of these animals suffered from diarrhea in consequence of poor sanitary conditions. Genes encoding fimbriae and enterotoxins were quantified by HRM-qPCR and/or qPCR. The multiplex HRM-qPCR allowed accurate analysis when the total gene copy number of targets was more than 1 × 10 5 / g wet feces and the HRM curves were able to simultaneously distinguish fimbriae genes in the fecal samples. The relative quantification of the most abundant F18 based on melting peak area was highly correlated (P < 0.001; r 2  = 0.956) with that of individual qPCR result but the correlation for less abundant fimbriae was much lower. The multiplex HRM assay identifies ETEC virulence factors specifically and efficiently. It correctly indicated the predominant fimbriae type and additionally provides information of presence/ absence of

Interlaboratory comparison of real-time pcr protocols for quantification of general fecal indicator bacteria

USGS Publications Warehouse

Shanks, O.C.; Sivaganesan, M.; Peed, L.; Kelty, C.A.; Blackwood, A.D.; Greene, M.R.; Noble, R.T.; Bushon, R.N.; Stelzer, E.A.; Kinzelman, J.; Anan'Eva, T.; Sinigalliano, C.; Wanless, D.; Griffith, J.; Cao, Y.; Weisberg, S.; Harwood, V.J.; Staley, C.; Oshima, K.H.; Varma, M.; Haugland, R.A.

2012-01-01

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 protocol requires information on the reproducibility and sources of variation associated with qPCR methodology across laboratories. This study examines interlaboratory variability in the measurement of enterococci and Bacteroidales concentrations from standardized, spiked, and environmental sources of DNA using the Entero1a and GenBac3 qPCR methods, respectively. Comparisons are based on data generated from eight different research facilities. Special attention was placed on the influence of the DNA isolation step and effect of simplex and multiplex amplification approaches on interlaboratory variability. Results suggest that a crude lysate is sufficient for DNA isolation unless environmental samples contain substances that can inhibit qPCR amplification. No appreciable difference was observed between simplex and multiplex amplification approaches. Overall, interlaboratory variability levels remained low (<10% coefficient of variation) regardless of qPCR protocol. ?? 2011 American Chemical Society.

Droplet Digital PCR for Minimal Residual Disease Detection in Mature Lymphoproliferative Disorders.

PubMed

Drandi, Daniela; Ferrero, Simone; Ladetto, Marco

2018-01-01

Minimal residual disease (MRD) detection has a powerful prognostic relevance for response evaluation and prediction of relapse in hematological malignancies. Real-time quantitative PCR (qPCR) has become the settled and standardized method for MRD assessment in lymphoid disorders. However, qPCR is a relative quantification approach, since it requires a reference standard curve. Droplet digital TM PCR (ddPCR TM ) allows a reliable absolute tumor burden quantification withdrawing the need for preparing, for each experiment, a tumor-specific standard curve. We have recently shown that ddPCR has a good concordance with qPCR and could be a feasible and reliable tool for MRD monitoring in mature lymphoproliferative disorders. In this chapter we describe the experimental workflow, from the detection of the clonal molecular marker to the MRD monitoring by ddPCR, in patients affected by multiple myeloma, mantle cell lymphoma and follicular lymphoma. However, standardization programs among different laboratories are needed in order to ensure the reliability and reproducibility of ddPCR-based MRD results.

Characterization of relative abundance of lactic acid bacteria species in French organic sourdough by cultural, qPCR and MiSeq high-throughput sequencing methods.

PubMed

Michel, Elisa; Monfort, Clarisse; Deffrasnes, Marion; Guezenec, Stéphane; Lhomme, Emilie; Barret, Matthieu; Sicard, Delphine; Dousset, Xavier; Onno, Bernard

2016-12-19

In order to contribute to the description of sourdough LAB composition, MiSeq sequencing and qPCR methods were performed in association with cultural methods. A panel of 16 French organic bakers and farmer-bakers were selected for this work. The lactic acid bacteria (LAB) diversity of their organic sourdoughs was investigated quantitatively and qualitatively combining (i) Lactobacillus sanfranciscensis-specific qPCR, (ii) global sequencing with MiSeq Illumina technology and (iii) molecular isolates identification. In addition, LAB and yeast enumeration, pH, Total Titratable Acidity, organic acids and bread specific volume were analyzed. Microbial and physico-chemical data were statistically treated by Principal Component Analysis (PCA) and Hierarchical Ascendant Classification (HAC). Total yeast counts were 6 log 10 to 7.6 log 10 CFU/g while LAB counts varied from 7.2 log 10 to 9.6 log 10 CFU/g. Values obtained by L. sanfranciscensis-specific qPCR were estimated between 7.2 and 10.3 log 10 CFU/g, except for one sample at 4.4 log 10 CFU/g. HAC and PCA clustered the sixteen sourdoughs into three classes described by their variables but without links to bakers' practices. L. sanfranciscensis was the dominant species in 13 of the 16 sourdoughs analyzed by Next Generation Sequencing (NGS), by the culture dependent method this species was dominant only in only 10 samples. Based on isolates identification, LAB diversity was higher for 7 sourdoughs with the recovery of L. curvatus, L. brevis, L. heilongjiangensis, L. xiangfangensis, L. koreensis, L. pontis, Weissella sp. and Pediococcus pentosaceus, as the most representative species. L. koreensis, L. heilongjiangensis and L. xiangfangensis were identified in traditional Asian food and here for the first time as dominant in organic sourdough. This study highlighted that L. sanfranciscensis was not the major species in 6/16 sourdough samples and that a relatively high LAB diversity can be observed in French organic

The Development of a Novel qPCR Assay-Set for Identifying Fecal Contamination Originating from Domestic Fowls and Waterfowl in Israel.

PubMed

Ohad, Shoshanit; Ben-Dor, Shifra; Prilusky, Jaime; Kravitz, Valeria; Dassa, Bareket; Chalifa-Caspi, Vered; Kashi, Yechezkel; Rorman, Efrat

2016-01-01

The emerging microbial source tracking (MST) methodologies aim to identify fecal contamination originating from domestic and wild animals, and from humans. Avian MST is especially challenging, primarily because the Aves class includes both domesticated and wild species with highly diverse habitats and dietary characteristics. The quest for specific fecal bacterial MST markers can be difficult with respect to attaining sufficient assay sensitivity and specificity. The present study utilizes high throughput sequencing (HTS) to screen bacterial 16S rRNA genes from fecal samples collected from both domestic and wild avian species. Operational taxonomic unit (OTU) analysis was then performed, from which sequences were retained for downstream quantitative polymerase chain reaction (qPCR) marker development. Identification of unique avian host DNA sequences, absent in non-avian hosts, was then carried out using a dedicated database of bacterial 16S rRNA gene taken from the Ribosomal Database Project. Six qPCR assays were developed targeting the 16S rRNA gene of Lactobacillus, Gallibacterium, Firmicutes, Fusobacteriaceae, and other bacteria. Two assays (Av4143 and Av163) identified most of the avian fecal samples and demonstrated sensitivity values of 91 and 70%, respectively. The Av43 assay only identified droppings from battery hens and poultry, whereas each of the other three assays (Av24, Av13, and Av216) identified waterfowl species with lower sensitivities values. The development of an MST assay-panel, which includes both domestic and wild avian species, expands the currently known MST analysis capabilities for decoding fecal contamination.

Fluorescence in situ hybridization and qPCR to detect Merkel cell polyomavirus physical status and load in Merkel cell carcinomas.

PubMed

Haugg, Anke M; Rennspiess, Dorit; zur Hausen, Axel; Speel, Ernst-Jan M; Cathomas, Gieri; Becker, Jürgen C; Schrama, David

2014-12-15

The Merkel cell polyomavirus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). Clonal integration and tumor-specific mutations in the large T antigen are strong arguments that MCPyV is a human tumor virus. However, the relationship between viral presence and cancer induction remains discussed controversially. Since almost all studies on virus prevalence are based on PCR techniques, we performed MCPyV fluorescence in situ hybridization (FISH) on MCC to gain information about the quality of the viral presence on the single cell level. MCPyV-FISH was performed on tissue microarrays containing 62 formalin-fixed and paraffin-embedded tissue samples including all tumor grades of 42 patients. The hybridization patterns were correlated to the qPCR data determined on corresponding whole tissue sections. Indeed, MCPyV-FISH and qPCR data were highly correlated, i.e. 83% for FISH-positive and 93% for FISH-negative cores. Accordingly, the mean of the qPCR values of all MCPyV-positive cores differed significantly from the mean of the negative cores (p = 0.0076). Importantly, two hybridization patterns were definable in the MCPyV-FISH: a punctate pattern (85%) indicating viral integration, which correlated with a moderate viral abundance and a combination of the punctate with a diffuse pattern (15%), suggesting a possible coexistence of integrated and episomal virus which was associated with very high viral load and VP1 expression. Thus, MCPyV-FISH adds important information on the single cell level within the histomorphological context and could therefore be an important tool to further elucidate MCPyV related carcinogenesis. © 2014 UICC.

Evaluation of bacterial pathogen diversity, abundance and health risks in urban recreational water by amplicon next-generation sequencing and quantitative PCR.

PubMed

Cui, Qijia; Fang, Tingting; Huang, Yong; Dong, Peiyan; Wang, Hui

2017-07-01

The microbial quality of urban recreational water is of great concern to public health. The monitoring of indicator organisms and several pathogens alone is not sufficient to accurately and comprehensively identify microbial risks. To assess the levels of bacterial pathogens and health risks in urban recreational water, we analyzed pathogen diversity and quantified four pathogens in 46 water samples collected from waterbodies in Beijing Olympic Forest Park in one year. The pathogen diversity revealed by 16S rRNA gene targeted next-generation sequencing (NGS) showed that 16 of 40 genera and 13 of 76 reference species were present. The most abundant species were Acinetobacter johnsonii, Mycobacterium avium and Aeromonas spp. Quantitative polymerase chain reaction (qPCR) of Escherichia coli (uidA), Aeromonas (aerA), M. avium (16S rRNA), Pseudomonas aeruginosa (oaa) and Salmonella (invA) showed that the aerA genes were the most abundant, occurring in all samples with concentrations of 10 4-6 genome copies/100mL, followed by oaa, invA and M. avium. In total, 34.8% of the samples harbored all genes, indicating the prevalence of these pathogens in this recreational waterbody. Based on the qPCR results, a quantitative microbial risk assessment (QMRA) showed that the annual infection risks of Salmonella, M. avium and P. aeruginosa in five activities were mostly greater than the U.S. EPA risk limit for recreational contacts, and children playing with water may be exposed to the greatest infection risk. Our findings provide a comprehensive understanding of bacterial pathogen diversity and pathogen abundance in urban recreational water by applying both NGS and qPCR. Copyright © 2016. Published by Elsevier B.V.

Use of propidium monoazide for the enumeration of viable Brettanomyces bruxellensis in wine and beer by quantitative PCR.

PubMed

Vendrame, Marco; Manzano, Marisa; Comi, Giuseppe; Bertrand, Julien; Iacumin, Lucilla

2014-09-01

Brettanomyces bruxellensis is a current problem in winemaking all over the world, and the question if B. bruxellensis has a positive or negative impact on wine is one of the most controversial discussions in the world. The presence of live B. bruxellensis cells represents the risk of growth and an increase in cell numbers, which is related to the potential production of volatile phenols. In this work, the optimisation of a PMA-quantitative PCR (qPCR) method to enumerate only viable cells was carried out using the standard strain B. bruxellensis DSMZ 70726. The obtained detection limits were 0.83 log CFU/mL in red wine, 0.63 log CFU/mL in white wine and 0.23 log CFU/mL in beer. Moreover, the quantification was also performed by Reverse Transcription quantitative PCR (RT-qPCR), and the results showed a higher detection limit for all of the trials. Copyright © 2014 Elsevier Ltd. All rights reserved.

Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR.

PubMed

Dobnik, David; Demšar, Tina; Huber, Ingrid; Gerdes, Lars; Broeders, Sylvia; Roosens, Nancy; Debode, Frederic; Berben, Gilbert; Žel, Jana

2018-01-01

Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. Graphical abstract The output of three different PCR-based platforms was assessed in an inter-laboratory comparison.

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

PubMed

Shanks, Orin C; Kelty, Catherine A; Oshiro, Robin; Haugland, Richard A; Madi, Tania; Brooks, Lauren; Field, Katharine G; Sivaganesan, Mano

2016-05-01

There is growing interest in the application of human-associated fecal source identification 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 quality across laboratories. Data quality is typically determined through a series of specifications that ensure good experimental practice and the absence of bias in the results due to DNA isolation and amplification interferences. However, there is currently a lack of consensus on how best to evaluate and interpret human fecal source identification qPCR experiments. This is, in part, due to the lack of standardized protocols and information on interlaboratory variability under conditions for data acceptance. The aim of this study is to provide users and reviewers with a complete series of conditions for data acceptance derived from a multiple laboratory data set using standardized procedures. To establish these benchmarks, data from HF183/BacR287 and HumM2 human-associated qPCR methods were generated across 14 laboratories. Each laboratory followed a standardized protocol utilizing the same lot of reference DNA materials, DNA isolation kits, amplification reagents, and test samples to generate comparable data. After removal of outliers, a nested analysis of variance (ANOVA) was used to establish proficiency metrics that include lab-to-lab, replicate testing within a lab, and random error for amplification inhibition and sample processing controls. Other data acceptance measurements included extraneous DNA contamination assessments (no-template and extraction blank controls) and calibration model performance (correlation coefficient, amplification efficiency, and lower limit of quantification). To demonstrate the implementation of the proposed standardized protocols and data acceptance criteria, comparable data from two additional laboratories were reviewed. The data acceptance criteria

Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks

PubMed Central

2010-01-01

Background Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum). Results Epaulette sharks were caught and exposed to hypoxia. Tissues were collected from 10 controls, 10 individuals with single hypoxic insult and 10 individuals with hypoxia preconditioning (8 hypoxic insults, 12 hours apart). We produced sequence information for reference gene candidates and monitored mRNA expression levels in four tissues: cerebellum, heart, gill and eye. The stability of the genes was examined with analysis of variance, geNorm and NormFinder. The best ranking genes in our study were eukaryotic translation elongation factor 1 beta (eef1b), ubiquitin (ubq) and polymerase (RNA) II (DNA directed) polypeptide F (polr2f). The performance of the ribosomal protein L6 (rpl6) was tissue-dependent. Notably, in one tissue the analysis of variance indicated statistically significant differences between treatments for genes that were ranked as the most stable candidates by reference gene software. Conclusions Our results indicate that eef1b and ubq are generally the most suitable reference genes for the conditions and tissues in the present epaulette shark studies. These genes could also be potential reference gene candidates for other physiological studies examining stress in elasmobranchs. The results emphasise the importance of inter-group variation in reference gene evaluation. PMID:20416043

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR.

PubMed

Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

2011-01-01

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch.

Analysis of a quantitative PCR assay for CMV infection in liver transplant recipients: an intent to find the optimal cut-off value.

PubMed

Martín-Dávila, P; Fortún, J; Gutiérrez, C; Martí-Belda, P; Candelas, A; Honrubia, A; Barcena, R; Martínez, A; Puente, A; de Vicente, E; Moreno, S

2005-06-01

Preemptive therapy required highly predictive tests for CMV disease. CMV antigenemia assay (pp65 Ag) has been commonly used for rapid diagnosis of CMV infection. Amplification methods for early detection of CMV DNA are under analysis. To compare two diagnostic methods for CMV infection and disease in this population: quantitative PCR (qPCR) performed in two different samples, plasma and leukocytes (PMNs) and using a commercial diagnostic test (COBAS Amplicor Monitor Test) versus pp65 Ag. Prospective study conducted in liver transplant recipients from February 2000 to February 2001. Analyses were performed on 164 samples collected weekly during early post-transplant period from 33 patients. Agreements higher than 78% were observed between the three assays. Optimal qPCR cut-off values were calculated using ROC curves for two specific antigenemia values. For antigenemia >or=10 positive cells, the optimal cut-off value for qPCR in plasma was 1330 copies/ml, with a sensitivity (S) of 58% and a specificity (E) of 98% and the optimal cut-off value for qPCR-cells was 713 copies/5x10(6) cells (S:91.7% and E:86%). Using a threshold of antigenemia >or=20 positive cells, the optimal cut-off values were 1330 copies/ml for qPCR-plasma (S 87%; E 98%) and 4755 copies/5x10(6) cells for qPCR-cells (S 87.5%; E 98%). Prediction values for the three assays were calculated in patients with CMV disease (9 pts; 27%). Considering the assays in a qualitative way, the most sensitive was CMV PCR in cells (S: 100%, E: 54%, PPV: 40%; NPV: 100%). Using specific cut-off values for disease detection the sensitivity, specificity, PPV and NPV for antigenemia >or=10 positive cells were: 89%; 83%; 67%; 95%, respectively. For qPCR-cells >or=713 copies/5x10(6) cells: 100%; 54%; 33% and 100% and for plasma-qPCR>or=1330 copies/ml: 78%, 77%, 47%, 89% respectively. Optimal cut-off for viral load performed in plasma and cells can be obtained for the breakpoint antigenemia value recommended for initiating

An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research

PubMed Central

Yan, Xu; Bishop, David J.

2018-01-01

Gene expression analysis by quantitative PCR in skeletal muscle is routine in exercise studies. The reproducibility and reliability of the data fundamentally depend on how the experiments are performed and interpreted. Despite the popularity of the assay, there is a considerable variation in experimental protocols and data analyses from different laboratories, and there is a lack of consistency of proper quality control steps throughout the assay. In this study, we present a number of experiments on various steps of quantitative PCR workflow, and demonstrate how to perform a quantitative PCR experiment with human skeletal muscle samples in an exercise study. We also tested some common mistakes in performing qPCR. Interestingly, we found that mishandling of muscle for a short time span (10 mins) before RNA extraction did not affect RNA quality, and isolated total RNA was preserved for up to one week at room temperature. Demonstrated by our data, use of unstable reference genes lead to substantial differences in the final results. Alternatively, cDNA content can be used for data normalisation; however, complete removal of RNA from cDNA samples is essential for obtaining accurate cDNA content. PMID:29746477

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

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.

SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

PubMed Central

Stadler, Julia; Eder, Johanna; Pratscher, Barbara; Brandt, Sabine; Schneller, Doris; Müllegger, Robert; Vogl, Claus; Trautinger, Franz; Brem, Gottfried; Burgstaller, Joerg P.

2015-01-01

Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent “gold standard”. Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients. PMID:26562020

Monitoring the freshness of fish: development of a qPCR method applied to MAP chilled whiting.

PubMed

Dehaut, Alexandre; Krzewinski, Frédéric; Grard, Thierry; Chollet, Marlène; Jacques, Philippe; Brisabois, Anne; Duflos, Guillaume

2016-04-01

Monitoring of early stages of freshness decay is a major issue for the fishery industry to guarantee the best quality for this highly perishable food matrix. Numerous techniques have been developed, but most of them have the disadvantage of being reliable only either in the last stages of fish freshness or for the analysis of whole fish. This study describes the development of a qPCR method targeting the torA gene harboured by fish spoilage microorganisms. torA encodes an enzyme that leads to the production of trimethylamine responsible for the characteristic spoiled-fish odour. A degenerate primer pair was designed. It amplified torA gene of both Vibrio and Photobacterium with good efficiencies on 7-log DNA dilutions. The primer pair was used during a shelf-life monitoring study achieved on modified atmosphere packed, chilled, whiting (Merlangius merlangus) fillets. The qPCR approach allows the detection of an increase of torA copies throughout the storage of fillets in correlation with the evolution of both total volatile basic nitrogen (-0.86) and trimethylamine concentrations (-0.81), known as spoilage markers. This study described a very promising, sensitive, reliable, time-effective, technique in the field of freshness characterisation of processed fish. © 2015 Society of Chemical Industry.

A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4.

PubMed

Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M

2014-01-01

Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes. Copyright © 2013 Elsevier B.V. All rights reserved.

Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.

PubMed

Li, Xiuying; Yang, Qiwei; Bai, Jinping; Xuan, Yali; Wang, Yimin

2015-01-01

Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, β-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.

Identification of Past and Recent Parvovirus B19 Infection in Immunocompetent Individuals by Quantitative PCR and Enzyme Immunoassays: a Dual-Laboratory Study

PubMed Central

Hedman, Lea; Dhanilall, Pravesh; Kantola, Kalle; Nurmi, Visa; Söderlund-Venermo, Maria; Brown, Kevin E.; Hedman, Klaus

2014-01-01

Parvovirus B19 (B19V) is a member of the family Parvoviridae, genus Erythrovirus. B19V-specific IgG and IgM react differently against conformational and linear epitopes of VP1 and VP2 antigens, leading to the development of IgG avidity and epitope type specificity (ETS) enzyme immunoassays (EIAs) for distinguishing past from recent infection. Additionally, B19V viral load determination (by quantitative PCR [qPCR]) is increasingly used in the staging of B19V infection. In this study, the utility of these methods is compared. A panel of 78 sera was jointly tested by the Virus Reference Department (VRD), London, United Kingdom, and the Haartman Institute (HI), Helsinki, Finland, using a number of EIAs, e.g., B19V-specific IgG and IgM, IgG avidity, and ETS EIAs. At VRD, the sera were also tested by a B19V viral load PCR (qPCR). By consensus analysis, 43 (55.1%) sera represented past infection, 28 (35.9%) sera represented recent infection, and 7 (9.0%) sera were indeterminate. Both VRD B19V qPCR and HI B19V VP2 IgM EIA gave the highest agreement with consensus interpretation for past or recent infection, with an overall agreement of 99% (95% confidence interval [CI], 92 to 100) and positive predictive value (PPV) of 100% (95% CI, 87 to 100). Nine sera designated as representing past infection by consensus analysis were B19V IgM positive by a commercial VRD B19V IgM EIA and B19V IgM negative by a new HI in-house B19V VP2 IgM EIA. A new VRD B19V IgG avidity EIA showed good (>95%) agreement (excluding equivocal results) with consensus interpretations for past or recent infection. Correct discrimination of past from recent B19V infection was achieved through application of qPCR or by appropriate selection of EIAs. PMID:24403307

Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism.

PubMed

Guo, Longhua; Qiu, Bin; Chi, Yuwu; Chen, Guonan

2008-09-01

In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.

Effect of the addition of phytosterols and tocopherols on Streptococcus thermophilus robustness during industrial manufacture and ripening of a functional cheese as evaluated by qPCR and RT-qPCR.

PubMed

Pega, J; Rizzo, S; Pérez, C D; Rossetti, L; Díaz, G; Ruzal, S M; Nanni, M; Descalzo, A M

2016-09-02

The quality of functional food products designed for the prevention of degenerative diseases can be affected by the incorporation of bioactive compounds. In many types of cheese, the performance of starter microorganisms is critical for optimal elaboration and for providing potential probiotic benefits. Phytosterols are plant lipophilic triterpenes that have been used for the design of functional dairy products because of their ability to lower serum cholesterol levels in humans. However, their effect on the starter culture behavior during cheesemaking has not yet been studied. Here, we followed DNA and RNA kinetics of the bacterium Streptococcus thermophilus, an extensively used dairy starter with probiotic potential, during industrial production of a functional, semi-soft, reduced-fat cheese containing phytosterol esters and alpha-tocopherol as bioactive compounds. For this purpose, real-time quantitative PCR (qPCR) and reverse transcription-qPCR (RT-qPCR) assays were optimized and applied to samples obtained during the manufacture and ripening of functional and control cheeses. An experimental set-up was used to evaluate the detection threshold of free nucleic acids for extraction protocols based on pelleted microorganisms. To our knowledge, this straight-forward approach provides the first experimental evidence indicating that DNA is not a reliable marker of cell integrity, whereas RNA may constitute a more accurate molecular signature to estimate both bacterial viability and metabolic activity. Compositional analysis revealed that the bioactive molecules were effectively incorporated into the cheese matrix, at levels considered optimal to exert their biological action. The starter S. thermophilus was detected by qPCR and RT-qPCR during cheese production at the industrial level, from at least 30min after its inoculation until 81days of ripening, supporting the possible role of this species in shaping organoleptic profiles. We also showed for the first time that

Validation of Reference Genes for Quantitative Expression Analysis by Real-Time RT-PCR in Four Lepidopteran Insects

PubMed Central

Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei

2012-01-01

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ΔCt analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2-ΔΔCt method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR. PMID:22938136

Validation of reference genes for quantitative expression analysis by real-time rt-PCR in four lepidopteran insects.

PubMed

Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei

2012-01-01

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ΔCt analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2(-ΔΔCt) method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR.

PCR Inhibition of a Quantitative PCR for Detection of Mycobacterium avium Subspecies Paratuberculosis DNA in Feces: Diagnostic Implications and Potential Solutions

PubMed Central

Acharya, Kamal R.; Dhand, Navneet K.; Whittington, Richard J.; Plain, Karren M.

2017-01-01

Molecular tests such as polymerase chain reaction (PCR) are increasingly being applied for the diagnosis of Johne’s disease, a chronic intestinal infection of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Feces, as the primary test sample, presents challenges in terms of effective DNA isolation, with potential for PCR inhibition and ultimately for reduced analytical and diagnostic sensitivity. However, limited evidence is available regarding the magnitude and diagnostic implications of PCR inhibition for the detection of MAP in feces. This study aimed to investigate the presence and diagnostic implications of PCR inhibition in a quantitative PCR assay for MAP (High-throughput Johne’s test) to investigate the characteristics of samples prone to inhibition and to identify measures that can be taken to overcome this. In a study of fecal samples derived from a high prevalence, endemically infected cattle herd, 19.94% of fecal DNA extracts showed some evidence of inhibition. Relief of inhibition by a five-fold dilution of the DNA extract led to an average increase in quantification of DNA by 3.3-fold that consequently increased test sensitivity of the qPCR from 55 to 80% compared to fecal culture. DNA extracts with higher DNA and protein content had 19.33 and 10.94 times higher odds of showing inhibition, respectively. The results suggest that the current test protocol is sensitive for herd level diagnosis of Johne’s disease but that test sensitivity and individual level diagnosis could be enhanced by relief of PCR inhibition, achieved by five-fold dilution of the DNA extract. Furthermore, qualitative and quantitative parameters derived from absorbance measures of DNA extracts could be useful for prediction of inhibitory fecal samples. PMID:28210245

Application of PCR and real-time PCR for monitoring cyanobacteria, Microcystis spp. and Cylindrospermopsis raciborskii in Macau freshwater reservoir

NASA Astrophysics Data System (ADS)

Zhang, Weiying; Lou, Inchio; Ung, Wai Kin; Kong, Yijun; Mok, Kai Meng

2014-06-01

Freshwater algal blooms have become a growing concern world-wide. They are caused by a high level of cyanobacteria, predominantly Microcystis spp. and Cylindrospermopsis raciborskii, which can produce microcystin and cylindrospermopsin, respectively. Longtime exposure to these cyanotoxins may affect public health, thus reliable detection, quantification, and enumeration of these harmful algae species has become a priority in water quality management. Traditional manual enumeration of algal bloom cells primarily involves microscopic identification which limited by inaccuracy and time-consumption.With the development of molecular techniques and an increasing number of microbial sequences available in the Genbank database, the use of molecular methods can be used for more rapid, reliable, and accurate detection and quantification. In this study, multiplex polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) techniques were developed and applied for monitoring cyanobacteria Microcystis spp. and C. raciborskii in the Macau Storage Reservoir (MSR). The results showed that the techniques were successful for identifying and quantifying the species in pure cultures and mixed cultures, and proved to be a potential application for water sampling in MSR. When the target species were above 1 million cells/L, similar cell numbers estimated by microscopic enumeration and qPCR were obtained. Further quantification in water samples indicated that the ratio of the estimated number of cell by microscopy and qPCR was 0.4-12.9 for cyanobacteria and 0.2-3.9 for C. raciborskii. However, Microcystis spp. was not observed by manual enumeration, while it was detected at low levels by qPCR, suggesting that qPCR is more sensitive and accurate. Thus the molecular approaches provide an additional reliable monitoring option to traditional microscopic enumeration for the ecosystems monitoring program.

quantGenius: implementation of a decision support system for qPCR-based gene quantification.

PubMed

Baebler, Špela; Svalina, Miha; Petek, Marko; Stare, Katja; Rotter, Ana; Pompe-Novak, Maruša; Gruden, Kristina

2017-05-25

Quantitative molecular biology remains a challenge for researchers due to inconsistent approaches for control of errors in the final results. Due to several factors that can influence the final result, quantitative analysis and interpretation of qPCR data are still not trivial. Together with the development of high-throughput qPCR platforms, there is a need for a tool allowing for robust, reliable and fast nucleic acid quantification. We have developed "quantGenius" ( http://quantgenius.nib.si ), an open-access web application for a reliable qPCR-based quantification of nucleic acids. The quantGenius workflow interactively guides the user through data import, quality control (QC) and calculation steps. The input is machine- and chemistry-independent. Quantification is performed using the standard curve approach, with normalization to one or several reference genes. The special feature of the application is the implementation of user-guided QC-based decision support system, based on qPCR standards, that takes into account pipetting errors, assay amplification efficiencies, limits of detection and quantification of the assays as well as the control of PCR inhibition in individual samples. The intermediate calculations and final results are exportable in a data matrix suitable for further statistical analysis or visualization. We additionally compare the most important features of quantGenius with similar advanced software tools and illustrate the importance of proper QC system in the analysis of qPCR data in two use cases. To our knowledge, quantGenius is the only qPCR data analysis tool that integrates QC-based decision support and will help scientists to obtain reliable results which are the basis for biologically meaningful data interpretation.

PCR detection of Streptococcus mutans and Aggregatibacter actinomycetemcomitans in dental plaque samples from Haitian adolescents.

PubMed

Psoter, Walter J; Ge, Yao; Russell, Stefanie L; Chen, Zhou; Katz, Ralph V; Jean-Charles, Germain; Li, Yihong

2011-08-01

Streptococcus mutans and Aggregatibacter actinomycetemcomitans are oral pathogens associated with dental caries and periodontitis, respectively. The aim of this study was to determine the colonization of these two microorganisms in the dental plaque of a group of Haitian adolescents using two different polymerase chain reaction (PCR) methods, standard PCR, and quantitative real-time PCR (qPCR) assays. Fifty-four pooled supra-gingival plaque samples and 98 pooled sub-gingival plaque samples were obtained from 104 12- to19-year-old rural-dwelling Haitians. The total genomic DNA of bacteria was isolated from these samples, and all participants also received caries and periodontal examinations. Caries prevalence was 42.2%, and the mean decayed, missing, and filled surface (DMFS) was 2.67 ± 5.3. More than half of the adolescents (53.3%) experienced periodontal pockets (Community Periodontal Index score ≥3). S. mutans was detected in 67.3% by qPCR and 38.8% by PCR of the supra-gingival plaque samples (p < 0.01), and 36.6% by qPCR and 8.1% by PCR of the sub-gingival samples (p < 0.01). A. actinomycetemcomitans was detected in 85.1% by qPCR and 44.0% by PCR of the sub-gingival samples (p < 0.01), but the prevalence was similar, 67.3% by qPCR and 59.2% by PCR, in the supra-gingival plaque samples. Neither age nor gender was significantly correlated to the bacterial colonization. The results demonstrated a moderate-to-high prevalence of S. mutans and A. actinomycetemcomitans in the Haitian adolescent population, and qPCR is more sensitive than standard PCR in field conditions. These findings suggest that qPCR should be considered for field oral epidemiologic studies and may be necessary in investigations having major logistic challenges.

Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia.

PubMed

Su, Y-L; Feng, J; Li, Y-W; Bai, J-S; Li, A-X

2016-02-01

Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real-time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS-s/IGS-a, which targets the 16S-23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post-injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post-injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish. © 2015 John Wiley & Sons Ltd.

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.

Development and validation of a citrate synthase directed quantitative PCR marker for soil bacterial communities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Castro Gonzalez, Hector F; Classen, Aimee T; Austin, Emily E

2012-01-01

Molecular innovations in microbial ecology are allowing scientists to correlate microbial community characteristics to a variety of ecosystem functions. However, to date the majority of soil microbial ecology studies target phylogenetic rRNA markers, while a smaller number target functional markers linked to soil processes. We validated a new primer set targeting citrate synthase (gtlA), a central enzyme in the citric acid cycle linked to aerobic respiration. Primers for a 225 bp fragment suitable for qPCR were tested for specificity and assay performance verified on multiple soils. Clone libraries of the PCR-amplified gtlA gene exhibited high diversity and recovered most majormore » groups identified in a previous 16S rRNA gene study. Comparisons among bacterial communities based on gtlA sequencing using UniFrac revealed differences among the experimental soils studied. Conditions for gtlA qPCR were optimized and calibration curves were highly linear (R2 > 0.99) over six orders of magnitude (4.56 10^5 to 4.56 10^11 copies), with high amplification efficiencies (>1.7). We examined the performance of the gtlA qPCR across a variety of soils and ecosystems, spanning forests, old fields and agricultural areas. We were able to amplify gtlA genes in all tested soils, and detected differences in gtlA abundance within and among environments. These results indicate that a fully developed gtlA-targeted qPCR approach may have potential to link microbial community characteristics with changes in soil respiration.« less

Quantitative CrAssphage PCR Assays for Human Fecal ...

EPA Pesticide Factsheets

Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal indicator bacteria are used; however, they cannot distinguish human fecal waste from pollution from other animals. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based upon initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against a large animal fecal reference library and were highly abundant in raw sewage and sewage impacted water samples. In addition, CPQ_056 and CPQ_064 assay performance was compared to HF183/BacR287 and HumM2 methods in paired experiments. Findings confirm viral crAssphage qPCR assays perform at a similar level to well established bacterial human-associated fecal source identification technologies. These new viral based assays could become important water quality management and research tools. To inform the public.

Flow cytometry and real-time quantitative PCR as tools for assessing plasmid persistence.

PubMed

Loftie-Eaton, Wesley; Tucker, Allison; Norton, Ann; Top, Eva M

2014-09-01

The maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in three Pseudomonas hosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Evaluation of Legionella real-time PCR against traditional culture for routine and public health testing of water samples.

PubMed

Collins, S; Stevenson, D; Walker, J; Bennett, A

2017-06-01

To evaluate the usefulness of Legionella qPCR alongside traditional culture for enumeration of Legionella from water samples as part of both routine and public health investigation testing. Routine water samples (n = 2002) and samples from public health investigations (n = 215) were analysed by culture and qPCR for Legionella spp., Legionella pneumophila and L. pneumophila sg-1. A negative qPCR result was highly predictive of a negative culture result for all water systems (negative predictive values, NPV from 97·4 to 100%). Positive predictive values (PPV) were lower (0-50%). Results for qPCR were generally larger than culture with average log 10 differences of 1·1 for Legionella spp. and 1·2 for L. pneumophila. Alert and action levels of 1000 and 10 000 GU per litre, respectively, are proposed for Legionella qPCR for hot and cold water systems (HCWS). The use of qPCR significantly reduced the time to results for public health investigations by rapidly identifying potential sources and ruling out others, thus enabling a more rapid and efficient response. The high NPV of qPCR supports its use to rapidly screen out negative samples without culture. Alert and action levels for Legionella qPCR for HCWS are proposed. Quantitative PCR will be a valuable tool for both routine and public health testing. This study generated comparative data of >2000 water samples by qPCR and culture. Action and alert levels have been recommended that could enable duty holders to interpret qPCR results to facilitate timely Legionella control and public health protection. © 2017 Crown copyright. Journal of Applied Microbiology © 2017 The Society for Applied Microbiology.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

PubMed

Ito, Takao; Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides

PubMed Central

Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays. PMID:28957362

Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data.

PubMed

Artico, Sinara; Nardeli, Sarah M; Brilhante, Osmundo; Grossi-de-Sa, Maria Fátima; Alves-Ferreira, Marcio

2010-03-21

Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1alpha5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhbetaTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene expression measures in

Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

PubMed Central

2010-01-01

Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. Results By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. Conclusion We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene

Babesia bovis and Babesia bigemina infection levels estimated by qPCR in Angus cattle from an endemic area of São Paulo state, Brazil.

PubMed

Giglioti, R; Oliveira, H N; Santana, C H; Ibelli, A M G; Néo, T A; Bilhassi, T B; Rabelo, M D; Machado, R Z; Brito, L G; Oliveira, M C S

2016-07-01

The levels of infection by Babesia bovis and Babesia bigemina were estimated by absolute quantification through the quantitative PCR technique (qPCR). Fifty-one contemporaneous Angus cattle were evaluated on two occasions. The number of standard female Rhipicephalus microplus ticks present on the left side of the body was counted and blood samples were drawn from the tail vein into tubes containing the anticoagulant EDTA. The blood samples were submitted to DNA extraction and used to quantify the number of copies (NC) of DNA from B. bovis and B. bigemina by qPCR. The data on tick count and number of DNA copies were transformed for normalization and analyzed by a mixed model method. A multivariate model with repeated measures of the same animal, including the effects of collection, parasite species and their interaction, was used. The repeatability values were obtained from the matrix of (co)variances and were expressed for each species. The correlations between the counts of different species on the same animal, in the same collection or different collections, were also estimated. The results showed the qPCR could distinguish the two between infection by the two Babesia species. Infection levels by B. bovis and B. bigemina were detected in 100% and 98% of the animals, respectively. Significant differences were found (P<0.05) between the NC of the two Babesia species, B. bovis 1.49±0.07 vs. B. bigemina 0.82±0.06. Low repeatabilities were found for the counts of R. microplus and NC of B. bovis and B. bigemina: 0.05, 0.10 and 0.02, respectively. The correlations between R. microplus count and NC of B. bovis and B. bigemina were both very near zero. However, an association was observed between the NC of the two species, with a correlation coefficient of 0.30 for measures from the same collection. The absence of associations between the quantity of DNA from B. bovis and B. bigemina and the tick counts suggests that the variation of parasitemia by the hemoparasites did

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.

GETPrime: a gene- or transcript-specific primer database for quantitative real-time PCR

PubMed Central

Gubelmann, Carine; Gattiker, Alexandre; Massouras, Andreas; Hens, Korneel; David, Fabrice; Decouttere, Frederik; Rougemont, Jacques; Deplancke, Bart

2011-01-01

The vast majority of genes in humans and other organisms undergo alternative splicing, yet the biological function of splice variants is still very poorly understood in large part because of the lack of simple tools that can map the expression profiles and patterns of these variants with high sensitivity. High-throughput quantitative real-time polymerase chain reaction (qPCR) is an ideal technique to accurately quantify nucleic acid sequences including splice variants. However, currently available primer design programs do not distinguish between splice variants and also differ substantially in overall quality, functionality or throughput mode. Here, we present GETPrime, a primer database supported by a novel platform that uniquely combines and automates several features critical for optimal qPCR primer design. These include the consideration of all gene splice variants to enable either gene-specific (covering the majority of splice variants) or transcript-specific (covering one splice variant) expression profiling, primer specificity validation, automated best primer pair selection according to strict criteria and graphical visualization of the latter primer pairs within their genomic context. GETPrime primers have been extensively validated experimentally, demonstrating high transcript specificity in complex samples. Thus, the free-access, user-friendly GETPrime database allows fast primer retrieval and visualization for genes or groups of genes of most common model organisms, and is available at http://updepla1srv1.epfl.ch/getprime/. Database URL: http://deplanckelab.epfl.ch. PMID:21917859

Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR.

PubMed

Lock, Martin; Alvira, Mauricio R; Chen, Shu-Jen; Wilson, James M

2014-04-01

Accurate titration of adeno-associated viral (AAV) vector genome copies is critical for ensuring correct and reproducible dosing in both preclinical and clinical settings. Quantitative PCR (qPCR) is the current method of choice for titrating AAV genomes because of the simplicity, accuracy, and robustness of the assay. However, issues with qPCR-based determination of self-complementary AAV vector genome titers, due to primer-probe exclusion through genome self-annealing or through packaging of prematurely terminated defective interfering (DI) genomes, have been reported. Alternative qPCR, gel-based, or Southern blotting titering methods have been designed to overcome these issues but may represent a backward step from standard qPCR methods in terms of simplicity, robustness, and precision. Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes. Here we compare a ddPCR-based AAV genome titer assay with a standard and an optimized qPCR assay for the titration of both single-stranded and self-complementary AAV genomes. We demonstrate absolute quantification of single-stranded AAV vector genomes by ddPCR with up to 4-fold increases in titer over a standard qPCR titration but with equivalent readout to an optimized qPCR assay. In the case of self-complementary vectors, ddPCR titers were on average 5-, 1.9-, and 2.3-fold higher than those determined by standard qPCR, optimized qPCR, and agarose gel assays, respectively. Droplet digital PCR-based genome titering was superior to qPCR in terms of both intra- and interassay precision and is more resistant to PCR inhibitors, a desirable feature for in-process monitoring of early-stage vector production and for vector genome biodistribution

Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR.

PubMed

Stokdyk, Joel P; Firnstahl, Aaron D; Spencer, Susan K; Burch, Tucker R; Borchardt, Mark A

2016-06-01

The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay alone rather than the entire sample process. Our objective was to develop an approach to determine the 95% LOD (lowest concentration at which 95% of positive samples are detected) for the entire process of waterborne pathogen detection. We began by spiking the lowest concentration that was consistently positive at the qPCR step (based on its standard curve) into each procedural step working backwards (i.e., extraction, secondary concentration, primary concentration), which established a concentration that was detectable following losses of the pathogen from processing. Using the fraction of positive replicates (n = 10) at this concentration, we selected and analyzed a second, and then third, concentration. If the fraction of positive replicates equaled 1 or 0 for two concentrations, we selected another. We calculated the LOD using probit analysis. To demonstrate our approach we determined the 95% LOD for Salmonella enterica serovar Typhimurium, adenovirus 41, and vaccine-derived poliovirus Sabin 3, which were 11, 12, and 6 genomic copies (gc) per reaction (rxn), respectively (equivalent to 1.3, 1.5, and 4.0 gc L(-1) assuming the 1500 L tap-water sample volume prescribed in EPA Method 1615). This approach limited the number of analyses required and was amenable to testing multiple genetic targets simultaneously (i.e., spiking a single sample with multiple microorganisms). An LOD determined this way can facilitate study design, guide the number of required technical replicates, aid method evaluation, and inform data interpretation. Published by Elsevier Ltd.

Determining the 95% limit of detection for waterborne pathogen analyses from primary concentration to qPCR

USGS Publications Warehouse

Stokdyk, Joel P.; Firnstahl, Aaron; Spencer, Susan K.; Burch, Tucker R; Borchardt, Mark A.

2016-01-01

The limit of detection (LOD) for qPCR-based analyses is not consistently defined or determined in studies on waterborne pathogens. Moreover, the LODs reported often reflect the qPCR assay alone rather than the entire sample process. Our objective was to develop an approach to determine the 95% LOD (lowest concentration at which 95% of positive samples are detected) for the entire process of waterborne pathogen detection. We began by spiking the lowest concentration that was consistently positive at the qPCR step (based on its standard curve) into each procedural step working backwards (i.e., extraction, secondary concentration, primary concentration), which established a concentration that was detectable following losses of the pathogen from processing. Using the fraction of positive replicates (n = 10) at this concentration, we selected and analyzed a second, and then third, concentration. If the fraction of positive replicates equaled 1 or 0 for two concentrations, we selected another. We calculated the LOD using probit analysis. To demonstrate our approach we determined the 95% LOD for Salmonella enterica serovar Typhimurium, adenovirus 41, and vaccine-derived poliovirus Sabin 3, which were 11, 12, and 6 genomic copies (gc) per reaction (rxn), respectively (equivalent to 1.3, 1.5, and 4.0 gc L−1 assuming the 1500 L tap-water sample volume prescribed in EPA Method 1615). This approach limited the number of analyses required and was amenable to testing multiple genetic targets simultaneously (i.e., spiking a single sample with multiple microorganisms). An LOD determined this way can facilitate study design, guide the number of required technical replicates, aid method evaluation, and inform data interpretation.

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.

Detection of nucleophosmin 1 mutations by quantitative real-time polymerase chain reaction versus capillary electrophoresis: a comparative study.

PubMed

Barakat, Fareed H; Luthra, Rajyalakshmi; Yin, C Cameron; Barkoh, Bedia A; Hai, Seema; Jamil, Waqar; Bhakta, Yaminiben I; Chen, Su; Medeiros, L Jeffrey; Zuo, Zhuang

2011-08-01

Nucleophosmin 1 (NPM1) is the most commonly mutated gene in acute myeloid leukemia. Detection of NPM1 mutations is useful for stratifying patients for therapy, predicting prognosis, and assessing for minimal residual disease. Several methods have been developed to rapidly detect NPM1 mutations in genomic DNA and/or messenger RNA specimens. To directly compare a quantitative real-time polymerase chain reaction (qPCR) assay with a widely used capillary electrophoresis assay for detecting NPM1 mutations. We adopted and modified a qPCR assay designed to detect the 6 most common NPM1 mutations and performed the assay in parallel with capillary electrophoresis assay in 207 bone marrow aspirate or peripheral blood samples from patients with a range of hematolymphoid neoplasms. The qPCR assay demonstrated a higher analytical sensitivity than the capillary electrophoresis 1/1000 versus 1/40, respectively. The capillary electrophoresis assay generated 10 equivocal results that needed to be repeated, whereas the qPCR assay generated only 1 equivocal result. After test conditions were optimized, the qPCR and capillary electrophoresis methods produced 100% concordant results, 85 positive and 122 negative. Given the higher analytical sensitivity and specificity of the qPCR assay, that assay is less likely to generate equivocal results than the capillary electrophoresis assay. Moreover, the qPCR assay is quantitative, faster, cheaper, less prone to contamination, and well suited for monitoring minimal residual disease.

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

PubMed Central

Bogema, D. R.; Deutscher, A. T.; Fell, S.; Collins, D.; Eamens, G. J.

2015-01-01

Theileria orientalis is an emerging pathogen of cattle in Asia, Australia, and New Zealand. This organism is a vector-borne hemoprotozoan that causes clinical disease characterized by anemia, abortion, and death, as well as persistent subclinical infections. Molecular methods of diagnosis are preferred due to their sensitivity and utility in differentiating between pathogenic and apathogenic genotypes. Conventional PCR (cPCR) assays for T. orientalis detection and typing are laborious and do not provide an estimate of parasite load. Current real-time PCR assays cannot differentiate between clinically relevant and benign genotypes or are only semiquantitative without a defined clinical threshold. Here, we developed and validated a hydrolysis probe quantitative PCR (qPCR) assay which universally detects and quantifies T. orientalis and identifies the clinically associated Ikeda and Chitose genotypes (UIC assay). Comparison of the UIC assay results with previously validated universal and genotype-specific cPCR results demonstrated that qPCR detects and differentiates T. orientalis with high sensitivity and specificiy. Comparison of quantitative results based on percent parasitemia, determined via blood film analysis and packed cell volume (PCV) revealed significant positive and negative correlations, respectively. One-way analysis of variance (ANOVA) indicated that blood samples from animals with clinical signs of disease contained statistically higher concentrations of T. orientalis DNA than animals with subclinical infections. We propose clinical thresholds to assist in classifying high-, moderate-, and low-level infections and describe how parasite load and the presence of the Ikeda and Chitose genotypes relate to disease. PMID:25588653

Construction of Genetically Engineered Streptococcus gordonii Strains to Provide Control in QPCR Assays for Assessing Microbiological Quality in Recreational Water.

EPA Science Inventory

Quantitative PCR (QPCR) methods for beach monitoring by estimating abundance of Enterococcus spp. in recreational waters use internal, positive controls which address only the amplification of target DNA. In this study two internal, positive controls were developed to control for...

PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

EPA Science Inventory

ABSTRACT

Palatal Dysmorphogenesis : Quantitative RT-PCR

Gary A. Held and Barbara D. Abbott

Reverse transcription PCR (RT-PCR) is a very sensitive method for detecting mRNA in tissue samples. However, as it is usually performed it is does not yield quantitativ...

Density measurement of Demodex canis by qPCR and analysis of serum cytokine levels in dogs with different clinical forms of demodicosis.

PubMed

Gasparetto, Naiani Domingos; Almeida, Arleana do Bom Parto Ferreira; Nakazato, Luciano; França, Eduardo Luzía; França, Adenilda Cristina Honorio; Fagundes, Danny Laura Gomes; Bortolini, Juliano; Sousa, Valéria Régia Franco

2018-06-15

To quantify (by qPCR) the density of Demodex canis mites in the skin of dogs with demodicosis and in healthy dogs, as well as measuring the serum concentrations of interleukin (IL)-1β, IL-6, IL-8, IL-10, IL-12, and tumour necrosis factor-alfa (TNF-α). Fifty-four dogs were divided into three groups: localized demodicosis (LD, n = 16), generalized demodicosis (GD, n = 22), and control group (CG, n = 16). All dogs were subjected to skin scraping, blood collection, and skin biopsy. DNA extraction was performed and the parasite density was established by qPCR. Serum cytokine concentrations were obtained by flow cytometry. The median number of mites in the skin of the GD (6.2 × 10 4 copies/μL) and LD dogs (1.2 × 10 4 copies/μL) was statistically higher than that in the CG dogs (8.7 × 10 2 copies/μL). Whereas there were no significant differences in median IL-1β, IL-8, IL-10, IL-12, and TNF-α levels among the study groups, there was a statistically higher IL-6 concentration in the LD dogs than in the healthy dogs. According to our results, qPCR is an effective method for measuring the density of D. canis in the canine integument. In addition, the activation of the acute-phase immune response in localized demodicosis can be induced by IL-6 activity. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantification of Endospore-Forming Firmicutes by Quantitative PCR with the Functional Gene spo0A

PubMed Central

Bueche, Matthieu; Wunderlin, Tina; Roussel-Delif, Ludovic; Junier, Thomas; Sauvain, Loic; Jeanneret, Nicole

2013-01-01

Bacterial endospores are highly specialized cellular forms that allow endospore-forming Firmicutes (EFF) to tolerate harsh environmental conditions. EFF are considered ubiquitous in natural environments, in particular, those subjected to stress conditions. In addition to natural habitats, EFF are often the cause of contamination problems in anthropogenic environments, such as industrial production plants or hospitals. It is therefore desirable to assess their prevalence in environmental and industrial fields. To this end, a high-sensitivity detection method is still needed. The aim of this study was to develop and evaluate an approach based on quantitative PCR (qPCR). For this, the suitability of functional genes specific for and common to all EFF were evaluated. Seven genes were considered, but only spo0A was retained to identify conserved regions for qPCR primer design. An approach based on multivariate analysis was developed for primer design. Two primer sets were obtained and evaluated with 16 pure cultures, including representatives of the genera Bacillus, Paenibacillus, Brevibacillus, Geobacillus, Alicyclobacillus, Sulfobacillus, Clostridium, and Desulfotomaculum, as well as with environmental samples. The primer sets developed gave a reliable quantification when tested on laboratory strains, with the exception of Sulfobacillus and Desulfotomaculum. A test using sediment samples with a diverse EFF community also gave a reliable quantification compared to 16S rRNA gene pyrosequencing. A detection limit of about 104 cells (or spores) per gram of initial material was calculated, indicating this method has a promising potential for the detection of EFF over a wide range of applications. PMID:23811505

Reference genes for quantitative PCR in the adipose tissue of mice with metabolic disease.

PubMed

Almeida-Oliveira, Fernanda; Leandro, João G B; Ausina, Priscila; Sola-Penna, Mauro; Majerowicz, David

2017-04-01

Obesity and diabetes are metabolic diseases and they are increasing in prevalence. The dynamics of gene expression associated with these diseases is fundamental to identifying genes involved in related biological processes. qPCR is a sensitive technique for mRNA quantification and the most commonly used method in gene-expression studies. However, the reliability of these results is directly influenced by data normalization. As reference genes are the major normalization method used, this work aims to identify reference genes for qPCR in adipose tissues of mice with type-I diabetes or obesity. We selected 12 genes that are commonly used as reference genes. The expression of these genes in the adipose tissues of mice was analyzed in the context of three different experimental protocols: 1) untreated animals; 2) high-fat-diet animals; and 3) streptozotocin-treated animals. Gene-expression stability was analyzed using four different algorithms. Our data indicate that TATA-binding protein is stably expressed across adipose tissues in control animals. This gene was also a useful reference when the brown adipose tissues of control and obese mice were analyzed. The mitochondrial ATP synthase F1 complex gene exhibits stable expression in subcutaneous and perigonadal adipose tissue from control and obese mice. Moreover, this gene is the best reference for qPCR normalization in adipose tissue from streptozotocin-treated animals. These results show that there is no perfect stable gene suited for use under all experimental conditions. In conclusion, the selection of appropriate genes is a prerequisite to ensure qPCR reliability and must be performed separately for different experimental protocols. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques.

PubMed

de Morais, Rayana Carla Silva; da Costa Oliveira, Cintia Nascimento; de Albuquerque, Suênia da Cunha Gonçalves; Mendonça Trajano Silva, Lays Adrianne; Pessoa-E-Silva, Rômulo; Alves da Cruz, Heidi Lacerda; de Brito, Maria Edileuza Felinto; de Paiva Cavalcanti, Milena

2016-06-01

Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques. qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains. Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing

Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay.

PubMed

Shen, Shu-Min; Chou, Ming-Yuan; Hsu, Bing-Mu; Ji, Wen-Tsai; Hsu, Tsui-Kang; Tsai, Hsiu-Feng; Huang, Yu-Li; Chiu, Yi-Chou; Kao, Erl-Shyh; Kao, Po-Min; Fan, Cheng-Wei

2015-07-01

Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 10(2) and 3.3 × 10(5) cells/l in river water and 72.1-5.7 × 10(6) cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors.

Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ

USDA-ARS?s Scientific Manuscript database

The pathogen causing corky root on lettuce, Sphingobium suberifaciens, is recalcitrant to standard epidemiological methods. Primers were selected from 16S rDNA sequences useful for the specific detection and quantification of S. suberifaciens. Conventional (PCR) and quantitative (qPCR) PCR protocols...

Evaluation of Faecalibacterium 16S rDNA genetic markers for accurate identification of swine faecal waste by quantitative PCR.

PubMed

Duan, Chuanren; Cui, Yamin; Zhao, Yi; Zhai, Jun; Zhang, Baoyun; Zhang, Kun; Sun, Da; Chen, Hang

2016-10-01

A genetic marker within the 16S rRNA gene of Faecalibacterium was identified for use in a quantitative PCR (qPCR) assay to detect swine faecal contamination in water. A total of 146,038 bacterial sequences were obtained using 454 pyrosequencing. By comparative bioinformatics analysis of Faecalibacterium sequences with those of numerous swine and other animal species, swine-specific Faecalibacterium 16S rRNA gene sequences were identified and Polymerase Chain Okabe (PCR) primer sets designed and tested against faecal DNA samples from swine and non-swine sources. Two PCR primer sets, PFB-1 and PFB-2, showed the highest specificity to swine faecal waste and had no cross-reaction with other animal samples. PFB-1 and PFB-2 amplified 16S rRNA gene sequences from 50 samples of swine with positive ratios of 86 and 90%, respectively. We compared swine-specific Faecalibacterium qPCR assays for the purpose of quantifying the newly identified markers. The quantification limits (LOQs) of PFB-1 and PFB-2 markers in environmental water were 6.5 and 2.9 copies per 100 ml, respectively. Of the swine-associated assays tested, PFB-2 was more sensitive in detecting the swine faecal waste and quantifying the microbial load. Furthermore, the microbial abundance and diversity of the microbiomes of swine and other animal faeces were estimated using operational taxonomic units (OTUs). The species specificity was demonstrated for the microbial populations present in various animal faeces. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum.

PubMed

Rubio, Marcela da Silva; Penha Filho, Rafael Antonio Casarin; Almeida, Adriana Maria de; Berchieri, Angelo

2017-12-01

Currently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host-specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97 bp) showed Tm of 78°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 10 8 -10 1 CFU of these biovars.

Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate.

PubMed

Hedrich, Sabrina; Guézennec, Anne-Gwenaëlle; Charron, Mickaël; Schippers, Axel; Joulian, Catherine

2016-01-01

Monitoring of the microbial community in bioleaching processes is essential in order to control process parameters and enhance the leaching efficiency. Suitable methods are, however, limited as they are usually not adapted to bioleaching samples and often no taxon-specific assays are available in the literature for these types of consortia. Therefore, our study focused on the development of novel quantitative real-time PCR (qPCR) assays for the quantification of Acidithiobacillus caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans , and Sulfobacillus benefaciens and comparison of the results with data from other common molecular monitoring methods in order to evaluate their accuracy and specificity. Stirred tank bioreactors for the leaching of copper concentrate, housing a consortium of acidophilic, moderately thermophilic bacteria, relevant in several bioleaching operations, served as a model system. The microbial community analysis via qPCR allowed a precise monitoring of the evolution of total biomass as well as abundance of specific species. Data achieved by the standard fingerprinting methods, terminal restriction fragment length polymorphism (T-RFLP) and capillary electrophoresis single strand conformation polymorphism (CE-SSCP) on the same samples followed the same trend as qPCR data. The main added value of qPCR was, however, to provide quantitative data for each species whereas only relative abundance could be deduced from T-RFLP and CE-SSCP profiles. Additional value was obtained by applying two further quantitative methods which do not require nucleic acid extraction, total cell counting after SYBR Green staining and metal sulfide oxidation activity measurements via microcalorimetry. Overall, these complementary methods allow for an efficient quantitative microbial community monitoring in various bioleaching operations.

Quantitative Monitoring of Microbial Species during Bioleaching of a Copper Concentrate

PubMed Central

Hedrich, Sabrina; Guézennec, Anne-Gwenaëlle; Charron, Mickaël; Schippers, Axel; Joulian, Catherine

2016-01-01

Monitoring of the microbial community in bioleaching processes is essential in order to control process parameters and enhance the leaching efficiency. Suitable methods are, however, limited as they are usually not adapted to bioleaching samples and often no taxon-specific assays are available in the literature for these types of consortia. Therefore, our study focused on the development of novel quantitative real-time PCR (qPCR) assays for the quantification of Acidithiobacillus caldus, Leptospirillum ferriphilum, Sulfobacillus thermosulfidooxidans, and Sulfobacillus benefaciens and comparison of the results with data from other common molecular monitoring methods in order to evaluate their accuracy and specificity. Stirred tank bioreactors for the leaching of copper concentrate, housing a consortium of acidophilic, moderately thermophilic bacteria, relevant in several bioleaching operations, served as a model system. The microbial community analysis via qPCR allowed a precise monitoring of the evolution of total biomass as well as abundance of specific species. Data achieved by the standard fingerprinting methods, terminal restriction fragment length polymorphism (T-RFLP) and capillary electrophoresis single strand conformation polymorphism (CE-SSCP) on the same samples followed the same trend as qPCR data. The main added value of qPCR was, however, to provide quantitative data for each species whereas only relative abundance could be deduced from T-RFLP and CE-SSCP profiles. Additional value was obtained by applying two further quantitative methods which do not require nucleic acid extraction, total cell counting after SYBR Green staining and metal sulfide oxidation activity measurements via microcalorimetry. Overall, these complementary methods allow for an efficient quantitative microbial community monitoring in various bioleaching operations. PMID:28066365

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis

USGS Publications Warehouse

Chase, D.M.; Elliott, D.G.; Pascho, R.J.

2006-01-01

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.

Validation of Reference Genes for Relative Quantitative Gene Expression Studies in Cassava (Manihot esculenta Crantz) by Using Quantitative Real-Time PCR

PubMed Central

Hu, Meizhen; Hu, Wenbin; Xia, Zhiqiang; Zhou, Xincheng; Wang, Wenquan

2016-01-01

Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes. PMID:27242878

Comparison of Real-Time PCR, Reverse Transcriptase Real-Time PCR, Loop-Mediated Isothermal Amplification, and the FDA Conventional Microbiological Method for the Detection of Salmonella spp. in Produce ▿ †

PubMed Central

Zhang, Guodong; Brown, Eric W.; González-Escalona, Narjol

2011-01-01

Contamination of foods, especially produce, with Salmonella spp. is a major concern for public health. Several methods are available for the detection of Salmonella in produce, but their relative efficiency for detecting Salmonella in commonly consumed vegetables, often associated with outbreaks of food poisoning, needs to be confirmed. In this study, the effectiveness of three molecular methods for detection of Salmonella in six produce matrices was evaluated and compared to the FDA microbiological detection method. Samples of cilantro (coriander leaves), lettuce, parsley, spinach, tomato, and jalapeno pepper were inoculated with Salmonella serovars at two different levels (105 and <101 CFU/25 g of produce). The inoculated produce was assayed by the FDA Salmonella culture method (Bacteriological Analytical Manual) and by three molecular methods: quantitative real-time PCR (qPCR), quantitative reverse transcriptase real-time PCR (RT-qPCR), and loop-mediated isothermal amplification (LAMP). Comparable results were obtained by these four methods, which all detected as little as 2 CFU of Salmonella cells/25 g of produce. All control samples (not inoculated) were negative by the four methods. RT-qPCR detects only live Salmonella cells, obviating the danger of false-positive results from nonviable cells. False negatives (inhibition of either qPCR or RT-qPCR) were avoided by the use of either a DNA or an RNA amplification internal control (IAC). Compared to the conventional culture method, the qPCR, RT-qPCR, and LAMP assays allowed faster and equally accurate detection of Salmonella spp. in six high-risk produce commodities. PMID:21803916

Quantitative polymerase chain reaction (PCR) assays for a bacterial thiaminase I gene and the thiaminase-producing bacterium Paenibacillus thiaminolyticus.

USGS Publications Warehouse

Richter, C.A.; Wright-Osment, Maureen K.; Zajicek, J.L.; Honeyfield, D.C.; Tillitt, D.E.

2009-01-01

The thiaminase I enzyme produced by the gram-positive bacterium Paenibacillus thiaminolyticus isolated from the viscera of Lake Michigan alewives Alosa pseudoharengus is currently the only defined source of the thiaminase activity linked to thiamine (vitamin B1) deficiency in early mortality syndrome (EMS) in the larvae of Great Lakes salmonines. Diets of alewife or isolated strains of P. thiaminolyticus mixed in a semipurified diet and fed to lake trout Salvelinus namaycush have been shown to produce EMS in fry. We utilized quantitative polymerase chain reaction (Q-PCR) to aid in studies of the sources of P. thiaminolyticus and thiaminase I. Quantitative PCR assays were established to detect the thiaminase I gene of P. thiaminolyticus, the 16S rRNA gene from most species of bacteria, and the 16S rRNA gene specifically from P. thiaminolyticus and a few closely related taxa. The Q-PCR assays are linear over at least six orders of magnitude and can detect the thiaminase I gene of P. thiaminolyticus from as few as 1,000 P. thiaminolyticus cells/g of sample or the Paenibacillus 16S rRNA gene from as few as 100 P. thiaminolyticus cells/g of sample. The initial results from alewife viscera samples with high thiaminase activity yielded unexpectedly low densities of P. thiaminolyticus cells; Paenibacillus thiaminolyticus was detectable in 2 of 6 alewife viscera tested at densities on the order of 100 cells/g out of 100,000,000 total bacterial cells/g. The low numbers of P. thiaminolyticus detected suggest that alewives contain additional non-P. thiaminolyticus sources of thiaminase activity.

NEW TARGET AND CONTROL ASSAYS FOR QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS OF ENTEROCOCCI IN WATER

EPA Science Inventory

Enterococci are frequently monitored in water samples as indicators of fecal pollution. Attention is now shifting from culture based methods for enumerating these organisms to more rapid molecular methods such as QPCR. Accurate quantitative analyses by this method requires highly...

Interlaboratory Comparison of Real-time PCR Protocols 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 proto...

Detection of Campylobacter jejuni in Lizard Faeces from Central Australia Using Quantitative PCR

PubMed Central

Whiley, Harriet; McLean, Ryan; Ross, Kirstin

2016-01-01

Worldwide, Campylobacter is a significant cause of gastrointestinal illness. It is predominately considered a foodborne pathogen, with human exposure via non-food transmission routes generally overlooked. Current literature has been exploring environmental reservoirs of campylobacteriosis including potential wildlife reservoirs. Given the close proximity between lizards and human habitats in Central Australia, this study examined the presence of Campylobacter jejuni from lizard faeces collected from this region. Of the 51 samples collected, 17 (33%) (this included 14/46 (30%) wild and 3/5 (60%) captive lizard samples) were positive for C. jejuni using quantitative PCR (qPCR). This was the first study to investigate the presence of C. jejuni in Australian lizards. This has public health implications regarding the risk of campylobacteriosis from handling of pet reptiles and through cross-contamination or contact with wild lizard faeces. Additionally this has implication for horizontal transmission via lizards of C. jejuni to food production farms. Further research is needed on this environmental reservoir and potential transmission routes to reduce the risk to public health. PMID:28025556

Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica.

PubMed

Wilbur, Ami E; Ford, Susan E; Gauthier, Julie D; Gomez-Chiarri, Marta

2012-12-27

The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.

[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.

Standardization of enterococci density estimates by EPA qPCR methods and comparison of beach action value exceedances in river waters with culture methods

EPA Science Inventory

The U.S.EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 Recreational Water Quality Criteria (RWQC). The CCE quantification unit stems from the calibration model used to ...

Multiplex Droplet Digital PCR Protocols for Quantification of GM Maize Events.

PubMed

Dobnik, David; Spilsberg, Bjørn; Bogožalec Košir, Alexandra; Štebih, Dejan; Morisset, Dany; Holst-Jensen, Arne; Žel, Jana

2018-01-01

The standard-curve based simplex quantitative polymerase chain reaction (qPCR) has been the gold standard for DNA target quantification for more than a decade. The large and growing number of individual analyses needed to test for genetically modified organisms (GMOs) is reducing the cost-effectiveness of qPCR. Droplet digital PCR (ddPCR) enables absolute quantification without standard curves, avoids the amplification efficiency bias observed with qPCR, allows more accurate estimations at low target copy numbers and, in combination with multiplexing, significantly improves cost efficiency. Here we describe two protocols for multiplex quantification of GM maize events: (1) nondiscriminating, with multiplex quantification of targets as a group (12 GM maize lines) and (2) discriminating, with multiplex quantification of individual targets (events). The first enables the quantification of twelve European Union authorized GM maize events as a group with only two assays, but does not permit determination of the individual events present. The second protocol enables the quantification of four individual targets (three GM events and one endogene) in a single reaction. Both protocols can be modified for quantification of any other DNA target.

Seasonal Dynamics of Microcystis spp. and Their Toxigenicity as Assessed by qPCR in a Temperate Reservoir

PubMed Central

Martins, António; Moreira, Cristiana; Vale, Micaela; Freitas, Marisa; Regueiras, Ana; Antunes, Agostinho; Vasconcelos, Vitor

2011-01-01

Blooms of toxic cyanobacteria are becoming increasingly frequent, mainly due to water quality degradation. This work applied qPCR as a tool for early warning of microcystin(MC)-producer cyanobacteria and risk assessment of water supplies. Specific marker genes for cyanobacteria, Microcystis and MC-producing Microcystis, were quantified to determine the genotypic composition of the natural Microcystis population. Correlations between limnological parameters, pH, water temperature, dissolved oxygen and conductivity and MC concentrations as well as Microcystis abundance were assessed. A negative significant correlation was observed between toxic (with mcy genes) to non-toxic (without mcy genes) genotypes ratio and the overall Microcystis density. The highest proportions of toxic Microcystis genotypes were found 4–6 weeks before and 8–10 weeks after the peak of the bloom, with the lowest being observed at its peak. These results suggest positive selection of non-toxic genotypes under favorable environmental growth conditions. Significant positive correlations could be found between quantity of toxic genotypes and MC concentration, suggesting that the method applied can be useful to predict potential MC toxicity risk. No significant correlation was found between the limnological parameters measured and MC concentrations or toxic genotypes proportions indicating that other abiotic and biotic factors should be governing MC production and toxic genotypes dynamics. The qPCR method here applied is useful to rapidly estimate the potential toxicity of environmental samples and so, it may contribute to the more efficient management of water use in eutrophic systems. PMID:22072994

A randomized and blinded comparison of qPCR and NGS-based detection of aneuploidy in a cell line mixture model of blastocyst biopsy mosaicism.

PubMed

Goodrich, David; Tao, Xin; Bohrer, Chelsea; Lonczak, Agnieszka; Xing, Tongji; Zimmerman, Rebekah; Zhan, Yiping; Scott, Richard T; Treff, Nathan R

2016-11-01

A subset of preimplantation stage embryos may possess mosaicism of chromosomal constitution, representing a possible limitation to the clinical predictive value of comprehensive chromosome screening (CCS) from a single biopsy. However, contemporary methods of CCS may be capable of predicting mosaicism in the blastocyst by detecting intermediate levels of aneuploidy within a trophectoderm biopsy. This study evaluates the sensitivity and specificity of aneuploidy detection by two CCS platforms using a cell line mixture model of a mosaic trophectoderm biopsy. Four cell lines with known karyotypes were obtained and mixed together at specific ratios of six total cells (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0). A female euploid and a male trisomy 18 cell line were used for one set, and a male trisomy 13 and a male trisomy 15 cell line were used for another. Replicates of each mixture were prepared, randomized, and blinded for analysis by one of two CCS platforms (quantitative polymerase chain reaction (qPCR) or VeriSeq next-generation sequencing (NGS)). Sensitivity and specificity of aneuploidy detection at each level of mosaicism was determined and compared between platforms. With the default settings for each platform, the sensitivity of qPCR and NGS were not statistically different, and 100 % specificity was observed (no false positives) at all levels of mosaicism. However, the use of previously published custom criteria for NGS increased sensitivity but also significantly decreased specificity (33 % false-positive prediction of aneuploidy). By demonstrating increased false-positive diagnoses when reducing the stringency of predicting an abnormality, these data illustrate the importance of preclinical evaluation of new testing paradigms before clinical implementation.

Reduced-cost Chlamydia trachomatis-specific multiplex real-time PCR diagnostic assay evaluated for ocular swabs and use by trachoma research programmes.

PubMed

Butcher, Robert; Houghton, Jo; Derrick, Tamsyn; Ramadhani, Athumani; Herrera, Beatriz; Last, Anna R; Massae, Patrick A; Burton, Matthew J; Holland, Martin J; Roberts, Chrissy H

2017-08-01

Trachoma, caused by the intracellular bacterium Chlamydia trachomatis (Ct), is the leading infectious cause of preventable blindness. Many commercial platforms are available that provide highly sensitive and specific detection of Ct DNA. However, the majority of these commercial platforms are inaccessible for population-level surveys in resource-limited settings typical to trachoma control programmes. We developed two low-cost quantitative PCR (qPCR) tests for Ct using readily available reagents on standard real-time thermocyclers. Each multiplex qPCR test targets one genomic and one plasmid Ct target in addition to an endogenous positive control for Homo sapiens DNA. The quantitative performance of the qPCR assays in clinical samples was determined by comparison to a previously evaluated droplet digital PCR (ddPCR) test. The diagnostic performance of the qPCR assays were evaluated against a commercial assay (artus C. trachomatis Plus RG PCR, Qiagen) using molecular diagnostics quality control standards and clinical samples. We examined the yield of Ct DNA prepared from five different DNA extraction kits and a cold chain-free dry-sample preservation method using swabs spiked with fixed concentrations of human and Ct DNA. The qPCR assay was highly reproducible (Ct plasmid and genomic targets mean total coefficients of variance 41.5% and 48.3%, respectively). The assay detected 8/8 core specimens upon testing of a quality control panel and performed well in comparison to commercially marketed comparator test (sensitivity and specificity>90%). Optimal extraction and sample preservation methods for research applications were identified. We describe a pipeline from collection to diagnosis providing the most efficient sample preservation and extraction with significant per test cost savings over a commercial qPCR diagnostic assay. The assay and its evaluation should allow control programs wishing to conduct independent research within the context of trachoma control

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...

MRPrimerW: a tool for rapid design of valid high-quality primers for multiple target qPCR experiments

PubMed Central

Kim, Hyerin; Kang, NaNa; An, KyuHyeon; Koo, JaeHyung; Kim, Min-Soo

2016-01-01

Design of high-quality primers for multiple target sequences is essential for qPCR experiments, but is challenging due to the need to consider both homology tests on off-target sequences and the same stringent filtering constraints on the primers. Existing web servers for primer design have major drawbacks, including requiring the use of BLAST-like tools for homology tests, lack of support for ranking of primers, TaqMan probes and simultaneous design of primers against multiple targets. Due to the large-scale computational overhead, the few web servers supporting homology tests use heuristic approaches or perform homology tests within a limited scope. Here, we describe the MRPrimerW, which performs complete homology testing, supports batch design of primers for multi-target qPCR experiments, supports design of TaqMan probes and ranks the resulting primers to return the top-1 best primers to the user. To ensure high accuracy, we adopted the core algorithm of a previously reported MapReduce-based method, MRPrimer, but completely redesigned it to allow users to receive query results quickly in a web interface, without requiring a MapReduce cluster or a long computation. MRPrimerW provides primer design services and a complete set of 341 963 135 in silico validated primers covering 99% of human and mouse genes. Free access: http://MRPrimerW.com. PMID:27154272

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.

[Evaluation of cytomegalovirus quantification in blood by the R-gene real-time PCR test].

PubMed

Marque-Juillet, S; Touzard, A; Monnier, S; Fernand-Laurent, C; Therby, A; Rigaudeau, S; Harzic, M

2010-04-01

Diagnosing the presence of cytomegalovirus (CMV) in the blood of immunodepressed patients is often done by quantitative polymerase chain reaction (Q-PCR) even though the reference method remains the antigenemia pp65 (Ag-pp65) test. To define the predictive value of the Q-PCR in the diagnosis of CMV disease and assess treatment efficacy using the CMV R-gene test. To compare the Q-PCR results and feasibility with those of the Ag-pp65 test. The Q-PCR was performed in 34 whole blood samples (frozen at -80 degrees C until use) from five patients diagnosed with CMV disease, defined as the presence of clinical signs and Ag-pp65 in the nuclei of more than two cells. After extraction, viral DNA was quantified in each sample using the Q-PCR CMV R-gene kit according to the manufacturer's instructions. Immediately after blood was drawn, the Ag-pp65 test had been performed in 32 samples using CINAkit (Argene). The 16 samples positive by the Ag-pp65 test were also positive by PCR; six samples negative by the Ag-pp65 test were positive by PCR; and the remaining 10 samples were negative by both techniques. During treatment, the two markers' kinetics were similar. The CMV R-gene test has a predictive value as good as that of the Ag-pp65 test but is fast and easier to use. A prospective study with a greater number of patients is needed to define the prediction threshold for CMV disease. Copyright 2009 Elsevier Masson SAS. All rights reserved.

3D printing and milling a real-time PCR device for infectious disease diagnostics.

PubMed

Mulberry, Geoffrey; White, Kevin A; Vaidya, Manjusha; Sugaya, Kiminobu; Kim, Brian N

2017-01-01

Diagnosing infectious diseases using quantitative polymerase chain reaction (qPCR) offers a conclusive result in determining the infection, the strain or type of pathogen, and the level of infection. However, due to the high-cost instrumentation involved and the complexity in maintenance, it is rarely used in the field to make a quick turnaround diagnosis. In order to provide a higher level of accessibility than current qPCR devices, a set of 3D manufacturing methods is explored as a possible option to fabricate a low-cost and portable qPCR device. The key advantage of this approach is the ability to upload the digital format of the design files on the internet for wide distribution so that people at any location can simply download and feed into their 3D printers for quick manufacturing. The material and design are carefully selected to minimize the number of custom parts that depend on advanced manufacturing processes which lower accessibility. The presented 3D manufactured qPCR device is tested with 20-μL samples that contain various concentrations of lentivirus, the same type as HIV. A reverse-transcription step is a part of the device's operation, which takes place prior to the qPCR step to reverse transcribe the target RNA from the lentivirus into complementary DNA (cDNA). This is immediately followed by qPCR which quantifies the target sequence molecules in the sample during the PCR amplification process. The entire process of thermal control and time-coordinated fluorescence reading is automated by closed-loop feedback and a microcontroller. The resulting device is portable and battery-operated, with a size of 12 × 7 × 6 cm3 and mass of only 214 g. By uploading and sharing the design files online, the presented low-cost qPCR device may provide easier access to a robust diagnosis protocol for various infectious diseases, such as HIV and malaria.

3D printing and milling a real-time PCR device for infectious disease diagnostics

PubMed Central

Mulberry, Geoffrey; White, Kevin A.; Vaidya, Manjusha; Sugaya, Kiminobu

2017-01-01

Diagnosing infectious diseases using quantitative polymerase chain reaction (qPCR) offers a conclusive result in determining the infection, the strain or type of pathogen, and the level of infection. However, due to the high-cost instrumentation involved and the complexity in maintenance, it is rarely used in the field to make a quick turnaround diagnosis. In order to provide a higher level of accessibility than current qPCR devices, a set of 3D manufacturing methods is explored as a possible option to fabricate a low-cost and portable qPCR device. The key advantage of this approach is the ability to upload the digital format of the design files on the internet for wide distribution so that people at any location can simply download and feed into their 3D printers for quick manufacturing. The material and design are carefully selected to minimize the number of custom parts that depend on advanced manufacturing processes which lower accessibility. The presented 3D manufactured qPCR device is tested with 20-μL samples that contain various concentrations of lentivirus, the same type as HIV. A reverse-transcription step is a part of the device’s operation, which takes place prior to the qPCR step to reverse transcribe the target RNA from the lentivirus into complementary DNA (cDNA). This is immediately followed by qPCR which quantifies the target sequence molecules in the sample during the PCR amplification process. The entire process of thermal control and time-coordinated fluorescence reading is automated by closed-loop feedback and a microcontroller. The resulting device is portable and battery-operated, with a size of 12 × 7 × 6 cm3 and mass of only 214 g. By uploading and sharing the design files online, the presented low-cost qPCR device may provide easier access to a robust diagnosis protocol for various infectious diseases, such as HIV and malaria. PMID:28586401

Implementation of Novel Design Features for qPCR-Based eDNA Assessment

PubMed Central

Veldhoen, Nik; Hobbs, Jared; Ikonomou, Georgios; Hii, Michael; Lesperance, Mary; Helbing, Caren C.

2016-01-01

Environmental stewardship requires timely, accurate information related to the status of a given ecosystem and the species that occupy it. Recent advances in the application of the highly sensitive real-time quantitative polymerase chain reaction (qPCR) towards identification of constituents within environmental DNA (eDNA) now allow targeted detection of the presence of species-specific biological material within a localized geographic region. However, as with all molecular techniques predicated on the specificity and sensitivity of the PCR assay, careful validation of each eDNA qPCR assay in development must be performed both under controlled laboratory conditions and when challenged with field-derived eDNA samples. Such a step-wise approach forms the basis for incorporation of innovative qPCR design features that strengthen the implementation and interpretation of the eDNA assay. This includes empirical determination that the qPCR assay is refractory to the presence of human DNA and the use of a tripartite assay approach comprised of 1) a primer set targeting plant chloroplast that evaluates the presence of amplifiable DNA from field samples to increase confidence in a negative result, 2) an animal group primer set to increase confidence in the assay result, and 3) a species-specific primer set to assess presence of DNA from the target species. To demonstrate this methodology, we generated eDNA assays specific for the North American bullfrog (Lithobates (Rana) catesbeiana) and the Rocky Mountain tailed frog (Ascaphus montanus) and characterized each with respect to detection sensitivity and specificity with demonstrated performance in a field survey scenario. The qPCR design features presented herein address specific challenges of eDNA assays thereby increasing their interpretative power. PMID:27802293

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 the efficiency of nested q-PCR in the detection of Mycobacterium tuberculosis complex directly from tuberculosis-suspected lesions in post-mortem macroscopic inspections of bovine carcasses slaughtered in the state of Mato Grosso, Brazil.

PubMed

Carvalho, Ricardo César Tavares; Furlanetto, Leone Vinícius; Maruyama, Fernanda Harumy; Araújo, Cristina Pires de; Barros, Sílvia Letícia Bomfim; Ramos, Carlos Alberto do Nascimento; Dutra, Valéria; Araújo, Flábio Ribeiro de; Paschoalin, Vânia Margaret Flosi; Nakazato, Luciano; Figueiredo, Eduardo Eustáquio de Souza

2015-08-01

Bovine tuberculosis (BTB) is a zoonotic disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). The quick and specific detection of this species is of extreme importance, since BTB may cause economic impacts, in addition to presenting imminent risks to human health. In the present study a nested real-time PCR test (nested q-PCR) was used in post-mortem evaluations to assess cattle carcasses with BTB-suspected lesions. A total of 41,193 cattle slaughtered in slaughterhouses located in the state of Mato Grosso, were examined. Of the examined animals, 198 (0.48%) showed BTB-suspected lesions. M. bovis was isolated in 1.5% (3/198) of the samples. Multiplex-PCR detected MTC in 7% (14/198) of the samples. The nested q-PCR test detected MTC in 28% (56/198) of the BTB-suspected lesions, demonstrating higher efficiency when compared to the multiplex-PCR and conventional microbiology. Nested q-PCR can therefore be used as a complementary test in the national program for control and eradication of bovine tuberculosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Detection of Brettanomyces spp. in red wines using real-time PCR.

PubMed

Tofalo, Rosanna; Schirone, Maria; Corsetti, Aldo; Suzzi, Giovanna

2012-09-01

The question if the "Brett character" is a favorable wine attribute is one of the most controversial issues and it is currently addressed by many researches. Actually, the presence of Brettanomyces/Dekkera in wine during barrel aging is often associated to detrimental organoleptic characteristics depending on the release of volatile phenols (for example, 4-ethylphenol and 4-ethylguaiacol); for that reason the possibility to rapidly detect the yeast at the early stage of wine production could allow preventive actions to reduce wine spoilage. In this work, 25 and 5 samples from conventional and organic vineyards, respectively, all suspected to be spoiled by Brettanomyces/Dekkera spp., were analyzed using both culture-dependent and culture-independent techniques. In particular, a DNA extraction protocol and a real-time quantitative PCR (qPCR) assay to directly detect and quantify B. bruxellensis were optimized. Results showed that B. bruxellensis was present in 22 of 30 samples, ranging from 10 to 10(4) CFU/mL, lower values being found in organic wines (10 to 10(2) CFU/mL). Overall, qPCR was proved to be a useful and valuable wine control system, since 12 samples were recorded as positive for yeast presence when analyzed by qPCR and negative in case of plate count analyses. Brettanomyces cells were detected using a qPCR method, optimized in this study, which allows to obtain results quickly. © 2012 Institute of Food Technologists®

Data on regulation of the gene for the adipocyte-enriched micropeptide Adig/Smaf1 by qPCR analysis and luciferase reporter assay.

PubMed

Ren, Gang; Cairl, Nicholas; Kim, Ji Young; Smas, Cynthia M

2016-12-01

This article describes qPCR analysis for the Adig/Smaf1 gene in multiple in vitro adipocyte differentiation models including white and brown adipogenesis, cell lines and primary cultures. The article also contains qPCR data for transcript levels of Adig/Smaf1 in a wide panel of murine tissues. Expression of Adig/Smaf1 transcript in white and brown adipose tissue in fasted and refed mice is reported and also data for Adig/Smaf1 transcript expression in genetically obese ob/ob mice. Data on the effects of siRNA-mediated knockdown of Srebp1c on Adig/Smaf1 transcript levels in 3T3-L1 adipocytes are shown. Luciferase reporter assays provide data for regulation of an ~ 2 kb fragment of the 5' flanking region of Adig/Smaf1 gene by PPARγ/RXRα. This data is related to a research article describing Adig/Smaf1 protein expression, "Expression, regulation and functional assessment of the 80 amino acid Small Adipocyte Factor 1 (Smaf1) protein in adipocytes" (G. Ren, P. Eskandari, S. Wang, C.M. Smas, 2016) [1].

Differentiation of five body fluids from forensic samples by expression analysis of four microRNAs using quantitative PCR.

PubMed

Sauer, Eva; Reinke, Ann-Kathrin; Courts, Cornelius

2016-05-01

Applying molecular genetic approaches for the identification of forensically relevant body fluids, which often yield crucial information for the reconstruction of a potential crime, is a current topic of forensic research. Due to their body fluid specific expression patterns and stability against degradation, microRNAs (miRNA) emerged as a promising molecular species, with a range of candidate markers published. The analysis of miRNA via quantitative Real-Time PCR, however, should be based on a relevant strategy of normalization of non-biological variances to deliver reliable and biologically meaningful results. The herein presented work is the as yet most comprehensive study of forensic body fluid identification via miRNA expression analysis based on a thoroughly validated qPCR procedure and unbiased statistical decision making to identify single source samples. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG™ cell line as a rapid and reliable tool for improved in vitro hazard assessment.

PubMed

Ates, Gamze; Mertens, Birgit; Heymans, Anja; Verschaeve, Luc; Milushev, Dimiter; Vanparys, Philippe; Roosens, Nancy H C; De Keersmaecker, Sigrid C J; Rogiers, Vera; Doktorova, Tatyana Y

2018-04-01

Although the value of the regulatory accepted batteries for in vitro genotoxicity testing is recognized, they result in a high number of false positives. This has a major impact on society and industries developing novel compounds for pharmaceutical, chemical, and consumer products, as afflicted compounds have to be (prematurely) abandoned or further tested on animals. Using the metabolically competent human HepaRG ™ cell line and toxicogenomics approaches, we have developed an upgraded, innovative, and proprietary gene classifier. This gene classifier is based on transcriptomic changes induced by 12 genotoxic and 12 non-genotoxic reference compounds tested at sub-cytotoxic concentrations, i.e., IC10 concentrations as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The resulting gene classifier was translated into an easy-to-handle qPCR array that, as shown by pathway analysis, covers several different cellular processes related to genotoxicity. To further assess the predictivity of the tool, a set of 5 known positive and 5 known negative test compounds for genotoxicity was evaluated. In addition, 2 compounds with debatable genotoxicity data were tested to explore how the qPCR array would classify these. With an accuracy of 100%, when equivocal results were considered positive, the results showed that combining HepaRG ™ cells with a genotoxin-specific qPCR array can improve (geno)toxicological hazard assessment. In addition, the developed qPCR array was able to provide additional information on compounds for which so far debatable genotoxicity data are available. The results indicate that the new in vitro tool can improve human safety assessment of chemicals in general by basing predictions on mechanistic toxicogenomics information.

Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vesper, Stephen; McKinstry, Craig A.; Hartmann, Chris

2007-11-28

A method is described to discriminate between live and dead cells of the infectious fungi Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Mucor racemosus, Rhizopus stolonifer and Paecilomyces variotii. To test the method, conidial suspensions were heat inactivated at 85 °C or held at 5 °C (controls) for 1 h. Polycarbonate filters (25 mm diameter, 0.8 μm pore size) were placed on "welled" slides (14 mm diameter) and the filters treated with either PBS or PMA. Propidium monoazide (PMA), which enters dead cells but not live cells, was incubated with cell suspensions, exposed to blue wavelength light-emitting diodes (LED) to inactivatemore » remaining PMA and secure intercalation of PMAwith DNA of dead cells. Treated cells were extracted and the live and dead cells evaluated with quantitative PCR (QPCR). After heat treatment and DNA modification with PMA, all fungal species tested showed an approximate 100- to 1000-fold difference in cell viability estimated by QPCR analysis which was consistent with estimates of viability based on culturing.« less

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.

Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus) by a Quantitative PCR (qPCR) Assay.

PubMed

Jarvi, Susan I; Pitt, William C; Farias, Margaret E; Shiels, Laura; Severino, Michael G; Howe, Kathleen M; Jacquier, Steven H; Shiels, Aaron B; Amano, Karis K; Luiz, Blaine C; Maher, Daisy E; Allison, Maureen L; Holtquist, Zachariah C; Scheibelhut, Neil T

2015-01-01

The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that causes human eosinophilic meningitis and ocular angiostrongyliasis characteristic of rat lungworm (RLW) disease. Definitive diagnosis is made by finding and identifying A. cantonensis larvae in the cerebral spinal fluid or by using a custom immunological or molecular test. This study was conducted to determine if genomic DNA from A. cantonensis is detectable by qPCR in the blood or tissues of experimentally infected rats. F1 offspring from wild rats were subjected to experimental infection with RLW larvae isolated from slugs, then blood or tissue samples were collected over multiple time points. Blood samples were collected from 21 rats throughout the course of two trials (15 rats in Trial I, and 6 rats in Trial II). In addition to a control group, each trial had two treatment groups: the rats in the low dose (LD) group were infected by approximately 10 larvae and the rats in the high dose (HD) group were infected with approximately 50 larvae. In Trial I, parasite DNA was detected in cardiac bleed samples from five of five LD rats and five of five HD rats at six weeks post-infection (PI), and three of five LD rats and five of five HD rats from tail tissue. In Trial II, parasite DNA was detected in peripheral blood samples from one of two HD rats at 53 minutes PI, one of two LD rats at 1.5 hours PI, one of two HD rats at 18 hours PI, one of two LD rats at five weeks PI and two of two at six weeks PI, and two of two HD rats at weeks five and six PI. These data demonstrate that parasite DNA can be detected in peripheral blood at various time points throughout RLW infection in rats.

Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR.

PubMed

Kim, Jeong-Soon; Wang, Nian

2009-03-06

Citrus Huanglongbing (HLB) is one of the most devastating diseases on citrus and is associated with Candidatus Liberibacter spp.. The pathogens are phloem limited and have not been cultured in vitro. The current management strategy of HLB is to remove infected citrus trees and reduce psyllid populations with insecticides to prevent the spreading. This strategy requires sensitive and reliable diagnostic methods for early detection. We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR) indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA. Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.

Development of a quantitative PCR assay for rapid detection of Lactobacillus plantarum and Lactobacillus fermentum in cocoa bean fermentation.

PubMed

Schwendimann, Livia; Kauf, Peter; Fieseler, Lars; Gantenbein-Demarchi, Corinne; Miescher Schwenninger, Susanne

2015-08-01

To monitor dominant species of lactic acid bacteria during cocoa bean fermentation, i.e. Lactobacillus plantarum and Lactobacillus fermentum, a fast and reliable culture-independent qPCR assay was developed. A modified DNA isolation procedure using a commercial kit followed by two species-specific qPCR assays resulted in 100% sensitivity for L. plantarum and L. fermentum. Kruskal-Wallis and post-hoc analyses of data obtained from experiments with cocoa beans that were artificially spiked with decimal concentrations of L. plantarum and L. fermentum strains allowed the calculation of a regression line suitable for the estimation of both species with a detection limit of 3 to 4 Log cells/g cocoa beans. This process was successfully tested for efficacy through the analyses of samples from laboratory-scale cocoa bean fermentations with both the qPCR assay and a culture-dependent method which resulted in comparable results. Copyright © 2015 Elsevier B.V. All rights reserved.

A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

PubMed Central

Rodríguez-Lázaro, David; Pla, Maria; Scortti, Mariela; Monzó, Héctor J.; Vázquez-Boland, José A.

2005-01-01

We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R2) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure. PMID:16332910