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Sample records for diagnostic real-time pcr

  1. Real-time PCR in Food Science: PCR Diagnostics.

    PubMed

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control. PMID:23513039

  2. Real-time PCR in Food Science: PCR Diagnostics.

    PubMed

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

  3. Overcoming inhibition in real-time diagnostic PCR.

    PubMed

    Hedman, Johannes; Rådström, Peter

    2013-01-01

    PCR is an important and powerful tool in several fields, including clinical diagnostics, food analysis, and forensic analysis. In theory, PCR enables the detection of one single cell or DNA molecule. However, the presence of PCR inhibitors in the sample affects the amplification efficiency of PCR, thus lowering the detection limit, as well as the precision of sequence-specific nucleic acid quantification in real-time PCR. In order to overcome the problems caused by PCR inhibitors, all the steps leading up to DNA amplification must be optimized for the sample type in question. Sampling and sample treatment are key steps, but most of the methods currently in use were developed for conventional diagnostic methods and not for PCR. Therefore, there is a need for fast, simple, and robust sample preparation methods that take advantage of the accuracy of PCR. In addition, the thermostable DNA polymerases and buffer systems used in PCR are affected differently by inhibitors. During recent years, real-time PCR has developed considerably and is now widely used as a diagnostic tool. This technique has greatly improved the degree of automation and reduced the analysis time, but has also introduced a new set of PCR inhibitors, namely those affecting the fluorescence signal. The purpose of this chapter is to view the complexity of PCR inhibition from different angles, presenting both molecular explanations and practical ways of dealing with the problem. Although diagnostic PCR brings together scientists from different diagnostic fields, end-users have not fully exploited the potential of learning from each other. Here, we have collected knowledge from archeological analysis, clinical diagnostics, environmental analysis, food analysis, and forensic analysis. The concept of integrating sampling, sample treatment, and the chemistry of PCR, i.e., pre-PCR processing, will be addressed as a general approach to overcoming real-time PCR inhibition and producing samples optimal for PCR

  4. Lab-on-a-chip PCR: real time PCR in miniaturized format for HLA diagnostics

    NASA Astrophysics Data System (ADS)

    Gaertner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Sewart, René; Frank, Rainer; Willems, Andreas

    2014-05-01

    In case of transplantation or the identification of special metabolic diseases like coeliac disease, HLA typing has to be done fast and reliably with easy-to-handle devices by using limited amount of sample. Against this background a lab-on-a-chip device was realized enabling a fast HLA typing via miniaturized Real-time PCR. Hereby, two main process steps were combined, namely the extraction of DNA from whole blood and the amplification of the target DNA by Real-time PCR giving rise-to a semi-quantitative analysis. For the implementation of both processes on chip, a sample preparation and a real-time module were used. Sample preparation was carried out by using magnetic beads that were stored directly on chip as dry powder, together with all lysis reagents. After purification of the DNA by applying a special buffer regime, the sample DNA was transferred into the PCR module for amplification and detection. Coping with a massively increased surface-to-volume ratio, which results in a higher amount of unspecific binding on the chip surface, special additives needed to be integrated to compensate for this effect. Finally the overall procedure showed a sensitivity comparable to standard Real-time PCR but reduced the duration of analysis to significantly less than one hour. The presented work demonstrates that the combination of lab-on-a-chip PCR with direct optical read-out in a real-time fashion is an extremely promising tool for molecular diagnostics.

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

    PubMed

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

    2010-09-01

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

  6. Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Evrard, A.; Boulle, N.; Lutfalla, G. S.

    Over the past few years there has been a considerable development of DNA amplification by polymerase chain reaction (PCR), and real-time PCR has now superseded conventional PCR techniques in many areas, e.g., the quantification of nucleic acids and genotyping. This new approach is based on the detection and quantification of a fluorescent signal proportional to the amount of amplicons generated by PCR. Real-time detection is achieved by coupling a thermocycler with a fluorimeter. This chapter discusses the general principles of quantitative real-time PCR, the different steps involved in implementing the technique, and some examples of applications in medicine. The polymerase chain reaction (PCR) provides a way of obtaining a large number of copies of a double-stranded DNA fragment of known sequence. This DNA amplification technique, developed in 1985 by K. Mullis (Cetus Corporation), saw a spectacular development over the space of a few years, revolutionising the methods used up to then in molecular biology. Indeed, PCR has many applications, such as the detection of small amounts of DNA, cloning, and quantitative analysis (assaying), each of which will be discussed further below.

  7. Diagnostic Real-Time PCR for Detection of Salmonella in Food

    PubMed Central

    Malorny, Burkhard; Paccassoni, Elisa; Fach, Patrick; Bunge, Cornelia; Martin, Annett; Helmuth, Reiner

    2004-01-01

    A robust 5′ nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 103 CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 104 CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method. PMID:15574899

  8. Harmonization of Bordetella pertussis real-time PCR diagnostics in the United States in 2012.

    PubMed

    Williams, Margaret M; Taylor, Thomas H; Warshauer, David M; Martin, Monte D; Valley, Ann M; Tondella, M Lucia

    2015-01-01

    Real-time PCR (rt-PCR) is an important diagnostic tool for the identification of Bordetella pertussis, Bordetella holmesii, and Bordetella parapertussis. Most U.S. public health laboratories (USPHLs) target IS481, present in 218 to 238 copies in the B. pertussis genome and 32 to 65 copies in B. holmesii. The CDC developed a multitarget PCR assay to differentiate B. pertussis, B. holmesii, and B. parapertussis and provided protocols and training to 19 USPHLs. The 2012 performance exercise (PE) assessed the capability of USPHLs to detect these three Bordetella species in clinical samples. Laboratories were recruited by the Wisconsin State Proficiency Testing program through the Association of Public Health Laboratories, in partnership with the CDC. Spring and fall PE panels contained 12 samples each of viable Bordetella and non-Bordetella species in saline. Fifty and 53 USPHLs participated in the spring and fall PEs, respectively, using a variety of nucleic acid extraction methods, PCR platforms, and assays. Ninety-six percent and 94% of laboratories targeted IS481 in spring and fall, respectively, in either singleplex or multiplex assays. In spring and fall, respectively, 72% and 79% of USPHLs differentiated B. pertussis and B. holmesii and 68% and 72% identified B. parapertussis. IS481 cycle threshold (CT) values for B. pertussis samples had coefficients of variation (CV) ranging from 10% to 28%. Of the USPHLs that differentiated B. pertussis and B. holmesii, sensitivity was 96% and specificity was 95% for the combined panels. The 2012 PE demonstrated increased harmonization of rt-PCR Bordetella diagnostic protocols in USPHLs compared to that of the previous survey.

  9. Evaluation of real-time PCR for Strongyloides stercoralis and hookworm as diagnostic tool in asymptomatic schoolchildren in Cambodia.

    PubMed

    Schär, Fabian; Odermatt, Peter; Khieu, Virak; Panning, Marcus; Duong, Socheat; Muth, Sinuon; Marti, Hanspeter; Kramme, Stefanie

    2013-05-01

    Diagnosis of soil-transmitted helminths such as Strongyloides stercoralis and hookworms (Ancylostoma duodenale and Necator americanus) is challenging due to irregular larval and egg output in infected individuals and insensitive conventional diagnostic procedures. Sensitive novel real-time PCR assays have been developed. Our study aimed to evaluate the real-time PCR assays as a diagnostic tool for detection of Strongyloides spp. and hookworms in a random stool sample of 218 asymptomatic schoolchildren in Cambodia. Overall prevalence of 17.4% (38/218) and 34.9% (76/218) were determined by real-time PCR for S. stercoralis and hookworms, respectively. Sensitivity and specificity of S. stercoralis specific real-time PCR as compared to the combination of Baermann/Koga Agar as gold standard were 88.9% and 92.7%, respectively. For hookworm specific real-time PCR a sensitivity of 78.9% and specificity of 78.9% were calculated. Co-infections were detectable by PCR in 12.8% (28/218) of individuals. S. stercoralis real-time PCR applied in asymptomatic cases showed a lower sensitivity compared to studies undertaken with symptomatic patients with the same molecular tool, yet it proved to be a valid supplement in the diagnosis of STH infection in Cambodia.

  10. Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

    PubMed Central

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

    2010-01-01

    Background 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. Methodology/Principal Findings 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 ϕA1122 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. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 103 CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl 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, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. Conclusions/Significance 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. PMID:20596528

  11. Accurate, fast and cost-effective diagnostic test for monosomy 1p36 using real-time quantitative PCR.

    PubMed

    Cunha, Pricila da Silva; Pena, Heloisa B; D'Angelo, Carla Sustek; Koiffmann, Celia P; Rosenfeld, Jill A; Shaffer, Lisa G; Stofanko, Martin; Gonçalves-Dornelas, Higgor; Pena, Sérgio Danilo Junho

    2014-01-01

    Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5-0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs.

  12. Accurate, Fast and Cost-Effective Diagnostic Test for Monosomy 1p36 Using Real-Time Quantitative PCR

    PubMed Central

    Cunha, Pricila da Silva; Pena, Heloisa B.; D'Angelo, Carla Sustek; Koiffmann, Celia P.; Rosenfeld, Jill A.; Shaffer, Lisa G.; Stofanko, Martin; Gonçalves-Dornelas, Higgor; Pena, Sérgio Danilo Junho

    2014-01-01

    Monosomy 1p36 is considered the most common subtelomeric deletion syndrome in humans and it accounts for 0.5–0.7% of all the cases of idiopathic intellectual disability. The molecular diagnosis is often made by microarray-based comparative genomic hybridization (aCGH), which has the drawback of being a high-cost technique. However, patients with classic monosomy 1p36 share some typical clinical characteristics that, together with its common prevalence, justify the development of a less expensive, targeted diagnostic method. In this study, we developed a simple, rapid, and inexpensive real-time quantitative PCR (qPCR) assay for targeted diagnosis of monosomy 1p36, easily accessible for low-budget laboratories in developing countries. For this, we have chosen two target genes which are deleted in the majority of patients with monosomy 1p36: PRKCZ and SKI. In total, 39 patients previously diagnosed with monosomy 1p36 by aCGH, fluorescent in situ hybridization (FISH), and/or multiplex ligation-dependent probe amplification (MLPA) all tested positive on our qPCR assay. By simultaneously using these two genes we have been able to detect 1p36 deletions with 100% sensitivity and 100% specificity. We conclude that qPCR of PRKCZ and SKI is a fast and accurate diagnostic test for monosomy 1p36, costing less than 10 US dollars in reagent costs. PMID:24839341

  13. Real-time PCR detection chemistry.

    PubMed

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

    2015-01-15

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

  14. Diagnostic real-time RT-PCR for the simultaneous detection of Citrus exocortis viroid and Hop stunt viroid.

    PubMed

    Papayiannis, Lambros C

    2014-02-01

    Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd) are two important viroids known to infect several plant species worldwide. In this study, a real-time reverse transcription (RT) TaqMan polymerase chain reaction (PCR) assay was developed and optimized for the simultaneous detection of CEVd and HSVd. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Two different RNA extraction methods and one rapid crude template preparation procedure were compared in terms of extraction purity and efficiency for PCR applications. Extraction method Q included a commercially available kit, whereas method C was a modified chloroform-phase extraction in house protocol. Procedure S involved blotting the sap extract on a positively charged nylon membrane and elution. The multiplex RT-TaqMan PCR assay successfully discriminated the two viroid species from all reference samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional RT-PCR tests, the overall Dse and Dsp were lower and estimated at 94 and 95% for CEVd, and 97 and 98% for HSVd, respectively. In a direct comparison, the developed assay presented 1000-fold more analytical sensitivity. Spectrophotometric results showed that RNA extraction methods Q and C, yielded the purest RNA, and gave the lowest mean Ct values. Alternative template preparation method S resulted in Ct values statistically similar to those obtained with methods Q to C when tested by RT-TaqMan PCR. The developed assay, using crude template preparation S, allows the simple, accurate and cost-effective testing of a large number of plant samples, and can be applied in surveys and certification schemes.

  15. Evaluation of Three Diagnostic Methods, Including Real-Time PCR, for Detection of Dientamoeba fragilis in Stool Specimens

    PubMed Central

    Stark, D.; Beebe, N.; Marriott, D.; Ellis, J.; Harkness, J.

    2006-01-01

    Dientamoeba fragilis is a protozoan parasite of humans that infects the mucosa of the large intestine and is associated with gastrointestinal disease. We developed a 5′ nuclease (TaqMan)-based real-time PCR assay, targeting the small subunit rRNA gene, for the detection of D. fragilis in human stool specimens and compared its sensitivity and specificity to conventional PCR and microscopic examination by a traditional modified iron-hematoxylin staining procedure. Real-time PCR exhibited 100% sensitivity and specificity. PMID:16390978

  16. Evaluation of inhibitor-resistant real-time PCR methods for diagnostics in clinical and environmental samples.

    PubMed

    Trombley Hall, Adrienne; McKay Zovanyi, Ashley; Christensen, Deanna Rose; Koehler, Jeffrey William; Devins Minogue, Timothy

    2013-01-01

    Polymerase chain reaction (PCR) is commonly used for pathogen detection in clinical and environmental samples. These sample matrices often contain inhibitors of PCR, which is a primary reason for sample processing; however, the purification process is highly inefficient, becoming unacceptable at lower signature concentrations. One potential solution is direct PCR assessment without sample processing. Here, we evaluated nine inhibitor-resistant PCR reagents for direct detection of Francisella tularensis in seven different clinical and environmental samples using an established real-time PCR assay to assess ability to overcome PCR inhibition. While several of these reagents were designed for standard PCR, the described inhibitor resistant properties (ex. Omni Klentaq can amplify target DNA samples of up to 20% whole blood or soil) led to our evaluation with real-time PCR. A preliminary limit of detection (LOD) was determined for each chemistry in whole blood and buffer, and LODs (20 replicates) were determined for the top five chemistries in each matrix (buffer, whole blood, sputum, stool, swab, soil, and sand). Not surprisingly, no single chemistry performed the best across all of the different matrices evaluated. For instance, Phusion Blood Direct PCR Kit, Phire Hot Start DNA polymerase, and Phire Hot Start DNA polymerase with STR Boost performed best for direct detection in whole blood while Phire Hot Start DNA polymerase with STR Boost were the only reagents to yield an LOD in the femtogram range for soil. Although not the best performer across all matrices, KAPA Blood PCR kit produced the most consistent results among the various conditions assessed. Overall, while these inhibitor resistant reagents show promise for direct amplification of complex samples by real-time PCR, the amount of template required for detection would not be in a clinically relevant range for most matrices. PMID:24040090

  17. Performance of a new gelled nested PCR test for the diagnosis of imported malaria: comparison with microscopy, rapid diagnostic test, and real-time PCR.

    PubMed

    Iglesias, Nuria; Subirats, Mercedes; Trevisi, Patricia; Ramírez-Olivencia, Germán; Castán, Pablo; Puente, Sabino; Toro, Carlos

    2014-07-01

    Microscopy and rapid diagnostic tests (RDTs) are the techniques commonly used for malaria diagnosis but they are usually insensitive at very low levels of parasitemia. Nested PCR is commonly used as a reference technique in the diagnosis of malaria due to its high sensitivity and specificity. However, it is a cumbersome assay only available in reference centers. We evaluated a new nested PCR-based assay, BIOMALAR kit (Biotools B&M Labs, Madrid, Spain) which employs ready-to-use gelled reagents and allows the identification of the main four species of Plasmodium. Blood samples were obtained from patients with clinical suspicion of malaria. A total of 94 subjects were studied. Fifty-two (55.3%) of them were malaria-infected subjects corresponding to 48 cases of Plasmodium falciparum, 1 Plasmodium malariae, 2 Plasmodium vivax, and 1 Plasmodium ovale. The performance of the BIOMALAR test was compared with microscopy, rapid diagnostic test (RDT) (BinaxNOW® Malaria) and real-time quantitative PCR (qPCR). The BIOMALAR test showed a sensitivity of 98.1% (95% confidence interval [CI], 89.7-100), superior to microscopy (82.7% [95% CI, 69.7-91.8]) and RDT (94.2% [95% CI, 84.1-98.8]) and similar to qPCR (100% [95% CI, 93.2-100]). In terms of specificity, the BIOMALAR assay showed the same value as microscopy and qPCR (100% [95% CI, 93.2-100]). Nine subjects were submicroscopic carriers of malaria. The BIOMALAR test identified almost all of them (8/9) in comparison with RDT (6/9) and microscopy (0/9). In conclusion, the BIOMALAR is a PCR-based assay easy to use with an excellent performance and especially useful for diagnosis submicroscopic malaria. PMID:24770719

  18. Application of real time PCR for diagnosis of Swine Dysentery.

    PubMed

    Akase, Satoru; Uchitani, Yumi; Sohmura, Yoshiko; Tatsuta, Keikichi; Sadamasu, Kenji; Adachi, Yoshikazu

    2009-03-01

    Evaluation of a genetic diagnostic technique using real time PCR of Swine Dysentery (SD) was performed using nox primers. Culture, ordinary PCR and real time PCR were compared in this experiment. Sixty-seven specimens from pigs with clinical signs of SD brought to a slaughterhouse in Shibaura, Tokyo, were used. B. hyodysenteriae was isolated from 49 of the pigs, was detected by ordinary PCR in 49 of the pigs and was detected by real time PCR in 54 of the pigs. Furthermore, we were able to determine the numbers of B. hyodysenteriae cells in all positive specimens by real time PCR. The rapid diagnostic technique established in this experiment was useful for detection of B. hyodysenteriae because it was more effective than ordinary PCR and culture.

  19. The Power of Real-Time PCR

    ERIC Educational Resources Information Center

    Valasek, Mark A.; Repa, Joyce J.

    2005-01-01

    In recent years, real-time polymerase chain reaction (PCR) has emerged as a robust and widely used methodology for biological investigation because it can detect and quantify very small amounts of specific nucleic acid sequences. As a research tool, a major application of this technology is the rapid and accurate assessment of changes in gene…

  20. Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens

    PubMed Central

    Fabian, Andrew W.; Barrette, Roger W.; Sayed, Abu

    2016-01-01

    Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates. PMID:26757142

  1. Real-Time Reverse Transcription PCR Assay for Detection of Senecavirus A in Swine Vesicular Diagnostic Specimens.

    PubMed

    Bracht, Alexa J; O'Hearn, Emily S; Fabian, Andrew W; Barrette, Roger W; Sayed, Abu

    2016-01-01

    Senecavirus A (SV-A), formerly, Seneca Valley virus (SVV), has been detected in swine with vesicular lesions and is thought to be associated with swine idiopathic vesicular disease (SIVD), a vesicular disease syndrome that lacks a defined causative agent. The clinical presentation of SIVD resembles that of other more contagious and economically devastating vesicular diseases, such as foot-and-mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS), that typically require immediate rule out diagnostics to lift restrictions on animal quarantine, movement, and trade. This study presents the development of a sensitive, SYBR Green RT-qPCR assay suitable for detection of SV-A in diagnostic swine specimens. After testing 50 pigs with clinical signs consistent with vesicular disease, 44 (88%) were found to be positive for SV-A by RT-qPCR as compared to none from a negative cohort of 35 animals without vesicular disease, indicating that the assay is able to successfully detect the virus in an endemic population. SV-A RNA was also detectable at a low level in sera from a subset of pigs that presented with (18%) or without (6%) vesicular signs. In 2015, there has been an increase in the occurrence of SV-A in the US, and over 200 specimens submitted to our laboratory for vesicular investigation have tested positive for the virus using this method. SV-A RNA was detectable in all common types of vesicular specimens including swabs and tissue from hoof lesions, oral and snout epithelium, oral swabs, scabs, and internal organ tissues such as liver and lymph node. Genome sequencing analysis from recent virus isolates was performed to confirm target amplicon specificity and was aligned to previous isolates. PMID:26757142

  2. Real-time PCR in Food Science: Introduction.

    PubMed

    Rodriguez-Lazaro, David; Hernandez, Marta

    2013-01-01

    Food safety and quality control programmes are increasingly applied throughout the production food chain in order to guarantee added value products as well as to minimize the risk of infection for the consumer. The development of real-time PCR has represented one of the most significant advances in food diagnostics as it provides rapid, reliable and quantitative results. These aspects become increasingly important for the agricultural and food industry. Different strategies for real-time PCR diagnostics have been developed including unspecific detection independent of the target sequence using fluorescent dyes such as SYBR Green, or by sequence-specific fluorescent oligonucleotide probes such as TaqMan probes or molecular beacons.

  3. Novel multiplex real-time PCR diagnostic assay for identification and differentiation of Mycobacterium tuberculosis, Mycobacterium canettii, and Mycobacterium tuberculosis complex strains.

    PubMed

    Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Maher, Majella; van Soolingen, Dick; Barry, Thomas

    2011-02-01

    Tuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosis complex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosis and M. canettii. The lepA gene was targeted for the detection of members of the MTC, the wbbl1 gene was used for the differentiation of M. tuberculosis and M. canettii from the remainder of the complex, and a unique region of the M. canettii genome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.

  4. Real-Time PCR for Diagnosing Helicobacter pylori Infection in Patients with Upper Gastrointestinal Bleeding: Comparison with Other Classical Diagnostic Methods

    PubMed Central

    Saez, Jesús; Belda, Sofía; Santibáñez, Miguel; Sola-Vera, Javier; Galiana, Antonio; Ruiz-García, Montserrat; Brotons, Alicia; López-Girona, Elena; Girona, Eva; Sillero, Carlos; Royo, Gloria

    2012-01-01

    The aim of this study was to determine the diagnostic usefulness of quantification of the H. pylori genome in detection of infection in patients with upper gastrointestinal bleeding (UGB). A total of 158 consecutive patients with digestive disorders, 80 of whom had clinical presentation of UGB, were studied. The number of microorganisms was quantified using a real-time PCR system which amplifies the urease gene with an internal control for eliminating the false negatives. A biopsy sample from the antrum and corpus of each patient was processed. The rapid urease test, culture, histological study, stool antigen test, and breath test were done. The gold standard was a positive culture or positive results in at least two of the other techniques. When a positive result was defined as any number of microorganisms/human cell, the sensitivity of real-time PCR was greater in bleeding patients, especially in the gastric corpus: 68.4% (95% confidence interval [CI], 52.3 to 84.5%) in non-UGB patients versus 91.5% (95% CI, 79.6 to 97.6%) in UGB patients. When a positive result was defined as a number of microorganisms/human cell above the optimal value that maximizes the Youden index (>3.56 microorganisms/human cell in the antrum and >2.69 in the corpus), the sensitivity and specificity in UGB patients were over 80% in both antrum and corpus. Our findings suggest that some bleeding patients with infection caused by H. pylori may not be correctly diagnosed by classical methods, and such patients could benefit from the improved diagnosis provided by real-time PCR. However, the clinical significance of a small number of microorganisms in patients with negative results in classical tests should be evaluated. PMID:22837325

  5. The clinical diagnostic accuracy of rapid detection of healthcare-associated bloodstream infection in intensive care using multipathogen real-time PCR technology

    PubMed Central

    Dunn, Graham; Chadwick, Paul; Young, Duncan; Bentley, Andrew; Carlson, Gordon; Warhurst, Geoffrey

    2011-01-01

    Background There is growing interest in the potential utility of real-time PCR in diagnosing bloodstream infection by detecting pathogen DNA in blood samples within a few hours. SeptiFast is a multipathogen probe-based real-time PCR system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection and has European regulatory approval. The SeptiFast pathogen panel is suited to identifying healthcare-associated bloodstream infection acquired during complex healthcare, and the authors report here the protocol for the first detailed health-technology assessment of multiplex real-time PCR in this setting. Methods/design A Phase III multicentre double-blinded diagnostic study will determine the clinical validity of SeptiFast for the rapid detection of healthcare-associated bloodstream infection, against the current service standard of microbiological culture, in an adequately sized population of critically ill adult patients. Results from SeptiFast and standard microbiological culture procedures in each patient will be compared at study conclusion and the metrics of clinical diagnostic accuracy of SeptiFast determined in this population setting. In addition, this study aims to assess further the preliminary evidence that the detection of pathogen DNA in the bloodstream using SeptiFast may have value in identifying the presence of infection elsewhere in the body. Furthermore, differences in circulating immune-inflammatory markers in patient groups differentiated by the presence/absence of culturable pathogens and pathogen DNA will help elucidate further the patho-physiology of infection developing in the critically ill. Ethics and dissemination Ethical approval has been granted by the North West 6 Research Ethics Committee (09/H1003/109). Based on the results of this first non-commercial study, independent recommendations will be made to The Department of Health (open-access health technology

  6. Cost-effectiveness of diagnostic strategies using quantitative real-time PCR and bacterial culture to identify contagious mastitis cases in large dairy herds.

    PubMed

    Murai, Kiyokazu; Lehenbauer, Terry W; Champagne, John D; Glenn, Kathy; Aly, Sharif S

    2014-03-01

    Diagnostic strategies to detect contagious mastitis caused by Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae in dairy herds during an outbreak have been minimally studied with regard to cost and diagnostic sensitivity. The objective of this cross-sectional study was to compare the cost-effectiveness of diagnostic strategies for identification of infected cows in two California dairy herds during contagious mastitis outbreaks. M. bovis was investigated in a subset of a herd (n=1210 cows) with an estimated prevalence of 2.8% (95% CI=1.9, 3.7), whereas Staph. aureus and Strep. agalactiae were studied in a second herd (n=351 cows) with an estimated prevalence of 3.4% (95% CI=1.5, 5.3) and 16.8% (95% CI=12.9, 20.7), respectively. Diagnostic strategies involved a combination of testing stages that utilized bacterial culture, quantitative real-time PCR (qPCR), or both. Strategies were applied to individual or pooled samples of 5, 10, 50 or 100 samples. Culture was considered the gold standard for sensitivity estimation of each strategy. The reference strategy was the strategy with the lowest cost per culture-positive cow which for both M. bovis and Strep. agalactiae consisted of 2 stages, culture of samples in pools of 5 followed by culture of individual samples in positive pools with a sensitivity of 73.5% (95% CI: 55.6, 87.1) and 96.6% (95% CI: 27.7, 84.8), respectively. The reference strategy for Staph. aureus consisted of 3 stages, culture of individual samples in pools of 100 (stage 1), culture constituents of those positive from stage 1 in pools of 5 (stage 2), culture constituents of those positive from stage 2 individually (stage 3) which resulted in a sensitivity of 58.3% (95% CI: 88.3, 99.6). The most cost-effective alternative to the reference strategy was whole herd milk culture for all 3 pathogens. QPCR testing was a component of the second most cost-effective alternative for M. bovis and the third most cost-effective alternatives for

  7. Influence of reagents formulation on real-time PCR parameters.

    PubMed

    Burgos, J S; Ramírez, C; Tenorio, R; Sastre, I; Bullido, M J

    2002-08-01

    Real-time polymerase chain reaction (PCR) techniques are increasingly used to quantify target sequences for diagnostic and research purposes. Due to its 'quantitative' character, it is very important to determine the variability of this technique correlating with several experimental conditions. The objective of this study was to analyse the effect of manufacturing lots of PCR reagents on two main PCR parameters, specificity and sensitivity. For this study, we used four different amplicons, using either mouse genomic DNA or viral DNA. Although a PCR product could be obtained in any of the conditions, we observed that there are relevant variations in sensitivity depending on the reagents formulation. We conclude that different lots of reagents may determine the analytical performance of PCR assays indicating that reagents testing are of special importance when the PCR protocol is used for quantitative purposes.

  8. Bayesian estimation of the diagnostic accuracy of a multiplex real-time PCR assay and bacteriological culture for 4 common bovine intramammary pathogens.

    PubMed

    Paradis, M-È; Haine, D; Gillespie, B; Oliver, S P; Messier, S; Comeau, J; Scholl, D T

    2012-11-01

    Bacteriological culture (BC) is the traditional method for intramammary infection diagnosis but lacks sensitivity and is time consuming. Multiplex real-time PCR (mr-PCR) enables testing the presence of several bacteria and reduces diagnosis time. Our objective was to estimate bacterial species-specific sensitivity (Se) and specificity of both BC and mr-PCR tests for detecting bacteria in milk samples from clinical mastitis cases and from apparently normal quarters, using a Bayesian latent class model. Milk samples from 1,014 clinical mastitis cases and 1,495 samples from apparently normal quarters were analyzed by BC and mr-PCR. Two positive culture definitions were used: ≥1 cfu/0.01 mL and ≥10 cfu/0.01 mL of the specified bacteria. The mr-PCR was designed to simultaneously detect Staphylococcus aureus, Streptococcus uberis, Escherichia coli, and Streptococcus agalactiae. The priors used in our Bayesian model were weakly informative, with BC priors using the best available error data. Results were compared with those obtained using uniform priors for mr-PCR to test robustness. Weak and uniform priors gave about the same posterior distributions except for Strep. uberis from normal quarters and Strep. agalactiae. Multiplex real-time PCR Se on milk from clinical mastitis were lower than mr-PCR Se on milk from normal quarters. Multiplex real-time PCR Se was higher than BC on milk from normal quarters. Multiplex real-time PCR Se was generally lower than BC on milk from clinical mastitis and it varied by clinical severity. The estimate specificities of detection for all pathogens were ≥99%, regardless of sample type. The effect of milk sample preservation before testing was evaluated and may have been a factor that affected our observed results. A significant association was observed between sample age and mr-PCR results leading to reduced detection of E. coli and Strep. agalactiae in nonclinical samples. Differences in sample age between conduct of BC and of mr-PCR

  9. Polymeric LabChip Real-Time PCR as a Point-of-Care-Potential Diagnostic Tool for Rapid Detection of Influenza A/H1N1 Virus in Human Clinical Specimens

    PubMed Central

    Song, Hyun-Ok; Kim, Je-Hyoung; Ryu, Ho-Sun; Lee, Dong-Hoon; Kim, Sun-Jin; Kim, Deog-Joong; Suh, In Bum; Choi, Du Young; In, Kwang-Ho; Kim, Sung-Woo; Park, Hyun

    2012-01-01

    It is clinically important to be able to detect influenza A/H1N1 virus using a fast, portable, and accurate system that has high specificity and sensitivity. To achieve this goal, it is necessary to develop a highly specific primer set that recognizes only influenza A viral genes and a rapid real-time PCR system that can detect even a single copy of the viral gene. In this study, we developed and validated a novel fluidic chip-type real-time PCR (LabChip real-time PCR) system that is sensitive and specific for the detection of influenza A/H1N1, including the pandemic influenza strain A/H1N1 of 2009. This LabChip real-time PCR system has several remarkable features: (1) It allows rapid quantitative analysis, requiring only 15 min to perform 30 cycles of real-time PCR. (2) It is portable, with a weight of only 5.5 kg. (3) The reaction cost is low, since it uses disposable plastic chips. (4) Its high efficiency is equivalent to that of commercially available tube-type real-time PCR systems. The developed disposable LabChip is an economic, heat-transferable, light-transparent, and easy-to-fabricate polymeric chip compared to conventional silicon- or glass-based labchip. In addition, our LabChip has large surface-to-volume ratios in micro channels that are required for overcoming time consumed for temperature control during real-time PCR. The efficiency of the LabChip real-time PCR system was confirmed using novel primer sets specifically targeted to the hemagglutinin (HA) gene of influenza A/H1N1 and clinical specimens. Eighty-five human clinical swab samples were tested using the LabChip real-time PCR. The results demonstrated 100% sensitivity and specificity, showing 72 positive and 13 negative cases. These results were identical to those from a tube-type real-time PCR system. This indicates that the novel LabChip real-time PCR may be an ultra-fast, quantitative, point-of-care-potential diagnostic tool for influenza A/H1N1 with a high sensitivity and specificity

  10. Quantitative Real-Time PCR: Recent Advances.

    PubMed

    Singh, Charanjeet; Roy-Chowdhuri, Sinchita

    2016-01-01

    Quantitative real-time polymerase chain reaction is a technique for simultaneous amplification and product quantification of a target DNA as the process takes place in real time in a "closed-tube" system. Although this technique can provide an absolute quantification of the initial template copy number, quantification relative to a control sample or second sequence is typically adequate. The quantification process employs melting curve analysis and/or fluorescent detection systems and can provide amplification and genotyping in a relatively short time. Here we describe the properties and uses of various fluorescent detection systems used for quantification. PMID:26843055

  11. Quantitative Real-Time PCR: Recent Advances.

    PubMed

    Singh, Charanjeet; Roy-Chowdhuri, Sinchita

    2016-01-01

    Quantitative real-time polymerase chain reaction is a technique for simultaneous amplification and product quantification of a target DNA as the process takes place in real time in a "closed-tube" system. Although this technique can provide an absolute quantification of the initial template copy number, quantification relative to a control sample or second sequence is typically adequate. The quantification process employs melting curve analysis and/or fluorescent detection systems and can provide amplification and genotyping in a relatively short time. Here we describe the properties and uses of various fluorescent detection systems used for quantification.

  12. Real-time PCR for detection of Strongyloides stercoralis in human stool samples from Côte d'Ivoire: diagnostic accuracy, inter-laboratory comparison and patterns of hookworm co-infection.

    PubMed

    Becker, Sören L; Piraisoody, Nivetha; Kramme, Stefanie; Marti, Hanspeter; Silué, Kigbafori D; Panning, Marcus; Nickel, Beatrice; Kern, Winfried V; Herrmann, Mathias; Hatz, Christoph F; N'Goran, Eliézer K; Utzinger, Jürg; von Müller, Lutz

    2015-10-01

    Human infections with the helminth species Strongyloides stercoralis encompass a wide clinical spectrum, ranging from asymptomatic carriage to life-threatening disease. The diagnosis of S. stercoralis is cumbersome and the sensitivity of conventional stool microscopy is low. New molecular tools have been developed to increase sensitivity. We compared the diagnostic accuracy of real-time PCR with microscopy for the detection of S. stercoralis and hookworm in human stool samples, and investigated the inter-laboratory agreement of S. stercoralis-specific real-time PCR in two European laboratories. Stool specimens from 256 randomly selected individuals in rural Côte d'Ivoire were examined using three microscopic techniques (i.e. Kato-Katz, Koga agar plate (KAP) and Baermann (BM)). Additionally, ethanol-fixed stool aliquots were subjected to molecular diagnosis. The prevalence of S. stercoralis and hookworm infection was 21.9% and 52.0%, respectively, whilst co-infections were detected in 35 (13.7%) participants. The diagnostic agreement between real-time PCR and microscopy was excellent when both KAP and BM tested positive for S. stercoralis, but was considerably lower when only one microscopic technique was positive. The sensitivity of KAP, BM and real-time PCR for detection of S. stercoralis as compared to a combination of all diagnostic techniques was 21.4%, 37.5% and 76.8%, respectively. The inter-laboratory agreement of S. stercoralis-specific PCR was substantial (κ=0.63, p<0.001). We conclude that a combination of real-time PCR and stool microscopy shows high accuracy for S. stercoralis diagnosis. Besides high sensitivity, PCR may also enhance specificity by reducing microscopic misdiagnosis of morphologically similar helminth larvae (i.e. hookworm and S. stercoralis) in settings where both helminth species co-exist.

  13. Bartonellae in domestic and stray cats from Israel: comparison of bacterial cultures and high-resolution melt real-time PCR as diagnostic methods.

    PubMed

    Gutiérrez, Ricardo; Morick, Danny; Gross, Ifat; Winkler, Ronen; Abdeen, Ziad; Harrus, Shimon

    2013-12-01

    To determine the occurrence of feline bartonellosis in Israel, blood samples were collected from 179 stray and 155 domestic cats from 18 cities or villages in central and northcentral Israel. Samples were screened for Bartonella infection by culture isolation and molecular detection using high-resolution melt (HRM) real-time PCR assay targeting the 16S-23S rRNA internal transcribed spacer (ITS). All positive samples were confirmed by two additional HRM real-time PCR assays targeting two fragments of the β-subunit of RNA polymerase (rpoB) and the 16S rRNA genes. The prevalence of Bartonella spp. infection in the general tested population was 25.1% (84/334). A higher prevalence was detected in the stray (30.7%; 55/179) than the domestic cats (18.7%; 29/155). Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae were highly prevalent in both cat populations, however their distribution among the two populations varied significantly (p=0.016). B. clarridgeiae and B. koehlerae were found to be more prevalent in stray than domestic cats, whereas B. henselae was evenly distributed. Co-infection with two or more different Bartonella spp. was determined in 2.1% (7) of the cats. The ITS HRM real-time PCR assay used in this study was shown to have a greater screening power than bacterial isolation, detecting 94.0% (79/84) compared to 35.7% (30/84), respectively, of all positive samples. The high prevalence of these zoonotic Bartonella species, coupled with the overpopulation of stray cats, and increased numbers of domestic cats in the major urban centers in Israel represent a significant threat for the public health in this country.

  14. [Epidemics of schistosomiasis in military staff assigned to endemic areas: standard diagnostic techniques and the development of real-time PCR techniques].

    PubMed

    Biance-Valero, E; De Laval, F; Delerue, M; Savini, H; Cheinin, S; Leroy, P; Soullié, B

    2013-05-01

    The authors report the results of molecular biology techniques for the early diagnosis of cases (invasion phase) of schistosomiasis during two epidemics occurring during French military projects in the Central African Republic and Madagascar. The use of these techniques in real time for subjects not residing in the endemic area significantly improves the sensitivity of screening. The attack rates of these episodes, according to a case definition that took positive specific PCR results into account, were 59% and 26%. These results are a concrete illustration of the proverb that "yaws begin where the trail stops".

  15. Real time PV manufacturing diagnostic system

    SciTech Connect

    Kochergin, Vladimir; Crawford, Michael A.

    2015-09-01

    The main obstacle Photovoltaic (PV) industry is facing at present is the higher cost of PV energy compared to that of fossil energy. While solar cell efficiencies continue to make incremental gains these improvements are so far insufficient to drive PV costs down to match that of fossil energy. Improved in-line diagnostics however, has the potential to significantly increase the productivity and reduce cost by improving the yield of the process. On this Phase I/Phase II SBIR project MicroXact developed and demonstrated at CIGS pilot manufacturing line a high-throughput in-line PV manufacturing diagnostic system, which was verified to provide fast and accurate data on the spatial uniformity of thickness, an composition of the thin films comprising the solar cell as the solar cell is processed reel-to-reel. In Phase II project MicroXact developed a stand-alone system prototype and demonstrated the following technical characteristics: 1) ability of real time defect/composition inconsistency detection over 60cm wide web at web speeds up to 3m/minute; 2) Better than 1mm spatial resolution on 60cm wide web; 3) an average better than 20nm spectral resolution resulting in more than sufficient sensitivity to composition imperfections (copper-rich and copper-poor regions were detected). The system was verified to be high vacuum compatible. Phase II results completely validated both technical and economic feasibility of the proposed concept. MicroXact’s solution is an enabling technique for in-line PV manufacturing diagnostics to increase the productivity of PV manufacturing lines and reduce the cost of solar energy, thus reducing the US dependency on foreign oil while simultaneously reducing emission of greenhouse gasses.

  16. Real-Time Quantitative PCR for Human Herpesvirus 6 DNA

    PubMed Central

    Locatelli, Giuseppe; Santoro, Fabio; Veglia, Fabrizio; Gobbi, Alberto; Lusso, Paolo; Malnati, Mauro S.

    2000-01-01

    The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection. We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids. The test uses a 5′ nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system. The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 106 viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule. The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions. Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys. PMID:11060066

  17. Multiplex real-time PCR assay for Legionella species.

    PubMed

    Kim, Seung Min; Jeong, Yoojung; Sohn, Jang Wook; Kim, Min Ja

    2015-12-01

    Legionella pneumophila serogroup 1 (sg1) accounts for the majority of infections in humans, but other Legionella species are also associated with human disease. In this study, a new SYBR Green I-based multiplex real-time PCR assay in a single reaction was developed to allow the rapid detection and differentiation of Legionella species by targeting specific gene sequences. Candidate target genes were selected, and primer sets were designed by referring to comparative genomic hybridization data of Legionella species. The Legionella species-specific groES primer set successfully detected all 30 Legionella strains tested. The xcpX and rfbA primers specifically detected L. pneumophila sg1-15 and L. pneumophila sg1, respectively. In addition, this assay was validated by testing clinical samples and isolates. In conclusion, this novel multiplex real-time PCR assay might be a useful diagnostic tool for the rapid detection and differentiation of Legionella species in both clinical and epidemiological studies.

  18. Lab-on-a-chip enabled HLA diagnostic: combined sample preparation and real time PCR for HLA-B57 diagnosis

    NASA Astrophysics Data System (ADS)

    Gärtner, Claudia; Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Schattschneider, Sebastian; Frank, Rainer; Willems, Andreas

    2015-05-01

    The diverse human HLA (human leukocyte antigen) system is responsible for antigen presentation and recognition. It is essential for the immune system to maintain a stable defense line, but also is also involved in autoimmunity as well as metabolic disease. HLA-haplotype (HLA-B27), for instance, is associated with inflammatory diseases such as Bechterew's disease. The administration of the HIV drug Abacavir in combination with another HLA-haplotype (HLAB57) is associated with severe hypersensitivity reactions. Accordingly, the HLA status has to be monitored for diagnosis or prior to start of therapy. Along this line, a miniaturized microfluidic platform has been developed allowing performing the complete analytical process from "sample-in" to "answer-out" in a point-of-care environment. The main steps of the analytical cascade inside the integrated system are blood cell lysis and DNA isolation, DNA purification, real-time PCR and quantitative monitoring of the rise of a fluorescent signal appearing during the PCR based sequence amplification. All bio-analytical steps were intended to be performed inside one chip and will be actuated, controlled and monitored by a matching device. This report will show that all required processes are established and tested and all device components work well and interact with the functional modules on the chips in a harmonized fashion.

  19. Identification of CD70 as a diagnostic biomarker for clear cell renal cell carcinoma by gene expression profiling, real-time RT-PCR and immunohistochemistry.

    PubMed

    Diegmann, Julia; Junker, Kerstin; Gerstmayer, Bernhard; Bosio, Andreas; Hindermann, Winfried; Rosenhahn, Julia; von Eggeling, Ferdinand

    2005-08-01

    The underlying molecular mechanisms of renal cell carcinoma (RCC) are poorly understood and more reliable markers for early diagnosis are needed. Hence, alternative strategies for biomarker discovery with appropriate validation technologies have to be performed. To elucidate genesis and progression of RCC we used high parallel chip based gene expression profiling comparing normal and tumour tissues. We compared corresponding control and tumour tissue samples from 10 patients with clear cell RCC. We isolated RNA from histologically well characterised tissue sections and performed reverse transcription, labelling and linear RNA amplification. Samples were hybridised on microarrays containing 642 human cDNAs. Of the 352 differentially expressed genes found, CD70 and FRA2 were selected for further evaluation by real-time RT-PCR. The analysis all showed a high potential to discriminate between normal and tumour tissue. Moreover, increased CD70 mRNA expression in tumour cells could be correlated to its expression at the protein level. Immunohistochemistry (IHC) showed very strong expression of CD70 in all tumour samples but no expression in adjacent normal kidney tissue. With our combined approach we were able to identify CD70 as a new marker for RCC, which may be useful in the future for improved immunohistochemical diagnosis. PMID:16043348

  20. [Real time PCR methodology for quantification of nucleic acids].

    PubMed

    Tse, C; Capeau, J

    2003-01-01

    The polymerase chain reaction (PCR) has become an essential tool for molecular biologists and its introduction into nucleic acids detection systems has revolutionized the quantitative analysis of DNA and RNA. The technique has rapidly evolved over the last few years and the growing interest in quantitative applications of the PCR has favoured the development of real-time quantitative PCR. In this paper, we review, after presentation of the theorical aspects of PCR, the basic principles of real-time PCR with the introduction of the concept of threshold cycle. More precisely, we describe the novel assay formats that greatly simplify the protocols used for the detection of specific nucleic acids. We focus on the actual four technologies that enable sequence detection in a closed tube and that are SYBR Green I, TaqMan probes, Hybridization probes and Molecular Beacon probes. We then discuss the different quantification strategies in real time PCR and compare the competiting instruments on the market. The most important real-time PCR applications in clinical biology are also described.

  1. Evaluation of tcdB real-time PCR in a three-step diagnostic algorithm for detection of toxigenic Clostridium difficile.

    PubMed

    Larson, Ann M; Fung, Angela M; Fang, Ferric C

    2010-01-01

    Clostridium difficile is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic C. difficile remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the tcdB gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for C. difficile glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified "gold standard" that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic C. difficile was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic C. difficile, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic C. difficile that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of

  2. Evaluation of tcdB Real-Time PCR in a Three-Step Diagnostic Algorithm for Detection of Toxigenic Clostridium difficile▿

    PubMed Central

    Larson, Ann M.; Fung, Angela M.; Fang, Ferric C.

    2010-01-01

    Clostridium difficile is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic C. difficile remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the tcdB gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for C. difficile glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified “gold standard” that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic C. difficile was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic C. difficile, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic C. difficile that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of

  3. Diagnostic performance and application of a real-time PCR assay for the detection of Salmonella in fecal samples collected from hospitalized horses with or without signs of gastrointestinal tract disease.

    PubMed

    Ekiri, A B; Long, M T; Hernandez, J A

    2016-02-01

    The main objective of this study was to assess the diagnostic performance of a real-time polymerase chain reaction (PCR) assay for the detection of Salmonella in fecal samples collected from hospitalized horses with or without signs of gastrointestinal (GI) tract disease. The PCR assay used primers and a probe that targeted the invA gene of Salmonella. Assuming a sensitivity of 100% and a specificity of 96.6%, and a disease prevalence of 2%, 5%, and 10-15% in study horses, the PCR assay had a high (100%) negative predictive value, and a positive predictive value that ranged from 37% in horses without signs of GI disease that tested Salmonella culture-negative, to 60% in horses with signs of GI disease that tested Salmonella culture-negative, to 76-83% in horses with signs of GI disease that tested Salmonella culture-positive. This study provides evidence that the real-time PCR that targets the Salmonella invA gene can be used as a screening test for the detection of Salmonella in feces of hospitalized horses with signs of GI disease. Horses that test PCR-positive can be tested in series using bacteriologic culture to reduce false positive results or to provide additional data (e.g., antibiogram and serotyping data) that can be used to identify potential nosocomial Salmonella infections.

  4. Monitoring gene expression: quantitative real-time rt-PCR.

    PubMed

    Wagner, Elke M

    2013-01-01

    Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR, or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in real time.RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in a specific tissue or cell type is calculated from the exponential phase of the reaction. Relative quantification or absolute quantification compared to standards that are run in parallel can be performed.This chapter describes the entire procedure from isolation of total RNA from liver and fatty tissues/cells to the use of RT-qPCR to study gene expression in these tissues. We perform relative quantification of transcripts to calculate the fold-difference of a certain mRNA level between different samples. In addition, tips for choosing primers and performing analyses are provided to help the beginner in understanding the technique.

  5. Nanodroplet real-time PCR system with laser assisted heating.

    PubMed

    Kim, Hanyoup; Dixit, Sanhita; Green, Christopher J; Faris, Gregory W

    2009-01-01

    We report the successful application of low-power (approximately 30 mW) laser radiation as an optical heating source for high-speed real-time polymerase chain reaction (PCR) amplification of DNA in nanoliter droplets dispersed in an oil phase. Light provides the heating, temperature measurement, and Taqman real-time readout in nanoliter droplets on a disposable plastic substrate. A selective heating scheme using an infrared laser appears ideal for driving PCR because it heats only the droplet, not the oil or plastic substrate, providing fast heating and completing the 40 cycles of PCR in 370 seconds. No microheaters or microfluidic circuitry were deposited on the substrate, and PCR was performed in one droplet without affecting neighboring droplets. The assay performance was quantitative and its amplification efficiency was comparable to that of a commercial instrument.

  6. Diagnostic Performance of a Multiple Real-Time PCR Assay in Patients with Suspected Sepsis Hospitalized in an Internal Medicine Ward

    PubMed Central

    Pasqualini, Leonella; Leli, Christian; Montagna, Paolo; Cardaccia, Angela; Cenci, Elio; Montecarlo, Ines; Pirro, Matteo; di Filippo, Francesco; Cistaro, Emma; Schillaci, Giuseppe; Bistoni, Francesco; Mannarino, Elmo

    2012-01-01

    Early identification of causative pathogen in sepsis patients is pivotal to improve clinical outcome. SeptiFast (SF), a commercially available system for molecular diagnosis of sepsis based on PCR, has been mostly used in patients hospitalized in hematology and intensive care units. We evaluated the diagnostic accuracy and clinical usefulness of SF, compared to blood culture (BC), in 391 patients with suspected sepsis, hospitalized in a department of internal medicine. A causative pathogen was identified in 85 patients (22%). Sixty pathogens were detected by SF and 57 by BC. No significant differences were found between the two methods in the rates of pathogen detection (P = 0.74), even after excluding 9 pathogens which were isolated by BC and were not included in the SF master list (P = 0.096). The combination of SF and BC significantly improved the diagnostic yield in comparison to BC alone (P < 0.001). Compared to BC, SF showed a significantly lower contamination rate (0 versus 19 cases; P < 0.001) with a higher specificity for pathogen identification (1.00, 95% confidence interval [CI] of 0.99 to 1.00, versus 0.94, 95% CI of 0.90 to 0.96; P = 0.005) and a higher positive predictive value (1.00, 95% CI of 1.00 to 0.92%, versus 0.75, 95% CI of 0.63 to 0.83; P = 0.005). In the subgroup of patients (n = 191) who had been receiving antibiotic treatment for ≥24 h, SF identified more pathogens (16 versus 6; P = 0.049) compared to BC. These results suggest that, in patients with suspected sepsis, hospitalized in an internal medicine ward, SF could be a highly valuable adjunct to conventional BC, particularly in patients under antibiotic treatment. PMID:22322348

  7. Introducing Undergraduate Students to Real-Time PCR

    ERIC Educational Resources Information Center

    Hancock, Dale; Funnell, Alister; Jack, Briony; Johnston, Jill

    2010-01-01

    An experiment is conducted, which in four 3 h laboratory sessions, introduces third year undergraduate Biochemistry students to the technique of real-time PCR in a biological context. The model used is a murine erythroleukemia cell line (MEL cells). These continuously cycling, immature red blood cells, arrested at an early stage in erythropoiesis,…

  8. DETECTION OF FECAL ENTEROCOCCI USING A REAL TIME PCR METHOD

    EPA Science Inventory

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

  9. Species identification in meat products using real-time PCR.

    PubMed

    Jonker, K M; Tilburg, J J H C; Hagele, G H; de Boer, E

    2008-05-01

    One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.

  10. Real-Time PCR for Gene Expression Quantification in Asthma.

    PubMed

    Segundo-Val, Ignacio San; García-Solaesa, Virginia; García-Sánchez, Asunción

    2016-01-01

    The quantitative real-time PCR (qPCR) has become the reference technique for studying gene expression in recent years. The application of qPCR to the study of asthma provides very useful information regarding the gene expression mechanisms. The quantification of RNA from cDNA can be performed by using fluorescent dyes or specific sequence probes. Here, we describe the protocol to quantify gene expression levels using SYBR Green as fluorescent dye. The protocol starts with the RNA extraction, followed by reverse transcription to obtain cDNA, quantification and finally data analysis.

  11. Real-Time PCR for Gene Expression Quantification in Asthma.

    PubMed

    Segundo-Val, Ignacio San; García-Solaesa, Virginia; García-Sánchez, Asunción

    2016-01-01

    The quantitative real-time PCR (qPCR) has become the reference technique for studying gene expression in recent years. The application of qPCR to the study of asthma provides very useful information regarding the gene expression mechanisms. The quantification of RNA from cDNA can be performed by using fluorescent dyes or specific sequence probes. Here, we describe the protocol to quantify gene expression levels using SYBR Green as fluorescent dye. The protocol starts with the RNA extraction, followed by reverse transcription to obtain cDNA, quantification and finally data analysis. PMID:27300530

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

    PubMed

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

    2014-12-01

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

  13. Widespread use of real-time PCR for rickettsial diagnosis.

    PubMed

    Renvoisé, Aurélie; Rolain, Jean-Marc; Socolovschi, Cristina; Raoult, Didier

    2012-02-01

    We report 2 years of experience with rickettsial molecular diagnosis using real-time PCR at the French National Reference Center. All Rickettsia genomes available were compared to discover specific sequences to design new sets of primers and probes. The specificity was verified in silico and against a panel of 30 rickettsial species. Sensitivity was determined using 10-fold serial dilutions. Finally, primers and probes that were both specific and sensitive were routinely used for the diagnosis of rickettsial infections from clinical specimens. We retained sets of primers and probes to detect spotted fever group Rickettsia, typhus group Rickettsia,Rickettsia conorii,Rickettsia slovaca,Rickettsia africae and Rickettsia australis; 643 clinical samples were screened for the presence of Rickettsia DNA. Overall, 45 positive samples were detected, including 15 Rickettsia africae, nine R. conorii, five Rickettsia sibirica mongolitimonae, four R. slovaca, two R. australis, four Rickettsia massiliae, one Rickettsia honei, one Rickettsia typhi and eight Rickettsia sp. Positive samples were detected mainly from cutaneous biopsies and swabs (31/45). Widespread use of real-time PCR is inexpensive and reduces delay in the diagnosis of rickettsial infections. These real-time PCR assays could be implemented easily in laboratories that have molecular facilities and may be added to existing molecular tools as a point-of-care strategy.

  14. Application of Reverse Transcription-PCR and Real-Time PCR in Nanotoxicity Research

    PubMed Central

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2016-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq® DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix. PMID:22975959

  15. PCR and real-time PCR assays to detect fungi of Alternaria alternata species.

    PubMed

    Kordalewska, Milena; Brillowska-Dąbrowska, Anna; Jagielski, Tomasz; Dworecka-Kaszak, Bożena

    2015-01-01

    Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens. PMID:26610309

  16. PCR and real-time PCR assays to detect fungi of Alternaria alternata species.

    PubMed

    Kordalewska, Milena; Brillowska-Dąbrowska, Anna; Jagielski, Tomasz; Dworecka-Kaszak, Bożena

    2015-01-01

    Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens.

  17. Expression profiling by real-time quantitative polymerase chain reaction (RT-qPCR).

    PubMed

    Lech, Maciej; Anders, Hans-Joachim

    2014-01-01

    Real-time quantitative PCR is a variation of the standard PCR technique that is commonly used to quantify nucleic acid. However, in this technique the amount of amplified specific sequence can be quantified at each stage of the PCR cycle. If investigated sequence is present in large number of copies in particular sample, amplification product is detected already in earlier cycles; if the sequence is rare, amplification is observed in later cycles. Quantification of amplified product is acquired using fluorescent probes or fluorescent DNA-binding dyes. Accumulation of fluorescent signal can be measured by real-time PCR instruments during each of 35-45 cycwwles of the PCR reaction, which simplify the procedure by eliminating the visualization of the amplified products using gel electrophoresis. Real-time-PCR allows quantifying the amount of product already during the PCR reaction as soon as it is detectable. Correctly performed, this method may be used for precise gene expression analysis in life science, medicine, and diagnostics and has become the standard method of choice for the quantification of mRNA. However in the past few years it became obvious that real-time PCR is complex and variability of RNA templates, assay designs, inappropriate data normalization, and data interpretation may cause diverse analytical problems.

  18. Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections

    PubMed Central

    Reddington, Kate; Schwenk, Stefan; Tuite, Nina; Platt, Gareth; Davar, Danesh; Coughlan, Helena; Personne, Yoann; Gant, Vanya; Enne, Virve I.; Zumla, Alimuddin

    2015-01-01

    Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens. PMID:26109443

  19. Comparison of Established Diagnostic Methodologies and a Novel Bacterial smpB Real-Time PCR Assay for Specific Detection of Haemophilus influenzae Isolates Associated with Respiratory Tract Infections.

    PubMed

    Reddington, Kate; Schwenk, Stefan; Tuite, Nina; Platt, Gareth; Davar, Danesh; Coughlan, Helena; Personne, Yoann; Gant, Vanya; Enne, Virve I; Zumla, Alimuddin; Barry, Thomas

    2015-09-01

    Haemophilus influenzae is a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzae diagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzae in RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpB gene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilus spp. Subsequently, 44 Haemophilus clinical isolates were collected, and 36 isolates were identified as H. influenzae using a gold standard methodology that combined the results of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and a fucK diagnostic assay. Using the novel smpB diagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n = 98) were blindly tested with the gold standard and smpB diagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpB assay when used directly on respiratory specimens. PMID:26109443

  20. Authentication of Atlantic cod (Gadus morhua) using real time PCR.

    PubMed

    Herrero, Beatriz; Madriñán, María; Vieites, Juan M; Espiñeira, Montserrat

    2010-04-28

    This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of Atlantic cod (Gadus morhua). Among the advantages of this technique, it is worth highlighting that this is reliable in terms of specificity and sensitivity. The TaqMan real-time PCR is the simplest, fastest testing process and has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of this species. The method can be applied to all kinds of products, fresh, frozen, and processed products, including those undergoing intensive processes of transformation. The developed methodology using specific primer-probe set was validated and further applied to 40 commercial samples labeled as cod in order to determinate if the species used for their manufacturing corresponded to G. morhua, detecting 20% that were incorrectly labeled. A C(t) value of about 19 was obtained when G. morhua was present. In samples with a species mixture, all samples that had a fluorescence signal were positive (C(t) < 30) for the presence of G. morhua by conventional end-point RT-PCR, and the estimated limit of detection for these type of samples was of 20 pg of DNA. The methodology herein developed is useful to check the fulfilment of labeling regulations for seafood products and verify the correct traceability in commercial trade and for fisheries control.

  1. Development and validation of real-time PCR for the detection of Yersinia ruckeri.

    PubMed

    Keeling, S E; Johnston, C; Wallis, R; Brosnahan, C L; Gudkovs, N; McDonald, W L

    2012-02-01

    Yersiniosis (enteric red mouth disease) is a contagious bacterial disease caused by Yersinia ruckeri, which primarily affects salmonids. A real-time PCR assay using a molecular beacon has been developed and validated to improve the detection of the causative biotypes of Y. ruckeri. The assay, which targets the glnA (glutamine synthetase) gene, proved to have 100% analytical specificity and analytical sensitivities of 5 fg and 3 × 10(3) CFU g(-1) for DNA and seeded kidney tissue, respectively. The assay was highly repeatable with low % CV for intra- and inter-run experiments, and the optimized parameters transferred easily between different real-time PCR platforms. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon (n = 750) from 10 farms during 2007/08. The real-time PCR was run in parallel with the bacterial culture detection method, and all fish tested were found to be negative by both methods for Y. ruckeri, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive, reproducible and a rapid method for the detection of Y. ruckeri and has the potential to be used for routine diagnostic testing, health certification and active surveillance programmes.

  2. Development and validation of a real-time PCR assay for the detection of Aeromonas salmonicida.

    PubMed

    Keeling, S E; Brosnahan, C L; Johnston, C; Wallis, R; Gudkovs, N; McDonald, W L

    2013-05-01

    A real-time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg (DNA), 2.2 × 10(4) ± 1 × 10(4) CFU g(-1) (without enrichment) and 40 ± 10 CFU g(-1) (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real-time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n = 750) and pink shubunkin, Carassius auratus (L.) (n = 157). The real-time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A. salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive and a reproducible method for the detection of A. salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.

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

    ERIC Educational Resources Information Center

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

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

  4. Molecular beacon real-time PCR detection of swine viruses.

    PubMed

    McKillen, John; Hjertner, Bernt; Millar, Andrena; McNeilly, Francis; Belák, Sándor; Adair, Brian; Allan, Gordon

    2007-03-01

    Rapid and reliable detection of viral pathogens is critical for the management of the diseases threatening the economic competitiveness of the swine farming industry worldwide. Molecular beacon assays are one type of real-time polymerase chain reaction (PCR) technology capable of fast, specific, sensitive, and reliable viral detection. In this paper, the development of molecular beacon assays as novel tools for the rapid detection of Aujeszky's disease virus, African swine fever virus, porcine circovirus type 2 and porcine parvovirus is described. The assays are capable of rapidly detecting 2 x 10(1) copies of target and are linear between 2 x 10(9) and 2 x 10(2) copies. They can detect virus specifically in clinical samples such as whole blood, serum and tissue. In comparison to conventional PCR they are either as sensitive or more sensitive. As such these molecular beacon assays represent a powerful tool for the detection of these viruses in swine.

  5. Development of real-time PCR assay for differential detection of Bordetella bronchiseptica and Bordetella parapertussis.

    PubMed

    Tizolova, Anette; Brun, Delphine; Guiso, Nicole; Guillot, Sophie

    2014-04-01

    Bordetella parapertussis is a causative agent of whooping cough in humans, and B. bronchiseptica is causing wide variety of respiratory infections in mammals, including humans. Specific diagnostic tests are not currently available. Our first objective was to develop a real-time PCR test for the specific detection of B. bronchiseptica based on the previously described end-point PCR, targeting an intergenomic sequence of the fla gene locus, but it has not been reached. However, there is cross-reactivity between B. parapertussis and B. bronchiseptica. Therefore, the targeted region of several clinical isolates of both species was sequenced, and alignment of the sequences allowed the development of a 2-step real-time PCR assay. The first PCR assay detected the DNA of all clinical isolates of both B. bronchiseptica and B. parapertussis tested. The second PCR assay detected only the DNA of B. parapertussis clinical isolates, thereby allowing discrimination between B. parapertussis and B. bronchiseptica.

  6. Monitoring the immune response using real-time PCR.

    PubMed

    Stordeur, Patrick

    2009-01-01

    Induction of an immune response to a particular antigen is the basis of vaccination. This has been done for years to prevent infectious diseases, and has the potential for the treatment of cancer. The immune response is nowadays more precisely modulated rather than simply induced, like in case of immunotherapy of allergic diseases. Likewise, autoimmune diseases are associated with an inappropriate immune response, and many efforts are made for specifically inhibiting this unwanted response. A possible line of attack is the induction of an antigen-specific immune tolerance, which also has a use in the field of transplantation, where allogeneic responses are deleterious for the graft. In all of these fields of fundamental and clinical medicine, the modulation of immune response requires the assistance of laboratory tests, among which real-time PCR appears more and more helpful. This chapter describes a protocol to quantify immune-related mRNAs using reverse transcription-real-time PCR. The transcripts can be quantified in cultured cells or in cultured whole blood, after an incubation period in the presence of the antigen to which the immune response is analyzed. This is the typical approach to evaluate the efficacy of a vaccine. The transcripts can also be quantified directly in the biological sample, giving information about the in vivo immune status of the individual. The techniques to achieve these different methods are described, and are illustrated by the analysis of the response against the toxoid tetanus antigen.

  7. Integrated real-time fracture-diagnostics instrumentation system

    SciTech Connect

    Engi, D

    1983-01-01

    The use of an integrated, real-time fracture-diagnostics instrumentation system for the control of the fracturing treatment during massive hydraulic fracturing is proposed. The proposed system consists of four subsystems: an internal-fracture-pressure measurement system, a fluid-flow measurement system, a borehole seismic system, and a surface-electric-potential measurement system. This use of borehole seismic and surface-electric-potential measurements, which are essentially away-from-the-wellbore measurements, in conjunction with the use of the more commonly used types of measurements, i.e., at-the-wellbore pressure and fluid-flow measurements, is a distinctive feature of the composite real-time diagnostics system. Currently, the real-time capabilities of the individual subsystems are being developed, and the problems associated with their integration into a complete, computer-linked instrumentation system are being addressed. 2 figures.

  8. Detection of Saccharopolyspora rectivirgula by quantitative real-time PCR.

    PubMed

    Schäfer, Jenny; Kämpfer, Peter; Jäckel, Udo

    2011-07-01

    The thermophilic actinomycete species Saccharopolyspora rectivirgula has been associated with the exogen allergic alveolitis (EAA). EAA is caused by the inhalation of high amounts of airborne spores that can be found for example in environments of agricultural production, compost facilities, mushroom cultivation rooms, or rooms with technical air moistening. Because of the medical relevance of S. rectivirgula, a reliable detection system is needed. Therefore, a quantitative real-time polymerase chain reaction (qPCR) primer system was designed, targeting the 16S rRNA gene of the type strain S. rectivirgula DSM 43747(T) and six other S. rectivirgula reference strains. Our investigation showed that S. rectivirgula presumably own four operons of the 16S rRNA gene, which has to be considered for estimation of cell equivalents. Furthermore, the DNA recovery efficiency from these strains was tested in combination with bioaerosol or material sample as well as the influence of non-target DNA to the recovery rate. Results showed a recovery DNA efficiency of 7-55%. The recovery rate of DNA in a mixture with non-target DNA resulted in ∼87%. In summary, a high amplification efficiency using real-time PCR was found, for which estimated concentrations revealed cell numbers of 2.7 × 10(5) cells m(-3) in bioaerosol and 2.8 × 10(6) cells g(-1) fw(-1) in material samples from a duck house. The specificity of the new developed quantification system was shown by generation of two clone libraries from bioarosol samples, from a duck house, and from a composting plant. Totally, the results clearly show the specificity and practicability of the established qPCR assay for detection of S. rectivirgula.

  9. Real-time PCR detection of ruminant DNA.

    PubMed

    Mendoza-Romero, Luis; Verkaar, Edward L C; Savelkoul, Paul H; Catsburg, Arnold; Aarts, Henk J M; Buntjer, Jaap B; Lenstra, Johannes A

    2004-03-01

    To control the spread of bovine spongiform encephalopathy, several DNA methods have been described for the detection of the species origin of meat and bone meal. Most of these methods are based on the amplification of a mitochondrial DNA segment. We have developed a semiquantitative method based on real-time PCR for detection of ruminant DNA, targeting an 88-bp segment of the ruminant short interspersed nuclear element Bov-A2. This method is specific for ruminants and is able to detect as little as 10 fg of bovine DNA. Autoclaving decreased the amount of detectable DNA, but positive signals were observed in feeding stuff containing 10% bovine material if this had not been rendered in accordance with the regulations, i.e., heated at 134 degrees C for 3 instead of 20 min. PMID:15035372

  10. Development and Application of Real-Time PCR for Detection of Subgroup J Avian Leukosis Virus

    PubMed Central

    Qin, Liting; Gao, Yulong; Ni, Wei; Sun, Meiyu; Wang, Yongqiang; Yin, Chunhong; Qi, Xiaole; Gao, Honglei

    2013-01-01

    Subgroup J avian leukosis virus (ALV-J) is an avian retrovirus that causes severe economic losses in the poultry industry. The early identification and removal of virus-shedding birds are important to reduce the spread of congenital and contact infections. In this study, a TaqMan-based real-time PCR method for the rapid detection and quantification of ALV-J with proviral DNA was developed. This method exhibited a high specificity for ALV-J. Moreover, the detection limit was as low as 10 viral DNA copies. The coefficients of variation (CVs) of both interassay and intra-assay reproducibility were less than 1%. The growth curves of ALV-J in DF-1 cells were measured by real-time PCR, yielding a trend line similar to those determined by 50% tissue culture infective dose (TCID50) and p27 antigen detection. Tissue samples suspected of ALV infection were evaluated using real-time PCR, virus isolation, and routine PCR, and the positivity rates were 60.1%, 41.6% and 44.5%, respectively. Our data indicated that the real-time PCR method provides a sensitive, specific, and reproducible diagnostic tool for the identification and quantification of ALV-J for clinical diagnosis and in laboratory research. PMID:23100340

  11. Towards real-time medical diagnostics using hyperspectral imaging technology

    NASA Astrophysics Data System (ADS)

    Bjorgan, Asgeir; Randeberg, Lise L.

    2015-07-01

    Hyperspectral imaging provides non-contact, high resolution spectral images which has a substantial diagnostic potential. This can be used for e.g. diagnosis and early detection of arthritis in finger joints. Processing speed is currently a limitation for clinical use of the technique. A real-time system for analysis and visualization using GPU processing and threaded CPU processing is presented. Images showing blood oxygenation, blood volume fraction and vessel enhanced images are among the data calculated in real-time. This study shows the potential of real-time processing in this context. A combination of the processing modules will be used in detection of arthritic finger joints from hyperspectral reflectance and transmittance data.

  12. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    PubMed

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

  13. Real-time PCR TaqMan assay for rapid screening of bloodstream infection

    PubMed Central

    2014-01-01

    Background Sepsis is one of the main causes of mortality and morbidity. The rapid detection of pathogens in blood of septic patients is essential for adequate antimicrobial therapy and better prognosis. This study aimed to accelerate the detection and discrimination of Gram-positive (GP) and Gram-negative (GN) bacteria and Candida species in blood culture samples by molecular methods. Methods The Real-GP®, -GN®, and -CAN® real-time PCR kit (M&D, Wonju, Republic of Korea) assays use the TaqMan probes for detecting pan-GP, pan-GN, and pan-Candida species, respectively. The diagnostic performances of the real-time PCR kits were evaluated with 115 clinical isolates, 256 positive and 200 negative blood culture bottle samples, and the data were compared to results obtained from conventional blood culture. Results Eighty-seven reference strains and 115 clinical isolates were correctly identified with specific probes corresponding to GP-bacteria, GN-bacteria and Candida, respectively. The overall sensitivity and specificity of the real-time PCR kit with blood culture samples were 99.6% and 89.5%, respectively. Conclusions The Real-GP®, -GN®, and -CAN® real-time PCR kits could be useful tools for the rapid and accurate screening of bloodstream infections (BSIs). PMID:24393579

  14. Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR.

    PubMed

    Keightley, Maria Cristina; Sillekens, Peter; Schippers, Wim; Rinaldo, Charles; George, Kirsten St

    2005-12-01

    Severe acute respiratory syndrome (SARS) exhibits a high mortality rate and the potential for rapid epidemic spread. Additionally, it has a poorly defined clinical presentation, and no known treatment or prevention methods. Collectively, these factors underscore the need for early diagnosis. Molecular tests have been developed to detect SARS coronavirus (SARS-CoV) RNA using real time reverse transcription polymerase chain reaction (RT-PCR) with varying levels of sensitivity. However, RNA amplification methods have been demonstrated to be more sensitive for the detection of some RNA viruses. We therefore developed a real-time nucleic acid sequence-based amplification (NASBA) test for SARS-CoV. A number of primer/beacon sets were designed to target different regions of the SARS-CoV genome, and were tested for sensitivity and specificity. The performance of the assays was compared with RT-PCR assays. A multi-target real-time NASBA application was developed for detection of SARS-CoV polymerase (Pol) and nucleocapsid (N) genes. The N targets were found to be consistently more sensitive than the Pol targets, and the real-time NASBA assay demonstrates equivalent sensitivity when compared to testing by real-time RT-PCR. A multi-target real-time NASBA assay has been successfully developed for the sensitive detection of SARS-CoV.

  15. Comparative detection of rabies RNA by NASBA, real-time PCR and conventional PCR.

    PubMed

    Wacharapluesadee, Supaporn; Phumesin, Patta; Supavonwong, Pornpun; Khawplod, Pakamatz; Intarut, Nirun; Hemachudha, Thiravat

    2011-08-01

    Five methods for the RNA detection of rabies virus were directly compared in this study. These included conventional nucleic acid sequence-based amplification with electrochemiluminescence (NASBA-ECL) assay, reverse transcription (RT)-heminested (hn) polymerase chain reaction (PCR) and TaqMan real-time RT-PCR using protocols as described previously. The first two methods have been routinely utilised for ante-mortem diagnosis of human rabies in Thailand and other rabies-endemic Asian and African countries. In addition, two real-time NASBA assays based on the use of a NucliSens EasyQ analyser (NASBA-Beacon-EQ) and LightCycler real-time PCR machine (NASBA-Beacon-LC) were studied in parallel. All methods target the N gene, whereas the L gene is used for RT-hnPCR. Using serial dilutions of purified RNA from rabies-infected dog brain tissue to assess sensitivity, all five methods had comparable degrees of sensitivities of detection. However, both real-time NASBA assays had slightly lower sensitivities by 10-fold than the other three assays. This finding was also true (except for TaqMan real-time RT-PCR due to a mismatch between the target and probe sequences) when laboratory-adapted (challenge virus standard-11) virus was used in the assays. Testing on previously NASBA-ECL positive clinical samples from 10 rabies patients (saliva [6] and brain [4]) and 10 rabies-infected dog brain tissues, similar results were obtained among the five methods; real-time NASBA assays yielded false-negative results on 2 saliva samples. None of the assays showed positive results on cerebrospinal fluid specimens of 10 patients without rabies encephalitis. Due to the unavailability of the NASBA-ECL assay, the results show that TaqMan real-time RT-PCR and RT-hnPCR can be useful for ante- and post-mortem diagnosis of rabies.

  16. Utility of real-time Taqman PCR for antemortem and postmortem diagnosis of human rabies.

    PubMed

    Mani, Reeta Subramaniam; Madhusudana, Shampur Narayan; Mahadevan, Anita; Reddy, Vijayalakshmi; Belludi, Ashwin Yajaman; Shankar, Susarla Krishna

    2014-10-01

    Rabies, a fatal zoonotic viral encephalitis remains a neglected disease in India despite a high disease burden. Laboratory confirmation is essential, especially in patients with paralytic rabies who pose a diagnostic dilemma. However, conventional tests for diagnosis of rabies have several limitations. In the present study the utility of a real-time TaqMan PCR assay was evaluated for antemortem/postmortem diagnosis of rabies. Human clinical samples received for antemortem rabies diagnosis (CSF, saliva, nuchal skin biopsy, serum), and samples obtained postmortem from laboratory confirmed rabies in humans (brain tissue, CSF, serum) and animals (brain tissue) were included in the study. All CSF and sera were tested for rabies viral neutralizing antibodies (RVNA) by rapid fluorescent focus inhibition test (RFFIT) and all samples (except sera) were processed for detection of rabies viral RNA by real-time TaqMan PCR. All the 29 (100%) brain tissues from confirmed cases of human and animal rabies, and 11/14 (78.5%) CSF samples obtained postmortem from confirmed human rabies cases were positive by real-time TaqMan PCR. Rabies viral RNA was detected in 5/11 (45.4%) CSF samples, 6/10 (60%) nuchal skin biopsies, and 6/7 (85.7%) saliva samples received for antemortem diagnosis. Real-time TaqMan PCR alone could achieve antemortem rabies diagnosis in 11/13 (84.6%) cases; combined with RVNA detection in CSF antemortem rabies diagnosis could be achieved in all 13 (100%) cases. Real-time TaqMan PCR should be made available widely as an adjunctive test for diagnosis of human rabies in high disease burden countries like India.

  17. Development and Evaluation of a Novel Real-Time PCR for Pan-Dermatophyte Detection in Nail Specimens.

    PubMed

    Gong, Jie; Ran, Menglong; Wang, Xiaowen; Wan, Zhe; Li, Ruoyu

    2016-02-01

    An accurate diagnosis of tinea unguium is necessary for the selection of antimycotics and successful treatment. To rapidly and accurately identify the aetiological agents causing tinea unguium, we improved upon the conventional boiling method for DNA extraction and developed a novel real-time PCR detection system that includes two assays. The two assays, based on the amplification of ribosomal internal transcribed spacer regions and 28S rDNA, were designed to detect pan-dermatophyte and Trichophyton rubrum, respectively. The analytical sensitivities of both assays permitted the detection of ten copies of plasmid DNA template. The analytical specificity of the detection system was confirmed using 11 dermatophyte strains and 25 non-dermatophyte strains. In total, 165 nail specimens were examined by microscopy, culture, conventional PCR, and the novel real-time PCR method. Real-time PCR gave positive results in 47.3 % of the specimens (78), a rate exceeding those obtained using microscopy (72, 43.6 %), conventional PCR (69, 41.8 %), and culture (49, 29.7 %). All conventional PCR-positive specimens were detected by real-time PCR, and real-time PCR detected nine specimens that were missed by conventional PCR. The results from latent class analysis, and further calculations, showed that real-time PCR was the most sensitive method, but the diagnostic specificity of the four approaches was equivalent. In particular, molecular approaches may be more effective than microscopy and culture when the clinical symptoms of tinea unguium are not evident.

  18. Advanced clinical monitoring: considerations for real-time hemodynamic diagnostics.

    PubMed

    Goldman, J M; Cordova, M J

    1994-01-01

    In an effort to ease staffing burdens and potentially improve patient outcome in an intensive care unit (ICU) environment, we are developing a real-time system to accurately and efficiently diagnose cardiopulmonary emergencies. The system is being designed to utilize all relevant routinely-monitored physiological data in order to automatically diagnose potentially fatal events. The initial stage of this project involved formulating the overall system design and appropriate methods for real-time data acquisition, data storage, data trending, waveform analysis, and implementing diagnostic rules. Initially, we defined a conceptual analysis of the minimum physiologic data set, and the monitoring time-frames (trends) which would be required to diagnose cardiopulmonary emergencies. Following that analysis, we used a fuzzy logic diagnostic engine to analyze physiological data during a simulated arrhythmic cardiac arrest (ACA) in order to assess the validity of our diagnostic methodology. We used rate, trend, and morphologic data extracted from the following signals: expired CO2 time-concentration curve (capnogram), electrocardiogram, and arterial blood pressure. The system performed well: The fuzzy logic engine effectively diagnosed the likelihood of ACA from the subtle hemodynamic trends which preceded the complete arrest. As the clinical picture worsened, the fuzzy logic-based system accurately indicated the change in patient condition. Termination of the simulated arrest was rapidly detected by the diagnostic engine. In view of the effectiveness of this fuzzy logic implementation, we plan to develop additional fuzzy logic modules to diagnose other cardiopulmonary emergencies.

  19. Universal reverse-transcriptase real-time PCR for infectious hematopoietic necrosis virus (IHNV)

    USGS Publications Warehouse

    Purcell, Maureen K.; Thompson, Rachel L.; Garver, Kyle A.; Hawley, Laura M.; Batts, William N.; Sprague, Laura; Sampson, Corie; Winton, James R.

    2013-01-01

    Infectious hematopoietic necrosis virus (IHNV) is an acute pathogen of salmonid fishes in North America, Europe and Asia and is reportable to the World Organization for Animal Health (OIE). Phylogenetic analysis has identified 5 major virus genogroups of IHNV worldwide, designated U, M, L, E and J; multiple subtypes also exist within those genogroups. Here, we report the development and validation of a universal IHNV reverse-transcriptase real-time PCR (RT-rPCR) assay targeting the IHNV nucleocapsid (N) gene. Properties of diagnostic sensitivity (DSe) and specificity (DSp) were defined using laboratory-challenged steelhead trout Oncorhynchus mykiss, and the new assay was compared to the OIE-accepted conventional PCR test and virus isolation in cell culture. The IHNV N gene RT-rPCR had 100% DSp and DSe and a higher estimated diagnostic odds ratio (DOR) than virus culture or conventional PCR. The RT-rPCR assay was highly repeatable within a laboratory and highly reproducible between laboratories. Field testing of the assay was conducted on a random sample of juvenile steelhead collected from a hatchery raceway experiencing an IHN epizootic. The RT-rPCR detected a greater number of positive samples than cell culture and there was 40% agreement between the 2 tests. Overall, the RT-rPCR assay was highly sensitive, specific, repeatable and reproducible and is suitable for use in a diagnostic setting.

  20. Differential diagnosis of Brucella abortus by real-time PCR based on a single-nucleotide polymorphisms.

    PubMed

    Kim, Ji-Yeon; Kang, Sung-Il; Lee, Jin Ju; Lee, Kichan; Sung, So-Ra; Erdenebaataar, Janchivdorj; Vanaabaatar, Batbaatar; Jung, Suk Chan; Park, Yong Ho; Yoo, Han-Sang; Her, Moon

    2016-05-01

    To diagnose brucellosis effectively, many genus- and species-specific detection methods based on PCR have been developed. With conventional PCR assays, real-time PCR techniques have been developed as rapid diagnostic tools. Among them, real-time PCR using hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology among species, with which it is possible to make an accurate diagnosis by means of an amplification curve and melting peak analysis. A hybprobe for B. abortus was designed from a specific single-nucleotide polymorphism (SNP) on the fbaA gene. This probe only showed specific amplification of B. abortus from approximately the 14th cycle, given a melting peak at 69°C. The sensitivity of real-time PCR was revealed to be 20 fg/µl by 10-fold DNA dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed greater sensitivity than that of conventional PCR and previous real-time PCR based on Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating B. abortus infection with rapidity and accuracy.

  1. Differential diagnosis of Brucella abortus by real-time PCR based on a single-nucleotide polymorphisms

    PubMed Central

    KIM, Ji-Yeon; KANG, Sung-Il; LEE, Jin Ju; LEE, Kichan; SUNG, So-Ra; ERDENEBAATAAR, Janchivdorj; VANAABAATAR, Batbaatar; JUNG, Suk Chan; PARK, Yong Ho; YOO, Han-Sang; HER, Moon

    2015-01-01

    To diagnose brucellosis effectively, many genus- and species-specific detection methods based on PCR have been developed. With conventional PCR assays, real-time PCR techniques have been developed as rapid diagnostic tools. Among them, real-time PCR using hybridization probe (hybprobe) has been recommended for bacteria with high DNA homology among species, with which it is possible to make an accurate diagnosis by means of an amplification curve and melting peak analysis. A hybprobe for B. abortus was designed from a specific single-nucleotide polymorphism (SNP) on the fbaA gene. This probe only showed specific amplification of B. abortus from approximately the 14th cycle, given a melting peak at 69°C. The sensitivity of real-time PCR was revealed to be 20 fg/µl by 10-fold DNA dilution, and the detection limit was 4 CFU in clinical samples. This real-time PCR showed greater sensitivity than that of conventional PCR and previous real-time PCR based on Taqman probe. Therefore, this new real-time PCR assay could be helpful for differentiating B. abortus infection with rapidity and accuracy. PMID:26666176

  2. Detection of Histoplasma capsulatum from clinical specimens by cycling probe-based real-time PCR and nested real-time PCR.

    PubMed

    Muraosa, Yasunori; Toyotome, Takahito; Yahiro, Maki; Watanabe, Akira; Shikanai-Yasuda, Maria Aparecida; Kamei, Katsuhiko

    2016-05-01

    We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens.

  3. Evaluation of IFN-γ polymorphism+874 T/A in patients with recurrent tonsillitis by PCR real time mismatch amplification mutation assay (MAMA real time PCR).

    PubMed

    Bergallo, Massimiliano; Gambarino, Stefano; Loiacono, Elisa; Vergano, Luca; Galliano, Ilaria; Montanari, Paola; Astegiano, Sara; Tavormina, Paolo; Tovo, Pier-Angelo

    2015-02-01

    Interferon gamma (IFN-γ) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-γ can give a predisposition to infectious disease, autoimmune pathologies and tumours. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP)+874∗T/A that may affect IFN-γ gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donor's blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p<0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-γ polymorphism when compared with control subject (p=0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard.

  4. Quantitation of HIV-1 RNA in breast milk by real time PCR.

    PubMed

    Becquart, Pierre; Foulongne, Vincent; Willumsen, Juana; Rouzioux, Christine; Segondy, Michel; Van de Perre, Philippe

    2006-04-01

    HIV-1 RNA in breast milk is a strong predictor of HIV-1 transmission through breastfeeding. In the present report, breast milk samples from HIV-1 uninfected donors were spiked with dilution of quantified culture supernatant from HIV-1(NDK) infected PBMC. Two RNA extraction techniques based on silica extraction, Nuclisens (BioMerieux) and Triazol (Qiagen), two techniques based on guanidine thiocynanate/chloroforme extraction, TRIzol (Life Technologie) and Amplicor HIV-1 Monitor (Roche Diagnostic Systems), and one technique based on electrostatic adsorption on iron oxide micro beads (Promega) were compared. HIV-1 RNA was quantitated by real time PCR (LTR gene) and Amplicor HIV-1 Monitor. Combining magnetic micro beads extraction and real time PCR quantitation allowed to correctly quantify breast milk HIV-1 RNA, with a difference between the expected and measured HIV-1 RNA levels always lower than 0.3 log copies/ml. The same combination was confirmed on 25 breast milk samples from HIV-1 infected women collected in Kwazulu-Natal, South Africa, by comparing measurements with those obtained by the Amplicor HIV-1 Monitor (r(2)=0.88). Nucleic acid extraction by magnetic micro beads followed by real time PCR is a reliable, sensitive, rapid and simple procedure to quantify HIV-1 RNA in breast milk and allows for PCR inhibitors found frequently in these samples.

  5. Real-time PCR with internal amplification control for detecting tuberculosis: method design and validation.

    PubMed

    Flores, E; Rodríguez, J C; Garcia-Pachón, E; Soto, J L; Ruiz, M; Escribano, I; Royo, G

    2009-08-01

    Real-time PCR has been a major development in the diagnosis of tuberculosis. However, most tests do not include an internal amplification control (IAC), which therefore limits it clinical application. In this study a new, easy to perform real-time PCR test with IAC was designed and validated in clinical samples. The primers amplified a 163-bp fragment of IS6110 of Mycobacterium tuberculosis and the IAC was designed with a fragment of a different microorganism (Chlamydia trachomatis). The interassay and intraassay variation of this test were very low (0.45-1.65% and 0.18-1.80%, respectively). The detection accuracy was validated in 50 samples (25 urine, 25 sputum) with different concentrations of M. tuberculosis, 18 clinical isolates of non-tuberculous mycobacteria and 148 samples with clinical suspicion of pulmonary tuberculosis. The specificity was 100%. The detection limit of this PCR test without IAC was approximately 15 bacteria and with IAC approximately 32 bacteria. This real-time PCR with IAC assay can improve the detection of M. tuberculosis and contribute to standardization of this diagnostic technique.

  6. Optical real-time defect-enhancement diagnostic system.

    PubMed

    Gaeta, C J; Mitchell, P V; Pepper, D M

    1992-12-15

    We have demonstrated an all-optical diagnostic system that enhances the observation of defects in periodic structures. This real-time technique employs a spatial light modulator as a smart-pixel array for information processing in the Fourier transform plane of a lens. The system also includes a phase-conjugate mirror for autoalignment and for correction of optical wave-front aberrations that are imparted on the object light by the smart-pixel processor and its associated optical train. PMID:19798320

  7. Comparison of six real-time PCR assays for qualitative detection of cytomegalovirus in clinical specimens.

    PubMed

    Binnicker, M J; Espy, M E

    2013-11-01

    In this study, we compared the performance of six real-time PCR assays for the qualitative detection of cytomegalovirus (CMV) in clinical samples other than plasma. Two hundred specimens (respiratory [n = 72], urine [n = 67], cerebrospinal fluid [CSF] [n = 25], tissue [n = 18], amniotic fluid [n = 10], and bone marrow [n = 8]) submitted for routine testing by CMV real-time PCR analyte-specific reagents (ASR) (Roche Diagnostics, Indianapolis, IN) were also tested by a laboratory-developed test (LDT) and 4 commercially available PCR assays: EraGen Multicode (Luminex, Austin, TX), Focus Simplexa (Focus Diagnostics, Cypress, CA), Elitech MGB Alert CMV (Fisher Scientific, Hanover Park, IL), and Abbott CMV (Abbott Park, IL). Nucleic acid was extracted using the MagNA Pure system (Roche Diagnostics) and subsequently tested by each PCR method. Results were analyzed by comparing each assay to a "consensus result," which was defined as the result obtained from at least 4 of the 6 assays. In addition to the prospective samples, 13 lower respiratory samples with known positive results by CMV shell vial were tested by each PCR method. Following testing of the 200 prospective specimens, the Abbott, Elitech, EraGen, and Focus PCR assays demonstrated a sensitivity of 100% (46/46), while the Roche analyte-specific reagents (ASR) and LDT showed sensitivities of 89% (41/46) and 98% (45/46), respectively. Percent specificities ranged from 97% (149/154) by Elitech to 100% (154/154) by the LDT. Among the 13 shell vial-positive lower respiratory samples, the percent sensitivities ranged from 69% (9/13) by Elitech to 92% (12/13) by the LDT. The Abbott, EraGen, Elitech, Focus, and LDT PCR assays performed similarly (κ ≥ 0.89) for the detection of CMV in clinical specimens and demonstrated increased sensitivity compared to the Roche ASR.

  8. Real-Time PCR Assays for Detection of Bocavirus in Human Specimens

    PubMed Central

    Lu, Xiaoyan; Chittaganpitch, Malinee; Olsen, Sonja J.; Mackay, Ian M.; Sloots, Theo P.; Fry, Alicia M.; Erdman, Dean D.

    2006-01-01

    The recently discovered human bocavirus (HBoV) is the first member of the family Parvoviridae, genus Bocavirus, to be potentially associated with human disease. Several studies have identified HBoV in respiratory specimens from children with acute respiratory disease, but the full spectrum of clinical disease and the epidemiology of HBoV infection remain unclear. The availability of rapid and reliable molecular diagnostics would therefore aid future studies of this novel virus. To address this, we developed two sensitive and specific real-time TaqMan PCR assays that target the HBoV NS1 and NP-1 genes. Both assays could reproducibly detect 10 copies of a recombinant DNA plasmid containing a partial region of the HBoV genome, with a dynamic range of 8 log units (101 to 108 copies). Eight blinded clinical specimen extracts positive for HBoV by an independent PCR assay were positive by both real-time assays. Among 1,178 NP swabs collected from hospitalized pneumonia patients in Sa Kaeo Province, Thailand, 53 (4.5%) were reproducibly positive for HBoV by one or both targets. Our data confirm the possible association of HBoV infection with pneumonia and demonstrate the utility of these real-time PCR assays for HBoV detection. PMID:16954253

  9. Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR.

    PubMed

    Huang, Shihai; Salituro, John; Tang, Ning; Luk, Ka-Cheung; Hackett, John; Swanson, Priscilla; Cloherty, Gavin; Mak, Wai-Bing; Robinson, John; Abravaya, Klara

    2007-01-01

    Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.

  10. Development and validation of a real-time PCR assay for the detection of anguillid herpesvirus 1.

    PubMed

    van Beurden, S J; Voorbergen-Laarman, M A; Roozenburg, I; van Tellingen, J; Haenen, O L M; Engelsma, M Y

    2016-01-01

    Anguillid herpesvirus 1 (AngHV1) causes a haemorrhagic disease with increased mortality in wild and farmed European eel, Anguilla anguilla (L.) and Japanese eel Anguilla japonica, Temminck & Schlegel). Detection of AngHV1 is currently based on virus isolation in cell culture, antibody-based typing assays or conventional PCR. We developed, optimized and concisely validated a diagnostic TaqMan probe based real-time PCR assay for the detection of AngHV1. The primers and probe target AngHV1 open reading frame 57, encoding the capsid protease and scaffold protein. Compared to conventional PCR, the developed real-time PCR is faster, less labour-intensive and has a reduced risk of cross-contamination. The real-time PCR assay was shown to be analytically sensitive and specific and has a high repeatability, efficiency and r(2) -value. The diagnostic performance of the assay was determined by testing 10% w/v organ suspensions and virus cultures from wild and farmed European eels from the Netherlands by conventional and real-time PCR. The developed real-time PCR assay is a useful tool for the rapid and sensitive detection of AngHV1 in 10% w/v organ suspensions from wild and farmed European eels.

  11. [Real-time PCR Detection Method for the Reston Subtype of the Ebola Virus].

    PubMed

    Xu, Lili; Bao, Linlin; Gu, Songzhi; Qin, Chuan

    2015-05-01

    We aimed to develop a real-time polymerase chain reaction (PCR) detection method for the Reston subtype of the Ebola virus. The NP gene of the Reston subtype of the Ebola virus was selected as the detection object. Sequences of different subtypes of Ebola viruses were aligned using Clustal W software. The most unique and conserved regions of the Reston subtype of the Ebola virus were recruited as candidate sequences for specific primers. Primer Express and Primer Premier 5. 0 software were used to filter the optimal pair of primers for detection. Real-time PCR was carried out using optimized parameters and positive DNA prepared by serial (tenfold) dilution of a recombinant plasmid and by plotting a standard curve. In addition, the reproducibility, accuracy, and specificity of the assay were tested. Results showed that the sensitivity of detection of the Reston subtype of the Ebola virus by real-time PCR could reached 10(2) copies/microL. The linear relationship (R2) reached 0.997, the slope of the standard curve was -0.3101, and amplification efficiency was 110.145%. A sharp and narrow melting peak appeared at 79.94 degrees C for all standards in different dilutions. In conclusion, a fast and sensitive real-time PCR detection system for the Reston subtype of the Ebola virus was developed. This system could be used as a supplementary diagnostic and monitoring approach for basic and clinical studies on the Reston subtype of the Ebola virus. The detection system does not require expensive technology or specialist operators. PMID:26470534

  12. Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes

    PubMed Central

    Deepak, SA; Kottapalli, KR; Rakwal, R; Oros, G; Rangappa, KS; Iwahashi, H; Masuo, Y; Agrawal, GK

    2007-01-01

    Invention of polymerase chain reaction (PCR) technology by Kary Mullis in 1984 gave birth to real-time PCR. Real-time PCR — detection and expression analysis of gene(s) in real-time — has revolutionized the 21st century biological science due to its tremendous application in quantitative genotyping, genetic variation of inter and intra organisms, early diagnosis of disease, forensic, to name a few. We comprehensively review various aspects of real-time PCR, including technological refinement and application in all scientific fields ranging from medical to environmental issues, and to plant. PMID:18645596

  13. Graph-based real-time fault diagnostics

    NASA Technical Reports Server (NTRS)

    Padalkar, S.; Karsai, G.; Sztipanovits, J.

    1988-01-01

    A real-time fault detection and diagnosis capability is absolutely crucial in the design of large-scale space systems. Some of the existing AI-based fault diagnostic techniques like expert systems and qualitative modelling are frequently ill-suited for this purpose. Expert systems are often inadequately structured, difficult to validate and suffer from knowledge acquisition bottlenecks. Qualitative modelling techniques sometimes generate a large number of failure source alternatives, thus hampering speedy diagnosis. In this paper we present a graph-based technique which is well suited for real-time fault diagnosis, structured knowledge representation and acquisition and testing and validation. A Hierarchical Fault Model of the system to be diagnosed is developed. At each level of hierarchy, there exist fault propagation digraphs denoting causal relations between failure modes of subsystems. The edges of such a digraph are weighted with fault propagation time intervals. Efficient and restartable graph algorithms are used for on-line speedy identification of failure source components.

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

    PubMed

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

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

  15. Low-cost, real-time, continuous flow PCR system for pathogen detection.

    PubMed

    Fernández-Carballo, B Leticia; McGuiness, Ian; McBeth, Christine; Kalashnikov, Maxim; Borrós, Salvador; Sharon, Andre; Sauer-Budge, Alexis F

    2016-04-01

    In this paper, we present a portable and low cost point-of-care (POC) PCR system for quantitative detection of pathogens. Our system is based on continuous flow PCR which maintains fixed temperatures zones and pushes the PCR solution between two heated areas allowing for faster heat transfer and as a result, a faster PCR. The PCR system is built around a 46.0 mm × 30.9 mm × 0.4 mm disposable thermoplastic chip. In order to make the single-use chip economically viable, it was manufactured by hot embossing and was designed to be compatible with roll-to-roll embossing for large scale production. The prototype instrumentation surrounding the chip includes two heaters, thermal sensors, and an optical system. The optical system allows for pathogen detection via real time fluorescence measurements. FAM probes were used as fluorescent reporters of the amplicons generated during the PCR. To demonstrate the function of the chip, two infectious bacteria targets were selected: Chlamydia trachomatis and Escherichia coli O157:H7. For both bacteria, the limit of detection of the system was determined, PCR efficiencies were calculated, and different flow velocities were tested. We have demonstrated successful detection for these two bacterial pathogens highlighting the versatility and broad utility of our portable, low-cost, and rapid PCR diagnostic device.

  16. Comparison of different standards for real-time PCR-based absolute quantification.

    PubMed

    Dhanasekaran, S; Doherty, T Mark; Kenneth, John

    2010-03-31

    Quantitative real-time PCR (qPCR) is a powerful tool used for both research and diagnostic, which has the advantage, compared to relative quantification, of providing an absolute copy number for a particular target. However, reliable standards are essential for qPCR. In this study, we have compared four types of commonly-used standards--PCR products (with and without purification) and cloned target sequences (circular and linear plasmid) for their stability during storage (using percentage of variance in copy numbers, PCR efficiency and regression curve correlation coefficient (R(2))) using hydrolysis probe (TaqMan) chemistry. Results, expressed as copy numbers/microl, are presented from a sample human system in which absolute levels of HuPO (reference gene) and the cytokine gene IFN-gamma were measured. To ensure the suitability and stability of the four standards, the experiments were performed at 0, 7 and 14 day intervals and repeated 6 times. We have found that the copy numbers vary (due to degradation of standards) over the period of time during storage at 4 degrees C and -20 degrees C, which affected PCR efficiency significantly. The cloned target sequences were noticeably more stable than the PCR product, which could lead to substantial variance in results using standards constructed by different routes. Standard quality and stability should be routinely tested for assays using qPCR.

  17. Low-cost, real-time, continuous flow PCR system for pathogen detection.

    PubMed

    Fernández-Carballo, B Leticia; McGuiness, Ian; McBeth, Christine; Kalashnikov, Maxim; Borrós, Salvador; Sharon, Andre; Sauer-Budge, Alexis F

    2016-04-01

    In this paper, we present a portable and low cost point-of-care (POC) PCR system for quantitative detection of pathogens. Our system is based on continuous flow PCR which maintains fixed temperatures zones and pushes the PCR solution between two heated areas allowing for faster heat transfer and as a result, a faster PCR. The PCR system is built around a 46.0 mm × 30.9 mm × 0.4 mm disposable thermoplastic chip. In order to make the single-use chip economically viable, it was manufactured by hot embossing and was designed to be compatible with roll-to-roll embossing for large scale production. The prototype instrumentation surrounding the chip includes two heaters, thermal sensors, and an optical system. The optical system allows for pathogen detection via real time fluorescence measurements. FAM probes were used as fluorescent reporters of the amplicons generated during the PCR. To demonstrate the function of the chip, two infectious bacteria targets were selected: Chlamydia trachomatis and Escherichia coli O157:H7. For both bacteria, the limit of detection of the system was determined, PCR efficiencies were calculated, and different flow velocities were tested. We have demonstrated successful detection for these two bacterial pathogens highlighting the versatility and broad utility of our portable, low-cost, and rapid PCR diagnostic device. PMID:26995085

  18. [Real-time PCR kits for the detection of the African Swine Fever virus].

    PubMed

    Latyshev, O E; Eliseeva, O V; Grebennikova, T V; Verkhovskiĭ, O A; Tsibezov, V V; Chernykh, O Iu; Dzhailidi, G A; Aliper, T I

    2014-01-01

    The results obtained using the diagnostic kit based on real-time polymerase chain reaction to detect the DNA of the African Swine Fever in the pathological material, as well as in the culture fluid, are presented. A high sensitivity and specificity for detection of the DNA in the organs and tissues of animals was shown to be useful for detection in the European Union referentiality reagent kits for DNA detection by real time PCR of ASFV. More rapid and effective method of DNA extraction using columns mini spin Quick gDNA(TM) MiniPrep was suggested and compared to the method of DNA isolation on the inorganic sorbent. High correlation of the results of the DNA detection of ASFV by real-time PCR and antigen detection results ASFV by competitive ELISA obtained with the ELISA SEROTEST/INGEZIM COMRAC PPA was demonstrated. The kit can be used in the veterinary services for effective monitoring of ASFV to contain, eliminate and prevent further spread of the disease.

  19. A FRET-Based Real-Time PCR Assay to Identify the Main Causal Agents of New World Tegumentary Leishmaniasis

    PubMed Central

    De Los Santos, Maxy; Soberón, Valeria; Lucas, Carmen M.; Matlashewski, Greg; Llanos-Cuentas, Alejandro; Ore, Marianela; Baldeviano, G. Christian; Edgel, Kimberly A.; Lescano, Andres G.; Graf, Paul C. F.; Bacon, David J.

    2013-01-01

    In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL). The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR) assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, L. (V.) peruviana and L. (V.) lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST). In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST) data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America. PMID:23301111

  20. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

    PubMed Central

    Espy, M. J.; Uhl, J. R.; Sloan, L. M.; Buckwalter, S. P.; Jones, M. F.; Vetter, E. A.; Yao, J. D. C.; Wengenack, N. L.; Rosenblatt, J. E.; Cockerill, F. R.; Smith, T. F.

    2006-01-01

    Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory. PMID:16418529

  1. Quantitative detection of Listeria monocytogenes in biofilms by real-time PCR.

    PubMed

    Guilbaud, Morgan; de Coppet, Pierre; Bourion, Fabrice; Rachman, Cinta; Prévost, Hervé; Dousset, Xavier

    2005-04-01

    A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 x 10(2) CFU/cm2.

  2. Role of real-time PCR (RT-PCR) in rapid diagnosis of tuberculous mycobacteria in different clinical samples.

    PubMed

    2014-02-01

    The study was aimed for molecular detection of mycobacterial DNA in different clinical samples using real-time polymerase chain reaction (RT-PCR) system and rapid diagnosis of tuberculosis. A total of 508 clinical specimens (blood 343, menstrual fluid 53, endometrial tissue 43, body fluid 36, pus from lymph nodes 18, sputum 8, urine 5 and semen 2) were included in this study. We extracted DNA using QIAamp DNA Mini Kit (QIAGEN, Germany) and performed real-time assay using Rotor-Gene Q machine from Corbett Research, Australia for specific amplification of IS6110 sequence of mycobacterial genome. The RT-PCR result was also compared with bacterial culture and acid-fast bacillus staining. RT-PCR assay showed positivity in 52 cases and negative in 456 cases. Corresponding positive results in culture and acid-fast bacillus staining methods were 49 cases and 24 cases respectively. The sensitivity and specificity of detecting Mycobacterium tuberculosis by RT-PCR were 93.87% and 98.69% respectively taking positive culture results as reference standards. The overall positive and negative predictive values were 88.46% and 99.34% respectively. RT-PCR is a useful diagnostic tool for rapid and sensitive detection of mycobacteria in different clinical samples. The easy processing, fast reporting and relative lack of contamination issues make it worthy as a possible replacement to time consuming culture techniques. Moreover, it has added advantage of quantification of mycobacterial DNA, hence bacterial load.

  3. Polymerase chain reaction and real-time PCR for diagnosing of Leishmania infantum chagasi in dogs.

    PubMed

    Ramos, Rafael Antonio do Nascimento; Ramos, Carlos Alberto do Nascimento; Jusi, Márcia Mariza Gomes; de Araújo, Flábio Ribeiro; Machado, Rosangela Zacarias; Faustino, Maria Aparecida da Glória; Alves, Leucio Câmara

    2012-01-01

    The importance of dogs as a reservoir for Leishmania infantumchagasi in urban environments has stimulated numerous studies assessing diagnostic techniques. When performed properly, such procedures are an important step in preventing leishmaniasis in humans. Molecular methods have become prominent for this purpose. The aim of the present study was to determine the performance of the polymerase chain reaction (PCR) and real-time PCR (qPCR) for diagnosing of canine visceral leishmaniasis (CVL) using different biological samples. For this, 35 dogs from an area endemic for CVL were used. Bone marrow aspirate and lymph node and spleen fragments from these dogs were used for the molecular diagnosis. In the present study, qPCR was able to detect a greater number of positive animals than seen with PCR. Among the different biological samples used, there was no significant difference in L. infantumchagasi DNA detection between PCR and qPCR. However, considering that lymph nodes are easy to acquire, these can be considered to be the best samples for making molecular diagnoses of L. infantum chagasi infection.

  4. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    NASA Astrophysics Data System (ADS)

    Huang, Su-Hua; Yang, Tsuey-Ching; Tsai, Ming-Hong; Tsai, I.-Shou; Lu, Huang-Chih; Chuang, Pei-Hsin; Wan, Lei; Lin, Ying-Ju; Lai, Chih-Ho; Lin, Cheng-Wen

    2008-10-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples.

  5. Critical analysis of rhinovirus RNA load quantification by real-time reverse transcription-PCR.

    PubMed

    Schibler, Manuel; Yerly, Sabine; Vieille, Gaël; Docquier, Mylène; Turin, Lara; Kaiser, Laurent; Tapparel, Caroline

    2012-09-01

    Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5'-untranslated regions (5'UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5'UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.

  6. [Development of a real-time RT-PCR for detection of equine influenza virus].

    PubMed

    Aeschbacher, S; Santschi, E; Gerber, V; Stalder, H P; Zanoni, R G

    2015-04-01

    Equine influenza is a highly contagious respiratory disease in horses caused by influenza A viruses. In this work a real-time RT-PCR for fast and sensitive diagnosis of equine influenza viruses (EIV) targeting a highly conserved region of the matrix gene was developed. In addition two RT-PCR methods for the amplification of large parts of the matrix- and HA gene were adapted for molecular-epidemiological characterization of viruses. The primers of the real-time RT-PCR had homologies of 99.4% to EIV- and 97.7% to all influenza A viral sequences, whereas the minor groove binder (MGB) probe showed homologies of 99.3% and 99.6%, respectively. These high values allow application of the assay for influenza viruses in other species. Using 20 equine, 11 porcine and 2 avian samples, diagnostic suitability of the assay was confirmed. High specificity for influenza viruses was shown both experimentally and by software simulation. The assay analytical sensitivity was at 10(2)-10(3) copies of RNA and 10(0)-10(1) copies of DNA, respectively. This allows virus detection also in circumstances of minor viral shedding. All amplified EIV sequences were classified phylogenetically within the known lineages. PMID:26757582

  7. [Research progress of real-time quantitative PCR method for group A rotavirus detection].

    PubMed

    Guo, Yan-Qing; Li, Dan-Di; Duan, Zhao-Jun

    2013-11-01

    Group A rotavirus is one of the most significant etiological agents which causes acute gastroenteritis among infants and young children worldwide. So far, several method which includes electron microscopy (EM), enzyme immunoassay (EIA), reverse transcription-polymerase chain reaction (RT-PCR)and Real-time Quantitative PCR has been established for the detection of rotavirus. Compared with other methods, Real-time quantitative PCR have advantages in specificity, sensitivity, genotyping and quantitative accuracy. This article shows a overview of the application of real-time quantitative PCR technique to detecte group A rotavirus.

  8. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)

    PubMed Central

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

    2012-01-01

    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. PMID:23105972

  9. Specific PCR and real-time PCR assays for detection and quantitation of 'Candidatus Phytoplasma phoenicium'.

    PubMed

    Jawhari, Maan; Abrahamian, Peter; Sater, Ali Abdel; Sobh, Hana; Tawidian, Patil; Abou-Jawdah, Yusuf

    2015-02-01

    Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.

  10. Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters.

    PubMed

    Wadle, Simon; Lehnert, Michael; Schuler, Friedrich; Köppel, René; Serr, Annerose; Zengerle, Roland; von Stetten, Felix

    2016-01-01

    Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2-5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength.

  11. Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters.

    PubMed

    Wadle, Simon; Lehnert, Michael; Schuler, Friedrich; Köppel, René; Serr, Annerose; Zengerle, Roland; von Stetten, Felix

    2016-01-01

    Mediator probe (MP) PCR is a real-time PCR approach that uses standardized universal fluorogenic reporter oligonucleotides (UR) in conjunction with label-free sequence-specific probes. To enable multiplex real-time MP PCR, we designed a set of five optimized URs with different fluorescent labels. Performance of the optimized URs was verified in multiplex real-time MP PCR for the detection of a pentaplex food panel and a quadruplex methicillin-resistant Staphylococcus aureus (MRSA) panel. Results were comparable to corresponding multiplex hydrolysis probe (HP) PCR, also designated as TaqMan PCR. Analyses of MRSA DNA standards and DNA extracted from patient swab samples showed improved lower limits of detection (LoDs) by a factor of 2-5 when using quadruplex real-time MP PCR instead of HP PCR. The novel set of standardized URs we present here simplifies development of multiplex real-time PCR assays by requiring only the design of label-free probes. In the future, real-time PCR master mixes could be augmented with up to five standardized fluorogenic URs, each emitting light at a different wavelength. PMID:27625206

  12. Real-time-PCR assay for diagnosis of Entamoeba histolytica infection.

    PubMed

    Roy, Shantanu; Kabir, Mamun; Mondal, Dinesh; Ali, Ibne Karim M; Petri, William A; Haque, Rashidul

    2005-05-01

    We developed a real-time-PCR assay utilizing a molecular-beacon probe for the detection of Entamoeba histolytica and compared its sensitivity to stool antigen detection and traditional PCR. A total of 205 stool and liver abscess pus specimens from patients and controls were used for this purpose, 101 (49%) of which were positive by the TechLab E. histolytica-specific antigen detection test, while the other 104 (51%) stool and liver abscess pus specimens were negative by the antigen detection test. DNA was extracted from the stool and liver abscess pus specimens by the QIAGEN method and the small-subunit rRNA gene of E. histolytica and then amplified by traditional and real-time PCR. Out of these 205 stool and liver abscess pus specimens, 124 were positive by the real-time-PCR assay and 90 were positive by the traditional-PCR test. Compared to the real-time-PCR assay, the antigen detection test was 79% sensitive and 96% specific. When the traditional-PCR test results were compared to the real-time-PCR assay, the sensitivity of traditional PCR was 72% and the specificity was 99%. In conclusion, all three methods for the detection of E. histolytica were highly specific, with real-time PCR being the most sensitive.

  13. Novel real-time PCR detection assay for Brucella suis

    PubMed Central

    Hänsel, C.; Mertens, K.; Elschner, M. C.; Melzer, F.

    2015-01-01

    Introduction Brucella suis is the causative agent of brucellosis in suidae and is differentiated into five biovars (bv). Biovars 1 and 3 possess zoonotic potential and can infect humans, whereas biovar 2 represents the main source of brucellosis in feral and domestic pigs in Europe. Both aspects, the zoonotic threat and the economic loss, emphasize the necessity to monitor feral and domestic pig populations. Available serological or PCR based methods lack sensitivity and specificity. Results Here a bioinformatics approach was used to identify a B. suis specific 17 bp repeat on chromosome II (BS1330_II0657 locus). This repeat is common for B. suis bv 1 to 4 and was used to develop a TaqMan probe assay. The average PCR efficiency was determined as 95% and the limit of detection as 12,5 fg/µl of DNA, equally to 3.7 bacterial genomes. This assay has the highest sensitivity of all previously described B. suis specific PCR assays, making it possible to detect 3-4 bacterial genomes per 1 µl of sample. The assay was tested 100% specific for B. suis and negative for other Brucella spp. and closely related non-Brucella species. Conclusions This novel qPCR assay could become a rapid, inexpensive and reliable screening method for large sample pools of B. suis 1 to 4. This method will be applicable for field samples after validation. PMID:26392898

  14. [Identification for genetically modified maize T14/T25 with real time fluorescent PCR method].

    PubMed

    Cao, Ji-Juan; Qin, Wen; Zhu, Shui-Fang; Cao, Yuan-Yin

    2004-09-01

    To identify genetically modified (GM) maize T14/T25 lines, a real-time fluorescent PCR (RTF PCR) assay was performed in this study. Primers and Taqman probes specific for inserted genes in the T14/T25 were used to conduct the real-time fluorescent (RTF) PCR and PCR assays. The RTF PCR method was established to detect and identify GM maize lines. The results show that the TaqMan probe could identify T14/T25 maize used, while other GM and NO-GM maize didn't be detected. The RTF PCR could be a new method for detecting other genetically modified organism.

  15. Optimal swab processing recovery method for detection of bioterrorism-related Francisella tularensis by real-time PCR.

    PubMed

    Walker, Roblena E; Petersen, Jeannine M; Stephens, Kenyatta W; Dauphin, Leslie A

    2010-10-01

    Francisella tularensis, the etiological agent of tularemia, is regarded as a potential bioterrorism agent. The advent of bioterrorism has heightened awareness of the need for validated methods for processing environmental samples. In this study we determined the optimal method for processing environmental swabs for the recovery and subsequent detection of F. tularensis by the use of real-time PCR assays. Four swab processing recovery methods were compared: heat, sonication, vortexing, and the Swab Extraction Tube System (SETS). These methods were evaluated using cotton, foam, polyester and rayon swabs spiked with six pathogenic strains of F. tularensis. Real-time PCR analysis using a multi-target 5'nuclease assay for F. tularensis showed that the use of the SETS method resulted in the best limit of detection when evaluated using multiple strains of F. tularensis. We demonstrated also that the efficiency of F. tularensis recovery from swab specimens was not equivalent for all swab processing methodologies and, thus, that this variable can affect real-time PCR assay sensitivity. The effectiveness of the SETS method was independent of the automated DNA extraction method and real-time PCR platforms used. In conclusion, diagnostic laboratories can now potentially incorporate the SETS method into specimen processing protocols for the rapid and efficient detection of F. tularensis by real-time PCR during laboratory bioterrorism-related investigations.

  16. Comprehensive GMO detection using real-time PCR array: single-laboratory validation.

    PubMed

    Mano, Junichi; Harada, Mioko; Takabatake, Reona; Furui, Satoshi; Kitta, Kazumi; Nakamura, Kosuke; Akiyama, Hiroshi; Teshima, Reiko; Noritake, Hiromichi; Hatano, Shuko; Futo, Satoshi; Minegishi, Yasutaka; Iizuka, Tayoshi

    2012-01-01

    We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports.

  17. Comprehensive GMO detection using real-time PCR array: single-laboratory validation.

    PubMed

    Mano, Junichi; Harada, Mioko; Takabatake, Reona; Furui, Satoshi; Kitta, Kazumi; Nakamura, Kosuke; Akiyama, Hiroshi; Teshima, Reiko; Noritake, Hiromichi; Hatano, Shuko; Futo, Satoshi; Minegishi, Yasutaka; Iizuka, Tayoshi

    2012-01-01

    We have developed a real-time PCR array method to comprehensively detect genetically modified (GM) organisms. In the method, genomic DNA extracted from an agricultural product is analyzed using various qualitative real-time PCR assays on a 96-well PCR plate, targeting for individual GM events, recombinant DNA (r-DNA) segments, taxon-specific DNAs, and donor organisms of the respective r-DNAs. In this article, we report the single-laboratory validation of both DNA extraction methods and component PCR assays constituting the real-time PCR array. We selected some DNA extraction methods for specified plant matrixes, i.e., maize flour, soybean flour, and ground canola seeds, then evaluated the DNA quantity, DNA fragmentation, and PCR inhibition of the resultant DNA extracts. For the component PCR assays, we evaluated the specificity and LOD. All DNA extraction methods and component PCR assays satisfied the criteria set on the basis of previous reports. PMID:22649939

  18. Rapid, sensitive, and specific detection of Mycobacterium tuberculosis complex by real-time PCR on paraffin-embedded human tissues.

    PubMed

    Lee, Hye Seung; Park, Kyoung Un; Park, Jung Ok; Chang, Ho Eun; Song, Junghan; Choe, Gheeyoung

    2011-07-01

    The detection of Mycobacterium tuberculosis complex (MTB) in clinical specimens is important for diagnosing and caring for patients in whom tuberculosis is clinically suspected. We collected 129 FFPE specimens, including 56 nontuberculosis cases, 63 MTB cases, and 10 nontuberculous mycobacteria (NTM) cases determined by acid-fast bacilli (AFB) culture. We performed AFB staining; nested MTB PCR, targeting the IS6110 gene; and real-time MTB PCR, targeting the senX3-regX3 intergenic region in the 129 FFPE specimens. The sensitivity and specificity of AFB staining were 37.0% and 98.2%, respectively, using AFB culture results as the reference standard. The sensitivity and specificity of detecting MTB were 68.3% and 98.5%, respectively, by nested PCR; and 74.6% and 98.5% by real-time PCR, respectively. Among the 129 specimens, four were positive by AFB staining but negative by nested or real-time PCR. NTM grew in all four of these cases by AFB culture. AFB density in FFPE tissue sections significantly correlated with MTB DNA load. Thus, real-time PCR is a useful diagnostic tool for rapid and sensitive MTB detection in FFPE specimens, whereas NTM should be included in differential diagnoses of cases positive by AFB staining but negative by PCR.

  19. Diagnostic Molecular Mycobacteriology in Regions With Low Tuberculosis Endemicity: Combining Real-time PCR Assays for Detection of Multiple Mycobacterial Pathogens With Line Probe Assays for Identification of Resistance Mutations.

    PubMed

    Deggim-Messmer, Vanessa; Bloemberg, Guido V; Ritter, Claudia; Voit, Antje; Hömke, Rico; Keller, Peter M; Böttger, Erik C

    2016-07-01

    Molecular assays have not yet been able to replace time-consuming culture-based methods in clinical mycobacteriology. Using 6875 clinical samples and a study period of 35months we evaluated the use of PCR-based assays to establish a diagnostic workflow with a fast time-to-result of 1-2days, for 1. detection of Mycobacterium tuberculosis complex (MTB), 2. detection and identification of nontuberculous mycobacteria (NTM), and 3. identification of drug susceptible MTB. MTB molecular-based detection and culture gave concordant results for 97.7% of the specimens. NTM PCR-based detection and culture gave concordant results for 97.0% of the specimens. Defining specimens on the basis of combined laboratory data as true positives or negatives with discrepant results resolved by clinical chart reviews, we calculated sensitivity, specificity, PPV and NPV for PCR-based MTB detection as 84.7%, 100%, 100%, and 98.7%; the corresponding values for culture-based MTB detection were 86.3%, 100%, 100%, and 98.8%. PCR-based detection of NTM had a sensitivity of 84.7% compared to 78.0% of that of culture-based NTM detection. Molecular drug susceptibility testing (DST) by line-probe assay was found to predict phenotypic DST results in MTB with excellent accuracy. Our findings suggest a diagnostic algorithm to largely replace lengthy culture-based techniques by rapid molecular-based methods.

  20. Sensitivity of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood

    PubMed Central

    da Costa Lima, Manoel Sebastião; Zorzenon, Denielly Christina Rodrigues; Dorval, Maria Elizabeth Cavalheiros; Pontes, Elenir Rose Jardim Cury; Oshiro, Elisa Teruya; Cunha, Rodrigo; Andreotti, Renato; Matos, Maria de Fatima Cepa

    2013-01-01

    Objective To evaluate the effectiveness of PCR and real-time PCR for the diagnosis of human visceral leishmaniasis using peripheral blood samples. Methods DNA extraction was performed using Promega Wizard® Genomic kits. PCR employing RV1/RV2 primers yielded 145-bp amplicons. Real-time PCR was performed with the same primers and SYBR Green ROX Plus mix. These techniques were used to analyze 100 peripheral blood samples from patients with clinical signs of the disease. Results The sensitivity and specificity levels were 91,3%% and 29,6%, respectively, for real-time PCR and 97.78% and 61.82%, respectively, for PCR. Conclusions Real-time PCR proved to be a satisfactory method for the diagnosis of human visceral leishmaniasis.

  1. In silico comparison of real-time PCR probes for detection of pathogens.

    PubMed

    Ram, Siya; Singh, Ram Lakhan; Shanker, Rishi

    2008-01-01

    Rapid diagnostics and risk assessment of the pathogens is possible by Real-Time Polymerase Chain Reaction (PCR) probes like TaqMan, Molecular Beacon (MB) and FRET. However, validation of such probes for real-life samples is an expensive and time consuming proposition. Hence, development and comparison of real-time probes in silico can be the first step in selection of most appropriate probe chemistry. The virulence genes specific for a model pathogen, Escherichia coli O157:H7, transmitted worldwide by contaminated water and food, were chosen to compare probe chemistries. MB was observed to be the best probe chemistry for virulence genes stx1, stx2 and eae, while FRET was preferred for hlyA gene, based on Tm and free energy values for self-dimer, hairpin and cross-dimer. Secondary structure analysis indicated that MB design was flexible and less dependent on nucleotide arrangement and repetitive sequences in the genes compared to TaqMan and FRET probes. In addition, multiplexed MB probes could be a feasible option using a single non-fluorescent quencher for high throughput diagnostics.

  2. Clinical evaluation of a type III secretion system real-time PCR assay for diagnosing melioidosis.

    PubMed

    Meumann, Ella M; Novak, Ryan T; Gal, Daniel; Kaestli, Mirjam E; Mayo, Mark; Hanson, Joshua P; Spencer, Emma; Glass, Mindy B; Gee, Jay E; Wilkins, Patricia P; Currie, Bart J

    2006-08-01

    A Burkholderia pseudomallei type III secretion system real-time PCR assay was evaluated on clinical specimens in a region where melioidosis is endemic. The PCR was positive in 30/33 (91%) patients with culture-confirmed melioidosis. All six patients with melioidosis septic shock were blood PCR positive, suggesting potential for rapid diagnosis and commencement of appropriate therapy.

  3. Improved Strategies and Optimization of Calibration Models for Real-time PCR Absolute Quantification

    EPA Science Inventory

    Real-time PCR absolute quantification applications rely on the use of standard curves to make estimates of DNA target concentrations in unknown samples. Traditional absolute quantification approaches dictate that a standard curve must accompany each experimental run. However, t...

  4. Goose Hemorrhagic polyomavirus detection in geese using real-time PCR assay.

    PubMed

    Leon, Olivier; Corrand, Léni; Bich, Tran Ngoc; Le Minor, Odile; Lemaire, Mylène; Guérin, Jean-Luc

    2013-12-01

    Goose hemorrhagic polyomavirus (GHPV) is the viral agent of hemorrhagic nephritis enteritis of geese (HNEG), a lethal disease of goslings. Although death is the most common outcome, geese that recover from HNEG are persistently infected. Here, we present the development of real-time SYBR Green real-time PCR targeted to GHPV and its use to assess the prevalence of GHPV infection in French geese flocks. When compared with classical end-point PCR, real-time PCR revealed a much better sensitivity and equivalent specificity. Real-time PCR could, therefore, be considered a gold standard for the detection of GHPV. Results of field investigations evidenced a very high prevalence of GHPV infections in French geese, largely associated with healthy carriage.

  5. Detection of Yersinia pestis using real-time PCR in patients with suspected bubonic plague.

    PubMed

    Riehm, Julia M; Rahalison, Lila; Scholz, Holger C; Thoma, Bryan; Pfeffer, Martin; Razanakoto, Léa Mamiharisoa; Al Dahouk, Sascha; Neubauer, Heinrich; Tomaso, Herbert

    2011-02-01

    Yersinia (Y.) pestis, the causative agent of plague, is endemic in natural foci of Asia, Africa, and America. Real-time PCR assays have been described as rapid diagnostic tools, but so far none has been validated for its clinical use. In a retrospective clinical study we evaluated three real-time PCR assays in two different assay formats, 5'-nuclease and hybridization probes assays. Lymph node aspirates from 149 patients from Madagascar with the clinical diagnosis of bubonic plague were investigated for the detection of Y. pestis DNA. Results of real-time PCR assays targeting the virulence plasmids pPCP1 (pla gene), and pMT1 (caf1, Ymt genes) were compared with an F1-antigen immunochromatographic test (ICT) and cultivation of the organism. Out of the 149 samples an infection with Y. pestis was confirmed by culture in 47 patients while ICT was positive in 88 including all culture proven cases. The best real-time PCR assay was the 5'-nuclease assay targeting pla which was positive in 120 cases. In conclusion, the 5'-nuclease assay targeting pla can be recommended as diagnostic tool for establishing a presumptive diagnosis when bubonic plague is clinically suspected.

  6. Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis.

    PubMed

    Sales, Mariana L; Fonseca Júnior, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Soares Filho, Paulo Martins; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2014-01-01

    Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 - 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% - 100%) and 100% (CI = 93.98% - 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method.

  7. Validation of a real-time PCR assay for the molecular identification of Mycobacterium tuberculosis

    PubMed Central

    Sales, Mariana L.; Fonseca, Antônio Augusto; Orzil, Lívia; Alencar, Andrea Padilha; Silva, Marcio Roberto; Issa, Marina Azevedo; Filho, Paulo Martins Soares; Lage, Andrey Pereira; Heinemann, Marcos Bryan

    2014-01-01

    Mycobacterium tuberculosis is the major cause of tuberculosis in humans. This bacillus gained prominence with the occurrence of HIV, presenting itself as an important opportunistic infection associated with acquired immunodeficiency syndrome (AIDS). The current study aimed to develop a real-time PCR using Eva Green technology for molecular identification of M. tuberculosis isolates. The primers were designed to Rv1510 gene. Ninety nine samples of M. tuberculosis and sixty samples of M. bovis were tested and no sample of the bovine bacillus was detected by the qPCR. Statistical tests showed no difference between the qPCR and biochemical tests used to identify the Mycobacterium tuberculosis. The correlation between tests was perfect with Kappa index of 1.0 (p < 0.001, CI = 0.84 – 1.0). The diagnostic sensitivity and specificity were 100% (CI = 95.94% – 100%) and 100% (CI = 93.98% – 100%). This qPCR was developed with the goal of diagnosing the bacillus M. tuberculosis in samples of bacterial suspension. TB reference laboratories (health and agriculture sectors), public health programs and epidemiological studies probably may benefit from such method. PMID:25763042

  8. Real time PCR on disposable PDMS chip with a miniaturized thermal cycler.

    PubMed

    Xiang, Q; Xu, B; Fu, R; Li, D

    2005-12-01

    This paper presents the design and implementation of a low-cost miniature PCR device consisting of a disposable reactor chip and a miniature thermal cycler. The simple fabrication of the PCR chip by PDMS (Polydimethylsiloxane) does not need micro-machining or photolithography processes. The thermal cycler was built with a thin film heater for heating and a fan for rapid cooling. This device can perform PCR tests in a single well chip or a multiple-well chip. It can run PCR reactions of different volumes to meet specific application requirements. The smallest reaction volume tested in this work is 0.9 microL. In addition, this device fits any standard fluorescence microscope for real time detection, which makes real time PCR affordable for most research labs and clinics with a fluorescence microscope. Real-time PCR of E. coli stx1 has been demonstrated with the device described. PMID:16404505

  9. Real-Time PCR Assay for the Identification of the Brown Marmorated Stink Bug (Halyomorpha halys)

    PubMed Central

    Dhami, Manpreet K.; Dsouza, Melissa; Waite, David W.; Anderson, Diane; Li, Dongmei

    2016-01-01

    The brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae), is a gregarious crop pest that has rapidly spread across the world in the last two decades. It is an excellent hitchhiker species, especially as an over-wintering adult. During this period it is often associated with non-biological commodities such as shipping containers and machinery that travel long distances. Inadequate identification keys and similarity to common species has assisted its spread across Europe, while accurate identification from immature stages or eggs is not possible. We developed a real-time TaqMan PCR assay for the accurate and sensitive detection of the brown marmorated stink bug from all life stages. The assay performance against required diagnostic criterion and within a quarantine framework are described. PMID:26955631

  10. Development of a single tube multiplex real-time PCR to detect the most clinically relevant Mucormycetes species.

    PubMed

    Bernal-Martínez, L; Buitrago, M J; Castelli, M V; Rodriguez-Tudela, J L; Cuenca-Estrella, M

    2013-01-01

    Mucormycetes infections are very difficult to treat and a delay in diagnosis could be fatal for the outcome of the patient. A molecular diagnostic technique based on Real Time PCR was developed for the simultaneous detection of Rhizopus oryzae, Rhizopus microsporus and the genus Mucor spp. in both culture and clinical samples. The methodology used was Molecular beacon species-specific probes with an internal control. This multiplex real-time PCR (MRT-PCR) was tested in 22 cultured strains and 12 clinical samples from patients suffering from a proven mucormycosis. Results showed 100% specificity and a detection limit of 1 fg of DNA per microlitre of sample. The sensitivity was 100% for clinical cultured strains and for clinical samples containing species detected by the PCR assay. Other mucormycetes species were not detected in clinical samples. This technique can be useful for clinical diagnosis and further studies are warranted.

  11. Towards real time diagnostics of Hybrid Welding Laser/GMAW

    SciTech Connect

    McJunkin, T. R.; Kunerth, D. C.; Nichol, C. I.; Todorov, E.; Levesque, S.

    2014-02-18

    Methods are currently being developed towards a more robust system real time feedback in the high throughput process combining laser welding with gas metal arc welding. A combination of ultrasonic, eddy current, electronic monitoring, and visual techniques are being applied to the welding process. Initial simulation and bench top evaluation of proposed real time techniques on weld samples are presented along with the concepts to apply the techniques concurrently to the weld process. Consideration for the eventual code acceptance of the methods and system are also being researched as a component of this project. The goal is to detect defects or precursors to defects and correct when possible during the weld process.

  12. Towards Real Time Diagnostics of Hybrid Welding Laser/GMAW

    SciTech Connect

    Timothy Mcjunkin; Dennis C. Kunerth; Corrie Nichol; Evgueni Todorov; Steve Levesque; Feng Yu; Robert Danna Couch

    2013-07-01

    Methods are currently being developed towards a more robust system real time feedback in the high throughput process combining laser welding with gas metal arc welding. A combination of ultrasonic, eddy current, electronic monitoring, and visual techniques are being applied to the welding process. Initial simulation and bench top evaluation of proposed real time techniques on weld samples are presented along with the concepts to apply the techniques concurrently to the weld process. Consideration for the eventual code acceptance of the methods and system are also being researched as a component of this project. The goal is to detect defects or precursors to defects and correct when possible during the weld process.

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

    PubMed

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

    2016-01-01

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

  14. Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil.

    PubMed

    Kim, Tae Gwan; Jeong, So-Yeon; Cho, Kyung-Suk

    2014-07-01

    The newly developed droplet digital PCR (DD-PCR) has shown promise as a DNA quantification technology in medical diagnostic fields. This study evaluated the applicability of DD-PCR as a quantitative tool for soil DNA using quantitative real-time PCR (qRT-PCR) as a reference technology. Cupriavidus sp. MBT14 and Sphingopyxis sp. MD2 were used, and a primer/TaqMan probe set was designed for each (CupMBT and SphMD2, respectively). Standard curve analyses on tenfold dilution series showed that both qRT-PCR and DD-PCR exhibited excellent linearity (R (2) = 1.00) and PCR efficiency (≥92 %) across their detectable ranges. However, DD-PCR showed a tenfold greater sensitivity than qRT-PCR. MBT14 and MD2 were added to non-sterile soil at 0 ~ 5 × 10(8) and 0 ~ 5 × 10(7) cells per gram of soil, respectively (n = 5). This bacterial load test indicated that DD-PCR was more sensitive and discriminating than qRT-PCR. For instance, DD-PCR showed a gradual DNA increase from 14 to 141,160 MBT14 rDNA copies μL DNA extract(-1) as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies μL DNA(-1)) at ≥5 × 10(5) MBT14 per gram of soil. When temporal DNA changes were monitored for 3 weeks in the amended soils, the two technologies exhibited nearly identical changes over time. Linearity tests (y = a · x) revealed excellent quantitative agreement between the two technologies (a = 0.98, R (2) = 0.97 in the CupMBT set and a = 0.90, R (2) = 0.94 in the SphMD2 set). These results suggest that DD-PCR is a promising tool to examine temporal dynamics of microorganisms in complex environments.

  15. 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.

  16. Microdroplet Sandwich Real-Time RT-PCR for Detection of Pandemic and Seasonal Influenza Subtypes

    PubMed Central

    Angione, Stephanie L.; Inde, Zintis; Beck, Christina M.; Artenstein, Andrew W.; Opal, Steven M.; Tripathi, Anubhav

    2013-01-01

    As demonstrated by the recent 2012/2013 flu epidemic, the continual emergence of new viral strains highlights the need for accurate medical diagnostics in multiple community settings. If rapid, robust, and sensitive diagnostics for influenza subtyping were available, it would help identify epidemics, facilitate appropriate antiviral usage, decrease inappropriate antibiotic usage, and eliminate the extra cost of unnecessary laboratory testing and treatment. Here, we describe a droplet sandwich platform that can detect influenza subtypes using real-time reverse-transcription polymerase chain reaction (rtRT-PCR). Using clinical samples collected during the 2010/11 season, we effectively differentiate between H1N1p (swine pandemic), H1N1s (seasonal), and H3N2 with an overall assay sensitivity was 96%, with 100% specificity for each subtype. Additionally, we demonstrate the ability to detect viral loads as low as 104 copies/mL, which is two orders of magnitude lower than viral loads in typical infected patients. This platform performs diagnostics in a miniaturized format without sacrificing any sensitivity, and can thus be easily developed into devices which are ideal for small clinics and pharmacies. PMID:24066051

  17. Quantitative detection of hazelnut (Corylus avellana) in cookies: ELISA versus real-time PCR.

    PubMed

    Platteau, Céline; De Loose, Marc; De Meulenaer, Bruno; Taverniers, Isabel

    2011-11-01

    Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted.

  18. Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia

    PubMed Central

    Chung, Yousun; Kim, Taek Soo; Min, Young Gi; Hong, Yun Ji; Park, Jeong Su; Hwang, Sang Mee; Song, Kyoung-Ho; Kim, Eu Suk; Kim, Hong Bin; Song, Junghan; Kim, Eui-Chong

    2016-01-01

    Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targets mecA, femA specific for S. aureus, femA specific for S. epidermidis, 16S rRNA for universal bacteria, and 16S rRNA specific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC). For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA), the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. epidermidis (MRSE), methicillin-susceptible S. epidermidis (MSSE), methicillin-resistant non-S. epidermidis CoNS (MRCoNS), and methicillin-susceptible non-S. epidermidis CoNS (MSCoNS) (κ = 0.9313). The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings. PMID:27403436

  19. Characterization of Chinook head salmon embryo phenotypes of infectious salmon anemia virus by real-time RT-PCR.

    PubMed

    Munir, Khalid

    2006-06-01

    We have previously described the development of a onetube SYBR Green real-time RT-PCR assay for the detection and quantitation of infectious salmon anemia virus (ISAV) in various biological samples. The twofold aim of the present study was to verify that the optimized SYBR Green real-time RT-PCR conditions could detect ISAV isolates of different geographic origins, and to analyze the growth patterns of the selected ISAV isolates in the Chinook head salmon embryo (CHSE) -214 cells by this assay to better characterize their CHSE-phenotypes. A total of 24 ISAV isolates were used in this study. The results indicated that the SYBR Green real-time RT-PCR could detect ISAV of different geographic origins or laboratory sources. The capacity of ISAV isolates to cause cytopathic effects (CPE) in the CHSE-214 cell line, viral titration of the infected CHSE-cell harvests, and analysis of viral RNA levels in CHSE-214 cells at post-infection day zero, 7 and 14 by SYBR Green real-time RT-PCR confirmed the existence of three CHSE-phenotypes of ISAV: replicating cytopathic, replicating non-cytopathic, and non-replicating non-cytopathic. The identification of these three CHSE- phenotypes of ISAV has important implications from diagnostic and biological points of view.

  20. Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia.

    PubMed

    Chung, Yousun; Kim, Taek Soo; Min, Young Gi; Hong, Yun Ji; Park, Jeong Su; Hwang, Sang Mee; Song, Kyoung-Ho; Kim, Eu Suk; Park, Kyoung Un; Kim, Hong Bin; Song, Junghan; Kim, Eui-Chong

    2016-01-01

    Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targets mecA, femA specific for S. aureus, femA specific for S. epidermidis, 16S rRNA for universal bacteria, and 16S rRNA specific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC). For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA), the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. epidermidis (MRSE), methicillin-susceptible S. epidermidis (MSSE), methicillin-resistant non-S. epidermidis CoNS (MRCoNS), and methicillin-susceptible non-S. epidermidis CoNS (MSCoNS) (κ = 0.9313). The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings. PMID:27403436

  1. 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...

  2. Ten hour real-time PCR technique for detection of Salmonella in meats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We evaluated the efficacy of real-time PCR assays to detect low levels of Salmonella in meats following 8 h of pre-enrichment. The sensitivity and accuracy of molecular beacon and TaqMan probe PCR assays were compared with the conventional USDA microbiological procedure using artificially contaminat...

  3. 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...

  4. Molecular-beacon multiplex real-time PCR assay for detection of Vibrio cholerae.

    PubMed

    Gubala, Aneta J; Proll, David F

    2006-09-01

    A multiplex real-time PCR assay was developed using molecular beacons for the detection of Vibrio cholerae by targeting four important virulence and regulatory genes. The specificity and sensitivity of this assay, when tested with pure culture and spiked environmental water samples, were high, surpassing those of currently published PCR assays for the detection of this organism.

  5. 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...

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

    PubMed Central

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

    2013-01-01

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

  7. Quantitation of viral load by real-time PCR-monitoring Invader reaction.

    PubMed

    Tadokoro, Kenichi; Yamaguchi, Toshikazu; Egashira, Toru; Hara, Takashi

    2009-02-01

    With its broad effective range for fluorescence detection, real-time PCR is one of the most valuable techniques for quantitation in molecular biology. A modified real-time PCR assay is described for determining viral load. The assay uses fluorescence to measure the number of PCR amplicons by monitoring the Invader reaction in four steps in the thermal cycle. The Invader reaction with its cleavase was performed at moderate temperature after the amplicon was denatured at a high temperature. The method was as effective as real-time PCR with a TaqMan probe in determining the quantity of virus in samples of human papillomavirus type 16. Importantly, the assay allows the use of a common probe for multiple reactions. Thus, this method is a rapid inexpensive assay with a common fluorescence probe that does not depend on the conformation of the target DNAs. PMID:19014973

  8. Comparison of Real-Time PCR Signal-Amplified In Situ Hybridization and Conventional PCR for Detection and Quantification of Human Papillomavirus in Archival Cervical Cancer Tissue

    PubMed Central

    Biedermann, Karin; Dandachi, Nadia; Trattner, Maria; Vogl, Georgia; Doppelmayr, Hildegard; Moré, Elena; Staudach, Alfons; Dietze, Otto; Hauser-Kronberger, Cornelia

    2004-01-01

    Archival paraffin-embedded tumor specimens offer a wealth of information for both cancer research and for routine clinical applications. However, the use of formalin-fixed, paraffin-embedded specimens for quantitative real-time PCR is not yet a standard diagnostic method in many laboratories, in particular for the quantification of human papillomavirus (HPV). Particularly high-risk HPV types are involved in almost 100% of the carcinogenesis of cervical cancer. We compared the diagnostic applicability and sensitivity of real-time PCR to that of chromogenic tyramide-signal-amplified in situ hybridization and conventional PCR for the detection of HPV from archival tissue in 164 cases of carcinoma in situ and cervical cancer. Furthermore, we examined whether the viral load of HPV is of prognostic relevance. Our findings indicate that patients in tumor stage I with a lower viral load of HPV type 16 (HPV16; up to 1,000 copies/ng of DNA) had a significantly better survival than HPV 16-negative patients (P = 0.037). We observed a greater sensitivity of both real-time PCR and conventional PCR for the detection of HPV16 and -18 compared to signal amplified in situ hybridization. We found a considerable concordance between HPV16 (κ = 0.661) and HPV18 (κ = 0.781) status as measured by real-time PCR and conventional PCR, indicating similar sensitivities. We recognized an inhibitory effect of formalin fixation and paraffin embedding on the evaluation of real-time PCR quantification. PMID:15297527

  9. [Real-time PCR detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae DNA in clinical specimens].

    PubMed

    Vacková, Z; Lžičařová, D; Stock, N K; Kozáková, J

    2015-10-01

    The study aim was to implement a molecular real-time polymerase chain reaction (PCR) assay recommended by the CDC (Centers for Disease Control and Prevention) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in clinical (culture negative) specimens from patients with suspected invasive bacterial disease. Clinical specimens are referred to the National Reference Laboratory (NRL) for Meningococcal Infections, Unit for Airborne Bacterial Infections, Centre for Epidemiology and Microbiology, National Institute of Public Health from various regions of the Czech Republic. Clinical specimens are, in particular, cerebrospinal fluid, anti-coagulated blood or serum and, exceptionally, post-mortem specimens. The NRL has implemented molecular diagnosis of these bacterial pathogens involved in meningitis and sepsis from clinical specimens since 1999. The first diagnostic method was semi-nested PCR followed by electrophoretic analysis. In 2014, a molecular qualitative real-time PCR assay was implemented.

  10. Real-time PCR assays compared to culture-based approaches for identification of aerobic bacteria in chronic wounds.

    PubMed

    Melendez, J H; Frankel, Y M; An, A T; Williams, L; Price, L B; Wang, N-Y; Lazarus, G S; Zenilman, J M

    2010-12-01

    Chronic wounds cause substantial morbidity and disability. Infection in chronic wounds is clinically defined by routine culture methods that can take several days to obtain a final result, and may not fully describe the community of organisms or biome within these wounds. Molecular diagnostic approaches offer promise for a more rapid and complete assessment. We report the development of a suite of real-time PCR assays for rapid identification of bacteria directly from tissue samples. The panel of assays targets 14 common, clinically relevant, aerobic pathogens and demonstrates a high degree of sensitivity and specificity using a panel of organisms commonly associated with chronic wound infection. Thirty-nine tissue samples from 29 chronic wounds were evaluated and the results compared with those obtained by culture. As revealed by culture and PCR, the most common organisms were methicillin-resistant Staphylococcus aureus (MRSA) followed by Streptococcus agalactiae (Group B streptococcus) and Pseudomonas aeruginosa. The sensitivities of the PCR assays were 100% and 90% when quantitative and qualitative culture results were used as the reference standard, respectively. The assays allowed the identification of bacterial DNA from ten additional organisms that were not revealed by quantitative or qualitative cultures. Under optimal conditions, the turnaround time for PCR results is as short as 4-6 h. Real-time PCR is a rapid and inexpensive approach that can be easily introduced into clinical practice for detection of organisms directly from tissue samples. Characterization of the anaerobic microflora by real-time PCR of chronic wounds is warranted.

  11. Detection of 12 respiratory viruses by duplex real time PCR assays in respiratory samples.

    PubMed

    Arvia, Rosaria; Corcioli, Fabiana; Ciccone, Nunziata; Della Malva, Nunzia; Azzi, Alberta

    2015-12-01

    Different viruses can be responsible for similar clinical manifestations of respiratory infections. Thus, the etiological diagnosis of respiratory viral diseases requires the detection of a large number of viruses. In this study, 6 duplex real-time PCR assays, using EvaGreen intercalating dye, were developed to detect 12 major viruses responsible for respiratory diseases: influenza A and B viruses, enteroviruses (including enterovirus spp, and rhinovirus spp), respiratory syncytial virus, human metapneumovirus, coronaviruses group I (of which CoV 229E and CoV NL63 are part) and II (including CoV OC43 and CoV HKU1), parainfluenza viruses type 1, 2, 3 and 4, human adenoviruses and human bocaviruses. The 2 target viruses of each duplex reaction were distinguishable by the melting temperatures of their amplicons. The 6 duplex real time PCR assays were applied for diagnostic purpose on 202 respiratory samples from 157 patients. One hundred fifty-seven samples were throat swabs and 45 were bronchoalveolar lavages. The results of the duplex PCR assays were confirmed by comparison with a commercial, validated, assay; in addition, the positive results were confirmed by sequencing. The analytical sensitivity of the duplex PCR assays varied from 10(3) copies/ml to 10(4) copies/ml. For parainfluenza virus 2 only it was 10(5) copies/ml. Seventy clinical samples (35%) from 55 patients (30 children and 25 adults) were positive for 1 or more viruses. In adult patients, influenza A virus was the most frequently detected respiratory virus followed by rhinoviruses. In contrast, respiratory syncytial virus was the most common virus in children, followed by enteroviruses, influenza A virus and coronavirus NL63. The small number of samples/patients does not allow us to draw any epidemiological conclusion. Altogether, the results of this study indicate that the 6 duplex PCR assays described in this study are sensitive, specific and cost-effective. Thus, this assay could be

  12. Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR.

    PubMed

    Liang, Ningjian; Dong, Jin; Luo, Laixin; Li, Yong

    2011-05-01

    Contamination of lettuce by Salmonella has caused serious public health problems. Polymerase chain reaction (PCR) allows rapid detection of pathogenic bacteria in food, but it is inaccurate as it might amplify DNA from dead target cells as well. This study aimed to investigate the stability of DNA of dead Salmonella cells in lettuce and to develop an approach to detecting viable Salmonella in lettuce. Salmonella-free lettuce was inoculated with heat-killed Salmonella Typhimurium cells and stored at 4 °C. Bacterial DNA extracted from the sample was amplified by real-time PCR targeting the invA gene. Our results indicate that DNA from the dead cells remained stable in lettuce for at least 8 d. To overcome this limitation, propidium monoazide (PMA), a dye that can selectively penetrate dead bacterial cells and cross-link their DNA upon light exposure, was combined with real-time PCR. Lettuce samples inoculated with different levels of dead or viable S. Typhimurium cells were treated or untreated with PMA before DNA extraction. Real-time PCR suggests that PMA treatment effectively prevented PCR amplification from as high as 10(8) CFU/g dead S. Typhimurium cells in lettuce. The PMA real-time PCR assay could detect viable Salmonella at as low as 10(2) CFU/mL in pure culture and 10(3) CFU/g in lettuce. With 12-h enrichment, S. Typhimurium of 10(1) CFU/g in lettuce was detectable. In conclusion, the PMA real-time PCR assay provides an alternative to real-time PCR assay for accurate detection of Salmonella in food.

  13. Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection.

    PubMed

    Hatch, Andrew C; Ray, Tathagata; Lintecum, Kelly; Youngbull, Cody

    2014-02-01

    High throughput automation is greatly enhanced using techniques that employ conveyor belt strategies with un-interrupted streams of flow. We have developed a 'conveyor belt' analog for high throughput real-time quantitative Polymerase Chain Reaction (qPCR) using droplet emulsion technology. We developed a low power, portable device that employs LED and fiber optic fluorescence excitation in conjunction with a continuous flow thermal cycler to achieve multi-channel fluorescence detection for real-time fluorescence measurements. Continuously streaming fluid plugs or droplets pass through tubing wrapped around a two-temperature zone thermal block with each wrap of tubing fluorescently coupled to a 64-channel multi-anode PMT. This work demonstrates real-time qPCR of 0.1-10 μL droplets or fluid plugs over a range of 7 orders of magnitude concentration from 1 × 10(1) to 1 × 10(7). The real-time qPCR analysis allows dynamic range quantification as high as 1 × 10(7) copies per 10 μL reaction, with PCR efficiencies within the range of 90-110% based on serial dilution assays and a limit of detection of 10 copies per rxn. The combined functionality of continuous flow, low power thermal cycling, high throughput sample processing, and real-time qPCR improves the rates at which biological or environmental samples can be continuously sampled and analyzed. PMID:24297040

  14. Interlaboratory Validation for a Real-Time PCR Salmonella Detection Method Using the ABI 7500 FAST Real-Time PCR System.

    PubMed

    Cheng, Chorng-Ming; Doran, Tara; Lin, Wen; Chen, Kai-Shun; Williams-Hill, Donna; Pamboukian, Ruiqing

    2015-06-01

    Sixteen FERN (Food Emergency Response Network) member laboratories collaborated in this study to verify extension of the real-time PCR Salmonella detection method originally designed for the single-tube Cepheid SmartCycler II and validated against the Salmonella method of the U. S. Food and Drug Administration Bacteriological Analytical Manual to the Applied Biosystems (ABI) 7500 FAST Real-Time PCR system multiwell plate platform. Four foods were selected for this study: chili powder, soft cheese, fish, and tomatoes; these foods represent products that are commonly analyzed for the presence of Salmonella for regulatory purposes. Each food consisted of six uninoculated control samples, six samples inoculated with low Salmonella levels (target 1 to 5 CFU/25 g), and six samples inoculated with high levels (target 10 to 50 CFU/25 g). All samples were tested for Salmonella using the 24-h quantitative PCR (qPCR) method for detecting Salmonella, which utilizes modified buffered peptone water as the sole enrichment medium and an internal control for the qPCR. Each of these 18 samples was individually analyzed for Salmonella by the collaborating laboratories using both the ABI 7500 FAST system (alternative method) and the SmartCycler II system (reference method). Statistical analysis of the data revealed no significant difference (P ≥ 0.05) between these two qPCR platforms except for the chili powder samples. The differences noted with chili powder (P = 0.0455) were attributed to the enhanced sensitivity of the ABI 7500 FAST system compared with the SmartCycler II system. The detection limit of both qPCR methods was 0.02 to 0.15 CFU/g. These results provide a solid basis for extending the 24-h qPCR Salmonella method to the ABI 7500 FAST system for high-throughput detection of Salmonella in foods. PMID:26038901

  15. Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum.

    PubMed

    Schneider, Petra; Wolters, Liselotte; Schoone, Gerard; Schallig, Henk; Sillekens, Peter; Hermsen, Rob; Sauerwein, Robert

    2005-01-01

    Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum, such as intervention or vaccine trials. To circumvent this problem, two more sensitive assays, real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) and real-time quantitative PCR (QT-PCR) were compared for quantification of P. falciparum parasites. QT-NASBA was adapted to molecular beacon real-time detection technology, which enables a reduction of the time of analysis and of contamination risk while retaining the specificity and sensitivity of the original assay. Both QT-NASBA and QT-PCR have a sensitivity of 20 parasites/ml of blood, but QT-PCR requires a complicated DNA extraction procedure and the use of 500 microl of venous blood to achieve this sensitivity, compared to 50 microl of finger prick blood for real-time QT-NASBA. Both techniques show a significant correlation to microscopic parasite counts, and the quantification results of the two real-time assays are significantly correlated for in vitro as well as in vivo samples. However, in comparison to real-time QT-PCR, the results of real-time QT-NASBA can be obtained 12 h earlier, with relatively easy RNA extraction and use of finger prick blood samples. The prospective development of multiplex QT-NASBA for detection of various P. falciparum developmental stages increases the value of QT-NASBA for malaria studies. Therefore, for studies requiring sensitive and accurate detection of P. falciparum parasites in large numbers of samples, the use of real-time QT-NASBA is preferred over that of real-time QT-PCR.

  16. Detection of orthopoxvirus DNA by real-time PCR and identification of variola virus DNA by melting analysis.

    PubMed

    Nitsche, Andreas; Ellerbrok, Heinz; Pauli, Georg

    2004-03-01

    Although variola virus was eradicated by the World Health Organization vaccination program in the 1970s, the diagnosis of smallpox infection has attracted great interest in the context of a possible deliberate release of variola virus in bioterrorist attacks. Obviously, fast and reliable diagnostic tools are required to detect variola virus and to distinguish it from orthopoxviruses that have identical morphological characteristics, including vaccinia virus. The advent of real-time PCR for the clinical diagnosis of viral infections has facilitated the detection of minute amounts of viral nucleic acids in a fast, safe, and precise manner, including the option to quantify and to genotype the target reliably. In this study a complete set of four hybridization probe-based real-time PCR assays for the specific detection of orthopoxvirus DNA is presented. Melting analysis following PCR enables the identification of variola virus by the PCR product's characteristic melting temperature, permitting the discrimination of variola virus from other orthopoxviruses. In addition, an assay for the specific amplification of variola virus DNA is presented. All assays can be performed simultaneously in the same cycler, and results of a PCR run are obtained in less than 1 h. The application of more than one assay for the same organism significantly contributes to the diagnostic reliability, reducing the risk of false-negative results due to unknown sequence variations. In conclusion, the assays presented will improve the speed and reliability of orthopoxvirus diagnostics and variola virus identification.

  17. Microsatellite Length Scoring by Single Molecule Real Time Sequencing - Effects of Sequence Structure and PCR Regime.

    PubMed

    Liljegren, Mikkel Meyn; de Muinck, Eric Jacques; Trosvik, Pål

    2016-01-01

    Microsatellites are DNA sequences consisting of repeated, short (1-6 bp) sequence motifs that are highly mutable by enzymatic slippage during replication. Due to their high intrinsic variability, microsatellites have important applications in population genetics, forensics, genome mapping, as well as cancer diagnostics and prognosis. The current analytical standard for microsatellites is based on length scoring by high precision electrophoresis, but due to increasing efficiency next-generation sequencing techniques may provide a viable alternative. Here, we evaluated single molecule real time (SMRT) sequencing, implemented in the PacBio series of sequencing apparatuses, as a means of microsatellite length scoring. To this end we carried out multiplexed SMRT sequencing of plasmid-carried artificial microsatellites of varying structure under different pre-sequencing PCR regimes. For each repeat structure, reads corresponding to the target length dominated. We found that pre-sequencing amplification had large effects on scoring accuracy and error distribution relative to controls, but that the effects of the number of amplification cycles were generally weak. In line with expectations enzymatic slippage decreased proportionally with microsatellite repeat unit length and increased with repetition number. Finally, we determined directional mutation trends, showing that PCR and SMRT sequencing introduced consistent but opposing error patterns in contraction and expansion of the microsatellites on the repeat motif and single nucleotide level. PMID:27414800

  18. Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR.

    PubMed

    Castillo, Marisol; Martín-Orúe, Susana M; Manzanilla, Edgar G; Badiola, Ignacio; Martín, Marga; Gasa, Josep

    2006-04-16

    Jejunum digesta samples were taken from weaning pigs in order to evaluate real-time PCR (qPCR) as a method for quantifying pig gut bacteria. Total bacteria, lactobacilli and enterobacteria were quantified by qPCR and the results were compared with those obtained with traditional methods: 4',6-diamidino-2-phenylindole (DAPI staining) for total bacteria, selective culture for lactobacilli and enterobacteria. Real-time PCR showed higher values in terms of 16S rRNA gene copies than DAPI counts or CFU. Despite the differences, the lactobacilli:enterobacteria ratio was similar between methods (2.5 +/- 0.58 for qPCR and 3.1 +/- 0.71 for selective culture, P = 0.39). Possible reasons for the higher PCR counts are discussed considering both an overestimation with PCR by quantification of dead bacteria or free DNA and also an underestimation with conventional methods. Inherent differences in the pre-treatment of the samples could partially explain the discrepancies observed. Regardless of the numerical differences between methods, values obtained by qPCR and traditional methods showed a significant correlation for lactobacilli and total bacteria. In the light of these results, real-time PCR seems a valid method to quantify microbial shifts in the gastrointestinal tract. PMID:16384658

  19. Detection of enteroviruses and parechoviruses by a multiplex real-time RT-PCR assay.

    PubMed

    Pabbaraju, Kanti; Wong, Sallene; Wong, Anita A; Tellier, Raymond

    2015-04-01

    Detection of all enteroviruses while excluding cross-detection of rhinoviruses is challenging because of sequence similarities in the commonly used conserved targets for molecular assays. In addition, simultaneous detection and differentiation of enteroviruses and parechoviruses would be beneficial because of a similar clinical picture presented by these viruses. A sensitive and specific real-time RT-PCR protocol that can address these clinical needs would be valuable to molecular diagnostic laboratories. Here we report a multiplex nucleic acid based assay using hydrolysis probes targeting the 5' non-translated region for the detection and differentiation of enteroviruses and parechoviruses without cross-detection of rhinoviruses. This assay has been shown to detect enteroviruses belonging to the different species in a variety of specimen types without detecting the different species of rhinoviruses. Laboratory validation shows the assay to be sensitive, specific, reproducible, easy to set up and uses generic cycling conditions. This assay can be implemented for diagnostic testing of patient samples in a high throughput fashion.

  20. Real-time PCR assay for detection and quantification of hepatitis B virus genotypes A to G.

    PubMed

    Welzel, Tania M; Miley, Wendell J; Parks, Thomas L; Goedert, James J; Whitby, Denise; Ortiz-Conde, Betty A

    2006-09-01

    The detection and quantification of hepatitis B virus (HBV) DNA play an important role in diagnosing and monitoring HBV infection as well as assessing therapeutic response. The great variability among HBV genotypes and the enormous range of clinical HBV DNA levels present challenges for PCR-based amplification techniques. In this study, we describe the development, evaluation, and validation of a novel real-time PCR assay designed to provide accurate quantification of DNA from all eight HBV genotypes in patient plasma specimens. A computer algorithm was used to design degenerate real-time PCR primers and probes based upon a large number (n = 340) of full-length genomic sequences including HBV genotypes A to H from Europe, Africa, Asia, and North and South America. Genotype performance was tested and confirmed using 59 genotype A to G specimens from two commercially available worldwide genotype panels. This assay has a dynamic range of at least 8 log(10) without the need for specimen dilution, good clinical intra- and interassay precision, and excellent correlation with the Bayer Diagnostics VERSANT HBV DNA 3.0 (branched DNA) assay (r = 0.93). Probit analysis determined the 95% detection level was 56 IU/ml, corresponding to 11 copies per PCR well. The high sensitivity, wide linear range, good reproducibility, and genotype inclusivity, combined with a small sample volume requirement and low cost, make this novel quantitative HBV real-time PCR assay particularly well suited for application to large clinical and epidemiological studies.

  1. Detection of Zika virus by SYBR green one-step real-time RT-PCR.

    PubMed

    Xu, Ming-Yue; Liu, Si-Qing; Deng, Cheng-Lin; Zhang, Qiu-Yan; Zhang, Bo

    2016-10-01

    The ongoing Zika virus (ZIKV) outbreak has rapidly spread to new areas of Americas, which were the first transmissions outside its traditional endemic areas in Africa and Asia. Due to the link with newborn defects and neurological disorder, numerous infected cases throughout the world and various mosquito vectors, the virus has been considered to be an international public health emergency. In the present study, we developed a SYBR Green based one-step real-time RT-PCR assay for rapid detection of ZIKV. Our results revealed that the real-time assay is highly specific and sensitive in detection of ZIKV in cell samples. Importantly, the replication of ZIKV at different time points in infected cells could be rapidly monitored by the real-time RT-PCR assay. Specifically, the real-time RT-PCR showed acceptable performance in measurement of infectious ZIKV RNA. This assay could detect ZIKV at a titer as low as 1PFU/mL. The real-time RT-PCR assay could be a useful tool for further virology surveillance and diagnosis of ZIKV. PMID:27444120

  2. Detection of Zika virus by SYBR green one-step real-time RT-PCR.

    PubMed

    Xu, Ming-Yue; Liu, Si-Qing; Deng, Cheng-Lin; Zhang, Qiu-Yan; Zhang, Bo

    2016-10-01

    The ongoing Zika virus (ZIKV) outbreak has rapidly spread to new areas of Americas, which were the first transmissions outside its traditional endemic areas in Africa and Asia. Due to the link with newborn defects and neurological disorder, numerous infected cases throughout the world and various mosquito vectors, the virus has been considered to be an international public health emergency. In the present study, we developed a SYBR Green based one-step real-time RT-PCR assay for rapid detection of ZIKV. Our results revealed that the real-time assay is highly specific and sensitive in detection of ZIKV in cell samples. Importantly, the replication of ZIKV at different time points in infected cells could be rapidly monitored by the real-time RT-PCR assay. Specifically, the real-time RT-PCR showed acceptable performance in measurement of infectious ZIKV RNA. This assay could detect ZIKV at a titer as low as 1PFU/mL. The real-time RT-PCR assay could be a useful tool for further virology surveillance and diagnosis of ZIKV.

  3. High Throughput Gene Expression Measurement with Real Time PCR in a Microfluidic Dynamic Array

    PubMed Central

    Spurgeon, Sandra L.; Jones, Robert C.; Ramakrishnan, Ramesh

    2008-01-01

    We describe a high throughput gene expression platform based on microfluidic dynamic arrays. This system allows 2,304 simultaneous real time PCR gene expression measurements in a single chip, while requiring less pipetting than is required to set up a 96 well plate. We show that one can measure the expression of 45 different genes in 18 tissues with replicates in a single chip. The data have excellent concordance with conventional real time PCR and the microfluidic dynamic arrays show better reproducibility than commercial DNA microarrays. PMID:18301740

  4. Quantitative assay of photoinduced DNA strand breaks by real-time PCR.

    PubMed

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-01

    Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies.

  5. Is real-time polymerase chain reaction (PCR) more useful than a conventional PCR for the clinical management of leishmaniasis?

    PubMed

    Antinori, Spinello; Calattini, Sara; Piolini, Roberta; Longhi, Erika; Bestetti, Giovanna; Cascio, Antonio; Parravicini, Carlo; Corbellino, Mario

    2009-07-01

    It is currently unknown if the use of a real-time polymerase chain reaction (PCR) adds value to the diagnosis and follow-up prognosis of patients affected by leishmaniasis. We performed a study using a real-time PCR directed against the alpha-polymerase gene and a semiquantitative PCR that target the SSU ribosomal RNA (rRNA) gene as control for the diagnosis and quantification of parasites in patients with visceral (VL) and cutaneous (CL) leishmaniasis. Our single copy real-time PCR missed one diagnosis of VL compared with the conventional PCR, whereas both PCR methods were able to detect Leishmania parasites in CL. Under anti-leishmania treatment the kinetics of parasitemia were comparable with the two methods. The real-time PCR directed against alpha-polymerase of Leishmania despite being able to make a more accurate quantification of parasites does not add to the decision-making management compared with a semiquantitative PCR, and it is comparatively expensive.

  6. Performance of two real-time PCR assays for hepatitis B virus DNA detection and quantitation.

    PubMed

    Kania, Dramane; Ottomani, Laure; Meda, Nicolas; Peries, Marianne; Dujols, Pierre; Bolloré, Karine; Rénier, Wendy; Viljoen, Johannes; Ducos, Jacques; Van de Perre, Philippe; Tuaillon, Edouard

    2014-06-01

    In-house developed real-time PCR (qPCR) techniques could be useful conjunctives to the management of hepatitis B virus (HBV) infection in resource-limited settings with high prevalence. Two qPCR assays (qPCR1 and qPCR2), based on primers/probes targeting conserved regions of the X and S genes of HBV respectively, were evaluated using clinical samples of varying HBV genotypes, and compared to the commercial Roche Cobas AmpliPrep/Cobas TaqMan HBV Test v2.0. The lower detection limit (LDL) was established at 104 IU/ml for qPCR1, and 91 IU/ml for qPCR2. Good agreement and correlation were obtained between the Roche assay and both qPCR assays (r = 0.834 for qPCR1; and r = 0.870 for qPCR2). Differences in HBV DNA load of > 0.5 Log10 IU/ml between the Roche and the qPCR assays were found in 49/122 samples of qPCR1, and 35/122 samples of qPCR2. qPCR1 tended to underestimate HBV DNA quantity in samples with a low viral load and overestimate HBV DNA concentration in samples with a high viral load when compared to the Roche test. Both molecular tools that were developed, used on an open real-time PCR system, were reliable for HBV DNA detection and quantitation. The qPCR2 performed better than the qPCR1 and had the additional advantage of various HBV genotype detection and quantitation. This low cost quantitative HBV DNA PCR assay may be an alternative solution when implementing national programmes to diagnose, monitor and treat HBV infection in low- to middle-income countries where testing for HBV DNA is not available in governmental health programmes.

  7. Whole blood Nested PCR and Real-time PCR amplification of Talaromyces marneffei specific DNA for diagnosis.

    PubMed

    Lu, Sha; Li, Xiqing; Calderone, Richard; Zhang, Jing; Ma, Jianchi; Cai, Wenying; Xi, Liyan

    2016-02-01

    Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients.

  8. Quantification of DNA Damage by Real-Time qPCR.

    PubMed

    Wang, Wei; Scheffler, Katja; Esbensen, Ying; Eide, Lars

    2016-01-01

    This chapter describes the use of real-time qPCR to quantify damages in genomic DNA. The method is based on the ability of a lesion in one strand to inhibit restriction enzyme digestion of double-stranded DNA. Subsequent amplification of the complementary strand after restriction cleavage gives a quantitative measure of the damage content in that site (Real-time qPCR Analysis of Damage Frequency; RADF). We compare the RADF assay with the commonly used technique to assess damages by their ability to inhibit amplification of a large PCR fragment relative to a short PCR fragment. The RADF method described here is quick, accurate and allows the detection of nuclear and mitochondrial DNA damage in detailed regions.

  9. Detection of Legionella pneumophila by Real-Time PCR for the mip Gene

    PubMed Central

    Wilson, Deborah A.; Yen-Lieberman, Belinda; Reischl, Udo; Gordon, Steve M.; Procop, Gary W.

    2003-01-01

    A real-time PCR assay for the mip gene of Legionella pneumophila was tested with 27 isolates of L. pneumophila, 20 isolates of 14 other Legionella species, and 103 non-Legionella bacteria. Eight culture-positive and 40 culture-negative clinical specimens were tested. This assay was 100% sensitive and 100% specific for L. pneumophila. PMID:12843084

  10. DEVELOPMENT OF A REAL-TIME FLUORESCENCE RESONANCE ENERGY TRANSFER (FRET) PCR TO DETECT ARCOBACTER SPECIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A real-time PCR targeting the gyrase A subunit gene outside the quinolone resistance-determining region has been developed to detect Arcobacter species. The species identification was made by probe hybridization and melting curve analysis, using the Fluorescence Resonance Energy Transfer technology...

  11. DETECTION OF PHAKOPSORA PACHYRHIZI SPORES IN RAIN USING REAL-TIME PCR ASSAY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In 2005, rain samples were collected weekly at selected National Atmospheric Deposition Program (NADP) sites in the eastern and central US and screened for Phakopsora pachyrhizi (Asian soybean rust) spores. A nested real-time PCR assay was used to detect P. pachyrhizi DNA in the filter residue. A su...

  12. Identification of Histoplasma capsulatum from culture extracts by real-time PCR.

    PubMed

    Martagon-Villamil, Jose; Shrestha, Nabin; Sholtis, Mary; Isada, Carlos M; Hall, Gerri S; Bryne, Terry; Lodge, Barbara A; Reller, L Barth; Procop, Gary W

    2003-03-01

    We designed and tested a real-time LightCycler PCR assay for Histoplasma capsulatum that correctly identified the 34 H. capsulatum isolates in a battery of 107 fungal isolates tested and also detected H. capsulatum in clinical specimens from three patients that were culture positive for this organism. PMID:12624071

  13. Protein Analysis Using Real-Time PCR Instrumentation: Incorporation in an Integrated, Inquiry-Based Project

    ERIC Educational Resources Information Center

    Southard, Jonathan N.

    2014-01-01

    Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein…

  14. REAL-TIME PCR METHOD TO DETECT ENTEROCOCCUS FAECALIS IN WATER

    EPA Science Inventory

    A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biof...

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

    EPA Science Inventory

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

  16. DEVELOPMENT OF A REAL-TIME FLUORESCENCE RESONANCE ENERGY TRANSFER PCR TO DETECT ARCOBACTER SPECIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A real-time PCR targeting the gyrase A subunit gene outside the quinolone resistance-determining region has been developed to detect Arcobacter species. The species identification was made by probe hybridization and melting curve analysis, using Fluorescence Resonance Energy Transfer technology. D...

  17. Quantification of viable Brochothrix thermosphacta in cooked shrimp and salmon by real-time PCR.

    PubMed

    Mamlouk, Kelthoum; Macé, Sabrina; Guilbaud, Morgan; Jaffrès, Emmanuel; Ferchichi, Mounir; Prévost, Hervé; Pilet, Marie-France; Dousset, Xavier

    2012-05-01

    Brochothrix thermosphacta, a Gram-positive bacterium, is considered as the predominant spoilage microbiota of modified atmosphere packing (MAP) shrimp and fish. Traditional methods currently used to detect B. thermosphacta in foods are time-consuming and labour-intensive. The aim of this study was to develop a real-time PCR quantification method combined with a propidium monoazide (PMA) sample treatment step to monitor the population of B. thermosphacta in cooked shrimp and salmon. The specificity of the two primers MO405 and MO404 used to amplify a 70 bp fragment of the 16S rRNA gene was demonstrated by using purified DNA from 30 strains, among 21 bacterial species including 22 reference strains. Using these primers for real-time PCR and in pure culture, a good correlation was obtained between real-time PCR and the conventional plating method. Quantification was linear over 7-log units using artificially inoculated samples. The method performed successfully when tested on naturally contaminated cooked shrimp and fresh salmon, with a minimum threshold of 1.9×10² CFU/g for accurate quantification of B. thermosphacta. The correlation between the B. thermosphacta counts obtained by real-time PCR and plate counts on naturally contaminated shrimp and salmon was high (R²=0.895). Thus, this study presents a rapid tool for producing reliable quantitative data on B. thermosphacta in cooked shrimp and fresh salmon.

  18. Identifying Haemophilus haemolyticus and Haemophilus influenzae by SYBR Green real-time PCR.

    PubMed

    Latham, Roger; Zhang, Bowen; Tristram, Stephen

    2015-05-01

    SYBR Green real time PCR assays for protein D (hpd), fuculose kinase (fucK) and [Cu, Zn]-superoxide dismutase (sodC) were designed for use in an algorithm for the identification of Haemophilus influenzae and H. haemolyticus. When tested on 127 H. influenzae and 60 H. haemolyticus all isolates were identified correctly. PMID:25753676

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

    PubMed

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

    2014-08-01

    The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories. PMID:24513055

  20. Multiplex SYBR Green Real-Time PCR Assay for Detection of Respiratory Viruses

    PubMed Central

    Sultani, Mozhdeh; Mokhtari Azad, Talat; Eshragian, Mohammadreza; Shadab, Azadeh; Naseri, Maryam; Eilami, Owrang; Yavarian, Jila

    2015-01-01

    Background: It is often difficult for a physician to distinguish between viral and bacterial causes of respiratory infections and this may result in overuse of antibiotics. In many cases of community-acquired respiratory infections, clinicians treat patients empirically. The development of molecular methods for direct detection of viruses has been progressed recently. Objectives: The objective of this study was recognizing the panel of respiratory RNA viruses by multiplex SYBR Green real-time polymerase chain reaction (PCR). Materials and Methods: Randomized 172 influenza-negative respiratory specimens of all age groups of hospitalized patients were collected. After RNA extraction, cDNA was synthesized. Three SYBR Green multiplex real-time PCR assays were developed for simultaneous detection of 12 respiratory RNA viruses. Each set of multiplex methods detected four viruses, the first set: respiratory syncytial virus, human metapneumovirus, rhinovirus, enterovirus; the second set: parainfluenza viruses 1 - 4 (PIV1-4); the third set: coronaviruses NL63, 229E, severe acute respiratory syndrome (SARS), and OC43. Results: Application of the multiplex SYBR Green real-time PCR in clinical samples from 172 patients in a one-year study resulted in detection of 19 (11.04%) PIV3, 9 (5.23%) PIV4, and 1 (0.58%) coronavirus NL63. All the positive samples were detected during December to March (2011 - 2012). Conclusions: Multiplex SYBR Green real-time PCR is a rapid and relatively inexpensive method for detection of respiratory viruses. PMID:26468358

  1. Real-time PCR validation of visually identified snapper Chrysophrys auratus (Sparidae) eggs.

    PubMed

    Dias, P J; Wakefield, C B; Fairclough, D V; Jackson, G; Travers, M J; Snow, M

    2016-02-01

    In this study, a total of 212 eggs were visually identified as snapper Chrysophrys auratus. Real-time PCR confirmed visual identification in 69% of cases but corroboration varied widely among plankton samples. The use of molecular tools to support visual identification prior to adopting daily egg production stock assessment methods should be considered.

  2. Detection of shrimp-derived components in food by real-time fluorescent PCR.

    PubMed

    Cao, Jijuan; Yu, Bing; Ma, Lidan; Zheng, Qiuyue; Zhao, Xin; Xu, Junyi

    2011-10-01

    Crustaceans such as shrimp and crabs and their products are important allergens in food, and allergic reactions due to the consumption of shrimp and crabs are frequently reported. However, the chemical properties of shrimp-derived allergens, except for Pen a I, are still unclear. Therefore, it is important to establish a more sensitive and specific method for detecting the composition of foods containing shrimp. In the present study, we developed a real-time fluorescent PCR to identify the specific shrimp-derived components in food. The primers and TaqMan probes for real-time fluorescent PCR were designed based on 16S rRNA genes through comparing a large number of nucleic acid sequences from different species of shrimp that have been published by the National Center for Biotechnology Information. In total, 56 kinds of samples, including different kinds of shrimp, crab, fish, shellfish, and octopus, were subjected to detection by real-time PCR. The results indicated that real-time fluorescent PCR could successfully identify the shrimp-derived components. In order to explore the effect of food processing on detection sensitivity, fish powder containing shrimp powder was treated by heating at 133°C for 30 min. The limit of detection of shrimp-derived components in fish powder was 0.05% (wt/wt). PMID:22004830

  3. Development of real-time PCR assays for detection of megalocytiviruses in imported ornamental fish.

    PubMed

    Gias, E; Johnston, C; Keeling, S; Spence, R P; McDonald, W L

    2011-08-01

    Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real-time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real-time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real-time PCR assays determined using genomic DNA templates from three target viruses, 12 non-target viruses and 25 aquatic bacterial species were 100%. The intra-run and inter-run coefficients of variation of both assays were <5%. The real-time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus.

  4. Use of real-time quantitative PCR to detect Chlamydophila felis infection.

    PubMed

    Helps, C; Reeves, N; Tasker, S; Harbour, D

    2001-07-01

    A real-time PCR assay was developed to detect and quantify Chlamydophila felis infection of cats. The assay uses a molecular beacon to specifically identify the major outer membrane protein gene, is highly reproducible, and is able to detect fewer than 10 genomic copies.

  5. Application of real-time PCR to postharvest physiology – DNA isolation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time PCR technology has been widely used in the postharvest plant physiology research. One of the difficulties to isolate DNA from plant martial and pathogen cells is the presence of rigid polysaccharide cell walls and capsules, which physically protect DNA from cell lysis. Many materials requi...

  6. A real-time PCR assay for the diagnosis of gastrointestinal nematode infections of small ruminants.

    PubMed

    Roeber, Florian; Jex, Aaron R; Gasser, Robin B

    2015-01-01

    The diagnosis of gastrointestinal nematode infections in small ruminants is central to studying the biology and epidemiology of these parasites and underpins their control. Traditional methods of diagnosis are inaccurate, time-consuming and laborious. Here, we describe a step-by-step protocol for the molecular-based diagnosis of infections by real-time PCR.

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

    PubMed

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

    2014-08-01

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

  8. Detection of shrimp-derived components in food by real-time fluorescent PCR.

    PubMed

    Cao, Jijuan; Yu, Bing; Ma, Lidan; Zheng, Qiuyue; Zhao, Xin; Xu, Junyi

    2011-10-01

    Crustaceans such as shrimp and crabs and their products are important allergens in food, and allergic reactions due to the consumption of shrimp and crabs are frequently reported. However, the chemical properties of shrimp-derived allergens, except for Pen a I, are still unclear. Therefore, it is important to establish a more sensitive and specific method for detecting the composition of foods containing shrimp. In the present study, we developed a real-time fluorescent PCR to identify the specific shrimp-derived components in food. The primers and TaqMan probes for real-time fluorescent PCR were designed based on 16S rRNA genes through comparing a large number of nucleic acid sequences from different species of shrimp that have been published by the National Center for Biotechnology Information. In total, 56 kinds of samples, including different kinds of shrimp, crab, fish, shellfish, and octopus, were subjected to detection by real-time PCR. The results indicated that real-time fluorescent PCR could successfully identify the shrimp-derived components. In order to explore the effect of food processing on detection sensitivity, fish powder containing shrimp powder was treated by heating at 133°C for 30 min. The limit of detection of shrimp-derived components in fish powder was 0.05% (wt/wt).

  9. Diagnosis of gastric cryptosporidiosis in birds using a duplex real-time PCR assay.

    PubMed

    Nakamura, Alex A; Homem, Camila G; da Silva, Adriana M J; Meireles, Marcelo V

    2014-09-15

    Three species and several genotypes of Cryptosporidium can infect the epithelial surface of the bursa of Fabricius, the respiratory tract, the proventriculus, the intestine, and the urinary tract in birds. There is reason to believe that gastric cryptosporidiosis in birds is caused by Cryptosporidium galli and Cryptosporidium avian genotype III, resulting in a chronic illness of the proventriculus that can lead to a debilitating and fatal clinical condition in birds of the orders Passeriformes and Psittaciformes. The objectives of the present study were to develop a duplex real-time polymerase chain reaction (PCR) that targets the 18S rRNA gene to simultaneously detect C. galli and Cryptosporidium avian genotype III DNA and to compare the duplex real-time PCR results to those of nested PCR targeting a partial fragment of the 18S rRNA gene, followed by sequencing of the amplified products (nPCR/S). A total of 1027 fecal samples were collected from birds of the orders Psittaciformes and Passeriformes originating either from captivity or the wild. Duplex real-time PCR results were positive in 580 (56.47%) and 21 (2.04%) samples, respectively, for C. galli and Cryptosporidium avian genotype III, whereas nPCR/S was positive in 28 (2.73%) and three (0.29%) samples, respectively, for C. galli and Cryptosporidium avian genotype III. Novel host birds were identified for both of the above gastric species, and it was also possible to identify Cryptosporidium baileyi and, for the first time in Brazil, Cryptosporidium avian genotype V. The duplex real-time PCR assay developed in the present study represents a sensitive and specific method for the detection of C. galli and Cryptosporidium avian genotype III in bird fecal samples. Moreover, this method may serve as an alternative to nPCR/S as a gold standard for the diagnosis of gastric cryptosporidiosis in birds.

  10. Extensible Multiplex Real-time PCR of MicroRNA Using Microparticles

    PubMed Central

    Jung, Seungwon; Kim, Junsun; Lee, Dong Jin; Oh, Eun Hae; Lim, Hwasup; Kim, Kwang Pyo; Choi, Nakwon; Kim, Tae Song; Kim, Sang Kyung

    2016-01-01

    Multiplex quantitative real-time PCR (qPCR), which measures multiple DNAs in a given sample, has received significant attention as a mean of verifying the rapidly increasing genetic targets of interest in single phenotype. Here we suggest a readily extensible qPCR for the expression analysis of multiple microRNA (miRNA) targets using microparticles of primer-immobilized networks as discrete reactors. Individual particles, 200~500 μm in diameter, are identified by two-dimensional codes engraved into the particles and the non-fluorescent encoding allows high-fidelity acquisition of signal in real-time PCR. During the course of PCR, the amplicons accumulate in the volume of the particles with high reliability and amplification efficiency over 95%. In a quick assay comprising of tens of particles holding different primers, each particle brings the independent real-time amplification curve representing the quantitative information of each target. Limited amount of sample was analyzed simultaneously in single chamber through this highly multiplexed qPCR; 10 kinds of miRNAs from purified extracellular vesicles (EVs). PMID:26964639

  11. ASSESSMENT OF DETECTION EFFICACY OF MYCOBACTERIUM TUBERCULOSIS IN SPUTUM SAMPLES BY REAL TIME PCR BASED METHOD.

    PubMed

    Upadhyaya, S K; Dixit, S M; Shrestha, S; Dangol, S D; Pokhrel, D; Banjara, S; Shrestha, K S; Hengoju, S; Dahal, B K

    2013-07-01

    Tuberculosis (TB) is a major public health problem in Nepal and ranks as one of the most prevalent communicable diseases throughout the country. In Nepal, 45% of total population is infected with TB and 40,000 people get TB every year. Twenty thousand new sputum positive cases are seen every year and 5000-7000 people die each year from TB. Thirty sputum samples were collected from Sukraraj Tropical and Infectious Disease Hospital, Teku, Kathmandu, Nepal and the comparative study of Acid-fast Bacilli (AFB) test and Real time PCR were conducted separately with the culture test which is regarded as gold standard by WHO. Detection of Mycobacterium tuberculosis through use of Real time PCR was found to be higher as compared to AFB and culture. Real-time PCR test showed higher sensitivity (100%) and specificity (94.11%) as compared to AFB test with sensitivity of 84.61% and specificity of 88.24%. Positive predictive value was found to be 84.61% and 92.86% for AFB and Q-PCR respectively. Negative predictive value was found to be 88.24%, and for Q-PCR, it was found to be 100%. Our statistics clearly show that TB diagnosis by Q-PCR is highly efficient and reliableover conventional methods of diagnosis and here we recommend its use in the hospitals and clinics of Nepal.

  12. A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus

    PubMed Central

    Do, Lien Anh Ha; van Doorn, H. Rogier; Bryant, Juliet E.; Nghiem, My Ngoc; Nguyen Van, Vinh Chau; Vo, Cong Khanh; Nguyen, Minh Dung; Tran, Tinh Hien; Farrar, Jeremy; de Jong, Menno D.

    2012-01-01

    Improved diagnostic tools for rapid detection, quantitation, and subgrouping of human respiratory syncytial virus (RSV) are needed to aid the development and evaluation of novel intervention strategies. A quantitative real-time RT-PCR using specific locked nucleic acid (LNA) probes was developed to identify RSV and to distinguish RSV subgroups A and B (RSV LNA assay). RSV subgroup diversity and the relationship between viral load and disease severity in confirmed RSV infections were also explored. 264 archived respiratory specimens from pediatric patients were tested in parallel using the commercial multiplex Seeplex™ RV detection kit (Seegene) and the novel RSV LNA assay. The LNA assay demonstrated a significantly higher sensitivity than Seeplex, improving overall detection rates from 24% (64/264) to 32% (84/264). Detection limits of 9.0 × 101 and 6.0 × 102 copies/mL were observed for RSV A and B, respectively. RSV A was detected in 53/84 (63%) cases, and 31/84 (37%) were positive for RSV B. This novel method offers a rapid, quantitative, highly specific and sensitive approach to laboratory diagnosis of RSV. PMID:22119628

  13. A real-time PCR signature to discriminate between tuberculosis and other pulmonary diseases.

    PubMed

    Laux da Costa, Lucas; Delcroix, Melaine; Dalla Costa, Elis R; Prestes, Isaías V; Milano, Mariana; Francis, Steve S; Unis, Gisela; Silva, Denise R; Riley, Lee W; Rossetti, Maria L R

    2015-07-01

    The goal of this study was to identify a host gene signature that can distinguish tuberculosis (TB) from other pulmonary diseases (OPD). We conducted real-time PCR on whole blood samples from patients in Brazil. TB and OPD patients (asthma and non-TB pneumonia) differentially expressed granzyme A (GZMA), guanylate binding protein 5 (GBP5) and Fc gamma receptor 1A (CD64). Receiver operating characteristic, tree classification and random forest analyses were applied to evaluate the discriminatory power of the three genes and find the gene panel most predictive of patients' disease classification. Tree classification produced a model based on GBP5 and CD64 expression. In random forest analysis, the combination of the three genes provided a robust biosignature to distinguish TB from OPD with 95% specificity and 93% sensitivity. Our results suggest that GBP5 and CD64 in tandem may be the most predictive combination. However, GZMA contribution to the prediction model requires further investigation. Regardless, these three genes show promise as a rapid diagnostic marker separating TB from OPD. PMID:26025597

  14. 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

  15. Development and Efficacy of Real-Time PCR in the Diagnosis of Vivax Malaria Using Field Samples in the Republic of Korea

    PubMed Central

    Ji, So-Young; Shin, Hyun-Il; Han, Eun-Taek; Hong, Yeonchul; Chung, Dong-Il; Cho, Shin-Hyung; Lee, Won-Ja

    2014-01-01

    The development of sensitive, rapid, and accurate diagnostic methods for vivax malaria is essential for the effective control of malaria in the Republic of Korea, where vivax malaria patients usually show low parasitemia. In this study, a TaqMan-based real-time polymerase chain reaction (PCR) method was established and compared with other PCR-based assays, including nested PCR, loop-mediated isothermal amplification, and multiplex PCR, using samples from febrile patients with suspected vivax malaria. The established real-time PCR had a high sensitivity (99.6%) and specificity (100%). Therefore, this sensitive, specific, rapid, and quantitative real-time PCR method could be used for the routine diagnosis of vivax malaria in the laboratory of the Korea National Institute of Health. PMID:25148038

  16. Real-time PCR for detection of Streptococcus suis serotype 2 in cerebrospinal fluid of human patients with meningitis

    PubMed Central

    Nga, Tran Vu Thieu; Nghia, Ho Dang Trung; Tu, Le Thi Phuong; Diep, To Song; Mai, Nguyen Thi Hoang; Chau, Tran Thi Hong; Sinh, Dinh Xuan; Phu, Nguyen Hoan; Nga, Tran Thi Thu; Chau, Nguyen Van Vinh; Campbell, James; Hoa, Ngo Thi; Chinh, Nguyen Tran; Hien, Tran Tinh; Farrar, Jeremy; Schultsz, Constance

    2011-01-01

    Streptococcus suis serotype 2 is an emerging zoonotic pathogen and is the main cause of acute bacterial meningitis in adult patients in Vietnam. We developed an internally controlled real-time PCR for detection of S. suis serotype 2 in cerebrospinal fluid (CSF) samples targeted at the cps2J gene. Sensitivity and specificity in culture-confirmed clinical samples were 100%. The PCR detected S. suis serotype 2 infection in 101 of 238 (42.4%) prospectively collected CSF samples, of which 55 (23%) were culture positive. Culture-negative but PCR-positive CSF samples were significantly associated with the use of antimicrobial agents before admission. S. suis serotype 2 infection was more common than infections with Streptococcus pneumoniae and Neisseria meningitidis combined. Our results strikingly illustrate the additional diagnostic value of PCR in patients who are pretreated with antimicrobial agents and demonstrate the extremely high prevalence of S. suis infections among Vietnamese adult patients with bacterial meningitis. PMID:21767702

  17. Thermal factors influencing detection of Vibrio vulnificus using real-time PCR.

    PubMed

    Wang, Shishan; Levin, Robert E

    2007-05-01

    Five thermal factors, including initial denaturation temperature, cycling denaturation temperature, annealing temperature, extension temperature and the temperature at which the intensity of the fluorescent signal is read, were evaluated for their effects on the detection of Vibrio vulnificus via real-time PCR. Fluorescent signal detection after extension was set between the Tm value of the primer-dimers (79 degrees C) and that of the PCR target amplicons (84 degrees C). This effectively eliminated the overestimation of the yield of PCR amplicons due to the presence of primer-dimers which otherwise led to erroneously lower Ct values (1.91+/-0.22 cycles lower). The annealing and extension steps were combined to convert a three-step PCR to a two-step PCR. This consisted of initial denaturation at 95 degrees C for 3 min, cycling denaturation at 94 degrees C for 15 s and a combined annealing and extension step at 60 degrees C for 5 s in each PCR cycle. One genomic target per real-time PCR reaction was detected with the simplified two-step PCR.

  18. Utility of real-time PCR for the detection of Paracoccidioides brasiliensis DNA in the diagnosis of imported paracoccidioidomycosis.

    PubMed

    Buitrago, M J; Merino, P; Puente, S; Gomez-Lopez, A; Arribi, A; Zancopé-Oliveira, R M; Gutierrez, M C; Rodriguez-Tudela, J L; Cuenca-Estrella, M

    2009-12-01

    An increase in immigration from endemic regions has resulted in a number of cases of paracoccidioidomycosis (PCM) being imported into Spain. A molecular diagnostic technique based on real-time PCR was developed for the detection of Paracoccidioides brasiliensis DNA in both culture and patients' clinical samples. A Molecular Beacon probe was used, labelled with FAM and directed at the ITS1 region of ribosomic DNA. The detection limit of the technique developed was 1 fg of fungal DNA per microl of sample. This procedure proved to be very reproducible and specific. The technique was tested with cultures of 12 clinical strains and on samples from two patients with proven PCM. Real-time PCR was positive for all the culture strains, as well as those from both patients. By samples, the technique was positive in sputum and tissue biopsies but less useful on blood samples. Samples were analyzed several months after patient treatment, detecting a small amount of fungal DNA in one respiratory sample. This technique of real-time PCR is a sensitive method for rapid diagnosis of paracoccidioidomycosis and could serve to monitor patients after treatment has begun.

  19. Development of a real-time RT-PCR method for detection of porcine rubulavirus (PoRV-LPMV).

    PubMed

    Cuevas-Romero, Sandra; Blomström, Anne-Lie; Alvarado, Arcelia; Hernández-Jauregui, Pablo; Rivera-Benitez, Francisco; Ramírez-Mendoza, Humberto; Berg, Mikael

    2013-04-01

    In order to provide a rapid and sensitive method for detection of the Porcine rubulavirus La Piedad-Michoacan-Mexico Virus (PoRV-LPMV), we have developed a specific real-time reverse transcriptase polymerase chain reaction assay. The detection of PoRV-LPMV, represents a diagnostic challenge due to the viral RNA being present in very small amounts in tissue samples. In this study, a TaqMan(®) real-time PCR assay was designed based on the phosphoprotein gene of PoRV-LPMV, to allow specific amplification and detection of viral RNA in clinical samples. Assay conditions for the primers and probe were optimized using infected PK15 cells and ten-fold serial dilutions of a plasmid containing the whole P-gene. The sensitivity of the developed TaqMan(®) assay was approximately 10 plasmid copies per reaction, and was shown to be 1000 fold better than a conventional nested RT-PCR. The performance of this real-time RT-PCR method enables studies of various aspects of PoRV-LPMV infection. Finally, the assay detects all current known variants of the virus.

  20. Comparison of the conventional multiplex RT-PCR, real time RT-PCR and Luminex xTAG® RVP fast assay for the detection of respiratory viruses.

    PubMed

    Choudhary, Manohar L; Anand, Siddharth P; Tikhe, Shamal A; Walimbe, Atul M; Potdar, Varsha A; Chadha, Mandeep S; Mishra, Akhilesh C

    2016-01-01

    Detection of respiratory viruses using polymerase chain reaction (PCR) is sensitive, specific and cost effective, having huge potential for patient management. In this study, the performance of an in-house developed conventional multiplex RT-PCR (mRT-PCR), real time RT-PCR (rtRT-PCR) and Luminex xTAG(®) RVP fast assay (Luminex Diagnostics, Toronto, Canada) for the detection of respiratory viruses was compared. A total 310 respiratory clinical specimens predominantly from pediatric patients, referred for diagnosis of influenza A/H1N1pdm09 from August 2009 to March 2011 were tested to determine performance characteristic of the three methods. A total 193 (62.2%) samples were detected positive for one or more viruses by mRT-PCR, 175 (56.4%) samples by real time monoplex RT-PCR, and 138 (44.5%) samples by xTAG(®) RVP fast assay. The overall sensitivity of mRT-PCR was 96.9% (95% CI: 93.5, 98.8), rtRT-PCR 87.9% (95% CI: 82.5, 92.1) and xTAG(®) RVP fast was 68.3% (95% CI: 61.4, 74.6). Rhinovirus was detected most commonly followed by respiratory syncytial virus group B and influenza A/H1N1pdm09. The monoplex real time RT-PCR and in-house developed mRT-PCR are more sensitive, specific and cost effective than the xTAG(®) RVP fast assay.

  1. Development plan for an advanced drilling system with real-time diagnostics (Diagnostics-While-Drilling)

    SciTech Connect

    FINGER,JOHN T.; MANSURE,ARTHUR J.; PRAIRIE,MICHAEL R.; GLOWKA,D.A.

    2000-02-01

    This proposal provides the rationale for an advanced system called Diagnostics-while-drilling (DWD) and describes its benefits, preliminary configuration, and essential characteristics. The central concept is a closed data circuit in which downhole sensors collect information and send it to the surface via a high-speed data link, where it is combined with surface measurements and processed through drilling advisory software. The driller then uses this information to adjust the drilling process, sending control signals back downhole with real-time knowledge of their effects on performance. The report presents background of related previous work, and defines a Program Plan for US Department of Energy (DOE), university, and industry cooperation.

  2. Detection and quantification of Enterococcus gilvus in cheese by real-time PCR.

    PubMed

    Zago, Miriam; Bonvini, Barbara; Carminati, Domenico; Giraffa, Giorgio

    2009-10-01

    The objective of this work was to investigate the occurrence of Enterococcus gilvus in cheese. For this purpose, a real-time PCR protocol using phenylalanyl-tRNA synthase (pheS) as a target gene was optimized to evaluate the presence and abundance of this microorganism in Italian artisan cheeses. The real-time assay unequivocally distinguished E. gilvus from 25 non-target LAB and non-LAB species, demonstrating its absolute specificity. The assay performed well not only with purified DNA but also with DNA extracted from cheese samples artificially contaminated with E. gilvus. The dynamic range of target determination of the method in the cheese matrix (from 10(7) to 10(4) cfu/ml, covering three orders of magnitude) was lower and the detection limit higher than in vitro conditions, but still high enough to obtain an excellent quantification accuracy in cheese. Twenty commercially available cheeses were analyzed by real-time PCR and approximately 40% of the cheese samples contained E. gilvus at levels ranging from 4.17+/-0.10 to 6.75+/-0.01 log cfu/g. Such levels represented 0.1-10% of the total enterococci counted on kanamycin aesculin azide agar (KAA) from the corresponding cheeses. The successful isolation of E. gilvus from cheeses containing high loads of this species, as detected by real-time PCR, provided definitive proof on both assay specificity and presence of this organism in cheeses. Despite the relatively low sensitivity in cheese (> or =4 log cfu/g), the real-time PCR described here may, however, be useful to detect E. gilvus rapidly when present at (sub)dominant levels within the enterococcal cheese microflora. The assay may be helpful to detect and quantify E. gilvus strains from food, thus enabling a better understanding of technological role, ecological and safety aspects in cheeses and other fermented food products of this infrequent species. PMID:19625150

  3. Real-time RT-PCR for quantitation of hepatitis C virus RNA.

    PubMed

    Yang, Ji Hong; Lai, Jian Ping; Douglas, Steven D; Metzger, David; Zhu, Xian Hua; Ho, Wen Zhe

    2002-04-01

    A newly developed real-time RT-polymerase chain reaction assay for quantitation of hepatitis C virus (HCV) RNA in human plasma and serum was applied. A pair of primers and a probe (molecular beacon) were designed that are specific for the recognition of a highly conservative 5'-non-coding region (5'-NCR) in HCV genome. HCV real-time RT-PCR assay had a sensitivity of 1000 RNA copies per reaction, with a dynamic range of detection between 10(3) and 10(7) RNA copies. The coefficient variation of threshold cycle (Ct) values in intra- and inter-runs were less than 1.37 and 4.66%, respectively. The real-time RT-PCR assay on the HCV sero-positive samples yielded reproducible data, with less than 2.09% of the inter-assay variation. In order to determine its potential for clinical diagnosis, real-time RT-PCR was used to examine the HCV RNA levels in plasma from sero-positive and negative subjects, showing that the assay is highly sensitive and has specificity of 100%. It was demonstrated that the real-time RT-PCR was able to amplify HCV RNA in reference sera with seven genotypes (1A, 1B, 2B, 3A, 4, 5A and 6A) that include six major HCV genotypes circulated in the world. Since HCV is a major pathogen of post-transfusion and community-transmitted non-A, non-B hepatitis, this assay has a broad application for basic and clinical investigations.

  4. Development of direct real-time PCR system applicable to a wide range of foods and agricultural products.

    PubMed

    Mano, Junichi; Hatano, Shuko; Futo, Satoshi; Minegishi, Yasutaka; Ninomiya, Kenji; Nakamura, Kosuke; Kondo, Kazunari; Teshima, Reiko; Takabatake, Reona; Kitta, Kazumi

    2014-01-01

    To improve the efficiency of DNA analysis of foods and agricultural products, we investigated a direct real-time PCR based on the real-time monitoring of DNA amplification directly from crude cell lysates of analytical samples. We established a direct real-time PCR system comprising sample pretreatment with a specified lysis buffer and real-time PCR using the developed master mix reagent. No PCR inhibition was observed in the analysis of crude cell lysates from 50 types of samples, indicating that the direct real-time PCR system is applicable to a wide range of materials. The specificity of the direct real-time PCR was evaluated by means of a model assay system for single nucleotide discrimination. Even when crude cell lysates coexisted in the reaction mixtures, the primer selectivity was not affected, suggesting that the sequence specificity of the direct real-time PCR was equivalent to that of PCR from purified DNA templates. We evaluated the sensitivity and quantitative performance of the direct real-time PCR using soybean flour samples including various amounts of genetically modified organisms. The results clearly showed that the direct real-time PCR system provides sensitive detection and precise quantitation. PMID:24598224

  5. Integrated real-time PCR for detection and monitoring of Legionella pneumophila in water systems.

    PubMed

    Yaradou, Diaraf Farba; Hallier-Soulier, Sylvie; Moreau, Sophie; Poty, Florence; Hillion, Yves; Reyrolle, Monique; André, Janine; Festoc, Gabriel; Delabre, Karine; Vandenesch, François; Etienne, Jerome; Jarraud, Sophie

    2007-03-01

    We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophila-contaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers. PMID:17194840

  6. A One-Step, Real-Time PCR Assay for Rapid Detection of Rhinovirus

    PubMed Central

    Do, Duc H.; Laus, Stella; Leber, Amy; Marcon, Mario J.; Jordan, Jeanne A.; Martin, Judith M.; Wadowsky, Robert M.

    2010-01-01

    One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids, which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes, which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID50 (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform. PMID:19948820

  7. Development of real-time RT-PCR for the detection of low concentrations of Rift Valley fever virus.

    PubMed

    Maquart, Marianne; Temmam, Sarah; Héraud, Jean-Michel; Leparc-Goffart, Isabelle; Cêtre-Sossah, Catherine; Dellagi, Koussay; Cardinale, Eric; Pascalis, Hervé

    2014-01-01

    In recent years, Madagascar and the Comoros archipelago have been affected by epidemics of Rift Valley fever (RVF), however detection of Rift Valley fever virus (RVFV) in zebu, sheep and goats during the post epidemic periods was frequently unsuccessful. Thus, a highly sensitive real-time RT-PCR assay was developed for the detection of RVFV at low viral loads. A new RVF SYBR Green RT-PCR targeting the M segment was tested on serum from different RVF seronegative ruminant species collected from May 2010 to August 2011 in Madagascar and the Comoros archipelago and compared with a RVF specific quantitative real time RT-PCR technique, which is considered as the reference technique. The specificity was tested on a wide range of arboviruses or other viruses giving RVF similar clinical signs. A total of 38 out of 2756 serum samples tested positive with the new RT-PCR, whereas the reference technique only detected 5 out of the 2756. The described RT-PCR is an efficient diagnostic tool for the investigation of enzootic circulation of the RVF virus. It allows the detection of low viral RNA loads adapted for the investigations of reservoirs or specific epidemiological situations such as inter-epizootic periods.

  8. Ultra fast miniaturized real-time PCR: 40 cycles in less than six minutes

    PubMed Central

    Neuzil, Pavel; Zhang, Chunyan; Pipper, Juergen; Oh, Sharon; Zhuo, Lang

    2006-01-01

    We have designed, fabricated and tested a real-time PCR chip capable of conducting one thermal cycle in 8.5 s. This corresponds to 40 cycles of PCR in 5 min and 40 s. The PCR system was made of silicon micromachined into the shape of a cantilever terminated with a disc. The thin film heater and a temperature sensor were placed on the disc perimeter. Due to the system's thermal constant of 0.27 s, we have achieved a heating rate of 175°C s−1 and a cooling rate of −125°C s−1. A PCR sample encapsulated with mineral oil was dispensed onto a glass cover slip placed on the silicon disc. The PCR cycle time was then determined by heat transfer through the glass, which took only 0.5 s. A real-time PCR sample with a volume of 100 nl was tested using a FAM probe. As the single PCR device occupied an area of only a few square millimeters, devices could be combined into a parallel system to increase throughput. PMID:16807313

  9. Development of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    PubMed

    Oguchi, Taichi; Onishi, Mari; Minegishi, Yasutaka; Kurosawa, Yasunori; Kasahara, Masaki; Akiyama, Hiroshi; Teshima, Reiko; Futo, Satoshi; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2009-06-01

    A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstuffs and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this. PMID:19602858

  10. Comparison of Culture, Conventional and Real-time PCR Methods for Listeria monocytogenes in Foods

    PubMed Central

    Moon, Jin-San

    2014-01-01

    We compared standard culture methods as well as conventional PCR and real-time PCR for the detection of Listeria monocytogenes (L. monocytogenes) in milk, cheese, fresh-cut vegetables, and raw beef that have different levels of background microflora. No statistical differences were observed in sensitivity between the two selective media in all foods. In total, real-time PCR assay exhibited statistically excellent detection sensitivity (p<0.05) and was less time consuming and laborious as compared with standard culture methods. Conventional culture methods showed poor performance in detecting L. monocytogenes in food with high levels of background microflora, generating numerous false negative results. While the detection of L. monocytogenes in fresh cut vegetable by culture methods was hindered only by L. innocua, various background microflora, such as L. innocua, L. welshimeri, L. grayi, and Enterococcus faecalis appeared on the two selective media as presumptive positive colonies in raw beef indicating the necessity of improvement of current selective media. It appears that real-time PCR is an effective and sensitive presumptive screening tool for L. monocytogenes in various types of foods, especially foods samples with high levels of background microflora, thus complementing standard culture methodologies. PMID:26761501

  11. Development of quantitative duplex real-time PCR method for screening analysis of genetically modified maize.

    PubMed

    Oguchi, Taichi; Onishi, Mari; Minegishi, Yasutaka; Kurosawa, Yasunori; Kasahara, Masaki; Akiyama, Hiroshi; Teshima, Reiko; Futo, Satoshi; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2009-06-01

    A duplex real-time PCR method was developed for quantitative screening analysis of GM maize. The duplex real-time PCR simultaneously detected two GM-specific segments, namely the cauliflower mosaic virus (CaMV) 35S promoter (P35S) segment and an event-specific segment for GA21 maize which does not contain P35S. Calibration was performed with a plasmid calibrant specially designed for the duplex PCR. The result of an in-house evaluation suggested that the analytical precision of the developed method was almost equivalent to those of simplex real-time PCR methods, which have been adopted as ISO standard methods for the analysis of GMOs in foodstuffs and have also been employed for the analysis of GMOs in Japan. In addition, this method will reduce both the cost and time requirement of routine GMO analysis by half. The high analytical performance demonstrated in the current study would be useful for the quantitative screening analysis of GM maize. We believe the developed method will be useful for practical screening analysis of GM maize, although interlaboratory collaborative studies should be conducted to confirm this.

  12. Determination of pig sex in meat and meat products using multiplex real time-PCR.

    PubMed

    Abdulmawjood, A; Krischek, C; Wicke, M; Klein, G

    2012-07-01

    For specific production lines, European retail companies demand exclusively female pork meat. To control the quality of their suppliers the identification and a quantitative detection of the animal sex origin of the meat is therefore of importance for meat processors. To enable a fast and reliable detection of male pig meat, a real time-PCR-system was designed in the present study. This was based on the genes AMEL-X and AMEL-Y. The real time-PCR assay allowed the detection of male pig meat at a concentration of 1% yielding a detection probability of 100% while the detection probability investigating meat samples containing 0.1% male pig meat was 44.4%. The analytic sensitivity of this system was assessed to be <5 pg DNA per PCR reaction. The assessment of the accuracy of the real time-PCR assay to correctly identify sex individuals was investigated with 62 pigs including males (n=29) and females (n=33) belonging to different breeds/lines. With the newly designed test all analysed animals were correctly sexed. No amplification was obtained with cow, goat, sheep, turkey and chicken genomic DNA. The presented assay can be used for sex diagnosis, for the detection of male pig meat and for meat quality control. PMID:22386444

  13. Monitoring HCV RNA viral load by locked nucleic acid molecular beacons real time PCR.

    PubMed

    Morandi, Luca; Ferrari, Daniela; Lombardo, Claudia; Pession, Annalisa; Tallini, Giovanni

    2007-03-01

    Locked nucleic acids (LNA) based real time PCR was used in particular situations where there are difficulties in primer design due to sequence complexity. In this study a new real time RT-PCR assay was developed using LNA modified primers and LNA molecular beacon probes to monitor hepatitis C virus (HCV) viral load in plasma and serum samples. The technique did not suffer from an heterogeneity of the HCV genome and, in addition, an internal RNA control was amplified in the same reaction tube with different short primers and beacon probe. Due to the short consensus LNA primers length, the PCR efficiency was close to 100% with no formation of hairpin loop structures. In summary a new LNA molecular beacon based real time RT-PCR assay was used successfully to measure quantitatively the total level of HCV RNA in both experimental and clinical specimens. The high sensitivity (50 IU/ml), the wide range of genotype detection, increased specificity and robustness obtained with this test are particularly useful for screening large number of specimens and measuring viral loads to monitor the progress of the disease.

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

    PubMed

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

    2016-04-01

    Diagnosis of avian botulism is based on clinical symptoms, which are indicative but not specific. Laboratory investigations are therefore required to confirm clinical suspicions and establish a definitive diagnosis. Real-time PCR methods have recently been developed for the detection of Clostridium botulinum group III producing type C, D, C/D or D/C toxins. However, no study has been conducted to determine which types of matrices should be analyzed for laboratory confirmation using this approach. This study reports on the comparison of different matrices (pooled intestinal contents, livers, spleens and cloacal swabs) for PCR detection of C. botulinum. Between 2013 and 2015, 63 avian botulism suspicions were tested and 37 were confirmed as botulism. Analysis of livers using real-time PCR after enrichment led to the confirmation of 97% of the botulism outbreaks. Using the same method, spleens led to the confirmation of 90% of botulism outbreaks, cloacal swabs of 93% and pooled intestinal contents of 46%. Liver appears to be the most reliable type of matrix for laboratory confirmation using real-time PCR analysis. PMID:26545739

  15. Multiplex real-time PCR assay for simultaneous detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri.

    PubMed

    Qvarnstrom, Yvonne; Visvesvara, Govinda S; Sriram, Rama; da Silva, Alexandre J

    2006-10-01

    Infections caused by Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris occur throughout the world and pose many diagnostic challenges. To date, at least 440 cases of severe central nervous system infections caused by these amebas have been documented worldwide. Rapid and specific identification of these free-living amebas in clinical samples is of crucial importance for efficient case management. We have developed a triplex real-time TaqMan PCR assay that can simultaneously identify Acanthamoeba spp., B. mandrillaris, and N. fowleri in the same PCR vessel. The assay was validated with 22 well-characterized amebic strains harvested from cultures and nine clinical specimens that were previously characterized by in vitro culture and/or immunofluorescence assay. The triplex assay demonstrated high specificity and a rapid test completion time of less than 5 h from the reception of the specimen in the laboratory. This assay was able to detect one single ameba per sample analyzed, as determined with cerebrospinal fluid spiked with diluted cultured amebas. This assay could become useful for fast laboratory diagnostic assessment of amebic infections (caused by free-living amebas) in laboratories with adequate infrastructure to perform real-time PCR testing.

  16. Detection of Thielaviopsis basicola in soil with real-time quantitative PCR assays.

    PubMed

    Huang, Junli; Kang, Zhenhui

    2010-07-20

    Thielaviopsis basicola is a soil-borne fungus with a wide host range and a cosmopolitan distribution. It causes disease on many agricultural crops and in China it is the causal agent of black root rot on tobacco plant. Early diagnosis and detection of the pathogen in soil are critical to control this disease in field. The objective of this study was to develop sensitive and effective methods suitable for large-scale detection and quantification of T. basicola. Based on the nucleotide sequences of the internal transcribed spacer (ITS) regions of rDNA genes of Thielaviopsis spp, primers and TaqMan probe were designed specifically to amplify DNA from T. basicola and real-time, quantitative PCR (qPCR) assays were developed for rapid, specific and sensitive detection and quantification of T. basicola. It was sensitive with the detection limit of 100 fg microl(-1) genomic DNA of T. basicola in qPCR assays. By combining the qPCR assays with the efficient protocol to extract DNA from soil, it was possible to achieve real-time detection of T. basicola in soil in 4-5 h and the detection limit of 3 conidia per reaction in qPCR was recorded. The assays were applied to survey soils from tobacco fields in China for the presence of T. basicola and the analyses of naturally infested soil showed the reliability of the qPCR assays.

  17. Quantification of mRNA Levels Using Real-Time Polymerase Chain Reaction (PCR).

    PubMed

    Li, Yiyi; Wang, Kai; Chen, Longhua; Zhu, Xiaoxia; Zhou, Jie

    2016-01-01

    Real-time quantitative reverse transcription PCR technique has advanced greatly over the past 20 years. Messenger RNA (mRNA) levels in cells or tissues can be quantified by this approach. It is well known that changes in mRNA expression in disease, and correlation of mRNA expression profiles with clinical parameters, serve as clinically relevant biomarkers. Hence, accurate determination of the mRNA levels is critically important in describing the biological, pathological, and clinical roles of genes in health and disease. This chapter describes a real-time PCR approach to detect and quantify mRNA expression levels, which can be used for both laboratorial and clinical studies in breast cancer research.

  18. Detection of allergen walnut component in food by an improved real-time PCR method.

    PubMed

    Wang, Haiyan; Yuan, Fei; Wu, Yajun; Yang, Hairong; Xu, Baoliang; Liu, Zhongxue; Chen, Ying

    2009-11-01

    A real-time PCR method aimed at the gene sequence of the walnut vicilin-like seed storage protein was established for the detection of the allergen walnut in food. The primers and probe were designed based on published methods. The method provided positive results for walnut and negative results for other tested agricultural plant materials including pecan. The intrinsic detection limit of the method was 0.00125 ng of walnut DNA, and the practical detection limit was 0.001% (wt/wt) walnut content in wheat; both of these values are lower than that of previously published methods. Therefore, this real-time PCR method is sufficiently specific and sensitive for the detection of walnut component in food.

  19. Single-channel multiplexing without melting curve analysis in real-time PCR.

    PubMed

    Lee, Young-Jo; Kim, Daeyoung; Lee, Kihoon; Chun, Jong-Yoon

    2014-01-01

    Multiplex real-time PCR with quantification of targets in a single fluorescence channel has been the demand in biotechnology industry. Here, we develop a novel analytical real-time PCR technique to detect multiple targets in a single fluorescence channel without melting curve analysis. In this technique, we show the intensity of the fluorescence signals of two discrete Tm targets is different at certain temperatures called detection temperatures, by which a high Tm target can be detected regardless of a low Tm target. We then identify the low Tm target by utilizing a change of the fluorescence signals between two different detection temperatures. Furthermore, it enables us to determine quantification of each target in a single channel, possibly facilitating convenient patient care for drug treatment in clinics. PMID:25501038

  20. Multiplex real-time PCR for identification of canine parvovirus antigenic types.

    PubMed

    Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Narang, Deepti

    2016-07-01

    Canine parvovirus (CPV) is an important disease causing gastroenteritis and/or haemorrhagic gastroenteritis in dogs. There are four antigenic types of CPV reported worldwide viz. CPV 2, CPV 2a, CPV 2b and CPV 2c. The diagnosis of CPV with the identification of the antigen type responsible remains problematic. In the present study, identification as well as antigenic typing of CPV was done using a de novo multiplex real time PCR to combat the problem of antigenic type identification. From the study it could be concluded that the here developed multiplex real time PCR assay could be used for rapid detection of CPV as well as typing of its three antigenic types.

  1. Fast real-time PCR for the detection of crustacean allergen in foods.

    PubMed

    Herrero, Beatriz; Vieites, Juan M; Espiñeira, Montserrat

    2012-02-29

    Crustaceans are one of the most common allergens causing severe food reaction. These food allergens are a health problem, and they have become very important; there are various regulations that establish that labeling must be present regarding these allergens to warn consumers. In the present work a fast real-time PCR, by a LNA probe, was developed. This allows the detection of crustaceans in all kinds of products, including processed products in which very aggressive treatments of temperature and pressure during the manufacturing process are used. This methodology provides greater sensitivity and specificity and reduces the analysis time of real-time PCR to 40 min. This methodology was further validated by means of simulating products likely to contain this allergen. For this, products present on the market were spiked with crustacean cooking water. The assay is a potential tool in issues related to the labeling of products and food security to protect the allergic consumer.

  2. Detection and quantification of cultured marine Alexandrium species by real-time PCR.

    PubMed

    Zhang, Fengli; Li, Zhiyong

    2012-12-01

    The occurrence of harmful algal blooms (HABs) throughout the world has increased and poses a large threat to human health, fishery resources and tourism industries. The genus Alexandrium includes a number of toxic species associated with HABs. Therefore, it is very important to rapidly detect and monitor the harmful algae, such as Alexandrium genus. In this study, a standard curve of plasmid containing 18S rDNA-28S rDNA region from Alexandrium catenella was constructed and 5.8S rDNA sequence served as the primer of the real-time PCR. Cultured A. catenella, Alexandrium affine, Alexandrium lusitanicum and Alexandrium minutum samples were analyzed by real-time PCR using the same set of primers simultaneously. Using microscopy cells counts, 5.8S rDNA copies per cell and total DNA per cell were estimated. This assay method is promising for rapid detection of large number of Alexandrium samples. PMID:22864601

  3. A Real-Time PCR for Detection and Quantification of Mycoplasma ovipneumoniae

    PubMed Central

    YANG, Falong; DAO, Xiaofang; RODRIGUEZ-PALACIOS, Alex; FENG, Xufei; TANG, Cheng; YANG, Xiaonong; YUE, Hua

    2014-01-01

    A real-time PCR for detection and quantification of M. ovipneumoniae was developed using 9 recently sequenced M. ovipneumoniae genomes and primers targeting a putative adhesin gene p113. The assay proved to be specific and sensitive (with a detection limit of 22 genomic DNA) and could quantify M. ovipneumoniae DNA over a wide linear range, from 2.2 × 102 to 2.2 × 107 genomes. PMID:25649947

  4. A real-time PCR for detection and quantification of Mycoplasma ovipneumoniae.

    PubMed

    Yang, Falong; Dao, Xiaofang; Rodriguez-Palacios, Alex; Feng, Xufei; Tang, Cheng; Yang, Xiaonong; Yue, Hua

    2014-12-01

    A real-time PCR for detection and quantification of M. ovipneumoniae was developed using 9 recently sequenced M. ovipneumoniae genomes and primers targeting a putative adhesin gene p113. The assay proved to be specific and sensitive (with a detection limit of 22 genomic DNA) and could quantify M. ovipneumoniae DNA over a wide linear range, from 2.2 × 10(2) to 2.2 × 10(7) genomes.

  5. A Novel Multiplex Real-Time PCR for the Identification of Mycobacteria Associated with Zoonotic Tuberculosis

    PubMed Central

    Reddington, Kate; O'Grady, Justin; Dorai-Raj, Siobhan; Niemann, Stefan; van Soolingen, Dick; Barry, Thomas

    2011-01-01

    Background Tuberculosis (TB) is the leading cause of death worldwide from a single infectious agent. An ability to detect the Mycobacterium tuberculosis complex (MTC) in clinical material while simultaneously differentiating its members is considered important. This allows for the gathering of epidemiological information pertaining to the prevalence, transmission and geographical distribution of the MTC, including those MTC members associated with zoonotic TB infection in humans. Also differentiating between members of the MTC provides the clinician with inherent MTC specific drug susceptibility profiles to guide appropriate chemotherapy. Methodology/Principal Findings The aim of this study was to develop a multiplex real-time PCR assay using novel molecular targets to identify and differentiate between the phylogenetically closely related M. bovis, M. bovis BCG and M. caprae. The lpqT gene was explored for the collective identification of M. bovis, M. bovis BCG and M. caprae, the lepA gene was targeted for the specific identification of M. caprae and a Region of Difference 1 (RD1) assay was incorporated in the test to differentiate M. bovis BCG. The multiplex real-time PCR assay was evaluated on 133 bacterial strains and was determined to be 100% specific for the members of the MTC targeted. Conclusions/Significance The multiplex real-time PCR assay developed in this study is the first assay described for the identification and simultaneous differentiation of M. bovis, M. bovis BCG and M. caprae in one internally controlled reaction. Future validation of this multiplex assay should demonstrate its potential in the rapid and accurate diagnosis of TB caused by these three mycobacteria. Furthermore, the developed assay may be used in conjunction with a recently described multiplex real-time PCR assay for identification of the MTC and simultaneous differentiation of M. tuberculosis, M. canettii resulting in an ability to differentiate five of the eight members of the

  6. Proportion of prey consumed can be determined from faecal DNA using real-time PCR.

    PubMed

    Bowles, Ella; Schulte, Patricia M; Tollit, Dominic J; Deagle, Bruce E; Trites, Andrew W

    2011-05-01

    Reconstructing the diets of pinnipeds by visually identifying prey remains recovered in faecal samples is challenging because of differences in digestion and passage rates of hard parts. Analysing the soft-matrix of faecal material using DNA-based techniques is an alternative means to identify prey species consumed, but published techniques are largely nonquantitative, which limits their usefulness for some applications. We further developed and validated a real-time PCR technique using species-specific mitochondrial DNA primers to quantify the proportion of prey in the diets of Steller sea lions (Eumetopias jubatus), a pinniped species thought to be facing significant diet related challenges in the North Pacific. We first demonstrated that the proportions of prey tissue DNA in mixtures of DNA isolated from four prey species could be estimated within a margin of ∼ 12% of the percent in the mix. These prey species included herring Clupea palasii, eulachon Thaleichthyes pacificus, squid Loligo opalescens and rosethorn rockfish Sebastes helvomaculatus. We then applied real-time PCR to DNA extracted from faecal samples obtained from Steller sea lions in captivity that were fed 11 different combinations of herring, eulachon, squid and Pacific ocean perch rockfish (Sebastes alutus), ranging from 7% to 75% contributions per meal (by wet weight). The difference between the average percentage estimated by real-time PCR and the percentage of prey consumed was generally <12% for all diets fed. Our findings indicate that real-time PCR of faecal DNA can detect the approximate relative quantity of prey consumed for complex diets and prey species, including cephalopods and fish. PMID:21481211

  7. Development of a high-speed real-time PCR system for rapid and precise nucleotide recognition

    NASA Astrophysics Data System (ADS)

    Terazono, Hideyuki; Takei, Hiroyuki; Hattori, Akihiro; Yasuda, Kenji

    2010-04-01

    Polymerase chain reaction (PCR) is a common method used to create copies of a specific target region of a DNA sequence and to produce large quantities of DNA. A few DNA molecules, which act as templates, are rapidly amplified by PCR into many billions of copies. PCR is a key technology in genome-based biological analysis, revolutionizing many life science fields such as medical diagnostics, food safety monitoring, and countermeasures against bioterrorism. Thus, many applications have been developed with the thermal cycling. For these PCR applications, one of the most important key factors is reduction in the data acquisition time. To reduce the acquisition time, it is necessary to decrease the temperature transition time between the high and low ends as much as possible. We have developed a novel rapid real-time PCR system based on rapid exchange of media maintained at different temperatures. This system consists of two thermal reservoirs and a reaction chamber for PCR observation. The temperature transition was achieved within 0.3 sec, and good thermal stability was achieved during thermal cycling with rapid exchange of circulating media. This system allows rigorous optimization of the temperatures required for each stage of the PCR processes. Resulting amplicons were confirmed by electrophoresis. Using the system, rapid DNA amplification was accomplished within 3.5 min, including initial heating and complete 50 PCR cycles. It clearly shows that the device could allow us faster temperature switching than the conventional conduction-based heating systems based on Peltier heating/cooling.

  8. Quantification of llama inflammatory cytokine mRNAs by real-time RT-PCR.

    PubMed

    Odbileg, Raadan; Konnai, Satoru; Usui, Tatsufumi; Ohashi, Kazuhiko; Onuma, Misao

    2005-02-01

    We have developed a method by which llama cytokine mRNAs can be quantified using real-time reverse transcription polymerase chain reaction (RT-PCR). Total RNA was extracted from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) of llama, reverse transcribed to cDNA, and cytokine profiles for interleukin (IL)-1alpha, IL-1beta, IL-6 and tumor necrosis factor (TNF) alpha were quantified by real-time PCR. The expressions of mRNAs of inflammatory cytokines IL-1alpha, IL-1beta, IL-6 and TNFalpha were upregulated upon stimulation with LPS in a dose- and time-dependent manner. Incubation of PBMCs with 100 and 1,000 pg/ml of LPS for 3 to 6 hr resulted in the acceleration of the mRNA levels of inflammatory cytokines. Here, we describe a highly sensitive and reproducible method to quantify the transcription of llama cytokine mRNAs by real-time RT-PCR with the double-stranded DNA-binding dye SYBR Green I.

  9. Development of a real-time PCR method for rapid sexing of human preimplantation embryos.

    PubMed

    Martinhago, Cd; Vagnini, Ld; Petersen, Cg; Mauri, Al; Baruffi, Rl; de Oliveira, Rm; Franco, Jg

    2010-01-01

    Genes on the X chromosome are known to be responsible for more than 200 hereditary diseases. After IVF, the simple selection of embryo sex before uterine transfer can prevent the occurrence of affected offspring among couples at risk for these genetic disorders. The aim of this investigation was to develop a rapid method of preimplantation genetic diagnosis (PGD) using real-time polymerase chain reaction (PCR) for the sexing of human embryos, and to compare it to the fluorescence in-situ hybridization technique, considered to be the gold standard. After biopsies were obtained from 40 surplus non-viable embryos for transfer, a total of 98 blastomeres were analysed. It was possible to analyse 24 embryos (60%) by both techniques, generating a total of 70 blastomeres (35 per technique), while 28 blastomeres from 16 embryos (40%) were analysed only by real-time PCR. A rapid and safe method was developed in the present study for the sexual diagnosis of a single human cell (blastomere and buccal cell) using the emerging technology of real-time PCR.

  10. Evaluation of Enrichment Protocols for Bacterial Endosymbionts of Ciliates by Real-Time PCR.

    PubMed

    Castelli, Michele; Lanzoni, Olivia; Rossi, Leonardo; Potekhin, Alexey; Schrallhammer, Martina; Petroni, Giulio

    2016-06-01

    Large-scale studies on obligate bacterial endosymbionts may frequently require preliminary purification and enrichment protocols, which are often elaborate to set up and to evaluate, especially if the host organism is a protist. The purpose of this study was to develop a real-time PCR-based strategy and employ it for assessing two of such enrichment protocols for Holospora caryophila, hosted by the ciliate Paramecium. Four SSU rRNA gene-targeted real-time PCR assays were designed, which allowed to compare the amount of H. caryophila to other organisms, namely the host, its food bacterium (Raoultella planticola), and free-living bacteria present in the culture medium. By the use of the real-time PCR assays in combination, it was possible to conclude that the "cell fractionation" protocol was quite successful in the enrichment of the symbiont, while the "Percoll gradient" protocol will need further refinements to be fully repeatable. The proposed approach has the potential to facilitate and encourage future studies on the yet underexplored field of bacterial endosymbionts of ciliates and other protists. It can also find valuable applications for experimental questions other than those tested, such as fast and precise assessment of symbiont abundance in natural populations and comparison among multiple coexisting symbionts. PMID:26894821

  11. A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates

    PubMed Central

    Svensson, Kerstin; Granberg, Malin; Karlsson, Linda; Neubauerova, Vera; Forsman, Mats; Johansson, Anders

    2009-01-01

    A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis. PMID:20027310

  12. Pathogens in COPD exacerbations identified by comprehensive real-time PCR plus older methods

    PubMed Central

    Shimizu, Kenichiro; Yoshii, Yutaka; Morozumi, Miyuki; Chiba, Naoko; Ubukata, Kimiko; Uruga, Hironori; Hanada, Shigeo; Saito, Nayuta; Kadota, Tsukasa; Ito, Saburo; Wakui, Hiroshi; Takasaka, Naoki; Minagawa, Shunsuke; Kojima, Jun; Hara, Hiromichi; Numata, Takanori; Kawaishi, Makoto; Saito, Keisuke; Araya, Jun; Kaneko, Yumi; Nakayama, Katsutoshi; Kishi, Kazuma; Kuwano, Kazuyoshi

    2015-01-01

    Respiratory infection is a major cause of exacerbation in chronic obstructive pulmonary disease (COPD). Infectious contributions to exacerbations remain incompletely described. We therefore analyzed respiratory tract samples by comprehensive real-time polymerase chain reaction (PCR) in combination with conventional methods. We evaluated multiple risk factors for prolonged hospitalization to manage COPD exacerbations, including infectious agents. Over 19 months, we prospectively studied 46 patients with 50 COPD exacerbations, collecting nasopharyngeal swab and sputum samples from each. We carried out real-time PCR designed to detect six bacterial species and eleven viruses, together with conventional procedures, including sputum culture. Infectious etiologies of COPD exacerbations were identified in 44 of 50 exacerbations (88%). Infections were viral in 17 of 50 exacerbations (34%). COPD exacerbations caused by Gram-negative bacilli, including enteric and nonfermenting organisms, were significantly associated with prolonged hospitalization for COPD exacerbations. Our results support the use of a combination of real-time PCR and conventional methods for determining both infectious etiologies and risk of extended hospitalization. PMID:26451098

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

    PubMed

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

    2006-09-01

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

  14. Evaluation of Enrichment Protocols for Bacterial Endosymbionts of Ciliates by Real-Time PCR.

    PubMed

    Castelli, Michele; Lanzoni, Olivia; Rossi, Leonardo; Potekhin, Alexey; Schrallhammer, Martina; Petroni, Giulio

    2016-06-01

    Large-scale studies on obligate bacterial endosymbionts may frequently require preliminary purification and enrichment protocols, which are often elaborate to set up and to evaluate, especially if the host organism is a protist. The purpose of this study was to develop a real-time PCR-based strategy and employ it for assessing two of such enrichment protocols for Holospora caryophila, hosted by the ciliate Paramecium. Four SSU rRNA gene-targeted real-time PCR assays were designed, which allowed to compare the amount of H. caryophila to other organisms, namely the host, its food bacterium (Raoultella planticola), and free-living bacteria present in the culture medium. By the use of the real-time PCR assays in combination, it was possible to conclude that the "cell fractionation" protocol was quite successful in the enrichment of the symbiont, while the "Percoll gradient" protocol will need further refinements to be fully repeatable. The proposed approach has the potential to facilitate and encourage future studies on the yet underexplored field of bacterial endosymbionts of ciliates and other protists. It can also find valuable applications for experimental questions other than those tested, such as fast and precise assessment of symbiont abundance in natural populations and comparison among multiple coexisting symbionts.

  15. Messenger RNA profiling: a novel method for body fluid identification by real-time PCR.

    PubMed

    Nussbaumer, Christa; Gharehbaghi-Schnell, Elisabeth; Korschineck, Irina

    2006-03-10

    Conventional methods for the identification of different body fluids like blood, semen and saliva from biological stains involve immunological or enzymatic detection of certain proteins. In this study, we investigated potential RNA markers with the aim of developing Real-Time polymerase chain reaction (PCR) based methods to allow differentiation between several body fluids. Total RNA samples from artificially stained swabs and from various pieces of evidence from case work were extracted, amplified and analyzed with several RNA markers. Three assays detecting the body fluids of interest were selected: hemoglobin-alpha locus 1 (HBA), kallikrein 3 (KLK) and mucin 4 (MUC). With this approach, we demonstrate that specific Real-Time PCR assays are useful in identifying the source of the biological stain. Furthermore, RNA profiling of various body fluids was even possible on samples stored over a long period of time at ambient temperature. The stability and sensitivity of the applied method outlines a novel application for Real-Time PCR within the forensic field.

  16. Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant.

    PubMed

    Harms, Gerda; Layton, Alice C; Dionisi, Hebe M; Gregory, Igrid R; Garrett, Victoria M; Hawkins, Shawn A; Robinson, Kevin G; Sayler, Gary S

    2003-01-15

    Real-time PCR assays using TaqMan or Molecular Beacon probes were developed and optimized for the quantification of total bacteria, the nitrite-oxidizing bacteria Nitrospira, and Nitrosomonas oligotropha-like ammonia oxidizing bacteria (AOB) in mixed liquor suspended solids (MLSS) from a municipal wastewater treatment plant (WWTP) using a single-sludge nitrification process. The targets for the real-time PCR assays were the 16S rRNA genes (16S rDNA) for bacteria and Nitrospira spp. and the amoA gene for N. oligotropha. A previously reported assay for AOB 16S rDNA was also tested for its application to activated sludge. The Nitrospira 16S rDNA, AOB 16S rDNA, and N. oligotropha-like amoA assays were log-linear over 6 orders of magnitude and the bacterial 16S rDNA real-time PCR assay was log-linear over 4 orders of magnitude with DNA standards. When these real-time PCR assays were applied to DNA extracted from MLSS, dilution of the DNA extracts was necessary to prevent PCR inhibition. The optimal DNA dilution range was broad for the bacterial 16S rDNA (1000-fold) and Nitrospira 16S rDNA assays (2500-fold) but narrow for the AOB 16S rDNA assay (10-fold) and N. oligotropha-like amoA real-time PCR assay (5-fold). In twelve MLSS samples collected over one year, mean cell per L values were 4.3 +/- 2.0 x 10(11) for bacteria, 3.7 +/- 3.2 x 10(10) for Nitrospira, 1.2 +/- 0.9 x 10(10) for all AOB, and 7.5 +/- 6.0 x 10(9) for N. oligotropha-like AOB. The percent of the nitrifying population was 1.7% N. oligotropha-like AOB based on the N. oligotropha amoA assay, 2.9% total AOB based on the AOB 16S rDNA assay, and 8.6% nitrite-oxidizing bacteria based on the Nitrospira 16S rDNA assay. Ammonia-oxidizing bacteria in the wastewater treatment plant were estimated to oxidize 7.7 +/- 6.8 fmol/hr/cell based on the AOB 16S rDNA assay and 12.4 +/- 7.3 fmol/hr/cell based on the N. oligotropha amoA assay.

  17. Real-Time Reverse Transcription-PCR Assay for Detection of Mumps Virus RNA in Clinical Specimens▿

    PubMed Central

    Boddicker, Jennifer D.; Rota, Paul A.; Kreman, Trisha; Wangeman, Andrea; Lowe, Louis; Hummel, Kimberly B.; Thompson, Robert; Bellini, William J.; Pentella, Michael; DesJardin, Lucy E.

    2007-01-01

    The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion. Laboratory confirmation of mumps infection can be made by the detection of immunoglobulin M-specific antibodies to mumps virus in acute-phase serum samples, the isolation of mumps virus in cell culture, or by detection of the RNA of the mumps virus by reverse transcription (RT)-PCR. We developed and validated a multiplex real-time RT-PCR assay for rapid mumps diagnosis in a clinical setting. This assay used oligonucleotide primers and a TaqMan probe targeting the mumps SH gene, as well as primers and a probe that targeted the human RNase P gene to assess the presence of PCR inhibitors and as a measure of specimen quality. The test was specific, since it did not amplify a product from near-neighbor viruses, as well as sensitive and accurate. Real-time RT-PCR results showed 100% correlation with results from viral culture, the gold standard for mumps diagnostic testing. Assay efficiency was over 90% and displayed good precision after performing inter- and intraassay replicates. Thus, we have developed and validated a molecular method for rapidly diagnosing mumps infection that may be used to complement existing techniques. PMID:17652480

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

    USGS Publications Warehouse

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

    2011-01-01

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

  19. Measuring Expression Levels of Endogenous Gli Genes by Immunoblotting and Real-Time PCR.

    PubMed

    Niewiadomski, Pawel; Rohatgi, Rajat

    2015-01-01

    Gli proteins are transcription factors that mediate the transcriptional effects of Hedgehog signaling in vertebrates. The activities of Gli2 and Gli3 are regulated primarily by posttranslational modifications, while Gli1 is mostly regulated at the transcriptional level. Detection of endogenous Gli proteins had been hampered by lack of good antibodies, but this problem has been mostly resolved in recent years. In this chapter we describe methods of detecting expression of endogenous Gli genes in whole-cell lysates and in subcellular fractions and also provide protocols for the measurement of Gli mRNA levels by quantitative real-time reverse transcriptase PCR (qPCR). PMID:26179041

  20. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.

    PubMed

    Bustin, S A

    2002-08-01

    The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

  1. Real-time quantitative PCR in the diagnosis of tuberculosis in formalin-fixed paraffin-embedded pleural tissue in patients from a high HIV endemic area.

    PubMed

    Baba, Kamaldeen; Pathak, Sharad; Sviland, Lisbeth; Langeland, Nina; Hoosen, Anwar A; Asjo, Birgitta; Dyrhol-Riise, Anne M; Mustafa, Tehmina

    2008-06-01

    The aim of the study was to improve the diagnosis of pleural tuberculosis (TB) based on formalin-fixed biopsies from patients living in high TB and human immunodeficiency virus (HIV) endemic areas. A real-time polymerase chain reaction (real-time PCR) assay targeting a segment of the gene for mycobacterial 65-kd heat shock protein was developed and evaluated on pleural biopsies from 25 patients clinically diagnosed as having TB, on the basis of the good response to treatment, and from 11 controls. A nested polymerase chain reaction (N-PCR) assay for the repetitive genetic sequence insert IS6110, common to Mycobacterium tuberculosis complex organisms, was performed for comparison. When compared with N-PCR, the real-time PCR assay gave a sensitivity and specificity of 83% and 72%, respectively. When compared with clinical diagnosis, the sensitivity and specificity of real-time PCR (68% and 73%, respectively) was comparable with the sensitivity and specificity of the N-PCR assay (64% and 82%, respectively). There were no major differences in the diagnostic validity for the confirmed TB/HIV coinfected patients compared with the results from the whole TB group. In conclusion, the overall accuracy of the real-time PCR assay was comparable with that of the N-PCR and both were equally useful as diagnostic tools in the setting of a HIV coinfection. The real-time PCR has the additional advantage of a short turn-around time, low risk of sample contamination, and offers the possibility to quantify bacterial load, making it a powerful tool for the rapid diagnosis of TB pleuritis.

  2. Real-time quantitative PCR in the diagnosis of tuberculosis in formalin-fixed paraffin-embedded pleural tissue in patients from a high HIV endemic area.

    PubMed

    Baba, Kamaldeen; Pathak, Sharad; Sviland, Lisbeth; Langeland, Nina; Hoosen, Anwar A; Asjo, Birgitta; Dyrhol-Riise, Anne M; Mustafa, Tehmina

    2008-06-01

    The aim of the study was to improve the diagnosis of pleural tuberculosis (TB) based on formalin-fixed biopsies from patients living in high TB and human immunodeficiency virus (HIV) endemic areas. A real-time polymerase chain reaction (real-time PCR) assay targeting a segment of the gene for mycobacterial 65-kd heat shock protein was developed and evaluated on pleural biopsies from 25 patients clinically diagnosed as having TB, on the basis of the good response to treatment, and from 11 controls. A nested polymerase chain reaction (N-PCR) assay for the repetitive genetic sequence insert IS6110, common to Mycobacterium tuberculosis complex organisms, was performed for comparison. When compared with N-PCR, the real-time PCR assay gave a sensitivity and specificity of 83% and 72%, respectively. When compared with clinical diagnosis, the sensitivity and specificity of real-time PCR (68% and 73%, respectively) was comparable with the sensitivity and specificity of the N-PCR assay (64% and 82%, respectively). There were no major differences in the diagnostic validity for the confirmed TB/HIV coinfected patients compared with the results from the whole TB group. In conclusion, the overall accuracy of the real-time PCR assay was comparable with that of the N-PCR and both were equally useful as diagnostic tools in the setting of a HIV coinfection. The real-time PCR has the additional advantage of a short turn-around time, low risk of sample contamination, and offers the possibility to quantify bacterial load, making it a powerful tool for the rapid diagnosis of TB pleuritis. PMID:18382372

  3. Real-time PCR probe optimization using design of experiments approach.

    PubMed

    Wadle, S; Lehnert, M; Rubenwolf, S; Zengerle, R; von Stetten, F

    2016-03-01

    Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3-14 target copies/10 μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7-11 copies/10 μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times.

  4. Real-time PCR probe optimization using design of experiments approach.

    PubMed

    Wadle, S; Lehnert, M; Rubenwolf, S; Zengerle, R; von Stetten, F

    2016-03-01

    Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3-14 target copies/10 μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7-11 copies/10 μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times. PMID:27077046

  5. Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR.

    PubMed

    Mhlanga, M M; Malmberg, L

    2001-12-01

    Detection of single-nucleotide polymorphisms (SNPs) in high-throughput studies promises to be an expanding field of molecular medicine in the near future. Highly specific, simple, and accessible methods are needed to meet the rigorous requirements of single-nucleotide detection needed in pharmacogenomic studies, linkage analysis, and the detection of pathogens. Molecular beacons present such a solution for the high-throughput screening of SNPs in homogeneous assays using the polymerase chain reaction (PCR). Molecular beacons are probes that fluoresce on hybridization to their perfectly complementary targets. In recent years they have emerged as a leading genetic analysis tool in a wide range of contexts from quantification of RNA transcripts, to probes on microarrays, to single-nucleotide polymorphism detection. The majority of these methods use PCR to obtain sufficient amounts of sample to analyze. The use of molecular beacons with other amplification schemes has been reliably demonstrated, though PCR remains the method of choice. Here we discuss and present how to design and use molecular beacons to achieve reliable SNP genotyping and allele discrimination in real-time PCR. In addition, we provide a new means of analyzing data outputs from such real-time PCR assays that compensates for differences between sample condition, assay conditions, variations in fluorescent signal, and amplification efficiency. The mechanisms by which molecular beacons are able to have extraordinary specificity are also presented. PMID:11846616

  6. Real-time PCR probe optimization using design of experiments approach

    PubMed Central

    Wadle, S.; Lehnert, M.; Rubenwolf, S.; Zengerle, R.; von Stetten, F.

    2015-01-01

    Primer and probe sequence designs are among the most critical input factors in real-time polymerase chain reaction (PCR) assay optimization. In this study, we present the use of statistical design of experiments (DOE) approach as a general guideline for probe optimization and more specifically focus on design optimization of label-free hydrolysis probes that are designated as mediator probes (MPs), which are used in reverse transcription MP PCR (RT-MP PCR). The effect of three input factors on assay performance was investigated: distance between primer and mediator probe cleavage site; dimer stability of MP and target sequence (influenza B virus); and dimer stability of the mediator and universal reporter (UR). The results indicated that the latter dimer stability had the greatest influence on assay performance, with RT-MP PCR efficiency increased by up to 10% with changes to this input factor. With an optimal design configuration, a detection limit of 3–14 target copies/10 μl reaction could be achieved. This improved detection limit was confirmed for another UR design and for a second target sequence, human metapneumovirus, with 7–11 copies/10 μl reaction detected in an optimum case. The DOE approach for improving oligonucleotide designs for real-time PCR not only produces excellent results but may also reduce the number of experiments that need to be performed, thus reducing costs and experimental times. PMID:27077046

  7. Detection of intestinal protozoa in paediatric patients with gastrointestinal symptoms by multiplex real-time PCR.

    PubMed

    Maas, L; Dorigo-Zetsma, J W; de Groot, C J; Bouter, S; Plötz, F B; van Ewijk, B E

    2014-06-01

    The performance of a multiplex real-time PCR for the detection of Blastocystis, Dientamoeba fragilis, Giardia lamblia, Cryptosporidium species and Entamoeba species in faecal samples was evaluated in an observational prospective study. Paediatric patients (0-18 years) presenting with gastrointestinal symptoms and suspected of having enteroparasitic disease were included. A questionnaire on gastrointestinal symptoms and the chosen treatment was completed at the start of the study and after 6 weeks. Of 163 paediatric patients (mean age, 7.8 years), 114 (70%) had a PCR-positive faecal sample. D. fragilis was detected most frequently, in 101 patients, followed by Blastocystis in 49. In faecal samples of 47 patients, more than one protozoan was detected, mainly the combination of D. fragilis and Blastocystis. Reported gastrointestinal symptoms were abdominal pain (78%), nausea (30%), and altered bowel habits (28%). Eighty-nine of the PCR-positive patients were treated with antibiotics. A significant reduction in abdominal pain was observed both in treated and in untreated patients. This study demonstrated that multiplex real-time PCR detects a high percentage of intestinal protozoa in paediatric patients with gastrointestinal symptoms. However, interpretation and determination of the clinical relevance of a positive PCR result in this population are still difficult.

  8. Identification of lactic acid bacteria isolated from wine using real-time PCR.

    PubMed

    Kántor, Attila; Kluz, Maciej; Puchalski, Czeslaw; Terentjeva, Margarita; Kačániová, Miroslava

    2016-01-01

    Different lactic acid bacteria strains have been shown to cause wine spoilage, including the generation of substances undesirable for the health of wine consumers. The aim of this study was to investigate the occurrence of selected species of heterofermentative lactobacilli, specifically Lactobacillus brevis, Lactobacillus hilgardii, and Lactobacillus plantarum in six different Slovak red wines following the fermentation process. In order to identify the dominant Lactobacillus strain using quantitative (real time) polymerized chain reaction (qPCR) method, pure lyophilized bacterial cultures from the Czech Collection of Microorganisms were used. Six different red wine samples following malolactic fermentation were obtained from selected wineries. After collection, the samples were subjected to a classic plate dilution method for enumeration of lactobacilli cells. Real-time PCR was performed after DNA extraction from pure bacterial strains and wine samples. We used SYBR® Green master mix reagents for measuring the fluorescence in qPCR. The number of lactobacilli ranged from 3.60 to 5.02 log CFU mL(-1). Specific lactobacilli strains were confirmed by qPCR in all wine samples. The number of lactobacilli ranged from 10(3) to 10(6) CFU mL(-1). A melting curve with different melting temperatures (T(m)) of DNA amplicons was obtained after PCR for the comparison of T(m) of control and experimental portions, revealing that the most common species in wine samples was Lactobacillus plantarum with a T(m) of 84.64°C.

  9. Quantitation of HIV-1 by real-time PCR with a unique fluorogenic probe.

    PubMed

    Saha, B K; Tian, B; Bucy, R P

    2001-04-01

    Quantitation of HIV-1 specific RNA and DNA is pivotal to understanding the pathophysiology of HIV-1 diseases. A method has been developed for quantitation of HIV-1 DNA/RNA by real-time PCR using a unique fluorogenic primer-probe adduct known as scorpion. The probe hybridises to the extension of the adjoining primer intramolecularly, a process kinetically and thermodynamically more favourable than the conventional bimolecular probe-target hybridisation. Data presented in this paper indicate that the scorpion assay is extremely robust and is quite comparable to beacon-based assays. The scorpion assay is also comparable to quantitative competitive PCR (QC--PCR) assays but requires only a fraction of time and effort. Additionally, the dynamic range of the scorpion assay is several log-fold higher than the conventional end point PCR assays. As few as ten copies of vDNA can be detected in the presence of a large excess of exogenously added genomic DNA. Limiting dilution analysis indicates that the assay is capable of detecting a single copy of the viral template. Thus, the scorpion assay presents a specific and sensitive approach for quantitation of DNA/RNA templates by real-time PCR.

  10. Detection and quantification of Histomonas meleagridis by real-time PCR targeting single copy genes.

    PubMed

    Hussain, Imtiaz; Jaskulska, Barbara; Hess, Michael; Bilic, Ivana

    2015-09-15

    Histomonas meleagridis, a protozoan parasite that can infect gallinaceous birds, affects mainly the liver and caeca of infected birds. As a consequence of the recent ban of chemotherapeuticals in Europe and the USA, histomonosis gained somewhat more attention due to its re-emergence and the fact that there is no effective treatment available. Therefore, special attention is now also given towards the diagnosis and the control of the disease. In the actual study we report the development of highly specific and sensitive real-time PCR methods for detection and quantification of the parasite, based on two protein coding genes, Fe-hydrogenase (FeHYD) and rpb1. Both genes seem to be in a single copy in H. meleagridis as shown by southern blotting and absolute quantification using real-time PCRs on samples containing a known amount of the parasite. The real-time PCR assays based on FeHYD and rpb1 genes were found to be an efficient method for the quantification and detection of H. meleagridis in in vitro grown cultures, tissues of infected birds and in faecal samples. Both real-time PCRs were able to detect up to a single cell in in vitro cultures of H. meleagridis and in fecal samples spiked with H. meleagridis. Finally, qPCR assays were shown to be highly specific for H. meleagridis as samples containing either of the two H. meleagridis genotypes were positive, whereas samples containing other protozoa such as Tetratrichomonas gallinarum, Trichomonas gallinae, Simplicimonas sp., Tritrichomonas sp., Parahistomonas wenrichi, Dientamoebidae sp. and Blastocystis sp. were all negative. PMID:26319200

  11. Neurotoxoplasmosis diagnosis for HIV-1 patients by real-time PCR of cerebrospinal fluid.

    PubMed

    Nogui, Fábio Luís Nascimento; Mattas, Sandro; Turcato Júnior, Gilberto; Lewi, David Salomão

    2009-02-01

    Encephalitis caused by Toxoplasma gondii is the most common cause of central nervous system damage in patients with acquired immunodeficiency syndrome (AIDS). Toxoplasma may infect any of the brain cells, thus leading to non-specific neurotoxoplasmosis clinical manifestations including focused or non-focused signs and symptoms of central nervous system malfunction. Clinical development ranges from insidious display during weeks to experiencing acute general confusion or ultimately fatal onset. Cerebral toxoplasmosis occurs in advanced stages of immunodeficiency, and the absence of anti-toxoplasmosis antibodies by the immunofluorescence method does not allow us to rule out its diagnosis. As specific therapy begins, diagnosis confirmation is sought through clinical and radiological response. There are few accurate diagnosis methods to confirm such cases. We present a method for T. gondii DNA detection by real time PCR-Multiplex. Fifty-one patients were evaluated; 16 patients had AIDS and a presumptive diagnosis for toxoplasmosis, 23 patients were HIV-positive with further morbidities except neurotoxoplasmosis, and 12 subjects were HIV-negative control patients. Real time PCR-Multiplex was applied to these patients' cephalorachidian liquid with a specific T. gondii genome sequence from the 529bp fragment. This test is usually carried out within four hours. Test sensitivity, specificity, positive predictive value, and negative predictive value were calculated according to applicable tables. Toxoplasma gondii assay by real time Multiplex of cephalorachidian fluid was positive for 11 out of 16 patients with AIDS and a presumptive diagnosis for cerebral toxoplasmosis, while none of the 35 control patients displayed such a result. Therefore, this method allowed us to achieve 68.8% sensitivity, 100% specificity, 100% positive predictive value, and 87.8% negative predictive value. Real time PCR on CSF allowed high specificity and good sensitivity among patients who

  12. Molecular detection of Puccinia horiana in Chrysanthemum x morifolium through conventional and real-time PCR.

    PubMed

    Alaei, Hossein; Baeyen, Steve; Maes, Martine; Höfte, Monica; Heungens, Kurt

    2009-02-01

    Puccinia horiana Henn. is a quarantine organism and one of the most important fungal pathogens of Chrysanthemum x morifolium cultivars grown for cut flower or potted plant production (florist's chrysanthemum) in several regions of the world. Highly specific primer pairs were identified for conventional, nested, and real-time PCR detection of P. horiana based on the specific and sensitive PCR amplification of selected regions in the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA). Using these different PCR versions, 10 pg, 10 fg, and 5 fg genomic DNA could be detected, respectively. When using cloned target DNA as template, the detection limits were 5000, 50, and 5 target copies, respectively. These detection limits were not affected by a background of chrysanthemum plant DNA. The DNA extraction method was optimized to maximize the recoverability of the pathogen from infected plant tissue. A CTAB extraction protocol or a selection of commercial DNA extraction methods allowed the use of 10 ng total (plant+pathogen) DNA without interference of PCR inhibitors. Due to the specificity of the primers, SYBR Green I technology enabled reliable real time PCR signal detection. However, an efficient TaqMan probe is available. The lowest proportion of infected plant material that could still be detected when mixed with healthy plant material was 0.001%. The real-time PCR assay could detect as few as eight pure P. horiana basidiospores, demonstrating the potential of the technique for aerial detection of the pathogen. The amount of P. horiana DNA in plant tissue was determined at various time points after basidiospore inoculation. Using the real-time PCR protocol, it was possible to detect the pathogen immediately after the inoculation period, even though the accumulation of pathogen DNA was most pronounced near the end of the latent period. The detection system proved to be accurate and sensitive and could help not only in pathogen diagnosis but

  13. Development of a novel detection system for microbes from bovine diarrhea by real-time PCR

    PubMed Central

    TSUCHIAKA, Shinobu; MASUDA, Tsuneyuki; SUGIMURA, Satoshi; KOBAYASHI, Suguru; KOMATSU, Natsumi; NAGAI, Makoto; OMATSU, Tsutomu; FURUYA, Tetsuya; OBA, Mami; KATAYAMA, Yukie; KANDA, Shuhei; YOKOYAMA, Tadashi; MIZUTANI, Tetsuya

    2015-01-01

    Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer–probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R2) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16–1.6 TCID50 (PFU/reaction), 1.3–13 CFU/reaction and 10–100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay’s clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea. PMID:26616156

  14. Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR.

    PubMed

    Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2015-06-01

    Multiplexed real-time PCR (qPCR) assays enable the detection of several target genes in a single reaction, which is applicable for simultaneous testing for the most important viral diseases in samples obtained from ruminants with unspecific clinical symptoms. Here, reverse transcription qPCR (RT-qPCR) systems for the detection of bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV) were combined with an internal control system based on the beta-actin gene. Additionally, a background screening for three further major pathogens of cloven-hoofed animals reportable to the World Organisation for Animal Health, namely foot-and-mouth disease virus, epizootic haemorrhagic disease virus, and Rift Valley fever virus, was integrated using the identical fluorophore for the respective RT-qPCR assays. Every pathogen-specific assay had an analytical sensitivity of at least 100 genome copies per reaction within the multiplex approach, and a series of reference samples and clinical specimens obtained from cattle, but also from small ruminants, were detected reliably. The qPCR systems integrated in the background screening were even not influenced by the simultaneous amplification of very high BVDV and BTV genome copy numbers. The newly developed multiplex qPCR allows the specific and sensitive detection of five of the most important diseases of ruminants and could be used in the context of monitoring programs or for differential diagnostics. PMID:25746154

  15. Simultaneous detection of five notifiable viral diseases of cattle by single-tube multiplex real-time RT-PCR.

    PubMed

    Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2015-06-01

    Multiplexed real-time PCR (qPCR) assays enable the detection of several target genes in a single reaction, which is applicable for simultaneous testing for the most important viral diseases in samples obtained from ruminants with unspecific clinical symptoms. Here, reverse transcription qPCR (RT-qPCR) systems for the detection of bovine viral diarrhoea virus (BVDV) and bluetongue virus (BTV) were combined with an internal control system based on the beta-actin gene. Additionally, a background screening for three further major pathogens of cloven-hoofed animals reportable to the World Organisation for Animal Health, namely foot-and-mouth disease virus, epizootic haemorrhagic disease virus, and Rift Valley fever virus, was integrated using the identical fluorophore for the respective RT-qPCR assays. Every pathogen-specific assay had an analytical sensitivity of at least 100 genome copies per reaction within the multiplex approach, and a series of reference samples and clinical specimens obtained from cattle, but also from small ruminants, were detected reliably. The qPCR systems integrated in the background screening were even not influenced by the simultaneous amplification of very high BVDV and BTV genome copy numbers. The newly developed multiplex qPCR allows the specific and sensitive detection of five of the most important diseases of ruminants and could be used in the context of monitoring programs or for differential diagnostics.

  16. Effects of bacterial contamination of media on the diagnosis of Tritrichomonas foetus by culture and real-time PCR.

    PubMed

    Clothier, Kristin A; Villanueva, Michelle; Torain, Andrea; Hult, Cynthia; Wallace, Rachel

    2015-03-15

    The venereal pathogen Tritrichomonas foetus causes early embryonic death and abortion in cattle. With no approved treatment, control involves detection of infected animals and their removal from the herd. Culture is the traditional diagnostic method; standard media are formulated to support protozoal growth while suppressing competing organisms which may prevent microscopic recognition of T. foetus. Real-time PCR increases diagnostic sensitivity and specificity over culture but requires intact T. foetus DNA for detection. The purposes of this study were 1) to evaluate the effects of resident preputial bacteria that are not suppressed by antimicrobials in a commercial culture medium (InPouch™) on T. foetus detection by culture and PCR, and 2) to determine the performance of a laboratory-prepared culture medium on T. foetus detection by culture and PCR in samples with and without this bacterial contamination. A known concentration of one of three different strains of T. foetus inoculated into InPouch™ (IP) or modified Diamonds-Plastridge media (DPM) were co-incubated with a smegma culture media (CONTAM) for 24h and examined microscopically for the presence of identifiable T. foetus. PCR was performed on IP samples to determine if CONTAM also affected T. foetus DNA detection. A PCR protocol was then validated in DPM that performed similarly to the established IP PCR method. IP and DPM with CONTAM were spiked with serial dilutions that mimic field infections of one of four T. foetus strains and evaluated by real-time PCR; cycles to threshold (Ct) values and "positive" classification were compared between media. T. foetus motility and morphology as well as media pH were severely altered in IP samples with CONTAM compared to those without as well as to DPM medium with and without CONTAM (P<0.0001). PCR testing demonstrated significantly greater Ct values were for T. foetus DNA (P<0.001) in IP contaminated with smegma bacteria compared to those without. When using T

  17. Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis

    PubMed Central

    Bode, Elizabeth; Hurtle, William; Norwood, David

    2004-01-01

    Real-time PCR has become an important method for the rapid identification of Bacillus anthracis since the 2001 anthrax mailings. Most real-time PCR assays for B. anthracis have been developed to detect virulence genes located on the pXO1 and pXO2 plasmids. In contrast, only two published chromosomal targets exist, the rpoB gene and the gyrA gene. In the present study, subtraction-hybridization with a plasmid-cured B. anthracis tester strain and a Bacillus cereus driver was used to find a unique chromosomal sequence. By targeting this region, a real-time assay was developed with the Ruggedized Advanced Pathogen Identification Device. Further testing has revealed that the assay has 100% sensitivity and 100% specificity, with a limit of detection of 50 fg of DNA. The results of a search for sequences with homology with the BLAST program demonstrated significant alignment to the recently published B. anthracis Ames strain, while an inquiry for protein sequence similarities indicated homology with an abhydrolase from B. anthracis strain A2012. The importance of this chromosomal assay will be to verify the presence of B. anthracis independently of plasmid occurrence. PMID:15583318

  18. [Outsourced real-time PCR diagnosis of cutaneous leishmaniasis in the outbreak region of Constatine, Algeria].

    PubMed

    Mihoubi, I; de Monbrison, F; Romeuf, N; Moulahem, T; Picot, S

    2006-02-01

    Taking into account the re-emergence of leishmaniasis in the world, the geographic variability of its epidemiology and the growing numbers of travellers, a pilot study on the diagnosis of cutaneous leishmaniasis was undertaken in Constatine, one of the outbreak regions in eastern Algeria. A total of 143 specimens were collected on blotters and tested by real-time PCR. Results were compared with those of direct examination. Diagnosis was positive for leishmaniasis in 81% of cases using PCR versus 48% of cases using microscopy. Real-time PCR showed a significant quantitative difference between patients for whom microscopic diagnosis was positive and those for whom direct examination was negative. The results presented in this study demonstrated the effectiveness and sensitivity of PCR in the diagnosis of cutaneous leishmaniasis from blotter specimens. This technique enabled in-field collection of specimens from each patient and provided prompt results. North-South cooperation based on the use of simple means for transmission of specimens for molecular diagnosis allowed creation of an effective partnership for daily diagnosis and promoted exchange between investigators in preparation for technology transfer.

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

    PubMed

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

    2010-01-01

    The Horwitz curve estimates interlaboratory precision as a function only of concentration, and is frequently used as a method performance criterion in food analysis with chemical methods. The quantitative biochemical methods based on real-time PCR require an analogous criterion to progressively promote method validation. We analyzed the tendency of precision using a simplex real-time PCR technique in 53 collaborative studies of seven genetically modified (GM) crops. Reproducibility standard deviation (SR) and repeatability standard deviation (Sr) of the genetically modified organism (GMO) amount (%) was more or less independent of GM crops (i.e., maize, soybean, cotton, oilseed rape, potato, sugar beet, and rice) and evaluation procedure steps. Some studies evaluated whole steps consisting of DNA extraction and PCR quantitation, whereas others focused only on the PCR quantitation step by using DNA extraction solutions. Therefore, SR and Sr for GMO amount (%) are functions only of concentration similar to the Horwitz curve. We proposed S(R) = 0.1971C 0.8685 and S(r) = 0.1478C 0.8424, where C is the GMO amount (%). We also proposed a method performance index in GMO quantitative methods that is analogous to the Horwitz Ratio. PMID:20480922

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

    PubMed

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

    2010-01-01

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

  1. Diagnosis of Listeria monocytogenes Meningoencephalitis by Real-Time PCR for the hly Gene ▿ §

    PubMed Central

    Le Monnier, Alban; Abachin, Eric; Beretti, Jean-Luc; Berche, Patrick; Kayal, Samer

    2011-01-01

    Listeria monocytogenes is a bacterial pathogen that can invade the central nervous system (CNS), causing meningoencephalitis and brain abscesses. The diagnosis of CNS listeriosis, based on the isolation of the bacteria in the cerebrospinal fluid (CSF), can be difficult because of previous antibiotic treatment and a low number of bacteria in the CSF. To improve the sensitivity of microbiological diagnosis, we have developed a real-time PCR assay for detecting and quantifying L. monocytogenes DNA in the CSF. The designed primers specifically amplify the L. monocytogenes hly gene, which encodes listeriolysin O, a pore-forming cytolysin. The PCR assay for the hly gene (PCR-hly) provides reproducible quantitative results over a wide dynamic range of concentrations and was highly sensitive while detecting a single gene copy/ml. By assaying a large panel of bacterial species, including species secreting pore-forming cytolysin, we determined the specificity of the PCR-hly, which exclusively detects the L. monocytogenes DNA. We then analyzed 214 CSF samples from patients suspected of having CNS listeriosis. PCR-hly was positive in all cases in which L. monocytogenes was isolated by culture. Positive PCR-hly of the CSF was also obtained for five additional, clinically confirmed cases of CNS listeriosis for which bacterial cultures were negative presumably due to previous treatment with antibiotics. As a complement to classical bacteriological CSF culture, our designed real-time PCR-hly assay proved to be valuable by enhancing the rapidity and the accuracy of the diagnosis of CNS infection by L. monocytogenes. In addition, the quantitative results provided may, in some instances, be useful for the follow-up of patients under treatment. PMID:21918022

  2. Clinical application of a multiplex real-time PCR assay for simultaneous detection of Legionella species, Legionella pneumophila, and Legionella pneumophila serogroup 1.

    PubMed

    Benitez, Alvaro J; Winchell, Jonas M

    2013-01-01

    We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.

  3. Real-time PCR using mycobacteriophage DNA for rapid phenotypic drug susceptibility results for Mycobacterium tuberculosis.

    PubMed

    Pholwat, Suporn; Ehdaie, Beeta; Foongladda, Suporn; Kelly, Kimberly; Houpt, Eric

    2012-03-01

    Managing drug-resistant Mycobacterium tuberculosis requires drug susceptibility testing, yet conventional drug susceptibility testing is slow, and molecular testing does not yield results for all antituberculous drugs. We addressed these challenges by utilizing real-time PCR of mycobacteriophage D29 DNA to evaluate the drug resistance of clinical M. tuberculosis isolates. Mycobacteriophages infect and replicate in viable bacterial cells faster than bacterial cells replicate and have been used for detection and drug resistance testing for M. tuberculosis either by using reporter cells or phages with engineered reporter constructs. Our primary protocol involved culturing M. tuberculosis isolates for 48 h with and without drugs at critical concentrations, followed by incubation with 10(3) PFU/ml of D29 mycobacteriophage for 24 h and then real-time PCR. Many drugs could be incubated instantly with M. tuberculosis and phage for 24 h alone. The change in phage DNA real-time PCR cycle threshold (C(T)) between control M. tuberculosis and M. tuberculosis treated with drugs was calculated and correlated with conventional agar proportion drug susceptibility results. Specifically, 9 susceptible clinical isolates, 22 multidrug-resistant (MDR), and 1 extensively drug-resistant (XDR) M. tuberculosis strains were used and C(T) control-C(T) drug cutoffs of between +0.3 and -6.0 yielded 422/429 (98%) accurate results for isoniazid, rifampin, streptomycin, ethambutol, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide, para-aminosalicylic acid, cycloserine, and linezolid. Moreover, the ΔC(T) values correlated with isolate MIC for most agents. This D29 quantitative PCR assay offers a rapid, accurate, 1- to 3-day phenotypic drug susceptibility test for first- and second-line drugs and may suggest an approximate MIC.

  4. Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR

    PubMed Central

    Eini, Maryam; Behzad-Behbahani, Abbas; Takhshid, Mohammad Ali; Ramezani, Amin; Rafiei Dehbidi, Gholam Reza; Okhovat, Mohammad Ali; Farhadi, Ali; Alavi, Parniyan

    2016-01-01

    Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time. Methods: A0 DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/500 μl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples. Results: Comparison of real time PCR threshold cycle (Ct) for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them. Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis. PMID:27141267

  5. Detection of Invasive Aspergillosis in Bone Marrow Transplant Recipients Using Real-Time PCR

    PubMed Central

    Nabili, Mojtaba; Shokohi, Tahereh; Janbabaie, Ghasem; Hashemi-Soteh, Mohammad Bagher; Ali-Moghaddam, Kamran; Aghili, Seyed Reza

    2013-01-01

    Objective: The invasive aspergillosis (IA) is a serious opportunistic infection caused by various species of Aspergillus in immunocompromised individuals. Basically, rapid and early diagnosis prevents IA progression. In this study we performed a Real Time PCR/ Fluorescence Resonance Energy Transfer (FRET) for diagnosis of IA in hematologic malignancies and bone marrow transplant recipients. Materials and Methods: Sixty two patients with hematologic malignancies and marrow transplant recipients were evaluated for IA in Sari and Tehran from 2009 to 2010. The primer and hybridization probe were designed to amplify the specific sequence of 18S rRNA genes using Light Cycler system and FRET. Galactomannan (GM) assay was performed on serums which obtained from selected patients using the Platelia Aspergillus kit. Results: According to the criteria defined by the European Organization for Research and Treatment of Cancer and Mycoses Study Group (EORTC/MSG) for IA, 18 (29%) patients out of 62 patients were stratified into probable and possible groups. The female-to-male ratio was 1:2; the mean age of the patients was 36 years. The most common malignancies in these patients were acute lymphoblastic leukemia (38.9%). The minimum detection limit was 10 conidia (101 CFU/ml) equivalents (100 fg) per PCR reaction. GM assay was positive in 20.9% and real-time PCR probe set assay were positive in 17.7% patients who had clinical signs and host factor according to the mentioned criteria. Conclusion: Using the Real-Time PCR/FRET assay in whole blood specimens seems to be a promising method for diagnosis of IA, especially when used in combination with the GM detection test. PMID:23853434

  6. Comparison of real-time PCR and microscopy to evaluate sclerotial colonisation by a biocontrol fungus.

    PubMed

    Kim, Tae Gwan; Knudsen, Guy R

    2011-01-01

    The biocontrol agent Trichoderma harzianum colonises sclerotia of the plant pathogenic fungus Sclerotinia sclerotiorum. Plating of sclerotia typically has been used to determine the incidence of mycoparasitism, but does not quantify the extent to which individual sclerotia are colonised. We developed a specific PCR primer/probe set for the green fluorescent protein (GFP)-transformant T. harzianum ThzID1-M3, which exhibited high precision and reproducibility. Quantitative real-time PCR was evaluated along with epifluorescence microscopy and image analysis to investigate dynamics of colonisation of sclerotia in non-sterile soil. Amounts of ThzID1-M3 DNA and S. sclerotiorum DNA from entire individual sclerotia were quantified using real-time PCR. Epifluorescence micrographs were captured from sclerotial thin-section samples, and GFP fluorescence from these was quantified using computer image analysis in order to estimate colonisation on a per-sclerotium basis. As determined by either method, ThzID1-M3 colonised sclerotia in soil, and both methods quantified colonisation dynamics over time. In a separate experiment, colonisation of sclerotia on agar plates was observed using confocal laser scanning microscopy to view the GFP-fluorescing hyphae of ThzID1-M3. This method, while highly labour-intensive, provided high spatial resolution of colonisation dynamics. Thus, each method has advantages: microscopy combined with image analysis can provide useful information on the spatial and temporal dynamics of colonisation, while real-time PCR can provide a more precise assessment of the extent of sclerotial colonisation over time and can more easily be used to sample entire sclerotia. PMID:21530913

  7. FPGA implementation of principal component regression (PCR) for real-time differentiation of dopamine from interferents.

    PubMed

    Bozorgzadeh, Bardia; Covey, Daniel P; Garris, Paul A; Mohseni, Pedram

    2015-01-01

    This paper reports on field-programmable gate array (FPGA) implementation of a digital signal processing (DSP) unit for real-time processing of neurochemical data obtained by fast-scan cyclic voltammetry (FSCV) at a carbonfiber microelectrode (CFM). The DSP unit comprises a decimation filter and two embedded processors to process the FSCV data obtained by an oversampling recording front-end and differentiate the target analyte from interferents in real time with a chemometrics algorithm using principal component regression (PCR). Interfaced with an integrated, FSCV-sensing front-end, the DSP unit successfully resolves the dopamine response from that of pH change and background-current drift, two common dopamine interferents, in flow injection analysis involving bolus injection of mixed solutions, as well as in biological tests involving electrically evoked, transient dopamine release in the forebrain of an anesthetized rat.

  8. FPGA implementation of principal component regression (PCR) for real-time differentiation of dopamine from interferents.

    PubMed

    Bozorgzadeh, Bardia; Covey, Daniel P; Garris, Paul A; Mohseni, Pedram

    2015-01-01

    This paper reports on field-programmable gate array (FPGA) implementation of a digital signal processing (DSP) unit for real-time processing of neurochemical data obtained by fast-scan cyclic voltammetry (FSCV) at a carbonfiber microelectrode (CFM). The DSP unit comprises a decimation filter and two embedded processors to process the FSCV data obtained by an oversampling recording front-end and differentiate the target analyte from interferents in real time with a chemometrics algorithm using principal component regression (PCR). Interfaced with an integrated, FSCV-sensing front-end, the DSP unit successfully resolves the dopamine response from that of pH change and background-current drift, two common dopamine interferents, in flow injection analysis involving bolus injection of mixed solutions, as well as in biological tests involving electrically evoked, transient dopamine release in the forebrain of an anesthetized rat. PMID:26737451

  9. On-Site DNA Extraction and Real-Time PCR for Detection of Phytophthora ramorum in the Field

    PubMed Central

    Tomlinson, J. A.; Boonham, N.; Hughes, K. J. D.; Griffin, R. L.; Barker, I.

    2005-01-01

    Phytophthora ramorum is a recently described pathogen causing oak mortality (sudden oak death) in forests in coastal areas of California and southern Oregon and dieback and leaf blight in a range of tree, shrub, and herbaceous species in the United States and Europe. Due to the threat posed by this organism, stringent quarantine regulations are in place, which restrict the movement of a number of hosts. Fast and accurate diagnostic tests are required in order to characterize the distribution of P. ramorum, prevent its introduction into pathogen-free areas, and minimize its spread within affected areas. However, sending samples to a laboratory for testing can cause a substantial delay between sampling and diagnosis. A rapid and simple DNA extraction method was developed for use at the point of sampling and used to extract DNAs from symptomatic foliage and stems in the field. A sensitive and specific single-round real-time PCR (TaqMan) assay for P. ramorum was performed using a portable real-time PCR platform (Cepheid SmartCycler II), and a cost-effective method for stabilizing PCR reagents was developed to allow their storage and transportation at room temperature. To our knowledge, this is the first description of a method for DNA extraction and molecular testing for a plant pathogen carried out entirely in the field, independent of any laboratory facilities. PMID:16269700

  10. Real-time quantitative PCR for the design of lentiviral vector analytical assays.

    PubMed

    Delenda, C; Gaillard, C

    2005-10-01

    From the recent and emerging concerns for approving lentiviral vector-mediated gene transfer in human clinical applications, several analytical methods have been applied in preclinical models to address the lentiviral vector load in batches, cells or tissues. This review points out the oldest generation methods (blots, RT activity, standard PCR) as well as a full description of the newest real-time quantitative PCR (qPCR) applications. Combinations of primer and probe sequences, which have worked in the lentiviral amplification context, have been included in the effort to dress an exhaustive list. Also, great variations have been observed from interlaboratory results, we have tempted to compare between them the different analytical methods that have been used to consider (i) the titration of lentiviral vector batches, (ii) the absence of the susceptible emerging replicative lentiviruses or (iii) the lentiviral vector biodistribution in the organism.

  11. Comparative Evaluation of Real-Time PCR Methods for Human Noroviruses in Wastewater and Human Stool.

    PubMed

    Masago, Yoshifumi; Konta, Yoshimitsu; Kazama, Shinobu; Inaba, Manami; Imagawa, Toshifumi; Tohma, Kentaro; Saito, Mayuko; Suzuki, Akira; Oshitani, Hitoshi; Omura, Tatsuo

    2016-01-01

    Selecting the best quantitative PCR assay is essential to detect human norovirus genome effectively from clinical and environmental samples because no cell lines have been developed to propagate this virus. The real-time PCR methods for noroviruses GI (4 assays) and GII (3 assays) were evaluated using wastewater (n = 70) and norovirus-positive stool (n = 77) samples collected in Japan between 2012 and 2013. Standard quantitative PCR assays recommended by the U.S. Environmental Protection Agency, International Organization for Standardization, and Ministry of Health, Labour and Welfare, Japan, together with recently reported assays were included. Significant differences in positive rates and quantification cycles were observed by non-parametric analysis. The present study identifies the best assay for norovirus GI and GII to amplify norovirus genomes efficiently. PMID:27525654

  12. Comparative Evaluation of Real-Time PCR Methods for Human Noroviruses in Wastewater and Human Stool

    PubMed Central

    Konta, Yoshimitsu; Kazama, Shinobu; Inaba, Manami; Imagawa, Toshifumi; Tohma, Kentaro; Saito, Mayuko; Suzuki, Akira; Oshitani, Hitoshi; Omura, Tatsuo

    2016-01-01

    Selecting the best quantitative PCR assay is essential to detect human norovirus genome effectively from clinical and environmental samples because no cell lines have been developed to propagate this virus. The real-time PCR methods for noroviruses GI (4 assays) and GII (3 assays) were evaluated using wastewater (n = 70) and norovirus-positive stool (n = 77) samples collected in Japan between 2012 and 2013. Standard quantitative PCR assays recommended by the U.S. Environmental Protection Agency, International Organization for Standardization, and Ministry of Health, Labour and Welfare, Japan, together with recently reported assays were included. Significant differences in positive rates and quantification cycles were observed by non-parametric analysis. The present study identifies the best assay for norovirus GI and GII to amplify norovirus genomes efficiently. PMID:27525654

  13. The workflow of single-cell expression profiling using quantitative real-time PCR

    PubMed Central

    Ståhlberg, Anders; Kubista, Mikael

    2014-01-01

    Biological material is heterogeneous and when exposed to stimuli the various cells present respond differently. Much of the complexity can be eliminated by disintegrating the sample, studying the cells one by one. Single-cell profiling reveals responses that go unnoticed when classical samples are studied. New cell types and cell subtypes may be found and relevant pathways and expression networks can be identified. The most powerful technique for single-cell expression profiling is currently quantitative reverse transcription real-time PCR (RT-qPCR). A robust RT-qPCR workflow for highly sensitive and specific measurements in high-throughput and a reasonable degree of multiplexing has been developed for targeting mRNAs, but also microRNAs, non-coding RNAs and most recently also proteins. We review the current state of the art of single-cell expression profiling and present also the improvements and developments expected in the next 5 years. PMID:24649819

  14. MicroRNA expression in formalin-fixed paraffin embedded tissue using real time quantitative PCR: the strengths and pitfalls

    PubMed Central

    Dijkstra, J R; Mekenkamp, L J M; Teerenstra, S; De Krijger, I; Nagtegaal, I D

    2012-01-01

    MicroRNAs (miRNAs) are a group of small non-coding RNAs with a huge impact in a wide range of biological processes, including cancer. The evidence collected to date demonstrates that miRNAs represent valid diagnostic, prognostic and predictive markers in cancer. The identification of these miRNA biomarkers in archived tissues has been facilitated by novel development and refinement of detection methodologies. Quantitative real-time reverse-transcription PCR (qRT-PCR) is one of the most common methods used to detect low levels of miRNAs with high sensitivity and specificity. However, several technical parameters should be identified and optimized in order to obtain meaningful and reproducible results. The purpose of this review is to describe some of these technical parameters and improve the validity and reliability of miRNA expression studies. PMID:22003827

  15. Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses.

    PubMed

    Cecilia, D; Kakade, M; Alagarasu, K; Patil, J; Salunke, A; Parashar, D; Shah, P S

    2015-01-01

    Dengue and chikungunya viruses co-circulate and cause infections that start with similar symptoms but progress to radically different outcomes. Therefore, an early diagnostic test that can differentiate between the two is needed. A single-step multiplex real-time RT-PCR assay was developed that can simultaneously detect and quantitate RNA of all dengue virus (DENV) serotypes and chikungunya virus (CHIKV). The sensitivity was 100 % for DENV and 95.8 % for CHIKV, whilst the specificity was 100 % for both viruses when compared with conventional RT-PCR. The detection limit ranged from 1 to 50 plaque-forming units. The assay was successfully used for differential diagnosis of dengue and chikungunya in Pune, where the viruses co-circulate.

  16. BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay

    PubMed Central

    2012-01-01

    Background Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay—BactQuant—for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant. Methods The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results A bacterial TaqMan® qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively. Conclusions The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions. PMID:22510143

  17. Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection

    PubMed Central

    Schunk, Mirjam; Kebede Mekonnen, Seleshi; Wondafrash, Beyene; Mengele, Carolin; Fleischmann, Erna; Herbinger, Karl-Heinz; Verweij, Jaco J.; Geldmacher, Christof; Bretzel, Gisela; Löscher, Thomas; Zeynudin, Ahmed

    2015-01-01

    Background In Schistosoma mansoni infection, diagnosis and control after treatment mainly rely on parasitological stool investigations which are laborious and have limited sensitivity. PCR methods have shown equal or superior sensitivity but preservation and storage methods limit their use in the field. Therefore, the use of occult blood detection cards (fecal cards) for easy sampling and storage of fecal samples for further PCR testing was evaluated in a pilot study. Methodology Stool specimens were collected in a highly endemic area for S. mansoni in Ethiopia and submitted in an investigator-blinded fashion to microscopic examination by Kato-Katz thick smear as well as to real-time PCR using either fresh frozen stool samples or stool smears on fecal cards which have been stored at ambient temperature for up to ten months. Principal Findings Out of 55 stool samples, 35 were positive by microscopy, 33 and 32 were positive by PCR of frozen samples and of fecal card samples, respectively. When microscopy was used as diagnostic “gold standard”, the sensitivity of PCR on fresh stool was 94.3% (95%-CI: 86.6; 100) and on fecal cards 91.4% (95%-CI: 82.2; 100). Conclusions The use of fecal cards proved to be a simple and useful method for stool collection and prolonged storage prior to PCR based diagnosis of S. mansoni infection. This technique may be a valuable approach for large scale surveillance and post treatment assessments PMID:26360049

  18. Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC).

    PubMed

    Muraosa, Yasunori; Schreiber, Angelica Zaninelli; Trabasso, Plínio; Matsuzawa, Tetsuhiro; Taguchi, Hideaki; Moretti, Maria Luiza; Mikami, Yuzuru; Kamei, Katsuhiko

    2014-05-01

    In the present study, we developed a new real-time PCR system based on the cycling probe technology (CPT), which is composed of two single tube real-time PCR assays: the Fusarium genus-specific assay and the Fusarium solani species complex (FSSC)-specific assay with primers targeting the 28s ribosomal RNA gene. The Fusarium genus-specific assay was shown to be highly specific, detecting all reference Fusarium strains with no cross-reaction with other reference fungal strains, such as Aspergillus spp. and human DNA. The FSSC-specific assay also reacted very specifically with FSSC, except for a cross-reaction with Fusarium lunatum. To validate the real-time PCR system, we tested 87 clinical isolates of Fusarium spp. Identification results from the real-time PCR system were found to be 100% concordant with those from DNA sequencing of EF-1α gene. The sensitivity testing also demonstrated high sensitivity, enabling detection of one copy of standard DNA with good reproducibility. Furthermore, both assays were shown to be extremely sensitive even when fungal cells were mixed with human cells, detecting 3 germinated conidia spiked in 3mL of human blood. To apply our new real-time PCR system to the molecular diagnosis of fusariosis, we evaluated its efficacy using a mouse model of invasive F. solani infection. Plasma and whole blood samples of infected mice were tested using the real-time PCR system. The sensitivity of the real-time PCR system was found to be 100% (n=4) in plasma samples. In contrast, no amplification signal was detected in whole blood samples. This system could provide a rapid and precise diagnostic tool for early diagnosis, which is necessary for appropriate treatment and improvement of prognosis of disseminated fusariosis.

  19. Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

    PubMed

    Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

    2013-01-01

    Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

  20. Evaluation of four endogenous reference genes and their real-time PCR assays for common wheat quantification in GMOs detection.

    PubMed

    Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

    2013-01-01

    Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat.

  1. Universal Probe Library based real-time PCR for rapid detection of bacterial pathogens from positive blood culture bottles.

    PubMed

    Zhu, Lingxiang; Shen, Ding-Xia; Zhou, Qiming; Liu, Chao-Jun; Li, Zexia; Fang, Xiangdong; Li, Quan-Zhen

    2014-03-01

    A set of real-time PCR based assays using the locked nucleic acid probes from Roche Universal ProbeLibrary were developed for rapid detection of eight bacterial species from positive blood culture bottles. Four duplex real-time PCR reactions targeting to one Gram-positive bacterium and one Gram-negative bacterium were optimized for species identification according to Gram stain results. We also included mecA-specific primers and probes in the assays to indicate the presence of methicillin resistance in the bacterial species. The analytical sensitivity was in the range of 1-10 CFU per PCR reaction mixture. The specificity and cross reactivity of the assay was validated by 28 ATCC reference strains and 77 negative blood culture specimens. No cross-reactivity was observed in these samples thus demonstrating 100 % specificity. 72 previously characterized clinical isolates were tested by the real-time PCR assay and validated the accuracy and feasibility of the real-time PCR assay. Furthermore, 55 positive blood culture samples were tested using real-time PCR and 50 (90.9 %) of them were identified as the same species as judged by biochemical analysis. In total, real-time PCR showed 98.2 % consistent to that of traditional methods. Real-time PCR can be used as a supplement for early detection of the frequently-occurred pathogens from the positive blood cultures.

  2. Real-time magnetic resonance imaging guidance improves the diagnostic yield of endomyocardial biopsy

    PubMed Central

    Rogers, Toby; Ratnayaka, Kanishka; Karmarkar, Parag; Campbell-Washburn, Adrienne E.; Schenke, William H.; Mazal, Jonathan R.; Kocaturk, Ozgur; Faranesh, Anthony Z.; Lederman, Robert J.

    2016-01-01

    Background Diagnostic yield of endomyocardial biopsy is low, particularly in disease that affects the myocardium in a non-uniform distribution. We hypothesized that real-time MRI guidance could improve the yield through targeted biopsy of focal myocardial pathology. Methods An animal model of focal myocardial pathology was created by infusing 3mL of fluorescent microspheres (NuFlow Hydrocoat, 15μm diameter, 5 million spheres/mL) followed by 2mL of 100% ethanol to a branch coronary artery. Animals were survived for minimum 14days, before undergoing MRI guided endomyocardial biopsy using a custom 6.5Fr active visualization MRI-conditional bioptome and X-ray guided biopsy using a commercial bioptome. Specimens were analyzed using a dissecting microscope under ultraviolet light to determine the proportion of ‘on-target’ specimens containing fluorescent microspheres. Results A total of 77 specimens were obtained using real-time MRI guidance and 87 using X-ray guidance, in five animals. Specimens obtained with the MRI-conditional bioptome were smaller compared with the commercial X-ray bioptome. Real-time MRI guidance significantly increased the diagnostic yield of endomyocardial biopsy (82% vs. 56% on-target biopsy specimens with real-time MRI vs. X-ray guidance, p<0.01). Conclusions Endomyocardial biopsy performed using real-time MRI guidance is feasible and significantly improves the diagnostic yield compared with X-ray fluoroscopy guidance. PMID:27631028

  3. Real-time magnetic resonance imaging guidance improves the diagnostic yield of endomyocardial biopsy

    PubMed Central

    Rogers, Toby; Ratnayaka, Kanishka; Karmarkar, Parag; Campbell-Washburn, Adrienne E.; Schenke, William H.; Mazal, Jonathan R.; Kocaturk, Ozgur; Faranesh, Anthony Z.; Lederman, Robert J.

    2016-01-01

    Background Diagnostic yield of endomyocardial biopsy is low, particularly in disease that affects the myocardium in a non-uniform distribution. We hypothesized that real-time MRI guidance could improve the yield through targeted biopsy of focal myocardial pathology. Methods An animal model of focal myocardial pathology was created by infusing 3mL of fluorescent microspheres (NuFlow Hydrocoat, 15μm diameter, 5 million spheres/mL) followed by 2mL of 100% ethanol to a branch coronary artery. Animals were survived for minimum 14days, before undergoing MRI guided endomyocardial biopsy using a custom 6.5Fr active visualization MRI-conditional bioptome and X-ray guided biopsy using a commercial bioptome. Specimens were analyzed using a dissecting microscope under ultraviolet light to determine the proportion of ‘on-target’ specimens containing fluorescent microspheres. Results A total of 77 specimens were obtained using real-time MRI guidance and 87 using X-ray guidance, in five animals. Specimens obtained with the MRI-conditional bioptome were smaller compared with the commercial X-ray bioptome. Real-time MRI guidance significantly increased the diagnostic yield of endomyocardial biopsy (82% vs. 56% on-target biopsy specimens with real-time MRI vs. X-ray guidance, p<0.01). Conclusions Endomyocardial biopsy performed using real-time MRI guidance is feasible and significantly improves the diagnostic yield compared with X-ray fluoroscopy guidance.

  4. Detection of Cryptococcus neoformans DNA in Tissue Samples by Nested and Real-Time PCR Assays

    PubMed Central

    Bialek, Ralf; Weiss, Michael; Bekure-Nemariam, Kubrom; Najvar, Laura K.; Alberdi, Maria B.; Graybill, John R.; Reischl, Udo

    2002-01-01

    Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 × 101 to 2.9 × 104 CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections. PMID:11874894

  5. Reliability of quantitative real-time PCR for bacterial detection in cystic fibrosis airway specimens.

    PubMed

    Zemanick, Edith T; Wagner, Brandie D; Sagel, Scott D; Stevens, Mark J; Accurso, Frank J; Harris, J Kirk

    2010-11-30

    The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities≥10(2) rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments.

  6. Reliability of Quantitative Real-Time PCR for Bacterial Detection in Cystic Fibrosis Airway Specimens

    PubMed Central

    Zemanick, Edith T.; Wagner, Brandie D.; Sagel, Scott D.; Stevens, Mark J.; Accurso, Frank J.; Harris, J. Kirk

    2010-01-01

    The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities ≥102 rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments. PMID:21152087

  7. Assessment of airborne microorganisms by real-time PCR: optimistic findings and research challenges.

    PubMed

    Oppliger, Anne; Masclaux, Frederic G; Niculita-Hirzel, Helene

    2011-01-01

    Most airborne microorganisms are natural components of our ecosystem. Soil, vegetation and animals, including humans, are sources for aerial release of these living or dead cells. In the past, assessment of airborne microorganisms was mainly restricted to occupational health concerns. Indeed, in several occupations, exposure to very high concentrations of non-infectious airborne bacteria and fungi, result in allergenic, toxic or irritant reactions. Recently, the threat of bioterrorism and pandemics have highlighted the urgent need to increase knowledge of bioaerosol ecology. More fundamentally, airborne bacterial and fungal communities begin to draw much more consideration from environmental microbiologists, who have neglected this area for a long time. This increased interest of scientists is to a great part due to the development and use of real-time PCR techniques to identify and quantify airborne microorganisms. Even if the advantages of the PCR technology are obvious, researchers are confronted with new problems. This review describes the methodological state of the art in bioaerosols field and emphasizes the future challenges and perspectives of the real-time PCR-based methods for airborne microorganism studies. PMID:21196388

  8. A real-time PCR method to rapidly titer adenovirus stocks.

    PubMed

    Thomas, Maria A; Lichtenstein, Drew L; Krajcsi, Peter; Wold, William S M

    2007-01-01

    A critical step in working with adenovirus (Ad) and its vectors is the accurate, reproducible, sensitive, and rapid measurement of the amount of virus present in a stock. Titration methods fall into one of two categories: determination of either the infectious or the particle (infectious plus noninfectious) titer. Determining the infectious titer of a virus stock by plaque assay has important limitations, including cell line-, researcher-, and laboratory-dependent variation in titer, and the length of time required to perform the assay (2-4 wk). A major drawback of particle titration methods is the lack of consistent correlation between the resultant titer and the infectious titer. To overcome these problems, a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay was developed that detects encapsidated full-length genomes. Importantly, there is a linear correlation between the titer determined by the realtime PCR assay and the infectious titer determined by a plaque assay. This chapter provides step-by-step guidance for preparing viral DNA, conducting the real-time PCR assay, and using the resultant data to calculate a viral titer.

  9. Quantification of rice blast disease progressions through Taqman real-time PCR.

    PubMed

    Su'udi, Mukhamad; Kim, Jinyeong; Park, Jong-Mi; Bae, Shin-Chul; Kim, Donghern; Kim, Yong-Hwan; Ahn, Il-Pyung

    2013-09-01

    Rice blast caused by Magnaporthe oryzae is a major disease in the paddy field and also a representative model system in the investigation of plant-microbe interactions. This study was undertaken to provide the quantitative evaluation method that specifically determines the amount of M. oryzae proliferation in planta. Real-time PCR was used as the detection strategy in combination with the primer pair and Taqman probe specific to MHP1, a unigene encoding HYDROPHOBIN that is indispensable for normal virulence expression. Based on the crossing point values from the PCR reactions containing a series of increasing concentration of cloned amplicon or fungal genomic DNA, correlation among the template's copy number or its amount and amplification pattern was calculated. Reliability of this equation was further confirmed using the DNA samples from the rice leaves infected with compatible or incompatible strains of M. oryzae. The primer pair used in the Taqman real-time PCR reaction can recognize the existence of fungal DNA as low as 1 pg. In sum, our quantitative evaluation system is applicable and reliable in the blast diagnosis and also in the estimation of objective blast disease progression.

  10. An immunomagnetic separation-real-time PCR method for quantification of Cryptosporidium parvum in water samples.

    PubMed

    Fontaine, Melanie; Guillot, Emmanuelle

    2003-07-01

    The protozoan parasite Cryptosporidium parvum is known to occur widely in both raw and drinking water and is the cause of waterborne outbreaks of gastroenteritis throughout the world. The routinely used method for the detection of Cryptosporidium oocysts in water is based on an immunofluorescence assay (IFA). It is both time-consuming and nonspecific for the human pathogenic species C. parvum. We have developed a TaqMan polymerase chain reaction (PCR) test that accurately quantifies C. parvum oocysts in treated and untreated water samples. The protocol consisted of the following successive steps: Envirochek capsule filtration, immunomagnetic separation (IMS), thermal lysis followed by DNA purification using Nanosep centrifugal devices and, finally, real-time PCR using fluorescent TaqMan technology. Quantification was accomplished by comparing the fluorescence signals obtained from test samples with those from standard dilutions of C. parvum oocysts. This IMS-real-time PCR assay permits rapid and reliable quantification over six orders of magnitude, with a detection limit of five oocysts for purified oocyst solutions and eight oocysts for spiked water samples. Replicate samples of spiked tap water and Seine River water samples (with approximately 78 and 775 oocysts) were tested. C. parvum oocyst recoveries, which ranged from 47.4% to 99% and from 39.1% to 68.3%, respectively, were significantly higher and less variable than those reported using the traditional US Environmental Protection Agency (USEPA) method 1622. This new molecular method offers a rapid, sensitive and specific alternative for C. parvum oocyst quantification in water.

  11. Detection of Helicobacter pylori in biofilms by real-time PCR.

    PubMed

    Linke, S; Lenz, J; Gemein, S; Exner, M; Gebel, J

    2010-06-01

    Helicobacter pylori is a cause of peptic ulcer disease and a causative agent of gastric cancer. Currently, a possible waterborne route of transmission or a possible survival in drinking water biofilms is discussed. H. pylori, like many other bacterial strains, has the ability to enter the viable but nonculturable state (vbnc) in case of unfavorable conditions. Therefore it is necessary to develop new analysis tools for vbnc bacteria. We established a fast and reliable method to detect H. pylori in drinking water biofilms by quantitative real-time PCR which makes it redundant to use difficult cultivation methods for nonculturable bacteria. With this method it was possible to identify water biofilms as a niche for H. pylori. The real-time PCR analysis targets the ureA subunit of the Helicobacter pylori urea gene which showed high specificity and sensitivity. The quantitative real-time PCR was used to detect H. pylori in biofilms of different age, unspiked and spiked with predetermined levels of cells. The drinking water biofilms were generated in a silicone-tube model. The DNA-sequences for probe and primers showed no cross-homologies to other related bacteria and it was possible to detect less than 10 genomic units of H. pylori. This novel method is a useful tool for a fast screening of drinking water biofilms for H. pylori. The results suggest that drinking water biofilms may act as a reservoir for H. pylori which raises new concerns about the role of biofilms as vectors for pathogens like Helicobacter pylori.

  12. Quantitative detection of perchlorate-reducing bacteria by real-time PCR targeting the perchlorate reductase gene.

    PubMed

    Nozawa-Inoue, Mamie; Jien, Mercy; Hamilton, Nicholas S; Stewart, Valley; Scow, Kate M; Hristova, Krassimira R

    2008-03-01

    A quantitative real-time PCR assay targeting the pcrA gene, encoding the catalytic subunit of perchlorate reductase, detected pcrA genes from perchlorate-reducing bacteria in three different genera and from soil microbial communities. Partial pcrA sequences indicated differences in the composition of perchlorate-reducing bacterial communities following exposure to different electron donors.

  13. Soft fruit traceability in food matrices using real-time PCR.

    PubMed

    Palmieri, Luisa; Bozza, Elisa; Giongo, Lara

    2009-02-01

    Food product authentication provides a means of monitoring and identifying products for consumer protection and regulatory compliance. There is a scarcity of analytical methods for confirming the identity of fruit pulp in products containing Soft Fruit. In the present work we have developed a very sensible qualitative and quantitative method to determine the presence of berry DNAs in different food matrices. To our knowledge, this is the first study that shows the applicability, to Soft Fruit traceability, of melting curve analysis and multiplexed fluorescent probes, in a Real-Time PCR platform. This methodology aims to protect the consumer from label misrepresentation.

  14. Quantitative Real-Time PCR Analysis of Gene Transcripts of Mosquito Follicles.

    PubMed

    Telang, Aparna

    2016-01-01

    Real-time (quantitative) PCR, or QPCR, has become an indispensible tool for characterizing gene expression. Depending on the experimental design, researchers can use either the relative or absolute (standard curve) method to quantify transcript abundance. Characterizing the expression of genes in mosquito ovaries will require use of the standard curve method of quantification. Here, I describe reagents and equipment necessary to run standard curve QPCR. I also provide details on the construction of the standard linear curve and calculations required to determine transcript abundance. PMID:27557577

  15. Soft Fruit Traceability in Food Matrices using Real-Time PCR

    PubMed Central

    Palmieri, Luisa; Bozza, Elisa; Giongo, Lara

    2009-01-01

    Food product authentication provides a means of monitoring and identifying products for consumer protection and regulatory compliance. There is a scarcity of analytical methods for confirming the identity of fruit pulp in products containing Soft Fruit. In the present work we have developed a very sensible qualitative and quantitative method to determine the presence of berry DNAs in different food matrices. To our knowledge, this is the first study that shows the applicability, to Soft Fruit traceability, of melting curve analysis and multiplexed fluorescent probes, in a Real-Time PCR platform. This methodology aims to protect the consumer from label misrepresentation. PMID:22253987

  16. Inhibition controls for qualitative real-time PCR assays: are they necessary for all specimen matrices?

    PubMed

    Buckwalter, S P; Sloan, L M; Cunningham, S A; Espy, M J; Uhl, J R; Jones, M F; Vetter, E A; Mandrekar, J; Cockerill, F R; Pritt, B S; Patel, R; Wengenack, N L

    2014-06-01

    A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤ 1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue.

  17. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    NASA Astrophysics Data System (ADS)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-05-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.

  18. Quantification of GPCR mRNA using real-time RT-PCR.

    PubMed

    Brattelid, Trond; Levy, Finn Olav

    2011-01-01

    Characterisation of G-protein-coupled receptor (GPCR) mRNA expression under normal, different pharmacological and pathological conditions in experimental animal models and human tissue biopsies by quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a valuable approach to understand the regulation of GPCR expression. RT-qPCR is specific and sensitive with a broad dynamic range, which allows precise quantification of mRNA species of interest. In addition to measuring the relative levels of mRNA in a tissue or changes in expression levels between groups of genes of interest, RT-qPCR is also used to identify splice variants and single nucleotide polymorphisms (SNPs) of GPCRs. Even though RT-qPCR has become the standard method for quantification of gene expression, RT-qPCR is sensitive to RNA quality, assay design, normalisation approach and data analysis. This protocol is meant as a guide to RT-qPCR methodology with references to the best standard methods available at present.

  19. Interlaboratory comparison of real-time PCR protocols for quantification of general fecal indicator bacteria.

    PubMed

    Shanks, Orin C; Sivaganesan, Mano; Peed, Lindsay; Kelty, Catherine A; Blackwood, A Denene; Greene, Monica R; Noble, Rachel T; Bushon, Rebecca N; Stelzer, Erin A; Kinzelman, Julie; Anan'eva, Tamara; Sinigalliano, Christopher; Wanless, David; Griffith, John; Cao, Yiping; Weisberg, Steve; Harwood, Valarie J; Staley, Christopher; Oshima, Kevin H; Varma, Manju; Haugland, Richard A

    2012-01-17

    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.

  20. Development of Quantitative Real-time PCR Assays for Different Clades of “Candidatus Accumulibacter”

    PubMed Central

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-01-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants. PMID:27142574

  1. 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.

  2. Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran.

    PubMed

    Molla Kazemiha, Vahid; Bonakdar, Shahin; Amanzadeh, Amir; Azari, Shahram; Memarnejadian, Arash; Shahbazi, Shirin; Shokrgozar, Mohammad Ali; Mahdian, Reza

    2016-08-01

    Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert(®) assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert(®) mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert(®), indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.

  3. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Gentiana macrophylla

    PubMed Central

    He, Yihan; Yan, Hailing; Hua, Wenping; Huang, Yaya; Wang, Zhezhi

    2016-01-01

    Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates – five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) – using the transcriptome data of Gentiana macrophylla. Common statistical algorithms ΔCt, GeNorm, NormFinder, and BestKeeper were run with samples collected from plants under various experimental conditions. For normalizing expression levels from tissues at different developmental stages, GAPC2 and UBC21 had the highest rankings. Both SAND1 and GAPC2 proved to be the optimal reference genes for roots from plants exposed to abiotic stresses while EF-1α4 and SAND1 were optimal when examining expression data from the leaves of stressed plants. Based on a comprehensive ranking of stability under different experimental conditions, we recommend that SAND1 and EF-1α4 are the most suitable overall. In this study, to find a suitable reference gene and its real-time PCR assay for G. macrophylla DNA content quantification, we evaluated three target genes including WRKY30, G10H, and SLS, through qualitative and absolute quantitative PCR with leaves under elicitors stressed experimental conditions. Arbitrary use of reference genes without previous evaluation can lead to a misinterpretation of the data. Our results will benefit future research on the expression of genes related to secoiridoid biosynthesis in this species under different experimental conditions. PMID:27446172

  4. Selection and Validation of Reference Genes for Quantitative Real-time PCR in Gentiana macrophylla.

    PubMed

    He, Yihan; Yan, Hailing; Hua, Wenping; Huang, Yaya; Wang, Zhezhi

    2016-01-01

    Real time quantitative PCR (RT-qPCR or qPCR) has been extensively applied for analyzing gene expression because of its accuracy, sensitivity, and high throughput. However, the unsuitable choice of reference gene(s) can lead to a misinterpretation of results. We evaluated the stability of 10 candidates - five traditional housekeeping genes (UBC21, GAPC2, EF-1α4, UBQ10, and UBC10) and five novel genes (SAND1, FBOX, PTB1, ARP, and Expressed1) - using the transcriptome data of Gentiana macrophylla. Common statistical algorithms ΔC t, GeNorm, NormFinder, and BestKeeper were run with samples collected from plants under various experimental conditions. For normalizing expression levels from tissues at different developmental stages, GAPC2 and UBC21 had the highest rankings. Both SAND1 and GAPC2 proved to be the optimal reference genes for roots from plants exposed to abiotic stresses while EF-1α4 and SAND1 were optimal when examining expression data from the leaves of stressed plants. Based on a comprehensive ranking of stability under different experimental conditions, we recommend that SAND1 and EF-1α4 are the most suitable overall. In this study, to find a suitable reference gene and its real-time PCR assay for G. macrophylla DNA content quantification, we evaluated three target genes including WRKY30, G10H, and SLS, through qualitative and absolute quantitative PCR with leaves under elicitors stressed experimental conditions. Arbitrary use of reference genes without previous evaluation can lead to a misinterpretation of the data. Our results will benefit future research on the expression of genes related to secoiridoid biosynthesis in this species under different experimental conditions. PMID:27446172

  5. PCR-based diagnostics for anaerobic infections.

    PubMed

    Song, Yuli

    2005-01-01

    Conventional methods to identify anaerobic bacteria have often relied on unique clinical findings, isolation of organisms, and laboratory identification by morphology and biochemical tests (phenotypic tests). Although these methods are still fundamental, there is an increasing move toward molecular diagnostics of anaerobes. In this review, some of the molecular approaches to anaerobic diagnostics based on the polymerase chain reaction (PCR) are discussed. This includes several technological advances in PCR-based methods for the detection, identification, and quantitation of anaerobes including real-time PCR which has been successfully used to provide rapid, quantitative data on anaerobic species on clinical samples. Since its introduction in the mid-1980s, PCR has provided many molecular diagnostic tools, some of which are discussed within this review. With the advances in micro-array technology and real-time PCR methods, the future is bright for the development of accurate, quantitative diagnostic tools that can provide information not only on individual anaerobic species but also on whole communities.

  6. Real-time PCR detection of telomerase activity using specific molecular beacon probes.

    PubMed

    Kong, Deming; Jin, Yawei; Yin, Yuji; Mi, Huaifeng; Shen, Hanxi

    2007-06-01

    Telomerase is a potentially important biomarker and a prognostic indicator of cancer. Several techniques for assessing telomerase activity, including the telomeric repeat amplification protocol (TRAP) and its modified versions, have been developed. Of these methods, real-time quantitative TRAP (RTQ-TRAP) is considered the most promising. In this work, a novel RTQ-TRAP method is developed in which a telomeric repeats-specific molecular beacon is used. The use of the molecular beacon can improve the specificity of the RTQ-TRAP assay, making the method suitable for studying the overall processivity results and the turnover rate of telomerase. In addition, the real-time, closed-tube protocol used obviates the need for post-amplification procedures, reduces the risk of carryover contamination, and supports high throughput. Its performance in synthetic telomerase products and cell extracts suggests that the developed molecular beacon assay can further enhance the clinical utility of telomerase activity as a biomarker/indicator in cancer diagnosis and prognosis. The method also provides a novel approach to the specific detection of some particular gene sequences to which sequence-specific fluorogenic probes cannot be applied directly. Figure Real-time PCR detection of telomerase activity using specific molecular beacon probes.

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

    PubMed

    Suwancharoen, Duangjai; Sittiwicheanwong, Busara; Wiratsudakul, Anuwat

    2016-09-01

    Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic Leptospira spp. Many molecular techniques have consecutively been developed to detect such pathogen including loop-mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold standard and to assess the agreement among these three tests using Cohen's kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5-99.8) and 97.0% (95% CI 94.9-98.2), respectively. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen's kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), respectively. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle. PMID:27150208

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

    PubMed

    Suwancharoen, Duangjai; Sittiwicheanwong, Busara; Wiratsudakul, Anuwat

    2016-09-01

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

  9. Detection of Brucella spp. in bottlenose dolphins Tursiops truncatus by a real-time PCR using blowhole swabs.

    PubMed

    Wu, Qingzhong; Conway, Jessica; Phillips, Kristen M; Stolen, Megan; Durden, Wendy N; Fauquier, Deborah; McFee, Wayne E; Schwacke, Lori

    2016-08-01

    Blowhole swabs are a simple and non-invasive method for collecting samples from cetaceans and can be used for screening large numbers of animals in the field. This study reports a real-time PCR assay for the detection of Brucella spp. using blowhole swab samples from bottlenose dolphins Tursiops truncatus stranded in the coastal region of Virginia, South Carolina and northern Florida, USA, between 2013 and 2015. We used real-time PCR results on lung samples from the same dolphins in order to estimate the relative sensitivity and specificity of real-time PCR of blowhole swabs. Brucella DNA was detected in lung tissue of 22% (18/81) and in blowhole swabs of 21% (17/81) of the sampled dolphins. The relative sensitivity and specificity of real-time PCR on blowhole swabs as compared to the real-time PCR on lung samples was 94% (17/18) and 100% (63/63), respectively. These results indicate that real-time PCR on blowhole swabs may be used as a non-invasive test for rapid detection of Brucella spp. in the respiratory tract of dolphins. To our knowledge, this is the first report on the use of blowhole swabs for detection of bacterial pathogens by real-time PCR in bottlenose dolphins.

  10. Detection of Brucella spp. in bottlenose dolphins Tursiops truncatus by a real-time PCR using blowhole swabs.

    PubMed

    Wu, Qingzhong; Conway, Jessica; Phillips, Kristen M; Stolen, Megan; Durden, Wendy N; Fauquier, Deborah; McFee, Wayne E; Schwacke, Lori

    2016-08-01

    Blowhole swabs are a simple and non-invasive method for collecting samples from cetaceans and can be used for screening large numbers of animals in the field. This study reports a real-time PCR assay for the detection of Brucella spp. using blowhole swab samples from bottlenose dolphins Tursiops truncatus stranded in the coastal region of Virginia, South Carolina and northern Florida, USA, between 2013 and 2015. We used real-time PCR results on lung samples from the same dolphins in order to estimate the relative sensitivity and specificity of real-time PCR of blowhole swabs. Brucella DNA was detected in lung tissue of 22% (18/81) and in blowhole swabs of 21% (17/81) of the sampled dolphins. The relative sensitivity and specificity of real-time PCR on blowhole swabs as compared to the real-time PCR on lung samples was 94% (17/18) and 100% (63/63), respectively. These results indicate that real-time PCR on blowhole swabs may be used as a non-invasive test for rapid detection of Brucella spp. in the respiratory tract of dolphins. To our knowledge, this is the first report on the use of blowhole swabs for detection of bacterial pathogens by real-time PCR in bottlenose dolphins. PMID:27503920

  11. Novel light-upon-extension real-time PCR assay for simultaneous detection, quantification, and genogrouping of group A rotavirus.

    PubMed

    Nordgren, Johan; Bucardo, Filemón; Svensson, Lennart; Lindgren, Per-Eric

    2010-05-01

    We have developed a light-upon-extension (LUX) real-time PCR assay for detection, quantification, and genogrouping of group A rotavirus (RV), the most common cause of acute gastroenteritis in children. The LUX system uses a fluorophore attached to one primer and having a self-quenching hairpin structure, making it cost-effective and specific. We designed genogroup-specific primers having different fluorophores, making it possible to differentiate between the two main genogroups of human group A RVs. The assay was applied on clinical stool specimens from Sweden and Central America (n=196) and compared to immunological and conventional PCR assays. The genogrouping ability was further validated against a subset of clinical specimens, which had been genogrouped using monoclonal antibodies. Our real-time PCR assay detected and quantified all positive specimens (n=145) and exhibited higher sensitivity than immunological assays and conventional PCR. The assay exhibited a wide dynamic range, detecting from 5 to >10(7) genes per PCR, resulting in a theoretical lower detection limit of <10,000 viruses per gram of stool. No cross-reaction was observed with specimens containing norovirus, sapovirus, astrovirus, or adenovirus. In total, 22 (15%) of the positive clinical specimens were identified as genogroup I, 122 (84%) were identified as genogroup II, and 1 specimen was found to contain a mix of both genogroups. All genogroup I-positive specimens were associated with capsid glycoprotein 2 (G2). No significant difference in viral load was found between genogroups or geographic region. The detection and quantification, combined with the genogrouping ability, make this assay a valuable tool both for diagnostics and for molecular epidemiological investigations.

  12. Validation of Learning Effort Algorithm for Real-Time Non-Interfering Based Diagnostic Technique

    ERIC Educational Resources Information Center

    Hsu, Pi-Shan; Chang, Te-Jeng

    2011-01-01

    The objective of this research is to validate the algorithm of learning effort which is an indicator of a new real-time and non-interfering based diagnostic technique. IC3 Mentor, the adaptive e-learning platform fulfilling the requirements of intelligent tutor system, was applied to 165 university students. The learning records of the subjects…

  13. Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR.

    PubMed

    Wang, Chong; Robles, Francisco; Ramirez, Saul; Riber, Anja Brinch; Bojesen, Anders Miki

    2016-10-01

    Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real-time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial strains. Twenty-two of the strains represented all of the presently available 13 phenotypic variants of G. anatis originating from different geographical locations. Nine strains represented each of the additional six Gallibacterium species and 21 strains represented other poultry associated bacterial species of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae. Regarding specificity none of non-G. anatis strains tested positive with the proposed assay. To test and compare the qPCR method's ability to detect G. anatis from field samples, the sensitivity was compared to a previously published conventional PCR method and culture-based identification, respectively. The detection rates were 97%, 78% and 34% for the current qPCR, the conventional PCR and the culture-based identification method, respectively. The qPCR assay was able to detect the gene gyrB in serial dilutions of 10(8) colony forming units (CFU)/ml to as low as 10(0) CFU/ml copies. The proposed qPCR method is thus highly specific, sensitive and reproducible. In conclusion, we have developed a qPCR method that allows species-specific identification of G. anatis.

  14. Real-time PCR improves detection of Trichomonas vaginalis infection compared with culture using self-collected vaginal swabs.

    PubMed Central

    Caliendo, A M; Jordan, J A; Green, A M; Ingersoll, J; Diclemente, R J; Wingood, G M

    2005-01-01

    OBJECTIVE: To compare a real-time polymerase chain reaction (PCR) assay with broth culture for the detection of Trichomonas vaginalis using self-collected vaginal swabs. METHODS: Self-collected vaginal swabs were obtained from adolescent and young adult African-American women participating in HIV-1 prevention programs. T. vaginalis culture was performed using the InPouch TV System. Samples for the real-time PCR assay were collected using the BDProbeTec ET Culturette Direct Dry Swab system and tested in a laboratory-developed assay which targeted a repeated sequence of the genome. Discrepant samples that were culture negative and positive in the real-time PCR assay were tested in a confirmatory PCR which targeted a different region of the T. vaginalis genome, the18S ribosomal DNA gene. RESULTS: Of the 524 specimens tested by both culture and real-time PCR, 36 were culture positive and 54 were positive in the real-time PCR assay; 16 of the 18 discrepant specimens were also positive in the confirmatory PCR assay. Using a modified gold standard of positive by culture or positive in both PCR assays, the sensitivity of the real-time PCR assay was 100% and the specificity was 99.6%, whereas culture had a sensitivity of 69.2% and a specificity of 100%. CONCLUSIONS: The real-time PCR assay was sensitive and specific for the detection of T. vaginalis DNA from self-collected vaginal swab specimens. The ability to use the BDProbeTec dry swab system for the real-time PCR testing allowed for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and T. vaginalis from a single specimen. PMID:16126499

  15. Ultra-rapid real-time PCR for the detection of Paenibacillus larvae, the causative agent of American Foulbrood (AFB).

    PubMed

    Han, Sang-Hoon; Lee, Do-Bu; Lee, Dong-Woo; Kim, Eul-Hwan; Yoon, Byoung-Su

    2008-09-01

    A novel micro-PCR-based detection method, termed ultra-rapid real-time PCR, was applied to the development of a rapid detection for Paenibacillus larvae (P. larvae) which is the causative agent of American Foulbrood (AFB). This method was designed to detect the 16S rRNA gene of P. larvae with a micro-scale chip-based real-time PCR system, GenSpector TMC-1000, which has uncommonly fast heating and cooling rates (10 degrees C per second) and small reaction volume (6microl). In the application of ultra-rapid real-time PCR detection to an AFB-infected larva, the minimum detection time was 7 min and 54s total reaction time (30 cycles), including the melting temperature analysis. To the best of our knowledge, this novel detection method is one of the most rapid real-time PCR-based detection tools. PMID:18571197

  16. Detection of sesame seed DNA in foods using real-time PCR.

    PubMed

    Brzezinski, Jennifer L

    2007-04-01

    The detection of potentially allergenic foods, such as sesame seeds, in food products is a major concern for the food-processing industry. A real-time PCR method was designed to determine if sesame seed DNA is present in food products. The PCR reaction amplifies a 66-bp fragment of the sesame seed 2S albumin gene, which is detected with a sesame-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other seeds present in baked goods, such as pumpkin, poppy, and sunflower seeds. Additionally, this assay will not cross-react with DNA from several tree nut species, such as almond, Brazil nut, cashew, hazelnut, and walnut, as well as four varieties of peanut. This assay is sensitive enough to detect 5 pg of purified sesame seed DNA, as well as sesame seed DNA in a spiked wheat cracker sample.

  17. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

    NASA Astrophysics Data System (ADS)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason

    2016-09-01

    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  18. Quantification of organellar DNA and RNA using real-time PCR.

    PubMed

    Weihe, Andreas

    2014-01-01

    Quantitative (real-time) polymerase chain reaction (PCR) allows the measurement of relative organellar gene copy numbers as well as transcript abundance of individual mitochondrial or plastidial genes. Requiring only minute amounts of total DNA or RNA, the described method can replace traditional analyses like Southern or Northern hybridization which require large amounts of organellar nucleic acids and usually provide only semiquantitative data. Here we describe prerequisites, reaction conditions, and data analysis principles, which should be applicable for a wide range of plant species and experimental situations where comparative and precise determination of gene copy numbers or transcript abundance is requested. Sequences of amplification primers for qPCR of organellar genes from Arabidopsis are provided.

  19. Detection of peanut (Arachis hypogaea) allergen by Real-time PCR method with internal amplification control.

    PubMed

    Zhang, Wen-Ju; Cai, Qin; Guan, Xiao; Chen, Qin

    2015-05-01

    Specific primer sets were designed based on the DNA sequence of Ara h 1, one of the major peanut (Arachis hypogaea) allergens, and a competitive internal amplification control (IAC) was designed by compound primer technology. By choosing 314 copies/PCR as the IAC dosage, a Real-time PCR method with IAC was established for detecting peanut allergen Ara h 1 DNA. The method showed high specificity with a detection limit of 0.005% peanut. A series of commercial food products with/without peanut components were tested. Among these products, the peanut allergen Ara h 1 DNA could be detected in 12 products labelled containing peanut ingredients, in two without a declaration of peanut and one labelled that was produced in a facility that produced peanut-containing foods. This indicates that the method is highly sensitive for the detection of peanut ingredients in foods.

  20. Development and evaluation of real-time PCR assays for bloodmeal identification in Culicoides midges.

    PubMed

    VAN DER Saag, M R; Gu, X; Ward, M P; Kirkland, P D

    2016-06-01

    Culicoides (Diptera: Ceratopogonidae) midges are the biological vectors of a number of arboviruses of veterinary importance. However, knowledge relating to the basic biology of some species, including their host-feeding preferences, is limited. Identification of host-feeding preferences in haematophagous insects can help to elucidate the transmission dynamics of the arboviruses they may transmit. In this study, a series of semi-quantitative real-time polymerase chain reaction (qPCR) assays to identify the vertebrate host sources of bloodmeals of Culicoides midges was developed. Two pan-reactive species group and seven species-specific qPCR assays were developed and evaluated. The assays are quick to perform and less expensive than nucleic acid sequencing of bloodmeals. Using these assays, it was possible to rapidly test nearly 700 blood-fed midges of various species from several geographic locations in Australia. PMID:26854008

  1. A Real-Time PCR Method to Detect the Population Level of Halovirus SNJ1

    PubMed Central

    Mei, Yunjun; He, Congcong; Deng, Wei; Ba, Dala; Yang, Ming; Zhang, Jian; Zhang, Shunxi; Shen, Ping; Chen, Xiangdong

    2016-01-01

    Although viruses of haloarchaea are the predominant predator in hypersaline ecosystem, the culture studies about halovirus-host systems are infancy. The main reason is the tradition methodology (plaque assay) for virus-host interaction depends on culturable and susceptible host. Actually, more than 90% of haloarchaea are unculturable. Therefore, it is necessary to establish an approach for detecting the dynamics of virus in hypersaline environment without culture. In this study, we report a convenient method to determine the dynamics of halovirus SNJ1 based on quantitative real-time PCR (qPCR). All findings showed that the qPCR method was specific (single peak in melt curves), accurate (a good linear relationship between the log of the PFU and the Ct values, R2 = 0.99), reproducible (low coefficient of variations, below 1%). Additionally, the physicochemical characteristics of the samples tested did not influence the stability of qPCR. Therefore, the qPCR method has the potential value in quantifying and surveying haloviruses in halophilic ecological system. PMID:27192212

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

    PubMed

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

    2013-05-01

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

  3. Brazilian spotted fever: real-time PCR for diagnosis of fatal cases.

    PubMed

    dos Santos, Fabiana Cristina Pereira; do Nascimento, Elvira Maria Mendes; Katz, Gizelda; Angerami, Rodrigo Nogueira; Colombo, Silvia; de Souza, Eliana Rodrigues; Labruna, Marcelo Bahia; da Silva, Marcos Vinicius

    2012-12-01

    Suspicion of Brazilian spotted fever (BSF) should occur in endemic regions upon surveillance of the acute febrile icteric hemorrhagic syndrome (AFIHS). However, limitations associated with currently available laboratory tests pose a challenge to early diagnosis, especially in fatal cases. Two real-time PCR (qPCR) protocols were evaluated to diagnose BSF in 110 fatal AFIHS cases, collected in BSF-endemic regions in 2009-2010. Of these, 24 were positive and 86 negative by indirect immunofluorescence (IFA) assay (cut-off IgG and/or IgM ≥ 128). DNA from these samples was used in the qPCR protocols: one to detect Rickettsia spp. (citrate synthase gene) and another to determine spotted fever group (SFG) Rickettsia species (OmpA gene). Of the 24 IFA-positive samples, 5 (21%) were positive for OmpA and 9 (38%) for citrate synthase. In the IFA-negative group (n=86), OmpA and citrate synthase were positive in 23 (27%) and 27 (31%), respectively. These results showed that the 2 qPCR protocols were about twice as sensitive as the IFA test alone (93% concordance). In conclusion, qPCR is a sensitive method for the diagnosis of fatal BSF cases and should be considered for routine surveillance of AFIHS in places like Brazil, where spotted fever-related lethality is high and other endemic diseases like dengue and leptospirosis can mislead diagnosis.

  4. Semiautomated clone verification by real-time PCR using molecular beacons.

    PubMed

    van Schie, R C; Marras, S A; Conroy, J M; Nowak, N J; Catanese, J J; de Jong, P J

    2000-12-01

    Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) detection system that swiftly and accurately confirms marker content in template containing common repeat elements. A simple, one-tube, real-time PCR assay system was developed to specifically detect regions containing CA and GATA repeats. Ninety-six samples can be confirmed for marker content in a closed-tube format in 3 h, eliminating product confirmation on agarose gels and avoiding crossover contamination. Multiple STSs can be detected simultaneously in the same reaction tube by utilizing molecular beacons labeled with multicolor fluorophores. Template DNA from 260 RPCI-11 bacterial artificial chromosome (BAC) clones was examined for the presence of CA and/or GATA repeats using molecular beacon PCR and compared with conventional PCR results of the same clones. Of the 205 clones containing CA and GATA repeats, we were able to identify 129 clones (CA, n = 99; GATA, n = 30) by using molecular beacons and only 121 clones (CA, n = 92; GATA, n = 29) by conventional PCR amplification. As anticipated, 55 clones that contained sequences other than CA or GATA failed molecular beacon detection. Molecular beacon PCR, employing beacons specific for tandem repeat elements, provides a fast, accurate, and sensitive multiplex detection assay that will expedite verification of marker content in a multitude of template containing these repeats.

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

    PubMed

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

    2015-05-01

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

  6. Multiplex real-time RT-PCR for the simultaneous detection and quantification of GI, GII and GIV noroviruses.

    PubMed

    Farkas, Tibor; Singh, Amy; Le Guyader, Françoise S; La Rosa, Giuseppina; Saif, Linda; McNeal, Monica

    2015-10-01

    Noroviruses are important causes of acute gastroenteritis and are classified into six genogroups with GI, GII and GIV containing human pathogens. This high genetic diversity represents a significant challenge for diagnostic assay development. Genogroup specific monoplex and multiplex real time RT-PCR assays are widely used for the detection of GI and GII noroviruses. On the other hand, GIV norovirus detection is not part of routine laboratory diagnosis. This study describes the development and evaluation of a one tube, real time RT-PCR assay for the simultaneous detection and quantification of GI, GII and GIV noroviruses, including both GIV.1 (human) and GIV.2 (animal) strains. Assay performance was evaluated on a panel of norovirus positive clinical samples by comparison of monoplex and multiplex standard curves and Ct values. The multiplex assay demonstrated equal sensitivity and specificity to the monoplex assays and was able to detect all GI, GII and GIV noroviruses with Ct values equal to that of the monoplex assays. The multiplex assay described in this study will be instrumental for the better understanding of GIV norovirus epidemiology, including their possible zoonotic nature. PMID:26248055

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

    PubMed Central

    2014-01-01

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

  8. Effect of ionizing radiation on the quantitative detection of Salmonella using real-time PCR

    NASA Astrophysics Data System (ADS)

    Lim, Sangyong; Jung, Jinwoo; Kim, Minjeong; Ryu, Sangryeol; Kim, Dongho

    2008-09-01

    Food irradiation is an economically viable technology for inactivating foodborne pathogens, but irradiation can mask pathogens in unhygienically prepared food. The aim of this study was to investigate the effect of irradiation treatment on the detection of Salmonella using real-time PCR. Three commercially available kits were tested, of which the InstaGene Matrix procedure was most effective in preparing template DNA from Salmonella exposed to radiation in broth culture. The minimum level of detection by real-time PCR combined with InstaGene Matrix was 3 log units of Salmonella per milliliter. However, when pure cultures of Salmonella were irradiated at 3 and 5 kGy, the cycle threshold ( CT) increased 1-1.5-fold compared to irradiation at 0 and 1 kGy. This indicated that irradiation treatment may result in an underestimation of bacterial counts due to radiation-induced DNA lesions. We also compared CT values in inoculated chicken homogenates before and after irradiation, which in this model caused a 1.3-3.3-fold underestimation of bacterial counts with respect to irradiation dose.

  9. Real-Time PCR Detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum

    PubMed Central

    Cobos, Rebeca; Martín, Laura; López-Enríquez, Lorena

    2012-01-01

    Phaeomoniella chlamydospora and Phaeoacremonium aleophilum are the two main fungal causal agents of Petri disease and esca. Both diseases cause significant economic losses to viticulturalists. Since no curative control measures are known, proactive defensive measures must be taken. An important aspect of current research is the development of sensitive and time-saving protocols for the detection and identification of these pathogens. Real-time PCR based on the amplification of specific sequences is now being used for the identification and quantification of many infective agents. The present work reports real-time PCR protocols for identification of P. chlamydospora and P. aleophilum. Specificity was demonstrated against purified DNA from 60 P. chlamydospora isolates or 61 P. aleophilum isolates, and no amplification was obtained with 54 nontarget DNAs. The limits of detection (i.e., DNA detectable in 95% of reactions) were around 100 fg for P. chlamydospora and 50 fg for P. aleophilum. Detection was specific and sensitive for P. chlamydospora and P. aleophilum. Spores of P. chlamydospora and P. aleophilum were detected without the need for DNA purification. The established protocols detected these fungi in wood samples after DNA purification. P. chlamydospora was detectable without DNA purification and isolation in 67% of reactions. The detection of these pathogens in wood samples has great potential for use in pathogen-free certification schemes. PMID:22447605

  10. Real-time PCR detection of Phaeomoniella chlamydospora and Phaeoacremonium aleophilum.

    PubMed

    Martín, Maria Teresa; Cobos, Rebeca; Martín, Laura; López-Enríquez, Lorena

    2012-06-01

    Phaeomoniella chlamydospora and Phaeoacremonium aleophilum are the two main fungal causal agents of Petri disease and esca. Both diseases cause significant economic losses to viticulturalists. Since no curative control measures are known, proactive defensive measures must be taken. An important aspect of current research is the development of sensitive and time-saving protocols for the detection and identification of these pathogens. Real-time PCR based on the amplification of specific sequences is now being used for the identification and quantification of many infective agents. The present work reports real-time PCR protocols for identification of P. chlamydospora and P. aleophilum. Specificity was demonstrated against purified DNA from 60 P. chlamydospora isolates or 61 P. aleophilum isolates, and no amplification was obtained with 54 nontarget DNAs. The limits of detection (i.e., DNA detectable in 95% of reactions) were around 100 fg for P. chlamydospora and 50 fg for P. aleophilum. Detection was specific and sensitive for P. chlamydospora and P. aleophilum. Spores of P. chlamydospora and P. aleophilum were detected without the need for DNA purification. The established protocols detected these fungi in wood samples after DNA purification. P. chlamydospora was detectable without DNA purification and isolation in 67% of reactions. The detection of these pathogens in wood samples has great potential for use in pathogen-free certification schemes.

  11. Identification of Legionella Pneumophila in Intubated Patients With TaqMan Real Time PCR

    PubMed Central

    Divan Khosroshahi, Nader; Naserpour Farivar, Taghi; Johari, Pouran

    2015-01-01

    Background: Legionellaceae contains Legionella genus with over 52 species and 64 serogroups. It is one of the most important causes of respiratory disease in human. More than 30% of hospital-acquired pneumonia is caused by Legionella. Ventilator-associated pneumonia (VAP) is an infection acquired in hospital wards, particularly in intensive care unit (ICU). This disease approximately affects 9% to 20% of intubated patients. Mortality in these patients varies between 8% and 76%. Legionella is one of the important factors for infection in intubated patients. Objectives: The present study was aimed to investigate the use of molecular methods in diagnosis of infection caused by Legionella pneumophila. Materials and Methods: In this study, 109 samples of lung secretions collected from intubated patients admitted to ICU wards of four university hospitals in a three-month period were examined. Cultivation and Real time Polymerase Chain Reaction (PCR) methods were used to assess L. pneumophila colonization in these samples. Results: Eleven samples had positive results using real time PCR analysis of 16s rRNA gene fragments specific for L. pneumophila, but according to culture method on specific buffered charcoal-yeast extract medium (BCYE), no positive cases were detected. Of the total positive cases, six were males, one female and four infants. The seven adults aged 40-65 years. Conclusions: Using molecular methods in diagnosis of infection caused by L. pneumophila has a great value because of its high specificity and rapid diagnosis potency. PMID:25834717

  12. Real-time PCR assays for genotyping of Cryptococcus gattii in North America

    PubMed Central

    2014-01-01

    Background Cryptococcus gattii has been the cause of an ongoing outbreak starting in 1999 on Vancouver Island, British Columbia and spreading to mainland Canada and the US Pacific Northwest. In the course of the outbreak, C. gattii has been identified outside of its previously documented climate, habitat, and host disease. Genotyping of C. gattii is essential to understand the ecological and geographical expansion of this emerging pathogen. Methods We developed and validated a mismatch amplification mutation assay (MAMA) real-time PCR panel for genotyping C. gattii molecular types VGI-VGIV and VGII subtypes a,b,c. Subtype assays were designed based on whole-genome sequence of 20 C. gattii strains. Publically available multilocus sequence typing (MLST) data from a study of 202 strains was used for the molecular type (VGI-VGIV) assay design. All assays were validated across DNA from 112 strains of diverse international origin and sample types, including animal, environmental and human. Results Validation revealed each assay on the panel is 100% sensitive, specific and concordant with MLST. The assay panel can detect down to 0.5 picograms of template DNA. Conclusions The (MAMA) real-time PCR panel for C. gattii accurately typed a collection of 112 diverse strains and demonstrated high sensitivity. This is a time and cost efficient method of genotyping C. gattii best suited for application in large-scale epidemiological studies. PMID:24886039

  13. Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum.

    PubMed

    Anniballi, Fabrizio; Auricchio, Bruna; Delibato, Elisabetta; Antonacci, Monia; De Medici, Dario; Fenicia, Lucia

    2012-01-27

    Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities.

  14. Using Real-Time PCR as a tool for monitoring the authenticity of commercial coffees.

    PubMed

    Ferreira, Thiago; Farah, Adriana; Oliveira, Tatiane C; Lima, Ivanilda S; Vitório, Felipe; Oliveira, Edna M M

    2016-05-15

    Coffee is one of the main food products commercialized in the world. Its considerable market value among food products makes it susceptible to adulteration, especially with cereals. Therefore, the objective of this study was to develop a method based on Real-Time Polymerase Chain Reaction (PCR) for detection of cereals in commercial ground roast and soluble coffees. After comparison with standard curves obtained by serial dilution of DNA extracted from barley, corn and rice, the method was sensitive and specific to quantify down to 0.6 pg, 14 pg and 16 pg of barley, corn and rice DNA, respectively. To verify the applicability of the method, 30 commercial samples obtained in different countries were evaluated and those classified as gourmets or superior did not present the tested cereals DNA. However, barley was detected in various traditional (cheaper) samples from South America. In addition, corn and rice were also detected in different samples. Real-Time PCR showed to be suitable for detection of food adulterants in commercial ground roast and soluble coffees.

  15. Using Real-Time PCR as a tool for monitoring the authenticity of commercial coffees.

    PubMed

    Ferreira, Thiago; Farah, Adriana; Oliveira, Tatiane C; Lima, Ivanilda S; Vitório, Felipe; Oliveira, Edna M M

    2016-05-15

    Coffee is one of the main food products commercialized in the world. Its considerable market value among food products makes it susceptible to adulteration, especially with cereals. Therefore, the objective of this study was to develop a method based on Real-Time Polymerase Chain Reaction (PCR) for detection of cereals in commercial ground roast and soluble coffees. After comparison with standard curves obtained by serial dilution of DNA extracted from barley, corn and rice, the method was sensitive and specific to quantify down to 0.6 pg, 14 pg and 16 pg of barley, corn and rice DNA, respectively. To verify the applicability of the method, 30 commercial samples obtained in different countries were evaluated and those classified as gourmets or superior did not present the tested cereals DNA. However, barley was detected in various traditional (cheaper) samples from South America. In addition, corn and rice were also detected in different samples. Real-Time PCR showed to be suitable for detection of food adulterants in commercial ground roast and soluble coffees. PMID:26775992

  16. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    PubMed

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections.

  17. A multiplex real-time PCR panel assay for simultaneous detection and differentiation of 12 common swine viruses.

    PubMed

    Shi, Xiju; Liu, Xuming; Wang, Qin; Das, Amaresh; Ma, Guiping; Xu, Lu; Sun, Qing; Peddireddi, Lalitha; Jia, Wei; Liu, Yanhua; Anderson, Gary; Bai, Jianfa; Shi, Jishu

    2016-10-01

    Mixed infection with different pathogens is common in swine production systems especially under intensive production conditions. Quick and accurate detection and differentiation of different pathogens are necessary for epidemiological surveillance, disease management and import and export controls. In this study, we developed and validated a panel of multiplex real-time PCR/RT-PCR assays composed of four subpanels, each detects three common swine pathogens. The panel detects 12 viruses or viral serotypes, namely, VSV-IN, VSV-NJ, SVDV, CSFV, ASFV, FMDV, PCV2, PPV, PRV, PRRSV-NA, PRRSV-EU and SIV. Correlation coefficients (R(2)) and PCR amplification efficiencies of all singular and triplex real-time PCR reactions are within the acceptable range. Comparison between singular and triplex real-time PCR assays of each subpanel indicates that there is no significant interference on assay sensitivities caused by multiplexing. Specificity tests on 226 target clinical samples or 4 viral strains and 91 non-target clinical samples revealed that the real-time PCR panel is 100% specific, and there is no cross amplification observed. The limit of detection of each triplex real-time PCR is less than 10 copies per reaction for DNA, and less than 16 copies per reaction for RNA viruses. The newly developed multiplex real-time PCR panel also detected different combinations of co-infections as confirmed by other means of detections. PMID:27506582

  18. Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife.

    PubMed

    Cuttell, Leigh; Corley, Sean W; Gray, Christian P; Vanderlinde, Paul B; Jackson, Louise A; Traub, Rebecca J

    2012-09-10

    Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking 10 g of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value=1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated

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

    PubMed Central

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

    2015-01-01

    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. PMID:25351780

  20. Development and calibration of real-time PCR for quantification of airborne microorganisms in air samples

    NASA Astrophysics Data System (ADS)

    An, Hey Reoun; Mainelis, Gediminas; White, Lori

    This manuscript describes the coupling of bioaerosol collection and the use of real-time PCR (RT-PCR) to quantify the airborne bacteria. The quantity of collected bacteria determined by RT-PCR is compared with conventional quantification techniques, such as culturing, microscopy and airborne microorganism counting by using optical particle counter (OPC). Our data show that an experimental approach used to develop standard curves for use with RT-PCR is critical for accurate sample quantification. Using universal primers we generated 12 different standard curves which were used to quantify model organism Escherichia coli (Migula) Catellani from air samples. Standard curves prepared using a traditional approach, where serially diluted genomic DNA extracted from pure cultured bacteria were used in PCR reaction as a template DNA yielded significant underestimation of sample quantities compared to airborne microorganism concentration as measured by an OPC. The underestimation was especially pronounced when standard curves were built using colony forming units (CFUs). In contrast, the estimate of cell concentration in an air sample by RT-PCR was more accurate (˜60% compared to the airborne microorganism concentration) when the standard curve was built using aerosolized E. coli. The accuracy improved even further (˜100%) when air samples used to build the standard curves were diluted first, then the DNA extracted from each dilution was amplified by the RT-PCR—to mimic the handling of air samples with unknown and possibly low concentration. Therefore, our data show that standard curves used for quantification by RT-PCR needs to be prepared using the same environmental matrix and procedures as handling of the environmental sample in question. Reliance on the standard curves generated with cultured bacterial suspension (a traditional approach) may lead to substantial underestimation of microorganism quantities in environmental samples.

  1. Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods.

    PubMed

    Rodríguez, Alicia; Rodríguez, Mar; Luque, M Isabel; Martín, Alberto; Córdoba, Juan J

    2012-08-01

    Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products. PMID:22475946

  2. Real-time PCR assays for detection and quantification of aflatoxin-producing molds in foods.

    PubMed

    Rodríguez, Alicia; Rodríguez, Mar; Luque, M Isabel; Martín, Alberto; Córdoba, Juan J

    2012-08-01

    Aflatoxins are among the most toxic mycotoxins. Early detection and quantification of aflatoxin-producing species is crucial to improve food safety. In the present work, two protocols of real-time PCR (qPCR) based on SYBR Green and TaqMan were developed, and their sensitivity and specificity were evaluated. Primers and probes were designed from the o-methyltransferase gene (omt-1) involved in aflatoxin biosynthesis. Fifty-three mold strains representing aflatoxin producers and non-producers of different species, usually reported in food products, were used as references. All strains were tested for aflatoxins production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the proposed qPCR method was demonstrated by the strong linear relationship of the standard curves constructed with the omt-1 gene copy number and Ct values for the different aflatoxin producers tested. The ability of the qPCR protocols to quantify aflatoxin-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 4 to 1 log cfu/g per reaction for all qPCR assays in the different food matrices (peanuts, spices and dry-fermented sausages). The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g for SYBR Green and TaqMan assays. No significant effect was observed due to the different equipment, operator, and qPCR methodology used in the tests of repeatability and reproducibility for different foods. The proposed methods quantified with high efficiency the fungal load in foods. These qPCR protocols are proposed for use to quantify aflatoxin-producing molds in food products.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  5. [Study of real-time PCR assays for rapid detection of food-borne pathogens].

    PubMed

    Fukushima, Hiroshi; Tsunomori, Yoshie

    2005-09-01

    A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using QIAamp DNA Stool Minikit was evaluated for detection of 8 of 19 species of food-borne pathogens, including Plesiomonas shigelloides, Providencia alcalifaciens, in five stool specimens. The time frame was within 2h or less. The protocol used the same LC-PCR with 22 pairs of specific primers. The rapid amplification and reliable detection of specific genes were determined by this LC-PCR assay from 10 cases of food-borne outbreaks in Shimane Prefecture from 2002 to 2005. These cases included Campylobacter jejuni (4), Clostridium perfringens (2), enteropathogenic Escherichia coli and astA positive E. coli (1), and astA positive E. coli, enterohemorrhagic E. coli 026, and Bacillus cereus (1 each). Rapid amplification and reliable detection of specific genes of food-or water-borne pathogenic bacteria in fecal samples should facilitate the diagnosis and management of food-borne outbreaks.

  6. Detection by real time PCR of walnut allergen coding sequences in processed foods.

    PubMed

    Linacero, Rosario; Ballesteros, Isabel; Sanchiz, Africa; Prieto, Nuria; Iniesto, Elisa; Martinez, Yolanda; Pedrosa, Mercedes M; Muzquiz, Mercedes; Cabanillas, Beatriz; Rovira, Mercè; Burbano, Carmen; Cuadrado, Carmen

    2016-07-01

    A quantitative real-time PCR (RT-PCR) method, employing novel primer sets designed on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences, was set up and validated. Its specificity, sensitivity, and applicability were evaluated. The DNA extraction method based on CTAB-phenol-chloroform was best for walnut. RT-PCR allowed a specific and accurate amplification of allergen sequence, and the limit of detection was 2.5pg of walnut DNA. The method sensitivity and robustness were confirmed with spiked samples, and Jug r 3 primers detected up to 100mg/kg of raw walnut (LOD 0.01%, LOQ 0.05%). Thermal treatment combined with pressure (autoclaving) reduced yield and amplification (integrity and quality) of walnut DNA. High hydrostatic pressure (HHP) did not produce any effect on the walnut DNA amplification. This RT-PCR method showed greater sensitivity and reliability in the detection of walnut traces in commercial foodstuffs compared with ELISA assays.

  7. Real-time quantitative PCR for Marek's disease vaccine virus in feather samples: applications and opportunities.

    PubMed

    Baigent, S; Nair, V; Currie, R

    2006-01-01

    Marek's disease, an economically-important lymphoid neoplasm of chickens, is controlled by vaccination with CVI988 strain of Marek's disease herpesvirus. Sub-optimal vaccinal protection can have multiple causes. Accurate quantification of CVI988 in vaccinated chickens will assist in understanding the causes of these vaccine breaks. We developed, optimised and validated a real-time PCR assay for quantification of CVI988 vaccine virus (in terms of CVI988 genomes per 10,000 cells) in the feather tips, a rich source of viral DNA which can easily be sampled in a non-invasive manner. CVI988 load in feathers was predictive of CVI988 load in spleen, so is anticipated to be a good predictor of protection. The optimal age of chicks for feather collection is between two to five weeks, and feathers are preferentially taken from the axillary tract. This PCR test is now used to monitor vaccine virus levels in commercial chicks. For each flock under test, fifty birds are feather sampled for q-PCR. We describe how the feather CVI988 genome profile can show the flock's response to vaccination, and the likelihood of vaccine breaks. Furthermore, the q-PCR test can be applied to researching optimal timing and vaccine delivery routes, and optimal vaccination regimes for different breeds of chick. PMID:17058503

  8. 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.

  9. Real-time quantitative PCR detection of Mycobacterium avium subspecies in meat products.

    PubMed

    Klanicova, B; Slana, I; Vondruskova, H; Kaevska, M; Pavlik, I

    2011-04-01

    The aim of this work was to examine various purchased meat products and to find out if any traces of Mycobacterium avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. paratuberculosis could be detected in these samples. Analysis of the meat products (raw, cooked, and fermented) was performed using a real-time quantitative PCR (qPCR) method for the detection of specific insertion sequences: duplex qPCR for the detection of IS900 specific for M. avium subsp. paratuberculosis, and triplex qPCR for the detection of IS901 specific for Mycobacterium avium subsp. avium and IS 1245 specific for M. avium subsp. hominissuis. Of the 77 analyzed meat samples, 17 (22%) were found to contain M. avium subsp. paratuberculosis DNA, 4 (5%) samples contained Mycobacterium avium subsp. avium DNA, and in 12 (16%) samples M. avium subsp. hominissuis DNA was detected. The concentration of M. avium subsp. paratuberculosis and M. avium subsp. hominissuis DNA in some meat products exceeded 10(4) genomes per g. Culture examination of these mycobacterial subspecies was negative. By analyzing a range of meat products, we have provided evidence to support the hypothesis that M. avium is present in everyday commodities sold to the general public.

  10. Development and Evaluation of an Enterovirus D68 Real-Time Reverse Transcriptase PCR Assay

    PubMed Central

    Wylie, Todd N.; Wylie, Kristine M.; Buller, Richard S.; Cannella, Maria

    2015-01-01

    We have developed and evaluated a real-time reverse transcriptase PCR (RT-PCR) assay for the detection of human enterovirus D68 (EV-D68) in clinical specimens. This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness. As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO. This sequence, along with other GenBank sequences from past EV-D68 occurrences, was used to computationally select a region of EV-D68 appropriate for targeting in a strain-specific RT-PCR assay. The RT-PCR assay amplifies a segment of the VP1 gene, with an analytic limit of detection of 4 copies per reaction, and it was more sensitive than commercially available assays that detect enteroviruses and rhinoviruses without distinguishing between the two, including three multiplex respiratory panels approved for clinical use by the FDA. The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962. This assay should be useful for identifying and studying current and future outbreaks of EV-D68 viruses. PMID:26063859

  11. Detection by real time PCR of walnut allergen coding sequences in processed foods.

    PubMed

    Linacero, Rosario; Ballesteros, Isabel; Sanchiz, Africa; Prieto, Nuria; Iniesto, Elisa; Martinez, Yolanda; Pedrosa, Mercedes M; Muzquiz, Mercedes; Cabanillas, Beatriz; Rovira, Mercè; Burbano, Carmen; Cuadrado, Carmen

    2016-07-01

    A quantitative real-time PCR (RT-PCR) method, employing novel primer sets designed on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences, was set up and validated. Its specificity, sensitivity, and applicability were evaluated. The DNA extraction method based on CTAB-phenol-chloroform was best for walnut. RT-PCR allowed a specific and accurate amplification of allergen sequence, and the limit of detection was 2.5pg of walnut DNA. The method sensitivity and robustness were confirmed with spiked samples, and Jug r 3 primers detected up to 100mg/kg of raw walnut (LOD 0.01%, LOQ 0.05%). Thermal treatment combined with pressure (autoclaving) reduced yield and amplification (integrity and quality) of walnut DNA. High hydrostatic pressure (HHP) did not produce any effect on the walnut DNA amplification. This RT-PCR method showed greater sensitivity and reliability in the detection of walnut traces in commercial foodstuffs compared with ELISA assays. PMID:26920302

  12. Sensitivity comparison of real-time PCR probe designs on a model DNA plasmid.

    PubMed

    Wang, L; Blasic, J R; Holden, M J; Pires, R

    2005-09-15

    We investigated three probe design strategies used in quantitative polymerase chain reaction (PCR) for sensitivity in detection of the PCR amplicon. A plasmid with a 120-bp insert served as the DNA template. The probes were TaqMan, conventional molecular beacon (MB), and shared-stem molecular beacon (ATssMB and GCssMB). A shared-stem beacon probe combines the properties of a TaqMan probe and a conventional molecular beacon. It was found that the overall sensitivities for the four PCR probes are in the order of MB>ATssMB>GCssMB>TaqMan. The fluorescence quantum yield measurements indicate that incomplete or partial enzymatic cleavage catalyzed by Taq polymerase is the likely cause of the low sensitivities of two shared-stem beacons when compared with the conventional beacon probe. A high-fluorescence background associated with the current TaqMan probe sequence contributes to the relatively low detection sensitivity and signal-to-background ratio. The study points out that the nucleotide environment surrounding the reporting fluorophore can strongly affect the probe performance in real-time PCR.

  13. Real-Time PCR Quantitation of Clostridia in Feces of Autistic Children

    PubMed Central

    Song, Yuli; Liu, Chengxu; Finegold, Sydney M.

    2004-01-01

    Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children. We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C. bolteae, in stool specimens. Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains. In this procedure, a linear relationship exists between the threshold cycle (CT) fluorescence value and the number of bacterial cells (CFU). The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C. bolteae could be detected per PCR. Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C. bolteae and the following Clostridium groups were statistically significant: mean counts of C. bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 × 108 CFU/g in autistic children and 4.8 × 108 CFU/g in controls; respectively). More subjects need to be studied. The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract. PMID:15528506

  14. Evaluation of absolute quantitation by nonlinear regression in probe-based real-time PCR

    PubMed Central

    Goll, Rasmus; Olsen, Trine; Cui, Guanglin; Florholmen, Jon

    2006-01-01

    Background In real-time PCR data analysis, the cycle threshold (CT) method is currently the gold standard. This method is based on an assumption of equal PCR efficiency in all reactions, and precision may suffer if this condition is not met. Nonlinear regression analysis (NLR) or curve fitting has therefore been suggested as an alternative to the cycle threshold method for absolute quantitation. The advantages of NLR are that the individual sample efficiency is simulated by the model and that absolute quantitation is possible without a standard curve, releasing reaction wells for unknown samples. However, the calculation method has not been evaluated systematically and has not previously been applied to a TaqMan platform. Aim: To develop and evaluate an automated NLR algorithm capable of generating batch production regression analysis. Results Total RNA samples extracted from human gastric mucosa were reverse transcribed and analysed for TNFA, IL18 and ACTB by TaqMan real-time PCR. Fluorescence data were analysed by the regular CT method with a standard curve, and by NLR with a positive control for conversion of fluorescence intensity to copy number, and for this purpose an automated algorithm was written in SPSS syntax. Eleven separate regression models were tested, and the output data was subjected to Altman-Bland analysis. The Altman-Bland analysis showed that the best regression model yielded quantitative data with an intra-assay variation of 58% vs. 24% for the CT derived copy numbers, and with a mean inter-method deviation of × 0.8. Conclusion NLR can be automated for batch production analysis, but the CT method is more precise for absolute quantitation in the present setting. The observed inter-method deviation is an indication that assessment of the fluorescence conversion factor used in the regression method can be improved. However, the versatility depends on the level of precision required, and in some settings the increased cost effectiveness of NLR

  15. Validation of a real-time reverse transcriptase-PCR assay for the detection of H7 avian influenza virus

    USGS Publications Warehouse

    Pedersen, J.; Killian, M.L.; Hines, N.; Senne, D.; Panigrahy, B.; Ip, H.S.; Spackman, Erica

    2010-01-01

    This report describes the validation of an avian influenza virus (AIV) H7 subtype-specific real-time reverse transcriptasePCR (rRT-PCR) assay developed at the Southeast Poultry Research Laboratory (SEPRL) for the detection of H7 AI in North and South American wild aquatic birds and poultry. The validation was a collaborative effort by the SEPRL and the National Veterinary Services Laboratories. The 2008 H7 rRT-PCR assay detects 101 50% embryo infectious doses per reaction, or 103104 copies of transcribed H7 RNA. Diagnostic sensitivity and specificity were estimated to be 97.5% and 82.4%, respectively; the assay was shown to be specific for H7 AI when tested with >270 wild birds and poultry viruses. Following validation, the 2008 H7 rRT-PCR procedure was adopted as an official U.S. Department of Agriculture procedure for the detection of H7 AIV. The 2008 H7 assay replaced the previously used (2002) assay, which does not detect H7 viruses currently circulating in wild birds in North and South America. ?? 2010 American Association of Avian Pathologists.

  16. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught Mosquitoes

    PubMed Central

    2013-01-01

    Background Zika virus (ZIKV), a mosquito borne flavivirus is a pathogen affecting humans in Asia and Africa. ZIKV infection diagnosis relies on serology–which is challenging due to cross-reactions with other flaviviruses and/or absence or low titer of IgM and IgG antibodies at early phase of infection- virus isolation, which is labor intensive, time consuming and requires appropriate containment. Therefore, real-time RT-PCR (rRT-PCR) is an appealing option as a rapid, sensitive and specific method for detection of ZIKV in the early stage of infection. So far, only one rRT-PCR assay has been described in the context of the outbreak in Micronesia in 2007. In this study, we described a one step rRT-PCR for ZIKV which can detect a wider genetic diversity of ZIKV isolates from Asia and Africa. Results The NS5 protein coding regions of African ZIKV isolates were sequenced and aligned with representative flaviviruses sequences from GenBank to design primers and probe from conserved regions. The analytical sensitivity of the assay was evaluated to be 32 genome-equivalents and 0.05 plaque forming unit (pfu). The assay was shown to detect 37 ZIKV isolates covering a wide geographic in Africa and Asia over 36 years but none of the 31 other flaviviruses tested showing high analytical specificity. The rRT-PCR could be performed in less than 3 hours. This method was used successfully to detect ZIKV strains from field-caught mosquitoes. Conclusion We have developed a rapid, sensitive and specific rRT – PCR for detection of ZIKV. This assay is a useful tool for detection of ZIKV infection in regions where a number of other clinically indistinguishable arboviruses like dengue or chikungunya co-circulate. Further studies are needed to validate this assay in clinical positive samples collected during acute ZIKV infection. PMID:24148652

  17. 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

  18. 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. PMID:25903566

  19. Quantitative real-time PCR (qPCR)--based tool for detection and quantification of Cordyceps militaris in soil.

    PubMed

    Saragih, Syaiful Amri; Takemoto, S; Hisamoto, Y; Fujii, M; Sato, H; Kamata, N

    2015-01-01

    A quantitative real-time PCR using a primer pair CM2946F/CM3160R was developed for specific detection and quantification of Cordyceps militaris from soil. Standard curves were obtained for genomic DNA and DNA extracts from autoclaved soil with a certain dose of C. militaris suspension. C. militaris was detected from two forest soil samples out of ten that were collected when fruit bodies of C. militaris were found. This method seemed effective in detection of C. militaris in the soil and useful for rapid and reliable quantification of C. militaris in different ecosystems. PMID:25446034

  20. Quantitative real-time PCR (qPCR)--based tool for detection and quantification of Cordyceps militaris in soil.

    PubMed

    Saragih, Syaiful Amri; Takemoto, S; Hisamoto, Y; Fujii, M; Sato, H; Kamata, N

    2015-01-01

    A quantitative real-time PCR using a primer pair CM2946F/CM3160R was developed for specific detection and quantification of Cordyceps militaris from soil. Standard curves were obtained for genomic DNA and DNA extracts from autoclaved soil with a certain dose of C. militaris suspension. C. militaris was detected from two forest soil samples out of ten that were collected when fruit bodies of C. militaris were found. This method seemed effective in detection of C. militaris in the soil and useful for rapid and reliable quantification of C. militaris in different ecosystems.

  1. Utility of a fecal real-time PCR protocol for detection of Mycobacterium bovis infection in African buffalo (Syncerus caffer).

    PubMed

    Roug, Annette; Geoghegan, Claire; Wellington, Elizabeth; Miller, Woutrina A; Travis, Emma; Porter, David; Cooper, David; Clifford, Deana L; Mazet, Jonna A K; Parsons, Sven

    2014-01-01

    A real-time PCR protocol for detecting Mycobacterium bovis in feces was evaluated in bovine tuberculosis-infected African buffalo (Syncerus caffer). Fecal samples spiked with 1.42 × 10(3) cells of M. bovis culture/g and Bacille Calmette-Guérin standards with 1.58 × 10(1) genome copies/well were positive by real-time PCR but all field samples were negative.

  2. Detection of dermcidin for sweat identification by real-time RT-PCR and ELISA.

    PubMed

    Sakurada, Koichi; Akutsu, Tomoko; Fukushima, Hisayo; Watanabe, Ken; Yoshino, Mineo

    2010-01-30

    We evaluated the performance of real-time RT-PCR and ELISA assays for detection of dermcidin (DCD) in sweat and body-fluid stains. DCD, a small antibiotic peptide secreted into human sweat, was detected by real-time RT-PCR in 7-day-old stains containing as small as 10 microL of sweat, and the assay showed high specificity when testing 7-day-old stains containing 30 microL of other body-fluid. ELISA using anti-human dermcidin mouse monoclonal antibody detected DCD sweat diluted up to approximately 10,000-fold and could specifically detect DCD in 10 microL of body-fluid stains. The performance of the two assays was tested during winter on samples that simulated forensic case samples: an undershirt and a sock worn for 20 h, a handkerchief used to wipe the brow several times within 12h, a cap and a cotton glove worn for 4h, and a white robe worn at intervals for 2 years. The result showed that the former assay detected DCD in all sites of the undershirt examined (armpit, back, and breast), and the latter gave a relatively high OD value in the armpit among the three sites. For the socks, although the latter assay gave very high OD values in both the center and toe of the foot sole, the former could not detect DCD in both of them. These results indicate that highly damp conditions, such as inside a shoe, might promote the degradation of mRNA in samples such as socks. In the other case samples, sweat was adequately detected by both assays. This study is the first demonstration of the use of real-time RT-PCR to sensitively identify sweat among body-fluid stains, and it confirmed that dermcidin was an excellent marker for sweat identification. In addition, the usefulness of ELISA was also verified. Positive sweat identification using these assays is expected to assist forensic practice.

  3. Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR.

    PubMed

    Demeler, Janina; Ramünke, Sabrina; Wolken, Sonja; Ianiello, Davide; Rinaldi, Laura; Gahutu, Jean Bosco; Cringoli, Giuseppe; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

    2013-01-01

    Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g). A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles - only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post

  4. DOUBLE-STEP MULTIPLEX REAL TIME PCR WITH MELTING CURVE ANALYSIS FOR DETECTION AND DIFFERENTIATION OF MYCOBACTERIA IN SPUTUM.

    PubMed

    Kasa, Sawinee; Faksri, Kiatichai; Kaewkes, Wanlop; Lulitanond, Viraphong; Namwat, Wises

    2015-01-01

    Mycobacterium tuberculosis (M. tb) is a causative agent of tuberculosis, a worldwide public health problem. In recent years, the incidence of human mycobacterial infection due to species other than M. tb has increased. However, the lack of specific, rapid, and inexpensive methods for identification of mycobacterial species remains a pressing problem. A diagnostic test was developed for mycobacterial strain differentiation utilizing a double-step multiplex real time PCR together with melting curve analysis for identifying and distinguishing among M. tb, M. bovis BCG, other members of M. tb. complex, M. avium, and non-tuberculosis mycobacteria. The assay was tested using 167 clinical sputum samples in comparison with acid-fast staining and culturing. Using only the first step (step A) the assay achieved sensitivity and specificity of 81% and 95%, respectively. The detection limit was equivalent to 50 genome copies.

  5. DOUBLE-STEP MULTIPLEX REAL TIME PCR WITH MELTING CURVE ANALYSIS FOR DETECTION AND DIFFERENTIATION OF MYCOBACTERIA IN SPUTUM.

    PubMed

    Kasa, Sawinee; Faksri, Kiatichai; Kaewkes, Wanlop; Lulitanond, Viraphong; Namwat, Wises

    2015-01-01

    Mycobacterium tuberculosis (M. tb) is a causative agent of tuberculosis, a worldwide public health problem. In recent years, the incidence of human mycobacterial infection due to species other than M. tb has increased. However, the lack of specific, rapid, and inexpensive methods for identification of mycobacterial species remains a pressing problem. A diagnostic test was developed for mycobacterial strain differentiation utilizing a double-step multiplex real time PCR together with melting curve analysis for identifying and distinguishing among M. tb, M. bovis BCG, other members of M. tb. complex, M. avium, and non-tuberculosis mycobacteria. The assay was tested using 167 clinical sputum samples in comparison with acid-fast staining and culturing. Using only the first step (step A) the assay achieved sensitivity and specificity of 81% and 95%, respectively. The detection limit was equivalent to 50 genome copies. PMID:26513906

  6. Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR.

    PubMed

    Basuni, M; Mohamed, Z; Ahmad, M; Zakaria, N Z; Noordin, R

    2012-09-01

    Intestinal parasites are the causative agents of a number of important human infections in developing countries. The objective of this study was to determine the prevalence of selected helminths and protozoan infections among patients admitted with gastrointestinal disorders at Hospital Universiti Sains Malaysia, Kelantan, Malaysia using multiplex real-time PCR. In addition microscopic examination was also performed following direct smear, zinc sulphate concentration and Kato-Katz thick smear techniques; and the presence of protozoan parasites was confirmed using trichrome and acid-fast stains. Of the 225 faecal samples analysed, 26.2% were positive for intestinal parasites by the multiplex real-time PCR, while 5.3% were positive by microscopy. As compared to microscopy, the multiplex real-time PCR detected 5.8 and 4.5 times more positives for the selected helminth and protozoan infections respectively. Among the selected helminths detected in this study, hookworm was the most prevalent by real-time PCR, while Ascaris lumbricoides was detected the most by microscopy. Meanwhile, among the selected protozoa detected in this study, Entamoeba histolytica was the most prevalent by real-time PCR, however microscopy detected equal number of cases with E. histolytica and Giardia lamblia. This study showed that real-time PCR can be used to obtain a more accurate prevalence data on intestinal helminths and protozoa.

  7. Real-time PCR quantification of human complement C4A and C4B genes

    PubMed Central

    Szilagyi, Agnes; Blasko, Bernadett; Szilassy, Denes; Fust, George; Sasvari-Szekely, Maria; Ronai, Zsolt

    2006-01-01

    Background The fourth component of human complement (C4), an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B) in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes. Results A novel quantitative real-time PCR (qPCR) technique was developed for rapid and accurate quantification of the C4A and C4B genes applying a duplex, TaqMan based methodology. The reliable, single-step analysis provides the determination of the copy number of the C4A and C4B genes applying a wide range of DNA template concentration (0.3–300 ng genomic DNA). The developed qPCR was applied to determine C4A and C4B gene dosages in a healthy Hungarian population (N = 118). The obtained data were compared to the results of an earlier study of the same population. Moreover a set of 33 samples were analyzed by two independent methods. No significant difference was observed between the gene dosages determined by the employed techniques demonstrating the reliability of the novel qPCR methodology. A Microsoft Excel worksheet and a DOS executable are also provided for simple and automated evaluation of the measured data. Conclusion This report describes a novel real-time PCR method for single-step quantification of C4A and C4B genes. The developed technique could facilitate studies investigating disease association of different C4 isotypes. PMID:16403222

  8. Evaluation of a new single-tube multiprobe real-time PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar.

    PubMed

    Liang, Shih-Yu; Hsia, Kan-Tai; Chan, Yun-Hsien; Fan, Chia-Kwung; Jiang, Donald Dah-Shyong; Landt, Olfert; Ji, Dar-Der

    2010-08-01

    A single-tube multiprobe real-time PCR assay for simultaneous detection of Entamoeba histolytica and Entamoeba dispar was developed. One primer pair with 2 species-specific probes was designed based on new SSU RNA regions of the ribosomal DNA-containing episome. The sensitivity is 1 parasite per milliliter of feces and thus superior to the conventional nested PCR and comparable to other published real-time PCR protocols. The applicability for clinical diagnosis was validated with 218 stool specimens from patients. A total of 51 E. histolytica and 39 E. dispar positive samples was detected by the multiprobe real-time PCR compared to 39 and 22 by routine nested PCR diagnosis. The detection rate of Entamoeba species for the multiprobe real-time PCR assays was significantly higher than the nested PCR (40.8% vs. 28.0%, P < 0.01). The test did not show cross reactivity with DNA from Entamoeba moshkovskii, Giardia lamblia , Cryptosporidium sp., Escherichia coli , or other nonpathogenic enteric parasites. The multiprobe real-time PCR assay is simple and rapid and has high specificity and sensitivity. The assay could streamline the laboratory diagnosis procedure and facilitate epidemiological investigation.

  9. Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation.

    PubMed

    Wei, Yongcheng; Liu, Qinghua; Dong, Hongyu; Zhou, Zhichun; Hao, Yanping; Chen, Xuelian; Xu, Liuyi

    2016-01-01

    Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana. PMID:26800152

  10. Selection of Reference Genes for Real-Time Quantitative PCR in Pinus massoniana Post Nematode Inoculation

    PubMed Central

    Wei, Yongcheng; Liu, Qinghua; Dong, Hongyu; Zhou, Zhichun; Hao, Yanping; Chen, Xuelian; Xu, Liuyi

    2016-01-01

    Pinus massoniaia Lamb has gained more and more attention as the most important tree species for timber and forestation in South China. Gene expression studies are of great importance to identify new and elite cultivars. Real-time quantitative PCR, a highly sensitive and specific method, is commonly used in the analysis of gene expression. The appropriate reference genes must be employed to normalize the calculation program for ascertaining repeatable and significant results. Herein, eleven housekeeping genes were evaluated during different stages of P. massoniana post nematode inoculation in this study. Three statistical approaches such as geNorm, NormFinder and BestKeeper were selected to analyze the stability of candidate genes. The results indicated that U2af and β-TUB were the most stable reference genes. These two genes could be used for the normalization in most of the experiments of P. massoniana, while Histone and AK were the least stable ones. In addition, EF expressed at the lowest average Ct value was the most abundant candidate gene. As an important gene associated with defense mechanisms, ABC transporter was analyzed by qRT-PCR, and the results were used to confirm the reliability of two genes. The selected reference genes in the present study will be conducive to future gene expression normalized by qRT-PCR in P. massoniana. PMID:26800152

  11. Looking for reference genes for real-time quantitative PCR experiments in Rhodnius prolixus (Hemiptera: Reduviidae).

    PubMed

    Majerowicz, D; Alves-Bezerra, M; Logullo, R; Fonseca-de-Souza, A L; Meyer-Fernandes, J R; Braz, G R C; Gondim, K C

    2011-12-01

    Quantitative real-time PCR (qPCR) has become one of the most used techniques to measure gene expression. However, normalization of gene expression data against reference genes is essential, although these are usually used without any kind of validation. The expression of seven genes was compared in organs of Rhodnius prolixus under diverse conditions, using published software to test gene expression stability. Rp18S and elongation factor 1 (RpEF -1) were the most reliable genes for normalization in qPCR when gene expression in different organs was compared. Moreover, both genes were found to be the best references when transcript levels were compared in the posterior midgut of insects infected with Trypanosoma cruzi. Rp18S was also the best reference gene in the fat bodies of unfed and fed insects. By contrast, RpEF-1 was found to be the best reference gene for comparison between posterior midguts, and RpMIP or RpActin should be used to compare gene expression in the ovaries. Although Rp18S is indicated here as the best reference in most cases, reports from the literature show that it is difficult to find an optimum reference gene. Nevertheless, validation of candidate genes to be taken as references is important when new experimental conditions are tested to avoid incorrect data interpretation. PMID:21929722

  12. Direct detection of salmonella cells in the air of livestock stables by real-time PCR.

    PubMed

    Fallschissel, Kerstin; Kämpfer, Peter; Jäckel, Udo

    2009-11-01

    A SYBR Green real-time quantitative polymerase chain reaction (qPCR) assay for specific detection and quantification of airborne Salmonella cells in livestock housings is presented. A set of specific primers was tested and validated for specific detection and quantification of Salmonella-specific invA genes of DNA extracted from bioaerosol samples. Application of the method to poultry house bioaerosol samples showed concentrations ranging from 2.2 x 10(1) to 3 x 10(6) Salmonella targets m(-3) of air. Salmonella were also detected by a cultivation-based approach in some samples, but concentrations were two to three magnitudes lower than the concentrations detected by molecular biological results. Specificity of results was demonstrated by cloning analyses of PCR products, which were exclusively assigned to the genus Salmonella. However, by molecular methods, microorganisms are detected independently of their viability status, leading to an overestimation of concentration. Hence, the survival rate of Salmonella cells was measured on filter surfaces during filtration samplings where 82% of the cells died within 20 min of filtration. The results clearly show the specificity and practicability of the established qPCR assay for analysis and quantification of salmonellae in bioaerosols. The results demonstrate airborne Salmonella workplace concentrations in poultry production of up to 3.3% of 4',6-Diamidino-2-phenylindole-counted total cell numbers.

  13. Detection of airborne genetically modified maize pollen by real-time PCR.

    PubMed

    Folloni, Silvia; Kagkli, Dafni-Maria; Rajcevic, Bojan; Guimarães, Nilson C C; Van Droogenbroeck, Bart; Valicente, Fernando H; Van den Eede, Guy; Van den Bulcke, Marc

    2012-09-01

    The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves. PMID:22805239

  14. Cancer-germline gene expression in pediatric solid tumors using quantitative real-time PCR.

    PubMed

    Jacobs, Joannes F M; Brasseur, Francis; Hulsbergen-van de Kaa, Christina A; van de Rakt, Mandy W M M; Figdor, Carl G; Adema, Gosse J; Hoogerbrugge, Peter M; Coulie, Pierre G; de Vries, I Jolanda M

    2007-01-01

    Cancer-germline genes (CGGs) code for immunogenic antigens that are present on various human tumors but not on normal tissues. The importance of CGGs in cancer immunotherapy has led to detailed studies of their expression in a range of human tumors. We measured the levels of expression of 12 CGGs in various pediatric solid tumors to identify targets for therapeutic cancer vaccines. Quantitative real-time PCR (qPCR) was used to measure the expression of 8 MAGE genes and of genes LAGE-2/NY-ESO-1 and GAGE-1, 2, 8 in 9 osteosarcomas, 10 neuroblastomas, 12 rhabdomyosarcomas and 18 Ewing's sarcomas. Nine tumors were also examined by immunohistochemistry with monoclonal antibodies specific for the MAGE-A1, MAGE-A4 and NY-ESO-1 proteins. All osteosarcoma and 80% of neuroblastoma samples expressed several CGGs at high levels. Six of 12 rhabdomyosarcomas and 11 of 18 Ewing's sarcomas expressed at least one CGG. Immunohistochemistry data correlated well with qPCR results and showed a homogeneous protein distribution pattern in most positive tumors. No correlation was found between the levels of CGG expression in the tumors and clinicopathological parameters of the patients. Pediatric solid tumors express several CGGs, which encode antigens that could be targeted in therapeutic vaccination trials. Several CGGs of the MAGE, GAGE and LAGE families are coexpressed in a large proportion of osteosarcoma and neuroblastoma samples. Some rhabdomyosarcomas express several of these genes at high levels. Ewing's sarcomas have an overall low CGG expression.

  15. Assessment of bluetongue viraemia in sheep by real-time PCR and correlation with viral infectivity.

    PubMed

    Chatzinasiou, E; Dovas, C I; Papanastassopoulou, M; Georgiadis, M; Psychas, V; Bouzalas, I; Koumbati, M; Koptopoulos, G; Papadopoulos, O

    2010-11-01

    Inoculation of embryonated chicken eggs is the standard method for the titration of infectious Bluetongue virus (BTV). Here, six RNA extraction methods coupled with optimised dsRNA denaturation and real-time RT-PCR were evaluated for the quantitation of BTV in blood samples from experimentally infected sheep and results were correlated to infectious virus titres. An exogenous dsRNA internal control (IC) from the closely related Epizootic hemorrhagic disease virus (EHDV) was used to assess the efficiency of BTV genome extraction, dsRNA denaturation, RT, and PCR amplification. Recovery rates of IC and BTV dsRNA copies from extracted blood samples were highly correlated. Adjustment of BTV concentrations according to the IC recovery reduced variation in sample analyses among the different extraction methods and improved the accuracy of BTV quantitation. The EID(50)/ml titre, determined in blood samples from sheep infected experimentally with BTV-1 or BTV-9, correlated highly with the assessed concentration of BTV dsRNA copies. However, this correlation was consistent only during the first 28 days post-infection. The optimised extraction methods and quantitative RT-PCR could be useful for experimental studies of BTV transmission, pathogenesis and vaccine efficacy, or adapted further for the detection and quantitation of EHDV, African horse sickness virus and other dsRNA viruses.

  16. Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR.

    PubMed

    Fittipaldi, Mariana; Rodriguez, Nancy J Pino; Codony, Francesc; Adrados, Bárbara; Peñuela, Gustavo A; Morató, Jordi

    2010-09-01

    The advent of quantitative PCR has improved the detection of human viral pathogens in the environment. However, a serious limitation of this method may arise from the inability to discriminate between viruses that are infectious and viruses that have been inactivated and do not represent a human health hazard. To assess whether propidium monoazide (PMA) pre-treatment is a good approach to inhibiting DNA amplification from non-infectious viruses, bacteriophage T4 survival was measured using cell culture titration and real-time PCR with and without PMA pre-treatment. Heat (85 degrees C) and proteolysis methods were carried out. After these inactivation treatments, the results indicated that the PMA pre-treatment approach is not appropriate for differentiating infectious viruses. However, when a heat treatment at 110 degrees C was undertaken, PMA pre-treatment did allow differentiation of non-infectious from infectious viruses. In this case, effective binding of PMA to bacteriophage T4 DNA could be taken to indicate capsid damage. Therefore, PMA pre-treatment may be appropriate for assessing effective disinfection treatments and for a more reliable understanding of the factors that contribute to viral inactivation through capsid damage monitoring. The PMA-PCR approach could be useful as a rapid and inexpensive analytical tool for screening and evaluation of the efficacy of disinfectants.

  17. Detection of airborne genetically modified maize pollen by real-time PCR.

    PubMed

    Folloni, Silvia; Kagkli, Dafni-Maria; Rajcevic, Bojan; Guimarães, Nilson C C; Van Droogenbroeck, Bart; Valicente, Fernando H; Van den Eede, Guy; Van den Bulcke, Marc

    2012-09-01

    The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves.

  18. [Diagnosis of whooping cough by serology and real-time PCR].

    PubMed

    Mikešová, Romana; Stiborová, Ivana; Richter, Josef; Rajnohová Dobiášová, Lucie; Král, Vlastimil

    2013-09-01

    The goal of this study is to summarize the results of the detection of Bordetella pertussis (BP) and Bordetella parapertussis (BPP) by a real-time polymerase chain reaction (RT-PCR) assay and serological methods. In 2008-2010, 73 patients of the Department of Clinical Immunology and Allergology of the Centre for Immunology and Microbiology, Public Health Institute in Ústí nad Labem were screened for pertussis. They were selected according to the WHO and ECDC criteria, i. e. they presented with a persistent cough lasting more than two weeks. Direct detection of BP and BPP DNA from nasopharyngeal wash specimens was performed using a RT PCR assay. The serological responses were evaluated by a direct agglutination test for the detection of total antibodies and by enzyme-linked immunosobent assay (ELISA) for the detection of IgG, IgA, and IgM antibodies against pertussis toxin. Forty-two patients were positive for BP and/or BPP, 19 of them by RT-PCR (group A) and 23 by serology (group B). Ten group A patients (52.6%) were also positive by serology. Our results show that pertussis needs to be a consideration in persistent cough. We believe that increased awareness of the medical community, along with improved laboratory tests will result in increased detection of pertussis that is still considered by many physicians as a childhood infection.

  19. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    PubMed

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR. PMID:27143320

  20. Identification of reference genes for real-time quantitative PCR experiments in the liverwort Marchantia polymorpha.

    PubMed

    Saint-Marcoux, Denis; Proust, Hélène; 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

  1. Evaluation of Housekeeping Genes for Quantitative Real-Time PCR Analysis of Bradysia odoriphaga (Diptera: Sciaridae)

    PubMed Central

    Shi, Caihua; Yang, Fengshan; Zhu, Xun; Du, Erxia; Yang, Yuting; Wang, Shaoli; Wu, Qingjun; Zhang, Youjun

    2016-01-01

    The soil insect Bradysia odoriphaga (Diptera: Sciaridae) causes substantial damage to Chinese chive. Suitable reference genes in B. odoriphaga (Bradysia odoriphaga) have yet to be identified for normalizing target gene expression among samples by quantitative real-time PCR (qRT-PCR). This study was focused on identifying the expression stability of 12 candidate housekeeping genes in B. odoriphaga under various experiment conditions. The final stability ranking of 12 housekeeping genes was obtained with RefFinder, and the most suitable number of reference genes was analyzed by GeNorm. The results revealed that the most appropriate sets of internal controls were RPS15, RPL18, and RPS18 across developmental phases; RPS15, RPL28, and GAPDH across temperatures; RPS15 and RPL18 across pesticide treatments; RSP5, RPS18, and SDHA across photoperiods; ACTb, RPS18, and RPS15 across diets; RPS13 and RPL28 across populations; and RPS15, ACTb, and RPS18 across all samples. The use of the most suitable reference genes versus an arbitrarily selected reference gene resulted in significant differences in the analysis of a target gene expression. HSP23 in B. odoriphaga was found to be up-regulated under low temperatures. These results will contribute to the standardization of qRT-PCR and will also be valuable for further research on gene function in B. odoriphaga. PMID:27399679

  2. Detection of Nicotiana DNA in Tobacco Products Using a Novel Multiplex Real-Time PCR Assay.

    PubMed

    Korchinski, Katie L; Land, Adrian D; Craft, David L; Brzezinski, Jennifer L

    2016-07-01

    Establishing that a product contains tobacco is a requirement for the U.S. Food and Drug Administration's regulation and/or prosecution of tobacco products. Therefore, a multiplex real-time PCR method was designed to determine if Nicotiana (tobacco) DNA is present in tobacco products. The PCR method simultaneously amplifies a 73 bp fragment of the cytochrome P450 monoxygenase CYP82E4 gene and 66 bp fragment in the nia-1 gene for nitrate reductase, which are detected using dual-labeled TaqMan probes. The assay is capable of detecting approximately 7.8 pg purified tobacco DNA, with a similar sensitivity for either gene target while incorporating an internal positive control (IPC). DNA was extracted from prepared tobacco products-including chewing tobacco, pipe tobacco, and snuff-or from the cut fill (no wrapper) of cigarettes and cigars. Of the 13 products analyzed, 12 were positive for both tobacco-specific markers and the IPC. DNA was also extracted from the fill of five varieties of herbal cigarettes, which were negative for both tobacco-specific gene targets and positive for the IPC. Our method expands on current assays by introducing a multiplex reaction, targeting two sequences in two different genes of interest, incorporating an IPC into the reaction, and lowering the LOD and LOQ while increasing the efficiency of the PCR.

  3. Development and Evaluation of a Novel Single-Nucleotide-Polymorphism Real-Time PCR Assay for Rapid Detection of Fluoroquinolone-Resistant Mycoplasma bovis▿ †

    PubMed Central

    Ben Shabat, M.; Mikula, I.; Gerchman, I.; Lysnyansky, I.

    2010-01-01

    Monitoring of the susceptibility of Mycoplasma bovis field isolates to antibiotics is important for the appropriate choice of treatment. However, in vitro susceptibility testing of mycoplasmas is technically demanding and time-consuming, especially for clinical isolates, and is rarely performed in mycoplasma diagnostic laboratories. Thus, the development of methods allowing rapid real-time detection of resistant strains of M. bovis in clinical samples is a high priority for successful treatment. In this study, a novel TaqMan single-nucleotide-polymorphism (SNP) real-time PCR assay, which enables the rapid identification of M. bovis strains with different susceptibilities to fluoroquinolones, was developed and evaluated. The TaqMan SNP real-time PCR assay is based on the amplification of a 97-bp fragment of the parC quinolone resistance-determining region (QRDR) and allows the specific detection of four possible genotypes: GAC or GAT (susceptible to fluoroquinolones) and AAC or AAT (resistant to fluoroquinolones). Four TaqMan minor groove binder (MGB) probes identifying 1-base mismatches were designed and applied in a dual-probe assay with two reaction tubes. The TaqMan SNP real-time PCRs developed are highly specific for M. bovis, with a detection limit of 5 fg/μl (about 5 M. bovis genomes). In addition, all four SNP real-time PCR tests have almost the same efficiency (97.7% [GAC], 94% [AAC], 99.99% [GAT], and 98% [AAT]). Taken together, the data suggest that this SNP real-time PCR assay has potential as a routine diagnostic test for the detection of decreased susceptibility of M. bovis to fluoroquinolones. PMID:20534803

  4. Monitoring Acidophilic Microbes with Real-Time Polymerase Chain Reaction (PCR) Assays

    SciTech Connect

    Frank F. Roberto

    2008-08-01

    Many techniques that are used to characterize and monitor microbial populations associated with sulfide mineral bioleaching require the cultivation of the organisms on solid or liquid media. Chemolithotrophic species, such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, or thermophilic chemolithotrophs, such as Acidianus brierleyi and Sulfolobus solfataricus can grow quite slowly, requiring weeks to complete efforts to identify and quantify these microbes associated with bioleach samples. Real-time PCR (polymerase chain reaction) assays in which DNA targets are amplified in the presence of fluorescent oligonucleotide primers, allowing the monitoring and quantification of the amplification reactions as they progress, provide a means of rapidly detecting the presence of microbial species of interest, and their relative abundance in a sample. This presentation will describe the design and use of such assays to monitor acidophilic microbes in the environment and in bioleaching operations. These assays provide results within 2-3 hours, and can detect less than 100 individual microbial cells.

  5. Single-tube real-time PCR assay for differentiation of Ixodes affinis and Ixodes scapularis

    PubMed Central

    Wright, Chelsea L.; Hynes, Wayne L.; White, Breanna T.; Marshall, Mindy N.; Gaff, Holly D.; Gauthier, David T.

    2013-01-01

    Ixodes affinis Neumann (1899) and Ixodes scapularis Say (1821) are tick vectors of the etiologic agent of Lyme disease, Borrelia burgdorferi sensu stricto. Ixodes affinis and I. scapularis are morphologically very similar, and as they are sympatric in the mid- and south-Atlantic U.S. coastal states, their accurate identification is crucial to studies of disease and vector ecology in this area. This work describes a rapid, single-tube SYBR® Green-based real-time PCR assay for differentiation of I. affinis and I. scapularis at all life stages. The assay employs 2 pairs of species-specific primers directed against the internal transcribed spacer 2 (ITS2) region of the nuclear rRNA operon. Amplification products for these primer pairs differ in size and may be differentiated with a melt curve analysis. This tool is intended as a supplement to morphological methods for accurate identification of these ticks. PMID:24192510

  6. Development of a real-time PCR method for the identification of Atlantic mackerel (Scomber scombrus).

    PubMed

    Velasco, Amaya; Sánchez, Ana; Martínez, Icíar; Santaclara, Francisco J; Pérez-Martín, Ricardo I; Sotelo, Carmen G

    2013-12-01

    A Real Time-PCR method based on TaqMan technology for the identification of Scomber scombrus has been developed. A system of specific primers and a Minor Groove Binding (MGB) TaqMan probe based on sequences of the mitochondrial cytochrome b region was designed. The method was successfully tested in 81 specimens of S. scombrus and related species and validated in 26 different commercial samples. An average Threshold Cycle (Ct) value of 15.3 was obtained with S. scombrus DNA. With the other species tested fluorescence signal was not detected or Ct was significantly higher (P<0.001). The efficiency of the assay was estimated to be 92.41%, with 100% specificity, and no cross reactivity was detected with any other species. These results reveal that the developed method is a rapid and efficient tool to unequivocally identify S. scombrus and may aid in the prevention of fraud or mislabelling in mackerel products.

  7. Diagnosis of Phytoplasmas by Real-Time PCR Using Locked Nucleic Acid (LNA) Probes.

    PubMed

    Palmano, Sabrina; Mulholland, Vincent; Kenyon, David; Saddler, Gerry S; Jeffries, Colin

    2015-01-01

    Phytoplasma infections are regularly reported worldwide, and concerns about their threats on agricultural production, especially in relation to global climate change, are increasing. Sensitive and reliable detection methods are important to ensure that propagation material is free of phytoplasma infection and for epidemiological studies that may provide information to limit the extent of phytoplasma diseases and to prevent large-scale crop losses. The detection method described here uses LNA chemistry in real-time PCR. It has been developed and validated for use on potatoes, and its sensitivity and specificity make it suitable for use in postentry potato quarantine and initiation of potato nuclear stocks to ensure that material is phytoplasma-free. PMID:25981250

  8. Assessment of bacterial pathogens in fresh rainwater and airborne particulate matter using Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Kaushik, Rajni; Balasubramanian, Rajasekhar

    2012-01-01

    Bacterial pathogens in airborne particulate matter (PM) and in rainwater (RW) were detected using a robust and sensitive Real-Time PCR method. Both RW and PM were collected simultaneously in the tropical atmosphere of Singapore, which were then subjected to analysis for the presence of selected bacterial pathogens and potential pathogen of health concern ( Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Aeromonas hydrophila). These pathogens were found to be prevalent in both PM and RW samples with E. coli being the most prevalent potential pathogen in both types of samples. The temporal distribution of these pathogens in PM and RW was found to be similar to each other. Using the proposed microbiological technique, the atmospheric deposition (dry and wet deposition) of bacterial pathogens to lakes and reservoirs can be studied in view of growing concerns about the outbreak of waterborne diseases.

  9. Integrated sorting, concentration and real time PCR based detection system for sensitive detection of microorganisms

    PubMed Central

    Nayak, Monalisha; Singh, Deepak; Singh, Himanshu; Kant, Rishi; Gupta, Ankur; Pandey, Shashank Shekhar; Mandal, Swarnasri; Ramanathan, Gurunath; Bhattacharya, Shantanu

    2013-01-01

    The extremely low limit of detection (LOD) posed by global food and water safety standards necessitates the need to perform a rapid process of integrated detection with high specificity, sensitivity and repeatability. The work reported in this article shows a microchip platform which carries out an ensemble of protocols which are otherwise carried in a molecular biology laboratory to achieve the global safety standards. The various steps in the microchip include pre-concentration of specific microorganisms from samples and a highly specific real time molecular identification utilizing a q-PCR process. The microchip process utilizes a high sensitivity antibody based recognition and an electric field mediated capture enabling an overall low LOD. The whole process of counting, sorting and molecular identification is performed in less than 4 hours for highly dilute samples. PMID:24253282

  10. Real-Time PCR Detection of Dogwood Anthracnose Fungus in Historical Herbarium Specimens from Asia

    PubMed Central

    Miller, Stephen; Masuya, Hayato; Zhang, Jian; Walsh, Emily; Zhang, Ning

    2016-01-01

    Cornus species (dogwoods) are popular ornamental trees and important understory plants in natural forests of northern hemisphere. Dogwood anthracnose, one of the major diseases affecting the native North American Cornus species, such as C. florida, is caused by the fungal pathogen Discula destructiva. The origin of this fungus is not known, but it is hypothesized that it was imported to North America with its host plants from Asia. In this study, a TaqMan real-time PCR assay was used to detect D. destructiva in dried herbarium and fresh Cornus samples. Several herbarium specimens from Japan and China were detected positive for D. destructiva, some of which were collected before the first report of the dogwood anthracnose in North America. Our findings further support that D. destructiva was introduced to North America from Asia where the fungus likely does not cause severe disease. PMID:27096929

  11. Real-time PCR for specific detection of three phytoplasmas from the apple proliferation group.

    PubMed

    Mehle, Nataša; Nikolić, Petra; Gruden, Kristina; Ravnikar, Maja; Dermastia, Marina

    2013-01-01

    In this chapter, we describe a real-time PCR detection system for fast, reliable, specific, and sensitive detection and discrimination of 'Candidatus Phytoplasma mali', 'Ca. P. prunorum', and 'Ca. P. pyri' from the 16SrX (apple proliferation-AP) group. These phytoplasmas are causal agents of fruit tree diseases within the Rosaceae family, namely apple proliferation, European stone fruit yellows, and pear decline. The assays use (hydrolysis) TaqMan(®) minor groove binder probes. The panel of assays comprises the same set of primers and specific probes for species-specific amplification, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The assays described can be used in routine phytoplasma surveys and in certification programmes.

  12. Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms.

    PubMed

    Petersen, Kenneth; Vogel, Ulla; Rockenbauer, Eszter; Nielsen, Kirsten Vang; Kølvraa, Steen; Bolund, Lars; Nexø, Bjørn

    2004-04-01

    The typing of a single nucleotide polymorphism with DNA probes is sometimes problematic because of the limited discriminating power of long DNA probes. As an alternative to existing assays, we have developed a real-time PCR assay for the genotyping of single nucleotide polymorphisms using short peptide nucleic acid (PNA) molecular beacons. A single nucleotide polymorphism in exon 6 of the XPD gene was chosen as the model system. The genotyping experiments were performed in the ABI 7700 using beacons labeled with either fluorescein or JOE, and in the Lightcycler using a fluorescein labeled beacon. QSY-7 was used as the quencher in all the beacons. The result of the genotyping was the same on both instruments and was in agreement with a previously performed RFLP genotyping of 79 samples. The length of PNA molecular beacons is significantly shorter than that of TaqMan or Lightcycler probes, making probe design and genotype discrimination easier.

  13. Recent innovation in microbial source tracking using bacterial real-time PCR markers in shellfish.

    PubMed

    Mauffret, A; Mieszkin, S; Morizur, M; Alfiansah, Y; Lozach, S; Gourmelon, M

    2013-03-15

    We assessed the capacity of real-time PCR markers to identify the origin of contamination in shellfish. Oyster, cockles or clams were either contaminated with fecal materials and host-associated markers designed from Bacteroidales or Catellicoccus marimammalium 16S RNA genes were extracted from their intravalvular liquid, digestive tissues or shellfish flesh. Extraction of bacterial DNA from the oyster intravalvular liquid with FastDNA spin kit for soil enabled the selected markers to be quantified in 100% of artificially contaminated samples, and the source of contamination to be identified in 13 out of 38 naturally contaminated batches from European Class B and Class C areas. However, this protocol did not enable the origin of the contamination to be identified in cockle or clam samples. Although results are promising for extracts from intravalvular liquid in oyster, it is unlikely that a single protocol could be the best across all bacterial markers and types of shellfish. PMID:23398745

  14. Real-Time PCR Detection of Dogwood Anthracnose Fungus in Historical Herbarium Specimens from Asia.

    PubMed

    Miller, Stephen; Masuya, Hayato; Zhang, Jian; Walsh, Emily; Zhang, Ning

    2016-01-01

    Cornus species (dogwoods) are popular ornamental trees and important understory plants in natural forests of northern hemisphere. Dogwood anthracnose, one of the major diseases affecting the native North American Cornus species, such as C. florida, is caused by the fungal pathogen Discula destructiva. The origin of this fungus is not known, but it is hypothesized that it was imported to North America with its host plants from Asia. In this study, a TaqMan real-time PCR assay was used to detect D. destructiva in dried herbarium and fresh Cornus samples. Several herbarium specimens from Japan and China were detected positive for D. destructiva, some of which were collected before the first report of the dogwood anthracnose in North America. Our findings further support that D. destructiva was introduced to North America from Asia where the fungus likely does not cause severe disease. PMID:27096929

  15. Seasonal variation in transcript abundance in cork tissue analyzed by real time RT-PCR.

    PubMed

    Soler, Marçal; Serra, Olga; Molinas, Marisa; García-Berthou, Emili; Caritat, Antònia; Figueras, Mercè

    2008-05-01

    The molecular processes underlying cork biosynthesis and differentiation are mostly unknown. Recently, a list of candidate genes for cork biosynthesis and regulation was made available opening new possibilities for molecular studies in cork oak (Quercus suber L.). Based on this list, we analyzed the seasonal variation in mRNA abundance in cork tissue of selected genes by real time reverse-transcriptase polymerase chain reaction (RT-PCR). Relative transcript abundance was evaluated by principal component analysis and genes were clustered in several functional subgroups. Structural genes of suberin pathways such as CYP86A1, GPAT and HCBT, and regulatory genes of the NAM and WRKY families showed highest transcript accumulation in June, a crucial month for cork development. Other cork structural genes, such as FAT and F5H, were significantly correlated with temperature and relative humidity. The stress genes HSP17.4 and ANN were strongly positively correlated to temperature, in accord with their protective role.

  16. Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles.

    PubMed

    Rohr, Ulrich-Peter; Heyd, Florian; Neukirchen, Judith; Wulf, Marc-Andre; Queitsch, Iris; Kroener-Lux, Gabriele; Steidl, Ulrich; Fenk, Roland; Haas, Rainer; Kronenwett, Ralf

    2005-07-01

    In this report, we present a fast, reliable and easy to perform method to quantify infectious titers of recombinant AAV-2 (rAAV-2) particles using the LightCycler technology, which is independent from the therapeutic transgene and without the presence of a marker gene. The method is based on the life cycle of AAV-2: after infection of the host cell, the single stranded (ss) AAV-2 genome is converted into a double stranded (ds) form. Following infection with rAAV-2, HeLa cells were lysed and ssDNA of transcriptionally inactive particles were efficiently removed by ssDNA-specific S1 nuclease digestion. The remaining viral dsDNA can be quantified by quantitative real-time PCR (qPCR). For validation of the new method, rAAV-2 preparations were analyzed by two other standard methods for titration of infectious particles in parallel, i.e. the infectious center assay (ICA) as well as flow cytometry using GFP as a marker. Comparing the infectious titers of 40 different AAV-2 fractions assessed by qPCR with the titers determined by FACS analysis a significant correlation (r=0.87, p<0.001) with a mean ratio of the titers assessed by qPCR and FACS of 1.92 (S.D.+/-1.59) was found. Further, the titers of seven rAAV-2 fractions using qPCR and ICA covering 5 log ranges were compared and a significant correlation was found between the results (r=0.80, p<0.001) with a mean ratio of 3.38 (S.D.+/-1.79), respectively.

  17. Multiplex real-time PCR for rapid Staphylococcal cassette chromosome mec typing.

    PubMed

    Chen, Liang; Mediavilla, José R; Oliveira, Duarte C; Willey, Barbara M; de Lencastre, Herminia; Kreiswirth, Barry N

    2009-11-01

    Rapid identification and typing of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is important for understanding the molecular epidemiology and evolution of MRSA and offers many advantages for controlling transmission in both health care and community settings. We developed a rapid molecular beacon real-time PCR (MB-PCR) assay for staphylococcal cassette chromosome mec (SCCmec) typing. The design of this system is based on the established definition of SCCmec types, namely, the combination of the mec class complex with the ccr allotype. The assay consists of two multiplex panels, the combination of which results in two targets (mec class, ccr) for each SCCmec type. MB-PCR panel I targets mecA, ccrB2, mecI, and the DeltamecR1-IS1272 junction (mec class B); it can definitively identify SCCmec types II and IV. MB-PCR panel II detects ccrC, ccrB1, ccrB3, ccrB4, and the DeltamecR1-IS431 junction (mec class C2) and is therefore capable of identifying SCCmec types I, III, V, and VI in combination with panel I. The method can also detect the recently described novel SCCmec type VIII (ccrAB4 with mec class A). Our assay demonstrated 100% concordance when applied to 162 MRSA strains previously characterized by traditional SCCmec typing schemes. Four geographically and temporally diverse S. aureus collections were also successfully classified by our assay, along with 1,683 clinical isolates comprising both hospital- and community-associated MRSA and methicillin-susceptible S. aureus strains. As many as 96 isolates can be classified easily within 3 to 4 h, including DNA isolation, PCR cycling, and analysis. The assay is rapid, robust, sensitive, and cost-effective, allowing for high-throughput SCCmec typing of MRSA isolates.

  18. Development and validation of a quantitative real time PCR assay for BK virus.

    PubMed

    Mitui, Midori; Leos, N Kristine; Lacey, Damon; Doern, Christopher; Rogers, Beverly B; Park, Jason Y

    2013-01-01

    Several studies have shown that BK viral load in plasma and urine are reliable markers for the detection of BK virus associated nephropathy (BKVAN) in renal transplant patients. We developed a quantitative real time PCR assay based on TaqMan technology for the measurement of BK viral load in plasma and urine. Considering the high similarity of the nucleotide sequence of the BK virus (BKV) with the JC virus (JCV), we designed this assay to specifically amplify BKV. We determined the viral DNA recovery rate on manual (QIAGEN's QIAamp DNA Blood Mini Kit) and automated (BioMerieux's NucliSENS EasyMAG) extraction methods. The comparison showed a higher viral DNA recovery rate on the automated extraction (61-76% in plasma and 52-65% in urine) as compared to the manual method (49-52% in plasma and 33-56% in urine). Quantitation of the viral load was performed using an external standard curve that was constructed with serial dilution of a plasmid containing the full length of the BKV genome. Commercially available quantitative BKV standards showed good correlation with the plasmid standard. The reproducibility of the assay was determined based on the Ct values of the amplified products as well as in BK copies per milliliter of sample. This assay is linear over a 7 log range (10 to 1 × 10(7) copies per reaction), no cross-reactivity was detected with the closest-related polyomavirus JCV, as well as other viruses that may be found in immunocompromised patients, and human genomic DNA. The limit of detection of the assay is 300 copies per milliliter in both plasma and urine and the limit of quantitation is 1000 copies per milliliter using the NATtrol BK Virus Linearity Panel (ZeptoMetrix). This real time PCR assay provides a reliable and sensitive method for the quantitation of BKV in plasma and urine samples.

  19. Performance of a real-time PCR assay for the rapid identification of Mycobacterium species.

    PubMed

    Wang, Hye-young; Kim, Hyunjung; Kim, Sunghyun; Kim, Do-kyoon; Cho, Sang-Nae; Lee, Hyeyoung

    2015-01-01

    Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis (MTB) from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. The diagnosis of diseases caused by NTM is difficult because NTM species are prevalent in the environment and because they have fastidious properties. In the present study, we evaluated 279 clinical isolates grown in liquid culture provided by The Catholic University of Korea, St. Vincent's Hospital using real-time PCR based on mycobacterial rpoB gene sequences. The positive rate of real-time PCR assay accurately discriminated 100% (195/195) and 100% (84/84) between MTB and NTM species. Comparison of isolates identified using the MolecuTech REBA Myco-ID(®) and Real Myco-ID® were completely concordant except for two samples. Two cases that were identified as mixed infection (M. intracellulare-M. massiliense and M. avium-M. massiliense co-infection) by PCRREBA assay were only detected using M. abscessus-specific probes by Real Myco-ID(®). Among a total of 84 cases, the most frequently identified NTM species were M. intracellulare (n=38, 45.2%), M. avium (n=18, 23.7%), M. massiliense (n=10, 13.2%), M. fortuitum (n=5, 6%), M. abscessus (n=3, 3.9%), M. gordonae (n=3, 3.9%), M. kansasii (n=2, 2.4%), M. mucogenicum (n=2, 2.4%), and M. chelonae (n= 1, 1.2%). Real Myco-ID(®) is an efficient tool for the rapid detection of NTM species as well as MTB and sensitive and specific and comparable to conventional methods.

  20. Development of Real-Time PCR Assays for Rapid Detection of Pfiesteria piscicida and Related Dinoflagellates†

    PubMed Central

    Bowers, Holly A.; Tengs, Torstein; Glasgow, Howard B.; Burkholder, JoAnn M.; Rublee, Parke A.; Oldach, David W.

    2000-01-01

    Pfiesteria complex species are heterotrophic and mixotrophic dinoflagellates that have been recognized as harmful algal bloom species associated with adverse fish and human health effects along the East Coast of North America, particularly in its largest (Chesapeake Bay in Maryland) and second largest (Albermarle-Pamlico Sound in North Carolina) estuaries. In response to impacts on human health and the economy, monitoring programs to detect the organism have been implemented in affected areas. However, until recently, specific identification of the two toxic species known thus far, Pfiesteria piscicida and P. shumwayae (sp. nov.), required scanning electron microscopy (SEM). SEM is a labor-intensive process in which a small number of cells can be analyzed, posing limitations when the method is applied to environmental estuarine water samples. To overcome these problems, we developed a real-time PCR-based assay that permits rapid and specific identification of these organisms in culture and heterogeneous environmental water samples. Various factors likely to be encountered when assessing environmental samples were addressed, and assay specificity was validated through screening of a comprehensive panel of cultures, including the two recognized Pfiesteria species, morphologically similar species, and a wide range of other estuarine dinoflagellates. Assay sensitivity and sample stability were established for both unpreserved and fixative (acidic Lugol's solution)-preserved samples. The effects of background DNA on organism detection and enumeration were also explored, and based on these results, we conclude that the assay may be utilized to derive quantitative data. This real-time PCR-based method will be useful for many other applications, including adaptation for field-based technology. PMID:11055905

  1. A real-time PCR assay for detection and quantification of Verticillium dahliae in spinach seed.

    PubMed

    Duressa, Dechassa; Rauscher, Gilda; Koike, Steven T; Mou, Beiquan; Hayes, Ryan J; Maruthachalam, Karunakaran; Subbarao, Krishna V; Klosterman, Steven J

    2012-04-01

    Verticillium dahliae is a soilborne fungus that causes Verticillium wilt on multiple crops in central coastal California. Although spinach crops grown in this region for fresh and processing commercial production do not display Verticillium wilt symptoms, spinach seeds produced in the United States or Europe are commonly infected with V. dahliae. Planting of the infected seed increases the soil inoculum density and may introduce exotic strains that contribute to Verticillium wilt epidemics on lettuce and other crops grown in rotation with spinach. A sensitive, rapid, and reliable method for quantification of V. dahliae in spinach seed may help identify highly infected lots, curtail their planting, and minimize the spread of exotic strains via spinach seed. In this study, a quantitative real-time polymerase chain reaction (qPCR) assay was optimized and employed for detection and quantification of V. dahliae in spinach germplasm and 15 commercial spinach seed lots. The assay used a previously reported V. dahliae-specific primer pair (VertBt-F and VertBt-R) and an analytical mill for grinding tough spinach seed for DNA extraction. The assay enabled reliable quantification of V. dahliae in spinach seed, with a sensitivity limit of ≈1 infected seed per 100 (1.3% infection in a seed lot). The quantification was highly reproducible between replicate samples of a seed lot and in different real-time PCR instruments. When tested on commercial seed lots, a pathogen DNA content corresponding to a quantification cycle value of ≥31 corresponded with a percent seed infection of ≤1.3%. The assay is useful in qualitatively assessing seed lots for V. dahliae infection levels, and the results of the assay can be helpful to guide decisions on whether to apply seed treatments.

  2. Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

    PubMed Central

    Ghelardini, Luisa; Belbahri, Lassaâd; Quartier, Marion; Santini, Alberto

    2013-01-01

    Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10−2 to 1.4 × 10−2 pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management. PMID:23811499

  3. Detection and quantification of Cladosporium in aerosols by real-time PCR.

    PubMed

    Zeng, Qing-Yin; Westermark, Sven-Olof; Rasmuson-Lestander, Asa; Wang, Xiao-Ru

    2006-01-01

    Cladosporium is one of the most common airborne molds found in indoor and outdoor environments. Cladosporium spores are important aeroallergens, and prolonged exposure to elevated spore concentrations can provoke chronic allergy and asthma. To accurately quantify the levels of Cladosporium in indoor and outdoor environments, two real-time PCR systems were developed in this study. The two real-time PCR systems are highly specific and sensitive for Cladosporium detection even in a high background of other fungal DNAs. These methods were employed to quantify Cladosporium in aerosols of five different indoor environments. The investigation revealed a high spore concentration of Cladosporium (10(7) m(-3)) in a cow barn that accounted for 28-44% of the airborne fungal propagules. In a countryside house that uses firewood for heating and in a paper and pulp factory, Cladosporium was detected at 10(4) spores m(-3), which accounted for 2-6% of the fungal propagules in the aerosols. The concentrations of Cladosporium in these three indoor environments far exceeded the medical borderline level (3000 spores m(-3)). In a power station and a fruit and vegetable storage, Cladosporium was found to be a minor component in the aerosols, accounted for 0.01-0.1% of the total fungal propagules. These results showed that monitoring Cladosporium in indoor environments is more important than in outdoor environments from the public health point of view. Cladosporium may not be the dominant fungi in some indoor environments, but its concentration could still be exceeding the threshold value for clinical significance. The methods developed in this study could facilitate accurate detection and quantification of Cladosporium for public health related risk assessment.

  4. Comparison of a Real-Time Multiplex PCR and Sequetyping Assay for Pneumococcal Serotyping

    PubMed Central

    Robberts, Lourens; Wolter, Nicole; Nicol, Paul; Mafofo, Joseph; Africa, Samantha; Zar, Heather J.; Nicol, Mark P.

    2015-01-01

    Background Pneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage. Methods A real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates. Results Of the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently

  5. Validation of a real time PCR for classical Swine Fever diagnosis.

    PubMed

    Dias, Natanael Lamas; Fonseca Júnior, Antônio Augusto; Oliveira, Anapolino Macedo; Sales, Erica Bravo; Alves, Bruna Rios Coelho; Dorella, Fernanda Alves; Camargos, Marcelo Fernandes

    2014-01-01

    The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5'NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5'NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF. PMID:24818039

  6. In-house validation and quality control of real-time PCR methods for GMO detection: a practical approach.

    PubMed

    Ciabatti, I; Froiio, A; Gatto, F; Amaddeo, D; Marchesi, U

    2006-01-01

    GMO detection and quantification methods in the EU are mainly based on real-time PCR. The analytical methods in use must be validated, first on an intra-laboratory scale and through a collaborative trial thereafter. Since a consensual protocol for intra-laboratory validation of real-time PCR methods is lacking, we provide a practical approach for the in-house validation of quantitative real-time PCR methods, establishing acceptability criteria and quality controls for PCR runs. Parameters such as limit of detection, limit of quantification, precision, trueness, linear dynamic range, PCR efficiency, robustness and specificity are considered. The protocol is sufficiently detailed to be directly applicable, increases the reliability of results and their harmonization among different laboratories, and represents a necessary preliminary step before proceeding to a time-consuming and costly full validation study.

  7. Rapid detection of group B streptococcus and Escherichia coli in amniotic fluid using real-time fluorescent PCR.

    PubMed Central

    Straka, Michele; Dela Cruz, Wifred; Blackmon, Camille; Johnson, Oswald; Stassen, Sara; Streitman, David; Golden, Stephen; Stamilio, David

    2004-01-01

    OBJECTIVE: To establish reliability and validity of real-time fluorescent PCR for early detection of bacterial invasion of the amniotic cavity. METHODS: Amniotic fluid samples from 40 patients undergoing mid-trimester genetic amniocentesis were incubated for 6 h at 37 degrees C and were cultured on media specific for group B streptococcus (GBS) and E. coli. Concurrently, samples were analyzed with real-time fluorescent PCR (Roche LightCycler) using DNA primers and probes designed to detect the CAMP factor encoding cfb gene and uidA gene of GBS and E. coli, respectively. For positive control and to simulate amniotic fluid colonization, 104 cfu/ml of GBS and E. coli were inoculated on sterile amniotic fluid and incubated for 6 h. Bacterial genomic DNA for the two organisms was extracted and purified via the two-step precipitation method using a commercial kit. The real-time PCR assays were also tested against 25 non-GBS and non-E. coli bacterial species. The lower limit of detection for each pathogen was established using serial dilution of bacterial genomic DNA. RESULTS: All patient samples were negative for evidence of GBS and E. coli with both culture and real-time PCR methods. Amniotic fluid samples inoculated with GBS and E. coli were positive with real-time PCR whereas the 25 bacterial species other than GBS or E. coli tested negative with the assay. Average total sample processing time including the pre-enrichment step was 7 h 40 min. The average cost for DNA extraction and PCR testing was 8.50 dollars per test. CONCLUSION: Real-time fluorescent PCR is a valid and reliable method for detection of specific pathogens in amniotic fluid. This technique is sensitive for low inoculation levels. Real-time fluorescent PCR has potential to impact clinical management as a rapid, reliable detection method for GBS and E. coli in chorioamnionitis. PMID:15763909

  8. Quantification of substance p mRNA in human immune cells by real-time reverse transcriptase PCR assay.

    PubMed

    Lai, Jian-Ping; Douglas, Steven D; Shaheen, Farida; Pleasure, David E; Ho, Wen-Zhe

    2002-01-01

    We have applied a newly developed real-time reverse transcriptase (RT) PCR (RT-PCR) assay for quantification of substance P (SP) mRNA expression (the SP real-time RT-PCR assay) in human blood monocyte-derived macrophages, peripheral blood lymphocytes, and microglia isolated from fetal brain. The SP real-time RT-PCR assay had a sensitivity of 60 mRNA copies, with a dynamic range of detection between 60 and 600,000 copies of the SP gene transcript per reaction mixture. The coefficient of variation of the threshold cycle number between the SP real-time RT-PCR assays was less than 1.16%. This assay with an SP-specific primer pair efficiently recognizes all four isoforms of preprotachykinin A (the SP precursor) gene transcripts. In order to use this assay to measure the levels of SP mRNA in the human immune cells quantitatively, we designed a specific probe (molecular beacon) derived from exon 3 of the SP gene. We demonstrated that the real-time RT-PCR quantitatively detected SP mRNA in the human immune cells, among which the microglia isolated from fetal brain had the highest levels of SP mRNA. The SP real-time PCR assay yielded reproducible data, as the intra-assay variation was less than 1%. Thus, it is feasible to apply the real-time RT-PCR assay for quantification of SP mRNA levels in human immune cells, as well as in other nonneuronal cells. Since SP is a major modulator of neuroimmunoregulation, this assay has the potential for widespread application for basic and clinical investigations.

  9. Utility of IgM ELISA, TaqMan real-time PCR, reverse transcription PCR, and RT-LAMP assay for the diagnosis of Chikungunya fever.

    PubMed

    Reddy, Vijayalakshmi; Ravi, Vasanthapuram; Desai, Anita; Parida, Manmohan; Powers, Ann M; Johnson, Barbara W

    2012-11-01

    Chikungunya fever a re-emerging infection with expanding geographical boundaries, can mimic symptoms of other infections like dengue, malaria which makes the definitive diagnosis of the infection important. The present study compares the utility of four laboratory diagnostic methods viz. IgM capture ELISA, an in house reverse transcription PCR for the diagnosis of Chikungunya fever, TaqMan real-time PCR, and a one step reverse transcription-loop mediated isothermal amplification assay (RT-LAMP). Out of the 70 serum samples tested, 29 (41%) were positive for Chikungunya IgM antibody by ELISA and 50 (71%) samples were positive by one of the three molecular assays. CHIKV specific nucleic acid was detected in 33/70 (47%) by reverse transcription PCR, 46/70 (66%) by TaqMan real-time PCR, and 43/70 (62%) by RT-LAMP assay. A majority of the samples (62/70; 89%) were positive by at least one of the four assays used in the study. The molecular assays were more sensitive for diagnosis in the early stages of illness (2-5 days post onset) when antibodies were not detectable. In the later stages of illness, the IgM ELISA is a more sensitive diagnostic test. In conclusion we recommend that the IgM ELISA be used as an initial screening test followed one of the molecular assays in samples that are collected in the early phase of illness and negative for CHIKV IgM antibodies. Such as approach would enable rapid confirmation of the diagnosis and implementation of public health measures especially during outbreaks.

  10. Evaluation of three real-time PCR assays for differential identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria species in liquid culture media.

    PubMed

    Jung, Yu Jung; Kim, Ji-Youn; Song, Dong Joon; Koh, Won-Jung; Huh, Hee Jae; Ki, Chang-Seok; Lee, Nam Yong

    2016-06-01

    We evaluated the analytical performance of M. tuberculosis complex (MTBC)/nontuberculous mycobacteria (NTM) PCR assays for differential identification of MTBC and NTM using culture-positive liquid media. Eighty-five type strains and 100 consecutive mycobacterial liquid media cultures (MGIT 960 system) were analyzed by a conventional PCR assay (MTB-ID(®) V3) and three real-time PCR assays (AdvanSure™ TB/NTM real-time PCR, AdvanSure; GENEDIA(®) MTB/NTM Detection Kit, Genedia; Real-Q MTB & NTM kit, Real-Q). The accuracy rates for reference strains were 89.4%, 100%, 98.8%, and 98.8% for the MTB-ID V3, AdvanSure, Genedia, and Real-Q assays, respectively. Cross-reactivity in the MTB-ID V3 assay was mainly attributable to non-mycobacterium Corynebacterineae species. The diagnostic performance was determined using clinical isolates grown in liquid media, and the overall sensitivities for all PCR assays were higher than 95%. In conclusion, the three real-time PCR assays showed better performance in discriminating mycobacterium species and non-mycobacterium Corynebacterineae species than the conventional PCR assay.

  11. Quantitative real-time PCR (qPCR) for Eimeria tenella replication — Implications for experimental refinement and animal welfare

    PubMed Central

    Nolan, Matthew J.; Tomley, Fiona M.; Kaiser, Pete; Blake, Damer P.

    2015-01-01

    The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R2 = 0.994) (p < 0.002) but not in those from day eight (after most oocyst shedding) (R2 = 0.006) (p > 0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R2 = 0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings

  12. Detection and identification of Rift Valley fever virus in mosquito vectors by quantitative real-time PCR.

    PubMed

    Mwaengo, D; Lorenzo, G; Iglesias, J; Warigia, M; Sang, R; Bishop, R P; Brun, A

    2012-10-01

    Diagnostic methods allowing for rapid identification of pathogens are crucial for controlling and preventing dissemination after disease outbreaks as well as for use in surveillance programs. For arboviruses, detection of the presence of virus in their arthropod hosts is important for monitoring of viral activity and quantitative information is useful for modeling of transmission dynamics. In this study, molecular detection of Rift Valley fever virus (RVFV) in mosquito samples from the 2006 to 2007 East African outbreaks was performed using quantitative real-time PCR assay (qRT-PCR). Specific RVFV sequence-based primer/fluorogenic (TaqMan) probe sets were derived from the L and S RNA segments of the virus. Both primer-probe L and S segment-based combinations detected genomic RVFV sequences, with generally comparable levels of sensitivity. Viral loads from three mosquito species, Aedes mcintoshi, Aedes ochraceus and Mansonia uniformis were estimated and significant differences of between 5- and 1000-fold were detected between Ae. mcintoshi and M. uniformis using both the L and S primer-probe-based assays. The genetic relationships of the viral sequences in mosquito samples were established by partial M segment sequencing and assigned to the two previously described viral lineages defined by analysis of livestock isolates obtained during the 2006-2007 outbreak, confirming that similar viruses were present in both the vector and mammalian host. The data confirms the utility of qRT-PCR for identification and initial quantification of virus in mosquito samples during RVFV outbreaks.

  13. Development of a real time quantitative PCR assay for the hard clam pathogen Quahog Parasite Unknown (QPX).

    PubMed

    Lyons, M Maille; Smolowitz, Roxanna; Dungan, Christopher F; Roberts, Steven B

    2006-09-14

    Quahog Parasite Unknown (QPX) is a thraustochytrid pathogen responsible for catastrophic mortalities of the northern quahog (hard clam) Mercenaria mercenaria. A real-time quantitative polymerase chain reaction (qPCR) assay was developed to assist research efforts on QPX ecology and pathology. Sensitivity of the assay was evaluated with serial dilutions of QPX-cultured cells to determine the lowest concentration of DNA that remained detectable in both the presence and absence of extraneous environmental substances. QPX cells were quantified before DNA extraction to calibrate standard curves to cell counts. Based on our results, the qPCR assay is able to quantify QPX within the range of 1 to several thousand organisms per reaction. Specificity of the assay was assessed by testing 29 thraustochytrid-like protists isolated from suspension-feeding bivalves from China, Oregon, Maryland, and Virginia. Application of the assay was demonstrated with positive qPCR results from naturally contaminated environmental samples including marine aggregates (i.e. marine snow), clam pseudofeces, and inflammatory nodules from infected clams. This quantitative assay for QPX will provide a valuable tool for characterizing QPX parasite abundances in coastal environments and for improving clam disease diagnostics.

  14. [Establishment and Preliminary Application of the SYBR Green I Real-time PCR Assay for Detection of the Bovine Enterovirus].

    PubMed

    Zhu, Tong; Zhao, Guimin; Shen, Furao; Hou Peili; Wang, Hongmei; Li, Jie; He, Hongbin

    2015-09-01

    The bovine enterovirus (BEV) is a pathogen found the digestive tracts of cattle. Recently, the BEV was discovered in cattle in a province in China. A rapid and effective detection method for the BEV is essential. An assay was carried out using two specific primers designed to amplify a highly conserved sequence of the 3D gene. A recombinant plasmid containing the target gene 3D was constructed as a standard control. The limit of detection of the reaction was 7.13 x 10(1) plasmid copies/μL of initial templates, which was tenfold more sensitive than the conventional reverse-transcription-polymerase chain reaction (RT-PCR). Moreover, the assay was highly specific because all negative controls and other viruses of clinical relevance did not develop positive results. Assay performance on field samples was evaluated on 44 (41 diarrhea and 3 aerosol) samples and compared with the conventional RT-PCR assay. Sixteen diarrhea samples were positive (16/41, 39. 02%) and 3 aerosol samples were positive (3/3, 100%). Preliminary results for clinical detection showed that the SYBR Green I real-time PCR assay was highly sensitive, specific and reproducible. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications for epidemics and in BEV research. PMID:26738285

  15. Increased efficacy for in-house validation of real-time PCR GMO detection methods.

    PubMed

    Scholtens, I M J; Kok, E J; Hougs, L; Molenaar, B; Thissen, J T N M; van der Voet, H

    2010-03-01

    To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision. Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML (residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility (RSD(r) and RSD(R)) were calculated. The results showed that not only the PCR reaction but also the factors 'DNA isolation' and 'PCR day' are important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methods. PMID:20012027

  16. Increased efficacy for in-house validation of real-time PCR GMO detection methods.

    PubMed

    Scholtens, I M J; Kok, E J; Hougs, L; Molenaar, B; Thissen, J T N M; van der Voet, H

    2010-03-01

    To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision. Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML (residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility (RSD(r) and RSD(R)) were calculated. The results showed that not only the PCR reaction but also the factors 'DNA isolation' and 'PCR day' are important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methods.

  17. 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.

  18. Enhanced detection of tuberculous mycobacteria in animal tissues using a semi-nested probe-based real-time PCR.

    PubMed

    Costa, Pedro; Ferreira, Ana S; Amaro, Ana; Albuquerque, Teresa; Botelho, Ana; Couto, Isabel; Cunha, Mónica V; Viveiros, Miguel; Inácio, João

    2013-01-01

    Bovine tuberculosis has been tackled for decades by costly eradication programs in most developed countries, involving the laboratory testing of tissue samples from allegedly infected animals for detection of Mycobacterium tuberculosis complex (MTC) members, namely Mycobacterium bovis. Definitive diagnosis is usually achieved by bacteriological culture, which may take up to 6-12 weeks, during which the suspect animal carcass and herd are under sanitary arrest. In this work, a user-friendly DNA extraction protocol adapted for tissues was coupled with an IS6110-targeted semi-nested duplex real-time PCR assay to enhance the direct detection of MTC bacteria in animal specimens, reducing the time to achieve a diagnosis and, thus, potentially limiting the herd restriction period. The duplex use of a novel β-actin gene targeted probe, with complementary targets in most mammals, allowed the assessment of amplification inhibitors in the tissue samples. The assay was evaluated with a group of 128 fresh tissue specimens collected from bovines, wild boars, deer and foxes. Mycobacterium bovis was cultured from 57 of these samples. Overall, the full test performance corresponds to a diagnostic sensitivity and specificity of 98.2% (CIP95% 89.4-99.9%) and 88.7% (CIP95% 78.5-94.7%), respectively. An observed kappa coefficient was estimated in 0.859 (CI P95% 0.771-0.948) for the overall agreement between the semi-nested PCR assay and the bacteriological culture. Considering only bovine samples (n = 69), the diagnostic sensitivity and specificity were estimated in 100% (CIP95% 84.0-100%) and 97.7% (CIP95% 86.2-99.9%), respectively. Eight negative culture samples exhibiting TB-like lesions were detected by the semi-nested real-time PCR, thus emphasizing the increased potential of this molecular approach to detect MTC-infected animal tissues. This novel IS6110-targeted assay allows the fast detection of tuberculous mycobacteria in animal specimens with very high sensitivity and

  19. Detection of methicillin-resistant Staphylococcus aureus directly from nasal swab specimens by a real-time PCR assay.

    PubMed

    Warren, David K; Liao, Robert S; Merz, Liana R; Eveland, Michael; Dunne, W Michael

    2004-12-01

    Screening for colonization with methicillin-resistant Staphylococcus aureus (MRSA) is a key aspect of infection control to limit the nosocomial spread of this organism. Current methods for the detection of MRSA in clinical microbiology laboratories, including molecularly based techniques, require a culture step and the isolation of pure colonies that result in a minimum of 20 to 24 h until a result is known. We describe a qualitative in vitro diagnostic test for the rapid detection of MRSA directly from nasal swab specimens (IDI-MRSA; Infectio Diagnostic, Inc., Sainte-Foy, Quebec, Canada), based upon a real-time PCR and direct detection of MRSA via amplicon hybridization with a fluorogenic target-specific molecular beacon probe. Samples from 288 patients were analyzed for the presence of MRSA with the IDI-MRSA assay, compared to detection by either direct plating or enrichment broth selective culture methods. The diagnostic values for this MRSA screening method were 91.7% sensitivity, 93.5% specificity, 82.5% positive predictive value, and 97.1% negative predictive value when compared to culture-based methods. The time from the start of processing of specimen to result was approximately 1.5 h. In our hands, the IDI-MRSA assay is a sensitive and specific test for detection of nasal colonization with MRSA and providing for same-day results, allowing more efficient and effective use of infection control resources to control MRSA in health care facilities.

  20. Real-time PCR assay for detection of a new simulant for poxvirus biothreat agents.

    PubMed

    Garnier, Laurence; Gaudin, Jean-Christophe; Bensadoun, Paul; Rebillat, Isabelle; Morel, Yannick

    2009-03-01

    Research and financial efforts spent on biodefense technologies highlight the current concern for biothreat event preparedness. Nonhazardous but relevant "simulant" microorganisms are typically used to simplify technological developments, testing, and staff training. The bacteriophage MS2, a small RNA virus, is classically used as the reference simulant for biothreat viruses within the biodefense community. However, variola virus, considered a major threat, displays very different features (size, envelope, and double-stranded DNA genome). The size parameter is critical for aerosol sampling, detection, and protection/filtration technologies. Therefore, a panel of relevant simulants should be used to cover the diversity of biothreat agents. Thus, we investigated a new virus model, the Cydia pomonella granulovirus (baculovirus), which is currently used as a biopesticide. It displays a size similar to that of poxviruses, is enveloped, and contains double-stranded DNA. To provide a molecular tool to detect and quantify this model virus, we developed an assay based on real-time PCR, with a limit of detection ranging from roughly 10 to a few tens of target copies per microl according to the sample matrix. The specificity of the assay against a large panel of potential cross-reactive microorganisms was checked, and the suitability of the assay for environmental samples, especially aerosol studies, was determined. In conclusion, we suggest that our PCR assay allows Cydia pomonella granulovirus to be used as a simulant for poxviruses. This assay may also be useful for environmental or crop treatment studies. PMID:19168659

  1. RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL-TIME PCR ASSAYS

    PubMed Central

    Bowers, Holly A.; Tomas, Carmelo; Tengs, Torstein; Kempton, Jason W.; Lewitus, Alan J.; Oldach, David W.

    2010-01-01

    Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real-time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small-subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species. PMID:20411032

  2. Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

    USGS Publications Warehouse

    Bushon, R.N.; Kephart, C.M.; Koltun, G.F.; Francy, D.S.; Schaefer, F. W.; Lindquist, H.D. Alan

    2010-01-01

    Aims: The aim of this study was to evaluate the variability in lots of a DNA extraction kit using real-time PCR assays for Bacillus anthracis, Francisella tularensis and Vibrio cholerae. Methods and Results: Replicate aliquots of three bacteria were processed in duplicate with three different lots of a commercial DNA extraction kit. This experiment was repeated in triplicate. Results showed that cycle threshold values were statistically different among the different lots. Conclusions: Differences in DNA extraction reagent lots were found to be a significant source of variability for qPCR results. Steps should be taken to ensure the quality and consistency of reagents. Minimally, we propose that standard curves should be constructed for each new lot of extraction reagents, so that lot-to-lot variation is accounted for in data interpretation. Significance and Impact of the Study: This study highlights the importance of evaluating variability in DNA extraction procedures, especially when different reagent lots are used. Consideration of this variability in data interpretation should be an integral part of studies investigating environmental samples with unknown concentrations of organisms. ?? 2010 The Society for Applied Microbiology.

  3. Development of real-time PCR methods to quantify patulin-producing molds in food products.

    PubMed

    Rodríguez, Alicia; Luque, M Isabel; Andrade, María J; Rodríguez, Mar; Asensio, Miguel A; Córdoba, Juan J

    2011-09-01

    Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g(-1) per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain. PMID:21645819

  4. Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples.

    PubMed

    Neumann, A P; Dunham, S M; Rehberger, T G; Siragusa, G R

    2010-08-01

    Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues.

  5. Real-time FRET PCR assay for Salmonella enterica serotype detection in food.

    PubMed

    Olsen, Eric V; Gibbins, Carl S; Grayson, J Kevin

    2009-09-01

    Salmonella enterica subsp. enterica serotypes are leading etiological agents of food-borne gastroenteritis. Traditional identification is laborious and time intensive. Faster molecular methods may allow early identification in contaminated food products. We developed a real-time, fluorescence resonance energy transfer hybridization probe polymerase chain reaction (PCR) assay for S. enterica serotypes on the basis of the exclusive presence of the apeE gene in Salmonella Typhimurium. Assay sensitivity for 12 S. enterica serotypes was as low as 1.87 x 10(2) genomic equivalents per milliliter. PCR efficiency was 94% and the dynamic range was linear over six orders of magnitude from 10(0) to 10(6) copies. The lower limit of detection for 12 different food matrices was between 1.5 x 10(2) and 1.5 x 10(5) CFU/mL without pre-enrichment. When combined with high-throughput automated DNA extraction, 32 food specimens were processed and assayed in less than 2 hours, allowing rapid, specific, sensitive detection of S. enterica serotypes in food products.

  6. [Evaluation of reference genes for quantitative real-time PCR normalization in cotton bollworm, Helicoverna armigera].

    PubMed

    Chandra, G Sharath; Asokan, R; Manamohan, M; Kumar, N K K; Sita, T

    2014-01-01

    Reverse-transcription quantitative real-time PCR (RT-qPCR), a sensitive technique is being extensively employed in quantification of gene expression. However this requires normalization with suitable reference gene (RG) which is crucial in minimizing inter sample variations. Information regarding suitable RG is scarce in general and more so in insects, including the cotton bollworm, Helicoverpa armigera, an economically important pest. In management of this pest RNA interference (RNAi), is perceived as a potential tool, which is achieved by double-stranded RNA (dsRNA) delivery. These studies demand accurate quantification of gene silencing. In this study we assessed the suitability of five RGs viz. β-actin (ACTB), 18S rRNA (18S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB) and elongation fator-1-alfa (EF1-α) for gene expression studies in dsRNA treatment and across different developmental stages of H. armigera and ranked using geNorm, NormFinder and BestKeeper software programs. Data analysis revealed that best ranked RGs were varied in dsRNA treatment and in developmental stages. Under dsRNA treatment, 18S and GAPDH were more stable whereas, TUB and GAPDH were more stable across developmental stages. We also demonstrate that inappropriate selection of RG led to erroneous estimation of the target gene, chymotrypsin, expression. These results facilitate accurate quantification of gene expression in H. armigera.

  7. Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

    PubMed Central

    Abt, Melissa A.; Grek, Christina L.; Ghatnekar, Gautam S.; Yeh, Elizabeth S.

    2016-01-01

    Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3–6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue. PMID:26862835

  8. Development of real-time PCR methods to quantify patulin-producing molds in food products.

    PubMed

    Rodríguez, Alicia; Luque, M Isabel; Andrade, María J; Rodríguez, Mar; Asensio, Miguel A; Córdoba, Juan J

    2011-09-01

    Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g(-1) per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain.

  9. Analysis of gene expression in emerald ash borer (Agrilus planipennis) using quantitative real time-PCR.

    PubMed

    Bhandary, Binny; Rajarapu, Swapna Priya; Rivera-Vega, Loren; Mittapalli, Omprakash

    2010-05-04

    Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1(st)-, 2(nd)-, 3(rd)-, 4(th)-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

  10. Inhibitor-free DNA for real-time PCR analysis of synovial fluid from horses, cattle and pigs.

    PubMed

    Schneeweiss, Wilfried; Stanek, Christian; Wagner, Martin; Hein, Ingeborg

    2007-03-31

    The potential of five different commercial DNA isolation methods to remove real-time PCR inhibitors from the synovial fluid of horses, cattle and pigs was investigated. All kits with the exception of one included a silica column-based purification of the DNA. With the fifth kit, DNA purification is achieved by removing contaminating macromolecules by a desalting process. We used a recently developed method based on comparison of the real-time PCR signal of an artificial target incorporated into each PCR reaction in the presence of the isolated DNA from the sample, and in control samples containing water instead of isolated DNA. This was followed by statistical analysis of the data. Inhibition and subsequent reduction of the endpoint fluorescence in the real-time PCR reaction was encountered in many cases. Less frequently, the target copy number in the samples was underestimated. However, we found no experimental evidence of a negative influence of the reduced endpoint fluorescence signal on the detection limit of the real-time PCR assay. All kits tested were useful for analyzing pelleted synovial fluid from horses, cattle and pigs. When analyzing non-pelleted synovial fluid, three kits - two based on silica columns and one employing a desalting process - yielded inhibitor-free DNA for real-time PCR analysis. PMID:17222992

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

    PubMed

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

    2015-01-01

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

  12. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses

    PubMed Central

    Parker, Jayme; Fowler, Nisha; Walmsley, Mary Louise; Schmidt, Terri; Scharrer, Jason; Kowaleski, James; Grimes, Teresa; Hoyos, Shanann; Chen, Jack

    2015-01-01

    Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID50/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. PMID:26569120

  13. Potential role of real-time PCR for detecting Mycobacterium avium subsp. paratuberculosis in chronically diseased milking cows: a case control study.

    PubMed

    Alajmi, Ahmad; Klein, Günter; Greiner, Matthias; Grabowski, Nils Th; Fohler, Svenja; Campe, Amely; Scheu, Theresa; Hoedemaker, Martina; Abdulmawjood, Amir

    2016-01-01

    Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) is economically important to dairy operations. In the present study, a real-time PCR kit (certified for use in Germany) was used to detect MAP in bovine fecal and milk samples within a case control study examining different factors for their association with chronic disease in Northern Germany.The aim of this study was to describe the suitability of the MAP real-time PCR kit to detect MAP in feces and milk of chronically diseased dairy cows. For MAP detection a total of 928 fecal, 922 composite foremilk, and 92 bulk milk samples obtained from 58 cases and 35 control dairy herds were investigated. The real-time PCR showed MAP positive results for 11 (18.96%) and 6 (17.14%) of the case and control herds, respectively. All bulk milk samples were MAP negative. The results of fecal and milk samples were moderately correlated (kappa = 0.27). These data indicate that real-time PCR results have diagnostic value for diagnosing MAP positive animals, and that fecal samples are more suitable than milk samples for assessing the reasons for chronic disease on dairy farms. PMID:27529992

  14. Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants

    PubMed Central

    Safeukui, Innocent; Millet, Pascal; Boucher, Sébastien; Melinard, Laurence; Fregeville, Frédéric; Receveur, Marie-Catherine; Pistone, Thierry; Fialon, Pierre; Vincendeau, Philippe; Fleury, Hervé; Malvy, Denis

    2008-01-01

    Background A simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) in febrile returning travellers and migrants was developed and evaluated. Methods Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth Plasmodium species, while the acceptor probe sequence was designed for P. falciparum over a region containing one mismatched, which allowed differentiation of the three other Plasmodium species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria. Results Blood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5°C (0.6°C) for P. falciparum infection and 64.6°C (1.8°C) for non-P. falciparum species. These Tms values of the P. falciparum or non-P. falciparum species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 – 1) or P. falciparum-specific (0.91, 0.8 – 1) primers, or with the microscopy results (0.70, 0.6 – 0.8). The real-time assay was 100% sensitive and specific for differentiation of P. falciparum to non-P. falciparum species, compared with conventional PCR or microscopy. The real-time PCR assay can also

  15. 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.

  16. Impact of Short-Time Urine Freezing on the Sensitivity of an Established Schistosoma Real-Time PCR Assay

    PubMed Central

    Kenguele, Hilaire M.; Adegnika, Ayola A.; Nkoma, Anne-Marie; Ateba-Ngoa, Ulysse; Mbong, Mirabeau; Zinsou, Jeannot; Lell, Bertrand; Verweij, Jaco J.

    2014-01-01

    Urogenital schistosomiaisis is a serious public health problem in sub-Saharan Africa. In this study, we have updated an established real-time polymerase chain reaction (PCR) routinely used in our laboratory. Schistosoma genus-specific real-time PCR was performed on DNA isolated from 85 urine samples and pellets obtained after centrifugation without and after frozen storage. The results revealed that concentration by centrifugation of the urine samples and freezing of the samples before extracting DNA improves the sensitivity of the PCR. PMID:24710612

  17. 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...

  18. 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...

  19. Undergraduate Virology Exercises Demonstrate Conventional and Real-Time PCR Using Commercially Available HIV Primers and Noninfectious Target

    ERIC Educational Resources Information Center

    Sulzinski, Michael A.; Wasilewski, Melissa A.; Farrell, James C.; Glick, David L.

    2009-01-01

    It is an extraordinary challenge to offer an undergraduate laboratory course in virology that teaches hands-on, relevant molecular biology techniques using nonpathogenic models of human virus detection. To our knowledge, there exists no inexpensive kits or reagent sets that are appropriate for demonstrating real-time PCR (RT-PCR) in an…

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

    EPA Science Inventory

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

  1. 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 ...

  2. 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...

  3. 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...

  4. 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)...

  5. 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...

  6. 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...

  7. 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...

  8. Duplex real-time PCR for rapid simultaneous detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in Amphibian samples.

    PubMed

    Blooi, M; Pasmans, F; Longcore, J E; Spitzen-van der Sluijs, A; Vercammen, F; Martel, A

    2013-12-01

    Chytridiomycosis is a lethal fungal disease contributing to declines and extinctions of amphibian species worldwide. The currently used molecular screening tests for chytridiomycosis fail to detect the recently described species Batrachochytrium salamandrivorans. In this study, we present a duplex real-time PCR that allows the simultaneous detection of B. salamandrivorans and Batrachochytrium dendrobatidis. With B. dendrobatidis- and B. salamandrivorans-specific primers and probes, detection of the two pathogens in amphibian samples is possible, with a detection limit of 0.1 genomic equivalent of zoospores of both pathogens per PCR. The developed real-time PCR shows high degrees of specificity and sensitivity, high linear correlations (r(2) > 0.995), and high amplification efficiencies (>94%) for B. dendrobatidis and B. salamandrivorans. In conclusion, the described duplex real-time PCR can be used to detect DNA of B. dendrobatidis and B. salamandrivorans with highly reproducible and reliable results.

  9. Identification of individual herbal drugs in tea mixtures using restriction analysis of ITS DNA and real-time PCR.

    PubMed

    Slanc, P; Ravnikar, M; Strukelj, B

    2006-11-01

    We have studied a sedative tea made of Valerianae radix (Valeriana officinalis L.), Lupuli strobuli (Humulus lupulus L.), Melissae folium (Melissa officinalis L.) and Menthae piperitae folium (Mentha piperita L.). In order to identify the constituent drugs a method was established involving amplification of the internal transcribed spacers (ITS) region of nuclear ribosomal DNA on the basis of restriction analysis and real-time PCR. ITS regions of individual drugs were amplified and sequenced. Restriction analysis was performed with selected restriction endonucleases Nae I, PshA I and Xcm I. Real-time PCR was carried out, using primers specifically designed for each individual herbal drug. Real-time PCR proved to be a method for identifying individual herbal drugs in a tea mixture with a single DNA extraction in a single PCR run, since its limit of detection is lower than that for restriction analysis. PMID:17152982

  10. Duplex Real-Time PCR for Rapid Simultaneous Detection of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans in Amphibian Samples

    PubMed Central

    Pasmans, F.; Longcore, J. E.; Spitzen-van der Sluijs, A.; Vercammen, F.; Martel, A.

    2013-01-01

    Chytridiomycosis is a lethal fungal disease contributing to declines and extinctions of amphibian species worldwide. The currently used molecular screening tests for chytridiomycosis fail to detect the recently described species Batrachochytrium salamandrivorans. In this study, we present a duplex real-time PCR that allows the simultaneous detection of B. salamandrivorans and Batrachochytrium dendrobatidis. With B. dendrobatidis- and B. salamandrivorans-specific primers and probes, detection of the two pathogens in amphibian samples is possible, with a detection limit of 0.1 genomic equivalent of zoospores of both pathogens per PCR. The developed real-time PCR shows high degrees of specificity and sensitivity, high linear correlations (r2 > 0.995), and high amplification efficiencies (>94%) for B. dendrobatidis and B. salamandrivorans. In conclusion, the described duplex real-time PCR can be used to detect DNA of B. dendrobatidis and B. salamandrivorans with highly reproducible and reliable results. PMID:24108616

  11. TaqMan real-time PCR for detection and quantitation of squash leaf curl virus in cucurbits.

    PubMed

    Kuan, Cheng-Ping; Huang, Hung-Chang; Chang, Chia-Che; Lu, Yi-Lin

    2012-02-01

    A real-time PCR assay based on the TaqMan chemistry was developed for reliable detection and quantitation of the squash leaf curl virus (SLCV) in melon and squash plants. This method was highly specific to SLCV and it was about one thousand times more sensitive than the conventional PCR method. The protocol of the real-time PCR established in this study enabled detection of as little as 10(2) copies of SLCV DNA with CP gene as the target. This TaqMan real-time PCR assay for detection and quantitation of SLCV would be a useful tool for application in quarantine and certification of SLCV in cucurbits as well as in the research of disease resistance and epidemiology.

  12. Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples.

    PubMed

    Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; van Doorn, Joop; Pham, Khanh; Cambra, Miguel A; Berruete, Isabel M; Pothier, Joël F; Duffy, Brion; Olmos, Antonio; López, María M

    2015-05-01

    Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample preparation were analyzed. Kappa coefficient, sensitivity, specificity, likelihood ratios and post-test probability of detection were estimated to manage the risk associated with the use of the two methods.

  13. Detection of HPV and co-infecting pathogens in healthy Italian women by multiplex real-time PCR.

    PubMed

    Camporiondo, Maria Pia; Farchi, Francesca; Ciccozzi, Massimo; Denaro, Aurelia; Gallone, Domenica; Maracchioni, Fabio; Favalli, Cartesio; Ciotti, Marco

    2016-01-01

    Several pathogens can be transmitted sexually and are an important cause of morbidity among sexually active women. The aim of the study was to detect the presence of human papillomavirus (HPV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Ureaplasma urealyticum (UU), and Ureaplasma parvum (UP) in a group of 309 healthy women enrolled at the San Camillo - Forlanini hospital of Rome by using two multiplex real-time PCR assays based on TOCE® technology. The women's ages ranged from 34 to 60 years, median 49 [IQR 45-54]. Of the 309 women tested, HPV DNA was detected in 77/309 (24.9%) patients. Of these, 44 (14.2%) harboured a single infection while 33 (10.7%) were infected by multiple genotypes. Prevalence of HPV infection was highest among females aged 40-50 years (15.2%). Of the other pathogens sought, CT, MG and NG were not detected while positive results were found for MH (12/309, 3.9%), TV (4/309, 1.3%), UP (89/309, 28.8%) and UU (14/309, 4.5%). Co-infections were as follows: 5 MH/HPV, 4 TV/HPV, 34 UP/HPV and 9 UU/HPV. In HPV-positive women, the probability of being infected by UP and UU was 2.5 (p=0.00045) and 6 fold higher (p=0.0016) than in HPV-negative women. The study supports the use of multiplex real-time PCR assays in a routine diagnostic setting. The high sensitivity and specificity of these assays along with the simultaneous detection of the most common sexually transmitted pathogens confers an advantage with respect to more obsolete methods reducing costs and time to diagnosis.

  14. Detection of HPV and co-infecting pathogens in healthy Italian women by multiplex real-time PCR.

    PubMed

    Camporiondo, Maria Pia; Farchi, Francesca; Ciccozzi, Massimo; Denaro, Aurelia; Gallone, Domenica; Maracchioni, Fabio; Favalli, Cartesio; Ciotti, Marco

    2016-01-01

    Several pathogens can be transmitted sexually and are an important cause of morbidity among sexually active women. The aim of the study was to detect the presence of human papillomavirus (HPV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Ureaplasma urealyticum (UU), and Ureaplasma parvum (UP) in a group of 309 healthy women enrolled at the San Camillo - Forlanini hospital of Rome by using two multiplex real-time PCR assays based on TOCE® technology. The women's ages ranged from 34 to 60 years, median 49 [IQR 45-54]. Of the 309 women tested, HPV DNA was detected in 77/309 (24.9%) patients. Of these, 44 (14.2%) harboured a single infection while 33 (10.7%) were infected by multiple genotypes. Prevalence of HPV infection was highest among females aged 40-50 years (15.2%). Of the other pathogens sought, CT, MG and NG were not detected while positive results were found for MH (12/309, 3.9%), TV (4/309, 1.3%), UP (89/309, 28.8%) and UU (14/309, 4.5%). Co-infections were as follows: 5 MH/HPV, 4 TV/HPV, 34 UP/HPV and 9 UU/HPV. In HPV-positive women, the probability of being infected by UP and UU was 2.5 (p=0.00045) and 6 fold higher (p=0.0016) than in HPV-negative women. The study supports the use of multiplex real-time PCR assays in a routine diagnostic setting. The high sensitivity and specificity of these assays along with the simultaneous detection of the most common sexually transmitted pathogens confers an advantage with respect to more obsolete methods reducing costs and time to diagnosis. PMID:27031891

  15. Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus.

    PubMed

    Odreman-Macchioli, María; Vielma, Silvana; Atchley, Daniel; Comach, Guillermo; Ramirez, Alvaro; Pérez, Saberio; Téllez, Luis; Quintero, Beatriz; Hernández, Erick; Muñoz, Maritza; Mendoza, José

    2013-03-01

    Early diagnosis of dengue virus (DENV) infection represents a key factor in preventing clinical complications attributed to the disease. The aim of this study was to evaluate the amplification efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, using the non-structural conserved genomic region protein-5 (NS5) versus two genomic regions usually employed for virus detection, the capsid/pre-membrane region (C-prM) and the 3'-noncoding region (3'NC). One-hundred sixty seven acute phase serum samples from febrile patients were used for validation purposes. Results showed that the three genomic regions had similar amplification profiles and correlation coefficients (0.987-0.999). When isolated viruses were used, the NS5 region had the highest qPCR efficiencies for the four serotypes (98-100%). Amplification from acute serum samples showed that 41.1% (67/167) were positive for the universal assay by at least two of the selected genomic regions. The agreement rates between NS5/C-prM and NS5/3'NC regions were 56.7% and 97%, respectively. Amplification concordance values between C-prM/NS5 and NS5/3'NC regions showed a weak (kappa = 0.109; CI 95%) and a moderate (kappa = 0.489; CI 95%) efficiencies in amplification, respectively. Serotyping assay using a singleplex NS5-TaqMan format was much more sensitive than the C-prM/SYBR Green I protocol (76%). External evaluation showed a high sensitivity (100%), specificity (78%) and high agreement between the assays. According to the results, the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples. PMID:23781709

  16. Exploring valid reference genes for quantitative real-time PCR analysis in Sesamia inferens (Lepidoptera: Noctuidae).

    PubMed

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2015-01-01

    The pink stem borer, Sesamia inferens, which is endemic in China and other parts of Asia, is a major pest of rice and causes significant yield loss in this host plant. Very few studies have addressed gene expression in S. inferens. Quantitative real-time PCR (qRT-PCR) is currently the most accurate and sensitive method for gene expression analysis. In qRT-PCR, data are normalized using reference genes, which help control for internal differences and reduce error between samples. In this study, seven candidate reference genes, 18S ribosomal RNA (18S rRNA), elongation factor 1 (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein S13 (RPS13), ribosomal protein S20 (RPS20), tubulin (TUB), and β-actin (ACTB) were evaluated for their suitability in normalizing gene expression under different experimental conditions. The results indicated that three genes (RPS13, RPS20, and EF1) were optimal for normalizing gene expression in different insect tissues (head, epidermis, fat body, foregut, midgut, hindgut, Malpighian tubules, haemocytes, and salivary glands). 18S rRNA, EF1, and GAPDH were best for normalizing expression with respect to developmental stages and sex (egg masses; first, second, third, fourth, fifth, and sixth instar larvae; male and female pupae; and one-day-old male and female adults). 18S rRNA, RPS20, and TUB were optimal for fifth instars exposed to different temperatures (-8, -6, -4, -2, 0, and 27°C). To validate this recommendation, the expression profile of a target gene heat shock protein 83 gene (hsp83) was investigated, and results showed the selection was necessary and effective. In conclusion, this study describes reference gene sets that can be used to accurately measure gene expression in S. inferens.

  17. Real-time PCR detection of environmental mycobacteria in house dust.

    PubMed

    Torvinen, Eila; Torkko, Pirjo; Rintala, Aino Nevalainen Helena

    2010-07-01

    Analysing the number and species of microbes in indoor dust is needed for assessment of human exposure to microbes in dwellings. Environmental mycobacteria are common heterotrophic bacteria in both natural and man-made environments and potential inducers of human immune system. Culture of mycobacteria from samples rich with other microbes is difficult due to the slow growth rate of mycobacteria and this has hampered the studies on their role in indoor human exposure. A quantitative, real-time 5'-nuclease (TaqMan) PCR assay was developed to detect environmental mycobacteria in indoor dust samples. The specificity of the primers and the probe targeting the 16S rDNA of mycobacteria, tested with 26 mycobacterial and 10 non-mycobacterial but related species, proved to be high. When tested on 20 indoor dust samples collected from five homes, the assay gave counts varying between 4.8 x 10(4) and 7.2 x 10(6)cell/g, being on average 1.1 x 10(3) times higher than culture. Seasonal variation in the dust counts of mycobacteria was observed by both culture and qPCR. Total of 140 isolates considered as mycobacteria by Ziehl-Neelsen staining and GLC-analyses were subjected to PCR analysis with the mycobacterial primers, and 39 isolates to partial 16S rDNA sequencing. All proved to be mycobacteria and revealed high diversity of mycobacterial species in the dust samples. Majority of the sequences were related to M. terrae and M. avium complexes. PMID:20434494

  18. Reference gene selection for quantitative real-time PCR normalization in Quercus suber.

    PubMed

    Marum, Liliana; Miguel, Andreia; Ricardo, Cândido P; Miguel, Célia

    2012-01-01

    The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1α, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

  19. A real-time diagnostic and performance monitor for UNIX. M.S. Thesis

    NASA Technical Reports Server (NTRS)

    Dong, Hongchao

    1992-01-01

    There are now over one million UNIX sites and the pace at which new installations are added is steadily increasing. Along with this increase, comes a need to develop simple efficient, effective and adaptable ways of simultaneously collecting real-time diagnostic and performance data. This need exists because distributed systems can give rise to complex failure situations that are often un-identifiable with single-machine diagnostic software. The simultaneous collection of error and performance data is also important for research in failure prediction and error/performance studies. This paper introduces a portable method to concurrently collect real-time diagnostic and performance data on a distributed UNIX system. The combined diagnostic/performance data collection is implemented on a distributed multi-computer system using SUN4's as servers. The approach uses existing UNIX system facilities to gather system dependability information such as error and crash reports. In addition, performance data such as CPU utilization, disk usage, I/O transfer rate and network contention is also collected. In the future, the collected data will be used to identify dependability bottlenecks and to analyze the impact of failures on system performance.

  20. Automated high-throughput flow-through real-time diagnostic system

    DOEpatents

    Regan, John Frederick

    2012-10-30

    An automated real-time flow-through system capable of processing multiple samples in an asynchronous, simultaneous, and parallel fashion for nucleic acid extraction and purification, followed by assay assembly, genetic amplification, multiplex detection, analysis, and decontamination. The system is able to hold and access an unlimited number of fluorescent reagents that may be used to screen samples for the presence of specific sequences. The apparatus works by associating extracted and purified sample with a series of reagent plugs that have been formed in a flow channel and delivered to a flow-through real-time amplification detector that has a multiplicity of optical windows, to which the sample-reagent plugs are placed in an operative position. The diagnostic apparatus includes sample multi-position valves, a master sample multi-position valve, a master reagent multi-position valve, reagent multi-position valves, and an optical amplification/detection system.

  1. A novel poisson distribution-based approach for testing boundaries of real-time PCR assays for food pathogen quantification.

    PubMed

    Rossmanith, Peter; Wagner, Martin

    2011-09-01

    The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in food pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.

  2. Procalcitonin Predicts Real-Time PCR Results in Blood Samples from Patients with Suspected Sepsis

    PubMed Central

    Mencacci, Antonella; Leli, Christian; Cardaccia, Angela; Meucci, Marta; Moretti, Amedeo; D'Alò, Francesco; Farinelli, Senia; Pagliochini, Rita; Barcaccia, Mariella; Bistoni, Francesco

    2012-01-01

    Background Early diagnosis and rapid bacterial identification are of primary importance for outcome of septic patients. SeptiFast® (SF) real-time PCR assay is of potential utility in the etiological diagnosis of sepsis, but it cannot replace blood culture (BC) for routine use in clinical laboratory. Procalcitonin (PCT) is a marker of sepsis and can predict bacteremia in septic patients. The aim of the present study was to investigate whether PCT serum levels could predict SF results, and could help screening febrile patients in which a SF assay can improve the etiological diagnosis of sepsis. Methods From 1009 febrile patients with suspected sepsis, 1009 samples for BC, SF real-time PCR, and PCT determination were obtained simultaneously, and results were compared and statistically analysed. Receiver operating characteristic (ROC) curves were generated to determine the area under the curve and to identify which cut-off of PCT value produced the best sensitivity to detect SF results. Results Mean PCT values of sera drawn simultaneously with samples SF positive (35.42±61.03 ng/ml) or BC positive (23.14±51.56 ng/ml) for a pathogen were statistically higher than those drawn simultaneously with SF negative (0.84±1.67 ng/ml) or BC negative (2.79±16.64 ng/ml) samples (p<0.0001). For SF, ROC analysis showed an area under the curve of 0.927 (95% confidence interval: 0.899–0.955, p<0.0001). The PCT cut-off value of 0.37 ng/ml showed a negative predictive value of 99%, reducing the number of SF assays of 53.9%, still identifying the 96.4% of the pathogens. Conclusion PCT can be used in febrile patients with suspected sepsis to predict SF positive or negative results. A cut-off value of 0.37 ng/ml can be considered for optimal sensitivity, so that, in the routine laboratory activity, SF assay should not be used for diagnosis of sepsis in an unselected patient population with a PCT value <0.37 ng/ml. PMID:23300907

  3. Rapid molecular typing of Prototheca zopfii by high resolution melting real-time PCR (PCR-HRM).

    PubMed

    Sobukawa, Hideto; Ibaraki, Masato; Kano, Rui; Ito, Takaaki; Suzuki, Kazuyuki; Kamata, Hiroshi; Hasegawa, Atsuhiko

    2013-01-01

    Prototheca zopfii is an achlorophyllic alga that is ubiquitous around cow sheds. The alga is associated with bovine mastitis, which causes a reduction in milk production and secretion of thin watery milk containing white flakes. Isolates of P. zopfii from bovine mastitis were almost all identified as P. zopfii genotype 2, suggesting that it is the main causative agent of bovine protothecal mastitis. The ability to differentiate between genotype 1 and genotype 2 is therefore very important for preventing bovine mastitis. In this study, high resolution melting real-time PCR (PCR-HRM) analysis of the protothecal 18S rDNA domain successfully differentiated between genotypes of P. zopfii in less than 3 hours, while conventional sequence analysis requires more than 48 hours to differentiate between genotypes. PCR-HRM analysis clustered P. zopfii genotype 1 isolates separately from P. zopfii genotype 2 isolates, indicating that this molecular typing method is an effective tool for rapidly diagnosing bovine protothecal mastitis. PMID:24292136

  4. Senior Thai fecal microbiota comparison between vegetarians and non-vegetarians using PCR-DGGE and real-time PCR.

    PubMed

    Ruengsomwong, Supatjaree; Korenori, Yuki; Sakamoto, Naoshige; Wannissorn, Bhusita; Nakayama, Jiro; Nitisinprasert, Sunee

    2014-08-01

    The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets.

  5. Senior Thai fecal microbiota comparison between vegetarians and non-vegetarians using PCR-DGGE and real-time PCR.

    PubMed

    Ruengsomwong, Supatjaree; Korenori, Yuki; Sakamoto, Naoshige; Wannissorn, Bhusita; Nakayama, Jiro; Nitisinprasert, Sunee

    2014-08-01

    The fecal microbiotas were investigated in 13 healthy Thai subjects using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Among the 186 DNA bands detected on the polyacrylamide gel, 37 bands were identified as representing 11 species: Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides uniformis, Bacteroides vulgatus, Clostridium colicanis, Eubacterium eligenes, E. rectale, Faecalibacterium prausnitzii, Megamonas funiformis, Prevotella copri, and Roseburia intestinalis, belonging mainly to the groups of Bacteroides, Prevotella, Clostridium, and F. prausnitzii. A dendrogram of the PCR-DGGE divided the subjects; vegetarians and non-vegetarians. The fecal microbiotas were also analyzed using a quantitative real-time PCR focused on Bacteroides, Bifidobacterium, Enterobacteriaceae, Clostrium coccoides-Eubacterium rectale, C. leptum, Lactobacillus, and Prevotella. The nonvegetarian and vegetarian subjects were found to have significant differences in the high abundance of the Bacteroides and Prevotella genera, respectively. No significant differences were found in the counts of Bifidabacterium, Enterobacteriaceae, C. coccoides-E. rectale group, C. leptum group, and Lactobacillus. Therefore, these findings on the microbiota of healthy Thais consuming different diets could provide helpful data for predicting the health of South East Asians with similar diets. PMID:24743571

  6. Detection of Grapevine leafroll-associated virus 7 using real time qRT-PCR and conventional RT-PCR.

    PubMed

    Al Rwahnih, Maher; Osman, Fatima; Sudarshana, Mysore; Uyemoto, Jerry; Minafra, Angelantonio; Saldarelli, Pasquale; Martelli, Giovanni; Rowhani, Adib

    2012-02-01

    Nine isolates of Grapevine leafroll-associated virus 7 (GLRaV-7) from diverse geographical regions were sequenced to design more sensitive molecular diagnostic tools. The coat protein (CP) and heat shock protein 70 homologue (HSP70h) genes of these nine isolates were sequenced. Sequences were then used to design more sensitive molecular diagnostic tools. Sequence identity among these isolates ranged between 90 to 100% at the nucleotide and amino acid levels. One RT-PCR and two qRT-PCR assays were used to survey 86 different grapevines from the University of California, Davis Grapevine Virus Collection, the Foundation Plant Services collection and the USDA National Clonal Germplasm Repository, Davis, CA with primers designed in conserved regions of the CP and HSP70h genes. Results revealed that qRT-PCR assays designed in the HSP70h gene was more sensitive (29.07% positives) than that designed in the CP gene (22.09% positives) and both qRT-PCR assays proved to be more sensitive than RT-PCR.

  7. Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR

    PubMed Central

    2010-01-01

    Background The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of Brucella DNA. Methods Five kits were evaluated using clinical specimens: QIAamp™ DNA Mini Kit (QIAGEN), peqGold™ Tissue DNA Mini Kit (PeqLab), UltraClean™ Tissue and Cells DNA Isolation Kit (MoBio), DNA Isolation Kit for Cells and Tissues (Roche), and NucleoSpin™ Tissue (Macherey-Nagel). DNA yield was determined using a quantitative real-time PCR assay targeting IS711 that included an internal amplification control. Results Kits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p < 0.05) and of diagnostic relevance. Sample volume, elution volume, and processing time were also compared. Conclusions We observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low. PMID:20403208

  8. Microsatellite Length Scoring by Single Molecule Real Time Sequencing – Effects of Sequence Structure and PCR Regime

    PubMed Central

    Liljegren, Mikkel Meyn; de Muinck, Eric Jacques; Trosvik, Pål

    2016-01-01

    Microsatellites are DNA sequences consisting of repeated, short (1–6 bp) sequence motifs that are highly mutable by enzymatic slippage during replication. Due to their high intrinsic variability, microsatellites have important applications in population genetics, forensics, genome mapping, as well as cancer diagnostics and prognosis. The current analytical standard for microsatellites is based on length scoring by high precision electrophoresis, but due to increasing efficiency next-generation sequencing techniques may provide a viable alternative. Here, we evaluated single molecule real time (SMRT) sequencing, implemented in the PacBio series of sequencing apparatuses, as a means of microsatellite length scoring. To this end we carried out multiplexed SMRT sequencing of plasmid-carried artificial microsatellites of varying structure under different pre-sequencing PCR regimes. For each repeat structure, reads corresponding to the target length dominated. We found that pre-sequencing amplification had large effects on scoring accuracy and error distribution relative to controls, but that the effects of the number of amplification cycles were generally weak. In line with expectations enzymatic slippage decreased proportionally with microsatellite repeat unit length and increased with repetition number. Finally, we determined directional mutation trends, showing that PCR and SMRT sequencing introduced consistent but opposing error patterns in contraction and expansion of the microsatellites on the repeat motif and single nucleotide level. PMID:27414800

  9. A novel method for diagnosis of smear-negative tuberculosis patients by combining a random unbiased Phi29 amplification with a specific real-time PCR.

    PubMed

    Pang, Yu; Lu, Jie; Yang, Jian; Wang, Yufeng; Cohen, Chad; Ni, Xin; Zhao, Yanlin

    2015-07-01

    In this study, we develop a novel method for diagnosis of smear-negative tuberculosis patients by performing a random unbiased Phi29 amplification prior to the use of a specific real-time PCR. The limit of detection (LOD) of the conventional real-time PCR was 100 colony-forming units (CFU) of MTB genome/reaction, while the REPLI real-time PCR assay could detect 0.4 CFU/reaction. In comparison with the conventional real-time PCR, REPLI real-time PCR shows better sensitivity for the detection of smear-negative tuberculosis (P = 0.015).

  10. Real time PCR assay for detection of all known lineages of West Nile virus.

    PubMed

    Vázquez, Ana; Herrero, Laura; Negredo, Anabel; Hernández, Lourdes; Sánchez-Seco, María Paz; Tenorio, Antonio

    2016-10-01

    West Nile virus (WNV) is one of the most widespread arbovirus and a large variety of WNV strains and lineages have been described. The molecular methods for the diagnosis of WNV target mainly lineages 1 and 2, which have caused outbreaks in humans, equines and birds. But the last few years new and putative WNV lineages of unknown pathogenicity have been described. Here we describe a new sensitive and specific real-time PCR assay for the detection and quantification of all the WNV lineages described until now. Primers and probe were designed in the 3'-untranslated region (3'-UTR) of the WNV genome and were designed to match all sequenced WNV strains perfectly. The sensitivity of the assay ranged from 1,5 to 15 copies per reaction depending on the WNV lineage tested. The method was validated for WNV diagnosis using different viral strains, human samples (cerebrospinal fluid, biopsies, serum and plasma) and mosquito pools. The assay did not amplify any other phylogenetically or symptomatically related viruses. All of the above make it a very suitable tool for the diagnosis of WNV and for surveillance studies. PMID:27481597

  11. Detection of Ochratoxin a Using Molecular Beacons and Real-Time PCR Thermal Cycler

    PubMed Central

    Sanzani, Simona Marianna; Reverberi, Massimo; Fanelli, Corrado; Ippolito, Antonio

    2015-01-01

    We developed a simple and cheap assay for quantitatively detecting ochratoxin A (OTA) in wine. A DNA aptamer available in literature was used as recognition probe in its molecular beacon form, i.e., with a fluorescence-quenching pair at the stem ends. Our aptabeacon could adopt a conformation allowing OTA binding, causing a fluorescence rise due to the increased distance between fluorophore and quencher. We used real-time PCR equipment for capturing the signal. With this assay, under optimized conditions, the entire process can be completed within 1 h. In addition, the proposed system exhibited a good selectivity for OTA against other mycotoxins (ochratoxin B and aflatoxin M1) and limited interference from aflatoxin B1 and patulin. A wide linear detection range (0.2–2000 µM) was achieved, with LOD = 13 nM, r = 0.9952, and R2 = 0.9904. The aptabeacon was also applied to detect OTA in red wine spiked with the same dilution series. A linear correlation with a LOD = 19 nM, r = 0.9843, and R2 = 0.9708 was observed, with recoveries in the range 63%–105%. Intra- and inter-day assays confirmed its reproducibility. The proposed biosensor, although still being finalized, might significantly facilitate the quantitative detection of OTA in wine samples, thus improving their quality control from a food safety perspective. PMID:25760080

  12. Microbiological quality indicators in waters of dairy farms: detection of pathogens by PCR in real time.

    PubMed

    Rodríguez, Diana Catalina; Pino, Nancy; Peñuela, Gustavo

    2012-06-15

    When contaminated water is used to wash the udders of dairy cattle and milking utensils, raw milk may become contaminated with pathogens. Washing with high quality water is essential to reduce the microbial contamination of milk. Furthermore, the wastewater generated in dairy herds also contains high populations of pathogens, antibiotics and nutrients that more often are thrown into the water bodies without any treatment. In this work, both supply water and wastewater from 20 dairy farms from Antioquia, Colombia was monitored for 10months to determine the presence of pathogenic microorganisms. Both Cryptosporidium and Fasciola were determined by the Polymerase Chain Reaction (PCR) technique in real time. The results showed that the supply water used for drinking and activities involving the herd, has high populations of Fasciola hepatica and Cryptosporidium parvum, with percentages of about 53.7% and 64.75% respectively. Additionally high populations of Pseudomonas aeruginosa, Shigella, Salmonella, total coliforms and Escherichia coli were found in both types of water, with values around 9.4×10(7), 2.1×10(7), 1.8×10(7), 1.9×10(10) and 1.5×10(10) UFC/100 ml respectively for the wastewater and 3.1×10(4), 1.9×10(4), 7.3×10(3), 1.2×10(5) and 6.2×10(3) UFC/100 ml for the supply water.

  13. Detection of ochratoxin A using molecular beacons and real-time PCR thermal cycler.

    PubMed

    Sanzani, Simona Marianna; Reverberi, Massimo; Fanelli, Corrado; Ippolito, Antonio

    2015-03-01

    We developed a simple and cheap assay for quantitatively detecting ochratoxin A (OTA) in wine. A DNA aptamer available in literature was used as recognition probe in its molecular beacon form, i.e., with a fluorescence-quenching pair at the stem ends. Our aptabeacon could adopt a conformation allowing OTA binding, causing a fluorescence rise due to the increased distance between fluorophore and quencher. We used real-time PCR equipment for capturing the signal. With this assay, under optimized conditions, the entire process can be completed within 1 h. In addition, the proposed system exhibited a good selectivity for OTA against other mycotoxins (ochratoxin B and aflatoxin M1) and limited interference from aflatoxin B1 and patulin. A wide linear detection range (0.2-2000 µM) was achieved, with LOD = 13 nM, r = 0.9952, and R2 = 0.9904. The aptabeacon was also applied to detect OTA in red wine spiked with the same dilution series. A linear correlation with a LOD = 19 nM, r = 0.9843, and R2 = 0.9708 was observed, with recoveries in the range 63%-105%. Intra- and inter-day assays confirmed its reproducibility. The proposed biosensor, although still being finalized, might significantly facilitate the quantitative detection of OTA in wine samples, thus improving their quality control from a food safety perspective. PMID:25760080

  14. Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis.

    PubMed

    Skottman, T; Piiparinen, H; Hyytiäinen, H; Myllys, V; Skurnik, M; Nikkari, S

    2007-03-01

    This report describes the development of in-house real-time PCR assays using minor groove binding probes for simultaneous detection of the Bacillus anthracis pag and cap genes, the Francisella tularensis 23 KDa gene, as well as the Yersinia pestis pla gene. The sensitivities of these assays were at least 1 fg, except for the assay targeting the Bacillus anthracis cap gene, which showed a sensitivity of 10 fg when total DNA was used as a template in a serial dilution. The clinical value of the Bacillus anthracis- and Francisella tularensis-specific assays was demonstrated by successful amplification of DNA from cases of cow anthrax and hare tularemia, respectively. No cross-reactivity between these species-specific assays or with 39 other bacterial species was noted. These assays may provide a rapid tool for the simultaneous detection and identification of the three category A bacterial species listed as biological threats by the Centers for Disease Control and Prevention. PMID:17294160

  15. Development of real-time PCR assay for genetic identification of the mottled skate, Beringraja pulchra.

    PubMed

    Hwang, In Kwan; Lee, Hae Young; Kim, Min-Hee; Jo, Hyun-Su; Choi, Dong-Ho; Kang, Pil-Won; Lee, Yang-Han; Cho, Nam-Soo; Park, Ki-Won; Chae, Ho Zoon

    2015-10-01

    The mottled skate, Beringraja pulchra is one of the commercially important fishes in the market today. However, B. pulchra identification methods have not been well developed. The current study reports a novel real-time PCR method based on TaqMan technology developed for the genetic identification of B. pulchra. The mitochondrial cytochrome oxidase subunit 1 (COI) nucleotide sequences of 29 B. pulchra, 157 skates and rays reported in GenBank DNA database were comparatively analyzed and the COI sequences specific to B. pulchra was identified. Based on this information, a system of specific primers and Minor Groove Binding (MGB) TaqMan probe were designed. The assay successfully discriminated in 29 specimens of B. pulchra and 27 commercial samples with unknown species identity. For B. pulchra DNA, an average Threshold Cycle (Ct) value of 19.1±0.1 was obtained. Among 27 commercial samples, two samples showed average Ct values 19.1±0.0 and 26.7±0.1, respectively and were confirmed to be B. pulchra based on sequencing. The other samples tested showed undetectable or extremely weak signals for the target fragment, which was also consistent with the sequencing results. These results reveal that the method developed is a rapid and efficient tool to identify B. pulchra and might prevent fraud or mislabeling during the distribution of B. pulchra products.

  16. The application of real-time PCR to the analysis of T cell repertoires.

    PubMed

    Wettstein, Peter; Strausbauch, Michael; Therneau, Terry; Borson, Nancy

    2008-12-01

    The diversity of T-cell populations is determined by the spectrum of antigen-specific T-cell receptors (TCRs) that a