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Sample records for pcr anidada para

  1. Mix-infection of S. Typhi and ParaTyphi A in Typhoid Fever and Chronic Typhoid Carriers: A Nested PCR Based Study in North India

    PubMed Central

    Pratap, Chandra Bhan; Kumar, Gopal; Patel, Saurabh Kumar; Shukla, Vijay K; Kumar, Kailash; Singh, Tej Bali

    2014-01-01

    Introduction: Enteric fever is a systemic disease caused by Salmonella organism such as serotypes Typhi and ParaTyphi A, B, C. Salmonella ParaTyphi A contributes more than 50% of all the enteric fever cases and it has recently been projected as an emerging pathogen. Materials and Methods: The present study was aimed to detect Salmonella Typhi and ParaTyphi A in urine, blood and stool specimens collected from cases of enteric fever (110), chronic typhoid carriers (46) and healthy controls (75) to explore the possibility of mixed infection by nested PCR. A new nested PCR primer was designed targeting putative fimbrial protein (stkG) gene which is one of the fimbrial gene families to Salmonella ParaTyphi A and for S. Typhi already reported primers targeting flagellin (fliC) gene. Results: Large volume of urine specimens (15 ml) was found to be the best for detection of Salmonella serotypes. The urine sample was found to have mixed-infection by both the serotypes in 40.9% of the cases but lower in blood (27.3%) and stool (13.6%). Conclusion: The present study concludes that occurrence of mixed infection may be quite frequent in typhoid and chronic typhoid carriers’ individuals, although the reported recent rise in ParaTyphi A incidence may not be real. PMID:25584217

  2. Virtual PCR

    SciTech Connect

    Gardner, S N; Clague, D S; Vandersall, J A; Hon, G; Williams, P L

    2006-02-23

    The polymerase chain reaction (PCR) stands among the keystone technologies for analysis of biological sequence data. PCR is used to amplify DNA, to generate many copies from as little as a single template. This is essential, for example, in processing forensic DNA samples, pathogen detection in clinical or biothreat surveillance applications, and medical genotyping for diagnosis and treatment of disease. It is used in virtually every laboratory doing molecular, cellular, genetic, ecologic, forensic, or medical research. Despite its ubiquity, we lack the precise predictive capability that would enable detailed optimization of PCR reaction dynamics. In this LDRD, we proposed to develop Virtual PCR (VPCR) software, a computational method to model the kinetic, thermodynamic, and biological processes of PCR reactions. Given a successful completion, these tools will allow us to predict both the sequences and concentrations of all species that are amplified during PCR. The ability to answer the following questions will allow us both to optimize the PCR process and interpret the PCR results: What products are amplified when sequence mixtures are present, containing multiple, closely related targets and multiplexed primers, which may hybridize with sequence mismatches? What are the effects of time, temperature, and DNA concentrations on the concentrations of products? A better understanding of these issues will improve the design and interpretation of PCR reactions. The status of the VPCR project after 1.5 years of funding is consistent with the goals of the overall project which was scoped for 3 years of funding. At half way through the projected timeline of the project we have an early beta version of the VPCR code. We have begun investigating means to improve the robustness of the code, performed preliminary experiments to test the code and begun drafting manuscripts for publication. Although an experimental protocol for testing the code was developed, the preliminary

  3. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2003-01-01

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  4. PCR thermocycler

    DOEpatents

    Benett, William J.; Richards, James B.

    2005-05-17

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  5. Sex Determination Using PCR

    ERIC Educational Resources Information Center

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  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. Overlap extension PCR cloning.

    PubMed

    Bryksin, Anton; Matsumura, Ichiro

    2013-01-01

    Rising demand for recombinant proteins has motivated the development of efficient and reliable cloning methods. Here we show how a beginner can clone virtually any DNA insert into a plasmid of choice without the use of restriction endonucleases or T4 DNA ligase. Chimeric primers encoding plasmid sequence at the 5' ends and insert sequence at the 3' ends are designed and synthesized. Phusion(®) DNA polymerase is utilized to amplify the desired insert by PCR. The double-stranded product is subsequently employed as a pair of mega-primers in a PCR-like reaction with circular plasmids. The original plasmids are then destroyed in restriction digests with Dpn I. The product of the overlap extension PCR is used to transform competent Escherichia coli cells. Phusion(®) DNA polymerase is used for both the amplification and fusion reactions, so both steps can be monitored and optimized in the same way. PMID:23996437

  8. QUALITY CONTROLS FOR PCR

    EPA Science Inventory

    The purpose of this presentation is to present an overview of the quality control (QC) sections of a draft EPA document entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples." This document has been prepared by th...

  9. QUALITY ASSURANCE FOR PCR

    EPA Science Inventory

    The U.S. Environmental Protection Agency (EPA) held a workshop in January 2003 on the detection of viruses in water using polymerase chain reaction (PCR)-based methods. Speakers were asked to address a series of specific questions, including whether a single standard method coul...

  10. MAMMALIAN DNA IN PCR REAGENTS

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high- cycle PCR amplification t...

  11. Digital droplet PCR on disk.

    PubMed

    Schuler, Friedrich; Trotter, Martin; Geltman, Marcel; Schwemmer, Frank; Wadle, Simon; Domínguez-Garrido, Elena; López, María; Cervera-Acedo, Cristina; Santibáñez, Paula; von Stetten, Felix; Zengerle, Roland; Paust, Nils

    2016-01-01

    Existing systems for digital droplet PCR (ddPCR) either suffer from low integration or are difficult to introduce to mass fabrication. Here we present an integrated system that is compatible to mass fabrication and combines emulsification, PCR, and fluorescence readout in a single chamber within a disposable cartridge (disk). Droplets are generated by injecting the sample into fluorinated oil via centrifugal step emulsification. The resulting emulsion is aligned in the PCR and readout zone by capillary action. During thermocycling, gas bubbles generated by degassing are removed by capillary driven transport through tapered regions in the PCR chamber. Thereby, the positioning of the emulsion within the readout zone of the PCR chamber is ensured at any time and no bubbles are present during readout. Manual handling of the disk solely requires pipetting of oil and PCR mix into the inlet structures, placing the disk into the thermocycler and subsequently into a microarray scanner. The functionality of the ddPCR process chain is demonstrated by quantitative detection of the cystic fibrosis causing mutation p.Phe508del, which is of interest for non-invasive prenatal testing (NIPT). The mutation was detected in a concentration range spanning four orders of magnitude. We envision that this work will lay the base for the development of highly integrated sample-to-digital-answer PCR systems that can be employed in routine clinical diagnosis. PMID:26610263

  12. [Rapid PCR authentication Lonicera japanica].

    PubMed

    Jiang, Chao; Hou, Jing-Yi; Huang, Lu-Qi; Yuan, Yuan; Chen, Min; Jin, Yan

    2014-10-01

    To simply and rapid authenticate Lonicera japanica. Rapid allele-specific PCR primer was designed base on trnL-trnF 625 G/T Single nucleotide polymorphism and the PCR reaction systems including annealing temperature was optimized; optimized results were performed to authenticate L. japanica and its 9 adulterants. When 100 x SYBR Green I was added in the PCR product of 87 degrees C initial denatured 1 min; 87 degrees C denatured 5 s, 68 degrees C annealing 5 s, 30 cycle; L. japanica visualize strong green fluorescence under 365 nm UV lamp whereas adulterants without. The results indicate rapid allele-specific PCR could authenticate L. japanica and its adulterants rapidly and simply. PMID:25612418

  13. Ligation-independent cloning of PCR products (LIC-PCR).

    PubMed Central

    Aslanidis, C; de Jong, P J

    1990-01-01

    A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones. Images PMID:2235490

  14. A study of PCR inhibition mechanisms using real time PCR.

    PubMed

    Opel, Kerry L; Chung, Denise; McCord, Bruce R

    2010-01-01

    In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co-extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall we find that amplicon size and melting temperature are important in some inhibition mechanisms and not in others and the key issue in understanding PCR inhibition is determining the identity of the interfering substance. PMID:20015162

  15. PALATAL DYSMORPHOGENESIS: QUANTITATIVE RT-PCR

    EPA Science Inventory

    ABSTRACT

    Palatal Dysmorphogenesis : Quantitative RT-PCR

    Gary A. Held and Barbara D. Abbott

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

  16. 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…

  17. 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

  18. Electrothermal modeling of silicon PCR chips

    NASA Astrophysics Data System (ADS)

    Cui, Zheng; Zhao, Zhan; Xia, Shanhong

    2001-04-01

    Polymerase chain reaction (PCR) on a microchip has drawn considerable attention in recent years. Although a microchip can have must fast heating and cooling rate, the delicacy in its structure makes the PCR experiment difficult and cracks often occurs particularly for the thin membrane type of PCR chips. Electrothermal modeling of PCR chips is presented using commercial MEMS software tool IntelliSuiteTM, with the aim of identifying the problems encountered in experiment and finding an optimum chip structure. Heating characteristics of four different heater designs have been compared, so have the PCR chambers with fixed frame and with suspended frame. The thermal stress analysis has shown that the structure and heater design can make significant difference in heating characteristics and in reducing the failure of PCR chips. The computer simulation has confirmed what has been found in experiment the reason of membrane cracks. Improvement in PCR chip design has been proposed.

  19. Multiplexed Primer Prediction for PCR

    Energy Science and Technology Software Center (ESTSC)

    2007-07-23

    MPP predicts sets of multiplex-compatible primers for Polymerase Chain Reaction (PCR), finding a near minimal set of primers such that at least one amplicon will be generated from every target sequence in the input file. The code finds highly conserved oligos that are suitable as primers, according to user-specified desired primer characteristics such as length, melting temperature, and amplicon length. The primers are predicted not to form unwanted dimer or hairpin structures. The target sequencesmore » used as input can be diverse, since no multiple sequence alighment is required. The code is scalable, taking up to tens of thousands of sequences as input, and works, for example, to find a "universal primer set" for all viral genomes provided as a single input file. The code generates a periodic check-point file, thus in the event of premature execution termination, the application can be restarted from the last check-point file.« less

  20. Real-time PCR in microfluidic devices

    NASA Astrophysics Data System (ADS)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

  1. Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus.

    PubMed

    Karim, Mohammad R; Fout, G Shay; Johnson, Clifford H; White, Karen M; Parshionikar, Sandhya U

    2015-07-01

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay. PMID:25796356

  2. Comparative analysis of conventional PCR and real-time PCR to diagnose shrimp WSD

    PubMed Central

    Leal, C.A.G.; Carvalho-Castro, G.A.; Cottorello, A.C.; Leite, R.C.; Figueiredo, H.C.P.

    2013-01-01

    The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples. PMID:24516428

  3. Testing for Genetically Modified Foods Using PCR

    ERIC Educational Resources Information Center

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  4. Methods for comparing multiple digital PCR experiments.

    PubMed

    Burdukiewicz, Michał; Rödiger, Stefan; Sobczyk, Piotr; Menschikowski, Mario; Schierack, Peter; Mackiewicz, Paweł

    2016-09-01

    The estimated mean copy per partition (λ) is the essential information from a digital PCR (dPCR) experiment because λ can be used to calculate the target concentration in a sample. However, little information is available how to statistically compare dPCR runs of multiple runs or reduplicates. The comparison of λ values from several runs is a multiple comparison problem, which can be solved using the binary structure of dPCR data. We propose and evaluate two novel methods based on Generalized Linear Models (GLM) and Multiple Ratio Tests (MRT) for comparison of digital PCR experiments. We enriched our MRT framework with computation of simultaneous confidence intervals suitable for comparing multiple dPCR runs. The evaluation of both statistical methods support that MRT is faster and more robust for dPCR experiments performed in large scale. Our theoretical results were confirmed by the analysis of dPCR measurements of dilution series. Both methods were implemented in the dpcR package (v. 0.2) for the open source R statistical computing environment. PMID:27551672

  5. Digital PCR for detection of citrus pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Assessment of the real-time PCR and different digital PCR platforms for DNA quantification.

    PubMed

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca

    2016-01-01

    Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method. PMID:26521179

  7. Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations.

    PubMed

    Kraytsberg, Yevgenya; Khrapko, Konstantin

    2005-09-01

    A critical review of the clone-by-clone approach to the analysis of complex spectra of somatic mutations is presented. The study of a priori unknown somatic mutations requires painstaking analysis of complex mixtures of multiple mutant and non-mutant DNA molecules. If mutant fractions are sufficiently high, these mixtures can be dissected by the cloning of individual DNA molecules and scanning of the individual clones for mutations (e.g., by sequencing). Currently, the majority of such cloning is performed using PCR fragments. However, post-PCR cloning may result in various PCR artifacts - PCR errors and jumping PCR - and preferential amplification of certain mutations. This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future. PMID:16149882

  8. Transgene Detection by Digital Droplet PCR

    PubMed Central

    Moser, Dirk A.; Braga, Luca; Raso, Andrea; Zacchigna, Serena; Giacca, Mauro; Simon, Perikles

    2014-01-01

    Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term ‘gene doping’ in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future. PMID:25375130

  9. Selecting optimal oligonucleotide primers for multiplex PCR.

    PubMed

    Nicodème, P; Steyaert, J M

    1997-01-01

    We investigate the problem of designing efficient multiplex PCR for medical applications. We show that the problem is NP-complete by transformation to the Multiple Choice Matching problem and give an efficient approximation algorithm. We developed this algorithm in a computer program that predicts which genomic regions may be simultaneously amplified by PCR. Practical use of the software shows that the method can treat 250 non-polymorphic loci with less than 5 simultaneous experiments. PMID:9322038

  10. A naked-eye colorimetric "PCR developer"

    NASA Astrophysics Data System (ADS)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated <10 billion reactions per year and a worldwide market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  11. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

    PubMed

    Gao, Song; He, Dan; Li, Guangquan; Zhang, Yanhua; Lv, Huiying; Wang, Li

    2016-09-15

    Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample. PMID:27393656

  12. A web server for performing electronic PCR

    PubMed Central

    Rotmistrovsky, Kirill; Jang, Wonhee; Schuler, Gregory D.

    2004-01-01

    ‘Electronic PCR’ (e-PCR) refers to a computational procedure that is used to search DNA sequences for sequence tagged sites (STSs), each of which is defined by a pair of primer sequences and an expected PCR product size. To gain speed, our implementation extracts short ‘words’ from the 3′ end of each primer and stores them in a sorted hash table that can be accessed efficiently during the search. One recent improvement is the use of overlapping discontinuous words to allow matches to be found despite the presence of a mismatch. Moreover, it is possible to allow gaps in the alignment between the primer and the sequence. The effect of these changes is to improve sensitivity without significantly affecting specificity. The new software provides a search mode using a query STS against a sequence database to augment the previously available mode using a query sequence against an STS database. Finally, e-PCR may now be used through a web service, with search results linked to other web resources such as the UniSTS database and the MapViewer genome browser. The e-PCR web server may be found at www.ncbi.nlm.nih.gov/sutils/e-pcr. PMID:15215361

  13. Chemically modified primers for improved multiplex PCR

    PubMed Central

    Shum, Jonathan; Paul, Natasha

    2009-01-01

    Multiplexed PCR, the amplification of multiple targets in a single reaction, presents a new set of challenges that further complicate more traditional PCR set-ups. These complications include a greater probability for non-specific amplicon formation and for imbalanced amplification of different targets, each of which can compromise quantification and detection of multiple targets. Despite these difficulties, multiplex PCR is frequently used in such applications as pathogen detection, RNA quantification, mutation analysis and now, next generation DNA sequencing. Herein, we investigate the utility of primers with one or two thermolabile 4-oxo-1-pentyl phosphotriester modifications in improving multiplex PCR performance. Initial endpoint and real-time analyses reveal a decrease in off-target amplification and subsequent increase in amplicon yield. Furthermore, the use of modified primers in multiplex set-ups revealed a greater limit of detection and more uniform amplification of each target as compared to unmodified primers. Overall, the thermolabile modified primers present a novel and exciting avenue in improving multiplex PCR performance. PMID:19258004

  14. PCR+ In Diesel Fuels and Emissions Research

    SciTech Connect

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  15. Diagnostic PCR of dermatophytes--an overview.

    PubMed

    Gräser, Yvonne; Czaika, Viktor; Ohst, Torsten

    2012-10-01

    The prevalence of onychomycosis is increasing steadily, sevenfold alone in the US within the last twenty years. An important aspect in this development is the demographic development of the human population of the industrial countries like Germany. A fast and accurate laboratory diagnosis is essential for successful treatment because 50% of the cases are misdiagnosed when relying on the clinical appearance only. The current diagnosis of dermatophytosis, based on direct microscopy and culture of the clinical specimen, is problematic given the lacking specificity of the former and the length of time needed for the latter. Molecular techniques can help to solve these problems. In recent years, a number of in-house PCR assays have been developed to identify dermatophytes directly from clinical specimens. Based on the "Mikrobiologisch-infektiologischen Qualitätsstandards (MIQ) für Nukleinsäure-Amplifikationstechniken" and the MIQE guideline (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) 11 studies are reviewed which were published between 2007 and 2010. The present article evaluates the quality of the PCR assays regarding false positive and false negative results due to contamination, PCR format, statistical analysis, and diagnostic performance of the studies. It shows that we are only at the beginning of providing high quality PCR diagnosis of dermatophytes. PMID:23013298

  16. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    EPA Science Inventory

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  17. Utility of PCR in diagnosing pulmonary tuberculosis.

    PubMed Central

    Bennedsen, J; Thomsen, V O; Pfyffer, G E; Funke, G; Feldmann, K; Beneke, A; Jenkins, P A; Hegginbothom, M; Fahr, A; Hengstler, M; Cleator, G; Klapper, P; Wilkins, E G

    1996-01-01

    At present, the rapid diagnosis of pulmonary tuberculosis rests with microscopy. However, this technique is insensitive and many cases of pulmonary tuberculosis cannot be initially confirmed. Nucleic acid amplification techniques are extremely sensitive, but when they are applied to tuberculosis diagnosis, they have given variable results. Investigators at six centers in Europe compared a standardized PCR system (Amplicor; Roche) against conventional culture methods. Defined clinical information was collected. Discrepant samples were retested, and inhibition assays and backup amplification with a separate primer pair were performed. Mycobacterium tuberculosis complex organisms were recovered from 654 (9.1%) of 7,194 samples and 293 (7.8%) of 3,738 patients. Four hundred fifty-two of the M. tuberculosis isolates from 204 patients were smear positive and culture positive. Among the culture-positive specimens, PCR had a sensitivity of 91.4% for smear-positive specimens and 60.9% for smear-negative specimens, with a specificity of 96.1%. Analysis of 254 PCR-positive, culture-negative specimens with discrepant results revealed that 130 were from patients with recently diagnosed tuberculosis and 94 represented a presumed laboratory error. Similar analysis of 118 PCR-negative, culture-positive specimens demonstrated that 27 discrepancies were due to presumed uneven aliquot distribution and 11 were due to presumed laboratory error; PCR inhibitors were detected in 8 specimens. Amplicor enables laboratories with little previous experience with nucleic acid amplification to perform PCR. Disease in more than 60% of the patients with tuberculosis with smear-negative, culture-positive specimens can be diagnosed at the time of admission, and potentially all patients with smear-positive specimens can immediately be confirmed as being infected with M. tuberculosis, leading to improved clinical management. PMID:8735089

  18. PCR microfluidic devices for DNA amplification.

    PubMed

    Zhang, Chunsun; Xu, Jinliang; Ma, Wenli; Zheng, Wenling

    2006-01-01

    The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055-1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92-98; Schneegabeta, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101-121; deMello, A.J. DNA amplification: does 'small' really mean 'efficient'? Lab Chip 2001; 1: 24N-29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77-87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28-29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820-825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such

  19. Real-time PCR: Advanced technologies and applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. PCR AS A DIAGNOSTIC TOOL FOR BRUCELLOSIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Numerous PCR-based assays have been developed for the identification of Brucella to improve diagnostic capabilities. Collectively, the repertoire of assays addresses several aspects of the diagnostic process. For some purposes, the simple identification of Brucella is adequate (e.g. diagnosis of ...

  1. 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…

  2. Handheld real-time PCR device.

    PubMed

    Ahrberg, Christian D; Ilic, Bojan Robert; Manz, Andreas; Neužil, Pavel

    2016-02-01

    Here we report one of the smallest real-time polymerase chain reaction (PCR) systems to date with an approximate size of 100 mm × 60 mm × 33 mm. The system is an autonomous unit requiring an external 12 V power supply. Four simultaneous reactions are performed in the form of virtual reaction chambers (VRCs) where a ≈200 nL sample is covered with mineral oil and placed on a glass cover slip. Fast, 40 cycle amplification of an amplicon from the H7N9 gene was used to demonstrate the PCR performance. The standard curve slope was -3.02 ± 0.16 cycles at threshold per decade (mean ± standard deviation) corresponding to an amplification efficiency of 0.91 ± 0.05 per cycle (mean ± standard deviation). The PCR device was capable of detecting a single deoxyribonucleic acid (DNA) copy. These results further suggest that our handheld PCR device may have broad, technologically-relevant applications extending to rapid detection of infectious diseases in small clinics. PMID:26753557

  3. 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. PMID:21445907

  4. Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.

    PubMed

    Mock, Ulrike; Hauber, Ilona; Fehse, Boris

    2016-03-01

    Genome editing using designer nucleases such as transcription activator-like effector nucleases (TALENs) or clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 nucleases is an emerging technology in basic and applied research. Whereas the application of editing tools, namely CRISPR-Cas9, has recently become very straightforward, quantification of resulting gene knockout rates still remains a bottleneck. This is particularly true if the product of a targeted gene is not easily detectable. To address this problem, we devised a novel gene-editing frequency digital PCR (GEF-dPCR) technique. GEF-dPCR exploits two differently labeled probes that are placed within one amplicon at the gene-editing target site to simultaneously detect wild-type and nonhomologous end-joining (NHEJ)-affected alleles. Taking advantage of the principle of dPCR, this enables concurrent quantification of edited and wild-type alleles in a given sample. We propose that our method is optimal for the monitoring of gene-edited cells in vivo, e.g., in clinical settings. Here we describe preparation, design of primers and probes, and setup and analysis of GEF-dPCR. The setup of GEF-dPCR requires up to 2 weeks (depending on the starting point); once the dPCR has been established, the protocol for sample analysis takes <1 d. PMID:26914317

  5. A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis.

    PubMed

    Funes-Huacca, Maribel E; Opel, Kerry; Thompson, Robyn; McCord, Bruce R

    2011-04-01

    In this paper we compare the effects of three representative PCR inhibitors using quantitative PCR (qPCR) and multiplex STR amplification in order to determine the effect of inhibitor concentration on allele dropout and to develop better ways to interpret forensic DNA data. We have used humic acid, collagen and calcium phosphate at different concentrations to evaluate the profiles of alleles inhibited in these amplifications. These data were correlated with previously obtained results from quantitative PCR including melt curve effects, efficiency changes and cycle threshold (Ct) values. Overall, the data show that there are two competing processes that result from PCR inhibition. The first process is a general loss of larger alleles. This appears to occur with all inhibitors. The second process is more sequence specific and occurs when the inhibitor binds DNA, altering the cycle threshold and the melt curve. This sequence-specific inhibition results in patterns of allele loss that occur in addition to the overall loss of larger alleles. The data demonstrate the applicability of utilizing real-time PCR results to predict the presence of certain types of PCR inhibition in STR analysis. PMID:21462225

  6. Identification of bacterial plant pathogens using multilocus PCR and electrospray ionization-mass spectrometry (PCR/ESI-MS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR/electrospray ionization-mass spectrometry (PCR/ESI-MS, previously known as “TIGER”) utilizes PCR with broad range primers to amplify products from wide array of organisms within a taxonomic group, followed by analysis of PCR amplicons using mass spectrometry. Computer analysis of precise masses ...

  7. Rapid PCR thermocycling using microscale thermal convection.

    PubMed

    Muddu, Radha; Hassan, Yassin A; Ugaz, Victor M

    2011-01-01

    Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed(1). Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format. Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations(2-9). Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument. One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time(10,11). Here we describe results of our efforts to probe the full 3-D velocity and

  8. Clinical Utility of Droplet Digital PCR for Human Cytomegalovirus

    PubMed Central

    Sedlak, Ruth Hall; Cook, Linda; Cheng, Anqi; Magaret, Amalia

    2014-01-01

    Human cytomegalovirus (CMV) has historically been the major infectious cause of morbidity and mortality among patients receiving hematopoietic cell or organ transplant. Standard care in a transplant setting involves frequent monitoring of CMV viral load over weeks to months to determine when antiviral treatment may be required. Quantitative PCR (qPCR) is the standard molecular diagnostic method for monitoring. Recently, digital PCR (dPCR) has shown promise in viral diagnostics, although current dPCR systems have lower throughput than qPCR systems. Here, we compare qPCR and droplet digital PCR (ddPCR) for CMV detection in patient plasma samples. Droplet digital PCR exhibits increased precision over qPCR at viral loads of ≥4 log10 with equivalent sensitivity. However, retrospective analysis of longitudinal samples from transplant patients with CMV viral loads near therapeutic thresholds did not provide evidence that the improved precision of ddPCR would be of clinical benefit. Given the throughput advantages of current qPCR systems, a widespread switch to dPCR for CMV monitoring would appear premature. PMID:24871214

  9. The Next-Generation PCR-Based Quantification Method for Ambient Waters: Digital PCR.

    PubMed

    Cao, Yiping; Griffith, John F; Weisberg, Stephen B

    2016-01-01

    Real-time quantitative PCR (qPCR) is increasingly being used for ambient water monitoring, but development of digital polymerase chain reaction (digital PCR) has the potential to further advance the use of molecular techniques in such applications. Digital PCR refines qPCR by partitioning the sample into thousands to millions of miniature reactions that are examined individually for binary endpoint results, with DNA density calculated from the fraction of positives using Poisson statistics. This direct quantification removes the need for standard curves, eliminating the labor and materials associated with creating and running standards with each batch, and removing biases associated with standard variability and mismatching amplification efficiency between standards and samples. Confining reactions and binary endpoint measurements to small partitions also leads to other performance advantages, including reduced susceptibility to inhibition, increased repeatability and reproducibility, and increased capacity to measure multiple targets in one analysis. As such, digital PCR is well suited for ambient water monitoring applications and is particularly advantageous as molecular methods move toward autonomous field application. PMID:27460373

  10. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri.

    PubMed

    Zhao, Yun; Xia, Qingyan; Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  11. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri

    PubMed Central

    Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  12. PCR identification of Mycobacterium bovis BCG.

    PubMed Central

    Talbot, E A; Williams, D L; Frothingham, R

    1997-01-01

    The attenuated bacillus Calmette-Guérin (BCG) vaccine strain is derived from a virulent strain of Mycobacterium bovis. BCG is difficult to differentiate from other strains of M. bovis and other members of the M. tuberculosis complex by conventional methods. Recently, a genomic region designated RD1 was found to be present in all virulent M. bovis and M. tuberculosis strains tested but deleted from all BCG strains tested. With this information, a multiplex PCR method was developed to detect the RD1 deletion. A large collection of BCG and other M. tuberculosis complex strains from diverse host and geographic origins was tested. RD1 was deleted in 23 of 23 BCG strains. RD1 was present in 129 of 129 other M. tuberculosis complex strains. This multiplex PCR method can be used as a tool for the rapid and specific identification of BCG. PMID:9041390

  13. Replaceable Microfluidic Cartridges for a PCR Biosensor

    NASA Technical Reports Server (NTRS)

    Francis, Kevin; Sullivan, Ron

    2005-01-01

    The figure depicts a replaceable microfluidic cartridge that is a component of a miniature biosensor that detects target deoxyribonucleic acid (DNA) sequences. The biosensor utilizes (1) polymerase chain reactions (PCRs) to multiply the amount of DNA to be detected, (2) fluorogenic polynucleotide probe chemicals for labeling the target DNA sequences, and (3) a high-sensitivity epifluorescence-detection optoelectronic subsystem. Microfluidics is a relatively new field of device development in which one applies techniques for fabricating microelectromechanical systems (MEMS) to miniature systems for containing and/or moving fluids. Typically, microfluidic devices are microfabricated, variously, from silicon or polymers. The development of microfluidic devices for applications that involve PCR and fluorescence-based detection of PCR products poses special challenges

  14. Digital PCR analysis of circulating nucleic acids.

    PubMed

    Hudecova, Irena

    2015-10-01

    Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date. PMID:25828047

  15. Clostridium difficile PCR Ribotypes in Calves, Canada

    PubMed Central

    Stämpfli, Henry R.; Duffield, Todd; Peregrine, Andrew S.; Trotz-Williams, Lise A.; Arroyo, Luis G.; Brazier, Jon S.; Weese, J. Scott

    2006-01-01

    We investigated Clostridium difficile in calves and the similarity between bovine and human C. difficile PCR ribotypes by conducting a case-control study of calves from 102 dairy farms in Canada. Fecal samples from 144 calves with diarrhea and 134 control calves were cultured for C. difficile and tested with an ELISA for C. difficile toxins A and B. C. difficile was isolated from 31 of 278 calves: 11 (7.6%) of 144 with diarrhea and 20 (14.9%) of 134 controls (p = 0.009). Toxins were detected in calf feces from 58 (56.8%) of 102 farms, 57 (39.6%) of 144 calves with diarrhea, and 28 (20.9%) of 134 controls (p = 0.0002). PCR ribotyping of 31 isolates showed 8 distinct patterns; 7 have been identified in humans, 2 of which have been associated with outbreaks of severe disease (PCR types 017 and 027). C. difficile may be associated with calf diarrhea, and cattle may be reservoirs of C. difficile for humans. PMID:17283624

  16. Quantitative PCR marker genes for endometrial adenocarcinoma.

    PubMed

    Kölbl, Alexandra C; Victor, Lisa-Marie; Birk, Amelie E; Jeschke, Udo; Andergassen, Ulrich

    2016-09-01

    Endometrial adenocarcinoma is a common malignancy in women worldwide, with formation of remote metastasis occurring following oncological treatment. Circulating tumor cells (CTCs) are regarded to be the origin of haematogenous metastasis formation. The present study aimed to identify suitable marker genes using a quantitative polymerase chain reaction (qPCR) approach to detect CTCs from blood samples of patients with endometrial carcinoma. Therefore, RNA was isolated from endometrial adenocarcinoma cell lines and from healthy endometrial tissue and reverse transcribed to cDNA, which was then used in qPCR on a number of marker genes. Cytokeratin 19 and claudin 4 were identified as suitable marker genes for CTCs in endometrial adenocarcinoma, due to their high expression in the majority of the cell lines investigated. The expression values of the genes examined varied widely between the different cell lines, which is similar to the variation in the patient samples. Therefore, the necessity for a set of genes for CTC detection and not one single marker gene is demonstrated. qPCR is a fast, cost‑efficient and easy to perform technique, which may be used in the detection of CTCs. Investigation of the occurrence of CTCs in cancer patients would aid in the prevention of metastasis and thereby refine treatment. PMID:27431566

  17. TqPCR: A Touchdown qPCR Assay with Significantly Improved Detection Sensitivity and Amplification Efficiency of SYBR Green qPCR

    PubMed Central

    Zhang, Qian; Wang, Jing; Deng, Fang; Yan, Zhengjian; Xia, Yinglin; Wang, Zhongliang; Ye, Jixing; Deng, Youlin; Zhang, Zhonglin; Qiao, Min; Li, Ruifang; Denduluri, Sahitya K.; Wei, Qiang; Zhao, Lianggong; Lu, Shun; Wang, Xin; Tang, Shengli; Liu, Hao; Luu, Hue H.; Haydon, Rex C.; He, Tong-Chuan; Jiang, Li

    2015-01-01

    The advent of fluorescence-based quantitative real-time PCR (qPCR) has revolutionized the quantification of gene expression analysis in many fields, including life sciences, agriculture, forensic science, molecular diagnostics, and medicine. While SYBR Green-based qPCR is the most commonly-used platform due to its inexpensive nature and robust chemistry, quantifying the expression of genes with low abundance or RNA samples extracted from highly restricted or limited sources can be challenging because the detection sensitivity of SYBR Green-based qPCR is limited. Here, we develop a novel and effective touchdown qPCR (TqPCR) protocol by incorporating a 4-cycle touchdown stage prior to the quantification amplification stage. Using the same cDNA templates, we find that TqPCR can reduce the average Cq values for Gapdh, Rps13, and Hprt1 reference genes by 4.45, 5.47, and 4.94 cycles, respectively, when compared with conventional qPCR; the overall average Cq value reduction for the three reference genes together is 4.95. We further find that TqPCR can improve PCR amplification efficiency and thus increase detection sensitivity. When the quantification of Wnt3A-induced target gene expression in mesenchymal stem cells is analyzed, we find that, while both conventional qPCR and TqPCR can detect the up-regulation of the relatively abundant target Axin2, only TqPCR can detect the up-regulation of the lowly-expressed targets Oct4 and Gbx2. Finally, we demonstrate that the MRQ2 and MRQ3 primer pairs derived from mouse reference gene Tbp can be used to validate the RNA/cDNA integrity of qPCR samples. Taken together, our results strongly suggest that TqPCR may increase detection sensitivity and PCR amplification efficiency. Overall, TqPCR should be advantageous over conventional qPCR in expression quantification, especially when the transcripts of interest are lowly expressed, and/or the availability of total RNA is highly restricted or limited. PMID:26172450

  18. Viral diagnostics in the era of digital PCR

    PubMed Central

    Sedlak, Ruth Hall; Jerome, Keith R.

    2012-01-01

    Unlike quantitative PCR (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR.Here we review current literature that demonstrates dPCR’s potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection. PMID:23182074

  19. Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR).

    PubMed

    Floren, C; Wiedemann, I; Brenig, B; Schütz, E; Beck, J

    2015-04-15

    Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems. PMID:25466124

  20. 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.

  1. Multiplex PCR serogrouping of Listeria monocytogenes isolated in Japan.

    PubMed

    Shimojima, Yukako; Ida, Miki; Nishino, Yukari; Ishitsuka, Rie; Kuroda, Sumiyo; Hirai, Akihiko; Sadamasu, Kenji; Nakama, Akiko; Kai, Akemi

    2016-04-01

    PCR serogrouping methods were used to examine strains of L. monocytogenes isolated in Japan. Among 187 strains, 99.5% were classified into 4 PCR serogroups corresponding to conventional serotypes. Only one isolate had a new PCR profile, which may be a variant of serogroup IVb. PMID:26537550

  2. Multiplex PCR serogrouping of Listeria monocytogenes isolated in Japan

    PubMed Central

    SHIMOJIMA, Yukako; IDA, Miki; NISHINO, Yukari; ISHITSUKA, Rie; KURODA, Sumiyo; HIRAI, Akihiko; SADAMASU, Kenji; NAKAMA, Akiko; KAI, Akemi

    2015-01-01

    PCR serogrouping methods were used to examine strains of L. monocytogenes isolated in Japan. Among 187 strains, 99.5% were classified into 4 PCR serogroups corresponding to conventional serotypes. Only one isolate had a new PCR profile, which may be a variant of serogroup IVb. PMID:26537550

  3. Determining Fungi rRNA Copy Number by PCR

    EPA Science Inventory

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

  4. Quantitative Real-Time PCR (qPCR) Workflow for Analyzing Staphylococcus aureus Gene Expression.

    PubMed

    Lewis, April M; Rice, Kelly C

    2016-01-01

    Quantitative real-time polymerase chain reaction (qPCR) is a sensitive tool that can be used to quantify and compare the amount of specific RNA transcripts between different biological samples. This chapter describes the use of a "two-step" qPCR method to calculate the relative fold change of expression of genes of interest in S. aureus. Using this work-flow, cDNA is synthesized from RNA templates (previously checked for the absence of significant genomic DNA contamination) using a cocktail of random primers and reverse-transcriptase enzyme. The cDNA pools generated can then be assessed for expression of specific genes of interest using SYBR Green-based qPCR and quantification of relative fold-change expression. PMID:25646613

  5. Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

    PubMed Central

    O'Sullivan, Matthew V. N.; Zhou, Fei; Sintchenko, Vitali; Kong, Fanrong; Gilbert, Gwendolyn L.

    2011-01-01

    Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific. Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms. The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane. PMID:21847083

  6. DNA fingerprinting of medically important microorganisms by use of PCR.

    PubMed Central

    van Belkum, A

    1994-01-01

    Selected segments of any DNA molecule can be amplified exponentially by PCR. This technique provides a powerful tool to detect and identify minimal numbers of microorganisms. PCR is applicable both in diagnosis and in epidemiology. By amplification of hypervariable DNA domains, differences can be detected even among closely related strains. PCR fingerprinting is a valuable tool for medical microbiologists, epidemiologists, and microbial taxonomists. The current state of PCR-mediated genotyping is reviewed, and a comparison with conventional molecular typing methods is included. Because of its speed and versatility, PCR fingerprinting will play an important role in microbial genetics, epidemiology, and systematics. Images PMID:8055466

  7. Denoising PCR-amplified metagenome data

    PubMed Central

    2012-01-01

    Background PCR amplification and high-throughput sequencing theoretically enable the characterization of the finest-scale diversity in natural microbial and viral populations, but each of these methods introduces random errors that are difficult to distinguish from genuine biological diversity. Several approaches have been proposed to denoise these data but lack either speed or accuracy. Results We introduce a new denoising algorithm that we call DADA (Divisive Amplicon Denoising Algorithm). Without training data, DADA infers both the sample genotypes and error parameters that produced a metagenome data set. We demonstrate performance on control data sequenced on Roche’s 454 platform, and compare the results to the most accurate denoising software currently available, AmpliconNoise. Conclusions DADA is more accurate and over an order of magnitude faster than AmpliconNoise. It eliminates the need for training data to establish error parameters, fully utilizes sequence-abundance information, and enables inclusion of context-dependent PCR error rates. It should be readily extensible to other sequencing platforms such as Illumina. PMID:23113967

  8. In silico PCR primer designing and validation.

    PubMed

    Kumar, Anil; Chordia, Nikita

    2015-01-01

    Polymerase chain reaction (PCR) is an enzymatic reaction whose efficiency and sensitivity largely depend on the efficiency of the primers that are used for the amplification of a concerned gene/DNA fragment. Selective amplification of nucleic acid molecules initially present in minute quantities provides a powerful tool for analyzing nucleic acids. In silico method helps in designing primers. There are various programs available for PCR primer design. Here we described designing of primers using web-based tools like "Primer3" and "Web Primer". For designing the primer, DNA template sequence is required that can be taken from any of the available sequence databases, e.g., RefSeq database. The in silico validation can be carried out using BLAST tool and Gene Runner software, which check their efficiency and specificity. Thereafter, the primers designed in silico can be validated in the wet lab. After that, these validated primers can be synthesized for use in the amplification of concerned gene/DNA fragment. PMID:25697657

  9. An evaluation of direct PCR amplification

    PubMed Central

    Hall, Daniel E.; Roy, Reena

    2014-01-01

    Aim To generate complete DNA profiles from blood and saliva samples deposited on FTA® and non-FTA® paper substrates following a direct amplification protocol. Methods Saliva samples from living donors and blood samples from deceased individuals were deposited on ten different FTA® and non-FTA® substrates. These ten paper substrates containing body fluids were kept at room temperature for varying lengths of time ranging from one day to approximately one year. For all assays in this research, 1.2 mm punches were collected from each substrate containing one type of body fluid and amplified with reagents provided in the nine commercial polymerase chain reaction (PCR) amplification kits. The substrates were not subjected to purification reagent or extraction buffer prior to amplification. Results Success rates were calculated for all nine amplification kits and all ten substrates based on their ability to yield complete DNA profiles following a direct amplification protocol. Six out of the nine amplification kits, and four out of the ten paper substrates had the highest success rates overall. Conclusion The data show that it is possible to generate complete DNA profiles following a direct amplification protocol using both standard (non-direct) and direct PCR amplification kits. The generation of complete DNA profiles appears to depend more on the success of the amplification kit rather than the than the FTA®- or non-FTA®-based substrates. PMID:25559837

  10. Accurate quantification of supercoiled DNA by digital PCR.

    PubMed

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  11. Accurate quantification of supercoiled DNA by digital PCR

    PubMed Central

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  12. Digital Droplet PCR: CNV Analysis and Other Applications.

    PubMed

    Mazaika, Erica; Homsy, Jason

    2014-01-01

    Digital droplet PCR (ddPCR) is an assay that combines state-of-the-art microfluidics technology with TaqMan-based PCR to achieve precise target DNA quantification at high levels of sensitivity and specificity. Because quantification is achieved without the need for standard assays in an easy to interpret, unambiguous digital readout, ddPCR is far simpler, faster, and less error prone than real-time qPCR. The basic protocol can be modified with minor adjustments to suit a wide range of applications, such as CNV analysis, rare variant detection, SNP genotyping, and transcript quantification. This unit describes the ddPCR workflow in detail for the Bio-Rad QX100 system, but the theory and data interpretation are generalizable to any ddPCR system. PMID:25042719

  13. Rapid micro-PCR system for hepatitis C virus amplification

    NASA Astrophysics Data System (ADS)

    Lin, Yu-Cheng; Huang, Ming-Yuan; Young, Kung-Chia; Chang, Ting-Tsung; Wu, Ching-Yi

    2000-08-01

    A rapid micro-polymerase chain reaction ((mu) -PCR) system was integrated to amplify the complementary DNA (cDNA) molecules of hepatitis C virus (HCV). This system consists of a rapid thermal cycling system and a (mu) PCR chip fabricated by MEMS fabrication techniques. This rapid (mu) PCR system is verified by using serum samples from patients with chronic hepatitis C. The HCV amplicon of the rapid (mu) PCR system was analyzed by slab gel electrophoresis with separation of DNA marker in parallel. The (mu) PCR chip was fabricated on silicon wafer and Pyrex glass using photolithography, wet etching, and anodic bonding methods. Using silicon material to fabricate the raction well improves the temperature uniformity of sample and helps to reach the desired temperature faster. The rapid close loop thermal cycling system comprises power supplies, a thermal generator, a computer control PID controller, and a data acquisition subsystem. The thermoelectric (T.E.) cooler is used to work as the thermal generator and a heat sink by controlling the polarity of supplied power. The (mu) PCR system was verified with traditional PCR equipment by loading the same PCR mixture with HCV cDNA and running the same cycle numbers, then comparing both HCV amplicon slab gel electrophoresis. The HCV amplicon from the (mu) PCR system shows a DNA fragment with an expected size of 145 base pairs. The background is lower with the (mu) PCR system than that with the tradional PCR equipment. Comparing the traditional PCR equipment which spends 5.5 hours for 30 cycles to gain the detectable amount of HCV amplicon in slab gel separation, this (mu) PCR system takes 30 minutes to finish the 30 thermal cycles. This work has demonstrated that this rapid (mu) PCR system can provide rapid heat generation and dissipation, improved temperature uniformity in DNA amplification.

  14. 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

  15. 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

  16. A quadruplex PCR (qxPCR) assay for adulteration in dairy products.

    PubMed

    Agrimonti, Caterina; Pirondini, Andrea; Marmiroli, Marta; Marmiroli, Nelson

    2015-11-15

    This study describes the development of a quadruplex quantitative Real Time PCR (qxPCR) based on SYBR®GreenER chemistry, for rapid identification of DNA of cow, goat, sheep and buffalo in dairy products, and for quantification of cow DNA in these products. The platform was applied to: (i) mixes of milks at fixed percentages; (ii) cheeses prepared with the same mixes; (iii) commercial dairy products. The methodology enabled the detection of DNA from cow in mixes of milk and cheeses with a limit of detection (LOD) of 0.1%. When applied to commercial dairy products the qxPCR gave results comparable with each single-plex Real Time PCR. A good correlation (R(2)>0.9) between peaks' area of derivative of melting curves of amplicons and percentages of cow milk in milk mixes and cheeses, allows for an estimation of cow DNA in a dynamic range varying from 0.1-5% to 1-25%. PMID:25976998

  17. 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.

  18. Identification of Actinobacillus actinomycetemcomitans in subgingival plaque by PCR.

    PubMed Central

    Flemmig, T F; Rüdiger, S; Hofmann, U; Schmidt, H; Plaschke, B; Strätz, A; Klaiber, B; Karch, H

    1995-01-01

    The purpose of this study was to assess the sensitivity and specificity of the PCR in detecting Actinobacillus actinomycetemcomitans. The PCR's detection capability was compared with those of three other methods: culture-enhanced PCR (CE-PCR), colony hybridization (CH), and conventional culture with presumptive biochemical identification. A 285-bp stretch of the leukotoxin gene lktA of A. actinomycetemcomitans was amplified by PCR with primers TT-15 and TT-16. For CH, the PCR product was labeled with digoxigenin and used as a hybridization probe. Nucleotide sequence analysis of the PCR product of A. actinomycetemcomitans 1D4 and 1664 and three clinical isolates revealed complete homology among the tested strains, with only one base substitution (at position 1344) in comparison with the published sequence. With artificially infected subgingival plaque, the detection limit of PCR for A. actinomycetemcomitans was 10(3) CFU/ml of plaque suspension. Culturing subgingival plaque on tryptic soy-serum-bacitracin-vancomycin agar prior to PCR (CE-PCR) improved the limit of detection to 10(2) CFU/ml. Analysis of subgingival plaque samples from 35 patients with periodontal disease and 10 periodontally healthy subjects revealed that CE-PCR and CH had the highest overall rate of A. actinomycetemcomitans detection (both 58%), followed by PCR and culture (both 42%). With CH as the "gold standard", the sensitivities of CE-PCR, PCR, and culture were 88, 65, and 58%, respectively; the specificities were 84, 89, and 79%, respectively. The CE-PCR provided acceptable positive and negative predictive values (> or = 70%) when the prevalence of A. actinomycetemcomitans varied between 30 and 70%. PCR alone provided comparable predictive values over a narrower range of prevalence rates (30 to 50%), while culture did not afford acceptable predictive values at any prevalence rate. PCR and CE-PCR were found to be superior to culture with presumptive biochemical identification and should be the

  19. STS-102 MPLM Leonardo moves into PCR

    NASA Technical Reports Server (NTRS)

    2001-01-01

    KENNEDY SPACE CENTER, Fla. -- In the payload changeout room on the Rotating Service Structure, Launch Pad 39B, workers move the Multi-Purpose Logistics Module Leonardo out of the payload canister. From the PCR Leonardo then will be transferred into Space Shuttle Discovery'''s payload bay. One of Italy'''s major contributions to the International Space Station program, Leonardo is a reusable logistics carrier. It is the primary delivery system used to resupply and return Station cargo requiring a pressurized environment. Leonardo is the primary payload on mission STS-102 and will deliver up to 10 tons of laboratory racks filled with equipment, experiments and supplies for outfitting the newly installed U.S. Laboratory Destiny. STS-102 is scheduled to launch March 8 at 6:45 a.m. EST.

  20. Reference genes in real-time PCR.

    PubMed

    Kozera, Bartłomiej; Rapacz, Marcin

    2013-11-01

    This paper aims to discuss various aspects of the use of reference genes in qPCR technique used in the thousands of present studies. Most frequently, these are housekeeping genes and they must meet several criteria so that they can lay claim to the name. Lots of papers report that in different conditions, for different organisms and even tissues the basic assumption—the constant level of the expression is not maintained for many genes that seem to be perfect candidates. Moreover, their transcription can not be affected by experimental factors. Sounds simple and clear but a great number of designed protocols and lack of consistency among them brings confusion on how to perform experiment properly. Since during selection of the most stable normalizing gene we can not use any reference gene, different ways and algorithms for their selection were developed. Such methods, including examples of best normalizing genes in some specific cases and possible mistakes are presented based on available sources. Numerous examples of reference genes applications, which are usually in too few numbers in relevant articles not allowing to make a solid fundament for a reader, will be shown along with instructive compilations to make an evidence for presented statements and an arrangement of future qPCR experiments. To include all the pitfalls and problems associated with the normalization methods there is no way not to begin from sample preparation and its storage going through candidate gene selection, primer design and statistical analysis. This is important because numerous short reviews available cover the topic only in lesser extent at the same time giving the reader false conviction of complete topic recognition. PMID:24078518

  1. Diagnostic utility of droplet digital PCR for HIV reservoir quantification.

    PubMed

    Trypsteen, Wim; Kiselinova, Maja; Vandekerckhove, Linos; De Spiegelaere, Ward

    2016-01-01

    Quantitative real-time PCR (qPCR) is implemented in many molecular laboratories worldwide for the quantification of viral nucleic acids. However, over the last two decades, there has been renewed interest in the concept of digital PCR (dPCR) as this platform offers direct quantification without the need for standard curves, a simplified workflow and the possibility to extend the current detection limit. These benefits are of great interest in terms of the quantification of low viral levels in HIV reservoir research because changes in the dynamics of residual HIV reservoirs will be important to monitor HIV cure efforts. Here, we have implemented a systematic literature screening and text mining approach to map the use of droplet dPCR (ddPCR) in the context of HIV quantification. In addition, several technical aspects of ddPCR were compared with qPCR: accuracy, sensitivity, precision and reproducibility, to determine its diagnostic utility. We have observed that ddPCR was used in different body compartments in multiple HIV-1 and HIV-2 assays, with the majority of reported assays focusing on HIV-1 DNA-based applications (i.e. total HIV DNA). Furthermore, ddPCR showed a higher accuracy, precision and reproducibility, but similar sensitivity when compared to qPCR due to reported false positive droplets in the negative template controls with a need for standardised data analysis (i.e. threshold determination). In the context of a low level of detection and HIV reservoir diagnostics, ddPCR can offer a valid alternative to qPCR-based assays but before this platform can be clinically accredited, some remaining issues need to be resolved. PMID:27482456

  2. IDH1 mutation detection by droplet digital PCR in glioma.

    PubMed

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; To, Shing shun Tony; Chen, Zhong-ping

    2015-11-24

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation. PMID:26485760

  3. IDH1 mutation detection by droplet digital PCR in glioma

    PubMed Central

    Wang, Jing; Zhao, Yi-ying; Li, Jian-feng; Guo, Cheng-cheng; Chen, Fu-rong; Su, Hong-kai; Zhao, Hua-fu; Long, Ya-kang; Shao, Jian-yong; Tony To, Shing-shun; Chen, Zhong-ping

    2015-01-01

    Glioma is the most frequent central nervous system tumor in adults. The overall survival of glioma patients is disappointing, mostly due to the poor prognosis of glioblastoma (Grade IV glioma). Isocitrate dehydrogenase (IDH) is a key factor in metabolism and catalyzes the oxidative decarboxylation of isocitrate. Mutations in IDH genes are observed in over 70% of low-grade gliomas and some cases of glioblastoma. As the most frequent mutation, IDH1(R132H) has been served as a predictive marker of glioma patients. The recently developed droplet digital PCR (ddPCR) technique generates a large amount of nanoliter-sized droplets, each of which carries out a PCR reaction on one template. Therefore, ddPCR provides high precision and absolute quantification of the nucleic acid target, with wide applications for both research and clinical diagnosis. In the current study, we collected 62 glioma tissue samples (Grade II to IV) and detected IDH1 mutations by Sanger direct sequencing, ddPCR, and quantitative real-time PCR (qRT-PCR). With the results from Sanger direct sequencing as the standard, the characteristics of ddPCR were compared with qRT-PCR. The data indicated that ddPCR was much more sensitive and much easier to interpret than qRT-PCR. Thus, we demonstrated that ddPCR is a reliable and sensitive method for screening the IDH mutation. Therefore, ddPCR is able to applied clinically in predicting patient prognosis and selecting effective therapeutic strategies. Our data also supported that the prognosis of Grade II and III glioma was better in patients with an IDH mutation than in those without mutation. PMID:26485760

  4. DNA rehybridization during PCR: the 'Cot effect' and its consequences.

    PubMed

    Mathieu-Daudé, F; Welsh, J; Vogt, T; McClelland, M

    1996-06-01

    The rate of amplification of abundant PCR products generally declines faster than that of less abundant products in the same tube in the later cycles of PCR. As a consequence, differences in product abundance diminish as the number of PCR cycles increases. Rehybridization of PCR products which may interfere with primer binding or extension can explain this significant feature in late cycles. Rehybridization occurs with a half-time dependent on the reciprocal of the DNA concentration. Thus, if multiple PCR products are amplified in the same tube, reannealing occurs faster for the more abundant PCR products. In RT-PCR using an internal control, this results in a systematic bias against the more abundant of the two PCR products. In RNA fingerprinting by arbitrarily primed PCR (or differentially display of cDNAs), very large or absolute differences in the expression of a transcript between samples are preserved but smaller real differences may be gradually erased as the PCR reaction proceeds. Thus, this 'Cot effect' may systematically cause an underestimate of the true difference in starting template concentrations. However, differences in starting template concentrations will be better preserved in the less abundant PCR products. Furthermore, the slow down in amplification of abundant products will allow these rarer products to become more visible in the fingerprint which may, in turn, allow rarer cDNAs to be sampled more efficiently. In some applications, where the object is to stochiometrically amplify a mixture of nucleic acids, the bias against abundant PCR products can be partly overcome by limiting the number of PCR cycles and, thus, the concentration of the products. In other cases, abundance normalization at later cycles may be useful, such as in the production of normalized libraries. PMID:8668539

  5. Real-time cdPCR opens a window into events occurring in the first few PCR amplification cycles.

    PubMed

    Duewer, David L; Kline, Margaret C; Romsos, Erica L

    2015-12-01

    Polymerase chain reaction (PCR) end-point limiting dilution techniques, collectively termed "digital PCR (dPCR)", have been proposed as providing a potentially primary method for DNA quantification. We are evaluating several commercially available dPCR systems for use in certifying mass concentration in human genomic DNA reference materials. To better understand observed anomalies among results from chamber- and droplet-dPCR (cdPCR and ddPCR) systems, we have developed a graphical tool for evaluating and documenting the performance of PCR assays in real-time cdPCR systems: the ogive plot, the cumulative distribution of crossing threshold values. The ogive structure appears to embed information about early amplification events. We have successfully simulated ogives observed with different assays and reaction conditions using a four-stage amplification model parameterized by the probability of creating an intact 1) first generation "long" amplicon of indeterminate length from an original DNA target, 2) second generation defined-length amplicon from a long amplicon, and 3) defined-length amplicon from another defined-length amplicon. We are using insights from this model to optimize dPCR assay design and reaction conditions and to help validate assays proposed for use in value-assigning DNA reference materials. PMID:26438478

  6. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

    PubMed Central

    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-01-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells. PMID:25437051

  7. Optimized MOL-PCR for Characterization of Microbial Pathogens.

    PubMed

    Wuyts, Véronique; Roosens, Nancy H C; Bertrand, Sophie; Marchal, Kathleen; De Keersmaecker, Sigrid C J

    2016-01-01

    Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium. PMID:26742655

  8. Variation in copy number of the 28S rDNA of Aspergillus fumigatus measured by droplet digital PCR and analog quantitative real-time PCR.

    PubMed

    Alanio, Alexandre; Sturny-Leclère, Aude; Benabou, Marion; Guigue, Nicolas; Bretagne, Stéphane

    2016-08-01

    Droplet digital PCR (ddPCR) after DNA digestion yielded a 28S rDNA copy number of 61 to 86 copies/genome when testing 10 unrelated Aspergillus fumigatus isolates, higher than with quantitative PCR. Unfortunately, ddPCR after DNA digestion did not improve the sensitivity of our PCR assay when testing serum patients with invasive aspergillosis. PMID:27316653

  9. Loop-linker PCR: an advanced PCR technique for genome walking.

    PubMed

    Trinh, Quoclinh; Shi, Hui; Xu, Wentao; Hao, Junran; Luo, Yunbo; Huang, Kunlun

    2012-10-01

    In this article, we developed a novel PCR method, termed loop-linker PCR, to isolate flanking sequences in transgenic crops. The novelty of this approach is its use of a stem-loop structure to design a loop-linker adapter. The adapter is designed to form a nick site when ligated with restricted DNA. This modification not only can prevent the self-ligation of adapters but also promotes the elongation of the 3' end of the loop-linker adapter to generate a stem-loop structure in the ligation products. Moreover, the suppressive effect of the stem-loop structure decreases nonspecific amplification and increases the success rate of the approach; all extension products will suppress exponential amplification except from the ligation product that contains the specific primer binding site. Using this method, 442, 1830, 107, and 512 bp left border flanking sequences were obtained from the transgenic maizes LY038, DAS-59122-7, Event 3272, and the transgenic soybean MON89788, respectively. The experimental results demonstrated that loop-linker PCR is an efficient, reliable, and cost-effective method for identifying flanking sequences in transgenic crops and could be applied for other genome walking applications. PMID:23008115

  10. A simple, universal, efficient PCR-based gene synthesis method: sequential OE-PCR gene synthesis.

    PubMed

    Zhang, Pingping; Ding, Yingying; Liao, Wenting; Chen, Qiuli; Zhang, Huaqun; Qi, Peipei; He, Ting; Wang, Jinhong; Deng, Songhua; Pan, Tianyue; Ren, Hao; Pan, Wei

    2013-07-25

    Herein we present a simple, universal, efficient gene synthesis method based on sequential overlap extension polymerase chain reactions (OE-PCRs). This method involves four key steps: (i) the design of paired complementary 54-mer oligonucleotides with 18 bp overlaps, (ii) the utilisation of sequential OE-PCR to synthesise full-length genes, (iii) the cloning and sequencing of four positive T-clones of the synthesised genes and (iv) the resynthesis of target genes by OE-PCR with correct templates. Mispriming and secondary structure were found to be the principal obstacles preventing successful gene synthesis and were easily identified and solved in this method. Compensating for the disadvantages of being laborious and time-consuming, this method has many attractive advantages, such as the ability to guarantee successful gene synthesis in most cases and good allowance for Taq polymerase, oligonucleotides, PCR conditions and a high error rate. Thus, this method provides an alternative tool for individual gene synthesis without strict needs of the high-specialised experience. PMID:23597923

  11. Evaluation of PCR based coprodiagnosis of human opisthorchiasis.

    PubMed

    Stensvold, C R; Saijuntha, W; Sithithaworn, P; Wongratanacheewin, S; Strandgaard, H; Ornbjerg, N; Johansen, M V

    2006-01-01

    In this study, a recently developed PCR test for the detection of Opisthorchis viverrini in human faecal samples was evaluated using two parasitological methods as references. During a survey of foodborne trematodes (FBT) in the Vientiane Province, Lao PDR, 85 samples were collected and evaluated for FBT eggs by the Kato Katz (KK) technique, the formalin ethyl acetate concentration technique (FECT) and a PCR analysis for the distinction between O. viverrini and other FBT. The two parasitological methods did not differ in the ability of detecting FBT eggs, and a single KK reading was characterized by a sensitivity of 85% when compared to two FECT readings. The PCR tested positive only in cases where eggs had been demonstrated by parasitological examination. However, the PCR tested negative in some samples with very high egg counts. Demonstrating a PCR sensitivity of approximately 50% in samples with faecal egg counts>1000, the previously reported PCR sensitivity based on in vitro studies was not supported. It is believed that technical problems rather than diagnostic reference related issues were responsible for the relatively low PCR performance. Further studies should aim at optimizing DNA extraction and amplification, and future PCR evaluation should include specificity control such as the scanning electron microscopy of eggs in test samples or the expulsion of adult trematodes from PCR tested individuals. PMID:16253202

  12. Detection of virulence genes of Clostridium difficile by multiplex PCR.

    PubMed

    Antikainen, Jenni; Pasanen, Tanja; Mero, Sointu; Tarkka, Eveliina; Kirveskari, Juha; Kotila, Saara; Mentula, Silja; Könönen, Eija; Virolainen-Julkunen, Anni-Riitta; Vaara, Martti; Tissari, Päivi

    2009-08-01

    Clostridium difficile strains belonging to the PCR ribotype 027, pulse-field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027. PMID:19664132

  13. Detection and quantification of chimerism by droplet digital PCR.

    PubMed

    George, David; Czech, Juliann; John, Bobby; Yu, Min; Jennings, Lawrence J

    2013-01-01

    Accurate quantification of chimerism and microchimerism is proving to be increasingly valuable for hematopoietic cell transplantation as well as non-transplant conditions. However, methods that are available to quantify low-level chimerism lack accuracy. Therefore, we developed and validated a method for quantifying chimerism based on digital PCR technology. We demonstrate accurate quantification that far exceeds what is possible with analog qPCR down to 0.01% with the potential to go even lower. Also, this method is inherently more informative than qPCR. We expect the advantages of digital PCR will make it the preferred method for chimerism analysis. PMID:23974275

  14. Antigenic typing of canine parvovirus using differential PCR.

    PubMed

    Kaur, Gurpreet; Chandra, Mudit; Dwivedi, P N; Sharma, N S

    2014-12-01

    Canine parvovirus (CPV) is an enteric pathogen causing hemorrhagic enteritis in pups of 3-6 months of age and is mainly transmitted via feco-oral route. In the present study, a total of 85 animals rectal swabs suspected of CPV were tested using a PCR, nested PCR and a newly designed differential PCR. Using PCR 7 (8.23 %) animals were positive whereas 39 (45.88 %) were positive by using nested PCR and 40 (47.05 %) were positive for either one or more than one antigenic types of CPV using differential PCR. Using differential PCR it was found that CPV-2a and CPV-2b were the most prevailing antigenic types. Also it was found that dogs that were vaccinated too yielded positive CPV indicating a possible presence of additional CPV antigenic types. Thus, the primers used in differential PCR can be used in a single PCR reaction to detect various antigenic types of CPV. PMID:25674626

  15. Buoyancy-Driven Polymerase Chain Reaction (PCR) Devices

    SciTech Connect

    Ness, K D; Wheeler, E K; Benett, W; Stratton, P; Christian, A; Chen, A; Ortega, J; Weisgraber, T H; Goodson, K E

    2004-09-28

    Polymerase chain reaction (PCR) facilitates DNA detection by significantly increasing the concentration of specific DNA segments. A new class of PCR instruments uses a buoyancy-driven re-circulating flow to thermally cycle the DNA sample and benefits from reduced cycle times, low sample volumes, a miniaturized format, and low power consumption. This paper analyzes a specific buoyancy PCR device in a micro-channel ''race-track'' geometry to determine key parameters about PCR cycle times and other figures of merit as functions of device dimensions. The 1-D model balances the buoyancy driving force with frictional losses. A hydrostatic pressure imbalance concept is used between the left and right sides of the fluid loop to calculate the buoyancy driving force. Velocity and temperature distributions within the channels are determined from two-dimensional analysis of the channel section, with developing region effects included empirically through scaled values of the local Nusselt number. Good agreement between four independent verification steps validate the 1-D simulation approach: (1) analytical expressions for the thermal entrance length are compared against, (2) comparison with a full 3-D finite element simulation, (3) comparison with an experimental flow field characterization, and (4) calculation of the minimum PCR runtime required to get a positive PCR signal from the buoyancy-driven PCR device. The 1-D approach closely models an actual buoyancy-driven PCR device and can further be used as a rapid design tool to simulate buoyancy PCR flows and perform detailed design optimizations studies.

  16. Highly precise measurement of HIV DNA by droplet digital PCR.

    PubMed

    Strain, Matthew C; Lada, Steven M; Luong, Tiffany; Rought, Steffney E; Gianella, Sara; Terry, Valeri H; Spina, Celsa A; Woelk, Christopher H; Richman, Douglas D

    2013-01-01

    Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it. PMID:23573183

  17. Highly Precise Measurement of HIV DNA by Droplet Digital PCR

    PubMed Central

    Strain, Matthew C.; Lada, Steven M.; Luong, Tiffany; Rought, Steffney E.; Gianella, Sara; Terry, Valeri H.; Spina, Celsa A.; Woelk, Christopher H.; Richman, Douglas D.

    2013-01-01

    Deoxyribonucleic acid (DNA) of the human immunodeficiency virus (HIV) provides the most sensitive measurement of residual infection in patients on effective combination antiretroviral therapy (cART). Droplet digital PCR (ddPCR) has recently been shown to provide highly accurate quantification of DNA copy number, but its application to quantification of HIV DNA, or other equally rare targets, has not been reported. This paper demonstrates and analyzes the application of ddPCR to measure the frequency of total HIV DNA (pol copies per million cells), and episomal 2-LTR (long terminal repeat) circles in cells isolated from infected patients. Analysis of over 300 clinical samples, including over 150 clinical samples assayed in triplicate by ddPCR and by real-time PCR (qPCR), demonstrates a significant increase in precision, with an average 5-fold decrease in the coefficient of variation of pol copy numbers and a >20-fold accuracy improvement for 2-LTR circles. Additional benefits of the ddPCR assay over qPCR include absolute quantification without reliance on an external standard and relative insensitivity to mismatches in primer and probe sequences. These features make digital PCR an attractive alternative for measurement of HIV DNA in clinical specimens. The improved sensitivity and precision of measurement of these rare events should facilitate measurements to characterize the latent HIV reservoir and interventions to eradicate it. PMID:23573183

  18. Global RT-PCR and RT-qPCR Analysis of the mRNA Expression of the Human PTPome.

    PubMed

    Nunes-Xavier, Caroline E; Pulido, Rafael

    2016-01-01

    Comprehensive comparative gene expression analysis of the tyrosine phosphatase superfamily members (PTPome) under cell- or tissue-specific growth conditions may help to define their individual and specific role in physiology and disease. Semi-quantitative and quantitative PCR are commonly used methods to analyze and measure gene expression. Here, we describe technical aspects of PTPome mRNA expression analysis by semi-quantitative RT-PCR and quantitative RT-PCR (RT-qPCR). We provide a protocol for each method consisting in reverse transcription followed by PCR using a global platform of specific PTP primers. The chapter includes aspects from primer validation to the setup of the PTPome RT-qPCR platform. Examples are given of PTP-profiling gene expression analysis using a human breast cancer cell line upon long-term or short-term treatment with cell signaling-activation agents. PMID:27514798

  19. Quantification of Bacterial Transcripts during Infection Using Competitive Reverse Transcription-PCR (RT-PCR) and LightCycler RT-PCR

    PubMed Central

    Goerke, Christiane; Bayer, Manfred G.; Wolz, Christiane

    2001-01-01

    Bacteria have evolved sophisticated regulatory circuits to modulate their gene expression in response to disparate environments. In order to monitor bacterial gene expression and regulation in the host, methods for direct transcript analysis from clinical specimens are needed. For most bacterial infections, amplification of the mRNAs of interest is necessary due to the low numbers of cells present and the low levels of specific transcripts. Here we compare two methods of quantitative reverse transcription-PCR (RT-PCR)—competitive RT-PCR using a one-tube system followed by standard gel analysis and the real-time detection of PCR product formation by fluorescence resonance energy transfer technology using the LightCycler unit. We isolated Staphylococcus aureus RNA directly from clinical specimens obtained from cystic fibrosis patients with chronic S. aureus lung infection and from an animal model of foreign-body infection with no further cultivation of the bacteria. Competitive RT-PCR and LightCycler RT-PCR were tested for their ability to quantify the transcription of a constitutively expressed gyrase gene (gyr) and a highly regulated α-toxin gene (hla) of S. aureus. Reproducible results were obtained with both methods. A sensitivity of 104 (gyr) and 103 (hla) copies, respectively, was reached, which was sufficient for the quantification of transcripts during bacterial infection. Overall, the competitive RT-PCR is a robust technique which does not need special RNA purification. On the negative side, it is labor intensive and time consuming, thus limiting the numbers of samples which can be analyzed at a given time. LightCycler RT-PCR is very susceptible to even traces of inhibitors, but it allows high-throughput processing of samples. PMID:11238208

  20. ANIMAL DNA IN PCR REAGENTS PLAGUES ANCIENT DNA RESEARCH

    EPA Science Inventory

    Ancient DNA analysis is becoming widespread. These studies use polymerase chain reaction (PCR) to amplify minute quantities of heavily damaged template. Unusual steps are taken to achieve the sensitivity necessary to detect ancient DNA, including high-cycle PCR amplification targ...

  1. REAL-TIME PCR ASSAY DEVELOPMENT FOR MULTIPLE MAIZE PATHOGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This talk presents updates on the development of real-time PCR assays for two seedborne pathogens of maize, Pantoea (Erwinia) stewartii, the causal agent of Stewart's bacterial wilt, and Stenocarpella (Diplodia) maydis, the causal agent of Diplodia ear rot. We developed primers and a real-time PCR p...

  2. Interlaboratory Comparison of Quantitative PCR Test Results for Dehalococcoides

    EPA Science Inventory

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

  3. DNA probes and PCR for diagnosis of parasitic infections.

    PubMed Central

    Weiss, J B

    1995-01-01

    DNA probe and PCR-based assays to identify and detect parasites are technically complex; however, they have high sensitivity, directly detect parasites independent of the immunocompetence or previous clinical history of the patient, and can distinguish between organisms that are morphologically similar. Diagnosis of parasites is often based on direct detection by microscopy, which is insensitive and laborious and can lack specificity. Most PCR-based assays were more sensitive than DNA probe assays. The development of PCR-based diagnostic assays requires multiple steps following the initial selection of oligonucleotide primers and reporter probe. Generally, the ability to detect the DNA of one parasite was attained by PCR; however, advances in the preparation of samples for PCR (extraction of DNA while removing PCR inhibitors) will be required to achieve that sensitivity with human specimens. Preliminary PCR systems have been developed for many different parasites, yet few have been evaluated with a large number of clinical specimens and/or under field conditions. Those evaluations are essential for determination of clinical and field utility and performance and of the most appropriate application of the assay. Several situations in which PCR-based diagnosis will result in epidemiologic, medical, or public health advances have been identified. PMID:7704890

  4. Quantitative PCR Method for Diagnosis of Citrus Bacterial Canker†

    PubMed Central

    Cubero, J.; Graham, J. H.; Gottwald, T. R.

    2001-01-01

    For diagnosis of citrus bacterial canker by PCR, an internal standard is employed to ensure the quality of the DNA extraction and that proper requisites exist for the amplification reaction. The ratio of PCR products from the internal standard and bacterial target is used to estimate the initial bacterial concentration in citrus tissues with lesions. PMID:11375206

  5. Predicting Salmonella enterica serotypes by repetitive sequence-based PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Repetitive extragenic palindromic sequence-based PCR (rep-PCR) utilizing a semi-automated system, was evaluated as a method to determine Salmonella serotypes. A group of 216 Salmonella isolates belonging to 13 frequently isolated serotypes and one rarer serotype from poultry were used to create a D...

  6. Urine sample used for congenital toxoplasmosis diagnosis by PCR.

    PubMed Central

    Fuentes, I; Rodriguez, M; Domingo, C J; del Castillo, F; Juncosa, T; Alvar, J

    1996-01-01

    The diagnosis of toxoplasmosis in congenitally infected infants can be difficult; serology is unreliable, and diagnosis must be based on the combination of symptomatology and direct demonstration of the parasite. Four infants suspected of having Toxoplasma gondii infection were studied by serological analysis, tissue culture, and PCR determination. T. gondii was isolated from the urine of one patient. The parasite was detected by PCR in the blood and cerebrospinal fluid of three infants and in the urine in all patients. Because nested PCR proved to be a sensitive, relatively rapid, and specific method and because it can be applied to a variety of different clinical samples, PCR can be a valuable technique for the identification of T. gondii infections in children. The present study indicates that PCR examination of urine, a fluid never before used for diagnosis in this age group, may be valuable in diagnosing cases of congenital toxoplasmosis. PMID:8880481

  7. Engineered DNA polymerase improves PCR results for plastid DNA1

    PubMed Central

    Schori, Melanie; Appel, Maryke; Kitko, AlexaRae; Showalter, Allan M.

    2013-01-01

    • Premise of the study: Secondary metabolites often inhibit PCR and sequencing reactions in extractions from plant material, especially from silica-dried and herbarium material. A DNA polymerase that is tolerant to inhibitors improves PCR results. • Methods and Results: A novel DNA amplification system, including a DNA polymerase engineered via directed evolution for improved tolerance to common plant-derived PCR inhibitors, was evaluated and PCR parameters optimized for three species. An additional 31 species were then tested with the engineered enzyme and optimized protocol, as well as with regular Taq polymerase. • Conclusions: PCR products and high-quality sequence data were obtained for 96% of samples for rbcL and 79% for matK, compared to 29% and 21% with regular Taq polymerase. PMID:25202519

  8. Investigation of vesicular rashes for HSV and VZV by PCR.

    PubMed

    Beards, G; Graham, C; Pillay, D

    1998-03-01

    Vesicular fluid from rashes of 132 patients was tested by a multiplex PCR shown to be specific for herpes simplex virus (HSV) type 1 and 2, and varicella zoster virus (VZV) genomic DNA. The results were compared with those obtained by examination by electron microscopy and virus isolation by cell culture. The PCR did not differentiate between HSV 1 and 2. By PCR, 64 HSV infections and 53 VZV infections were identified, with presumed 100% sensitivity and specificity. Fifteen specimens tested negative by PCR, electron microscopy, and virus isolation for herpes viruses. The sensitivities of virus isolation and electron microscopy for detection of herpes simplex virus were 56% and 80%. For varicella zoster virus, the sensitivities of virus isolation and electron microscopy were 47% and 60%. These data illustrate the advantage of rapid PCR diagnosis of herpes simplex virus and varicella zoster virus in vesicle fluids. PMID:9515761

  9. A thermally baffled device for highly stabilized convective PCR.

    PubMed

    Chang, Hsiao-Fen Grace; Tsai, Yun-Long; Tsai, Chuan-Fu; Lin, Ching-Ko; Lee, Pei-Yu; Teng, Ping-Hua; Su, Chen; Jeng, Chien-Chung

    2012-05-01

    Rayleigh-Bénard convective PCR is a simple and effective design for amplification of DNA. Convective PCR is, however, extremely sensitive to environmental temperature fluctuations, especially when using small- diameter test tubes. Therefore, this method is inherently unstable with limited applications. Here, we present a convective PCR device that has been modified by adding thermal baffles. With this thermally baffled device the influence from fluctuations in environmental temperature were significantly reduced, even in a wind tunnel (1 m/s). The thermally baffled PCR instrument described here has the potential to be used as a low-cost, point-of-care device for PCR-based molecular diagnostics in the field. PMID:22241586

  10. Epidemiological typing of Stenotrophomonas (Xanthomonas) maltophilia by PCR.

    PubMed Central

    Chatelut, M; Dournes, J L; Chabanon, G; Marty, N

    1995-01-01

    We used two PCR methods for epidemiological typing of Stenotrophomonas (Xanthomonas) maltophilia with either arbitrary primers (random amplified polymorphic DNA) or enterobacterial repetitive intergenic consensus sequences as primers (ERIC-PCR). The analysis was performed with 38 isolates of S. maltophilia, comprising 9 nosocomial isolates from a burn unit, 20 other clinical isolates epidemiologically unrelated, and 9 isolates from one cystic fibrosis patient. Both methods indicated that all of the nosocomial episodes were independent. In contrast, the nine isolates from the cystic fibrosis patient were assigned to very closely related profiles, especially by ERIC-PCR. We conclude that random amplified polymorphic DNA and ERIC-PCR have comparable reproducible and discriminatory powers for epidemiological typing of S. maltophilia, but ERIC-PCR profiles can be more easily evaluated. PMID:7790459

  11. Multiplex PCR for rapid diagnosis of tuberculous meningitis.

    PubMed

    Kusum, Sharma; Aman, Sharma; Pallab, Ray; Kumar, Sharma Shiv; Manish, Modi; Sudesh, Prabhakar; Subhash, Varma; Meera, Sharma

    2011-10-01

    Rapid and specific diagnosis of tubercular meningitis is of paramount importance to decrease morbidity and mortality. The aim of the study was to evaluate multiplex PCR using protein b, MPB 64, and IS6110 primers directed against M. tuberculosis complex for the diagnosis of tuberculous meningitis (TBM). Multiplex PCR was performed on 18 TBM confirmed cases (culture was positive), 92 clinically suspected TBM cases and 100 non-TBM (control group) patients. Multiplex PCR had a sensitivity of 94.4% for confirmed cases and specificity of 100% for confirmed TBM cases. In 92 clinically diagnosed but unconfirmed TBM cases, multiplex PCR was positive in 84.78% cases. The overall sensitivity of microscopy, culture and multiplex cases were 1.81, 16.73, and 86.63% and specificity was 100, 100, and 100% respectively. Multiplex PCR using protein b, MPB 64, and IS6110 primers has a high sensitivity and specificity in diagnosis of tubercular meningitis. PMID:21455603

  12. Is real-time PCR-based diagnosis similar in performance to routine parasitological examination for the identification of Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica from stool samples? Evaluation of a new commercial multiplex PCR assay and literature review.

    PubMed

    Laude, A; Valot, S; Desoubeaux, G; Argy, N; Nourrisson, C; Pomares, C; Machouart, M; Le Govic, Y; Dalle, F; Botterel, F; Bourgeois, N; Cateau, E; Leterrier, M; Le Pape, P; Morio, F

    2016-02-01

    Microscopy is the reference standard for routine laboratory diagnosis in faecal parasitology but there is growing interest in alternative methods to overcome the limitations of microscopic examination, which is time-consuming and highly dependent on an operator's skills and expertise. Compared with microscopy, DNA detection by PCR is simple and can offer a better turnaround time. However, PCR performances remain difficult to assess as most studies have been conducted on a limited number of positive clinical samples and used in-house PCR methods. Our aim was to evaluate a new multiplex PCR assay (G-DiaParaTrio; Diagenode Diagnostics), targeting Giardia intestinalis, Cryptosporidium parvum/Cryptosporidium hominis and Entamoeba histolytica. To minimize the turnaround time, PCR was coupled with automated DNA extraction (QiaSymphony; Qiagen). The PCR assay was evaluated using a reference panel of 185 samples established by routine microscopic examination using a standardized protocol including Ziehl-Neelsen staining and adhesin detection by ELISA (E. histolytica II; TechLab). This panel, collected from 12 French parasitology laboratories, included 135 positive samples for G. intestinalis (n = 38), C. parvum/C. hominis (n = 26), E. histolytica (n = 5), 21 other gastrointestinal parasites, together with 50 negative samples. In all, the G-DiaParaTrio multiplex PCR assay identified 38 G. intestinalis, 25 C. parvum/C. hominis and five E. histolytica leading to sensitivity/specificity of 92%/100%, 96%/100% and 100%/100% for G. intestinalis, C. parvum/C. hominis and E. histolytica, respectively. This new multiplex PCR assay offers fast and reliable results, similar to microscopy-driven diagnosis for the detection of these gastrointestinal protozoa, allowing its implementation in routine clinical practice. PMID:26548509

  13. Use of Repetitive Element Palindromic-PCR (rep-PCR) for the Epidemiologic Discrimination of Food-Borne Pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of defined primers for polymerase chain reactions (PCR) amplicifcations of interspersed repetitive DNA elements present at distinct locations in prokaryotic genomes is referred to as Repetitive Element Palindromic Sequences Based-Polymerase Chain Reactions, rep-PCR. The initial discovery of...

  14. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments.

    PubMed

    Svec, David; Tichopad, Ales; Novosadova, Vendula; Pfaffl, Michael W; Kubista, Mikael

    2015-03-01

    We have examined the imprecision in the estimation of PCR efficiency by means of standard curves based on strategic experimental design with large number of technical replicates. In particular, how robust this estimation is in terms of a commonly varying factors: the instrument used, the number of technical replicates performed and the effect of the volume transferred throughout the dilution series. We used six different qPCR instruments, we performed 1-16 qPCR replicates per concentration and we tested 2-10 μl volume of analyte transferred, respectively. We find that the estimated PCR efficiency varies significantly across different instruments. Using a Monte Carlo approach, we find the uncertainty in the PCR efficiency estimation may be as large as 42.5% (95% CI) if standard curve with only one qPCR replicate is used in 16 different plates. Based on our investigation we propose recommendations for the precise estimation of PCR efficiency: (1) one robust standard curve with at least 3-4 qPCR replicates at each concentration shall be generated, (2) the efficiency is instrument dependent, but reproducibly stable on one platform, and (3) using a larger volume when constructing serial dilution series reduces sampling error and enables calibration across a wider dynamic range. PMID:27077029

  15. Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA.

    PubMed

    Kiselinova, Maja; Pasternak, Alexander O; De Spiegelaere, Ward; Vogelaers, Dirk; Berkhout, Ben; Vandekerckhove, Linos

    2014-01-01

    Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used

  16. Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA

    PubMed Central

    De Spiegelaere, Ward; Vogelaers, Dirk; Berkhout, Ben; Vandekerckhove, Linos

    2014-01-01

    Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used

  17. Detection of beet yellows virus by RT-PCR and immunocapture RT-PCR in Tetragonia expansa and Beta vulgaris.

    PubMed

    Kundu, K; Rysánek, P

    2004-01-01

    Two sensitive methods, RT-PCR with phenol-extracted RNA or Triton X-100-released RNA and immunocapture RT-PCR (IR-RT-PCR) were used for the detection of Beet yellows virus (BYV) in young and old leaves of Tetragonia expansa and sugar beet (Beta vulgaris) and in sugar beet roots. Four oligonucleotide primer pairs proved suitable for the detection of BYV. The release of BYV RNA with Triton X-100 was shown to be a very effective and easy as compared to isolation of total RNA by phenol extraction with the same or higher sensitivity of subsequent PCR. Using the Triton X-100 release of RNA and IC-RT-PCR the sensitivity of detection was so high that pg amounts of BYV RNA occurring in dilutions up to 10(-6) of saps from young Tetragonia and sugar beet leaves could be detected. PMID:15595212

  18. Field effect sensors for PCR applications

    NASA Astrophysics Data System (ADS)

    Taing, Meng-Houit; Sweatman, Denis R.

    2004-03-01

    The use of field effect sensors for biological and chemical sensing is widely employed due to its ability to make detections based on charge and surface potential. Because proteins and DNA almost always carry a charge [1], silicon can be used to micro fabricate such a sensor. The EIS structure (Electrolyte on Insulator on Silicon) provides a novel, label-free and simple to fabricate way to make a field effect DNA detection sensor. The sensor responds to fluctuating capacitance caused by a depletion layer thickness change at the surface of the silicon substrate through DNA adsorption onto the dielectric oxide/PLL (Poly-L-Lysine) surface. As DNA molecules diffuse to the sensor surface, they are bound to their complimentary capture probes deposited on the surface. The negative charge exhibited by the DNA forces negative charge carriers in the substrate to move away from the surface. This causes an n-type depletion layer substrate to thicken and a p-type to thin. The depletion layer thickness can be measured by its capacitance using an LCR meter. This experiment is conducted using the ConVolt (constant voltage) approach. Nucleic acids are amplified by an on chip PCR (Polymerase Chain Reaction) system and then fed into the sensor. The low ionic solution strength will ensure that counter-ions do not affect the sensor measurements. The sensor surface contains capture probes that bind to the pathogen. The types of pathogens we"ll be detecting include salmonella, campylobacter and E.Coli DNA. They are held onto the sensor surface by the positively charged Poly-L-Lysine layer. The electrolyte is biased through a pseudo-reference electrode. Pseudo reference electrodes are usually made from metals such as Platinum or Silver. The problem associated with "floating" biasing electrodes is they cannot provide stable biasing potentials [2]. They drift due to surface charging effects and trapped charges on the surface. To eliminate this, a differential system consisting of 2 sensors

  19. Tiempo para un cambio

    NASA Astrophysics Data System (ADS)

    Woltjer, L.

    1987-06-01

    En la reunion celebrada en diciembre dei ano pasado informe al Consejo de mi deseo de terminar mi contrato como Director General de la ESO una vez que fuera aprobado el proyecto dei VLT, que se espera sucedera hacia fines de este aAo. Cuando fue renovada mi designacion hace tres aAos, el Consejo conocia mi intencion de no completar los cinco aAos dei contrato debido a mi deseo de disponer de mas tiempo para otras actividades. Ahora, una vez terminada la fase preparatoria para el VLT, Y habiendose presentado el proyecto formalmente al Consejo el dia 31 de marzo, y esperando su muy probable aprobacion antes dei termino de este ano, me parece que el 10 de enero de 1988 presenta una excelente fecha para que se produzca un cambio en la administracion de la ESO.

  20. PCR in laboratory diagnosis of human Borrelia burgdorferi infections.

    PubMed Central

    Schmidt, B L

    1997-01-01

    The laboratory diagnosis of Lyme borreliosis, the most prevalent vector-borne disease in the United States and endemic in parts of Europe and Asia, is currently based on serology with known limitations. Direct demonstration of Borrelia burgdorferi by culture may require weeks, while enzyme-linked immunosorbent assays for antigen detection often lack sensitivity. The development of the PCR has offered a new dimension in the diagnosis. Capable of amplifying minute amounts of DNA into billions of copies in just a few hours, PCR facilitates the sensitive and specific detection of DNA or RNA of pathogenic organisms. This review is restricted to applications of PCR methods in the diagnosis of human B. burgdorferi infections. In the first section, methodological aspects, e.g., sample preparation, target selection, primers and PCR methods, and detection and control of inhibition and contamination, are highlighted. In the second part, emphasis is placed on diagnostic aspects, where PCR results in patients with dermatological, neurological, joint, and ocular manifestations of the disease are discussed. Here, special attention is given to monitoring treatment efficacy by PCR tests. Last, specific guidelines on how to interpret PCR results, together with the advantages and limitations of these new techniques, are presented. PMID:8993863

  1. A comparison of four methods for PCR inhibitor removal.

    PubMed

    Hu, Qingqing; Liu, Yuxuan; Yi, Shaohua; Huang, Daixin

    2015-05-01

    Biological samples collected from the crime scenes often contain some compounds that can inhibit the polymerase chain reaction (PCR). The removal of PCR inhibitors from the extracts prior to the PCR amplification is vital for successful forensic DNA typing. This paper aimed to evaluate the ability of four different methods (PowerClean® DNA Clean-Up kit, DNA IQ™ System, Phenol-Chloroform extraction and Chelex®-100 methods) to remove eight commonly encountered PCR inhibitors including: melanin, humic acid, collagen, bile salt, hematin, calcium ions, indigo and urea. Each of these PCR inhibitors was effectively removed by the PowerClean® DNA Clean-Up kit and DNA IQ™ System as demonstrated by generating more complete short tandem repeat (STR) profiles from the cleaned up inhibitor samples than from the raw inhibitor samples. The Phenol-Chloroform extraction and Chelex®-100 methods, however, could only remove some of eight PCR inhibitors. Our results demonstrated that the PowerClean® DNA Clean-Up kit and DNA IQ™ System were very effective for the removal of known PCR inhibitors that are routinely found in DNA extracts from forensic samples. PMID:25553520

  2. Interaction of quantitative PCR components with polymeric surfaces.

    PubMed

    Gonzalez, Asensio; Grimes, Ronan; Walsh, Edmond J; Dalton, Tara; Davies, Mark

    2007-04-01

    This study investigated the effect of exposing a polymerase chain reaction (PCR) mixture to capillary tubing of different materials and lengths, at different contact times and flow rates and the adsorption of major reaction components into the tubing wall. Using 0.5 mm ID tubing, lengths of 40 cm and residence times up to 45 min, none of the tested polymeric materials was found to affect subsequent PCR amplification. However, after exposure of the mixture to tubing lengths of 3 m or reduction of sample volume, PCR inhibition occurred, increasing with the volume to length ratio. Different flow velocities did not affect PCR yield. When the adsorption of individual PCR components was studied, significant DNA adsorption and even more significant adsorption of the fluorescent dye Sybr Green I was found. The results indicate that PCR inhibition in polymeric tubing results from adsorption of reaction components to wall surfaces, increasing substantially with tubing length or sample volume reduction, but not with contact time or flow velocities typical in dynamic PCR amplification. The data also highlight that chemical compatibility of polymeric capillaries with DNA dyes should be carefully considered for the design of quantitative microfluidic devices. PMID:17180709

  3. PCR assays for detection of Baylisascaris procyonis eggs and larvae.

    PubMed

    Dangoudoubiyam, Sriveny; Vemulapalli, Ramesh; Kazacos, Kevin R

    2009-06-01

    The objective of this study was to develop polymerase chain reaction (PCR) assays for detection of Baylisascaris procyonis eggs and larvae in fecal, environmental, and tissue samples. We have optimized conventional and real-time PCR assays for B. procyonis using the mitochondrial cytochrome oxidase 2 gene as the target for amplification. The lower limit of detection of the parasite genomic DNA was 10 pg in the conventional PCR and 100 fg in the real-time PCR. In both PCR assays, specific amplification of a 146 bp product was achieved with DNA extracted from a single in vitro hatched B. procyonis larva and also from canine fecal samples spiked with as few as 20 unembryonated B. procyonis eggs per gram of feces. The PCR assays were successfully used for detection of B. procyonis eggs and larvae in fecal, environmental, and tissue samples. No DNA amplification was seen when the genomic DNA of related ascarids (including B. transfuga) and a hookworm was used as template in the PCR; however, amplification was seen with the very closely related B. columnaris. PMID:19090651

  4. 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. PMID:25850372

  5. Sensitive detection of sample interference in environmental qPCR.

    PubMed

    Green, Hyatt C; Field, Katharine G

    2012-06-15

    Sample interference in environmental applications of quantitative PCR (qPCR) can prevent accurate estimations of molecular markers in the environment. We developed a spike-and-recovery approach using a mutant strain of Escherichia coli that contains a chromosomal insertion of a mutant GFP gene. The method was tested in water samples by separately reducing extraction efficiency or adding humic acids and ethanol, compounds that often contaminate environmental DNA extracts, and analyzing qPCR amplification of the spiked E. coli control and human fecal Bacteroides markers (HF183 and HF134). This approach, coupled with previously developed kinetic outlier detection (KOD) methods, allowed sensitive detection of PCR inhibition at much lower inhibitor concentrations than alternative approaches using Cq values or amplification efficiencies. Although HF183 was more sensitive to the effects of qPCR inhibitors than the E. coli control assay, KOD methods correctly identified inhibition of both control and HF183 assays in samples containing as little as 0.1 ng humic acids per reaction or 5% ethanol. Because sigmoidal modeling methods allow distinction of qPCR inhibition from poor DNA recovery, we were able to simultaneously identify qPCR-inhibited reactions and estimate recovery of nucleic acids in environmental samples using a single control assay. Since qPCR is currently used to estimate important water quality parameters that have serious economic and human health outcomes, these results are timely. While we demonstrate the methods in the context of water quality regulation, they will be useful in all areas of environmental research that use qPCR. PMID:22560896

  6. Predicting Gene Structures from Multiple RT-PCR Tests

    NASA Astrophysics Data System (ADS)

    Kováč, Jakub; Vinař, Tomáš; Brejová, Broňa

    It has been demonstrated that the use of additional information such as ESTs and protein homology can significantly improve accuracy of gene prediction. However, many sources of external information are still being omitted from consideration. Here, we investigate the use of product lengths from RT-PCR experiments in gene finding. We present hardness results and practical algorithms for several variants of the problem and apply our methods to a real RT-PCR data set in the Drosophila genome. We conclude that the use of RT-PCR data can improve the sensitivity of gene prediction and locate novel splicing variants.

  7. Efficient PCR amplification by an unnatural base pair system.

    PubMed

    Kimoto, Michiko; Kawai, Rie; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, Ichiro

    2008-01-01

    Expansion of the genetic alphabet by an unnatural base pair system enables the site-specific incorporation of extra functional components into nucleic acids and proteins. In this system, PCR amplification of DNA templates containing unnatural base pairs is essential for modern biotechnology. We present a new unnatural base pair system, in which DNA duplexes containing the unnatural base pairs can be efficiently amplified by PCR. The system also provides a method for the site-specific incorporation of functional components into amplified DNA fragments by PCR, using unnatural base substrates linked with functional groups of interest. PMID:18776457

  8. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  9. Preparation of DNA-containing extract for PCR amplification

    DOEpatents

    Dunbar, John M.; Kuske, Cheryl R.

    2006-07-11

    Environmental samples typically include impurities that interfere with PCR amplification and DNA quantitation. Samples of soil, river water, and aerosol were taken from the environment and added to an aqueous buffer (with or without detergent). Cells from the sample are lysed, releasing their DNA into the buffer. After removing insoluble cell components, the remaining soluble DNA-containing extract is treated with N-phenacylthiazolium bromide, which causes rapid precipitation of impurities. Centrifugation provides a supernatant that can be used or diluted for PCR amplification of DNA, or further purified. The method may provide a DNA-containing extract sufficiently pure for PCR amplification within 5–10 minutes.

  10. Analysis of mutations using PCR and denaturing gradient gel electrophoresis

    SciTech Connect

    Cariello, N.F.; Swenberg, J.A. Duke Univ., Durham, NC ); DeBellis, A.; Skopek, T.R. )

    1991-01-01

    Denaturing gradient gel electrophoresis (DGGE) separates DNA molecules based on primary sequence. Under the appropriate conditions, all base pair (bp) substitutions, frameshifts, and deletions less than about 10 bp can be resolved from the wild type sequence using DGGE. Polymerase chain reaction (PCR) permits facile amplification of a given region of the genome. The authors have combined PCR and DGGE to: (1) localize mutations in the X-linked human androgen receptor gene; (2) analyze thousands of thioguanine-resistant mutants simultaneously; (3) examine the fidelity of several DNA polymerases used in PCR.

  11. Identification and quantification of ice nucleation active microorganisms by digital droplet PCR (ddPCR)

    NASA Astrophysics Data System (ADS)

    Linden, Martin; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2015-04-01

    Several bioaerosol types, including bacteria, fungi, pollen and lichen, have been identified as sources of biological ice nucleators (IN) which induce ice formation already at temperatures as high as -10 °C or above. Accordingly, they potentially contribute widely to environmental ice nucleation in the atmosphere and are of great interest in the study of natural heterogenous ice nucleation processes. Ice nucleation active microorganisms have been found and studied among bacteria (Proteobacteria) and fungi (phyla Basidiomycota and Ascomycota). The mechanisms enabling the microorganisms to ice nucleation are subject to ongoing research. While it has been demonstrated that whole cells can act as ice nucleators in the case of bacteria due to the presence of specific membrane proteins, cell-free ice nucleation active particles seem to be responsible for this phenomenon in fungi and lichen. The identification and quantification of these ice nucleation active microorganisms and their IN in atmospheric samples is crucial to understand their contribution to the pool of atmospheric IN. This is not a trivial task since the respective microorganisms are often prevalent in lowest concentrations and a variety of states, be it viable cells, spores or cell debris from dead cells. Molecular biology provides tools to identify and quantify ice nucleation active microorganisms independent of their state by detecting genetic markers specific for the organism of interest. Those methods are not without their drawbacks in terms of sample material concentration required or reliable standardization. Digital Droplet Polymerase Chain Reaction (ddPCR) was chosen for our demands as a more elegant, quick and specific method in the investigation of ice nucleation active microorganisms in atmospheric samples. The advantages of ddPCR lie in the simultaneous detection and quantification of genetic markers and their original copy numbers in a sample. This is facilitated by the fractionation of the

  12. Sensitive Simultaneous Detection of Seven Sexually Transmitted Agents in Semen by Multiplex-PCR and of HPV by Single PCR

    PubMed Central

    de Abreu, André Luelsdorf Pimenta; Irie, Mary Mayumi Taguti; Esquiçati, Isis Baroni; Malagutti, Natália; Vasconcellos, Vinícius Rodrigo Bulla; Discacciati, Michele Garcia; Bonini, Marcelo Gialluisi; Maria-Engler, Silvya Stuchi; Consolaro, Marcia Edilaine Lopes

    2014-01-01

    Sexually transmitted diseases (STDs) may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR) assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV) −1 and −2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV) and genotypes by single PCR (sPCR) in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%), sensitivity (100.00%), specificity (99.70%), positive (96.40%) and negative predictive values (100.00%) and accuracy (99.80%). The prevalence of STDs was very high (55.3%). Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks. PMID:24921247

  13. Oligoribonucleotide (ORN) Interference-PCR (ORNi-PCR): A Simple Method for Suppressing PCR Amplification of Specific DNA Sequences Using ORNs

    PubMed Central

    Tanigawa, Naoki; Fujita, Toshitsugu; Fujii, Hodaka

    2014-01-01

    Polymerase chain reaction (PCR) amplification of multiple templates using common primers is used in a wide variety of molecular biological techniques. However, abundant templates sometimes obscure the amplification of minor species containing the same primer sequences. To overcome this challenge, we used oligoribonucleotides (ORNs) to inhibit amplification of undesired template sequences without affecting amplification of control sequences lacking complementarity to the ORNs. ORNs were effective at very low concentrations, with IC50 values for ORN-mediated suppression on the order of 10 nM. DNA polymerases that retain 3′–5′ exonuclease activity, such as KOD and Pfu polymerases, but not those that retain 5′–3′ exonuclease activity, such as Taq polymerase, could be used for ORN-mediated suppression. ORN interference-PCR (ORNi-PCR) technology should be a useful tool for both molecular biology research and clinical diagnosis. PMID:25405983

  14. The clinical potential of Enhanced-ice-COLD-PCR.

    PubMed

    Tost, Jörg

    2016-01-01

    Enhanced-ice-COLD-PCR (E-ice-COLD-PCR) is a novel assay format that allows for the efficient enrichment and sensitive detection of all mutations in a region of interest using a chemically modified blocking oligonucleotide, which impedes the amplification of wild-type sequences. The assay is compatible with DNA extracted from tissue and cell-free circulating DNA. The main features of E-ice-COLD-PCR are the simplicity of the setup and the optimization of the assay, the use of standard laboratory equipment and the very short time to results (~4 h including DNA extraction, enrichment and sequence-based identification of mutations). E-ice-COLD-PCR is therefore a highly promising technology for a number of basic research as well as clinical applications including detection of clinically relevant mutated subclones and monitoring of treatment response or disease recurrence. PMID:26589575

  15. Quantitative PCR for Genetic Markers of Human Fecal Pollution

    EPA Science Inventory

    Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantificationapproach. We report the development of quantitative PCR assays for quantification of two recently described human-...

  16. Quantitative PCR for genetic markers of human fecal pollution

    EPA Science Inventory

    Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for enumeration of two recently described hum...

  17. DNA Microarray-Based PCR Ribotyping of Clostridium difficile

    PubMed Central

    Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2014-01-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. PMID:25411174

  18. MPprimer: a program for reliable multiplex PCR primer design

    PubMed Central

    2010-01-01

    Background Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. Results A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2× to 5× plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes to detect underlying exon deletions. Conclusions MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays. PMID:20298595

  19. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces

    PubMed Central

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-01-01

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan® Environmental Master Mix 2.0; UMM: TaqMan® Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material. PMID:27213452

  20. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces.

    PubMed

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-01-01

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan(®) Environmental Master Mix 2.0; UMM: TaqMan(®) Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material. PMID:27213452

  1. Quantification of HEV RNA by Droplet Digital PCR.

    PubMed

    Nicot, Florence; Cazabat, Michelle; Lhomme, Sébastien; Marion, Olivier; Sauné, Karine; Chiabrando, Julie; Dubois, Martine; Kamar, Nassim; Abravanel, Florence; Izopet, Jacques

    2016-01-01

    The sensitivity of real-time PCR for hepatitis E virus (HEV) RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR) assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF)3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU)/mL and linearities of HEV genotype 1, 3 and 4 were very similar. Clinical validation based on 45 samples of genotype 1, 3 or 4 gave results that correlated well with a validated reverse transcription quantitative PCR (RT-qPCR) assay (Spearman rs = 0.89, p < 0.0001). The RT-ddPCR assay is a sensitive method and could be a promising tool for standardizing HEV RNA quantification in various sample types. PMID:27548205

  2. Partially overlapping primer-based PCR for genome walking.

    PubMed

    Li, Haixing; Ding, Dongqin; Cao, Yusheng; Yu, Bo; Guo, Liang; Liu, Xiaohua

    2015-01-01

    Current genome walking methods are cumbersome to perform and can result in non-specific products. Here, we demonstrate the use of partially overlapping primer-based PCR (POP-PCR), a direct genome walking technique for the isolation of unknown flanking regions. This method exploits the partially overlapping characteristic at the 3' ends of a set of POP primers (walking primers), which guarantees that the POP primer only anneals to the POP site of the preceding PCR product at relatively low temperatures. POP primer adaptation priming at the genomic DNA/POP site occurs only once due to one low-/reduced-stringency cycle in each nested PCR, resulting in the synthesis of a pool of single-stranded DNA molecules. Of this pool, the target single-stranded DNA is replicated to the double-stranded form bound by the specific primer and the POP primer in the subsequent high-stringency cycle due to the presence of the specific primer-binding site. The non-target single stranded DNA does not become double stranded due to the absence of a binding site for any of the primers. Therefore, the POP-PCR enriches target DNA while suppressing non-target products. We successfully used POP-PCR to retrieve flanking regions bordering the gadA locus in Lactobacillus brevis NCL912, malQ in Pichia pastoris GS115, the human aldolase A gene, and hyg in rice. PMID:25811779

  3. Improved PCR Amplification of Broad Spectrum GC DNA Templates

    PubMed Central

    Guido, Nicholas; Starostina, Elena; Leake, Devin; Saaem, Ishtiaq

    2016-01-01

    Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10–90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content. PMID:27271574

  4. Rapid diagnosis of goose viral infections by multiplex PCR.

    PubMed

    Chen, Zongyan; Li, Chuanfeng; Li, Guoxin; Yu, Hai; Jiang, Yifeng; Yan, Liping; Meng, Chunchun; Zhou, Yanjun; Tong, Guangzhi; Liu, Guangqing

    2013-08-01

    Goose parvovirus (GPV), newcastle disease virus (NDV), goose herpesvirus (GHV) and goose adenovirus (GAV) are considered collectively to be four of the most important and widespread viruses of geese. Because all of these viruses cause similar pathological changes, histological differentiation among these viruses is difficult. A reliable, specific and sensitive multiplex PCR (mPCR) assay was developed for the combined detection of GPV, NDV, GHV and GAV in clinical samples of geese. Using the mPCR technique, single infections with GPV (28/76; 36.8%), NDV (9/76; 11.8%), GHV (3/76; 3.9%) and GAV (12/76; 15.8%) were identified in the samples; co-infections with GAV and either GPV or NDV (31.6%; 24/76) were also identified with this approach. The results for all of the samples tested were the same in both the uPCR and mPCR systems. The mPCR approach is considered to be useful for routine molecular diagnosis and epidemiological applications in geese. PMID:23518397

  5. Quantification of HEV RNA by Droplet Digital PCR

    PubMed Central

    Nicot, Florence; Cazabat, Michelle; Lhomme, Sébastien; Marion, Olivier; Sauné, Karine; Chiabrando, Julie; Dubois, Martine; Kamar, Nassim; Abravanel, Florence; Izopet, Jacques

    2016-01-01

    The sensitivity of real-time PCR for hepatitis E virus (HEV) RNA quantification differs greatly among techniques. Standardized tools that measure the real quantity of virus are needed. We assessed the performance of a reverse transcription droplet digital PCR (RT-ddPCR) assay that gives absolute quantities of HEV RNA. Analytical and clinical validation was done on HEV genotypes 1, 3 and 4, and was based on open reading frame (ORF)3 amplification. The within-run and between-run reproducibilities were very good, the analytical sensitivity was 80 HEV RNA international units (IU)/mL and linearities of HEV genotype 1, 3 and 4 were very similar. Clinical validation based on 45 samples of genotype 1, 3 or 4 gave results that correlated well with a validated reverse transcription quantitative PCR (RT-qPCR) assay (Spearman rs = 0.89, p < 0.0001). The RT-ddPCR assay is a sensitive method and could be a promising tool for standardizing HEV RNA quantification in various sample types. PMID:27548205

  6. Detection of Treponema pallidum in the vitreous by PCR

    PubMed Central

    Müller, M; Ewert, I; Hansmann, F; Tiemann, C; Hagedorn, H J; Solbach, W; Roider, J; Nölle, B; Laqua, H; Hoerauf, H

    2007-01-01

    Background Ocular involvement of syphilis still poses a clinical challenge due to the chameleonic behaviour of the disease. As the serodiagnosis has significant limitations, the direct detection of Treponema pallidum (TP) in the vitreous represents a desirable diagnostic tool. Methods Real‐time polymerase chain reaction (PCR) for the detection of TP was applied in diagnostic vitrectomies of two patients with acute chorioretinitis. Qualitative verification of TP by real‐time PCR and melting point analysis according to a modified protocol was ruled out. Patients underwent complete ophthalmological examination with fundus photographs, fluorescein angiography, serological examination, antibiotic treatment and follow‐up. Results In two cases of acute chorioretinitis of unknown origin, real‐time PCR of vitreous specimens of both patients provided evidence of TP and was 100% specific. Initial diagnosis of presumed viral retinitis was ruled out by PCR of vitreous specimen. Patients were treated with systemic antibiotics and showed prompt improvement in visual function and resolution of fundus lesions. Conclusions With real‐time PCR, detection of TP in the vitreous was possible and delivered a sensitive, quick and inexpensive answer to a disease rather difficult to assess. In cases of acute chorioretinitis, the use of PCR‐based assays of vitreous specimens in the diagnostic evaluation of patients is advisable. Although syphilitic chorioretinitis is a rare disease, PCR should include search for TP, as diagnostic dilemmas prolong definitive treatment in a sight‐threatening disease. PMID:17108014

  7. Real-time PCR for sexing Schistosoma mansoni cercariae.

    PubMed

    Chevalier, Frédéric D; Le Clec'h, Winka; Alves de Mattos, Ana Carolina; LoVerde, Philip T; Anderson, Timothy J C

    2016-01-01

    The gender of cercarial larvae can only be determined using molecular methods. End point PCR methods that amplify repetitive markers on the W chromosome of the female (ZW) parasites have been developed, but sometimes results are ambiguous or incorrect. To more effectively distinguish sexes, and to determine why end point PCR can be incorrect, we quantified the W6 repeat sequence and a specific Z chromosome gene using real-time PCR. The ratio between copy number of W6 and a Z chromosome marker unambiguously identifies gender: females have higher ratios (421-4371) than males (0-21). However, some males have low numbers of W6 elements in their genome, and qPCR demonstrated significantly higher W6/Z marker ratios for male genotypes giving ambiguous end point PCR results compared with males giving clear end point results. The quantitative PCR sexing method developed will be particularly useful where reliable sexing of cercariae is critical, for example when staging genetic crosses. PMID:27021570

  8. Simulation of between Repeat Variability in Real Time PCR Reactions

    PubMed Central

    Lievens, Antoon; Van Aelst, Stefan; Van den Bulcke, Marc; Goetghebeur, Els

    2012-01-01

    While many decisions rely on real time quantitative PCR (qPCR) analysis few attempts have hitherto been made to quantify bounds of precision accounting for the various sources of variation involved in the measurement process. Besides influences of more obvious factors such as camera noise and pipetting variation, changing efficiencies within and between reactions affect PCR results to a degree which is not fully recognized. Here, we develop a statistical framework that models measurement error and other sources of variation as they contribute to fluorescence observations during the amplification process and to derived parameter estimates. Evaluation of reproducibility is then based on simulations capable of generating realistic variation patterns. To this end, we start from a relatively simple statistical model for the evolution of efficiency in a single PCR reaction and introduce additional error components, one at a time, to arrive at stochastic data generation capable of simulating the variation patterns witnessed in repeated reactions (technical repeats). Most of the variation in values was adequately captured by the statistical model in terms of foreseen components. To recreate the dispersion of the repeats' plateau levels while keeping the other aspects of the PCR curves within realistic bounds, additional sources of reagent consumption (side reactions) enter into the model. Once an adequate data generating model is available, simulations can serve to evaluate various aspects of PCR under the assumptions of the model and beyond. PMID:23189123

  9. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    PubMed

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number. PMID:27451195

  10. Detection of DNA double-strand breaks and chromosome translocations using ligation-mediated PCR and inverse PCR.

    PubMed

    Villalobos, Michael J; Betti, Christopher J; Vaughan, Andrew T M

    2005-01-01

    Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks introduced into any identifiable genomic location. PMID:15502230

  11. Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

    PubMed Central

    Wang, Xiaoyu; Seo, Dong Joo; Lee, Min Hwa

    2014-01-01

    This study aimed to develop a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Arcobacter species. Specific primers targeting the 23S ribosomal RNA gene were used to detect Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The specificity of the LAMP primer set was assessed using DNA samples from a panel of Arcobacter and Campylobacter species, and the sensitivity was determined using serial dilutions of Arcobacter species cultures. LAMP showed a 10- to 1,000-fold-higher sensitivity than multiplex PCR, with a detection limit of 2 to 20 CFU per reaction in vitro. Whereas multiplex PCR showed cross-reactivity with Campylobacter species, the LAMP method developed in this study was more sensitive and reliable than conventional PCR or multiplex PCR for the detection of Arcobacter species. PMID:24478488

  12. Comparing Rapid and Specific Detection of Brucella in Clinical Samples by PCR-ELISA and Multiplex-PCR Method

    PubMed Central

    Mohammad Hasani, Sharareh; Mirnejad, Reza; Amani, Jafar; Vafadar, Mohamad javad

    2016-01-01

    Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests. Methods: In this experimental and sectional study, during March 2014 to Sep 2015, 52 blood samples of suspected patients with clinical symptoms of brucellosis were evaluated in medical centers all over Iran with serum titers higher than 1:80. Using two pairs of specific primers of Brucella abortus, B. melitensis and DIG-dUTP, Fragment IS711 (The common gene fragment in B. melitensis and B. abortus) was amplified. DIG-ELISA was performed using specific probes of these 2 species of Brucella and patterns were subsequently analyzed, then positive responses were compared by detecting gel electrophoresis. Results: PCR-ELISA method detected all 28 samples from 52 positive samples. Its sensitivity was 6.0 pg concentration of genomic DNA of Brucella. In gel electrophoresis method, 22 samples of all positive samples were detected. PCR-ELISA was more efficient than PCR and bacterial culture method at P-value <0.05. Conclusion: PCR-ELISA molecular method is more sensitive than other molecular methods, lack of mutagenic color and also a semi-quantitative ability. This method is more effective and more accurate compared to PCR, serology and culture of bacteria. PCR-ELISA does not have false responses. The limitation of this method is detection of bacteria in the genus compared to Multiplex PCR and Gel Electrophoresis. PMID:27499776

  13. DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus)

    PubMed Central

    Li, Xiaolei; Zeng, Weiping; Liao, Jing; Liang, Zhenbiao; Huang, Shuhua

    2015-01-01

    We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes of Bungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites of SpeI and BstEII in the COI sequence of B. multicinctus to allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion with SpeI and BstEII (except for that of Zaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA of B. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS. PMID:26078770

  14. DNA Barcode-Based PCR-RFLP and Diagnostic PCR for Authentication of Jinqian Baihua She (Bungarus Parvus).

    PubMed

    Li, Xiaolei; Zeng, Weiping; Liao, Jing; Liang, Zhenbiao; Huang, Shuhua; Chao, Zhi

    2015-01-01

    We established polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and diagnostic PCR based on cytochrome C oxidase subunit I (COI) barcodes of Bungarus multicinctus, genuine Jinqian Baihua She (JBS), and adulterant snake species. The PCR-RFLP system utilizes the specific restriction sites of SpeI and BstEII in the COI sequence of B. multicinctus to allow its cleavage into 3 fragments (120 bp, 230 bp, and 340 bp); the COI sequences of the adulterants do not contain these restriction sites and therefore remained intact after digestion with SpeI and BstEII (except for that of Zaocys dhumnades, which could be cleaved into a 120 bp and a 570 bp fragment). For diagnostic PCR, a pair of species-specific primers (COI37 and COI337) was designed to amplify a specific 300 bp amplicon from the genomic DNA of B. multicinctus; no such amplicons were found in other allied species. We tested the two methods using 11 commercial JBS samples, and the results demonstrated that barcode-based PCR-RFLP and diagnostic PCR both allowed effective and accurate authentication of JBS. PMID:26078770

  15. Determining Fungi rRNA Copy Number by PCR

    PubMed Central

    Black, Jonathan; Dean, Timothy; Byfield, Grace; Foarde, Karin; Menetrez, Marc

    2013-01-01

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within a standard qPCR reaction. The first control developed was the internal standard control gene, benA. This gene encodes for β-tubulin and was selected based on its single-copy nature. The second control developed was the standard control plasmid, which contained a fragment of the ribosomal RNA (rRNA) gene and produced a specific PCR product. The results confirm the multicopy nature of the rRNA region in several filamentous fungi and show that we can quantify fungi of unknown genome size over a range of spore extractions by inclusion of these two standard controls. Advances in qPCR have led to extremely sensitive and quantitative methods for single-copy genes; however, it has not been well established that the rRNA can be used to quantitate fungal contamination. We report on the use of qPCR, combined with two controls, to identify and quantify indoor fungal contaminants with a greater degree of confidence than has been achieved previously. Advances in indoor environmental health have demonstrated that contamination of the built environment by the filamentous fungi has adverse impacts on the health of building occupants. This study meets the need for more accurate and reliable methods for fungal identification and quantitation in the indoor environment. PMID:23543828

  16. Inverse PCR for subtyping of Acinetobacter baumannii carrying ISAba1.

    PubMed

    Kim, Shukho; Park, Yun-Ju; Kim, Jungmin

    2016-05-01

    Acinetobacter baumannii has been prevalent in nosocomial infections, often causing outbreaks in intensive care units. ISAba1 is an insertion sequence that has been identified only in A. baumannii and its copy number varies among strains. It has been reported that ISAba1 provides a promoter for bla OXA-51-like, bla OXA-23-like, and bla ampC, which are associated with the resistance of A. baumannii to carbapenems and cephalosporins. The main purpose of this study was to develop a novel inverse PCR method capable of typing A. baumannii strains. The method involves three major steps: cutting of genomic DNA with a restriction enzyme, ligation, and PCR. In the first step, bacterial genomic DNA was digested with DpnI. In the second step, the digested genomic DNAs were ligated to form intramolecular circular DNAs. In the last step, the ligated circular DNAs were amplified by PCR with primers specific for ISAba1 and the amplified PCR products were electrophoresed. Twenty-two clinical isolates of A. baumannii were used for the evaluation of the inverse PCR (iPCR) typing method. Dendrogram analysis revealed two major clusters, similar to pulsed-field gel electrophoresis (PFGE) results. Three ISAba1-associated genes - bla ampC, bla OXA-66-like, and csuD - were amplified and detected in the clinical isolates. This novel iPCR typing method is comparable to PFGE in its ability to discriminate A. baumannii strains, and is a promising molecular epidemiological tool for investigating A. baumannii carrying ISAba1. PMID:27095456

  17. Droplet-based micro oscillating-flow PCR chip

    NASA Astrophysics Data System (ADS)

    Wang, Wei; Li, Zhi-Xin; Luo, Rong; Lü, Shu-Hai; Xu, Ai-Dong; Yang, Yong-Jun

    2005-08-01

    Polymerase chain reactions (PCR), thermally activated chemical reactions which are widely used for nucleic acid amplification, have recently received much attention in microelectromechanical systems and micro total analysis systems because a wide variety of DNA/RNA molecules can be enriched by PCR for further analyses. In the present work, a droplet-based micro oscillating-flow PCR chip was designed and fabricated by the silicon microfabrication technique. Three different temperature zones, which were stable at denaturation, extension and annealing temperatures and isolated from each other by a thin-wall linkage, were integrated with a single, simple and straight microchannel to form the chip's basic functional structure. The PCR mixture was injected into the chip as a single droplet and flowed through the three temperature zones in the main microchannel in an oscillating manner to achieve the temperature maintenance and transitions. The chip's thermal performance was theoretically analyzed and numerically simulated. The results indicated that the time needed for the temperature of the droplet to change to the target value is less than 1 s, and the root mean square error of temperature is less than 0.2 °C. A droplet of 1 µl PCR mixture with standard HPV (Human Papilloma Virus)-DNA sample inside was amplified by the present chip and the results were analyzed by slab gel electrophoresis with separation of DNA markers in parallel. The electrophoresis results demonstrated that the micro oscillating-flow PCR chip successfully amplified the HPV-DNA, with a processing time of about 15 min which is significantly reduced compared to that for the conventional PCR instrument.

  18. The methylation status of plant genomic DNA influences PCR efficiency.

    PubMed

    Kiselev, K V; Dubrovina, A S; Tyunin, A P

    2015-03-01

    During the polymerase chain reaction (PCR), which is a versatile and widely used method, certain DNA sequences are rapidly amplified through thermocycling. Although there are numerous protocols of PCR optimization for different applications, little is known about the effect of DNA modifications, such as DNA methylation, on PCR efficiency. Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR. In the present study, using plant DNA, we found that highly methylated plant DNA genomic regions were amplified with lower efficiencies compared to that for the regions methylated at a lower level. The correlation was observed when amplifying stilbene synthase (STS1, STS10) genes of Vitis amurensis, the Actin2 gene of Arabidopsis thaliana, the internal transcribed spacer (AtITS), and tRNAPro of A. thaliana. The level of DNA methylation within the analyzed DNA regions has been analyzed with bisulfite sequencing. The obtained data show that efficient PCRs of highly methylated plant DNA regions can be hampered. Proteinase K treatment of the plant DNA prior to PCR and using HotTaq DNA polymerase improved amplification of the highly methylated plant DNA regions. We suggest that increased DNA denaturation temperatures of the highly methylated DNA and contamination with DNA-binding proteins contribute to the hampered PCR amplification of highly methylated DNA. The data show that it is necessary to use current DNA purification protocols and commercial kits with caution to ensure appropriate PCR product yield and prevent bias toward unmethylated DNA amplification in PCRs. PMID:25506767

  19. Early diagnosis of Lassa fever by reverse transcription-PCR.

    PubMed Central

    Demby, A H; Chamberlain, J; Brown, D W; Clegg, C S

    1994-01-01

    We developed a method based on a coupled reverse transcription-PCR (RT-PCR) for the detection of Lassa virus using primers specific for regions of the S RNA segment which are well conserved between isolates from Sierra Leone, Liberia, and Nigeria. The specificity of the assay was confirmed by Southern blotting with a chemiluminescent probe. The assay was able to detect 1 to 10 copies of a plasmid or an RNA transcript containing the target sequence. There was complete concordance between RT-PCR and virus culture for the detection of Lassa virus in a set of 29 positive and 32 negative serum samples obtained on admission to the hospital from patients suspected of having Lassa fever in Sierra Leone. Specificity was confirmed by the failure of amplification of specific products from serum samples collected from 129 healthy blood donors in Sierra Leone or from tissue culture supernatants from cells infected with related arenaviruses (Mopeia, lymphocytic choriomeningitis, Tacaribe, and Pichinde viruses). Sequential serum samples from 29 hospitalized patients confirmed to have Lassa fever were tested by RT-PCR and for Lassa virus-specific antibodies by indirect immunofluorescence (IF). RT-PCR detected virus RNA in 79% of the patients at the time of admission, comparing favorably with IF, which detected antibodies in only 21% of the patients. Lassa virus RNA was detected by RT-PCR in all 29 patients by the third day of admission, whereas antibody was detectable by IF in only 52% of the patients. These results point to an important role for RT-PCR in the management of suspected cases of Lassa fever. Images PMID:7883875

  20. Anti-PcrV antibody strategies against virulent Pseudomonas aeruginosa.

    PubMed

    Sawa, Teiji; Ito, Emi; Nguyen, Vinh Huu; Haight, Matthew

    2014-01-01

    Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes fatal acute lung infections in critically ill individuals. Its pathogenesis is associated with bacterial virulence conferred by the type III secretion system (TTSS), through which P. aeruginosa causes necrosis of the lung epithelium and disseminates into the circulation, resulting in bacteremia, sepsis, and mortality. TTSS allows P. aeruginosa to directly translocate cytotoxins into eukaryotic cells, inducing cell death. The P. aeruginosa V-antigen PcrV, a homolog of the Yersinia V-antigen LcrV, is an indispensable contributor to TTS toxin translocation. Vaccination against PcrV ensures the survival of challenged mice and decreases lung inflammation and injury. Both the rabbit polyclonal anti-PcrV antibody and the murine monoclonal anti-PcrV antibody, mAb166, inhibit TTS toxin translocation. mAb166 IgG was cloned, and a molecular engineered humanized anti-PcrV IgG antigen-binding fragment, KB001, was developed for clinical use. KB001 is currently undergoing Phase-II clinical trials for ventilator-associated pneumonia in France and chronic pneumonia in cystic fibrosis in USA. In these studies, KB001 has demonstrated its safety, a favorable pharmacokinetic profile, and promising potential as a nonantibiotic strategy to reduce airway inflammation and damage in P. aeruginosa pneumonia. PMID:25483637

  1. Reproducibility problems with the AMPLICOR PCR Chlamydia trachomatis test.

    PubMed Central

    Peterson, E M; Darrow, V; Blanding, J; Aarnaes, S; de la Maza, L M

    1997-01-01

    In an attempt to use an expanded "gold standard" in an evaluation of an antigen detection test for Chlamydia trachomatis, the AMPLICOR (Roche Diagnostics Systems, Inc., Branchburg, N.J.) PCR Chlamydia trachomatis test and culture were used with 591 sets of cervical specimens. Of the 591 specimens assayed, 35 were retested due to either an equivocal result by the PCR (19 samples) or a discrepancy between the results of culture, PCR, and the antigen detection method. During the repeat testing of the samples with equivocal and discrepant results, all but one interpretation change was due to the PCR result. In addition, upon repeat testing the PCR assay value measured in optical density units varied widely for 13 of these specimens. These 13 specimens were then tested in triplicate by the manufacturer with primers to the chlamydia plasmid and in duplicate with primers to the major outer membrane protein. Only 3 of the 13 specimens gave the same interpretation with these five replicates. In summary, reproducibility problems with the AMPLICOR test should be considered before it is incorporated as part of routine testing or used as an expanded gold standard for chlamydia testing. PMID:9157161

  2. Multiplex PCR: Optimization and Application in Diagnostic Virology

    PubMed Central

    Elnifro, Elfath M.; Ashshi, Ahmed M.; Cooper, Robert J.; Klapper, Paul E.

    2000-01-01

    PCR has revolutionized the field of infectious disease diagnosis. To overcome the inherent disadvantage of cost and to improve the diagnostic capacity of the test, multiplex PCR, a variant of the test in which more than one target sequence is amplified using more than one pair of primers, has been developed. Multiplex PCRs to detect viral, bacterial, and/or other infectious agents in one reaction tube have been described. Early studies highlighted the obstacles that can jeopardize the production of sensitive and specific multiplex assays, but more recent studies have provided systematic protocols and technical improvements for simple test design. The most useful of these are the empirical choice of oligonucleotide primers and the use of hot start-based PCR methodology. These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. This article reviews the principles, optimization, and application of multiplex PCR for the detection of viruses of clinical and epidemiological importance. PMID:11023957

  3. Performance of Nanoliter-Sized Droplet-based Microfluidic PCR

    PubMed Central

    Wang, Fang; Burns, Mark A.

    2010-01-01

    A microfluidic device was used to characterize PCR in aqueous-in-oil droplets for potential point-of-care applications. Droplets with a volume range of 5–250nL can be formed on-chip reproducibly, and PCR in the droplets shows amplification efficiencies comparable to benchtop reactions with no evaporation loss. A higher polymerase concentration is required in the reaction droplet while the optimal Magnesium ion concentration is the same for both on-chip and benchtop systems. The optimal hold time is 9 and 30 seconds for denaturation and annealing/extension in thermal cycling, respectively. With the optimized cycling parameters, the total reaction time is reduced to half of that required for benchtop PCR. For the droplets containing the same quantity of template DNA, the PCR yield is approximately the same with either fixed droplet size or fixed template DNA concentration. The droplet-based PCR can be monitored in real time with FRET probes, and provide amplification with a cycle threshold of ~10 cycles earlier than the benchtop instruments. PMID:19479169

  4. [Pnemocystis jiroveci pneumonia: Comparison between conventional PCR and staining techniques].

    PubMed

    Kaouech, E; Kallel, K; Anane, S; Belhadj, S; Abdellatif, S; Mnif, K; Ben Othmane, T; Ben Lakhal, S; Kilani, B; Ben Châabane, T; Chaker, E

    2009-07-01

    Diagnosis of pneumocystis pneumonia is usually based on clinical features and X-rays photography and confirmed in the laboratory by visualisation of Pneumocystis organisms in stained preparations of respiratory specimens using several techniques (Gomori-Grocott, May-Grünwald Giemsa, bleu de toluidine O). Actually, PCR has considerably increased sensitivity of detection of Pneumocystis. The aim of this study is to compare conventional PCR results to those of staining techniques (Gomori-Grocott, May-Grünwald Giemsa) in addition to the X-ray and clinical findings in order to evaluate the contribution of each method. Sixty-four respiratory specimens were collected from 54 immuno-compromised patients with clinical symptoms of pulmonary infection. We diagnosed pneumocystis pneumonia in 16 patients according to staining techniques and/or typical clinical and radiological findings and/or response to treatment. Of the 15 patients, 14 were positive by PCR and only five were positive by direct examination, yielding a sensitivity and specificity of 93.3 and 87.1% for PCR and 33.3 and 100% for staining techniques. Conventional PCR provides a sensitive and objective method for the detection Pneumocystis jiroveci from less invasive sample. PMID:19038508

  5. Digital PCR using micropatterned superporous absorbent array chips.

    PubMed

    Wang, Yazhen; Southard, Kristopher M; Zeng, Yong

    2016-06-21

    Digital PCR (dPCR) is an emerging technology for genetic analysis and clinical diagnostics. To facilitate the widespread application of dPCR, here we developed a new micropatterned superporous absorbent array chip (μSAAC) which consists of an array of microwells packed with highly porous agarose microbeads. The packed beads construct a hierarchically porous microgel which confers superior water adsorption capacity to enable spontaneous filling of PDMS microwells for fluid compartmentalization without the need of sophisticated microfluidic equipment and operation expertise. Using large λ-DNA as the model template, we validated the μSAAC for stochastic partitioning and quantitative digital detection of DNA molecules. Furthermore, as a proof-of-concept, we conducted dPCR detection and single-molecule sequencing of a mutation prevalent in blood cancer, the chromosomal translocation t(14;18), demonstrating the feasibility of the μSAAC for analysis of disease-associated mutations. These experiments were carried out using the standard molecular biology techniques and instruments. Because of its low cost, ease of fabrication, and equipment-free liquid partitioning, the μSAAC is readily adaptable to general lab settings, which could significantly facilitate the widespread application of dPCR technology in basic research and clinical practice. PMID:27010726

  6. A QUANTITATIVE MODEL OF ERROR ACCUMULATION DURING PCR AMPLIFICATION

    PubMed Central

    Pienaar, E; Theron, M; Nelson, M; Viljoen, HJ

    2006-01-01

    The amplification of target DNA by the polymerase chain reaction (PCR) produces copies which may contain errors. Two sources of errors are associated with the PCR process: (1) editing errors that occur during DNA polymerase-catalyzed enzymatic copying and (2) errors due to DNA thermal damage. In this study a quantitative model of error frequencies is proposed and the role of reaction conditions is investigated. The errors which are ascribed to the polymerase depend on the efficiency of its editing function as well as the reaction conditions; specifically the temperature and the dNTP pool composition. Thermally induced errors stem mostly from three sources: A+G depurination, oxidative damage of guanine to 8-oxoG and cytosine deamination to uracil. The post-PCR modifications of sequences are primarily due to exposure of nucleic acids to elevated temperatures, especially if the DNA is in a single-stranded form. The proposed quantitative model predicts the accumulation of errors over the course of a PCR cycle. Thermal damage contributes significantly to the total errors; therefore consideration must be given to thermal management of the PCR process. PMID:16412692

  7. Optimizing methods for PCR-based analysis of predation

    PubMed Central

    Sint, Daniela; Raso, Lorna; Kaufmann, Rüdiger; Traugott, Michael

    2011-01-01

    Molecular methods have become an important tool for studying feeding interactions under natural conditions. Despite their growing importance, many methodological aspects have not yet been evaluated but need to be considered to fully exploit the potential of this approach. Using feeding experiments with high alpine carabid beetles and lycosid spiders, we investigated how PCR annealing temperature affects prey DNA detection success and how post-PCR visualization methods differ in their sensitivity. Moreover, the replicability of prey DNA detection among individual PCR assays was tested using beetles and spiders that had digested their prey for extended times postfeeding. By screening all predators for three differently sized prey DNA fragments (range 116–612 bp), we found that only in the longest PCR product, a marked decrease in prey detection success occurred. Lowering maximum annealing temperatures by 4 °C resulted in significantly increased prey DNA detection rates in both predator taxa. Among the three post-PCR visualization methods, an eightfold difference in sensitivity was observed. Repeated screening of predators increased the total number of samples scoring positive, although the proportion of samples testing positive did not vary significantly between different PCRs. The present findings demonstrate that assay sensitivity, in combination with other methodological factors, plays a crucial role to obtain robust trophic interaction data. Future work employing molecular prey detection should thus consider and minimize the methodologically induced variation that would also allow for better cross-study comparisons. PMID:21507208

  8. Multiplex PCR Tests for Detection of Pathogens Associated with Gastroenteritis

    PubMed Central

    Zhang, Hongwei; Morrison, Scott; Tang, Yi-Wei

    2016-01-01

    Synopsis A wide range of enteric pathogens can cause infectious gastroenteritis. Conventional diagnostic algorithms including culture, biochemical identification, immunoassay and microscopic examination are time consuming and often lack sensitivity and specificity. Advances in molecular technology have as allowed its use as clinical diagnostic tools. Multiplex PCR based testing has made its way to gastroenterology diagnostic arena in recent years. In this article we present a review of recent laboratory developed multiplex PCR tests and current commercial multiplex gastrointestinal pathogen tests. We will focus on two FDA cleared commercial syndromic multiplex tests: Luminex xTAG GPP and Biofire FimArray GI test. These multiplex tests can detect and identify multiple enteric pathogens in one test and provide results within hours. Multiplex PCR tests have shown superior sensitivity to conventional methods for detection of most pathogens. The high negative predictive value of these multiplex tests has led to the suggestion that they be used as screening tools especially in outbreaks. Although the clinical utility and benefit of multiplex PCR test are to be further investigated, implementing these multiplex PCR tests in gastroenterology diagnostic algorithm has the potential to improve diagnosis of infectious gastroenteritis. PMID:26004652

  9. Distinguishing human and possum faeces using PCR markers.

    PubMed

    Devane, M; Robson, B; Nourozi, F; Wood, D; Gilpin, B J

    2013-09-01

    Specificity testing of two published polymerase chain reaction (PCR) markers for the detection of human faecal pollution, revealed 100% false-positive rates to brush-tailed possum faeces (n = 10), but low false-positive rates against other potential pollution sources. Cross-reaction with possums could be a problem with other human-specific markers; therefore, a possum PCR marker was developed for use in conjunction with human PCR markers. The possum PCR marker was based on Bacteroidales 16S ribosomal ribonucleic acid sequences, and was tested on 233 individual faecal samples from 11 other animal species. Sensitivity of the possum marker in possum faeces (n = 36) was high at 83.3%. Cross-reactivity of the possum marker was limited to black swan (7/20 samples), human (2/48 samples) and rabbit (1/10) faecal samples, all at marker concentrations at least four orders of magnitude lower than possum faeces. The possum marker was not detected in human sewage or the faeces of other animal species. Specificity of the possum PCR marker, therefore, was high at 95.7%. To exclude the possibility that only possum pollution is being detected, additional testing by other faecal source tracking methods is required where the water sample is positive for both human and possum markers. PMID:23981869

  10. 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.

  11. An improved, PCR-based strategy for the detection of Trypanosoma cruzi in human blood samples.

    PubMed

    Ribeiro-dos-Santos, G; Nishiya, A S; Sabino, E C; Chamone, D F; Saez-Alquézar, A

    1999-10-01

    Attempts were made to improve the PCR-based detection of Trypanosoma cruzi in blood samples, primarily for screening blood donors. Samples were obtained from candidate donors who were reactive in one or two of three serological tests for Chagas disease (and therefore considered 'indeterminate') or in all three tests (3+). Each sample was then examined using three different, PCR-based techniques: 'PCR-I' (in which the target DNA is a nuclear repetitive sequence); 'PCR-II' [amplifying a conserved region of the T. cruzi kinetoplast DNA (kDNA)]; and 'PCR-III' (a new strategy in which the target kDNA is amplified by 'nested' PCR). Among the samples from 3+ individuals, PCR-I, PCR-II and PCR-III amplified two (3.8%) out of 52, four (4.5%) out of 88, and 27 (25.7%) out of 105 samples tested, respectively. Seven, 69 and 70 samples from 'indeterminate' subjects were tested by PCR-I, PCR-II and PCR-III, respectively; there was not a single positive result by PCR-I or PCR-II, but three (4.3%) of the samples tested by PCR-III were positive. In a reconstruction experiment, in conditions in which PCR-I and PCR-II could not detect 10,000 parasites/ml, PCR-III was able to detect one parasite/ml. Although all three PCR-based strategies examined had rather poor sensitivities, PCR-III was far more sensitive than PCR-I or PCR-II. PMID:10715696

  12. Using PCR to Target Misconceptions about Gene Expression †

    PubMed Central

    Wright, Leslie K.; Newman, Dina L.

    2013-01-01

    We present a PCR-based laboratory exercise that can be used with first- or second-year biology students to help overcome common misconceptions about gene expression. Biology students typically do not have a clear understanding of the difference between genes (DNA) and gene expression (mRNA/protein) and often believe that genes exist in an organism or cell only when they are expressed. This laboratory exercise allows students to carry out a PCR-based experiment designed to challenge their misunderstanding of the difference between genes and gene expression. Students first transform E. coli with an inducible GFP gene containing plasmid and observe induced and un-induced colonies. The following exercise creates cognitive dissonance when actual PCR results contradict their initial (incorrect) predictions of the presence of the GFP gene in transformed cells. Field testing of this laboratory exercise resulted in learning gains on both knowledge and application questions on concepts related to genes and gene expression. PMID:23858358

  13. Identification of neotropical felid faeces using RCP-PCR.

    PubMed

    Roques, S; Adrados, B; Chavez, C; Keller, C; Magnusson, W E; Palomares, F; Godoy, J A

    2011-01-01

    Faeces similarity among sympatric felid species has generally hampered their use in distributional, demographic and dietary studies. Here, we present a new and simple approach based on a set of species-specific primers, for the unambiguous identification of faeces from sympatric neotropical felids (i.e. puma, jaguar, jaguarundi and ocelot/ margay). This method, referred to as rapid classificatory protocol-PCR (RCP-PCR), consists of a single-tube multiplex PCR yielding species-specific banding patterns on agarose gel. The method was optimized with samples of known origin (14 blood and 15 fresh faeces) and validated in faecal samples of unknown origin (n = 138), for some of which (n = 40) we also obtained species identification based on mtDNA sequencing. This approach proved reliable and provides high identification success rates from faeces. Its simplicity and cost effectiveness should facilitate its application for routine surveys of presence and abundance of these species. PMID:21429118

  14. Development of a PCR Assay for Rapid Detection of Enterococci

    PubMed Central

    Ke, Danbing; Picard, François J.; Martineau, Francis; Ménard, Christian; Roy, Paul H.; Ouellette, Marc; Bergeron, Michel G.

    1999-01-01

    Enterococci are becoming major nosocomial pathogens, and increasing resistance to vancomycin has been well documented. Conventional identification methods, which are based on culturing, require 2 to 3 days to provide results. PCR has provided a means for the culture-independent detection of enterococci in a variety of clinical specimens and is capable of yielding results in just a few hours. However, all PCR-based assays developed so far are species specific only for clinically important enterococci. We have developed a PCR-based assay which allows the detection of enterococci at the genus level by targeting the tuf gene, which encodes elongation factor EF-Tu. Initially, we compared the nucleotide sequences of the tuf gene from several bacterial species (available in public databases) and designed degenerate PCR primers derived from conserved regions. These primers were used to amplify a target region of 803 bp from four enterococcal species (Enterococcus avium, E. faecalis, E. faecium, and E. gallinarum). Subsequently, the complete nucleotide sequences of these amplicons were determined. The analysis of a multiple alignment of these sequences revealed regions conserved among enterococci but distinct from those of other bacteria. PCR primers complementary to these regions allowed amplification of genomic DNAs from 14 of 15 species of enterococci tested (E. solitarius DNA could not be amplified). There was no amplification with a majority of 79 nonenterococcal bacterial species, except for 2 Abiotrophia species and several Listeria species. Furthermore, this assay efficiently amplified all 159 clinical isolates of enterococci tested (61 E. faecium, 77 E. faecalis, 9 E. gallinarum, and 12 E. casseliflavus isolates). Interestingly, the preliminary sequence comparison of the amplicons for four enterococcal species demonstrated that there were some sequence variations which may be used to generate species-specific internal probes. In conclusion, this rapid PCR

  15. 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.

  16. Detection and Quantification of Wallemia sebi in Aerosols by Real-Time PCR, Conventional PCR, and Cultivation

    PubMed Central

    Zeng, Qing-Yin; Westermark, Sven-Olof; Rasmuson-Lestander, Åsa; Wang, Xiao-Ru

    2004-01-01

    Wallemia sebi is a deuteromycete fungus commonly found in agricultural environments in many parts of the world and is suspected to be a causative agent of farmer's lung disease. The fungus grows slowly on commonly used culture media and is often obscured by the fast-growing fungi. Thus, its occurrence in different environments has often been underestimated. In this study, we developed two sets of PCR primers specific to W. sebi that can be applied in either conventional PCR or real-time PCR for rapid detection and quantification of the fungus in environmental samples. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. These methods were employed to investigate the presence of W. sebi in the aerosols of a farm. The results revealed a high concentration of W. sebi spores, 107 m−3 by real-time PCR and 106 m−3 by cultivation, which indicates the prevalence of W. sebi in farms handling hay and grain and in cow barns. The methods developed in this study could serve as rapid, specific, and sensitive means of detecting W. sebi in aerosol and surface samples and could thus facilitate investigations of its distribution, ecology, clinical diagnosis, and exposure risk assessment. PMID:15574929

  17. Revealing the Diversity and Quantity of Peritrich Ciliates in Environmental Samples Using Specific Primer-based PCR and Quantitative PCR

    PubMed Central

    Liu, Xihan; Gong, Jun

    2012-01-01

    Peritrichs are a diverse, ecologically important ciliate group usually with a complex life cycle. To date, the community of the peritrichs has been investigated by using morphology-based methods such as living observation and silver staining. Here we show a molecular approach for characterizing the diversity and quantity of free-living peritrichs in environmental samples. We newly designed four peritrich-specific primers targeting 18S rRNA genes that allow clone library construction, screening and analysis. A quantitative real-time PCR (qPCR) assay was developed to quantify peritrichs in environmental samples by using rDNA copy number as an indicator. DNA extracted from four water samples of contrasting environmental gradients was analysed. The results showed that the peritrich community was differentiated among these samples, and that the diversity decreased with the increase of water salinity. The qPCR results are consistent with the library sequence analysis in terms of quantity variations from sample to sample. The development of peritrich-specific primers, for the first time, for conventional PCR and qPCR assays, provides useful molecular tools for revealing the diversity and quantity of peritrich ciliates in environmental samples. Also, our study illustrates the potential of these molecular tools to ecological studies of other ciliate groups in diverse environments. PMID:23100023

  18. Optimized 5-hour multiplex PCR test for the detection of tinea unguium: performance in a routine PCR laboratory.

    PubMed

    Brillowska-Dabrowska, Anna; Nielsen, Sanne Søgaard; Nielsen, Henrik Vedel; Arendrup, Maiken Cavling

    2010-09-01

    We recently reported the development of a 5-hour multiplex PCR test for the detection of tinea unguium and the optimization of this test by the inclusion of an inhibition control. Here we report the performance of this procedure as used in a routine clinical laboratory as compared to conventional microscopy and culture-based techniques performed in a mycology reference laboratory. We found in processing 109 samples that 22 (20.2%) yielded fungi in culture while the suspected etiologic agents were noted microscopically in 15 (13.8%) that were negative in culture. Fungi were detected by PCR in 37 (33.9%) samples, of which only three were positive in culture. Since the majority of PCR positive but culture negative samples were positive in microscopic examinations, the increased sensitivity was not due to contamination. PCR inhibitors were present in 5% of the samples, but this was overcome by re-running the samples with a 50% reduction of sample DNA. In conclusion, the PCR test performance in the routine setting was excellent and provided a markedly reduced time to diagnosis with a higher sensitivity. PMID:20105101

  19. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    SciTech Connect

    Gardner, S. N.

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether there are also TaqMan/Luminex probe matches within predicted amplicons.

  20. PCR Amplicon Prediction from Multiplex Degenerate Primer and Probe Sets

    Energy Science and Technology Software Center (ESTSC)

    2013-08-08

    Assessing primer specificity and predicting both desired and off-target amplification products is an essential step for robust PCR assay design. Code is described to predict potential polymerase chain reaction (PCR) amplicons in a large sequence database such as NCBI nt from either singleplex or a large multiplexed set of primers, allowing degenerate primer and probe bases, with target mismatch annotates amplicons with gene information automatically downloaded from NCBI, and optionally it can predict whether theremore » are also TaqMan/Luminex probe matches within predicted amplicons.« less

  1. [Do Multiplex PCR techniques displace classical cultures in microbiology?].

    PubMed

    Auckenthaler, Raymond; Risch, Martin

    2015-02-01

    Multiplex PCR technologies progressively find their way in clinical microbiology. This technique allows the simultaneous amplification of multiple DNA targets in a single test run for the identification of pathogens up to the species level. Various pathogens of infectious diseases can be detected by a symptom-oriented approach clearly and quickly with high reliability. Essentially multiplex PCR panels are available for clarification of gastrointestinal, respiratory, sexually transmitted infections and meningitis. Today's offer from industry, university hospitals and large private laboratories of Switzerland is tabulated and commented. PMID:25630288

  2. Use of Treponema pallidum PCR in Testing of Ulcers for Diagnosis of Primary Syphilis1

    PubMed Central

    Sednaoui, Patrice; Lautenschlager, Stephan; Ferry, Tristan; Toutous-Trellu, Laurence; Cavassini, Matthias; Yassir, Fatima; Martinez de Tejada, Begoña; Emonet, Stéphane; Combescure, Christophe; Schrenzel, Jacques; Perneger, Thomas

    2015-01-01

    Treponema pallidum PCR (Tp-PCR) has been noted as a valid method for diagnosing syphilis. We compared Tp-PCR to a combination of darkfield microscopy (DFM), the reference method, and serologic testing in a cohort of 273 patients from France and Switzerland and found the diagnostic accuracy of Tp-PCR was higher than that for DFM. PMID:25531672

  3. Use of Treponema pallidum PCR in testing of ulcers for diagnosis of primary syphilis.

    PubMed

    Gayet-Ageron, Angèle; Sednaoui, Patrice; Lautenschlager, Stephan; Ferry, Tristan; Toutous-Trellu, Laurence; Cavassini, Matthias; Yassir, Fatima; Martinez de Tejada, Begoña; Emonet, Stéphane; Combescure, Christophe; Schrenzel, Jacques; Perneger, Thomas

    2015-01-01

    Treponema pallidum PCR (Tp-PCR) has been noted as a valid method for diagnosing syphilis. We compared Tp-PCR to a combination of darkfield microscopy (DFM), the reference method, and serologic testing in a cohort of 273 patients from France and Switzerland and found the diagnostic accuracy of Tp-PCR was higher than that for DFM. PMID:25531672

  4. Use of Droplet Digital PCR for Estimation of Fish Abundance and Biomass in Environmental DNA Surveys

    PubMed Central

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

    2015-01-01

    An environmental DNA (eDNA) analysis method has been recently developed to estimate the distribution of aquatic animals by quantifying the number of target DNA copies with quantitative real-time PCR (qPCR). A new quantitative PCR technology, droplet digital PCR (ddPCR), partitions PCR reactions into thousands of droplets and detects the amplification in each droplet, thereby allowing direct quantification of target DNA. We evaluated the quantification accuracy of qPCR and ddPCR to estimate species abundance and biomass by using eDNA in mesocosm experiments involving different numbers of common carp. We found that ddPCR quantified the concentration of carp eDNA along with carp abundance and biomass more accurately than qPCR, especially at low eDNA concentrations. In addition, errors in the analysis were smaller in ddPCR than in qPCR. Thus, ddPCR is better suited to measure eDNA concentration in water, and it provides more accurate results for the abundance and biomass of the target species than qPCR. We also found that the relationship between carp abundance and eDNA concentration was stronger than that between biomass and eDNA by using both ddPCR and qPCR; this suggests that abundance can be better estimated by the analysis of eDNA for species with fewer variations in body mass. PMID:25799582

  5. A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples.

    PubMed

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

    2015-05-18

    Sensitive and quantitative detection of foodborne enteric viruses is classically achieved by quantitative RT-PCR (RT-qPCR). Recently, digital PCR (dPCR) was described as a novel approach to genome quantification without need for a standard curve. The performance of microfluidic digital RT-PCR (RT-dPCR) was compared to RT-qPCR for detecting the main viruses responsible for foodborne outbreaks (human Noroviruses (NoV) and Hepatitis A virus (HAV)) in spiked lettuce and bottled water. Two process controls (Mengovirus and Murine Norovirus) were used and external amplification controls (EAC) were added to examine inhibition of RT-qPCR and RT-dPCR. For detecting viral RNA and cDNA, the sensitivity of the RT-dPCR assays was either comparable to that of RT-qPCR (RNA of HAV, NoV GI, Mengovirus) or slightly (around 1 log10) decreased (NoV GII and MNV-1 RNA and of HAV, NoV GI, NoV GII cDNA). The number of genomic copies determined by dPCR was always from 0.4 to 1.7 log10 lower than the expected numbers of copies calculated by using the standard qPCR curve. Viral recoveries calculated by RT-dPCR were found to be significantly higher than by RT-qPCR for NoV GI, HAV and Mengovirus in water, and for NoV GII and HAV in lettuce samples. The RT-dPCR assay proved to be more tolerant to inhibitory substances present in lettuce samples. This absolute quantitation approach may be useful to standardize quantification of enteric viruses in bottled water and lettuce samples and may be extended to quantifying other human pathogens in food samples. PMID:25725459

  6. Methods for producing partially digested restriction DNA fragments and for producing a partially modified PCR product

    DOEpatents

    Wong, Kwong-Kwok

    2000-01-01

    The present invention is an improved method of making a partially modified PCR product from a DNA fragment with a polymerase chain reaction (PCR). In a standard PCR process, the DNA fragment is combined with starting deoxynucleoside triphosphates, a primer, a buffer and a DNA polymerase in a PCR mixture. The PCR mixture is then reacted in the PCR producing copies of the DNA fragment. The improvement of the present invention is adding an amount of a modifier at any step prior to completion of the PCR process thereby randomly and partially modifying the copies of the DNA fragment as a partially modified PCR product. The partially modified PCR product may then be digested with an enzyme that cuts the partially modified PCR product at unmodified sites thereby producing an array of DNA restriction fragments.

  7. Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution.

    PubMed

    Weaver, Suzanne; Dube, Simant; Mir, Alain; Qin, Jian; Sun, Gang; Ramakrishnan, Ramesh; Jones, Robert C; Livak, Kenneth J

    2010-04-01

    This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies, which corresponds to a 1.25-fold difference in relative quantity. Digital PCR is considered as an alternative form of qPCR. For digital PCR, an error model is shown that relates the precision of CNV determination to the number of reaction chambers. The quantitative capability of digital PCR is illustrated with an experiment distinguishing four and five copies of the human gene MRGPRX1. For either real-time qPCR or digital PCR, practical application of these models to achieve enhanced quantitative resolution requires use of a high throughput PCR platform that can simultaneously perform thousands of reactions. Comparing the two methods, real-time qPCR has the advantage of throughput and digital PCR has the advantage of simplicity in terms of the assumptions made for data analysis. PMID:20079846

  8. Use of multiplex PCR and PCR restriction enzyme analysis for detection and exploration of the variability in the free-living amoeba Naegleria in the environment.

    PubMed

    Pélandakis, Michel; Pernin, Pierre

    2002-04-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. PMID:11916734

  9. Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living Amoeba Naegleria in the Environment

    PubMed Central

    Pélandakis, Michel; Pernin, Pierre

    2002-01-01

    A multiplex PCR was developed to simultaneously detect Naegleria fowleri and other Naegleria species in the environment. Multiplex PCR was also capable of identifying N. fowleri isolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilic Naegleria species from the sampling sites. PMID:11916734

  10. PCR detection of bovine herpesviruses from nonbovine ruminants in Hungary.

    PubMed

    Kálmán, Dóra; Egyed, László

    2005-07-01

    Polymerase chain reaction (PCR) was used to test six different nonbovine ruminant species for five bovine herpesviruses including infectious bovine rhinotracheitis virus (BoHV-1), bovine herpes mammillitis virus (BoHV-2), Movar-type herpesvirus (BoHV-4), bovine herpesvirus type 5 (BoHV-5), and alcelaphine herpesvirus 1 (AlHV-1). Species tested included 56 roe deer (Capreolus capreolus), 66 red deer (Cervus elaphus), 20 fallow deer (Dama dama), 16 mouflon (Ovis musimon), 34 domestic sheep, and 44 domestic goats, which were sampled in Hungary in 2003. Tracheal and popliteal lymph nodes collected from these animals were tested for the presence of the five bovine herpesviruses using three nested (two of which were duplex) PCR assays. Three bovine herpesviruses (BoHV-1, -2, and -4) were detected, whereas no evidence of AlHV-1 or BoHV-5 was observed. Prevalence of BoHV-1 ranged from 12% to 47%, and PCR-positive results were observed in all species tested. BoHV-2 was detected from roe deer, red deer, fallow deer, mouflon, and domestic sheep, and the prevalence in these species ranged from 3% to 50%. BoHV-4 was detected in all species, with prevalence ranging from 12% to 69%. Sequenced PCR products were 99-100% identical to bovine herpesviral sequences deposited in the GenBank. PMID:16244057

  11. PCR-BASED IDENTIFICATION OF BACTERIA IN ENDODONTIC INFECTIONS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR primers, that target the bacterial 16S rRNA genes (or the tuf gene for Enterococcus genus), were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were invest...

  12. Quantitative Detection of Spiroplasma Citri by Real Time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is a need to develop an accurate and rapid method to detect Spiroplasma citri, the causal agent of citrus stubborn disease for use in epidemiology studies. Quantitative real-time PCR was developed for detection of S. citri. Two sets of primers based on sequences from the P58 putative adhesin ...

  13. A Trio of Human Molecular Genetics PCR Assays

    ERIC Educational Resources Information Center

    Reinking, Jeffrey L.; Waldo, Jennifer T.; Dinsmore, Jannett

    2013-01-01

    This laboratory exercise demonstrates three different analytical forms of the polymerase chain reaction (PCR) that allow students to genotype themselves at four different loci. Here, we present protocols to allow students to a) genotype a non-coding polymorphic Variable Number of Tandem Repeat (VNTR) locus on human chromosome 5 using conventional…

  14. MOLD SPECIFIC QUANTITATIVE PCR: THE EMERGING STANDARD IN MOLD ANALYSIS

    EPA Science Inventory

    Today I will talk about the use of quantitative or Real time PCR for the standardized identification and quantification of molds. There are probably at least 100,000 species of molds or fungi. But there are actually about 100 typically found indoors. Some pose a threat to human...

  15. Qualitative PCR method for Roundup Ready soybean: interlaboratory study.

    PubMed

    Kodama, Takashi; Kasahara, Masaki; Minegishi, Yasutaka; Futo, Satoshi; Sawada, Chihiro; Watai, Masatoshi; Akiyama, Hiroshi; Teshima, Reiko; Kurosawa, Yasunori; Furui, Satoshi; Hino, Akihiro; Kitta, Kazumi

    2011-01-01

    Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots. PMID:21391499

  16. UTILIZATION OF PCR TO DETECT SALMONELLA ON TURKEY CARCASSES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The risk which is presented by food-borne pathogens to the consumer demonstrates the need to utilize rapid methods for the detection of these microbes. This study compared conventional microbiology with the application of PCR assays to detect Salmonella on turkey carcasses at a processing plant in ...

  17. Real-time PCR for Strongyloides stercoralis-associated meningitis.

    PubMed

    Nadir, Eyal; Grossman, Tamar; Ciobotaro, Pnina; Attali, Malka; Barkan, Daniel; Bardenstein, Rita; Zimhony, Oren

    2016-03-01

    Four immunocompromised patients, immigrants from Ethiopia, presented with diverse clinical manifestations of meningitis associated with Strongyloides stercoralis dissemination as determined by identification of intestinal larvae. The cerebrospinal fluid of 3 patients was tested by a validated (for stool) real-time PCR for S. stercoralis and was found positive, establishing this association. PMID:26704620

  18. Multiplex PCR Method for Identifying Recombinant Vaccine-Related Polioviruses

    PubMed Central

    Kilpatrick, David R.; Ching, Karen; Iber, Jane; Campagnoli, Ray; Freeman, Christopher J.; Mishrik, Nada; Liu, Hong-Mei; Pallansch, Mark A.; Kew, Olen M.

    2004-01-01

    The recent discovery of recombinant circulating vaccine-derived poliovirus (recombinant cVDPV) has highlighted the need for enhanced global poliovirus surveillance to assure timely detection of any future cVDPV outbreaks. Six pairs of Sabin strain-specific recombinant primers were designed to permit rapid screening for VDPV recombinants by PCR. PMID:15365031

  19. Detection of major diarrheagenic bacterial pathogens by multiplex PCR panels.

    PubMed

    Sjöling, Åsa; Sadeghipoorjahromi, Leila; Novak, Daniel; Tobias, Joshua

    2015-03-01

    Diarrheal diseases remain a major threat to the youngest population in low- and middle-income countries. The main bacterial pathogens causing diarrhea are diarrheagenic Escherichia coli (DEC) that consists of enteroaggregative (EAEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorrhagic EHEC and enteroinvasive E. coli (EIEC), Salmonella, Shigella spp. (S. dysenteria, S. sonnei, S. flexneri) Campylobacter (C. coli, C. jejuni), Vibrio (V. vulnificus, V. parahaemolyticusm, V. cholerae), Yersinia enterocolitica and Aeromonas hydrophila. The aim of this study was to set up rapid multiplex PCR (mPCR) panels to identify these diarrheagenic pathogens based on their specific virulence genes. Primers against specific target genes were combined into three mPCR panels: one for diarrheal E. coli, one for pathogens causing mainly bloody diarrhea, and the third for the remaining pathogens. The panels were tested against a set of stool samples from Swedish children with diarrhea and controls and the analysis identified bacterial pathogens in 14/54 (26%) of the samples. These results show that our three developed mPCR panels can detect main bacterial diarrheagenic pathogens in clinical samples. PMID:25542594

  20. 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) ...

  1. Citrus stubborn disease incidence determined by quantitative real time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time (q) PCR was developed for detection of Spiroplasma citri, the causal agent of citrus stubborn disease (CSD), using the DNA binding fluorophore SYBR Green I. The primer pair, P58-3f/4r, developed based on sequences from the P58 putative adhesin multigene of the pathogen result...

  2. Detection of Treponema pallidum by a sensitive reverse transcriptase PCR.

    PubMed Central

    Centurion-Lara, A; Castro, C; Shaffer, J M; Van Voorhis, W C; Marra, C M; Lukehart, S A

    1997-01-01

    Syphilis is diagnosed by serologic testing or by identification of the causative agent, Treponema pallidum. The bacterium has historically been detected in clinical specimens by dark-field microscopy, immunostaining with polyclonal or monoclonal antibodies, or the rabbit inoculation test (RIT). RIT is considered to be very sensitive and specific, although it is available only in research settings and is not clinically useful due to the length of time required to obtain a result. In recent years, several PCR methods have been developed for the detection of T. pallidum, but none of these has shown a clear advantage in sensitivity over RIT. We have developed a specific and highly sensitive reverse transcriptase PCR (RT-PCR) that targets a 366 bp region of the 16S rRNA of T. pallidum. This RT-PCR can detect a single organism by Southern analysis when whole organisms are diluted and 10(-2) to 10(-3) T. pallidum organisms when RNA equivalents are used to make cDNA. The test was demonstrated to detect 10(-2) T. pallidum RNA equivalents in cerebrospinal fluid. Twenty different strains of T. pallidum, isolated from cerebrospinal fluids, aqueous humor, blood, and chancres, were shown to be detectable by this test. This efficient and sensitive technique could be more useful than existing methods for detecting very low numbers of organisms in clinical samples. PMID:9163442

  3. PCR-Based Identification of Bacteria Associated with Endodontic Infections

    PubMed Central

    Fouad, Ashraf F.; Barry, Jody; Caimano, Melissa; Clawson, Michael; Zhu, Qiang; Carver, Rachaele; Hazlett, Karsten; Radolf, Justin D.

    2002-01-01

    PCR primers that target the bacterial 16S rRNA genes (or the tuf gene for the genus Enterococcus) were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samples from the root canals of 24 teeth with necrotic pulp were included in the study. PCR with universal bacterial primers identified bacterial DNA in 22 specimens; the remaining 2 specimens were from intact teeth that had been traumatized 6 months prior to treatment. PCR with specific primers showed that preoperative symptoms were significantly associated with the presence of Streptococcus spp. (P < 0.001 by chi-square analysis). There was also a nonsignificant trend for symptoms to be associated with Fusobacterium nucleatum and Porphyromonas gingivalis (odds ratio, >2) and for diabetes mellitus to be associated with P. gingivalis and Porphyromonas endodontalis (odds ratio, >2). Cloning and sequencing of the universal PCR product in one specimen revealed the presence of an organism related to the genus Olsenella, which has not previously been described in endodontic infections. PMID:12202557

  4. FTA card utility for PCR detection of Mycobacterium leprae.

    PubMed

    Aye, Khin Saw; Matsuoka, Masanori; Kai, Masanori; Kyaw, Kyaw; Win, Aye Aye; Shwe, Mu Mu; Thein, Min; Htoo, Maung Maung; Htoon, Myo Thet

    2011-01-01

    The suitability of the FTA® elute card for the collection of slit skin smear (SSS) samples for PCR detection of Mycobacterium leprae was evaluated. A total of 192 SSS leprosy samples, of bacillary index (BI) 1 to 5, were collected from patients attending two skin clinics in Myanmar and preserved using both FTA® elute cards and 70% ethanol tubes. To compare the efficacy of PCR detection of DNA from each BI class, PCR was performed to amplify an M. leprae-specific repetitive element. Of the 192 samples, 116 FTA® elute card and 112 70% ethanol samples were PCR positive for M. leprae DNA. When correlated with BI, area under the curve (AUC) values of the respective receiver-operating characteristic curves were similar for the FTA® elute card and ethanol collection methods (AUC=0.6). Taken together, our results indicate that the FTA® elute card, which enables the collection, transport, and archiving of clinical samples, is an attractive alternative to ethanol preservation for the detection of M. leprae DNA. PMID:21617312

  5. Facilitated Molecular Typing of Shigella Isolates Using ERIC-PCR

    PubMed Central

    Kosek, Margaret; Yori, Pablo Peñataro; Gilman, Robert H.; Vela, Henry; Olortegui, Maribel Paredes; Chavez, Cesar Banda; Calderon, Maritza; Bao, Juan Perez; Hall, Eric; Maves, Ryan; Burga, Rosa; Sanchez, Graciela Meza

    2012-01-01

    To evaluate the performance of enterobacterial repetitive intergenic sequence-based polymerase chain reaction (ERIC-PCR) typing versus the current standard for the typing of Shigella pulsed gel electrophoresis (PFGE), we typed 116 Shigella isolates from a village in an endemic setting over a 20-month period using both methods. PFGE identified 37 pulse types and had a discrimination index of 0.925 (95% confidence interval = 0.830–1.00), whereas ERIC-PCR identified 42 types and had a discrimination index of 0.961 (95% confidence interval = 0.886–1.00). PFGE and ERIC-PCR showed a 90.4% correlation in the designation of isolates as clonal or non-clonal in pairwise comparisons. Both systems were highly reproducible and provided highly similar and supplementary data compared with serotyping regarding the transmission dynamics of shigellosis in this community. ERIC-PCR is considerably more rapid and inexpensive than PFGE and may have a complementary role to PFGE for initial investigations of hypothesized outbreaks in resource-limited settings. PMID:22665611

  6. Human anaplasmosis: the first Spanish case confirmed by PCR.

    PubMed

    García, J C; Núñez, M J; Castro, B; Fraile, F J; López, A; Mella, M C; Blanco, A; Sieira, C; Loureiro, E; Portillo, A; Oteo, J A

    2006-10-01

    We report a case of human anaplasmosis (HA) fulfilling the confirmation criteria: epidemiologic data and clinical picture compatible with HA; presence of a morulae within polymorphonuclear leukocyte; and positive PCR assay for Anaplasma phagocytophilum: This case report shows the presence of HA in Spain. PMID:17114773

  7. PCR detection of Helicobacter pylori in clinical samples.

    PubMed

    Rimbara, Emiko; Sasatsu, Masanori; Graham, David Y

    2013-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etiologically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (gastric biopsy for histology, culture, or PCR) tests are used for diagnosis. PCR detection of H. pylori has been reported using a variety of clinical samples including gastric biopsy, gastric juice, saliva, dental plaque, and stools as well as environmental samples. Whenever possibly, noninvasive tests are preferred over invasive tests. H. pylori are excreted in the stool. Culture from stool is variable whereas stool antigen testing is widely used. Stool consists of a complicated mixture of commensal bacteria and chemicals and often includes inhibitors of PCR. Nevertheless, simple extraction methods are available to efficiently extract DNA from human stools and nested-PCR targeting the 23S rRNA gene have proven to be highly sensitive for the detection of H. pylori. Detection of clarithromycin susceptibility/resistance is important clinically and the mutation of the 23S rRNA gene responsible for resistance can also be detected using stool. This described method can be modified for other clinical samples such as gastric juice or biopsy material. PMID:23104297

  8. Detection of carbapenemases in Enterobacteriaceae by a commercial multiplex PCR.

    PubMed

    Kaase, Martin; Szabados, Florian; Wassill, Lars; Gatermann, Sören G

    2012-09-01

    A commercial multiplex PCR (hyplex SuperBug ID) was tested with a collection of 132 clinical Enterobacteriaceae strains producing different carbapenemases. The sensitivity for the detection of KPC-, VIM-, NDM-, and OXA-48-encoding genes was 100%, whereas two IMP variants were missed. PMID:22785190

  9. Detection of viable Cryptosporidium parvum oocysts by PCR.

    PubMed Central

    Wagner-Wiening, C; Kimmig, P

    1995-01-01

    PCR was used to detect and specifically identify a gene fragment from Cryptosporidium parvum. An 873-bp region of a 2,359-bp DNA fragment encoding a repetitive oocyst protein of C. parvum was shown to be specifically amplified in C. parvum. An excystation protocol before DNA extraction allowed the differentiation between live and dead Cryptosporidium parvum oocysts. PMID:8534121

  10. Halal authenticity of gelatin using species-specific PCR.

    PubMed

    Shabani, Hessam; Mehdizadeh, Mehrangiz; Mousavi, Seyed Mohammad; Dezfouli, Ehsan Ansari; Solgi, Tara; Khodaverdi, Mahdi; Rabiei, Maryam; Rastegar, Hossein; Alebouyeh, Mahmoud

    2015-10-01

    Consumption of food products derived from porcine sources is strictly prohibited in Islam. Gelatin, mostly derived from bovine and porcine sources, has many applications in the food and pharmaceutical industries. To ensure that food products comply with halal regulations, development of valid and reliable analytical methods is very much required. In this study, a species-specific polymerase chain reaction (PCR) assay using conserved regions of mitochondrial DNA (cytochrome b gene) was performed to evaluate the halal authenticity of gelatin. After isolation of DNA from gelatin powders with known origin, conventional PCR using species-specific primers was carried out on the extracted DNA. The amplified expected PCR products of 212 and 271 bp were observed for porcine and bovine gelatin, respectively. The sensitivity of the method was tested on binary gelatin mixtures containing 0.1%, 1%, 10%, and 100% (w/w) of porcine gelatin within bovine gelatin and vice versa. Although most of the DNA is degraded due to the severe processing steps of gelatin production, the minimum level of 0.1% w/w of both porcine and bovine gelatin was detected. Moreover, eight food products labeled as containing bovine gelatin and eight capsule shells were subjected to PCR examination. The results showed that all samples contained bovine gelatin, and the absence of porcine gelatin was verified. This method of species authenticity is very useful to verify whether gelatin and gelatin-containing food products are derived from halal ingredients. PMID:25872445

  11. 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

  12. A PCR test for avian malaria in Hawaiian birds.

    PubMed

    Feldman, R A; Freed, L A; Cann, R L

    1995-12-01

    The decline of native Hawaiian forest birds since European contact is attributed to factors ranging from habitat destruction to interactions with introduced species. Remaining populations of Hawaiian honeycreepers (Fringillidae: Drepanidinae) are most abundant and diverse in high elevation refuges above the normal range of disease-carrying mosquitoes. Challenge experiments suggest that honeycreepers are highly susceptible to avian malaria (Plasmodium sp.) but resistance exists in some species. In order to detect low levels of malarial infection and quantify prevalence of Plasmodium in high elevation natural populations of Hawaiian birds, a polymerase chain reaction (PCR) based diagnostic test was developed that identifies rRNA genes of Plasmodium in avian blood samples. Quantitative competitive PCR (QC-PCR) experiments indicate that the detection limit of our test is an order of magnitude greater than that reported for human malaria DNA blot tests. Compared with standard histological methods, the PCR test detected a higher prevalence of diseased birds at mid-elevations. Malaria was detected in three species of native birds living in a high elevation wildlife refuge on the island of Hawaii and in four species from Maui. Our results show that avian malaria is more widespread in Hawaiian forests than previously thought, a finding that has important conservation implications for these threatened species. PMID:8564006

  13. 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,…

  14. QUANTITATIVE PCR OF SELECTED ASPERGILLUS, PENICILLIUM AND PAECILOMYCES SPECIES

    EPA Science Inventory

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

  15. Genus identification of toxic plant by real-time PCR.

    PubMed

    Matsuyama, Shuji; Nishi, Katsuji

    2011-03-01

    Some plants have toxicities that are dangerous for humans. In the case of poisoning by toxic plants, a rapid and easy screening test is required for accurate medical treatment or forensic investigation. In this study, we designed specific primer pairs for identification of toxic plants, such as subgenus Aconitum, genus Ricinus, genus Illicium, and genus Scopolia, by internal transcribed spacer sequences of nuclear ribosomal DNA. Allied species of target plants, foods, and human DNA were not detected, but each primer pair provided a specific PCR product from the target plant using real-time PCR. This method can detect the subgenus Aconitum, genus Ricinus, and genus Scopolia with template DNA of 10 pg, respectively, and genus Illicium with 1 pg. Furthermore, each primer pair provided the exact PCR product from digested target plants in artificial gastric fluid. When a trace unknown plant sample in forensic investigation is collected from stomach contents, this PCR assay may be useful for screening toxic plants. PMID:20623131

  16. Using qPCR for Water Microbial Risk Assessments

    EPA Science Inventory

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

  17. Screening ancient tuberculosis with qPCR: challenges and opportunities

    PubMed Central

    Harkins, Kelly M.; Buikstra, Jane E.; Campbell, Tessa; Bos, Kirsten I.; Johnson, Eric D.; Krause, Johannes; Stone, Anne C.

    2015-01-01

    The field of ancient DNA (aDNA) has rapidly accelerated in recent years as a result of new methods in next-generation sequencing, library preparation and targeted enrichment. Such research is restricted, however, by the highly variable DNA preservation within different tissues, especially when isolating ancient pathogens from human remains. Identifying positive candidate samples via quantitative PCR (qPCR) for downstream procedures can reduce reagent costs, increase capture efficiency and maximize the number of sequencing reads of the target. This study uses four qPCR assays designed to target regions within the Mycobacterium tuberculosis complex (MTBC) to examine 133 human skeletal samples from a wide geographical and temporal range, identified by the presence of skeletal lesions typical of chronic disseminated tuberculosis. Given the inherent challenges working with ancient mycobacteria, strict criteria must be used and primer/probe design continually re-evaluated as new data from bacteria become available. Seven samples tested positive for multiple MTBC loci, supporting them as strong candidates for downstream analyses. Using strict and conservative criteria, qPCR remains a fast and effective screening tool when compared with screening by more expensive sequencing and enrichment technologies. PMID:25487341

  18. A Ribeiroia spp. (Class: Trematoda) - Specific PCR-based diagnostic

    USGS Publications Warehouse

    Reinitz, D.M.; Yoshino, T.P.; Cole, R.A.

    2007-01-01

    Increased reporting of amphibian malformations in North America has been noted with concern in light of reports that amphibian numbers and species are declining worldwide. Ribeiroia ondatrae has been shown to cause a variety of types of malformations in amphibians. However, little is known about the prevalence of R. ondatrae in North America. To aid in conducting field studies of Ribeiroia spp., we have developed a polymerase chain reaction (PCR)-based diagnostic. Herein, we describe the development of an accurate, rapid, simple, and cost-effective diagnostic for detection of Ribeiroia spp. infection in snails (Planorbella trivolvis). Candidate oligonucleotide primers for PCR were designed via DNA sequence analyses of multiple ribosomal internal transcribed spacer-2 regions from Ribeiroia spp. and Echinostoma spp. Comparison of consensus sequences determined from both genera identified areas of sequence potentially unique to Ribeiroia spp. The PCR reliably produced a diagnostic 290-base pair (bp) product in the presence of a wide concentration range of snail or frog DNA. Sensitivity was examined with DNA extracted from single R. ondatrae cercaria. The single-tube PCR could routinely detect less than 1 cercariae equivalent, because DNA isolated from a single cercaria could be diluted at least 1:50 and still yield a positive result via gel electrophoresis. An even more sensitive nested PCR also was developed that routinely detected 100 fg of the 290-bp fragment. The assay did not detect furcocercous cercariae of certain Schistosomatidae, Echinostoma sp., or Sphaeridiotrema globulus nor adults of Clinostomum sp. or Cyathocotyle bushiensis. Field testing of 137 P. trivolvis identified 3 positives with no overt environmental cross-reactivity, and results concurred with microscopic examinations in all cases. ?? American Society of Parasitologists 2007.

  19. Application of multiplex PCR, pulsed-field gel electrophoresis (PFGE), and BOX-PCR for molecular analysis of enterococci

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of the study was to use band-based molecular methods including BOX-PCR (Polymerase Chain Reaction) and Pulsed-Field Gel Electrophoresis (PFGE) to determine if genetically related enterococci were found among different stores, food types, or years. Enterococci were also characterized f...

  20. Nested-PCR and TaqMan real-time quantitative PCR assays for human adenoviruses in environmental waters.

    PubMed

    Huang, Wen-Chien; Chou, Yi-Pen; Kao, Po-Min; Hsu, Tsui-Kang; Su, Hung-Chang; Ho, Ying-Ning; Yang, Yi-Chun; Hsu, Bing-Mu

    2016-01-01

    Human adenovirus (HAdV) infections can occur throughout the year. Cases of HAdV-associated respiratory disease have been more common in the late winter, spring, and early summer. In this study, to provide viral pollution data for further epidemiological studies and governmental actions, the presence of HAdV in the aquatic environment was quantitatively surveyed in the summer. This study was conducted to compare the efficiencies of nested-PCR (polymerase chain reaction) and qPCR (quantitative PCR) for detecting HAdV in environmental waters. A total of 73 water samples were collected from Puzi River in Taiwan and subjected to virus concentration methods. In the results, qPCR had much better efficiency for specifying the pathogen in river sample. HAdV41 was detected most frequently in the river water sample (10.9%). The estimated HAdV concentrations ranged between 6.75 × 10(2) and 2.04 × 10(9) genome copies/L. Significant difference was also found in heterotrophic plate counts, conductivity, water temperature, and water turbidity between presence/absence of HAdV. HAdV in the Puzi River may pose a significant health risk. PMID:27120637

  1. 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

  2. Quantitative real-time PCR (qPCR) for Eimeria tenella replication--Implications for experimental refinement and animal welfare.

    PubMed

    Nolan, Matthew J; Tomley, Fiona M; Kaiser, Pete; Blake, Damer P

    2015-10-01

    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) (R(2)=0.994) (p<0.002) but not in those from day eight (after most oocyst shedding) (R(2)=0.006) (p>0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R(2)=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. PMID:26141544

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

    PubMed Central

    Te, Shu Harn; Chen, Enid Yingru

    2015-01-01

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

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

    PubMed

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

    2015-08-01

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

  5. PCR detection and characterization of type-2 porcine circovirus.

    PubMed Central

    Hamel, A L; Lin, L L; Sachvie, C; Grudeski, E; Nayar, G P

    2000-01-01

    A polymerase chain reaction (PCR) assay was developed for detecting porcine circovirus (PCV). The assay readily detected type-2 PCV (PCV-2) and type-1 PCV (PCV-1). The PCR primers were designed based on DNA sequences conserved in all reported PCV genomes. Type 1 PCV and type 2 PCV both produced 438 bp amplification products, which were easily identified and differentiated from one another by restriction fragment length polymorphism (RFLP) analysis. Porcine circovirus was detected in 55% (931/1693) of randomly tested pigs with various clinical signs and lesions, most of which were difficult to differentiate from those associated with porcine reproductive and respiratory syndrome (PRRS). The PCR products from all positive clinical samples were identified by RFLP to be only PCV-2; DNA tested by PCR was extracted directly from one or more of lung, mesenteric or mediastinal lymph nodes, and tonsil. Type 2 PCV was also detected in 6% (2/34) of DNA extracted directly from semen of randomly chosen healthy boars. Positive PCR reactions from 554 diseased pigs were characterized by RFLP and categorized into 5 different profiles (A-E), of which 82.8% were PCV-2A (456/554), 3.0% were PCV-2B (17/554), 9.9% were PCV-2C (55/554), 1.1% were PCV-2D (6/554), and 3.2% were PCV-2E (18/554). The complete genomic nucleotide sequences of PCV-2A, B, C, D, and E were determined and found to have at least 95% homology compared with one another and with all other PCV-2 found in the GenBank database. All PCV-2 had less than 76% homology with PCV-1. This PCR assay will hopefully be useful to veterinary diagnostic laboratories for routine testing and surveillance of infection with PCV-2. The RFLP profiling system might be useful for preliminary characterization and identification of PCV isolates and might also benefit studies on the molecular epidemiology of PCV. Images Figure 1. PMID:10680656

  6. Computational tradeoffs in multiplex PCR assay design for SNP genotyping

    PubMed Central

    Rachlin, John; Ding, Chunming; Cantor, Charles; Kasif, Simon

    2005-01-01

    Background Multiplex PCR is a key technology for detecting infectious microorganisms, whole-genome sequencing, forensic analysis, and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays requires the consideration of multiple competing objectives and physical constraints, and extensive computational analysis must be performed in order to identify the possible formation of primer-dimers that can negatively impact product yield. Results This paper examines the computational design limits of multiplex PCR in the context of SNP genotyping and examines tradeoffs associated with several key design factors including multiplexing level (the number of primer pairs per tube), coverage (the % of SNP whose associated primers are actually assigned to one of several available tube), and tube-size uniformity. We also examine how design performance depends on the total number of available SNPs from which to choose, and primer stringency criterial. We show that finding high-multiplexing/high-coverage designs is subject to a computational phase transition, becoming dramatically more difficult when the probability of primer pair interaction exceeds a critical threshold. The precise location of this critical transition point depends on the number of available SNPs and the level of multiplexing required. We also demonstrate how coverage performance is impacted by the number of available snps, primer selection criteria, and target multiplexing levels. Conclusion The presence of a phase transition suggests limits to scaling Multiplex PCR performance for high-throughput genomics applications. Achieving broad SNP coverage rapidly transitions from being very easy to very hard as the target multiplexing level (# of primer pairs per tube) increases. The onset of a phase transition can be "delayed" by having a larger pool of SNPs, or loosening primer selection constraints so as to increase the number of candidate primer pairs per SNP, though the latter

  7. Sporulation properties and antimicrobial susceptibility in endemic and rare Clostridium difficile PCR ribotypes.

    PubMed

    Zidaric, Valerija; Rupnik, Maja

    2016-06-01

    Increased sporulation and antibiotic resistance have been proposed to be associated with certain Clostridium difficile epidemic strains such as PCR ribotype 027. In this study we examined these properties in another widespread PCR ribotype, 014/020, in comparison to prevalent PCR ribotype 002 and a group of rarely represented PCR ribotypes. Highest sporulation was observed in 014/020 strains at 24 h, while after 72 h PCR ribotype 002 and rare PCR ribotypes formed higher total number of spores. PCR ribotype 014/020 strains exhibited slightly higher resistance to tested antimicrobials, followed by group of rare PCR ribotypes and less common PCR ribotype 002. Neither sporulation properties nor antibiotic resistance clearly differed in endemic and rare strains. PMID:27095618

  8. A comparative analysis of different molecular targets using PCR for diagnosis of old world leishmaniasis.

    PubMed

    Koltas, Ismail S; Eroglu, Fadime; Uzun, Soner; Alabaz, Derya

    2016-05-01

    The different sensitivity values were obtained in each study conducted for the diagnosis of leishmaniasis with the polymerase chain reaction (PCR). However, a standardized PCR target for the diagnosis of leishmaniasis does not exist. The aim of the current study, the most ideal PCR target was determined for diagnosis of leishmaniasis. A total of 72 smear and 48 bone marrow samples were analyzed with six different molecular targets to determine their potential as a tool for the specific molecular diagnosis of leishmaniasis using PCR. The positivity-negativity value and the sensitivity-specificity of each PCR targets were calculated. The positivity value of PCR targets were sequenced in different levels in the diagnosis of leishmaniasis from highest to lowest in the order of kDNA-PCR > SSU rRNA-PCR > ITS2-PCR > ITS1-PCR > ME-PCR > HSP70-PCR. The sensitivities of PCR targets except ITS1-PCR, ME-PCR and HSP70-PCR were found to be 100% in cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) cases as compared to microscopic examination accepted as a gold standard. The sensitivities of ITS1-PCR, ME-PCR and HSP70-PCR were found 96.6%, 90.0% and 86.6%, respectively, in CL-cases. In addition, the sensitivities of ITS1-PCR, ME-PCR and HSP70-PCR were found 90.0%, 70.0% and 60.0%, respectively, in VL-cases. The kDNA genomic region was the most sensitive for routine diagnosis of leishmaniasis. ITS1-PCR restriction fragment length polymorphism, the alternative method for the identification of Old World Leishmania species, did not require culturing of the parasites. PMID:26896641

  9. Adaptive DNA Computing Algorithm by Using PCR and Restriction Enzyme

    NASA Astrophysics Data System (ADS)

    Kon, Yuji; Yabe, Kaoru; Rajaee, Nordiana; Ono, Osamu

    In this paper, we introduce an adaptive DNA computing algorithm by using polymerase chain reaction (PCR) and restriction enzyme. The adaptive algorithm is designed based on Adleman-Lipton paradigm[3] of DNA computing. In this work, however, unlike the Adleman- Lipton architecture a cutting operation has been introduced to the algorithm and the mechanism in which the molecules used by computation were feedback to the next cycle devised. Moreover, the amplification by PCR is performed in the molecule used by feedback and the difference concentration arisen in the base sequence can be used again. By this operation the molecules which serve as a solution candidate can be reduced down and the optimal solution is carried out in the shortest path problem. The validity of the proposed adaptive algorithm is considered with the logical simulation and finally we go on to propose applying adaptive algorithm to the chemical experiment which used the actual DNA molecules for solving an optimal network problem.

  10. Direct identification and typing of Mycobacterium tuberculosis by PCR.

    PubMed Central

    Neimark, H; Ali Baig, M; Carleton, S

    1996-01-01

    We have developed a rapid PCR assay that types strains of Mycobacterium tuberculosis by generating distinct DNA fingerprints directly from primary cultures. This assay allows strain identification analogous to that achieved by the standard restriction fragment length polymorphism method, and fingerprints are obtained in less than 8 h. This assay does not require subculturing, DNA purification, restriction digestion, Southern blotting, or nucleic acid hybridization. Rapid and precise identification of M. tuberculosis strains permits immediate molecular epidemiologic studies. The assay can be converted to a computer-automated system by employing fluorescently labeled PCR primers and the Perkin-Elmer DNA sequencer so that unknown-specimen fingerprints are identified by computer comparison to a database of M. tuberculosis strain fingerprints. PMID:8880499

  11. Actuation method and apparatus, micropump, and PCR enhancement method

    SciTech Connect

    Ullakko, Kari; Mullner, Peter; Hampikian, Greg; Smith, Aaron

    2015-07-28

    An actuation apparatus includes at least one magnetic shape memory (MSM) element containing a material configured to expand and/or contract in response to exposure to a magnetic field. Among other things, the MSM element may be configured to pump fluid through a micropump by expanding and/or contracting in response to the magnetic field. The magnetic field may rotate about an axis of rotation and exhibit a distribution having a component substantially perpendicular to the axis of rotation. Further, the magnetic field distribution may include at least two components substantially orthogonal to one another lying in one or more planes perpendicular to the axis of rotation. The at least one MSM element may contain nickel, manganese, and gallium. A polymerase chain reaction (PCR) may be enhanced by contacting a PCR reagent and DNA material with the MSM element.

  12. Measuring Digital PCR Quality: Performance Parameters and Their Optimization.

    PubMed

    Lievens, A; Jacchia, S; Kagkli, D; Savini, C; Querci, M

    2016-01-01

    Digital PCR is rapidly being adopted in the field of DNA-based food analysis. The direct, absolute quantification it offers makes it an attractive technology for routine analysis of food and feed samples for their composition, possible GMO content, and compliance with labelling requirements. However, assessing the performance of dPCR assays is not yet well established. This article introduces three straightforward parameters based on statistical principles that allow users to evaluate if their assays are robust. In addition, we present post-run evaluation criteria to check if quantification was accurate. Finally, we evaluate the usefulness of Poisson confidence intervals and present an alternative strategy to better capture the variability in the analytical chain. PMID:27149415

  13. Measuring Digital PCR Quality: Performance Parameters and Their Optimization

    PubMed Central

    Lievens, A.; Jacchia, S.; Kagkli, D.; Savini, C.; Querci, M.

    2016-01-01

    Digital PCR is rapidly being adopted in the field of DNA-based food analysis. The direct, absolute quantification it offers makes it an attractive technology for routine analysis of food and feed samples for their composition, possible GMO content, and compliance with labelling requirements. However, assessing the performance of dPCR assays is not yet well established. This article introduces three straightforward parameters based on statistical principles that allow users to evaluate if their assays are robust. In addition, we present post-run evaluation criteria to check if quantification was accurate. Finally, we evaluate the usefulness of Poisson confidence intervals and present an alternative strategy to better capture the variability in the analytical chain. PMID:27149415

  14. [PCR-based detection of pathogens in clinical rheumatology].

    PubMed

    Ehrenstein, B; Reischl, U

    2016-05-01

    In the differential diagnostics of autoimmune-mediated rheumatic diseases, rheumatologists often have to consider infections (e. g. Lyme arthritis) or reactive diseases (e. g. reactive arthritis after urogenital bacterial infections). Furthermore, infections with an atypical presentation or caused by atypical pathogens (opportunistic infections) can complicate the immunosuppressive therapy of autoimmune diseases. For this purpose not only conventional microbiological culture methods but also PCR-based methods are increasingly being applied for the direct detection of pathogens in clinical specimens. The aim of this overview is to present commonly used PCR methods in the clinical practice of rheumatology and to describe their benefits and limitations compared to culture-based detection methods. PMID:26892924

  15. Multiplex PCR for detection of acquired carbapenemase genes.

    PubMed

    Poirel, Laurent; Walsh, Timothy R; Cuvillier, Vincent; Nordmann, Patrice

    2011-05-01

    A rapid and reliable PCR-based technique was developed for detection of genes encoding carbapenemases belonging to different classes. Primers were designed to amplify the following 11 genes: bla(IMP), bla(VIM), bla(NDM), bla(SPM), bla(AIM), bla(DIM), bla(GIM), bla(SIM)bla(KPC), bla(BIC), and bla(OXA-48). Three different multiplex reaction mixtures were defined and evaluated for the detection of all these 11 genes. Using optimized conditions, each reaction mixture allowed to identify the respective genes, with PCR giving distinct amplicon sizes corresponding to the different genes for each mixture. We reported here a rapid and reliable technique for screening all clinically relevant carbapenemase genes. PMID:21398074

  16. An improved PCR method for gender identification of eagles.

    PubMed

    Chang, Hsueh-Wei; Chou, Ta-Ching; Gu, De-Leung; Cheng, Chun-An; Chang, Chia-Che; Yao, Cheng-Te; Chuang, Li-Yeh; Wen, Cheng-Hao; Chou, Yii-Cheng; Tan, Kock-Yee; Cheng, Chien-Chung

    2008-06-01

    Eagles are sexually monomorphic and therefore it is difficult to determine their gender, which is a crucial need for management purposes. In this study, we have developed an improved gender identification method by exploiting length differences between the Chromo-Helicase-DNA binding protein (CHD)-Z and CHD-W genes of Spilornis cheela hoya. By comparing DNA sequences for CHD-W and CHD-Z from 10 species of Falconiformes eagles we designed universal gender identification PCR primers that exploit differences in product size. Standard agarose gels were shown to easily distinguish between the 148-bp CHD-ZW and the 258-bp CHD-W PCR products. When used with 28 samples of S. cheela hoya, our improved universal primers provided a fast and precise gender identification assay. PMID:18385011

  17. Rapid detection of Serpulina hyodysenteriae in diagnostic specimens by PCR.

    PubMed Central

    Elder, R O; Duhamel, G E; Schafer, R W; Mathiesen, M R; Ramanathan, M

    1994-01-01

    A PCR assay for the detection of Serpulina hyodysenteriae in diagnostic specimens was developed on the basis of sequence analysis of a recombinant clone designated pRED3C6. Clone pRED3C6, which contained a 2.3-kb DNA fragment unique to S. hyodysenteriae, was identified by screening a plasmid library of S. hyodysenteriae isolate B204 genomic DNA in Escherichia coli by colony immunoblot with the mouse monoclonal antibody 10G6/G10, which was produced against cell-free supernatant antigens from the same isolate. Southern blot analysis of HindIII-digested genomic DNA of S. hyodysenteriae serotypes 1 through 7 and of four weakly beta-hemolytic intestinal spirochetes, including Serpulina innocens, with the 2.3-kb DNA fragment of pRED3C6 indicated that the cloned sequence was present exclusively in the seven serotypes of S. hyodysenteriae. An oligonucleotide primer pair for PCR amplification of a 1.55-kb fragment and an internal oligonucleotide probe were designed and synthesized on the basis of sequence analysis of the 2.3-kb DNA fragment of pRED3C6. Purified genomic DNAs from reference isolates of S. hyodysenteriae serotypes 1 through 9, S. innocens, weakly beta-hemolytic intestinal spirochetes belonging to genotypic groups distinct from those of reference Serpulina spp., other cultivable reference isolates of the order Spirochaetales, and enteric bacteria including Escherichia coli, Salmonella spp., Campylobacter spp., and Bacteroides vulgatus were amplified with the oligonucleotide primer pair in a hot-start PCR. The 1.55-kb products were obtained only in the presence of genomic DNA from each of the nine serotypes of S. hyodysenteriae. The specificity of the 1.55-kb products for S. hyodysenteriae was confirmed on the basis of production of a restriction endonuclease pattern of the PCR products identical to the predicted restriction map analysis of pRED3C6 and positive hybridization signal with the S. hyodysenteriae-specific internal oligonucleotide probe. By using

  18. Species identification of cattle and buffalo fat through PCR assay.

    PubMed

    Vaithiyanathan, S; Kulkarni, V V

    2016-04-01

    A method was standardized to isolate quality DNA from cattle and buffalo fat for species identification using QIAamp DNA stool mini kit. The quality of the DNA was sufficient enough to amplify universal primers viz., mt 12S rRNA and mt 16S rRNA, and species specific D loop primers for cattle and buffalo. The sensitivity of the PCR assay in the species specific D loop primer amplification was with a detection level of 0. 47 ng cattle DNA and 0.23 ng buffalo DNA in simplex and, 0. 47 ng cattle DNA and 0.12 ng buffalo DNA in duplex PCR. It is a potentially reliable method for DNA detection to authenticate animal fat. PMID:27413237

  19. Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model.

    PubMed

    Monteiro, L; Bonnemaison, D; Vekris, A; Petry, K G; Bonnet, J; Vidal, R; Cabrita, J; Mégraud, F

    1997-04-01

    A model was developed to study inhibitors present in feces which prevent the use of PCR for the detection of Helicobacter pylori. A DNA fragment amplified with the same primers as H. pylori was used to spike samples before extraction by a modified QIAamp tissue method. Inhibitors, separated on an Ultrogel AcA44 column, were characterized. Inhibitors in feces are complex polysaccharides possibly originating from vegetable material in the diet. PMID:9157172

  20. Underwater application of quantitative PCR on an ocean mooring.

    PubMed

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

    2011-01-01

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

  1. Extraction of PCR-amplifiable genomic DNA from Bacillus anthracisspores

    SciTech Connect

    Torok, Tamas

    2003-05-19

    Bacterial endospore disruption and nucleic acid extractionresulting in DNA of PCR-amplifiable quality and quantity are not trivial.Responding to the needs of the Hazardous Materials Response Unit (HMRU),Laboratory Division, Federal Bureau of Investigation, protocols weredeveloped to close these gaps. Effectiveness and reproducibility of thetechniques were validated with laboratory grown pure spores of Bacillusanthracis and its close phylogenetic neighbors, and with spiked soils anddamaged samples.

  2. Underwater Application of Quantitative PCR on an Ocean Mooring

    PubMed Central

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

    2011-01-01

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

  3. Stabilized, Freeze-Dried PCR Mix for Detection of Mycobacteria

    PubMed Central

    Klatser, Paul R.; Kuijper, Sjoukje; van Ingen, Cor W.; Kolk, Arend H. J.

    1998-01-01

    We report here the development of a freeze-drying procedure allowing stabilization at ambient temperature of preoptimized, premixed, and predispensed PCR mixes aimed at the detection of mycobacteria in clinical materials. The freeze-dried mixes retained activity at 4°C and at 20°C for 1 year and for 3 months at 37°C, as judged by their performance with 50 and 500 fg of purified Mycobacterium bovis BCG target DNA. PMID:9620427

  4. Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses.

    PubMed

    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

  5. 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

  6. Clinical differences among PCR-proven dengue serotype infections.

    PubMed

    Limkittikul, Kriengsak; Yingsakmongkon, Sangchai; Jittmittraphap, Akanitt; Chuananon, Somchai; Kongphrai, Yuphin; Kowasupathr, Surasak; Rojanawatsirivit, Chaiyaporn; Mammen, Mammen P; Jampangern, Wipawee

    2005-11-01

    The objective of this study was to compare the clinical spectra of the dengue serotypes proven by the PCR technique. This retrospective study reviewed the clinical information of dengue-infected patients who were admitted to northeastern provincial hospitals in Thailand from June to September 2002. Dengue infection and viral serotypes were confirmed by polymerase chain reaction (PCR). Paired anti-dengue immunoglobulin G (IgG) and IgM from paired sera were analyzed by enzyme-linked immunosorbent assay (ELISA). Ninety-nine PCR-proven dengue-infected Thai patients were studied. Their ages ranged from 3-30 years. They were infected with DEN1, DEN2, DEN3 and DEN4 in 21, 55, 12, and 12%, respectively. Twenty-two percent had primary and 78% had secondary infections. Dengue fever was the most common presentation for both primary (77.2%) and secondary infections (46.7%). The ratios of dengue fever:dengue hemorrhagic fever (DF:DHF) and non-dengue shock syndrome:dengue shock syndrome (non-DSS:DSS) for DEN2 was the lowest of the dengue serotypes. There was no difference in the duration of fever, percentage of hepatomegaly and bleeding among the serotypes in both DF and DHF. The trends in the white blood cells, lymphocyte and atypical lymphocyte counts in DEN3 were the highest, while those of DEN1 were the lowest of the dengue serotypes. PMID:16610645

  7. Detection and counting of Nitrobacter populations in soil by PCR.

    PubMed Central

    Degrange, V; Bardin, R

    1995-01-01

    Although the biological conversion of nitrite to nitrate is a well-known process, studies of Nitrobacter populations are hindered by their physiological characteristics. This report describes a new method for detecting and counting Nitrobacter populations in situ with the PCR. Two primers from the 16S rRNA gene were used to generate a 397-bp fragment by amplification of Nitrobacter species DNA. No signal was detected from their phylogenetic neighbors or the common soil bacteria tested. Extraction and purification steps were optimized for minimal loss and maximal purity of soil DNA. The detection threshold and accuracy of the molecular method were determined from soil inoculated with 10, 10(2), or 10(3) Nitrobacter hamburgensis cells per g of soil. Counts were also done by the most-probable-number (MPN)-Griess and fluorescent antibody methods. PCR had a lower detection threshold (10(2) Nitrobacter cells per g of soil) than did the MPN-Griess or fluorescent antibody method. When PCR amplification was coupled with the MPN method, the counting rate reached 65 to 72% of inoculated Nitrobacter cells. Tested on nonsterile soil, this rapid procedure was proved efficient. PMID:7793930

  8. Rat Gene Mapping Using Pcr-Analyzed Microsatellites

    PubMed Central

    Serikawa, T.; Kuramoto, T.; Hilbert, P.; Mori, M.; Yamada, J.; Dubay, C. J.; Lindpainter, K.; Ganten, D.; Guenet, J. L.; Lathrop, G. M.; Beckmann, J. S.

    1992-01-01

    One hundred and seventy-four rat loci which contain short tandem repeat sequences were extracted from the GenBank or EMBL data bases and used to define primers for amplification by the polymerase chain reaction (PCR) of the microsatellite regions, creating PCR-formatted sequence-tagged microsatellite sites (STMSs). One hundred and thirty-four STMSs for 118 loci, including 6 randomly cloned STMSs, were characterized: (i) PCR-analyzed loci were assigned to specific chromosomes using a panel of rat X mouse somatic cell hybrid clones. (ii) Length variation of the STMSs among 8 inbred rat strains could be visualized at 85 of 107 loci examined (79.4%). (iii) A genetic map, integrating biochemical, coat color, mutant and restriction fragment length polymorphism loci, was constructed based on the segregation of 125 polymorphic markers in seven rat backcrosses and in two F(2) crosses. Twenty four linkage groups were identified, all of which were assigned to a defined chromosome. As a reflection of the bias for coding sequences in the public data bases, the STMSs described herein are often associated with genes. Hence, the genetic map we report coincides with a gene map. The corresponding map locations of the homologous mouse and human genes are also listed for comparative mapping purposes. PMID:1628813

  9. [Using inter-SINE-PCR to study mammalian phylogeny].

    PubMed

    Bannikova, A A; Matveev, V A; Kramerov, D A

    2002-06-01

    Results of the use of the fingerprinting method related to short interspersed elements (SINEs), inter-SINE-PCR, in the study of phylogenetic and taxonomic relationship in mammals from orders Chiroptera (family Vespertilionidae) and Lipotyphla (family Erinaceidae) are reported. The inter-SINE-PCR method is based on the amplification of fragments situated between copies of SINEs, which are short retroposons spaced 100 to 1000 bp apart. Specifically selected primers were used, which are complementary to consensus sequences of two short retroposons: the mammalian interspersed repeat (MIR), which is typical of all mammals and some other vertebrates, was used in the cases of bats and Erinaceidae, and the ERI-1 element recently isolated from the genome of the Daurian hedgehog was used in the case of Erinaceidae. The results support the current view on phylogenetic relationship between hedgehogs belonging to genera Erinaceus, Hemiechinus, and Paraechinus (but not the genus Atelerix). In bats, the phylogenetic reconstruction revealed a statistically valid topology only at lower taxonomic levels, whereas the topology for the genus and supragenus ranks was unresolved and fan-shaped. The benefits and limitations of the inter-SINE-PCR method are discussed. PMID:12138785

  10. Km typing with PCR: application to population screening.

    PubMed Central

    Kurth, J H; Bowcock, A M; Erlich, H A; Nevo, S; Cavalli-Sforza, L L

    1991-01-01

    The immunoglobulin kappa light chain (IgK) locus may play a significant role in the pathology of both infectious and autoimmune diseases. Most of the work on IgK genetics has been conducted using immunological techniques for allelic typing and sequence analysis. This is restricted by availability of reagents and can be both expensive and time-consuming. PCR primers were designed to amplify the kappa constant gene (Ck), and four allele-specific oligonucleotides (ASOs) were used to distinguish the alleles in the amplified PCR products. Direct sequencing of PCR products was performed to confirm that the primers specifically amplified the Ck region and the ASOs differentiated the Km alleles. Sequencing of an average of 209 nucleotides of DNA from 50 individuals revealed no variation except at codon 191, which is known to be involved in a frequent polymorphism. An analysis of 347 different individual DNAs from 10 human populations was conducted to determine Km allelic frequencies within these populations and to apply this type of data collection to population studies. Images Figure 4 Figure 5 PMID:1900145

  11. Clinically Applicable Multiplex PCR for Four Middle Ear Pathogens

    PubMed Central

    Hendolin, Panu H.; Paulin, Lars; Ylikoski, Jukka

    2000-01-01

    The multiplex PCR method for the detection of Alloiococcus otitidis, Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae (P. H. Hendolin, A. Markkanen, J. Ylikoski, and J. J. Wahlfors, J. Clin. Microbiol. 35:2854–2858, 1997) in middle ear effusions (MEEs) was modified to be better suited for clinical use. To detect false-negative results, an internal amplification was added to the reaction, and to prevent carryover contamination, the dUTP–uracil-N-glycosidase system was incorporated into the procedure. Labor was minimized by using the heat-activatable AmpliTaq Gold polymerase in order to circumvent manual hot start and by detecting the amplification products on an automated sequencer. The performance of the improved protocol was verified with MEEs from patients with otitis media with effusion. In addition, a ligase detection reaction (LDR) was developed for confirmation of the PCR products. The modifications increased the reliability of the protocol and the hands-off time significantly. However, when two DNA extraction protocols were compared, gram-negative bacteria were detected more often in phenol-treated MEEs (94 versus 46%; P < 0.001), and gram-positive bacteria were detected more often in MEEs dissolved in sodium dodecyl sulfate-NaOH-chaotropic salt (83 versus 27%; P < 0.001). The LDR was found to be 100% specific. In all, the results demonstrate the feasibility of the rapid (7-h) multiplex PCR method for routine laboratory use. PMID:10618075

  12. Sex identification from exfoliated primary teeth--a PCR study.

    PubMed

    Kumar, Manju Gopa; Hegde, Amitha M

    2005-01-01

    Teeth endure postmortem degradation and extreme changes in ambient temperature and pressure better than most human tissues. In the present day scenario the growing number of crime against children in the form of battering, physical/sexual abuse, abduction and kidnapping, the use of exfoliated primary teeth, become many times the only evidence available at the crime scene. Despite exposure of the body to burial, mutilation, explosion or incineration, it is usually possible to extract DNA from pulp tissue of tooth with sufficient quality and quantity. Hence the present study was undertaken to find out the sex of a child from exfoliated/extracted deciduous teeth using a Polymerase Chain reaction (PCR) based analysis. Tooth samples were stored in room temperature after double coding for various periods. Dental pulp tissue was collected from each sample and DNA was isolated by proteinase-k digestion and phenol chloroform extraction methods. PCR amplification was done with two sets of oligonucleiotide primers. Amplification of X (131bp) and Y-specific sequences (172bp) in males and that of the X-specific sequence in females was observed and compared with the template DNA showing male and female controls. Determination of sexes of all freshly collected samples within 24 hours and after 1 month of extraction respectively gave 100% result. However, PCR was not found to be an effective method for sex determination after 6 months post extraction. PMID:16302594

  13. Development of a multiplex PCR for identification of vineyard mealybugs.

    PubMed

    Daane, Kent M; Middleton, Mathew C; Sforza, René; Cooper, Monica L; Walton, Vaughn M; Walsh, Douglas B; Zaviezo, Tania; Almeida, Rodrigo P P

    2011-12-01

    A simple molecular tool was developed and tested to identify seven mealybug species found in North American vineyards: Pseudococcus maritimus Ehrhorn, Pseudococcus viburni (Signoret), Pseudococcus longispinus (Targioni-Tozzeti), Pseudococcus calceolariae (Maskell), Planococcus ficus (Signoret), Planococcus citri (Risso), and Ferrisia gilli Gullan. The developed multiplex PCR is based on the mitochondrial cytochrome c oxidase subunit one gene. In tests, this single-step multiplex PCR correctly identified 95 of 95 mealybug samples, representing all seven species and collected from diverse geographic regions. To test the sensitivity, single specimen samples with different Pl. ficus developmental stages (egg to adult female and adult male) were processed PCR and the resulting output provided consistent positive identification. To test the utility of this protocol for adult males caught in sex baited pheromone traps, Pl. ficus adult males were placed in pheromone traps, aged at a constant temperature of 26±2°C, and processed with the multiplex each day thereafter for 8 d. Results showed consistent positive identification for up to 6 d (range, 6-8 d). Results are discussed with respect to the usefulness of this molecular tool for the identification of mealybugs in pest management programs and biosecurity of invasive mealybugs. PMID:22217778

  14. Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR.

    PubMed Central

    Folgueira, L; Delgado, R; Palenque, E; Aguado, J M; Noriega, A R

    1996-01-01

    A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population. PMID:8904404

  15. Improved purification and PCR amplification of DNA from environmental samples.

    PubMed

    Arbeli, Ziv; Fuentes, Cilia L

    2007-07-01

    Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA. PMID:17521406

  16. Electrochemiluminescence-PCR detection of genetically modified organisms

    NASA Astrophysics Data System (ADS)

    Liu, Jinfeng; Xing, Da; Shen, Xingyan; Zhu, Debin

    2005-01-01

    The detection methods for genetically modified (GM) components in foods have been developed recently. But many of them are complicated and time-consuming; some of them need to use the carcinogenic substance, and can"t avoid false-positive results. In this study, an electrochemiluminescence polymerase chain reaction (ECL-PCR) method for detection GM tobaccos is proposed. The Cauliflower mosaic virus 35S (CaMV35S) promoter was amplified by PCR, Then hybridized with a Ru(bpy)32+ (TBR)-labeled and a biotinylated probe. The hybridization products were captured onto streptavidin-coated paramagnetic beads, and detected by measuring the electrochemiluminescence (ECL) signal of the TBR label. Whether the tobaccos contain GM components was discriminated by detecting the ECL signal of CaMV35S promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM tobaccos. The ECL-PCR method provide a new means in GMOs detection due to its safety, simplicity and high efficiency.

  17. Analysis of copy number variation using quantitative interspecies competitive PCR.

    PubMed

    Williams, Nigel M; Williams, Hywel; Majounie, Elisa; Norton, Nadine; Glaser, Beate; Morris, Huw R; Owen, Michael J; O'Donovan, Michael C

    2008-10-01

    Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls. PMID:18697816

  18. Pure chromosome-specific PCR libraries from single sorted chromosomes.

    PubMed Central

    VanDevanter, D R; Choongkittaworn, N M; Dyer, K A; Aten, J; Otto, P; Behler, C; Bryant, E M; Rabinovitch, P S

    1994-01-01

    Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted chromosome or chromosome fragment. Previously reported methods for the development of chromosome libraries require larger numbers of chromosomes, with preparation of pure chromosomes sorted by flow cytometry, generation of somatic cell hybrids containing targeted chromosomes, or a combination of both procedures. These procedures are labor intensive, especially when hybrid cell lines are not already available, and this has limited the generation of chromosome-specific DNA libraries from nonhuman species. In contrast, a single sorted chromosome is a pure source of DNA for library production even when flow cytometric resolution of chromosome populations is poor. Furthermore, any sorting cytometer may be used with this technique. Using this approach, we demonstrate the generation of PCR libraries suitable for both molecular and fluorescence in situ hybridization studies from individual baboon and canine chromosomes, separate human homologues, and a rearranged marker chromosome from a transformed cell line. PCR libraries specific to subchromosomal regions have also been produced by sorting a small chromosome fragment. This simple and rapid technique will allow generation of nonhuman linkage maps and probes for fluorescence in situ hybridization and the characterization of marker chromosomes from solid tumors. In addition, allele-specific libraries generated by this strategy may also be useful for mapping genetic diseases. Images PMID:8016078

  19. Determination of Arabidopsis thaliana telomere length by PCR.

    PubMed

    Vaquero-Sedas, María I; Vega-Palas, Miguel A

    2014-01-01

    In humans, telomere length studies have acquired great relevance because the length of telomeres has been related to natural processes like disease, aging and cancer. However, very little is known about the influence of telomere length on the biology of wild type plants. The length of plant telomeres has been usually studied by Terminal Restriction Fragment (TRF) analyses. This technique requires high amounts of tissue, including multiple cell types, which might be the reason why very little is known about the influence of telomere length on plant natural processes. In contrast, many of the human telomere length studies have focused on homogenous cell populations. Most of these studies have been performed by PCR, using telomeric degenerated primers, which allow the determination of telomere length from small amounts of human cells. Here, we have adapted the human PCR procedure to analyze the length of Arabidopsis thaliana telomeres. This PCR approach will facilitate the analysis of telomere length from low amounts of tissue. We have used it to determine that CG and non CG DNA methylation positively regulates Arabidopsis telomere length. PMID:24986269

  20. Determination of Arabidopsis thaliana telomere length by PCR

    PubMed Central

    Vaquero-Sedas, María I.; Vega-Palas, Miguel A.

    2014-01-01

    In humans, telomere length studies have acquired great relevance because the length of telomeres has been related to natural processes like disease, aging and cancer. However, very little is known about the influence of telomere length on the biology of wild type plants. The length of plant telomeres has been usually studied by Terminal Restriction Fragment (TRF) analyses. This technique requires high amounts of tissue, including multiple cell types, which might be the reason why very little is known about the influence of telomere length on plant natural processes. In contrast, many of the human telomere length studies have focused on homogenous cell populations. Most of these studies have been performed by PCR, using telomeric degenerated primers, which allow the determination of telomere length from small amounts of human cells. Here, we have adapted the human PCR procedure to analyze the length of Arabidopsis thaliana telomeres. This PCR approach will facilitate the analysis of telomere length from low amounts of tissue. We have used it to determine that CG and non CG DNA methylation positively regulates Arabidopsis telomere length. PMID:24986269

  1. Nested PCR for detection of HSV-1 in oral mucosa

    PubMed Central

    Jalouli, Miranda-Masoumeh; Jalouli, Jamshid; Hasséus, Bengt; Öhman, Jenny; Hirsch, Jan-Michaél

    2015-01-01

    Background It has been estimated that 15%-20% of human tumours are driven by infection and inflammation, and viral infections play an important role in malignant transformation. The evidence that herpes simplex virus type 1 (HSV-1) could be involved in the aetiology of oral cancer varies from weak to persuasive. This study aimed to investigate by nested PCR (NPCR) the prevalence of HSV-1 in samples from normal oral mucosa, oral leukoplakia, and oral squamous cell carcinoma (OSCC). Material and Methods We investigated the prevalence of HSV-1 in biopsies obtained from 26 fresh, normal oral mucosa from healthy volunteers as well as 53 oral leukoplakia and 27 OSCC paraffin-embedded samples. DNA was extracted from the specimens and investigated for the presence of HSV-1 by nested polymerase chain reaction (NPCR) and DNA sequencing. Results HSV-1 was detected in 14 (54%) of the healthy samples, in 19 (36%) of the oral leukoplakia samples, and in 14 (52%) of the OSCC samples. The differences were not statistically significant. Conclusions We observed a high incidence of HSV-1 in healthy oral mucosa, oral leukoplakia, and OSCC tissues. Thus, no connection between OSCC development and presence of HSV-1 was detected. Key words:HSV-1, nested PCR, PCR. PMID:26449432

  2. Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP)

    PubMed Central

    2016-01-01

    Antibodies are widely used biomarkers for the diagnosis of many diseases. Assays based on solid-phase immobilization of antigens comprise the majority of clinical platforms for antibody detection, but can be undermined by antigen denaturation and epitope masking. These technological hurdles are especially troublesome in detecting antibodies that bind nonlinear or conformational epitopes, such as anti-insulin antibodies in type 1 diabetes patients and anti-thyroglobulin antibodies associated with thyroid cancers. Radioimmunoassay remains the gold standard for these challenging antibody biomarkers, but the limited multiplexability and reliance on hazardous radioactive reagents have prevented their use outside specialized testing facilities. Here we present an ultrasensitive solution-phase method for detecting antibodies, termed antibody detection by agglutination-PCR (ADAP). Antibodies bind to and agglutinate synthetic antigen–DNA conjugates, enabling ligation of the DNA strands and subsequent quantification by qPCR. ADAP detects zepto- to attomoles of antibodies in 2 μL of sample with a dynamic range spanning 5–6 orders of magnitude. Using ADAP, we detected anti-thyroglobulin autoantibodies from human patient plasma with a 1000-fold increased sensitivity over an FDA-approved radioimmunoassay. Finally, we demonstrate the multiplexability of ADAP by simultaneously detecting multiple antibodies in one experiment. ADAP’s combination of simplicity, sensitivity, broad dynamic range, multiplexability, and use of standard PCR protocols creates new opportunities for the discovery and detection of antibody biomarkers. PMID:27064772

  3. Direct-to-PCR tissue preservation for DNA profiling.

    PubMed

    Sorensen, Amy; Berry, Clare; Bruce, David; Gahan, Michelle Elizabeth; Hughes-Stamm, Sheree; McNevin, Dennis

    2016-05-01

    Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition. PMID:26530406

  4. Simultaneous multiplex PCR detection of seven cucurbit-infecting viruses.

    PubMed

    Kwon, Ji Yeon; Hong, Jin Sung; Kim, Min Jea; Choi, Sun Hee; Min, Byeong Eun; Song, Eun Gyeong; Kim, Hyun Hee; Ryu, Ki Hyun

    2014-09-01

    Two multiplex polymerase chain reaction (PCR) systems using dual priming oligonucleotide (DPO) primers were developed for the simultaneous detection of seven cucurbit-infecting viruses. One system allows for the detection of papaya ringspot virus, watermelon mosaic virus, and zucchini yellow mosaic virus, whereas the other permits the detection of cucumber green mottle mosaic virus, cucumber fruit mottle mosaic virus, kyuri green mottle mosaic virus, and zucchini green mottle mosaic virus. Viral species-specific DPO primers developed in this study detected as little as 10 fg/μl of viral RNA under monoplex conditions and 10 pg/μl of viral RNA under multiplex conditions. Multiplex PCR using the DPO primer sets was capable of amplifying viral genes at annealing temperatures ranging from 53 °C to 63 °C. Whereas the use of conventional primers gave rise to non-specific bands, the DPO primers detected target viral genes in the absence of non-specific amplification. When these DPO multiplex primer sets were applied to virus-infected cucurbit samples obtained in the field, multiple infection as well as single infection was accurately identified. This novel approach could also detect multiple viruses in infected seeds. The reliability of multiplex PCR systems using DPO primers for plant virus detection is discussed. PMID:24937806

  5. Usefulness of PCR method for detection of Leishmania in Poland.

    PubMed

    Myjak, Przemysław; Szulta, Joanna; de Almeida, Marcos E; da Silva, Alexandre J; Steurer, Francis; Lass, Anna; Pietkiewicz, Halina; Nahorski, Wacław L; Goljan, Jolanta; Knap, Józef; Szostakowska, Beata

    2009-01-01

    Leishmania parasites are the etiological agents of leishmaniosis, with severe course and often fatal prognosis, and the global number of cases has increased in recent decades. The gold standards for the diagnosis of leishmaniosis are microscopic examinations and culture in vitro of the different clinical specimens. The sensitivity of these methods is insufficient. Recent development in specific and sensitive molecular methods (PCR) allows for detection as well as identification of the parasite species (subspecies). The aim of the study was to estimate the usefulness of molecular methods (PCR) for detection of Leishmania species and consequently for the implementation of such methods in routine diagnostics of leishmaniosis in Polish patients returning from endemic areas of the disease. In our investigations we used 54 known Leishmania positive DNA templates (from culture and clinical specimens) received from the CDC (Atlanta, GA, USA). Moreover, 25 samples of bone marrow, blood or other tissues obtained from 18 Polish individuals suspected of leishmaniosis were also examined. In PCR we used two pairs of primers specific to the conserved region of Leishmania kinetoplast DNA (kDNA) minicircle (13A/13B and F/R). Using these primers we obtained amplicons in all DNA templates from the CDC and in three Polish patients suspected for Leishmania infection. In one sample from among these cases we also obtained positive results with DNA isolated from a blood specimen which was previously negative in microscopic examinations. PMID:19899614

  6. Molecular Serogrouping of Listeria monocytogenes from Brazil Using PCR.

    PubMed

    Camargo, Anderson Carlos; Vallim, Deyse Christina; Hofer, Ernesto; Nero, Luís Augusto

    2016-01-01

    We assessed the serotype distribution of Listeria monocytogenes isolates from clinical, beef, and environment samples using two PCR-based protocols for serogrouping. A panel of 134 isolates (22 clinical samples, 79 samples of beef cuts, and 33 samples from the beef processing environment) were subjected to conventional serology and identified as serotypes 1/2a (n = 12), 1/2b (n = 21), 1/2c (n = 71), and 4b (n = 30). Isolates from clinical samples were predominantly serotype 4b, and the most prevalent serotype among the beef cut and environment samples was 1/2c. The protocol described by M. Doumith, C. Buchrieser, P. Glaser, C. Jacquet, and P. Martin (J. Clin. Microbiol. 42:3819-3822, 2004) produced contradictory results for seven 1/2a isolates, which were positive for lmo1118 and had the profile IIc (serotypes 1/2c and 3c). Fifteen serotype 4b isolates amplified the target lmo0737, with the atypical profile IVb variant 1. The results obtained with the protocol described by M. K. Borucki and D. R. Call (J. Clin. Microbiol. 41:5537-5540, 2003) were in full agreement with those of the conventional serology. We recommend using this multiplex PCR approach by adding one pair of the reported primers to the panel to reduce total effort by one PCR while maintaining specificity. We present additional recommendations to improve the efficiency and reproducibility of this serogrouping assay. PMID:26735041

  7. Rapid diagnosis of aneuploidy using segmental duplication quantitative fluorescent PCR.

    PubMed

    Kong, Xiangdong; Li, Lin; Sun, Lei; Fu, Kepeng; Long, Ju; Weng, Xunjin; Ye, Xuehe; Liu, Xinxiong; Wang, Bo; Yan, Shanhuo; Ye, Haiming; Fan, Zuqian

    2014-01-01

    The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR), for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36); trisomy 18 (n = 6); trisomy 13 (n = 4); 45, X (n = 5); 47, XXX (n = 3); 48, XXYY (n = 2); and unaffected controls (n = 40). We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use. PMID:24625828

  8. An unusual presentation of herpes simplex encephalitis with negative PCR.

    PubMed

    Buerger, Kelly J; Zerr, Kayleigh; Salazar, Richard

    2015-01-01

    A 74-year-old man presented with acute right-sided hemiparesis and epilepsia partialis continua in association with fever and confusion. Initial workup revealed possible cerebritis in the left medial frontal lobe without involvement of the temporal lobes. Cerebrospinal fluid (CSF) analysis revealed minimal lymphocytic pleocytosis but negative real-time herpes simplex virus (HSV) PCR. Acyclovir was discontinued on day 5 due to a negative infectious workup and clinical improvement. On day 9 his condition deteriorated and he was transferred to a higher level of acuity for advanced supportive care. Worsening encephalopathy and refractory status epilepticus ensued despite medical care. Repeat CSF analysis showed mild lymphocytic pleocytosis with negative real-time HSV PCR. Brain MRI revealed progression of cortical enhancement. Immunosuppressive therapy and plasma exchange were attempted without clinical response. On day 24, another lumbar puncture showed only mild lymphocytic pleocytosis. Brain MRI showed involvement of the right medial temporal lobe. Subsequently, acyclovir was resumed. The HSV-1 PCR result was positive on day 30. Unfortunately, the patient expired. PMID:26243746

  9. Primer Extension Reactions for the PCR- based α- complementation Assay

    PubMed Central

    Achuthan, Vasudevan; DeStefano, Jeffrey J.

    2016-01-01

    The PCR- based- α- complementation assay is an effective technique to measure the fidelity of polymerases, especially RNA-dependent RNA polymerases (RDRP) and Reverse Transcriptases (RT). It has been successfully employed to determine the fidelity of the poliovirus polymerase 3D-pol (DeStefano, 2010) as well as the human immunodeficiency virus Reverse Transcriptase (HIV RT) (Achuthan et al., 2014). A major advantage of the assay is that since the PCR step is involved, even the low yield of products obtained after two rounds of low yield of RNA synthesis (for RDRP) or reverse transcription (for RT) can be measured using the assay. The assay also mimics the reverse transcription process, since both RNA- and DNA- directed RT synthesis steps are performed. We recently used this assay to show that the HIV RT, at physiologically relevant magnesium concentration, has accuracy in the same range as other reverse transcriptases (Achuthan et al., 2014). Here, we describe in detail how to prepare the inserts using the primer extension reactions. The prepared inserts are then processed further in the PCR- based- α- complementation assay.

  10. Milk adulteration: Detection of bovine milk in bulk goat milk produced by smallholders in northeastern Brazil by a duplex PCR assay.

    PubMed

    Rodrigues, N P A; Givisiez, P E N; Queiroga, R C R E; Azevedo, P S; Gebreyes, W A; Oliveira, C J B

    2012-05-01

    The aim of this study was to investigate the adulteration of goat milk produced by smallholders in semiarid northeastern Brazil with bovine milk as an adulterant. The study was requested by the association of smallholder producers in the region to investigate and to inhibit adulteration practices as a need to ensure the quality and safety of goat milk. A duplex PCR assay has been developed and standardized. Further validation was performed in 160 fresh bulk goat milk samples. The detection limit of the duplex PCR was 0.5% bovine milk in goat milk and the results indicated that 41.2% of the goat milk presented to market was positive for bovine milk. Making the test available to the association of producers, together with extension activities, have been applied to reduce adulteration in goat milk sold to small-scale dairy plants and to ensure the species origin for goat milk in the state of Paraíba. PMID:22541505

  11. Optimization of Droplet Digital PCR from RNA and DNA extracts with direct comparison to RT-qPCR: Clinical implications for quantification of Oseltamivir-resistant subpopulations.

    PubMed

    Taylor, Sean C; Carbonneau, Julie; Shelton, Dawne N; Boivin, Guy

    2015-11-01

    The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and ΔCq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery. PMID:26315318

  12. Single-step PCR in molecular diagnosis of hepatitis C virus infection.

    PubMed Central

    Farma, E; Boeri, E; Bettini, P; Repetto, C M; McDermott, J; Lillo, F B; Varnier, O E

    1996-01-01

    The diagnostic utility of two PCR systems and three PCR detection methods for hepatitis C virus (HCV) RNA was evaluated in serum samples. A nested PCR was considered the reference assay and was compared with two single-step PCR methods: the first is based on the detection of PCR products by liquid hybridization with a 32P-end-labeled probe, and the second is the Roche Amplicor colorimetric assay using microwell plate hybridization with a specific nucleic acid probe. Using the Pelicheck HCV RNA Eurohep genotype 1 proficiency panel, our laboratory achieved medium-high levels of performance with all three methods. The highest sensitivity was, however, observed with the isotopic single-step PCR (ss-PCR) method. The analytical sensitivity of ss-PCR with isotopic detection and ss-PCR with colorimetric detection was identical to that of nested PCR, with a 100% result concordance. Comparison of ss-PCR with enzyme-linked immunosorbent and RIBA assays in the analysis of clinical samples showed a high concordance. ss-PCR methods appear more suitable for diagnostic application. Nevertheless, HCV RNA PCR cannot be considered a screening assay; it should be requested in the presence of reactive serology or specific clinical symptomatology with altered liver parameters, and it is a potential tool for the follow-up of patients with HCV infection. PMID:8940466

  13. Nucleic acid extraction from polluted estuarine water for detection of viruses and bacteria by PCR and RT-PCR analysis.

    PubMed

    Petit, F; Craquelin, S; Guespin-Michel, J; Buffet-Janvresse, C

    1999-03-01

    We describe an extraction protocol for genomic DNA and RNA of both viruses and bacteria from polluted estuary water. This procedure was adapted to the molecular study of microflora of estuarine water where bacteria and viruses are found free, forming low-density biofilms, or intimately associated with organo-mineral particles. The sensitivity of the method was determined with seeded samples for RT-PCR and PCR analysis of viruses (10 virions/mL), and bacteria (1 colony-forming unit mL). We report an example of molecular detection of both poliovirus and Salmonella in the Seine estuary (France) and an approach to studying their association with organo-mineral particles. PMID:10209769

  14. Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta

    PubMed Central

    Rueda-Martínez, Carmen; Lamas, Oscar; Mataró, María José; Robledo-Carmona, Juan; Sánchez-Espín, Gemma; Jiménez-Navarro, Manuel; Such-Martínez, Miguel; Fernández, Borja

    2014-01-01

    Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1β, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples. PMID:24841551

  15. Detection of feline calicivirus, feline herpesvirus 1 and Chlamydia psittaci mucosal swabs by multiplex RT-PCR/PCR.

    PubMed

    Sykes, J E; Allen, J L; Studdert, V P; Browning, G F

    2001-07-26

    A single tube, multiplex reverse transcription (RT)-polymerase chain reaction (PCR)/PCR assay was developed for detection of feline herpesvirus 1 (FHV1), Chlamydia psittaci and feline calicivirus (FCV) in cats with upper respiratory tract disease (URTD), incorporating a simple, rapid extraction procedure capable of extracting both DNA and RNA. The assay was found to be as sensitive in vitro as simplex assays that have previously been shown to be as sensitive as, or more sensitive than, culture for each pathogen in experimentally infected cats. Conjunctival alone or both conjunctival and oropharyngeal swabs were collected from cats in 104 households with URTD. FHV1 was detected in 18 (17.3%) and C. psittaci was detected in 12 (11.5%) households. The prevalence of C. psittaci was not significantly different to that determined using a duplex PCR assay for C. psittaci and FHV1. The prevalence of FCV was affected by sample storage temperature. Of samples stored at -70 degrees C, 0/31 were positive for FCV but FCV was detected in 10/73 (13.7%) samples stored at 4 degrees C (P=0.006). Of the samples stored at 4 degrees C, 3/19 (15.8%) conjunctival swabs were positive for FCV and 6/32 (18.8%) oropharyngeal/conjunctival swabs were positive for FCV (P=0.79). The potential utility of restriction endonuclease analysis of RT-PCR products resulting from amplification of the hypervariable region of the capsid protein gene of FCV in field samples, without prior cultivation, was also examined. The assay may have considerable importance for diagnosis and epidemiological surveys of feline upper respiratory tract pathogens. PMID:11376956

  16. 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

  17. Use of an arbitrarily primed PCR product in the development of a Campylobacter jejuni-specific PCR.

    PubMed Central

    Day, W A; Pepper, I L; Joens, L A

    1997-01-01

    Development of a PCR assay for Campylobacter jejuni is based on the isolation of species-specific DNA. An arbitrarily primed PCR incorporating 10-mer primers was used to generate fingerprints of C. jejuni M129 genomic DNA. Fingerprint products were then screened individually for their species specificity in dot blot hybridizations with 6 C. jejuni isolates, 4 Campylobacter species other than C. jejuni, and 27 enteric bacterial species other than Campylobacter spp. A 486-bp fingerprint product hybridized specifically to C. jejuni DNA under stringent conditions; no binding to Campylobacter DNA other than that of C. jejuni or to DNA from enteric bacteria was detected. The 486-bp fingerprint product was sequenced, and primers corresponding to three overlapping regions of the DNA probe were synthesized. Evaluation of the three primer pairs for specificity to C. jejuni DNA identified an oligonucleotide primer pair which amplified a 265-bp product from six C. jejuni isolates only. In sensitivity studies using a crude M129 lysate as the template, the C. jejuni-specific PCR amplified the 265-bp product in a lysate with as few as 100 bacteria. PMID:9055418

  18. Microbial Pollution Tracking of Dairy Farm with a Combined PCR-DGGE and qPCR Approach.

    PubMed

    Xi, Xiaoxia; Zhang, Jiachao; Kwok, Laiyu; Huo, Dongxue; Feng, Shuzhen; Zhang, Heping; Sun, Tiansong

    2015-12-01

    Animal husbandry is a traditional industry with regional characteristic in the Inner Mongolia of China. Recent years, animal breeding has been one of the main pollution sources in this area, followed by domestic sewage and industrial wastewater. The pollution of livestock farm feces may accelerate the development of pathogens and antibiotic resistance genes which pose health risks to humans and animals. In present research, culture-independent molecular ecological methods based on DGGE combined with qPCR were used to investigate the pollution to surrounding environment with different degrees of livestock farm. The cluster analysis of DGGE patterns showed that the livestock farm feces from point pollution source flowed with wastewater discharge has resulted in an impacted range of at least 3000 m, but it did not cause pollution to residential water delivered from upstream of sewage drain outlet. qPCR results revealed that 5 common pathogens (Escherichia coli, Enterococcus, Staphylococcus aureus, Shigella, and Salmonella) presented decreased trend as the sampled distance from point pollution source increased. Also, qPCR assays of 10 common antibiotic resistance genes (tetO, tetL, rpp, rpoB, sul2, sulA, floR, yidY, mphA, and ermC) which cause resistance to tetracycline, rifampicin, fluoroquinolone, quinolone, and erythromycin have been found in the environmental samples. This study clearly indicates the livestock farm discharge pollutants contaminated to the surrounding environment. Our data have provided important information to pollution control in the future. PMID:26341923

  19. Type A influenza virus detection from horses by real-time RT-PCR and insulated isothermal RT-PCR.

    PubMed

    Balasuriya, Udeni B R

    2014-01-01

    Equine influenza (EI) is a highly contagious disease of horses caused by the equine influenza virus (EIV) H3N8 subtype. EI is the most important respiratory virus infection of horses and can disrupt major equestrian events and cause significant economic losses to the equine industry worldwide. Influenza H3N8 virus spreads rapidly in susceptible horses and can result in very high morbidity within 24-48 h after exposure to the virus. Therefore, rapid and accurate diagnosis of EI is critical for implementation of prevention and control measures to avoid the spread of EIV and to reduce the economic impact of the disease. The probe-based real-time reverse transcriptase polymerase chain reaction (rRT-PCR) assays targeting various EIV genes are reported to be highly sensitive and specific compared to the Directigen Flu A(®) test and virus isolation in embryonated hens' eggs. Recently, a TaqMan(®) probe-based insulated isothermal RT-PCR (iiRT-PCR) assay for the detection of EIV H3N8 subtype has been described. These molecular based diagnostic assays provide a fast and reliable means of EIV detection and disease surveillance. PMID:24899448

  20. 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

  1. FUNGAL-SPECIFIC PCR PRIMERS DEVELOPED FOR ANALYSIS OF THE ITS REGION OF ENVIRONMENTAL DNA EXTRACTS

    EPA Science Inventory

    Background The Internal Transcribed Spacer (ITS) regions of fungal ribosomal DNA (rDNA) are highly variable sequences of great importance in distinguishing fungal species by PCR analysis. Previously published PCR primers available for amplifying these sequences from environmenta...

  2. 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

  3. A-T linker adapter polymerase chain reaction for determining flanking sequences by rescuing inverse PCR or thermal asymmetric interlaced PCR products.

    PubMed

    Trinh, Quoclinh; Zhu, Pengyu; Shi, Hui; Xu, Wentao; Hao, Junran; Luo, Yunbo; Huang, Kunlun

    2014-12-01

    The polymerase chain reaction (PCR)-based genome walking method has been extensively used to isolate unknown flanking sequences, whereas nonspecific products are always inevitable. To resolve these problems, we developed a new strategy to isolate the unknown flanking sequences by combining A-T linker adapter PCR with inverse PCR (I-PCR) or thermal asymmetric interlaced PCR (TAIL-PCR). The result showed that this method can be efficiently achieved with the flanking sequence from the Arabidopsis mutant and papain gene. Our study provides researchers with an additional method for determining genomic DNA flanking sequences to identify the target band from bulk of bands and to eliminate the cloning step for sequencing. PMID:25086366

  4. Specific PCR to identify the heavy-metal-resistant bacterium Cupriavidus metallidurans.

    PubMed

    Ryan, Michael P; Adley, Catherine C

    2011-09-01

    The aim of this study is to develop a polymerase chain reaction (PCR) assay for rapid detection of Cupriavidus metallidurans. PCR primers targeting the Signal transduction histidine kinase gene were designed and designated Cm-F1/Cm-R1. Strains of C. metallidurans were positively identified. The size of the PCR products was 437 bp, as expected. This PCR method enables monitoring of industrial, environmental and clinical sources for presence of C. metallidurans. PMID:21720772

  5. Comparative analysis of human papillomavirus detection by PCR and non-isotopic in situ hybridisation.

    PubMed Central

    Herrington, C S; Anderson, S M; Bauer, H M; Troncone, B; de Angelis, M L; Noell, H; Chimera, J A; Van Eyck, S L; McGee, J O

    1995-01-01

    AIMS--To assess the relative diagnostic performance of the polymerase chain reaction (PCR) and non-isotopic in situ hybridisation (NISH) and to correlate these data with cytopathological assessment. METHODS--Paired analysis of human papillomavirus (HPV) detection was performed by PCR and NISH on exfoliated cervical cells from 122 women attending a routine gynaecological examination. PCR amplification followed by generic and HPV type specific hybridisation was compared with NISH on a parallel cervical smear. RESULTS--Overall, 32 cases were positive by NISH and 61 positive by PCR. Of the 105 cases in which both PCR and NISH were interpretable, 76 (26%) were normal smears, 20 of which were HPV positive by NISH and 37 (49%) by PCR. Of 17 borderline smears, two were NISH positive and 12 PCR positive. Eight of nine smears containing koilocytes were positive by NISH and seven by PCR. Of three dyskaryotic smears, none were NISH and two were PCR positive. The concordance of NISH and PCR in these samples was 57%. To assess sampling error, NISH and PCR were performed on an additional 50 cases using aliquots from the same sample. This increased the concordance between assays to 74%. Filter hybridisation of PCR products with the cocktail of probes used in NISH (under low and high stringency conditions) demonstrated that several cases of NISH positivity could be accounted for by cross-hybridisation to HPV types identified by PCR but not present in the NISH probe cocktail. CONCLUSIONS--Sampling error and potential cross-hybridisation of probe and target should be considered in interpretation of these techniques. PCR is more sensitive because it provides for the amplification of target DNA sequences. In addition, the PCR assay utilised in this study detects a wider range of HPV types than are contained in the cocktails used for NISH. However, PCR assays detect viral DNA present both within cells and in cervical fluid whereas NISH permits morphological localisation. Images PMID

  6. Comparative analysis of human papillomavirus detection by PCR and non-isotopic in situ hybridisation.

    PubMed

    Herrington, C S; Anderson, S M; Bauer, H M; Troncone, B; de Angelis, M L; Noell, H; Chimera, J A; Van Eyck, S L; McGee, J O

    1995-05-01

    AIMS--To assess the relative diagnostic performance of the polymerase chain reaction (PCR) and non-isotopic in situ hybridisation (NISH) and to correlate these data with cytopathological assessment. METHODS--Paired analysis of human papillomavirus (HPV) detection was performed by PCR and NISH on exfoliated cervical cells from 122 women attending a routine gynaecological examination. PCR amplification followed by generic and HPV type specific hybridisation was compared with NISH on a parallel cervical smear. RESULTS--Overall, 32 cases were positive by NISH and 61 positive by PCR. Of the 105 cases in which both PCR and NISH were interpretable, 76 (26%) were normal smears, 20 of which were HPV positive by NISH and 37 (49%) by PCR. Of 17 borderline smears, two were NISH positive and 12 PCR positive. Eight of nine smears containing koilocytes were positive by NISH and seven by PCR. Of three dyskaryotic smears, none were NISH and two were PCR positive. The concordance of NISH and PCR in these samples was 57%. To assess sampling error, NISH and PCR were performed on an additional 50 cases using aliquots from the same sample. This increased the concordance between assays to 74%. Filter hybridisation of PCR products with the cocktail of probes used in NISH (under low and high stringency conditions) demonstrated that several cases of NISH positivity could be accounted for by cross-hybridisation to HPV types identified by PCR but not present in the NISH probe cocktail. CONCLUSIONS--Sampling error and potential cross-hybridisation of probe and target should be considered in interpretation of these techniques. PCR is more sensitive because it provides for the amplification of target DNA sequences. In addition, the PCR assay utilised in this study detects a wider range of HPV types than are contained in the cocktails used for NISH. However, PCR assays detect viral DNA present both within cells and in cervical fluid whereas NISH permits morphological localisation. PMID:7629286

  7. Detection sensitivity and quantitation of Plasmodium falciparum var gene transcripts by real-time RT-PCR in comparison with conventional RT-PCR.

    PubMed

    Gatton, Michelle L; Peters, Jennifer M; Gresty, Karryn; Fowler, Elizabeth V; Chen, Nanhua; Cheng, Qin

    2006-08-01

    Antigenic variation in Plasmodium falciparum erythrocyte membrane protein 1, caused by a switch in transcription of the encoding var gene, is an important feature of malaria. In this study, we quantified the relative abundance of var gene transcripts present in P. falciparum parasite clones using real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional RT-PCR combined with cloning and sequencing, with the aim of directly comparing the results obtained. When there was sufficient abundance of RNA for the real-time RT-PCR assay to be operating within the region of good reproducibility, RT-PCR and real-time RT-PCR tended to identify the same dominant transcript, although some transcript-specific issues were identified. When there were differences in the estimated relative amounts of minor transcripts, the RT-PCR assay tended to produce higher estimates than real-time RT-PCR. These results provide valuable information comparing RT-PCR and real-time RT-PCR analysis of samples with small quantities of RNA as might be expected in the analysis of field or clinical samples. PMID:16896121

  8. Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach

    PubMed Central

    Hořínek, Aleš; Novotná, Michaela; Calda, Pavel; Korabečná, Marie

    2015-01-01

    Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods’ performance parameters—standard curve linearity, detection limit and measurement precision—were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438). PMID:26562517

  9. Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

    PubMed

    Odongo, Steven; Delespaux, Vincent; Ngotho, Maina; Bekkele, Serkalem Mindaye; Magez, Stefan

    2016-09-01

    We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05). PMID:27423733

  10. Real-time PCR as a tool to study weed biology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time polymerase chain reaction (Real-time PCR) also known as quantitative PCR is used to determine relative gene expression or to quantify exact levels of mRNA in cells or tissues. Before the advent of Real-time PCR, the major difficulty associated with traditional quantitative or semi-quantita...

  11. Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms.

    PubMed

    Krawczyk, Beata; Kur, Józef; Stojowska-Swędrzyńska, Karolina; Śpibida, Marta

    2016-01-01

    A significant number of DNA-based techniques has been introduced into the field of microorganisms' characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. In recent years, some alternative techniques based on ligation of oligonucleotide adapters before DNA amplification by PCR, known as Ligation-Mediated PCR methods (LM PCR), have been successfully applied for the typing of microorganisms below the species level. These molecular methods include: Amplified Fragment Length Polymorphism (AFLP), Amplification of DNA fragments Surrounding Rare Restriction Sites (ADSRRS), PCR Melting Profiles (PCR MP), Ligation Mediated PCR/Shifter (LM PCR/Shifter), Infrequent-Restriction-Site Amplification (IRS PCR), double digestion Ligation Mediated Suppression PCR (ddLMS PCR). These techniques are now applied more and more often because they involve less time, are comparably inexpensive, and require only standard lab equipment. Here, we present a general review of this group of methods showing their possibilities and limitations. We also identify questions and propose solutions which may be helpful in choosing a particular LM PCR method for the achievement of the required goal. PMID:26885774

  12. Underdiagnosing of Mycoplasma pneumoniae infections as revealed by use of a respiratory multiplex PCR panel.

    PubMed

    Dalpke, Alexander; Zimmermann, Stefan; Schnitzler, Paul

    2016-09-01

    We compared a multiplex PCR diagnostic approach against specific PCR diagnosis for detection of Mycoplasma pneumoniae infection. Seventy-five percent of all M. pneumoniae infections were only detected "unintentionally" by the use of the multiplex PCR indicating underdiagnosing of M. pneumoniae due to absence of clinical suspicion. PMID:27377674

  13. Quantitative PCR for glucose transporter and tristetraprolin family gene expression in cultured mouse adipocytes and macrophages

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time PCR (qPCR) such as TaqMan and SYBR Green qPCR are widely used for gene expression analysis. The drawbacks of SYBR Green assay are that the dye binds to any double-stranded DNA which can generate falsepositive signals and that the length of the amplicon affects the intensity of...

  14. Detection and quantification of Bacillus cereus group in milk by droplet digital PCR.

    PubMed

    Porcellato, Davide; Narvhus, Judith; Skeie, Siv Borghild

    2016-08-01

    Droplet digital PCR (ddPCR) is one of the newest and most promising methods for the detection and quantification of molecular targets by PCR. Here, we optimized and used a new ddPCR assay for the detection and quantification of the Bacillus cereus group in milk. We also compared the ddPCR to a standard qPCR assay. The new ddPCR assay showed a similar coefficient of determination and a better limit of detection compared to the qPCR assay during quantification of the target molecules in the samples. However, the ddPCR assay has a limitation during quantification of a high number of target molecules. This new assay was then tested for the quantification of the B. cereus group in 90 milk samples obtained over three months from two different dairies and the milk was stored at different temperatures before sampling. The ddPCR assay showed good agreement with the qPCR assay for the quantification of the B. cereus group in milk, and due to its lower detection limit more samples were detected as positive. The new ddPCR assay is a promising method for the quantification of target bacteria in low concentration in milk. PMID:27211508

  15. Type-A influenza virus detection and quantitation by real-time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (RRT-PCR) is a relatively new technology which has been used for AIV detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of RRT-PCR are: quantitative nature, scalability, cost, high sensitivity, high specificity, and ...

  16. Exogeneous controls to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. A SOE-PCR method of introducing multiple mutations into Mycoplasma gallisepticum neuraminidase.

    PubMed

    Tan, Lei; Chen, Hongjun; Yu, Shengqing; Qiu, Xusheng; Song, Cuiping; Chen, Danqing; Zhang, Shilei; Zhang, Fanqing; He, Suibin; Shen, Xinyue; Hu, Meirong; Ding, Chan

    2013-08-01

    A modified splicing with overlap extension PCR (SOE-PCR) was generated to introduce 21 TGA to TGG at Mycoplasma gallisepticum MGA_0329 gene. The recombinant protein was successfully expressed and retained neuraminidase activities, indicating that SOE-PCR is a rapid and highly efficient method of introducing multiple mutations into large M. gallisepticum genes. PMID:23707236

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. Effects of prolonged chlorine exposures upon PCR detection of Helicobacter pylori DNA.

    EPA Science Inventory

    The effect of low doses of free chlorine on the detection by qPCR of Helicobacter pylori (H. pylori) cells by qPCR in tap water was monitored. H. pylori target sequences (within suspended, intact cells at densities of 102 to 103 cells /ml) were rendered undetectable by qPCR an...

  20. A SIMPLE VERSATILE HIGH THROUPHPUT DNA EXTRACTION METHOD SUITABLE FOR PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR has become the most popular technique in functional genomics. Both forward and reverse genetic projects routinely require PCR amplification of thousands of samples. Processing the samples for DNA suitable for PCR is usually the limiting step. We have developed a simple high throughput DNA extra...

  1. Evaluation of Quantitative Real-Time PCR Assays for Detection of Citrus Greening

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus huanglongbing (HLB), or citrus greening, is a serious and industry-limiting disease. Preliminary diagnoses can be made through visual symptoms, and greater certainty can be achieved through quantitative real-time PCR (qPCR). Several qPCR procedures are available including those by designed by...

  2. A proline racemase based PCR for identification of Trypanosoma vivax in cattle blood.

    PubMed

    Fikru, Regassa; Hagos, Ashenafi; Rogé, Stijn; Reyna-Bello, Armando; Gonzatti, Mary Isabel; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe

    2014-01-01

    A study was conducted to develop a Trypanosoma vivax (T. vivax) specific PCR based on the T. vivax proline racemase (TvPRAC) gene. Forward and reverse primers were designed that bind at 764-783 bp and 983-1002 bp of the gene. To assess its specificity, TvPRAC PCR was conducted on DNA extracted from different haemotropic pathogens: T. vivax from Nigeria, Ethiopia and Venezuela, T. congolense Savannah type, T. brucei brucei, T. evansi, T. equiperdum, T. theileri, Theileria parva, Anaplasma marginale, Babesia bovis and Babesia bigemina and from bovine, goat, mouse, camel and human blood. The analytical sensitivity of the TvPRAC PCR was compared with that of the ITS-1 PCR and the 18S PCR-RFLP on a dilution series of T. vivax DNA in water. The diagnostic performance of the three PCRs was compared on 411 Ethiopian bovine blood specimens collected in a former study. TvPRAC PCR proved to be fully specific for T. vivax, irrespective of its geographical origin. Its analytical sensitivity was lower than that of ITS-1 PCR. On these bovine specimens, TvPRAC PCR detected 8.3% T. vivax infections while ITS-1 PCR and 18S PCR-RFLP detected respectively 22.6 and 6.1% T. vivax infections. The study demonstrates that a proline racemase based PCR could be used, preferably in combination with ITS-1 PCR, as a species-specific diagnostic test for T. vivax infections worldwide. PMID:24416292

  3. DEVELOPMENT OF AN IMPROVED PCR-BASED TECHNIQUE FOR DETECTION OF PHYTOPHTHORA CACTORUM IN STRAWBERRY PLANTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Specific and rapid plant pathogen detection methods can aid in strawberry disease management decisions. PCR-based diagnostics for Phytophthora cactorum and other strawberry pathogens are hindered by PCR inhibitors and lack of species-specific PCR primers. We developed a DNA extraction and purificati...

  4. The PCR-GLOBWB global hydrological reanalysis product

    NASA Astrophysics Data System (ADS)

    Bierkens, M. F.; Wanders, N.; Sutanudjaja, E.; Van Beek, L. P.

    2013-12-01

    Accurate and long time series of hydrological data are important for understanding land surface water and energy budgets in many parts of the world, as well as for improving real-time hydrological monitoring and climate change anticipation. The ultimate goal of the present work is to produce a multi-decadal land surface hydrological reanalysis with retrospective and updated hydrological states and fluxes that are constrained to available in-situ river discharge measurements. Here we used PCR-GLOBWB (van Beek et al., 2011), which is a large-scale hydrological model intended for global to regional studies. PCR-GLOBWB provides a grid-based representation of terrestrial hydrology with a typical spatial resolution of approximately 50×50 km (currently 0.5° globally) on a daily basis. For each grid cell, PCR-GLOBWB is basically a leaky bucket type of water balance model with a process-based simulation of moisture storage in two vertically stacked soil layers as well as the water exchange between the soil and the atmosphere and the underlying groundwater reservoir. Exchange to the atmosphere comprises precipitation, evaporation and transpiration, as well as snow accumulation and melt, which are all simulated by considering vegetation phenology and sub-grid distributions of elevation, land cover and soil saturation distribution. The model thus includes detailed schemes for runoff-infiltration partitioning, interflow, groundwater recharge and baseflow, as well as river routing of discharge. . By embedding the PCR-GLOBWB model in an Ensemble Kalman Filter framework, we calibrated the model parameters based on the discharge observations from the Global Runoff Data Centre. The parameters calibrated are related to snow module, runoff-infiltration partitioning, groundwater recharge, channel discharge and baseflow processes, as well as pre-factors to correct forcing precipitation fields due to local topographic and orographic effects. Results show that the model parameters can

  5. The PCR-GLOBWB global hydrological reanalysis product

    NASA Astrophysics Data System (ADS)

    Wanders, Niko; Bierkens, Marc; Sutanudjaja, Edwin; van Beek, Rens

    2014-05-01

    Accurate and long time series of hydrological data are important for understanding land surface water and energy budgets in many parts of the world, as well as for improving real-time hydrological monitoring and climate change anticipation. The ultimate goal of the present work is to produce a multi-decadal "land surface hydrological reanalysis" dataset with retrospective and updated hydrological states and fluxes that are constrained to available in-situ river discharge measurements. Here we use PCR-GLOBWB (van Beek et al., 2011), which is a large-scale hydrological model intended for global to regional studies. PCR-GLOBWB provides a grid-based representation of terrestrial hydrology with a typical spatial resolution of approximately 50×50 km (currently 0.5° globally) on a daily basis. For each grid cell, PCR-GLOBWB simulates moisture storage in two vertically stacked soil layers as well as the water exchange between the soil and the atmosphere and the underlying groundwater reservoir. Exchange to the atmosphere comprises precipitation, evaporation and transpiration, as well as snow accumulation and melt, which are all simulated by considering vegetation phenology and sub-grid variations of elevation, land cover and soil saturation distribution. The model includes improved schemes for runoff-infiltration partitioning, interflow, groundwater recharge and baseflow, as well as river routing of discharge. It also dynamically simulates water storage in reservoirs, water demand and the withdrawal, allocation and consumptive use of surface water and groundwater resources. By embedding the PCR-GLOBWB model in an Ensemble Kalman Filter framework, we calibrate the model parameters based on the discharge observations from the Global Runoff Data Centre. The parameters calibrated are related to snow accumulation and melt, runoff-infiltration partitioning, groundwater recharge, channel discharge and baseflow processes, as well as pre-factors to correct forcing precipitation

  6. PCR and Genotyping for HPV in Cervical Cancer Patients

    PubMed Central

    Prakash, Pradyot; Patne, Shashikant C U; Singh, Ashish Kumar; Kumar, Mohan; Mishra, Mukti Nath; Gulati, Anil Kumar

    2016-01-01

    Aims: To devise nested multiplex polymerase chain reaction (NMPCR) protocol for detection of mucosal human papilloma viruses (HPVs) and typing of HPV-16 and -18 in formalin-fixed, paraffin-embedded (FFPE) tissues of carcinoma cervix (CaCx). Settings and Design: Cross-sectional observational study. Materials and Methods: NMPCR was done for simultaneous detection of HPV, targeting 134 bp L1 capsid gene employing GP+/mGP+ primers and typing of genotypes-16 and -18, targeting E6/E7 gene from 34 FFPE tissue blocks of CaCx and cervical intraepithelial neoplasia (CIN). Detection of 142 bp consensus sequence of L1 capsid gene was performed by nested PCR employing MY/GP+ primers. Sequencing of selected PCR amplicons of the later protocol obtained from control cell line DNA and 5 select samples were done for validation of the NMPCR protocol. Statistical Analysis Used: Calculation of percentage from the Microsoft Excel Software. Results: Of 26 FFPE samples of CaCx, 17 (65.3%) samples were found positive for HPV by NMPCR. Amplicons of 142 bp L1 capsid gene employing MY/GP+ primers were observed in 11 (42.3%) samples of CaCx. Nearly 25% samples of CIN were positive for HPV. On sequence analysis, it was observed that the sample typed as HPV-16 by NMPCR was found to be the same on sequencing of amplicons obtained after MY/GP+ nested PCR. Conclusions: This study indicates the usefulness of our NMPCR protocol for detection of mucosal HPVs and typing of HPV-16 and -18 from FFPE tissue samples of CaCx. The NMPCR protocol may be used to detect HPV and type common genotypes-16 and -18 in fresh tissue of cervical biopsy or scrape samples for screening of CaCx. PMID:27621560

  7. PCR-based polymorphisms in neurofibromatosis type 1 (NFI)

    SciTech Connect

    Lai, P.S.; Chee, S.; Low, P.S.

    1994-09-01

    Neurofibromatosis type 1 (NF1) is one of the most common genetic disorders in humans with an incidence of 1 in 3,000. The NF1 gene is located on chromosome 17q 11.2 and encodes an ubiquitously expressed transcript of about 13kb. Direct mutation detection is difficult in this disorder due to the large gene size, high mutation rate and variety of mutations. We have studied the allele frequencies of seven PCR-based polymorphisms. Six of the probes used flank the NF1 gene, namely p11.3C4.2/Msp I (proximal), pEW206/Msp I (distal), p2.f9.8/Rsa I (distal), pEW207/Bgl II (distal), pEW207/Hind III (distal) and pHHH202/Rsa I (proximal). An intragenic RFLP, pEvi 2B-B/Eco R1 polymorphism in intron 27, was also analyzed by PCR. Allele frequencies for 48 normal unrelated individuals were obtained as follows: A1 = 0.40, A2 = 0.6 (p11.3C4.2/Msp I), A1 = 0.44, A2 = 0.56 (pEW206/Msp I), A1 = 0.17, A2 = 0.83 (p2.F9.8/Rsa I), A1 = 0.64, A2 = 0.36 (pEW207/Bgl I), A1 = 0.45, A2 = 0.55 (pEvi 2B-B/Eco RI). Heterozygosity rates of the alleles ranged from 20.8% to 51.7%. Using a combination of these markers, seven local families with NF1 were studied. Normal Mendelian segregation of alleles was observed in these families and no recombination was detected so far. These PCR-based markers were found to be useful for linkage analysis in our families.

  8. [Detection of Staphylococcus aureus toxins using immuno-PCR].

    PubMed

    Maerle, A V; Riazantsev, D Iu; Dmitrenko, O A; Petrova, E Ia; Komaleva, R L; Sergeev, I V; Trofimov, D Iu; Zavriev, S K

    2014-01-01

    A highly sensitive test system, based on the method of immuno-PCR, was developed for the detection of two staphylococcal toxins: enterotoxin A (SEA) and toxic shock syndrome toxin (TSST). A key element of the developed systems was obtaining supramolecular complexes of bisbiotinylated oligodeoxynucleotides and streptavidin, which were used as DNA tags. Specificity studies showed no cross-reactivity when determining SEA and TSST. The sensitivity of detection of these toxins in the culture supernatants S. aureus was not less than 10 pg/mL. PMID:25895352

  9. [Seal finger in Denmark diagnosed by PCR-technique].

    PubMed

    Jansen, Louise Charlotte; Justesen, Ulrik Stenz; Roos, Signe Moeslund; Dargis, Rimtas; Jensen, Jørgen Skov; Christensen, Jens Jørgen; Kemp, Michael

    2012-02-13

    Seal finger is an unusual infection in Denmark but is seen quite often in Greenland. A 69 year-old Danish man developed severe infection after cutting his finger on a sea urchin while handling a fishing net. Treatment with beta-lactam antibiotics had no effect. Standard culture from the lesion was negative. A Mycoplasma species was detected by PCR and DNA sequencing and subsequently cultured on special media. Specifically asked about exposure to sea mammals the patient could inform that a dead seal had also been trapped in the fishing net. PMID:22331047

  10. PCR amplification of crude microbial DNA extracted from soil.

    PubMed

    Yeates, C; Gillings, M R; Davison, A D; Altavilla, N; Veal, D A

    1997-10-01

    A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types. DNA was extracted from 100 g of soil using direct lysis with glass beads and sodium dodecyl sulphate (SDS) followed by polyethylene glycol precipitation, potassium acetate precipitation, phenol extraction and isopropanol precipitation. The crude extract could be used in PCR directed at high-copy number (bacterial small subunit rRNA) and single-copy (fungal beta-tubulin) genes. PMID:9351282

  11. Methods for microbial DNA extraction from soil for PCR amplification.

    PubMed

    Yeates, C; Gillings, M R; Davison, A D; Altavilla, N; Veal, D A

    1998-05-14

    Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size. PMID:12734590

  12. A PCR amplification strategy for unrestricted generation of chimeric genes.

    PubMed

    Vos, Michel J; Kampinga, Harm H

    2008-09-15

    For analyzing protein function, protein dynamics, or protein-protein interactions, the use of chimeric proteins has become an indispensable tool. The generation of DNA constructs coding for such fused proteins can, however, be a tedious process. Currently used strategies often make use of available endonuclease sites, leading to limitations in the choice of the site of fusion between two genes and problems in maintaining protein secondary structure. We have developed a cloning strategy to get around these disadvantages that is based on a single round of PCR amplification followed by antibiotic-resistant gene complementation. PMID:18555003

  13. First Australian isolation of epidemic Clostridium difficile PCR ribotype 027.

    PubMed

    Riley, Thomas V; Thean, Sarah; Hool, Graham; Golledge, Clayton L

    2009-06-15

    We report the first isolation in Australia of a hypervirulent epidemic strain of Clostridium difficile, PCR ribotype 027. It was isolated from a 43-year-old woman with a permanent ileostomy, who appears to have been infected while travelling in the United States. The isolate was positive for toxin A, toxin B and binary toxin, and resistant to fluoroquinolone antimicrobials, and had characteristic deletions in the tcdC gene. All diagnostic laboratories and health care facilities in Australia should now be on high alert for this organism. PMID:19527210

  14. Quantitative analysis of food and feed samples with droplet digital PCR.

    PubMed

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

    2013-01-01

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

  15. Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR

    PubMed Central

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

    2013-01-01

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

  16. Development of PCR and TaqMan PCR Assays to Detect Pseudomonas coronafaciens, a Causal Agent of Halo Blight of Oats

    PubMed Central

    An, Ji-Hye; Noh, Young-Hee; Kim, Yong-Eon; Lee, Hyok-In; Cha, Jae-Soon

    2015-01-01

    Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens. PMID:25774107

  17. Absolute Quantification of Enterococcal 23S rRNA Gene Using Digital PCR.

    PubMed

    Wang, Dan; Yamahara, Kevan M; Cao, Yiping; Boehm, Alexandria B

    2016-04-01

    We evaluated the ability of chip-based digital PCR (dPCR) to quantify enterococci, the fecal indicator recommended by the United States Environmental Protection Agency (USEPA) for water-quality monitoring. dPCR uses Poisson statistics to estimate the number of DNA fragments in a sample with a specific sequence. Underestimation may occur when a gene is redundantly encoded in the genome and multiple copies of that gene are on one DNA fragment. When genomic DNA (gDNA) was extracted using two commercial DNA extraction kits, we confirmed that dPCR could discern individual copies of the redundant 23s rRNA gene in the enterococcal genome. dPCR quantification was accurate when compared to the nominal concentration inferred from fluorometer measurements (linear regression slope = 0.98, intercept = 0.03, R(2) = 0.99, and p value <0.0001). dPCR quantification was also consistent with quantitative PCR (qPCR) measurements as well as cell counts for BioBall reference standard and 24 environmental water samples. qPCR and dPCR quantification of enterococci in the 24 environmental samples were significantly correlated (linear regression slope =1.08, R(2) of 0.96, and p value <0.0001); the group mean of the qPCR measurements was 0.19 log units higher than that of the dPCR measurements. At environmentally relevant concentrations, dPCR quantification was more precise (i.e., had narrower 95% confidence intervals than qPCR quantification). We observed that humic acid caused a similar level of inhibition in both dPCR and qPCR, but calcium inhibited dPCR to a lesser degree than qPCR. Inhibition of dPCR was partially relieved when the number of thermal cycles was increased. Based on these results, we conclude that dPCR is a viable option for enumerating enterococci in ambient water. PMID:26903207

  18. Identifying of meat species using polymerase chain reaction (PCR)

    SciTech Connect

    Foong, Chow Ming; Sani, Norrakiah Abdullah

    2013-11-27

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one’s diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing.

  19. Quantitative PCR for genetic markers of human fecal pollution.

    PubMed

    Shanks, Orin C; Kelty, Catherine A; Sivaganesan, Mano; Varma, Manju; Haugland, Richard A

    2009-09-01

    Assessment of health risk and fecal bacterial loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface-associated genes. Each assay exhibited a range of quantification from 10 to 1 x 10(6) copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker in primary effluent wastewater samples collected from 20 geographically distinct locations was measured and compared to quantities estimated by real-time PCR assays specific for rRNA gene sequences from total Bacteroidales and enterococcal fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters. PMID:19592537

  20. One-step PCR amplification of complete arthropod mitochondrial genomes.

    PubMed

    Hwang, U W; Park, C J; Yong, T S; Kim, W

    2001-06-01

    A new PCR primer set which enables one-step amplification of complete arthropod mitochondrial genomes was designed from two conserved 16S rDNA regions for the long PCR technique. For this purpose, partial 16S rDNAs amplified with universal primers 16SA and 16SB were newly sequenced from six representative arthropods: Armadillidium vulgare and Macrobrachium nipponense (Crustacea), Anopheles sinensis (Insecta), Lithobius forficatus and Megaphyllum sp. (Myriapoda), and Limulus polyphemus (Chelicerata). The genomic locations of two new primers, HPK16Saa and HPK16Sbb, correspond to positions 13314-13345 and 12951-12984, respectively, in the Drosophila yakuba mitochondrial genome. The usefulness of the primer set was experimentally examined and confirmed with five of the representative arthropods, except for A. vulgare, which has a linearized mitochondrial genome. With this set, therefore, we could easily and rapidly amplify complete mitochondrial genomes with small amounts of arthropod DNA. Although the primers suggested here were examined only with arthropod groups, a possibility of successful application to other invertebrates is very high, since the high degree of sequence conservation is shown on the primer sites in other invertebrates. Thus, this primer set can serve various research fields, such as molecular evolution, population genetics, and molecular phylogenetics based on DNA sequences, RFLP, and gene rearrangement of mitochondrial genomes in arthropods and other invertebrates. PMID:11399145

  1. PCR Primers for Metazoan Mitochondrial 12S Ribosomal DNA Sequences

    PubMed Central

    Machida, Ryuji J.; Kweskin, Matthew; Knowlton, Nancy

    2012-01-01

    Background Assessment of the biodiversity of communities of small organisms is most readily done using PCR-based analysis of environmental samples consisting of mixtures of individuals. Known as metagenetics, this approach has transformed understanding of microbial communities and is beginning to be applied to metazoans as well. Unlike microbial studies, where analysis of the 16S ribosomal DNA sequence is standard, the best gene for metazoan metagenetics is less clear. In this study we designed a set of PCR primers for the mitochondrial 12S ribosomal DNA sequence based on 64 complete mitochondrial genomes and then tested their efficacy. Methodology/Principal Findings A total of the 64 complete mitochondrial genome sequences representing all metazoan classes available in GenBank were downloaded using the NCBI Taxonomy Browser. Alignment of sequences was performed for the excised mitochondrial 12S ribosomal DNA sequences, and conserved regions were identified for all 64 mitochondrial genomes. These regions were used to design a primer pair that flanks a more variable region in the gene. Then all of the complete metazoan mitochondrial genomes available in NCBI's Organelle Genome Resources database were used to determine the percentage of taxa that would likely be amplified using these primers. Results suggest that these primers will amplify target sequences for many metazoans. Conclusions/Significance Newly designed 12S ribosomal DNA primers have considerable potential for metazoan metagenetic analysis because of their ability to amplify sequences from many metazoans. PMID:22536450

  2. Quantitative PCR analysis of laryngeal muscle fiber types

    PubMed Central

    Van Daele, Douglas J.

    2013-01-01

    Voice and swallowing dysfunction as a result of recurrent laryngeal nerve paralysis can be improved with vocal fold injections or laryngeal framework surgery. However, denervation atrophy can cause late-term clinical failure. A major determinant of skeletal muscle physiology is myosin heavy chain (MyHC) expression, and previous protein analyses have shown changes in laryngeal muscle fiber MyHC isoform with denervation. RNA analyses in this setting have not been performed, and understanding RNA levels will allow interventions better designed to reverse processes such as denervation in the future. Total RNA was extracted from bilateral rat thyroarytenoid (TA), posterior cricoarytenoid (PCA), and cricothyroid (CT) muscles in rats. Primers were designed using published MyHC isoform sequences. SYBR Green real time reverse transcription-polymerase chain reaction (SYBR-RT-PCR) was used for quantification. The electropherogram showed a clear separation of total RNA to 28S and 18S subunits. Melting curves illustrated single peaks for all type MyHC primers. All MyHC isoforms were identified in all muscles with various degrees of expression. Quantitative PCR is a sensitive method to detect MyHC isoforms in laryngeal muscle. Isoform expression using mRNA analysis was similar to previous analyses but showed some important differences. This technique can be used to quantitatively assess response to interventions targeted to maintain muscle bulk after denervation. PMID:20430402

  3. Fast detection of deletion breakpoints using quantitative PCR.

    PubMed

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-06-16

    The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27333265

  4. Fast detection of deletion breakpoints using quantitative PCR

    PubMed Central

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-01-01

    Abstract The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27560363

  5. Fast detection of deletion breakpoints using quantitative PCR.

    PubMed

    Abildinova, Gulshara; Abdrakhmanova, Zhanara; Tuchinsky, Helena; Nesher, Elimelech; Pinhasov, Albert; Raskin, Leon

    2016-01-01

    The routine detection of large and medium copy number variants (CNVs) is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories. PMID:27560363

  6. Identifying of meat species using polymerase chain reaction (PCR)

    NASA Astrophysics Data System (ADS)

    Foong, Chow Ming; Sani, Norrakiah Abdullah

    2013-11-01

    Meat has been widely consumed as an important protein source in daily life of human. Furthermore, with busy and intense urban lifestyle, processed food is now one of the main protein sources of one's diet. Consumers rely on the food labeling to decide if the meat product purchased is safe and reliable. Therefore, it is important to ensure the food labeling is done in a correct manner to avoid consumer fraud. More consumers are now concern about the food quality and safety as compared to before. This study described the meat species identification and detection method using Polymerase Chain Reaction (PCR) in 8 types of meats (cattle, buffalo, goat, sheep, chicken, duck, pork and horse). The objective of this study is to decide on the specificity of oligonucleotide sequences obtained from previous study. There were 5 proposed oligonucleotide primer in this study. The main important finding in this work is the specificity of oligonucleotide primers to raw meats. It if found that the oligonucleotide primers proposed were not specific to the local raw meat species. Therefore, further study is needed to obtain a species-specific oligonucletide primers for PCR, in order to be applied in food product testing.

  7. PCR-based identification of drowning: four case reports.

    PubMed

    Rácz, Evelin; Könczöl, Franciska; Tóth, Dénes; Patonai, Zoltán; Porpáczy, Zoltán; Kozma, Zsolt; Poór, Viktor S; Sipos, Katalin

    2016-09-01

    Proper diagnosis in drowning victims is often difficult due to the lack of signs specific to drowning. The diatom test is a widely used procedure for the diagnosis. Some types of water contain only minimal amounts of diatom cells which may provide false-negative results, while a negative diatom test result does not exclude drowning. In proving drowning, we used a polymerase chain reaction (PCR)-based biological method in addition to the conventional methods. DNA was extracted from postmortem spleen tissues and water of the drowning site. Samples were tested with algae (diatoms and small green algae)- and cyanobacteria (blue-green algae)-specific primers. We present here multiple drowning cases in which diatom tests of the postmortem tissue samples and the water were negative. In each case, the presence of phytoplanktonic DNA strengthened the autopsy diagnosis of drowning even in the absence of visible diatoms. In the future, the PCR method may be of consideration as a possible supplement of the diatom test in the examination of presumed drowning cases. PMID:27080711

  8. Analysis of PCR Thermocycling by Rayleigh-Bénard Convection

    NASA Astrophysics Data System (ADS)

    Sharma, Ruchi; Ugaz, Victor

    2004-03-01

    In previous studies, we demonstrated a novel device employing the circulatory flow field established by Rayleigh-Bénard convection to perform amplification of a 295 base target region from a human genomic DNA template inside a 35 uL cylindrical cavity using the polymerase chain reaction (PCR) [Krishnan, Ugaz & Burns, Science, Vol. 298, 2002, p. 793]. This design eliminates the need for dynamic external temperature control required in conventional thermocyclers that repeatedly heat and cool static sample volumes to denaturation, annealing, and extension temperatures. In this paper, we extend these studies by demonstrating the design and operation of a multiwell convective flow device capable of achieving amplification of a 191 base pair fragment associated with membrane channel proteins M1 and M2 of the influenza-A virus in as little as 15 minutes with performance comparable to a conventional thermocycler. We also study the effect of initial template concentration and observe no degradation in performance over four orders of magnitude of initial template loading dilution, consistent with conventional thermocycler results. These results illustrate the ability of convective flow PCR systems to achieve performance equal to or exceeding conventional thermocycling hardware, and demonstrate their suitability for use in rapid biodetection assays.

  9. 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

  10. Value of PCR in surgically treated patients with staphylococcal infective endocarditis: a 4-year retrospective study.

    PubMed

    Zaloudíková, B; Němcová, E; Pol, J; Sorm, Z; Wurmová, S; Novotná, K; Vaněrková, M; Holá, V; Růžička, F; Dušek, L; Němec, P; Freiberger, T

    2012-06-01

    The aim of the study was to establish a diagnostic value for broad-range polymerase chain reaction (br-PCR) and staphylococci-specific multiplex PCR (ssm-PCR) performed on surgical material from patients with staphylococcal infective endocarditis (IE). Data were analysed retrospectively from 60 patients with suspected staphylococcal IE and 59 controls who were surgically treated at three cardiosurgery centres over 4 years. Both PCR tests showed high agreement and could be aggregated. In patients with definite and rejected IE, the clinical sensitivity and specificity of PCR reached 89 and 95%, respectively. Tissue culture (TC) and PCR agreed with blood culture (BC) in 29% and 67% of IE cases. TC helped to determine aetiology in five BC negative cases while PCR aided in nine cases. Out of 52 patients with conclusive staphylococcal IE, 40 were diagnosed with S. aureus and 12 with coagulase-negative staphylococci. PCR was shown to be highly superior to TC in confirming preoperative diagnosis of IE. In addition to aid in culture negative patients, PCR helped to establish or refine aetiology in inconclusive cases. We suggest that simultaneous br-PCR and ssm-PCR performed on surgical material together with histopathology could significantly increase the performance of current Duke criteria. PMID:21964590

  11. Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

    PubMed Central

    Barbano, Raffaela; Pasculli, Barbara; Coco, Michelina; Fontana, Andrea; Copetti, Massimiliano; Rendina, Michelina; Valori, Vanna Maria; Graziano, Paolo; Maiello, Evaristo; Fazio, Vito Michele; Parrella, Paola

    2015-01-01

    BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method. PMID:26690267

  12. EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

    EPA Science Inventory

    EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. The viral ribonucleic acid (RNA) from water sample concentrates is extracted and tested for enterovirus and norovirus RNA using reverse transcription-quantitative PCR (RT-qPCR). V...

  13. Separating para and ortho water.

    PubMed

    Horke, Daniel A; Chang, Yuan-Pin; Długołęcki, Karol; Küpper, Jochen

    2014-10-27

    Water exists as two nuclear-spin isomers, para and ortho, determined by the overall spin of its two hydrogen nuclei. For isolated water molecules, the conversion between these isomers is forbidden and they act as different molecular species. Yet, these species are not readily separated, and no pure para sample has been produced. Accordingly, little is known about their specific physical and chemical properties, conversion mechanisms, or interactions. The production of isolated samples of both spin isomers is demonstrated in pure beams of para and ortho water in their respective absolute ground state. These single-quantum-state samples are ideal targets for unraveling spin-conversion mechanisms, for precision spectroscopy and fundamental symmetry-breaking studies, and for spin-enhanced applications, for example laboratory astrophysics and astrochemistry or hypersensitized NMR experiments. PMID:25196938

  14. Comparison of Leishmania OligoC-TesT PCR with conventional and real-time PCR for Diagnosis of canine Leishmania infection.

    PubMed

    Carson, Connor; Quinnell, Rupert J; Holden, Jennifer; Garcez, Lourdes M; Deborggraeve, Stijn; Courtenay, Orin

    2010-09-01

    There is a need for standardization and simplification of the existing methods for molecular detection of Leishmania infantum in the canine reservoir host. The commercially available OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic dipstick detection of PCR products and is highly sensitive for use in humans but not yet independently validated for use in dogs. Here we compare the sensitivity of OligoC-TesT with those of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer 1 (ITS-1) PCR, and a PCR-hybridization protocol, using longitudinal naturally infected canine bone marrow samples whose parasite burdens were measured by real-time quantitative PCR (qPCR). The sensitivity of OligoC-TesT for infected dogs was 70% (95% confidence interval [CI], 63 to 78%), similar to that of kDNA PCR (72%; 95% CI, 65 to 80%; P = 0.69) but significantly greater than those of PCR-hybridization (61%; 95% CI, 53 to 69%; P = 0.007) and ITS-1 nested PCR (54%; 95% CI, 45 to 62%; P < 0.001); real-time qPCR had the highest sensitivity (91%; 95% CI, 85 to 95%; P < 0.001). OligoC-TesT sensitivity was greater for polysymptomatic and oligosymptomatic dogs than for asymptomatic dogs (93%, 74%, and 61%, respectively; P = 0.005), a trend also observed for the other qualitative PCR methods tested (P PCR: OligoC-TesT and kDNA PCR detected 100% and 99% of positive samples when parasite burdens exceeded 74 and 49 parasites/ml, respectively. OligoC-TesT has high sensitivity for detection of canine Leishmania infections; its ease of operation and ease of interpretation are further advantages for veterinary diagnostic laboratories and for large-scale survey work in developing countries. PMID:20631112

  15. Sensitivity and reproducibility of standardized-competitive RT-PCR for transcript quantification and its comparison with real time RT-PCR

    PubMed Central

    Pagliarulo, Vincenzo; George, Ben; Beil, Stephen J; Groshen, Susan; Laird, Peter W; Cai, Jie; Willey, James; Cote, Richard J; Datar, Ram H

    2004-01-01

    Background Probe based detection assays form the mainstay of transcript quantification. Problems with these assays include varying hybridization efficiencies of the probes used for transcript quantification and the expense involved. We examined the ability of a standardized competitive RT-PCR (StaRT PCR) assay to quantify transcripts of 4 cell cycle associated genes (RB, E2F1, CDKN2A and PCNA) in two cell lines (T24 & LD419) and compared its efficacy with the established Taqman real time quantitative RT-PCR assay. We also assessed the sensitivity, reproducibility and consistency of StaRT PCR. StaRT PCR assay is based on the incorporation of competitive templates (CT) in precisely standardized quantities along with the native template (NT) in a PCR reaction. This enables transcript quantification by comparing the NT and CT band intensities at the end of the PCR amplification. The CT serves as an ideal internal control. The transcript numbers are expressed as copies per million transcripts of a control gene such as β-actin (ACTB). Results The NT and CT were amplified at remarkably similar rates throughout the StaRT PCR amplification cycles, and the coefficient of variation was least (<3.8%) when the NT/CT ratio was kept as close to 1:1 as possible. The variability between the rates of amplification in different tubes subjected to the same StaRT PCR reaction was very low and within the range of experimental noise. Further, StaRT PCR was sensitive enough to detect variations as low as 10% in endogenous actin transcript quantity (p < 0.01 by the paired student's t-test). StaRT PCR correlated well with Taqman real time RT-PCR assay in terms of transcript quantification efficacy (p < 0.01 for all 4 genes by the Spearman Rank correlation method) and the ability to discriminate between cell types and confluence patterns. Conclusion StaRT PCR is thus a reliable and sensitive technique that can be applied to medium-high throughput quantitative transcript measurement. Further

  16. Detection of Methicillin-Resistant Staphylococcus aureus by a Duplex Droplet Digital PCR Assay

    PubMed Central

    Kelley, KaShonda; Cosman, Angela; Belgrader, Phillip; Chapman, Brenda

    2013-01-01

    Health care-associated infections with methicillin-resistant Staphylococcus aureus (MRSA) contribute to significant hospitalization costs. We report here a droplet digital PCR (ddPCR) assay, which is a next-generation emulsion-based endpoint PCR assay for high-precision MRSA analysis. Reference cultures of MRSA, methicillin-susceptible S. aureus (MSSA), and confounders were included as controls. Copan swabs were used to sample cultures and collect specimens for analysis from patients at a large teaching hospital. Swab extraction and cell lysis were accomplished using magnetic-driven agitation of silica beads. Quantitative PCR (qPCR) (Roche Light Cycler 480) and ddPCR (Bio-Rad QX100 droplet digital PCR system) assays were used to detect genes for the staphylococcal protein SA0140 (SA) and the methicillin resistance (mecA) gene employing standard TaqMan chemistries. Both qPCR and ddPCR assays correctly identified culture controls for MRSA (76), MSSA (12), and confounder organisms (36) with 100% sensitivity and specificity. Analysis of the clinical samples (211 negative and 186 positive) collected during a study of MRSA nasal carriage allowed direct comparison of the qPCR and ddPCR assays to the Cepheid MRSA GeneXpert assay. A total of 397 clinical samples were examined in this study. Cepheid MRSA GeneXpert values were used to define negative and positive samples. Both the qPCR and ddPCR assays were in good agreement with the reference assay. The sensitivities for the qPCR and ddPCR assays were 96.8% (95% confidence interval [CI], 93.1 to 98.5%) and 96.8% (95% CI, 93.1 to 98.5%), respectively. Both the qPCR and ddPCR assays had specificities of 91.9% (95% CI, 87.5 to 94.9%) for qPCR and 91.0% (95% CI, 86.4 to 94.2%) for ddPCR technology. PMID:23596244

  17. Development of nested PCR assays for detection of bovine respiratory syncytial virus in clinical samples.

    PubMed Central

    Vilcek, S; Elvander, M; Ballagi-Pordány, A; Belák, S

    1994-01-01

    Two nested PCR assays were developed for the detection of bovine respiratory syncytial virus (BRSV). Primers were selected from the gene encoding the F fusion protein (PCR-F) and the gene encoding the G attachment protein (PCR-G). Biotinylated oligonucleotide probes, termed F and G, were selected for the hybridization of the respective PCR products. The sensitivities of the PCR-F and PCR-G assays were similar, both detecting 0.1 tissue culture infective dose of the virus. The PCR-F assay amplified all bovine strains and one human strain (RS32) tested. No cross-reactions were observed with nine heterologous respiratory viruses. PCR-F products of bovine and human RSV strains were discriminated by using endonuclease restriction enzyme ScaI, which specifically cleaved, products of BRSV. Oligonucleotide probe F was also specific for products of BRSV. The PCR-G assay detected all bovine strains and none of the human strains tested. A faint electrophoretic band was also observed with products of Sendai virus. However, probe G did not hybridize with this product, only with products of BRSV. Nasal swabs collected from cattle with no symptoms and cattle in the acute stage of respiratory disease were analyzed for BRSV by the immunofluorescence (IF) method and by the PCR-F and PCR-G assays. The virus was detected by the PCR assays in 31 of 35 (89%) samples tested. Only 23 samples (66%) were positive by the IF method, and these samples were also positive by both the PCR-F and PCR-G assays. The 31 samples detected as positive by PCR originated from cattle presenting clinical signs of acute respiratory disease; the four PCR-negative samples originated from clinically asymptomatic neighboring cattle. All sampled animals subsequently seroconverted and became reactive to BRSV. Thus, the detection of BRSV by PCR correlated with clinical observations and was considerably more sensitive (66 versus 89%) than IF. These results indicate that both nested PCR assays provide rapid and

  18. Source identification of airborne Escherichia coli of swine house surroundings using ERIC-PCR and REP-PCR.

    PubMed

    Duan, Huiyong; Chai, Tongjie; Liu, Jianzhu; Zhang, Xingxiao; Qi, Chunhua; Gao, Jing; Wang, Yaling; Cai, Yumei; Miao, Zengmin; Yao, Meiling; Schlenker, Gerd

    2009-07-01

    Evidence is mounting that microorganisms originating from livestock impact the air quality of the animal houses themselves and the public in the surrounding neighborhoods. The aim of this study was to develop efficient bacterial source tracking capabilities to identify sources of Escherichia coli aerosol pollution caused by pigs. Airborne E. coli were isolated from indoor air, upwind air (10 and 50 m away) and downwind air samples (10, 50, 100, 200 and 400 m away) for five swine houses using six-stage Andersen microbial samplers and Reuter-Centrifugal samplers (RCS). E. coli strains from pig fecal samples were also collected simultaneously. The enterobacterial repetitive intergenic consensus polymerize chain reaction (ERIC-PCR) and the repetitive extragenic palindromic (REP-PCR) approaches were used to study the genetic variability and to determine the strain relationships among E. coli isolated from different sites in each swine house. Results showed that 35.1% (20/57) of the bacterial DNA fingerprints from the fecal isolates matched with the corresponding strains isolated from indoor and downwind air samples (similarity > or = 90%). E. coli strains from the indoor and downwind air samples were closely related to the E. coli strains isolated from feces, while those isolated from upwind air samples (swine house C) had low similarity (61-69%). Our results suggest that some strains isolated from downwind and indoor air originated in the swine feces. Effective hygienic measures should be taken in animal farms to prevent or minimize the downwind spread of microorganism aerosol. PMID:19349045

  19. Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.

    PubMed

    Burns, Cara C; Kilpatrick, David R; Iber, Jane C; Chen, Qi; Kew, Olen M

    2016-01-01

    Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures. PMID:26983735

  20. Rapid and sensitive detection of Feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform.

    PubMed

    Wilkes, Rebecca Penrose; Kania, Stephen A; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chang, Hsiu-Hui; Ma, Li-Juan; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2015-07-01

    Feline immunodeficiency virus (FIV) is an important infectious agent of cats. Clinical syndromes resulting from FIV infection include immunodeficiency, opportunistic infections, and neoplasia. In our study, a 5' long terminal repeat/gag region-based reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) was developed to amplify all known FIV strains to facilitate point-of-need FIV diagnosis. The RT-iiPCR method was applied in a point-of-need PCR detection platform--a field-deployable device capable of generating automatically interpreted RT-iiPCR results from nucleic acids within 1 hr. Limit of detection 95% of FIV RT-iiPCR was calculated to be 95 copies standard in vitro transcription RNA per reaction. Endpoint dilution studies with serial dilutions of an ATCC FIV type strain showed that the sensitivity of lyophilized FIV RT-iiPCR reagent was comparable to that of a reference nested PCR. The established reaction did not amplify any nontargeted feline pathogens, including Felid herpesvirus 1, feline coronavirus, Feline calicivirus, Feline leukemia virus, Mycoplasma haemofelis, and Chlamydophila felis. Based on analysis of 76 clinical samples (including blood and bone marrow) with the FIV RT-iiPCR, test sensitivity was 97.78% (44/45), specificity was 100.00% (31/31), and agreement was 98.65% (75/76), determined against a reference nested-PCR assay. A kappa value of 0.97 indicated excellent correlation between these 2 methods. The lyophilized FIV RT-iiPCR reagent, deployed on a user-friendly portable device, has potential utility for rapid and easy point-of-need detection of FIV in cats. PMID:26185125

  1. Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

    PubMed Central

    2012-01-01

    Background The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR. Results A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose–response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was <6 % for repeatability and <2 % for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed. Conclusions The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the

  2. Fluorescent-BOX-PCR for resolving bacterial genetic diversity, endemism and biogeography

    PubMed Central

    Brusetti, Lorenzo; Malkhazova, Iana; Gtari, Maher; Tamagnini, Isabella; Borin, Sara; Merabishvili, Maya; Chanishvili, Nina; Mora, Diego; Cappitelli, Francesca; Daffonchio, Daniele

    2008-01-01

    Background BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) is one of the most used techniques in biogeography studies of microbial isolates. However the traditional separation of BOX-PCR patterns by agarose gel electrophoresis suffers many limitations. The aim of this research was to set up a fluorescent BOX-PCR (F-BOX-PCR) assay in which separation of PCR products is automated in a capillary electrophoresis system. F-BOX-PCR was compared with the traditional BOX-PCR using bacterial strains with different G+C content (Bacillus cereus; Escherichia coli; isolates of the family Geodermatophilaceae). Resolution, discriminatory power and reproducibility were evaluated by assaying different electrophoretic runs, PCR reactions and independent DNA extractions. BOX-PCR and F-BOX-PCR were compared for the analysis of 29 strains of Modestobacter multiseptatus isolated from three different microsites in an altered carbonatic wall from Cagliari, Italy, and 45 strains of Streptococcus thermophilus isolated from 34 samples of the hand-made, yogurt-like product Matsoni, collected in different locations in Georgia. Results Fluorophore 6-FAM proved more informative than HEX and BOX-PCR both in agarose gel electrophoresis (p < 0.004 and p < 0.00003) and in capillary electrophoresis (compared only with HEX, p < 2 × 10-7). 6-FAM- and HEX-based F-BOX-PCR respectively detected up to 12.0 and 11.3 times more fragments than BOX-PCR. Replicate separations of F-BOX-PCR showed an accuracy of the size calling of ± 0.5 bp until 500 bp, constantly decreasing to ± 10 bp at 2000 bp. Cluster analysis of F-BOX-PCR profiles grouped M. multiseptatus strains according to the microsite of isolation and S. thermophilus strains according to the geographical origin of Matsoni, but resulted intermixed when a BOX-PCR dataset was used. Conclusion F-BOX-PCR represents an improved method for addressing bacterial biogeography studies both in term of sensitivity, reproducibility and data analysis

  3. PCR detection of groundwater bacteria associated with colloidal transport

    SciTech Connect

    Cruz-Perez, P.; Stetzenbach, L.D.; Alvarez, A.J.

    1996-02-29

    Colloidal transport may increase the amount of contaminant material than that which could be transported by water flow alone. The role of colloids in groundwater contaminant transport is complicated and may involve many different processes, including sorption of elements onto colloidal particles, coagulation/dissolution, adsorption onto solid surfaces, filtration, and migration. Bacteria are known to concentrate minerals and influence the transport of compounds in aqueous environments and may also serve as organic colloids, thereby influencing subsurface transport of radionuclides and other contaminants. The initial phase of the project consisted of assembling a list of bacteria capable of sequestering or facilitating mineral transport. The development and optimization of the PCR amplification assay for the detection of the organisms of interest, and the examination of regional groundwaters for those organisms, are presented for subsequent research.

  4. Single-cell PCR profiling of gene expression in hematopoiesis.

    PubMed

    Teles, José; Enver, Tariq; Pina, Cristina

    2014-01-01

    Single-cell analysis of gene expression offers the possibility of exploring cellular and molecular heterogeneity in stem and developmental cell systems, including cancer, to infer routes of cellular specification and their respective gene regulatory modules. PCR-based technologies, although limited to the analysis of a predefined set of genes, afford a cost-effective balance of throughput and biological information and have become a method of choice in stem cell laboratories. Here we describe an experimental and analytical protocol based on the Fluidigm microfluidics platform for the simultaneous expression analysis of 48 or 96 genes in multiples of 48 or 96 cells. We detail wet laboratory procedures and describe clustering, principal component analysis, correlation, and classification tools for the inference of cellular pathways and gene networks. PMID:25062620

  5. Simultaneous detection of bee viruses by multiplex PCR.

    PubMed

    Sguazza, Guillermo Hernán; Reynaldi, Francisco José; Galosi, Cecilia Mónica; Pecoraro, Marcelo Ricardo

    2013-12-01

    Honey bee mortality is a serious problem that beekeepers in Argentina have had to face during the last 3 years. It is known that the consequence of the complex interactions between environmental and beekeeping parameters added to the effect of different disease agents such as viruses, bacteria, fungi and parasitic mites may result in a sudden collapse of the colony. In addition, multiple viral infections are detected frequently concomitantly in bee colonies. The aim of this study was to establish a multiplex polymerase chain reaction method for rapid and simultaneous detection of the most prevalent bee viruses. This multiplex PCR assay will provide specific, rapid and reliable results and allow for the cost effective detection of a particular virus as well as multiple virus infections in a single reaction tube. This method could be a helpful tool in the surveillance of the most frequently found bee viruses and to study the dynamics and the interactions of the virus populations within colonies. PMID:23948157

  6. Detection of Legionella Contamination in Tabriz Hospitals by PCR Assay

    PubMed Central

    Ghotaslou, Reza; Yeganeh Sefidan, Fatemeh; Akhi, Mohammad Taghi; Soroush, Mohammad Hussein; Hejazi, Mohammad Saeid

    2013-01-01

    Purpose: The present study was designed to evaluate the occurrence of Legionella contamination in the tap water of Tabriz hospitals, Azerbaijan, Iran. Methods: One hundred and forty water samples from diverse water supply systems of 17 hospitals were collected and analyzed for the presence of Legionella spp. by PCR assay. Results: In this study, 10 of 140 (7.1%) samples were positive for Legionella which L. pneumophila was detected in 4 (2.85%) water samples. Conclusion: In conclusion, hospital potable systems are the primary reservoirs for Legionnaires’ disease. This study concludes that Legionella spp. are present in aquatic hospitals environment of Tabriz. Due to the serious risk of infections, it is better to make efforts to eliminate Legionella spp. in water supplies. PMID:24312825

  7. [Detection of fish DNA in ruminant feed by PCR amplification].

    PubMed

    Nomura, Tetsuya; Kusama, Toyoko; Kadowaki, Koh-ichi

    2006-10-01

    The Japanese Government has prohibited the use of seafood protein, as well as mammalian protein, in ruminant feed. There is an official method to detect meat and bone meal, but no method is yet available to detect fishmeal in ruminant feed. We tried to develop a suitable method to detect fishmeal in ruminant feed, similar to the official method "PCR detection of animal-derived DNA in feed". Our previously reported primers (fishcon5 and fishcon3-1) showed low sensitivity, so we designed new primers based on a DNA sequence from yellowfin tuna mitchondrial DNA. Among the primers, FM5 and FM3 specifically detected fish DNA (sardine, yellowfin tuna, skipjack tuna, chub mackerel, Pacific saury, salmon, rainbow trout, Japanese anchovy, codfish and Japanese horse mackerel) from fish meat, and did not amplify DNA from animals and plants. The sensitivity for detection of the presence of fishmeal in ruminant feed was 0.01-0.001%. PMID:17128872

  8. STS-79 payload SPACEHAB in PCR at LC39A

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Workers in the Payload Changeout Room (PCR) at Launch Pad 39A are preparing to close the payload doors for flight on the Space Shuttle Atlantis, targeted for liftoff on Mission STS-79 around September 12. The SPACEHAB Double Module located in the aft area of the payload bay is filled with supplies that will be transferred to the Russian Space Station Mir. STS-79 marks the second flight of a SPACEHAB in support of the Shuttle-Mir dockings, and the first flight of the double-module configuration. The SPACEHAB is connected by tunnel to the Orbiter Docking System (ODS), with the Androgynous Peripheral Docking System (APDS) clearly visible on top of the ODS. The APDS provides the docking interface for the linkup with Mir, while the ODS provides a passageway from the orbiter to the Russian space station and the SPACEHAB.

  9. Hungarian population data on seven PCR-based loci.

    PubMed

    Budowle, B; Woller, J; Koons, B W; Furedi, S; Errera, J D; Padar, Z

    1996-07-01

    Hungarian population data for the loci LDLR, GYPA, HBGG, D7S8, Gc, HLA-DQA1, and D1S80 were generated. The genotype frequency distributions for the loci do not deviate from Hardy Weinberg expectations. Furthermore, there was little evidence for departures from expectations of independence between the loci. Using a test for homogeneity all the loci were similar between two Hungarian population samples and only the HLA-DQA1 locus was statistically different between Hungarians and US Caucasians. There generally would be little forensic differences, whether a Hungarian or a US Caucasian database was used, for estimating multiple locus profile frequencies for the seven PCR-based loci. PMID:8754580

  10. 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

  11. High-throughput single-cell PCR using microfluidic emulsions

    NASA Astrophysics Data System (ADS)

    Guo, Mira; Mazutis, Linas; Agresti, Jeremy; Sommer, Morten; Dantas, Gautam; Church, George; Turnbaugh, Peter; Weitz, David

    2012-02-01

    The human gut and other environmental samples contain large populations of diverse bacteria that are poorly characterized and unculturable, yet have many functions relevant to human health. Our goal is to identify exactly which species carry some gene of interest, such as a carbohydrate metabolism gene. Conventional metagenomic assays sequence DNA extracted in bulk from populations of mixed cell types, and are therefore unable to associate a gene of interest with a species-identifying 16S gene, to determine that the two genes originated from the same cell. We solve this problem by microfluidically encapsulating single bacteria cells in drops, using PCR to amplify the two genes inside any drop whose encapsulated cell contains both genes, and sequencing the DNA from those drops that contain both amplification products.

  12. Enhancement of Borrelia burgdorferi PCR by uracil N-glycosylase.

    PubMed Central

    Loewy, Z G; Mecca, J; Diaco, R

    1994-01-01

    Uracil DNA glycosylases are DNA repair enzymes present in virtually every organism. These enzymes function by excising from DNA uracil residues resulting from either misincorporation of dUMP residues by a DNA polymerase or deamination of cytosine. Recently, the enzyme has been exploited in PCRs as a means for controlling carryover contamination from previously amplified DNA. When the enzyme is used in amplifications of Borrelia burgdorferi target sequences, we have observed an enhancement in signal detected by a microwell plate DNA hybridization assay. This increase in signal is dependent upon the length of the target, is titratable with enzyme concentration, and has been observed with amplifications performed with both symmetric and asymmetric PCR profiles. The enhancement is shown to occur at the level of the target genomic DNA. PMID:8126168

  13. Detection of Bacteroides fragilis Enterotoxin Gene by PCR

    PubMed Central

    Shetab, Razeq; Cohen, Stuart H.; Prindiville, Thomas; Tang, Yajarayma J.; Cantrell, Mary; Rahmani, Darush; Silva, Joseph

    1998-01-01

    Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases. PMID:9620408

  14. Multiplex PCR method for detection of three Aeromonas enterotoxin genes.

    PubMed

    Kingombe, Cesar I Bin; D'Aoust, Jean-Yves; Huys, Geert; Hofmann, Lisa; Rao, Mary; Kwan, Judy

    2010-01-01

    A novel multiplex PCR method using three sets of specific primers was developed for the detection of the cytotoxic (act), heat-labile (alt), and heat-stable (ast) enterotoxin genes in Aeromonas spp. This assay was used to characterize 35 reference strains as well as 537 food-borne isolates. A total of seven gene pattern combinations were encountered, including act, alt, act/alt, act/alt/ast, act/alt/148-bp amplicon, alt/ast, and alt/148-bp amplicon. The alt gene was detected with 34 reference strains (97%) and occurred singly in 14% of these strains. The frequency of occurrence of the act/alt, act/alt/ast, and alt/ast gene patterns in reference strains was 14 (40%), 2 (6%), and 2 (6%), respectively. An unpredicted amplicon was detected in 11 reference strains (31%). Characterization of this amplicon showed that its size was 148 bp, as generated by the AHLF and AHLR primers, and that it uniquely aligned with the Aeromonas salmonicida A449 genome sequence (GenBank accession number CP000644). This amplicon was named Aeromonas salmonicida subsp. salmonicida hypothetical protein amplicon (AssHPA). In the 537 food-borne isolates, the act and alt genes were most dominant and were detected in 349 (65%) and 452 (84%) isolates, respectively, either alone or in combinations. The act and alt genes occurred singly in 30 (6%) and 128 (24%) of these strains, respectively. The act/alt gene pattern occurred in 315 isolates (59%), whereas the ast gene was always linked to strains exhibiting the act/alt/ast and alt/ast gene combinations in 4 (0.7%) and 5 (0.9%) isolates, respectively. The uniplex amplification of three enterotoxin genes separately confirms the specificity of the unique selected primers. This multiplex PCR is rapid and simple and can detect the presence of three Aeromonas enterotoxin genes in a single assay. PMID:19933350

  15. 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

  16. Modified Real-Time PCR for Detecting, Differentiating, and Quantifying Ureaplasma urealyticum and Ureaplasma parvum

    PubMed Central

    Vancutsem, Ellen; Soetens, Oriane; Breugelmans, Maria; Foulon, Walter; Naessens, Anne

    2011-01-01

    We evaluated a previously described quantitative real-time PCR (qPCR) for quantifying and differentiating Ureaplasma parvum and U. urealyticum. Because of nonspecific reactions with Staphylococcus aureus DNA in the U. parvum PCR, we developed a modified qPCR and designed new primers. These oligonucleotides eradicated cross-reactions, indicating higher specificity. The detection limits of the qPCR were determined at 1 and 3 colony-forming units/ml for U. parvum and U. urealyticum, respectively. The quantification limits of the assay for both Ureaplasma species ranged from 2.106 to 2.101 copy numbers per PCR. A total of 300 patient samples obtained from the lower genital tract were tested with this newly designed qPCR assay and compared with culture results. Of the samples, 132 (44.0%) were culture positive, whereas 151 (50.3%) tested positive using qPCR. The U. parvum and U. urealyticum species were present in 79.5% and 12.6% of the qPCR-positive samples, respectively. Both species were found in 7.9% of those samples. Quantification of U. parvum and U. urealyticum in the samples ranged from less than 2.5 × 103 to 7.4 × 107 copies per specimen. In conclusion, the modified qPCR is a suitable method for rapid detection, differentiation, and quantification of U. parvum and U. urealyticum. PMID:21354056

  17. PCR biocompatibility of lab-on-a-chip and MEMS materials

    NASA Astrophysics Data System (ADS)

    Christensen, T. B.; Pedersen, C. M.; Gröndahl, K. G.; Jensen, T. G.; Sekulovic, A.; Bang, D. D.; Wolff, A.

    2007-08-01

    DNA amplification using the polymerase chain reaction (PCR) is an important tool in biotechnology, pathogen surveillance in food, medical and forensic science etc. The PCR technique is now an important part of the research in and development of miniaturized biochemical analysis systems. However, reduced or no DNA amplification at all is an important challenge for microfabricated PCR devices due to a negative interaction between PCR chemicals and the surrounding environment, i.e. the materials encapsulating the PCR mix. Materials of special interest regarding PCR compatibility are silicon, glass and polymers, which are important in the fabrication of microelectromechanical systems (MEMS), micro total analysis systems (µTAS) and lab-on-a-chip (LOC) systems. The PCR inhibition effect is a particularly important phenomenon in microsystems due to an increased surface-to-volume ratio which enhances the possibility of interaction between the surfaces and ingredients in the PCR mixture. By proper surface treatment the PCR reaction can be facilitated and in this paper we present a systematic and quantitative study of the impact on the PCR compatibility of a chemical and a biological surface treatment. The chemical treatments are based on the silanizing agent dichlordimethylsilane [(CH3)2SiCl2

  18. On-chip quantitative detection of pathogen genes by autonomous microfluidic PCR platform.

    PubMed

    Tachibana, Hiroaki; Saito, Masato; Shibuya, Shogo; Tsuji, Koji; Miyagawa, Nobuyuki; Yamanaka, Keiichiro; Tamiya, Eiichi

    2015-12-15

    Polymerase chain reaction (PCR)-based genetic testing has become a routine part of clinical diagnoses and food testing. In these fields, rapid, easy-to-use, and cost-efficient PCR chips are expected to be appeared for providing such testing on-site. In this study, a new autonomous disposable plastic microfluidic PCR chip was created, and was utilized for quantitative detection of pathogenic microorganisms. To control the capillary flow of the following solution in the PCR microchannel, a driving microchannel was newly designed behind the PCR microchannel. This allowed the effective PCR by simply dropping the PCR solution onto the inlet without any external pumps. In order to achieve disposability, injection-molded cyclo-olefin polymer (COP) of a cost-competitive plastic was used for the PCR chip. We discovered that coating the microchannel walls with non-ionic surfactant produced a suitable hydrophilic surface for driving the capillary flow through the 1250-mm long microchannel. As a result, quantitative real-time PCR with the lowest initial concentration of human, Escherichia coli (E. coli), and pathogenic E. coli O157 genomic DNA of 4, 0.0019, 0.031 pg/μl, respectively, was successfully achieved in less than 18 min. Our results indicate that the platform presented in this study provided a rapid, easy-to-use, and low-cost real-time PCR system that could be potentially used for on-site gene testing. PMID:26210470

  19. Comparison of PCR-based approaches to molecular epidemiologic analysis of Clostridium difficile.

    PubMed Central

    Collier, M C; Stock, F; DeGirolami, P C; Samore, M H; Cartwright, C P

    1996-01-01

    Representative isolates of the 10 serogroups of Clostridium difficile and 39 clinical isolates (30 toxigenic and 9 nontoxigenic), including 5 isolates from a confirmed nosocomial outbreak, were analyzed by using two previously described arbitrary-primer PCR (AP-PCR) molecular typing methodologies (AP-PG05 and AP-ARB11) and PCR ribotyping. The two AP-PCR methods investigated gave comparable results; AP-PG05 and AP-ARB11 identified 8 and 7 groups among the serogroup isolates and classified the clinical isolates into 21 and 20 distinct groups, respectively. PCR ribotyping also identified 8 unique groups among the serogroup isolates but classified the clinical isolates into 23 groups. In addition, when results obtained by the PCR methods were compared with typing data generated by pulsed-field gel electrophoresis (PFGE), PCR ribotyping and PFGE were found to be in agreement for 83% (29 of 35) of isolates typeable by both techniques while AP-PG05 was in agreement with PFGE for 60% (20 of 33) and AP-ARB11 was in agreement with PFGE for only 44% (17 of 36). These results indicate that PCR ribotyping is a more discriminatory approach than AP-PCR for typing C. difficile and, furthermore, that this technique generates results that are in higher concordance with those obtained by using an established method for differentiating isolates of this organism on a molecular level than are results generated by using AP-PCR. PMID:8727893

  20. A multicenter study of viable PCR using propidium monoazide to detect Legionella in water samples.

    PubMed

    Scaturro, Maria; Fontana, Stefano; Dell'eva, Italo; Helfer, Fabrizia; Marchio, Michele; Stefanetti, Maria Vittoria; Cavallaro, Mario; Miglietta, Marilena; Montagna, Maria Teresa; De Giglio, Osvalda; Cuna, Teresa; Chetti, Leonarda; Sabattini, Maria Antonietta Bucci; Carlotti, Michela; Viggiani, Mariagabriella; Stenico, Alberta; Romanin, Elisa; Bonanni, Emma; Ottaviano, Claudio; Franzin, Laura; Avanzini, Claudio; Demarie, Valerio; Corbella, Marta; Cambieri, Patrizia; Marone, Piero; Rota, Maria Cristina; Bella, Antonino; Ricci, Maria Luisa

    2016-07-01

    Legionella quantification in environmental samples is overestimated by qPCR. Combination with a viable dye, such as Propidium monoazide (PMA), could make qPCR (named then vPCR) very reliable. In this multicentre study 717 artificial water samples, spiked with fixed concentrations of Legionella and interfering bacterial flora, were analysed by qPCR, vPCR and culture and data were compared by statistical analysis. A heat-treatment at 55 °C for 10 minutes was also performed to obtain viable and not-viable bacteria. When data of vPCR were compared with those of culture and qPCR, statistical analysis showed significant differences (P < 0.001). However, although the heat-treatment caused an abatement of CFU/mL ≤1 to 1 log10 unit, the comparison between untreated and heat-treated samples analysed by vPCR highlighted non-significant differences (P > 0.05). Overall this study provided a good experimental reproducibility of vPCR but also highlighted limits of PMA in the discriminating capability of dead and live bacteria, making vPCR not completely reliable. PMID:27133308

  1. Development and validation of a quantitative PCR assay for Ichthyophonus spp.

    PubMed

    White, Vanessa C; Morado, J Frank; Crosson, Lisa M; Vadopalas, Brent; Friedman, Carolyn S

    2013-04-29

    Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus. PMID:23670081

  2. Comparison of arbitrarily primed PCR with restriction endonuclease and immunoblot analyses for typing Clostridium difficile isolates.

    PubMed Central

    Tang, Y J; Houston, S T; Gumerlock, P H; Mulligan, M E; Gerding, D N; Johnson, S; Fekety, F R; Silva, J

    1995-01-01

    Arbitrarily primed PCR (AP-PCR) was used to genotype 26 clinical isolates of Clostridium difficile previously analyzed by immunoblotting (IB) and 20 isolates typed by restriction endonuclease analysis (REA) with HindIII. Two levels of differentiation were achieved with the AP-PCR approach by use of two different arbitrary primers. With the 19-mer arbitrary primer T-7 (first level of differentiation), a good correlation was found between IB and AP-PCR typing. Twenty isolates grouped into six IB types were separated into seven major AP-PCR types. These seven AP-PCR groups were further discriminated into 12 subtypes after genotyping with the arbitrary primer PG-05 (second level of differentiation). The remaining six isolates, all of different IB types, showed a unique and distinct DNA banding pattern with both of the arbitrary primers, T-7 and PG-05. Twenty isolates representing 20 REA types from 15 REA groups were resolved into 13 AP-PCR DNA profiles with the arbitrary primer T-7. A good correlation was found at this level of differentiation between the major REA groups, Y and M, and AP-PCR typing. While AP-PCR with this primer failed to differentiate isolates in REA groups J, G, R, and B, AP-PCR with PG-05 resolved these four isolates into four distinct AP-PCR types. In addition, one of three M strains and one of four Y strains displayed a slightly different DNA banding pattern by AP-PCR (with PG-05) from that of the other strains in the group. We conclude that AP-PCR is a rapid and sensitive method which not only complements other typing schemes but also may be a substitute and prove to be especially suited for immediate epidemiological tracking of nosocomial infections due to C. difficile. PMID:8586695

  3. Mensaje para alumnos y padres

    NASA Video Gallery

    El astronauta de la NASA José Hernández alienta a los estudiantes a que sigan sus sueños. Hernández también habla acerca del papel que juegan los padres para ayudar a que sus hijos hagan realidad s...

  4. Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir

    PubMed Central

    Bosman, K. J.; Nijhuis, M.; van Ham, P. M.; Wensing, A. M. J.; Vervisch, K.; Vandekerckhove, L.; De Spiegelaere, W.

    2015-01-01

    HIV persists in latently infected cells of patients on antiretroviral therapy (ART). This persistent proviral DNA reservoir is an important predictor of viral rebound upon therapy failure or interruption and forms a major obstacle towards cure. Accurate quantification of the low levels of persisting HIV DNA may aid patient monitoring and cure research. Digital PCR is a promising tool that enables direct absolute quantification with high sensitivity. With recent technological advances, several platforms are available to implement digital PCR in a clinical setting. Here, we compared two digital PCR platforms, the Quantstudio 3D (Life Technologies) and the QX100 (Bio-Rad) with a semi-nested qPCR on serial HIV DNA dilutions and DNA isolated from PBMCs of ART-suppressed patients. All three methods were able to detect target to the lowest levels of 2.5 HIV DNA copies. The QX100 excelled in having the least bias and highest precision, efficiency and quantitative linearity. Patient sample quantifications by the QX100 and semi-nested qPCR were highly agreeable by Bland-Altman analysis (0.01 ± 0.32 log10). Due to the observation of false-positive signals with current digital PCR platforms however, semi-nested qPCR may still be preferred in a setup of low quantity detection to discriminate between presence or absence of HIV DNA. PMID:26350506

  5. 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. PMID:26705837

  6. Comparative analysis between RQ-PCR and digital-droplet-PCR of immunoglobulin/T-cell receptor gene rearrangements to monitor minimal residual disease in acute lymphoblastic leukaemia.

    PubMed

    Della Starza, Irene; Nunes, Vittorio; Cavalli, Marzia; De Novi, Lucia Anna; Ilari, Caterina; Apicella, Valerio; Vitale, Antonella; Testi, Anna Maria; Del Giudice, Ilaria; Chiaretti, Sabina; Foà, Robin; Guarini, Anna

    2016-08-01

    Real-time quantitative polymerase chain reaction (RQ-PCR) is a standardized tool for minimal residual disease (MRD) monitoring in acute lymphoblastic leukaemia (ALL). The applicability of this technology is limited by the need of a standard curve based on diagnostic DNA. The digital droplet PCR (ddPCR) technology has been recently applied to various medical fields, but its use in MRD monitoring is under investigation. In this study, we analysed 50 ALL cases by both methods in two phases: in the first, we established analytical parameters to investigate the applicability of this new technique; in the second, we analysed MRD levels in 141 follow-up (FU) samples to investigate the possible use of ddPCR for MRD monitoring in ALL patients. We documented that ddPCR has sensitivity and accuracy at least comparable to those of RQ-PCR. Overall, the two methods gave concordant results in 124 of the 141 analysed MRD samples (88%, P = 0·94). Discordant results were found in 12% borderline cases. The results obtained prove that ddPCR is a reliable method for MRD monitoring in ALL, with the advantage of quantifying without the need of the calibration curves. Its application in a cohort of patients with a longer FU will conclusively define its clinical predictive value. PMID:27172403

  7. Comparison of a PNA clamp PCR and an ARMS/Scorpion PCR assay for the detection of K-ras mutations.

    PubMed

    Nordgård, Oddmund; Oltedal, Satu; Janssen, Emiel A M; Gilje, Bjørnar; Kørner, Hartwig; Tjensvoll, Kjersti; Smaaland, Rune

    2012-03-01

    Point mutations in the K-ras gene have been shown to confer resistance against epidermal growth factor receptor-directed therapy of metastatic colorectal cancer. Accordingly, K-ras mutation testing has become mandatory in hospitals offering such treatment. We compared the performance and reagent costs of 2 sensitive methods for detection of K-ras mutations: a peptide nucleic acid (PNA) clamp polymerase chain reaction (PCR) assay and a commercially available amplification refractory mutation system/Scorpion (ARMS/S) PCR assay. Both methods were applied in parallel to 101 formalin-fixed, paraffin-embedded tumor and metastasis samples from patients with colon cancer. The PNA clamp PCR assay detected K-ras mutations in 35% (35 of 101) of the samples, whereas the ARMS/S PCR assay detected mutations in 27% (27 of 101) of them. There was 92% (93 of 101) concordance between the 2 methods and the κ coefficient for the comparison was 0.82. The 8 discordant cases were exclusively positive by PNA clamp PCR. Finally, the reagent costs of the PNA clamp PCR assay were estimated to be at least 20 times lower than the ARMS/S assay. We concluded that the high performance and low costs associated with the PNA clamp PCR assay encourage its use in the administration of personalized epidermal growth factor receptor-directed therapy. PMID:22306670

  8. Further improvement and validation of MagMAX-96 AI/ND viral RNA isolation for efficient removal of RT-PCR inhibitors from cloacal swabs and tissues for rapid diagnosis of avian influenza virus by RT reverse transcription PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real time RT-PCR (RRT-PCR) is a high throughput molecular diagnostic test used for rapid detection of avian influenza virus (AIV) in clinical samples. However the performance of RRT-PCR can be adversely affected by RT-PCR inhibitors present in the sample. The tested commercial RNA extraction kits ...

  9. Comparison of Simplexa HSV 1 & 2 PCR with culture, immunofluorescence, and laboratory-developed TaqMan PCR for detection of herpes simplex virus in swab specimens.

    PubMed

    Gitman, Melissa R; Ferguson, David; Landry, Marie L

    2013-11-01

    The Simplexa HSV 1 & 2 direct PCR assay was compared with conventional cell culture, cytospin-enhanced direct fluorescent antibody (DFA), and a laboratory-developed real-time TaqMan PCR (LDT HSV PCR) using extracted nucleic acid for the detection of herpes simplex virus (HSV) in dermal, genital, mouth, ocular, and other swab samples. One hundred seventy-one swabs were tested prospectively, and 58 were positive for HSV (34 HSV-1 and 24 HSV-2). Cytospin-DFA detected 50 (86.2%), conventional cell culture 51 (87.9%), Simplexa direct 55 (94.8%), and LDT HSV PCR 57 (98.3%) of 58 true positives. Simplexa direct detected more positives than DFA and culture, but the differences were not significant (P = 0.0736 and P = 0.3711, respectively, by the McNemar test). Samples that were positive by all methods (n = 48) were strong positives (LDT cycle threshold [CT] value, 14.4 to 26.1). One strongly positive sample was falsely negative by LDT HSV PCR due to a failure of TaqMan probe binding. Three samples falsely negative by Simplexa direct had high CT values by LDT HSV PCR (LDT CT, 35.8 to 38.2). Omission of the DNA extraction step by Simplexa direct led to a drop in sensitivity compared to the sensitivity of LDT HSV PCR using extracted samples (94.8% versus 98.3%, respectively), but the difference was not significant (P = 0.6171). Simplexa HSV 1 & 2 direct PCR was the most expensive but required the least training of the assays used, had the lowest hands-on time and fastest assay time (75 min, versus 3 h by LDT HSV PCR), and provided the HSV type. PMID:24006008

  10. Nanoparticles Affect PCR Primarily via Surface Interactions with PCR Components: Using Amino-Modified Silica-Coated Magnetic Nanoparticles as a Main Model.

    PubMed

    Bai, Yalong; Cui, Yan; Paoli, George C; Shi, Chunlei; Wang, Dapeng; Shi, Xianming

    2015-06-24

    Nanomaterials have been widely reported to affect the polymerase chain reaction (PCR). However, many studies in which these effects were observed were not comprehensive, and many of the proposed mechanisms have been primarily speculative. In this work, we used amino-modified silica-coated magnetic nanoparticles (ASMNPs, which can be collected very easily using an external magnetic field) as a model and compared them with gold nanoparticles (AuNPs, which have been studied extensively) to reveal the mechanisms by which nanoparticles affect PCR. We found that nanoparticles affect PCR primarily by binding to PCR components: (1) inhibition, (2) specifity, and (3) efficiency and yield of PCR are impacted. (1) Excess nanomaterials inhibit PCR by adsorbing to DNA polymerase, Mg(2+), oligonucleotide primers, or DNA templates. Nanoparticle surface-active groups are particularly important to this effect. (2, a) Nanomaterials do not inhibit nonspecific amplification products caused by false priming as previously surmised. It was shown that relatively low concentrations of nanoparticles inhibited the amplification of long amplicons, and increasing the amount of nanoparticles inhibited the amplification of short amplicons. This concentration phenomenon appears to be the result of the formation of "joints" upon the adsorption of ASMNPs to DNA templates. (b) Nanomaterials are able to inhibit nonspecific amplification products due to incomplete amplification by preferably adsorbing single-stranded incomplete amplification products. (3) Some types of nanomaterials, such as AuNPs, enhance the efficiency and yield of PCR because these types of nanoparticles can adsorb to single-stranded DNA more strongly than to double-stranded DNA. This behavior assists in the rapid and thorough denaturation of double-stranded DNA templates. Therefore, the interaction between the surface of nanoparticles and PCR components is sufficient to explain most of the effects of nanoparticles on PCR. PMID

  11. The analysis of para-cresol production and tolerance in Clostridium difficile 027 and 012 strains

    PubMed Central

    2011-01-01

    Background Clostridium difficile is the major cause of antibiotic associated diarrhoea and in recent years its increased prevalence has been linked to the emergence of hypervirulent clones such as the PCR-ribotype 027. Characteristically, C. difficile infection (CDI) occurs after treatment with broad-spectrum antibiotics, which disrupt the normal gut microflora and allow C. difficile to flourish. One of the relatively unique features of C. difficile is its ability to ferment tyrosine to para-cresol via the intermediate para-hydroxyphenylacetate (p-HPA). P-cresol is a phenolic compound with bacteriostatic properties which C. difficile can tolerate and may provide the organism with a competitive advantage over other gut microflora, enabling it to proliferate and cause CDI. It has been proposed that the hpdBCA operon, rarely found in other gut microflora, encodes the enzymes responsible for the conversion of p-HPA to p-cresol. Results We show that the PCR-ribotype 027 strain R20291 quantitatively produced more p-cresol in-vitro and was significantly more tolerant to p-cresol than the sequenced strain 630 (PCR-ribotype 012). Tyrosine conversion to p-HPA was only observed under certain conditions. We constructed gene inactivation mutants in the hpdBCA operon in strains R20291 and 630Δerm which curtails their ability to produce p-cresol, confirming the role of these genes in p-cresol production. The mutants were equally able to tolerate p-cresol compared to the respective parent strains, suggesting that tolerance to p-cresol is not linked to its production. Conclusions C. difficile converts tyrosine to p-cresol, utilising the hpdBCA operon in C. difficile strains 630 and R20291. The hypervirulent strain R20291 exhibits increased production of and tolerance to p-cresol, which may be a contributory factor to the virulence of this strain and other hypervirulent PCR-ribotype 027 strains. PMID:21527013

  12. Quantitative nucleic acid amplification by digital PCR for clinical viral diagnostics.

    PubMed

    Zhang, Kuo; Lin, Guigao; Li, Jinming

    2016-09-01

    In the past few years, interest in the development of digital PCR (dPCR) as a direct nucleic acid amplification technique for clinical viral diagnostics has grown. The main advantages of dPCR over qPCR include: quantification of nucleic acid concentrations without a calibration curve, comparable sensitivity, superior quantitative precision, greater resistance to perturbations by inhibitors, and increased robustness to the variability of the target sequence. In this review, we address the application of dPCR to viral nucleic acid quantification in clinical applications and for nucleic acid quantification standardization. Further development is required to overcome the current limitations of dPCR in order to realize its widespread use for viral load measurements in clinical diagnostic applications. PMID:26845722

  13. 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

  14. HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

    PubMed Central

    Noda, Angel Alberto; Rodríguez, Islay; Rodríguez, Yaindrys; Govín, Anamays; Fernández, Carmen; Obregón, Ana Margarita

    2014-01-01

    This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries. PMID:25229221

  15. An integrated software solution for real-time PCR analysis based on microfluidic biochip

    NASA Astrophysics Data System (ADS)

    Wang, Qinghui; Gong, Haiqing

    2003-04-01

    In this paper, we present an integrated and automated prototype system which has been developed for real-time polymerase chain reaction (PCR) analysis based on microfluidic PCR array chips. The system integrates the PCR thermal cycling and optical detection capabilities to enable real-time fluorescence imaging and image processing for data analysis. The main advantage of the system is that it provides a solution that can rapidly perform and evaluate PCR experiment simultaneously on microfluidic PCR array chips. The system has demonstrated fast and efficient on-chip real-time PCR analysis using human genomic DNA samples. The implementation of the system integration is a multi-thread Windows software with component structure which is written in Visual C++.

  16. Integrated Real-Time PCR for Detection and Monitoring of Legionella pneumophila in Water Systems▿

    PubMed Central

    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

    2007-01-01

    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

  17. Evaluation of the use of PCR and reverse transcriptase PCR for detection of pathogenic bacteria in biosolids from anaerobic digestors and aerobic composters.

    PubMed

    Burtscher, Carola; Wuertz, Stefan

    2003-08-01

    A PCR-based method and a reverse transcriptase PCR (RT-PCR)-based method were developed for the detection of pathogenic bacteria in organic waste, using Salmonella spp., Listeria monocytogenes, Yersinia enterocolitica, and Staphylococcus aureus as model organisms. In seeded organic waste samples, detection limits of less than 10 cells per g of organic waste were achieved after one-step enrichment of bacteria, isolation, and purification of DNA or RNA before PCR or RT-PCR amplification. To test the reproducibility and reliability of the newly developed methods, 46 unseeded samples were collected from diverse aerobic (composting) facilities and anaerobic digestors and analyzed by both culture-based classical and newly developed PCR-based procedures. No false-positive but some false-negative results were generated by the PCR- or RT-PCR-based methods after one-step enrichment when compared to the classical detection methods. The results indicated that the level of activity of the tested bacteria in unseeded samples was very low compared to that of freshly inoculated cells, preventing samples from reaching the cell density required for PCR-based detection after one-step enrichment. However, for Salmonella spp., a distinct PCR product could be obtained for all 22 nonamended samples that tested positive for Salmonella spp. by the classical detection procedure when a selective two-step enrichment (20 h in peptone water at 37 degrees C and 24 h in Rappaport Vassiliadis medium at 43 degrees C) was performed prior to nucleic acid extraction and PCR. Hence, the classical procedure was shortened, since cell plating and further differentiation of isolated colonies can be omitted, substituted for by highly sensitive and reliable detection based on nucleic acid extraction and PCR. Similarly, 2 of the 22 samples in which Salmonella spp. were detected also tested positive for Listeria monocytogenes according to a two-step enrichment procedure followed by PCR, compared to 3 samples

  18. A new nanoPCR molecular assay for detection of porcine bocavirus.

    PubMed

    Wang, Xiaoling; Bai, Aiquan; Zhang, Jing; Kong, Miaomiao; Cui, Yuchao; Ma, Xingjie; Ai, Xia; Tang, Qinghai; Cui, Shangjin

    2014-06-01

    Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a novel method for the rapid amplification of DNA and has been used for the detection of virus. For detection of porcine bocavirus (PBoV), a sensitive and specific nanoPCR assay was developed with a pair of primers that were designed based on NS1 gene sequences available in GenBank. Under the optimized conditions of the PBoV nanoPCR assay, the nanoPCR assay was 100-fold more sensitive than a conventional PCR assay. The lower detection limit of the nanoPCR assay was about 6.70×10(1) copies. The nanoPCR assay amplified the specific 482-bp fragment of the PBoV NS1 recombinant plasmid but did not produce any product with genomic DNA or cDNA of porcine parvovirus, porcine circovirus type II, porcine reproductive and respiratory syndrome virus, pseudorabies virus, classic swine fever virus, Encephalomyocarditis virus, Porcine Teschovirus or African swine fever virus plasmid. Of 65 clinical samples collected from diseased pigs, 73.8% and 86.2% were determined to be PBoV positive by PBoV conventional PCR and PBoV nanoPCR assay, respectively. Of 36 clinical samples from healthy pigs, 27.8% and 44.4% were PBoV positive by PBoV conventional PCR and PBoV nanoPCR assay, respectively. The nanoPCR assay will be useful for diagnosing PBoV and for studying its epidemiology and pathology. PMID:24642242

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

    PubMed

    Winn-Deen

    1998-12-01

    Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the PCR product liberated during the detection step. The author has combined the PCR amplification and detection steps into a single procedure suitable for closed-tube analysis. Methods and Results: Allele-specific PCR primers were designed as Sunrise energy-transfer primers and contained a 3' terminal mismatch to distinguish between normal and mutant DNA. Cloned normal (W64) and mutant (R64) templates of the beta3-adrenergic receptor gene were tested to verify amplification specificity and yield. A no-target negative control was also run with each reaction. After PCR, each reaction was tested for fluorescence yield by measuring fluorescence on a spectrofluorimeter or fluorescent microtitreplate reader. The cloned controls and 24 patient samples were tested for the W64R mutation by two methods. The direct fluorescence results with the Sunrise allele-specific PCR method gave comparable genotypes to those obtained with the PCR/ restriction digest/gel electrophoresis control method. No PCR artifacts were observed in the negative controls or in the PCR reactions run with the mismatched target. Conclusions: The results of this pilot study indicate good PCR product and fluorescence yield from allele-specific energy-transfer labeled primers, and the capability of distinguishing between normal and mutant alleles based on fluorescence alone, without the need for restriction digestion, gel electrophoresis, or hybridization with an allele-specific probe. PMID:10089280

  20. Detection of salmonella using a real-time PCR based on molecular beacons

    NASA Astrophysics Data System (ADS)

    Chen, Wilfred; Martinez, Grisselle; Mulchandani, Ashok

    2000-03-01

    Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization. We developed a new approach to detect the presence of Salmonella species using these fluorogenic reporter molecules and demonstrated their ability to discriminate between similar E. coli species in real-time PCR assays. A detection limit of 1 CFU per PCR reaction was obtained. The assays were carried out entirely in sealed PCR tubes, enabling fast and direct detection in a semiautomated format.

  1. The need for transparency and good practices in the qPCR literature.

    PubMed

    Bustin, Stephen A; Benes, Vladimir; Garson, Jeremy; Hellemans, Jan; Huggett, Jim; Kubista, Mikael; Mueller, Reinhold; Nolan, Tania; Pfaffl, Michael W; Shipley, Gregory; Wittwer, Carl T; Schjerling, Peter; Day, Philip J; Abreu, Mónica; Aguado, Begoña; Beaulieu, Jean-François; Beckers, Anneleen; Bogaert, Sara; Browne, John A; Carrasco-Ramiro, Fernando; Ceelen, Liesbeth; Ciborowski, Kate; Cornillie, Pieter; Coulon, Stephanie; Cuypers, Ann; De Brouwer, Sara; De Ceuninck, Leentje; De Craene, Jurgen; De Naeyer, Hélène; De Spiegelaere, Ward; Deckers, Kato; Dheedene, Annelies; Durinck, Kaat; Ferreira-Teixeira, Margarida; Fieuw, Annelies; Gallup, Jack M; Gonzalo-Flores, Sandra; Goossens, Karen; Heindryckx, Femke; Herring, Elizabeth; Hoenicka, Hans; Icardi, Laura; Jaggi, Rolf; Javad, Farzad; Karampelias, Michael; Kibenge, Frederick; Kibenge, Molly; Kumps, Candy; Lambertz, Irina; Lammens, Tim; Markey, Amelia; Messiaen, Peter; Mets, Evelien; Morais, Sofia; Mudarra-Rubio, Alberto; Nakiwala, Justine; Nelis, Hilde; Olsvik, Pal A; Pérez-Novo, Claudina; Plusquin, Michelle; Remans, Tony; Rihani, Ali; Rodrigues-Santos, Paulo; Rondou, Pieter; Sanders, Rebecca; Schmidt-Bleek, Katharina; Skovgaard, Kerstin; Smeets, Karen; Tabera, Laura; Toegel, Stefan; Van Acker, Tim; Van den Broeck, Wim; Van der Meulen, Joni; Van Gele, Mireille; Van Peer, Gert; Van Poucke, Mario; Van Roy, Nadine; Vergult, Sarah; Wauman, Joris; Tshuikina-Wiklander, Marina; Willems, Erik; Zaccara, Sara; Zeka, Fjoralba; Vandesompele, Jo

    2013-11-01

    Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not. PMID:24173381

  2. Performances of Four Real-Time PCR Assays for Diagnosis of Pneumocystis jirovecii Pneumonia.

    PubMed

    Sasso, Milène; Chastang-Dumas, Elsa; Bastide, Sophie; Alonso, Sandrine; Lechiche, Catherine; Bourgeois, Nathalie; Lachaud, Laurence

    2016-03-01

    Pneumonia due to Pneumocystis jirovecii (PCP) is a frequent infection among HIV-positive or other immunocompromised patients. In the past several years, PCR on pulmonary samples has become an essential element for the laboratory diagnosis of PCP. Nevertheless, very few comparative studies of available PCR assays have been published. In this work, we evaluated the concordance between four real-time PCR assays, including three commercial kits, AmpliSens, MycAssay, and Bio-Evolution PCR, and an in-house PCR (J. Fillaux et al. 2008, J Microbiol Methods 75:258-261, doi:http://dx.doi.org/10.1016/j.mimet.2008.06.009), on 148 pulmonary samples. The results showed concordance rates ranging from 81.6% to 96.6% (kappa, 0.64 to 0.93). Concordance was excellent between three assays: the in-house assay, AmpliSens, and the MycAssay PCR (kappa, >0.8). The performances of these PCR assays were also evaluated according to the classification of the probability of PCP (proven, probable, possible, or no final diagnosis of PCP) based on clinical and radiological signs as well as on the direct examination of bronchoalveolar lavage samples. In the proven PCP category, Pneumocystis jirovecii DNA was detected with all four assays. In the probable PCP category, the in-house PCR, AmpliSens, and the MycAssay PCR were positive for all samples, while the Bio-Evolution PCR failed to detect Pneumocystis jirovecii DNA in two samples. In the possible PCP category, the percentage of positive samples according to PCR varied from 54.5% to 86.4%. Detection of colonized patients is discussed. Finally, among the four evaluated PCR assays, one was not suitable for colonization detection but showed good performance in the proven and probable PCP groups. For the three other assays, performances were excellent and allowed detection of a very low fungal burden. PMID:26719435

  3. Recovery of Bordetella pertussis from PCR-Positive Nasopharyngeal Samples Is Dependent on Bacterial Load

    PubMed Central

    Steinbakk, Martin; Bjørnstad, Martha L.; Moghaddam, Amir; Reinton, Nils; Dahl, Mette L.; Grude, Nils; Sandven, Per

    2012-01-01

    Viable Bordetella pertussis isolates are essential for surveillance purposes. We performed culture of 223 PCR-positive nasopharyngeal samples. B. pertussis was recovered from 45 (20.2%) of the samples. Growth was associated with a high bacterial load, as determined by PCR. Culture from PCR-positive samples is a feasible approach to recover B. pertussis isolates, and culture can be limited to samples with a high bacterial load. PMID:23035189

  4. Recovery of Bordetella pertussis from PCR-positive nasopharyngeal samples is dependent on bacterial load.

    PubMed

    Vestrheim, Didrik F; Steinbakk, Martin; Bjørnstad, Martha L; Moghaddam, Amir; Reinton, Nils; Dahl, Mette L; Grude, Nils; Sandven, Per

    2012-12-01

    Viable Bordetella pertussis isolates are essential for surveillance purposes. We performed culture of 223 PCR-positive nasopharyngeal samples. B. pertussis was recovered from 45 (20.2%) of the samples. Growth was associated with a high bacterial load, as determined by PCR. Culture from PCR-positive samples is a feasible approach to recover B. pertussis isolates, and culture can be limited to samples with a high bacterial load. PMID:23035189

  5. Evaluation of Pan-Dermatophyte Nested PCR in Diagnosis of Onychomycosis▿

    PubMed Central

    Garg, Jaya; Tilak, Ragini; Singh, Sanjay; Gulati, Anil Kumar; Garg, Atul; Prakash, Pradyot; Nath, Gopal

    2007-01-01

    In this study, nested PCR using novel primers targeting the pan-dermatophyte-specific sequence of the chitin synthase 1 gene (CHS1) was compared with KOH microscopy, culture isolation, and single-round PCR for diagnosis of 152 patients with clinically suspected onychomycosis. Results indicate that nested PCR may be considered the gold standard for the diagnosis of cases of onychomycosis for which the etiological agents are dermatophytes. PMID:17699656

  6. Evidence for a functional dimeric form of the PcrA helicase in DNA unwinding

    PubMed Central

    Yang, Ye; Dou, Shuo-Xing; Ren, Hua; Wang, Peng-Ye; Zhang, Xing-Dong; Qian, Min; Pan, Bing-Yi

    2008-01-01

    PcrA helicase, a member of the superfamily 1, is an essential enzyme in many bacteria. The first crystal structures of helicases were obtained with PcrA. Based on structural and biochemical studies, it was proposed and then generally believed that PcrA is a monomeric helicase that unwinds DNA by an inchworm mechanism. But a functional state of PcrA from unwinding kinetics studies has been lacking. In this work, we studied the kinetic mechanism of PcrA-catalysed DNA unwinding with fluorometric stopped-flow method under both single- and multiple-turnover conditions. It was found that the PcrA-catalysed DNA unwinding depended strongly on the PcrA concentration as well as on the 3′-ssDNA tail length of the substrate, indicating that an oligomerization was indispensable for efficient unwinding. Study of the effect of ATP concentration on the unwinding rate gave a Hill coefficient of ∼2, suggesting strongly that PcrA functions as a dimer. It was further determined that PcrA unwound DNA with a step size of 4 bp and a rate of ∼9 steps per second. Surprisingly, it was observed that PcrA unwound 12-bp duplex substrates much less efficiently than 16-bp ones, highlighting the importance of protein-DNA duplex interaction in the helicase activity. From the present studies, it is concluded that PcrA is a dimeric helicase with a low processivity in vitro. Implications of the experimental results for the DNA unwinding mechanism of PcrA are discussed. PMID:18276648

  7. Direct Detection of Erythromycin-Resistant Bordetella pertussis in Clinical Specimens by PCR.

    PubMed

    Wang, Zengguo; Han, Ruijun; Liu, Ying; Du, Quanli; Liu, Jifeng; Ma, Chaofeng; Li, Hengxin; He, Qiushui; Yan, Yongping

    2015-11-01

    Resistance of Bordetella pertussis to erythromycin has been increasingly reported. We developed an allele-specific PCR method for rapid detection of erythromycin-resistant B. pertussis directly from nasopharyngeal (NP) swab samples submitted for diagnostic PCR. Based on the proven association of erythromycin resistance with the A2047G mutation in the 23S rRNA of B. pertussis, four primers, two of which were designed to be specific for either the wild-type or the mutant allele, were used in two different versions of the allele-specific PCR assay. The methods were verified with results obtained by PCR-based sequencing of 16 recent B. pertussis isolates and 100 NP swab samples submitted for diagnostic PCR. The detection limits of the two PCR assays ranged from 10 to 100 fg per reaction for both erythromycin-susceptible and -resistant B. pertussis. Two amplified fragments of each PCR, of 286 and 112 bp, respectively, were obtained from a mutant allele of the isolates and/or NP swab samples containing B. pertussis DNAs. For the wild-type allele, only a 286-bp fragment was visible when the allele-specific PCR assay 1 was performed. No amplification was found when a number of non-Bordetella bacterial pathogens and NP swab samples that did not contain the DNAs of B. pertussis were examined. This assay can serve as an alternative for PCR-based sequencing, especially for local laboratories in resource-poor countries. PMID:26224847

  8. Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

    PubMed Central

    Requena, Teresa; Burton, Jeremy; Matsuki, Takahiro; Munro, Karen; Simon, Mary Alice; Tanaka, Ryuichiro; Watanabe, Koichi; Tannock, Gerald W.

    2002-01-01

    Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations. PMID:11976117

  9. 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

  10. A new method of pebrine inspection of silkworm egg using multiprimer PCR.

    PubMed

    Hatakeyama, Yoshinori; Hayasaka, Shoji

    2003-03-01

    Using a mixture of several PCR primers, we evaluated whether multiprimer PCR is practically useful for the early and simultaneous detection of several kinds of microsporidia that cause silkworm pebrine. When genomic DNA extracted from silkworm eggs infected with Nosema bombycis was used as the DNA template, the specific DNA sequences were amplified by multiprimer PCR. In addition, similar results were obtained even when genomic DNA extracted from silkworms infected with N. bombycis was used as the DNA template. These findings suggest that multiprimer PCR using several primers designed for this study is practically useful for pebrine inspection of silkworm eggs. PMID:12676550

  11. 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

  12. Improved thermal cycling durability and PCR compatibility of polymer coated quantum dot

    NASA Astrophysics Data System (ADS)

    Xun, Zhe; Zhao, Xiaoyun; Guan, Yifu

    2013-09-01

    Quantum dots have experienced rapid development in imaging, labeling and sensing in medicine and life science. To be suitable for polymerase chain reaction (PCR) assay, we have tested QD thermal cycling durability and compatibility, which have not been addressed in previous reports. In this study, we synthesized CdSe/ZnS QDs with a surface modification with high-MW amphiphilic copolymers and observed that Mg2+ ions in the PCR reaction could induce the QDs to precipitate and reduce their fluorescence signal significantly after thermal cycling. To overcome this problem, we used mPEG2000 to conjugate the QD surface for further protection, and found that this modification enables QDs to endure 40 thermal cycles in the presence of other components essential for PCR reactions. We have also identified that QDs have different effects on rTaq and Ex Taq polymerization systems. A high QD concentration could apparently reduce the PCR efficiency, but this inhibition was relieved significantly in the Ex PCR system as the concentration of Ex Taq polymerase was increased. Real-time PCR amplification results showed that QDs could provide a sufficiently measurable fluorescence signal without excessively inhibiting the DNA amplification. Based on this improved thermal cycling durability and compatibility with the PCR system, QDs have the potential to be developed as stable fluorescent sensors in PCR and real-time PCR amplification.

  13. Functional integration of PCR amplification and capillary electrophoresis in a microfabricated DNA analysis device.

    PubMed

    Woolley, A T; Hadley, D; Landre, P; deMello, A J; Mathies, R A; Northrup, M A

    1996-12-01

    Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10 degrees C/s heating, 2.5 degrees C/s cooling) with the high-speed (< 120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an "electrophoretic valve" to couple the PCR and CE devices on-chip. To demonstrate the functionality of this system, a 15 min PCR amplification of a beta-globin target cloned in M13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. Amplification of the beta-globin target as a function of cycle number was directly monitored for two different reactions starting with 4 x 10(7) and 4 x 10(5) copies of DNA template. This work establishes the feasibility of performing high-speed DNA analyses in microfabricated integrated fluidic systems. PMID:8946790

  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. Improved thermal cycling durability and PCR compatibility of polymer coated quantum dot.

    PubMed

    Xun, Zhe; Zhao, Xiaoyun; Guan, Yifu

    2013-09-01

    Quantum dots have experienced rapid development in imaging, labeling and sensing in medicine and life science. To be suitable for polymerase chain reaction (PCR) assay, we have tested QD thermal cycling durability and compatibility, which have not been addressed in previous reports. In this study, we synthesized CdSe/ZnS QDs with a surface modification with high-MW amphiphilic copolymers and observed that Mg²⁺ ions in the PCR reaction could induce the QDs to precipitate and reduce their fluorescence signal significantly after thermal cycling. To overcome this problem, we used mPEG2000 to conjugate the QD surface for further protection, and found that this modification enables QDs to endure 40 thermal cycles in the presence of other components essential for PCR reactions. We have also identified that QDs have different effects on rTaq and Ex Taq polymerization systems. A high QD concentration could apparently reduce the PCR efficiency, but this inhibition was relieved significantly in the Ex PCR system as the concentration of Ex Taq polymerase was increased. Real-time PCR amplification results showed that QDs could provide a sufficiently measurable fluorescence signal without excessively inhibiting the DNA amplification. Based on this improved thermal cycling durability and compatibility with the PCR system, QDs have the potential to be developed as stable fluorescent sensors in PCR and real-time PCR amplification. PMID:23924819

  16. Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA

    PubMed Central

    Gabriel, Richard; Kutschera, Ina; Bartholomae, Cynthia C; von Kalle, Christof; Schmidt, Manfred

    2014-01-01

    Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3’- and 5’- sequences adjacent to the integrated lentiviral vector. PMID:24998871

  17. Surmounting a PCR challenge using a Contradictory matrix from the Theory of Inventive Problem Solving (TRIZ).

    PubMed

    Drábek, Jiří

    2016-01-01

    In this paper I tested whether Contradictory Matrix with 40 Inventive Principles, the simplest instrument from the Theory of Inventive Problem Solving (TRIZ), is a useful approach to a real-life PCR scenario. The PCR challenge consisted of standardization of fluorescence melting curve measurements in Competitive Amplification of Differentially Melting Amplicons (CADMA) PCR for multiple targets. Here I describe my way of using the TRIZ Matrix to generate seven alternative solutions from which I can choose the successful solution, consisting of repeated cycles of amplification and melting in a single PCR run. PMID:26835236

  18. Evaluation of ERIC-PCR as Genotyping Method for Corynebacterium pseudotuberculosis Isolates

    PubMed Central

    Dorneles, Elaine M. S.; Santana, Jordana A.; Ribeiro, Dayana; Dorella, Fernanda Alves; Guimarães, Alessandro S.; Moawad, Mohamed S.; Selim, Salah A.; Garaldi, Ana Luiza M.; Miyoshi, Anderson; Ribeiro, Márcio G.; Gouveia, Aurora M. G.; Azevedo, Vasco; Heinemann, Marcos B.; Lage, Andrey P.

    2014-01-01

    The aim of this study was to evaluate the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis isolates from eight different hosts in twelve countries. Ninety-nine C. pseudotuberculosis field strains, one type strain (ATCC 19410T) and one vaccine strain (1002) were fingerprinted using the ERIC-1R and ERIC-2 primers, and the ERIC-1R+ERIC-2 primer pair. Twenty-nine different genotypes were generated by ERIC 1-PCR, 28 by ERIC 2-PCR and 35 by ERIC 1+2-PCR. The discriminatory index calculated for ERIC 1, ERIC 2, and ERIC 1+2-PCR was 0.89, 0.86, and 0.92, respectively. Epidemiological concordance was established for all ERIC-PCR assays. ERIC 1+2-PCR was defined as the best method based on suitability of the amplification patterns and discriminatory index. Minimal spanning tree for ERIC 1+2-PCR revealed three major clonal complexes and clustering around nitrate-positive (biovar Equi) and nitrate-negative (biovar Ovis) strains. Therefore, ERIC 1+2-PCR proved to be the best technique evaluated in this study for genotyping C. pseudotuberculosis strains, due to its usefulness for molecular epidemiology investigations. PMID:24901343

  19. Quantitative PCR analysis of salivary pathogen burden in periodontitis

    PubMed Central

    Salminen, Aino; Kopra, K. A. Elisa; Hyvärinen, Kati; Paju, Susanna; Mäntylä, Päivi; Buhlin, Kåre; Nieminen, Markku S.; Sinisalo, Juha; Pussinen, Pirkko J.

    2015-01-01

    Our aim was to investigate the value of salivary concentrations of four major periodontal pathogens and their combination in diagnostics of periodontitis. The Parogene study included 462 dentate subjects (mean age 62.9 ± 9.2 years) with coronary artery disease (CAD) diagnosis who underwent an extensive clinical and radiographic oral examination. Salivary levels of four major periodontal bacteria were measured by quantitative real-time PCR (qPCR). Median salivary concentrations of Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia, as well as the sum of the concentrations of the four bacteria, were higher in subjects with moderate to severe periodontitis compared to subjects with no to mild periodontitis. Median salivary Aggregatibacter actinomycetemcomitans concentrations did not differ significantly between the subjects with no to mild periodontitis and subjects with moderate to severe periodontitis. In logistic regression analysis adjusted for age, gender, diabetes, and the number of teeth and implants, high salivary concentrations of P. gingivalis, T. forsythia, and P. intermedia were significantly associated with moderate to severe periodontitis. When looking at different clinical and radiographic parameters of periodontitis, high concentrations of P. gingivalis and T. forsythia were significantly associated with the number of 4–5 mm periodontal pockets, ≥6 mm pockets, and alveolar bone loss (ABL). High level of T. forsythia was associated also with bleeding on probing (BOP). The combination of the four bacteria, i.e., the bacterial burden index, was associated with moderate to severe periodontitis with an odds ratio (OR) of 2.40 (95% CI 1.39–4.13). When A. actinomycetemcomitans was excluded from the combination of the bacteria, the OR was improved to 2.61 (95% CI 1.51–4.52). The highest OR 3.59 (95% CI 1.94–6.63) was achieved when P. intermedia was further excluded from the combination and only the levels of P. gingivalis and

  20. Comparison of Simplexa Flu A/B & RSV PCR with Cytospin-Immunofluorescence and Laboratory-Developed TaqMan PCR in Predominantly Adult Hospitalized Patients

    PubMed Central

    Ferguson, David

    2014-01-01

    To compare Simplexa Flu A/B & RSV PCR with cytospin-immunofluorescence and laboratory-developed TaqMan PCR methods, 445 nasopharyngeal samples were tested. Of these, 199 were positive (46 for respiratory syncytial virus [RSV], 120 for influenza A, and 33 for influenza B) and 246 were negative. The direct fluorescent-antibody assay (DFA) detected 132 (66.3%) positive samples, Simplexa direct detected 162 (81.4%), Simplexa using extracts detected 177 (88.9%), and lab-developed TaqMan PCR reference methods detected 199 (100%). The specificities were 99.6%, 100%, 100%, and 100%, respectively. The two Simplexa methods were more sensitive than the DFA (P = 0.0001) but less sensitive than the TaqMan reverse transcriptase PCR (P = 0.0001). PMID:24850350

  1. One-Step RT-PCR protocols improve the rate of dengue diagnosis compared to Two-Step RT-PCR approaches.

    PubMed

    De Paula, Sérgio Oliveira; de Melo Lima, Cristiane; Torres, Maria Paula; Pereira, Márcia Rodrigues Garbin; Lopes da Fonseca, Benedito Antônio

    2004-08-01

    Dengue is the most important arboviral disease transmitted to humans. In our laboratory, we have been working on the standardization of the polymerase chain reaction (PCR) diagnosis of this disease. In this work, we compared five commercial kits regularly used on reverse-transcription polymerase chain reaction (RT-PCR) protocols: two Two-Step kits (SuperScript II RT/Super Mix kit and reverse transcription system/Taq DNA polymerase) and three One-Step kits (ready-to-go RT-PCR Beads kit, QIAGEN One-Step RT-PCR kit, and AcessQuick RT-PCR system). Thirty-one serum samples of patients with clinical diagnosis of dengue fever (DF) were analyzed by RT-PCR and serology. RNA extraction was done with the QIAamp Viral RNA kit, and cDNA synthesis and PCR done according to the manufacturer's protocol for the five kits. Out of the 31 serum samples collected from patients suspected of having dengue, 27 were IgM-positive, confirming the dengue diagnosis. Out of those, 24 were positive by the ready-to-go RT-PCR Beads kit, 25 were positive by AcessQuick RT-PCR system and 27 were positive by QIAGEN One-Step RT-PCR kit. On the other hand, only six samples were positive by the SuperScript II RT/Super Mix kits and 10 were positive by reverse transcription system/Taq DNA polymerase kit. The best performance observed with the One-Step kits was confirmed in spiked samples with known quantities of dengue-1 virus since they detected up 1 x 10(2) PFU/ml, while the most sensitive Two-Step kit detected up 1 x 10(4) PFU/ml. These data show that One-Step RT-PCR kits yielded a higher rate of dengue virus detection than the Two-Step kits and correlated well with the serological diagnosis. PMID:15163417

  2. Comparison of a quantitative Real-Time PCR assay and droplet digital PCR for copy number analysis of the CCL4L genes.

    PubMed

    Bharuthram, Avani; Paximadis, Maria; Picton, Anabela C P; Tiemessen, Caroline T

    2014-07-01

    The controversy surrounding the findings that copy number variation, of the CCL3 encoding genes, influences HIV-1 infection and disease progression has been in part attributed to the variable results obtained from methods used for copy number evaluation. Like CCL3, the genes encoding the CC chemokine CCL4, also a natural ligand of the CCR5 receptor, are found to occur in population-specific multiple copy number and have been shown to play a protective role against HIV-1. This study evaluated the standard method of quantitative Real-Time PCR (qPCR) and droplet digital PCR (ddPCR) for CCL4L gene copy number determination. The CCL4 encoding genes are CCL4, occurring in two copies per diploid genome (pdg), and the non-allelic CCL4L genes, comprised of CCL4L1 and CCL4L2, which are both found in multiple copies pdg. Copy number of CCL4L, CCL4L1 and CCL4L2 was determined in a cohort of HIV-1-uninfected individuals from the South African Black (n=23) and Caucasian (n=32) population groups using qPCR and ddPCR. A stronger correlation between the number of CCL4L copies and the sum of CCL4L1 and CCL4L2 copies generated by ddPCR (r=0.99, p<0.0001) compared to qPCR (r=0.87, p<0.0001) was observed. Real-Time qPCR exhibited greater inaccuracy at higher copy numbers which is particularly relevant to our cohort of Black individuals who have a higher range of CCL4L copies (3-6) compared to Caucasians (0-4) and a higher population median (4 and 2, respectively). Medians and ranges of CCL4L1 (Black: 2, 0-4, Caucasian: 0, 0-2) and CCL4L2 (Black: 2, 1-5, Caucasian: 2, 0-3) were also higher in the Black population. Droplet digital PCR was shown to be a far superior method to qPCR for assessment of CCL4 gene copy number variation, the accuracy of which is essential for studies of the contribution of variable gene copy number to phenotypic outcomes of host infection and disease course. PMID:24727646

  3. Applicability of integrated cell culture quantitative PCR (ICC-qPCR) for the detection of infectious adenovirus type 2 in UV disinfection studies.

    PubMed

    Ryu, Hodon; Cashdollar, Jennifer L; Fout, G Shay; Schrantz, Karen A; Hayes, Samuel

    2015-01-01

    Practical difficulties of the traditional adenovirus infectivity assay such as intensive labor requirements and longer turnaround period limit the direct use of adenovirus as a testing microorganism for systematic, comprehensive disinfection studies. In this study, we attempted to validate the applicability of integrated cell culture quantitative PCR (ICC-qPCR) as an alternative to the traditional cell culture method with human adenovirus type 2 (HAdV2) in a low-pressure UV disinfection study and to further optimize the procedures of ICC-qPCR for 24-well plate format. The relatively high stability of the hexon gene of HAdV2 was observed after exposure to UV radiation, resulting in a maximum gene copy reduction of 0.5 log10 at 280 mJ cm(-2). Two-day post-inoculation incubation period and a maximum spiking level of 10(5) MPN mL(-1) were selected as optimum conditions of ICC-qPCR with the tested HAdV2. An approximate 1:1 correlation of virus quantities by the traditional and ICC-qPCR cell culture based methods suggested that ICC-qPCR is a satisfactory alternative for practical application in HAdV2 disinfection studies. ICC-qPCR results, coupled with a first-order kinetic model (i.e., the inactivation rate constant of 0.0232 cm(2) mJ(-1)), showed that an UV dose of 172 mJ cm(-2) achieved a 4-log inactivation credit for HAdV2. This estimate is comparable to other studies with HAdV2 and other adenovirus respiratory types. The newly optimized ICC-qPCR shows much promise for further study on its applicability of other slow replicating viruses in disinfection studies. PMID:26030683

  4. [Identification of SHV-type extended spectrum beta-lactamase genes in Pseudomonas aeruginosa by PCR-restriction fragment length polymorphism and insertion site restriction-PCR].

    PubMed

    Kalai Blagui, S; Achour, W; Abdeladhim, A; Ben Hassen, A

    2009-07-01

    We propose a simple and rapid method to discriminate SHV-type extended spectrum beta-lactamase (ESBL) genes in P. aeruginosa based on PCR techniques (PCR-RFLP and RSI-PCR). We studied 22 producing ESBL P. aeruginosa strains isolated from seven immunocompromised patients (19 isolates) and from environmental swabs (three isolates) at the Bone Marrow Transplantation Center of Tunis. Screening PCR with primer pairs designed to detect gene encoding TEM, SHV, OXA group I, OXA group II, OXA-18 and PER-1 ESBL was positive for bla(OXA18) and bla(SHV) genes in all isolates. Pulsed field gel electrophoresis using SpeI endonuclease defined five genotypic groups. For at least one isolate corresponding to each genotype observed, restriction of PCR products by DdeI and BsrI revealed the same restriction pattern that the bla(SHV-1) negative control; in the same way, RSI-PCR products digestion by NruI, thus excluding 35, 238 and 240 mutations characterizing reported ESBL in P. aeruginosa (SHV-2a, SHV5 et SHV12), and suggesting that studied bla(SHV) genes were not ESBL ones. Genomic DNA hybridization by southern blot with probe consisting in bla(SHV-1) gene was positive in these isolates. Sequencing the full-length open reading frame revealed nucleotide sequence of the bla(SHV-1). PCR-RFLP and RSI-PCR results were then confirmed. This approach is effective for screening P. aeruginosa for ESBL genes carriage in epidemiological studies and for detecting new variants. PMID:18838231

  5. 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

  6. APPLICATION OF POLYMERASE CHAIN REACTION (PCR) AND PCR BASED RESTRICTION FRAGMENT LENGTH POLYMORPHISM FOR DETECTION AND IDENTIFICATION OF DERMATOPHYTES FROM DERMATOLOGICAL SPECIMENS

    PubMed Central

    Bagyalakshmi, R; Senthilvelan, B; Therese, K L; Murugusundram, S; Madhavan, H N

    2008-01-01

    Objective: To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes. Materials and Methods: Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods. Results: PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%. Conclusion: PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes. PMID:19967012

  7. Clostridium difficile PCR Ribotype 018, a Successful Epidemic Genotype

    PubMed Central

    Trovato, Alberto; Bianchini, Valentina; Biancardi, Anna; Cichero, Paola; Mazzotti, Maria; Nizzero, Paola; Moro, Matteo; Ossi, Cristina; Scarpellini, Paolo

    2015-01-01

    Clostridium difficile infection (CDI) became a public health problem for the global spreading of the so-called hypervirulent PCR ribotypes (RTs) 027 and 078, associated with increases in the transmission and severity of the disease. However, especially in Europe, several RTs are prevalent, and the concept of hypervirulence is currently debated. We investigated the toxin and resistance profiles and the genetic relatedness of 312 C. difficile strains isolated in a large Italian teaching hospital during a 5-year period. We evaluated the role of CDI-related antibiotic consumption and infection control practices on the RT predominance in association with their molecular features and transmission capacity. Excluding secondary cases due to nosocomial transmission, RT018 was the predominant genotype (42.4%) followed by RT078 (13.6%), while RT027 accounted for 0.8% of the strains. RT078 was most frequently isolated from patients in intensive care units. Its prevalence significantly increased over time, but its transmission capacity was very low. In contrast, RT018 was highly transmissible and accounted for 95.7% of the secondary cases. Patients with the RT018 genotype were significantly older than those with RT078 and other RTs, indicating an association between epidemic RT and age. We provide here the first epidemiological evidence to consider RT018 as a successful epidemic genotype that deserves more attention in clinical practice. PMID:26041894

  8. Amplification and characterization of the retinoblastoma gene VNTR by PCR.

    PubMed Central

    Scharf, S J; Bowcock, A M; McClure, G; Klitz, W; Yandell, D W; Erlich, H A

    1992-01-01

    VNTR regions are informative genetic markers for linkage mapping and individual identification. Using PCR, we have developed a procedure for the enzymatic amplification of the VNTR located in the 16th intron of the human retinoblastoma (RB1) gene. We have also prepared a nonisotopically labeled oligonucleotide probe which facilitates detection of the amplification products. In examining 250 individuals from four different populations, we have detected 11 alleles ranging from 650 to 1,800 bp in size. The core repeat is approximately 50 bp in length. On the basis of the observed allele frequencies for Caucasian, African-American, and Hispanic populations from the United States and for the Mexican Hispanic population, the heterozygosities have been calculated to be 62%, 75%, 61%, and 50%, respectively. The observed genotype frequencies do not deviate from the values expected under Hardy-Weinberg equilibrium. The effect of varying primer sequences, annealing temperature, and cycle number on the amplification are also discussed. Amplification of this marker may also prove useful for detecting the heterozygosity loss that is associated with tumor formation in retinoblastoma. Images Figure 4 Figure 2 Figure 3 Figure 5 PMID:1734717

  9. DNA profiles from clothing fibers using direct PCR.

    PubMed

    Blackie, Renée; Taylor, Duncan; Linacre, Adrian

    2016-09-01

    We report on the successful use of direct PCR amplification of single fibers from items of worn clothing. Items of clothing were worn throughout the course of a day, with the individual commencing regular activities. Single fibers were taken from the cuff of the clothing at regular intervals and amplified directly. The same areas were subjected to tape-lifting, and also amplified directly for comparison. The NGM™ kit that amplifies 15 STR loci plus amelogenin was used. A total of 35 single fiber samples were processed and analyzed from five items of clothing, with 81 % of samples returning a profile of 14 alleles or more. All tape-lift samples amplified directly produced DNA profiles of 15 alleles or more. The aim was to develop a simple, operational method that could be used routinely in forensic science casework and that has the potential to generate more complete profiles, which would not be detected using standard extraction methods on this type of sample. For ease of implementation, the process also adheres to standard methods with no increase in the cycle number. PMID:27421265

  10. A high-plex PCR approach for massively parallel sequencing.

    PubMed

    Nguyen-Dumont, Tú; Pope, Bernard J; Hammet, Fleur; Southey, Melissa C; Park, Daniel J

    2013-08-01

    Current methods for targeted massively parallel sequencing (MPS) have several drawbacks, including limited design flexibility, expense, and protocol complexity, which restrict their application to settings involving modest target size and requiring low cost and high throughput. To address this, we have developed Hi-Plex, a PCR-MPS strategy intended for high-throughput screening of multiple genomic target regions that integrates simple, automated primer design software to control product size. Featuring permissive thermocycling conditions and clamp bias reduction, our protocol is simple, cost- and time-effective, uses readily available reagents, does not require expensive instrumentation, and requires minimal optimization. In a 60-plex assay targeting the breast cancer predisposition genes PALB2 and XRCC2, we applied Hi-Plex to 100 ng LCL-derived DNA, and 100 ng and 25 ng FFPE tumor-derived DNA. Altogether, at least 86.94% of the human genome-mapped reads were on target, and 100% of targeted amplicons were represented within 25-fold of the mean. Using 25 ng FFPE-derived DNA, 95.14% of mapped reads were on-target and relative representation ranged from 10.1-fold lower to 5.8-fold higher than the mean. These results were obtained using only the initial automatically-designed primers present in equal concentration. Hi-Plex represents a powerful new approach for screening panels of genomic target regions. PMID:23931594

  11. Flow Through PCR Module of BioBriefcase

    SciTech Connect

    Arroyo, E S; Wheeler, E K; Hindson, B; Nasarabadi, S; Vrankovich, G; Bell, P; Bailey, C; Sheppod, T; Christian, A

    2005-09-19

    The BioBriefcase is an integrated briefcase-sized aerosol collection and analysis system for autonomous monitoring of the environment, which is currently being jointly developed by Lawrence Livermore and Sandia National Laboratories. This poster presents results from the polymerase chain reaction (PCR) module of the system. The DNA must be purified after exiting the aerosol collector to prevent inhibition of the enzymatic reaction. Traditional solid-phase extraction results in a large loss of sample. In this flow-through system, we perform sample purification, concentration and amplification in one reactor, which minimizes the loss of material. The sample from the aerosol collector is mixed with a denaturation solution prior to flowing through a capillary packed with silica beads. The DNA adheres to the silica beads allowing the environmental contaminants to be flushed to waste while effectively concentrating the DNA on the silica matrix. The adhered DNA is amplified while on the surface of the silica beads, resulting in a lower limit of detection than an equivalent eluted sample. Thus, this system is beneficial since more DNA is available for amplification, less reagents are utilized, and contamination risks are reduced.

  12. Flow through PCR module of BioBriefcase

    NASA Astrophysics Data System (ADS)

    Arroyo, E.; Wheeler, E. K.; Shediac, R.; Hindson, B.; Nasarabadi, S.; Vrankovich, G.; Bell, P.; Bailey, C.; Sheppod, T.; Christian, A. T.

    2005-11-01

    The BioBriefcase is an integrated briefcase-sized aerosol collection and analysis system for autonomous monitoring of the environment, which is currently being jointly developed by Lawrence Livermore and Sandia National Laboratories. This poster presents results from the polymerase chain reaction (PCR) module of the system. The DNA must be purified after exiting the aerosol collector to prevent inhibition of the enzymatic reaction. Traditional solid-phase extraction results in a large loss of sample. In this flow-through system, we perform sample purification, concentration and amplification in one reactor, which minimizes the loss of material. The sample from the aerosol collector is mixed with a denaturation solution prior to flowing through a capillary packed with silica beads. The DNA adheres to the silica beads allowing the environmental contaminants to be flushed to waste while effectively concentrating the DNA on the silica matrix. The adhered DNA is amplified while on the surface of the silica beads, resulting in a lower limit of detection than an equivalent eluted sample. Thus, this system is beneficial since more DNA is available for amplification, less reagents are utilized, and contamination risks are reduced.

  13. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    DOEpatents

    Nolan, John P.; White, P. Scott

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  14. PCR Assays That Identify the Grapevine Dieback Fungus Eutypa lata

    PubMed Central

    Lecomte, Pascal; Péros, Jean-Pierre; Blancard, Dominique; Bastien, Nicolas; Délye, Christophe

    2000-01-01

    Eutypa lata is the causal fungal agent of Eutypa dieback, a serious grapevine necrotic disease. The erratic and delayed (1 to 2 months) appearance of characteristic conidia on culture media and the presence of numerous microorganisms in decaying wood make it difficult either to identify or to detect E. lata in grapevine wood samples. We designed six pairs of PCR primers for diagnosis of E. lata. Three primer pairs were derived from ribosomal DNA internal transcribed spacer sequences, and three pairs were derived from randomly amplified polymorphic DNA fragments. The six primer pairs could be used to amplify DNAs extracted from all of the E. lata isolates tested. They did not amplify DNAs from fungi and bacteria representing more than 50 different species of microorganisms associated with grapevine. We developed a simple protocol, leading to a rapid release of DNA, that enabled us to identify E. lata from pure or mixed cultures as well as from grapevine wood samples. Identification of E. lata in wood was achieved within a few hours, instead of the several weeks required for classical cultures on agar medium. We believe that the procedure described here can be adapted to detect other microorganisms involved in woody plant diseases. PMID:11010901

  15. Detection of North American orthopoxviruses by real time-PCR

    PubMed Central

    2011-01-01

    The prevalence of North American orthopoxviruses in nature is unknown and may be more difficult to ascertain due to wide spread use of vaccinia virus recombinant vaccines in the wild. A real time PCR assay was developed to allow for highly sensitive and specific detection of North American orthopoxvirus DNA in animal tissues and bodily fluids. This method is based on the amplification of a 156 bp sequence within a myristylated protein, highly conserved within the North American orthopoxviruses but distinct from orthologous genes present in other orthopoxviruses. The analytical sensitivity was 1.1 fg for Volepox virus DNA, 1.99 fg for Skunkpox virus DNA, and 6.4 fg for Raccoonpox virus DNA with a 95% confidence interval. Our assay did not cross-react with other orthopoxviruses or ten diverse representatives of the Chordopoxvirinae subfamily. This new assay showed more sensitivity than tissue culture tests, and was capable of differentiating North American orthopoxviruses from other members of Orthopoxvirus. Thus, our assay is a promising tool for highly sensitive and specific detection of North American orthopoxviruses in the United States and abroad. PMID:21689420

  16. PCR diagnostic system in the treatment of prosthetic joint infections.

    PubMed

    Jahoda, D; Landor, I; Benedík, J; Pokorný, D; Judl, T; Barták, V; Jahodová, I; Fulín, P; Síbek, M

    2015-09-01

    In our prospective study, we examined whether a multiplex PCR diagnostic method is suitable for the primary detection of pathogens. We also examined the possibility and sensitivity of detecting genes responsible for biofilm production and methicillin resistance. From 2007 to 2009, 94 patients were included in the study. A UNB (universal detection of 16S ribosomal bacterial DNA) and UNF (universal detection of pathogenic fungi) were used in the primary detection. A multiplex assay for biofilm production, methicillin resistance allowed us to distinguish between Gram positivity and negativity and to detect Staphylococci. From all the samples, the culture was positive in 53.2 % of cases, and by using the UNB method, we detected bacteria in 79.8 % of cases-the UNF detection of fungi was positive in 10.6 % of cases. In 75 % of positive findings, we detected a Gram-negative bacterium in 65.3 % of cases. In 47.2 % of Staphylococci detected, the ability to produce biofilm was confirmed. 61.1 % of the Staphylococci exhibited a methicillin resistance. Our multiplex scheme cannot yet fully replace microbial cultivation but can be a rational guide when choosing an appropriate antibiotic therapy in cases where the microbial culture is negative. PMID:25523034

  17. A new pentaplex PCR system for forensic casework analysis.

    PubMed

    Lederer, T; Seidl, S; Graham, B; Betz, P

    2000-01-01

    In 1998 the Federal Criminal Police Office of Germany (BKA) established a central genetic database of offenders and suspects to facilitate comparisons with biological samples from future criminal offences. The five obligatory short tandem repeat (STR) loci in this database (TH01, SE33, vWA, FGA and D21S11) were co-amplified in a new PCR pentaplex analysing system together with the sex-specific locus amelogenin. Due to overlapping fragment sizes, amplification products were fluorescent dye-labelled with different colours, separated by electrophoresis and detected directly using the ABI PRISM 310 Genetic Analyzer. Reproducible and reliable results were obtained from as low as 125 pg template DNA, indicating high specificity and sensitivity of the assay. Environmental studies and enzymatic digest with DNase I revealed an excellent stability of the pentaplex system with typeable results even in cases of partially degraded DNA. Complete and reproducible DNA typing was possible in blood-stain mixtures with the minor component as low as 10%. Mean stutter peak intensities were analysed for all loci and ranged from 2.7 +/- 0.8% (TH01) to 10.6 +/- 1.6% (vWA) of the main signal intensity. Allele frequencies were determined in a North Bavarian population sample (n = 121). The combination of five systems resulted in a mean exclusion chance of 99.86% and a power of discrimination of 99.999996%. No deviation from Hardy-Weinberg equilibrium could be found. PMID:11197636

  18. Disentangling mite predator-prey relationships by multiplex PCR.

    PubMed

    Pérez-Sayas, Consuelo; Pina, Tatiana; Gómez-Martínez, María A; Camañes, Gemma; Ibáñez-Gual, María V; Jaques, Josep A; Hurtado, Mónica A

    2015-11-01

    Gut content analysis using molecular techniques can help elucidate predator-prey relationships in situations in which other methodologies are not feasible, such as in the case of trophic interactions between minute species such as mites. We designed species-specific primers for a mite community occurring in Spanish citrus orchards comprising two herbivores, the Tetranychidae Tetranychus urticae and Panonychus citri, and six predatory mites belonging to the Phytoseiidae family; these predatory mites are considered to be these herbivores' main biological control agents. These primers were successfully multiplexed in a single PCR to test the range of predators feeding on each of the two prey species. We estimated prey DNA detectability success over time (DS50), which depended on the predator-prey combination and ranged from 0.2 to 18 h. These values were further used to weight prey detection in field samples to disentangle the predatory role played by the most abundant predators (i.e. Euseius stipulatus and Phytoseiulus persimilis). The corrected predation value for E. stipulatus was significantly higher than for P. persimilis. However, because this 1.5-fold difference was less than that observed regarding their sevenfold difference in abundance, we conclude that P. persimilis is the most effective predator in the system; it preyed on tetranychids almost five times more frequently than E. stipulatus did. The present results demonstrate that molecular tools are appropriate to unravel predator-prey interactions in tiny species such as mites, which include important agricultural pests and their predators. PMID:25824504

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

    PubMed

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

    2015-12-15

    Waterborne transmission of Toxoplasma gondii is a potential public health risk and there are currently no agreed optimised methods for the recovery, processing and detection of T. gondii oocysts in water samples. In this study modified methods of T. gondii oocyst recovery and DNA extraction were applied to 1427 samples collected from 147 public water supplies throughout Scotland. T. gondii DNA was detected, using real time PCR (qPCR) targeting the 529bp repeat element, in 8.79% of interpretable samples (124 out of 1411 samples). The samples which were positive for T. gondii DNA originated from a third of the sampled water sources. The samples which were positive by qPCR and some of the negative samples were reanalysed using ITS1 nested PCR (nPCR) and results compared. The 529bp qPCR was the more sensitive technique and a full analysis of assay performance, by Bayesian analysis using a Markov Chain Monte Carlo method, was completed which demonstrated the efficacy of this method for the detection of T. gondii in water samples. PMID:26408950

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

    PubMed

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

    2016-07-01

    The aim of this study was to compare the microbial analysis methods of polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in terms of detection of five selected potentially periodontal pathogenic bacteria in peri-implant disease. Therefore 45 samples of healthy, mucositis and peri-implantitis (n = 15 each) were assessed according to presence of the following bacteria using PCR (DNA-strip technology) and RT-PCR (fluorescent dye SYBR green-system): Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tanerella forsythia (Tf), and Fusobacterium nucleatum (Fn). There were no significant correlations between the bacterial and disease patterns, so the benefit of using microbiological tests for the diagnosis of peri-implant diseases is questionable. Correlations between the methods were highest for Tf (Kendall's Tau: 0.65, Spearman: 0.78), Fn (0.49, 0.61) and Td (0.49, 0.59). For Aa (0.38, 0.42) and Pg (0.04, 0.04), lower correlation values were detected. Accordingly, conventional semi-quantitative PCR seems to be sufficient for analyzing potentially periodontal pathogenic bacterial species. PMID:27142589

  1. Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants.

    PubMed

    Stefano, Biricolti; Patrizia, Bogani; Matteo, Cerboneschi; Massimo, Gori

    2016-06-01

    One of the major unanswered questions with respect to the commercial use of genetic transformation in woody plants is the stability of the transgene expression over several decades within the same individual. Gene expression is strongly affected by the copy number which has been integrated into the plant genome and by the local DNA features close to the integration sites. Because woody plants cannot be subjected to selfing or backcrossing to modify the transgenic allelic structure without affecting the valuable traits of the cultivar, molecular characterization of the transformation event is therefore crucial. After assessing the transgene copy number of a set of apple transgenic clones with Southern blotting, we describe two alternative methods: the first is based on inverse PCR (i-PCR) and the second on the quantitative PCR (q-PCR). The methods produced comparable results with the exception of the data regarding a high copy number clone, but while the q-PCR-based system is rapid and easily adaptable to high throughput systems, the i-PCR-based method can provide information regarding the transformation event and the characteristics of the sequences flanking the transgenic construct. PMID:26895172

  2. Evaluation of the Broad-Range PCR-Electrospray Ionization Mass Spectrometry (PCR/ESI-MS) System and Virus Microarrays for Virus Detection

    PubMed Central

    Taliaferro, Lanyn P.; Galvin, Teresa A.; Ma, Hailun; Shaheduzzaman, Syed; Williams, Dhanya K.; Glasner, Dustin R.; Khan, Arifa S.

    2014-01-01

    Advanced nucleic acid-based technologies are powerful research tools for novel virus discovery but need to be standardized for broader applications such as virus detection in biological products and clinical samples. We have used well-characterized retrovirus stocks to evaluate the limit of detection (LOD) for broad-range PCR with electrospray ionization mass spectrometry (PCR/ESI-MS or PLEX-ID), RT-PCR assays, and virus microarrays. The results indicated that in the absence of background cellular nucleic acids, PLEX-ID and RT-PCR had a similar LOD for xenotropic murine retrovirus-related virus (XMRV; 3.12 particles per µL) whereas sensitivity of virus detection was 10-fold greater using virus microarrays. When virus was spiked into a background of cellular nucleic acids, the LOD using PLEX-ID remained the same, whereas virus detection by RT-PCR was 10-fold less sensitive, and no virus could be detected by microarrays. Expected endogenous retrovirus (ERV) sequences were detected in cell lines tested and known species-specific viral sequences were detected in bovine serum and porcine trypsin. A follow-up strategy was developed using PCR amplification, nucleotide sequencing, and bioinformatics to demonstrate that an RD114-like retrovirus sequence that was detected by PLEX-ID in canine cell lines (Madin-Darby canine kidney (MDCK) and Cf2Th canine thymus) was due to defective, endogenous gammaretrovirus-related sequences. PMID:24777034

  3. Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalgae Ankistrodesmus convolutus.

    PubMed

    Thanh, Tran; Chi, Vu Thi Quynh; Abdullah, Mohd Puad; Omar, Hishamuddin; Napis, Suhaimi

    2012-01-01

    Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus. PMID:22642102

  4. Comparison of real-time PCR techniques to cytotoxigenic culture methods for diagnosing Clostridium difficile infection.

    PubMed

    Knetsch, C W; Bakker, D; de Boer, R F; Sanders, I; Hofs, S; Kooistra-Smid, A M D; Corver, J; Eastwood, K; Wilcox, M H; Kuijper, E J

    2011-01-01

    In the past decade, the incidence of Clostridium difficile infections (CDI) with a more severe course has increased in Europe and North America. Assays that are capable of rapidly diagnosing CDI are essential. Two real-time PCRs (LUMC and LvI) targeting C. difficile toxin genes (tcdB, and tcdA and tcdB, respectively) were compared with the BD GeneOhm PCR (targeting the tcdB gene), using cytotoxigenic culture as a gold standard. In addition, a real-time PCR targeting the tcdC frameshift mutation at position 117 (Δ117 PCR) was evaluated for detecting toxigenic C. difficile and the presence of PCR ribotype 027 in stool samples. In total, 526 diarrheal samples were prospectively collected and included in the study. Compared with those for cytotoxigenic culture, sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were for PCR LUMC 96.0%, 88.0%, 66.0%, and 98.9%, for PCR LvI 100.0%, 89.4%, 69.7%, and 100.0%, for PCR Δ117 98.0%, 90.7%, 71.9%, and 99.5%, and for PCR BD GeneOhm 88.3%, 96.9%, 86.5%, and 97.4%. Compared to those with feces samples cultured positive for C. difficile type 027, the sensitivity, specificity, PPV, and NPV of the Δ117 PCR were 95.2%, 96.2%, 87.0%, and 98.7%. We conclude that all real-time PCRs can be applied as a first screening test in an algorithm for diagnosing CDI. However, the low PPVs hinder the use of the assays as stand-alone tests. Furthermore, the Δ117 PCR may provide valuable information for minimizing the spread of the epidemic C. difficile PCR ribotype 027. PMID:20980562

  5. Pre-PCR processing in bioterrorism preparedness: improved diagnostic capabilities for laboratory response networks.

    PubMed

    Hedman, Johannes; Knutsson, Rickard; Ansell, Ricky; Rådström, Peter; Rasmusson, Birgitta

    2013-09-01

    Diagnostic DNA analysis using polymerase chain reaction (PCR) has become a valuable tool for rapid detection of biothreat agents. However, analysis is often challenging because of the limited size, quality, and purity of the biological target. Pre-PCR processing is an integrated concept in which the issues of analytical limit of detection and simplicity for automation are addressed in all steps leading up to PCR amplification--that is, sampling, sample treatment, and the chemical composition of PCR. The sampling method should maximize target uptake and minimize uptake of extraneous substances that could impair the analysis--so-called PCR inhibitors. In sample treatment, there is a trade-off between yield and purity, as extensive purification leads to DNA loss. A cornerstone of pre-PCR processing is to apply DNA polymerase-buffer systems that are tolerant to specific sample impurities, thereby lowering the need for expensive purification steps and maximizing DNA recovery. Improved awareness among Laboratory Response Networks (LRNs) regarding pre-PCR processing is important, as ineffective sample processing leads to increased cost and possibly false-negative or ambiguous results, hindering the decision-making process in a bioterrorism crisis. This article covers the nature and mechanisms of PCR-inhibitory substances relevant for agroterrorism and bioterrorism preparedness, methods for quality control of PCR reactions, and applications of pre-PCR processing to optimize and simplify the analysis of various biothreat agents. Knowledge about pre-PCR processing will improve diagnostic capabilities of LRNs involved in the response to bioterrorism incidents. PMID:23971826

  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. [Selective detection of viable pathogenic bacteria in water using reverse transcription quantitative PCR].

    PubMed

    Lin, Yi-Wen; Li, Dan; Wu, Shu-Xu; He, Miao; Yang, Tian

    2012-11-01

    A reverse transcription q quantitative PCR (RT-qPCR) assay method was established, which can quantify the copy numbers of RNA in pathogenic bacteria of E. coli and Enterococcus faecium. The results showed that cDNA was generated with the RT-PCR reagents, target gene was quantified with the qPCR, the copy numbers of RNA were stable at about 1 copies x CFU(-1) for E. coli and 7.98 x 10(2) copies x CFU(-1) for Enterococcus faecium respectively during the stationary grow phase for the both indicator bacteria [E. coli (6-18 h) and Enterococcus faecium (10-38 h)]. The established RT-qPCR method can quantify the numbers of viable bacteria through detecting bacterial RNA targets. Through detecting the heat-treated E. coli and Enterococcus faecium by three methods (culture method, qPCR, RT-qPCR), we found that the qPCR and RT-qPCR can distinguish 1.43 lg copy non-viable E. coli and 2.5 lg copy non-viable Enterococcus faecium. These results indicated that the established methods could effectively distinguish viable bacteria from non-viable bacteria. Finally we used this method to evaluate the real effluents of the secondary sedimentation of wastewater treatment plant (WWTP), the results showed that the correlation coefficients (R2) between RT-qPCR and culture method were 0.930 (E. coli) and 0.948 (Enterococcus faecium), and this established RT-PCR method can rapidly detect viable pathogenic bacteria in genuine waters. PMID:23323443

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

    PubMed

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

    2013-01-01

    Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25 × 16 × 8 cm(3) in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample. PMID:24349341

  9. Real-Time PCR Improves Helicobacter pylori Detection in Patients with Peptic Ulcer Bleeding

    PubMed Central

    Casalots, Alex; Sanfeliu, Esther; Boix, Loreto; García-Iglesias, Pilar; Sánchez-Delgado, Jordi; Montserrat, Antònia; Bella-Cueto, Maria Rosa; Gallach, Marta; Sanfeliu, Isabel; Segura, Ferran; Calvet, Xavier

    2011-01-01

    Background and Aims Histological and rapid urease tests to detect H. pylori in biopsy specimens obtained during peptic ulcer bleeding episodes (PUB) often produce false-negative results. We aimed to examine whether immunohistochemistry and real-time PCR can improve the sensitivity of these biopsies. Patients and Methods We selected 52 histology-negative formalin-fixed paraffin-embedded biopsy specimens obtained during PUB episodes. Additional tests showed 10 were true negatives and 42 were false negatives. We also selected 17 histology-positive biopsy specimens obtained during PUB to use as controls. We performed immunohistochemistry staining and real-time PCR for 16S rRNA, ureA, and 23S rRNA for H. pylori genes on all specimens. Results All controls were positive for H. pylori on all PCR assays and immunohistochemical staining. Regarding the 52 initially negative biopsies, all PCR tests were significantly more sensitive than immunohistochemical staining (p<0.01). Sensitivity and specificity were 55% and 80% for 16S rRNA PCR, 43% and 90% for ureA PCR, 41% and 80% for 23S rRNA PCR, and 7% and 100% for immunohistochemical staining, respectively. Combined analysis of PCR assays for two genes were significantly more sensitive than ureA or 23S rRNA PCR tests alone (p<0.05) and marginally better than 16S rRNA PCR alone. The best combination was 16S rRNA+ureA, with a sensitivity of 64% and a specificity of 80%. Conclusions Real-time PCR improves the detection of H. pylori infection in histology-negative formalin-fixed paraffin-embedded biopsy samples obtained during PUB episodes. The low reported prevalence of H. pylori in PUB may be due to the failure of conventional tests to detect infection. PMID:21625499

  10. Sources of PCR-induced distortions in high-throughput sequencing data sets

    PubMed Central

    Kebschull, Justus M.; Zador, Anthony M.

    2015-01-01

    PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

  11. DEVELOPMENT OF MULTIPLEX REAL-TIME RT-PCR AS A DIAGNOSTIC TOOL FOR AVIAN INFLUENZA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex real-time RT-PCR (RRT-PCR) assay for the simultaneous detection of the H5 and H7 hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro tran...

  12. An integrated microfluidic system for bovine DNA purification and digital PCR detection.

    PubMed

    Tian, Qingchang; Mu, Ying; Xu, Yanan; Song, Qi; Yu, Bingwen; Ma, Congcong; Jin, Wei; Jin, Qinhan

    2015-12-15

    In this paper, we described an integrated modularized microfluidic system that contained two distinct functional modules, one for nucleic acids (NA) extraction and the other for digital PCR (dPCR), allowing for detecting the bovine DNA in ovine tissue. PMID:26364950

  13. Quantitative Analysis of Copy Number Variants Based on Real-Time LightCycler PCR

    PubMed Central

    Ma, Lijiang; Chung, Wendy K.

    2014-01-01

    Quantitative real-time PCR is PCR visualized in real time by the use of fluorescent or intercalating dyes used to measure gene expression or gene quantification including including contiguous gene deletions or duplications. A simple method is described to quantify DNA copy number from human samples. PMID:24510682

  14. Straightforward and sensitive RT-qPCR based gene expression analysis of FFPE samples

    PubMed Central

    Zeka, Fjoralba; Vanderheyden, Katrien; De Smet, Els; Cuvelier, Claude A.; Mestdagh, Pieter; Vandesompele, Jo

    2016-01-01

    Fragmented RNA from formalin-fixed paraffin-embedded (FFPE) tissue is a known obstacle to gene expression analysis. In this study, the impact of RNA integrity, gene-specific reverse transcription and targeted cDNA preamplification was quantified in terms of reverse transcription polymerase chain reaction (RT-qPCR) sensitivity by measuring 48 protein coding genes on eight duplicate cultured cancer cell pellet FFPE samples and twenty cancer tissue FFPE samples. More intact RNA modestly increased gene detection sensitivity by 1.6 fold (earlier detection by 0.7 PCR cycles, 95% CI = 0.593–0.850). Application of gene-specific priming instead of whole transcriptome priming during reverse transcription further improved RT-qPCR sensitivity by a considerable 4.0 fold increase (earlier detection by 2.0 PCR cycles, 95% CI = 1.73–2.32). Targeted cDNA preamplification resulted in the strongest increase of RT-qPCR sensitivity and enabled earlier detection by an average of 172.4 fold (7.43 PCR cycles, 95% CI = 6.83–7.05). We conclude that gene-specific reverse transcription and targeted cDNA preamplification are adequate methods for accurate and sensitive RT-qPCR based gene expression analysis of FFPE material. The presented methods do not involve expensive or complex procedures and can be easily implemented in any routine RT-qPCR practice. PMID:26898768

  15. A PCR-ELISA for detecting Shiga toxin-producing Escherichia coli.

    PubMed

    Ge, Beilei; Zhao, Shaohua; Hall, Robert; Meng, Jianghong

    2002-03-01

    A sensitive and specific PCR-ELISA was developed to detect Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in food. The assay was based on the incorporation of digoxigenin-labeled dUTP and a biotin-labeled primer specific for Shiga toxin genes during PCR amplification. The labeled PCR products were bound to streptavidin-coated wells of a microtiter plate and detected by an ELISA. The specificity of the PCR was determined using 39 bacterial strains, including STEC, enteropathogenic E. coli, E. coli K12, and Salmonella. All of the STEC strains were positive, and non-STEC organisms were negative. The ELISA detecting system was able to increase the sensitivity of the PCR assay by up to 100-fold, compared with a conventional gel electrophoresis. The detection limit of the PCR-ELISA was 0.1-10 CFU dependent upon STEC serotypes, and genotypes of Shiga toxins. With the aid of a simple DNA extraction system, PrepMan, the PCR-ELISA was able to detect ca. 10(5) CFU of STEC per gram of ground beef without any culture enrichment. The entire procedure took about 6 h. Because of its microtiter plate format, PCR-ELISA is particularly suitable for large-scale screening and compatible with future automation. PMID:11909738

  16. One-PCR-tube approach for in situ DNA isolation and detection.

    PubMed

    Huang, Xin; Hou, Lihua; Xu, Xiaohe; Chen, Hongjun; Ji, Haifeng; Zhu, Shuifang

    2011-10-21

    Traditional real-time polymerase chain reaction (PCR) requires a purified DNA sample for PCR amplification and detection. This requires PCR tests be conducted in clean laboratories, and limits its applications for field tests. This work developed a method that can carry out DNA purification, amplification and detection in a single PCR tube. The polypropylene PCR tube was first treated with chromic acid and peptide nucleic acids (PNA) as DNA-capturer were immobilized on the internal surface of the tube. Cauliflower mosaic virus 35S (CaMV-35S) promoter in the crude extract was hybridized with the PNA on the tube surface, and the inhibitors, interfering agents and irrelevant DNA in the crude extract were effectively removed by rinsing with buffer solutions. The tube that has captured the target DNA can be used for the following real-time PCR (RT-PCR). By using this approach, the detection of less than 2500 copies of 35S plasmids in a complex sample could be completed within 3 hours. Chocolate samples were tested for real sample analysis, and 35S plasmids in genetically modified chocolate samples have been successfully identified with this method in situ. The novel One-PCR-tube method is competitive for commercial kits with the same time and simpler operation procedure. This method may be widely used for identifying food that contains modified DNA and specific pathogens in the field. PMID:21879029

  17. Integrating PCR Theory and Bioinformatics into a Research-oriented Primer Design Exercise

    ERIC Educational Resources Information Center

    Robertson, Amber L.; Phillips, Allison R.

    2008-01-01

    Polymerase chain reaction (PCR) is a conceptually difficult technique that embodies many fundamental biological processes. Traditionally, students have struggled to analyze PCR results due to an incomplete understanding of the biological concepts (theory) of DNA replication and strand complementarity. Here we describe the design of a novel…

  18. Designing Polymerase Chain Reaction (PCR) Primer Multiplexes in the Forensic Laboratory

    ERIC Educational Resources Information Center

    Elkins, Kelly M.

    2011-01-01

    The polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry, molecular biology, and forensic laboratory courses as reagents and thermocyclers have become more affordable for institutions. Typically, instructors design PCR primers to amplify the region of interest and the students prepare their samples for…

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

    EPA Science Inventory

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

  20. 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...

  1. Molecular detection and identification of intimin alleles in pathogenic Escherichia coli by multiplex PCR.

    PubMed

    Reid, S D; Betting, D J; Whittam, T S

    1999-08-01

    A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenic Escherichia coli. The method was tested on 87 strains representing the diarrheagenic E. coli clones. The results show that the PCR assay accurately detects eae and resolves alleles encoding the alpha, beta, and gamma intimin variants. PMID:10405431

  2. Molecular Detection and Identification of Intimin Alleles in Pathogenic Escherichia coli by Multiplex PCR

    PubMed Central

    Reid, Sean D.; Betting, David J.; Whittam, Thomas S.

    1999-01-01

    A multiplex PCR was designed to detect the eae gene and simultaneously identify specific alleles in pathogenic Escherichia coli. The method was tested on 87 strains representing the diarrheagenic E. coli clones. The results show that the PCR assay accurately detects eae and resolves alleles encoding the α, β, and γ intimin variants. PMID:10405431

  3. Extensive Recombination Due to Heteroduplexes Generates Large Amounts of Artificial Gene Fragments during PCR

    PubMed Central

    Liu, Jia; Song, Hongshuo; Liu, Donglai; Zuo, Tao; Lu, Fengmin; Zhuang, Hui; Gao, Feng

    2014-01-01

    Artificial recombinants can be generated during PCR when more than two genetically distinct templates coexist in a single PCR reaction. These recombinant amplicons can lead to the false interpretation of genetic diversity and incorrect identification of biological phenotypes that do not exist in vivo. We investigated how recombination between 2 or 35 genetically distinct HIV-1 genomes was affected by different PCR conditions using the parallel allele-specific sequencing (PASS) assay and the next generation sequencing method. In a standard PCR condition, about 40% of amplicons in a PCR reaction were recombinants. The high recombination frequency could be significantly reduced if the number of amplicons in a PCR reaction was below a threshold of 1013–1014 using low thermal cycles, fewer input templates, and longer extension time. Heteroduplexes (each DNA strand from a distinct template) were present at a large proportion in the PCR products when more thermal cycles, more templates, and shorter extension time were used. Importantly, the majority of recombinants were identified in heteroduplexes, indicating that the recombinants were mainly generated through heteroduplexes. Since prematurely terminated extension fragments can form heteroduplexes by annealing to different templates during PCR amplification, recombination has a better chance to occur with samples containing different genomes when the number of amplicons accumulate over the threshold. New technologies are warranted to accurately characterize complex quasispecies gene populations. PMID:25211143

  4. Development of a real-time PCR assay for detection and quantification of Anaplasma ovis infection.

    PubMed

    Chi, Q; Liu, Z; Li, Y; Yang, J; Chen, Z; Yue, C; Luo, J; Yin, H

    2013-11-01

    Anaplasma ovis is a tick-borne intra-erythrocytic rickettsial pathogen of small ruminants. Real-time PCR possesses merits of rapidity, accuracy, reliability, automation and ease of standardization, but has not been used for detection of A. ovis, to the best of our knowledge. In this study, a real-time PCR assay was developed for detection and quantification of A. ovis. Species-specific primers and TaqMan probe were designed based on the gltA gene. No cross-reactions were observed with Anaplasma marginale, Anaplasma bovis, Anaplasma phagocytophilum, Borrelia burgdorferi s. l., Chlamydia psittaci, Mycoplasma mycoides, Theileria luwenshuni and Babesia sp. Xinjiang isolate. Analytic sensitivity results revealed that real-time PCR could detect as few as 10 copies of the gltA gene. The performance of real-time PCR was assessed by testing 254 blood samples from goats and comparing with the results from conventional PCR. This demonstrated that the real-time PCR assay was significantly more sensitive than conventional PCR. Our results indicated that real-time PCR is a useful approach for detecting A. ovis infections and has potential as an alternative tool for ecological and epidemiological surveillance of ovine anaplasmosis. PMID:24589111

  5. Detection of Toxoplasma gondii oocysts in water sample concentrates by real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR techniques in combination with conventional parasite concentration procedures have potential for sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were compared for detection of T. gondii tachyz...

  6. PCR-based Detection of Spiroplasma Citri Associated with Citrus Stubborn Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improvements in polymerase chain reaction (PCR) detection of Spiroplasma citri, the causal agent of citrus stubborn disease, made PCR more reliable than culturing for S. citri detection. Primer sequences from the P89 putative adhesin gene, which is present on a plasmid as well as in the S. citri gen...

  7. 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

  8. PCR Conditions for 16S Primers for Analysis of Microbes in the Colon of Rats

    PubMed Central

    Camacho, H.; Tuero, A. D.; Bacardí, D.; Palenzuela, D. O.; Aguilera, A.; Silva, J. A.; Estrada, R.; Gell, O.; Suárez, J.; Ancizar, J.; Brown, E.; Colarte, A. B.; Castro, J.; Novoa, L. I.

    2016-01-01

    The study of the composition of the intestinal flora is important to the health of the host, playing a key role in maintaining intestinal homeostasis and the evolution of the immune system. For these studies, various universal primers of the 16S rDNA gene are used in microbial taxonomy. Here, we report an evaluation of 5 universal primers to explore the presence of microbial DNA in colon biopsies preserved in RNAlater solution. The DNA extracted was used for the amplification of PCR products containing the variable (V) regions of the microbial 16S rDNA gene. The PCR products were studied by restriction fragment length polymorphism (RFLP) analysis and DNA sequence, whose percent of homology with microbial sequences reported in GenBank was verified using bioinformatics tools. The presence of microbes in the colon of rats was quantified by the quantitative PCR (qPCR) technique. We obtained microbial DNA from rat, useful for PCR analysis with the universal primers for the bacteria 16S rDNA. The sequences of PCR products obtained from a colon biopsy of the animal showed homology with the classes bacilli (Lactobacillus spp) and proteobacteria, normally represented in the colon of rats. The proposed methodology allowed the attainment of DNA of bacteria with the quality and integrity for use in qPCR, sequencing, and PCR-RFLP analysis. The selected universal primers provided knowledge of the abundance of microorganisms and the formation of a preliminary test of bacterial diversity in rat colon biopsies. PMID:27382362

  9. Sources of PCR-induced distortions in high-throughput sequencing data sets.

    PubMed

    Kebschull, Justus M; Zador, Anthony M

    2015-12-01

    PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error-bias, stochasticity, template switches and polymerase errors-on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

  10. Advantages and disadvantages of using PCR techniques to characterize transgenic plants.

    PubMed

    Wassenegger, M

    2001-01-01

    The polymerase chain reaction (PCR) revolutionized molecular biology to a similar extent as the discovery of plasmids and restriction endonucleases. However, there are some limitations to the use of PCR. Transgenic plants containing potato spindle tuber viroid (PSTVd) cDNA constructs, demonstrated to become de novo methylated upon PSTVd infection, represent a good example to illustrate the advantages of PCR. PSTVd is a 359 nt long autonomously replicating plant pathogenic RNA where all of its enzymatic requirements are entirely provided by the host cell. In addition, viroids that propagate without a DNA intermediate barely tolerate nucleotide substitutions of their RNA genome without losing infectivity. PCR is the method of choice to characterize the sequence context of genome-integrated viroid cDNA or of reverse transcribed PSTVd RNA, and can hardly be replaced by any alternative procedure. Furthermore, the precise examination of DNA methylation patterns (genomic sequencing) is entirely dependent on PCR. In contrast, the use of PCR is critical for the determination of copy number and arrangement of transgene constructs. Here, the advantages and disadvantages of PCR are discussed and protocols for PCR amplification of cDNA, genomic DNA, and bisulfite-treated DNA from transgenic plants are presented. PMID:11280933

  11. Real time RT-PCR for the H5 HA subtype

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (RRT-PCR) for type A influenza detection is widely used with subsequent tests for subtype identification. Due to the importance of the H5 HA subtype, rapid and sensitive detection of the H5 HA subtype is particularly useful due to the importance of the recent Asian H5N1 HPAI viruse...

  12. PCR detection and quantitation of predominant anaerobic bacteria in human and animal fecal samples

    SciTech Connect

    Wang, Rong-Fu; Cao, Wei-Wen; Cerniglia, C.E.

    1996-04-01

    PCR procedures based on 16S rRNA genen sequence specific for 12 anaerobic bacteria that predominate in the human intestinal tract were developed and used for quantitative detection of these species in human feces and animal feces. The reported PCR procedure including the fecal sample preparation method is simplified and rapid and eliminates the DNA isolation steps.

  13. 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...

  14. PMA treatment is an effective means to reduce false positive PCR testing results

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Traditional and real time PCR are widely used in detecting bacterial pathogens in various food matrix and environmental samples. Sometimes a positive detection using PCR can not be confirmed by subsequent culture isolation of the targeted pathogen, resulting in a potential “false positive.” False po...

  15. Nested PCR for ultrasensitive detection of the potato ring rot bacterium, Clavibacter michiganensis subsp. sepedonicus.

    PubMed Central

    Lee, I M; Bartoszyk, I M; Gundersen, D E; Mogen, B; Davis, R E

    1997-01-01

    Oligonucleotide primers derived from sequences of the 16S rRNA gene (CMR16F1, CMR16R1, CMR16F2, and CMR16R2) and insertion element IS1121 of Clavibacter michiganensis subsp. sepedonicus (CMSIF1, CMSIR1, CMSIF2, and CMISR2) were used in nested PCR to detect the potato ring rot bacterium C. michiganensis subsp. sepedonicus. Nested PCR with primer pair CMSIF1-CMSIR1 followed by primer pair CMSIF2-CMSIR2 specifically detected C. michiganensis subsp. sepedonicus, while nested PCR with CMR16F1-CMR16R1 followed by CMR16F2-CMR16R2 detected C. michiganensis subsp. sepedonicus and the other C. michiganensis subspecies. In the latter case, C. michiganensis subsp. sepedonicus can be differentiated from the other subspecies by restriction fragment length polymorphism (RFLP) analyses of the nested PCR products (16S rDNA sequences). The nested PCR assays developed in this work allow ultrasensitive detection of very low titers of C. michiganensis subsp. sepedonicus which may be present in symptomiess potato plants or tubers and which cannot be readily detected by direct PCR (single PCR amplification). RFLP analysis of PCR products provides for an unambiguous confirmation of the identify of C. michiganensis subsp. sepedonicus. PMID:9212412

  16. Improved detection of episomal Banana streak viruses by multiplex immunocapture PCR.

    PubMed

    Le Provost, Grégoire; Iskra-Caruana, Marie-Line; Acina, Isabelle; Teycheney, Pierre-Yves

    2006-10-01

    Banana streak viruses (BSV) are currently the main viral constraint to Musa germplasm movement, genetic improvement and mass propagation. Therefore, it is necessary to develop and implement BSV detection strategies that are both reliable and sensitive, such as PCR-based techniques. Unfortunately, BSV endogenous pararetrovirus sequences (BSV EPRVs) are present in the genome of Musa balbisiana. They interfere with PCR-based detection of episomal BSV in infected banana and plantain, such as immunocapture PCR. Therefore, a multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of BSV. Musa sequence tagged microsatellite site (STMS) primers were selected and used in combination with BSV species-specific primers in order to monitor possible contamination by Musa genomic DNA, using multiplex PCR. Furthermore, immunocapture conditions were optimized in order to prevent Musa DNA from interfering with episomal BSV DNA during the PCR step. This improved detection method successfully allowed the accurate, specific and sensitive detection of episomal DNA only from distinct BSV species. Its implementation should benefit PCR-based detection of viruses for which homologous sequences are present in the genome of their hosts, including transgenic plants expressing viral sequences. PMID:16857272

  17. Development of a multiplex PCR assay to detect gastroenteric pathogens in the feces of Mexican children.

    PubMed

    Tolentino-Ruiz, R; Montoya-Varela, D; García-Espitia, M; Salas-Benito, M; Gutiérrez-Escolano, A; Gómez-García, C; Figueroa-Arredondo, P; Salas-Benito, J; De Nova-Ocampo, M

    2012-10-01

    Acute gastroenteritis (AGE) is a major cause of childhood morbidity and mortality worldwide; the etiology of AGE includes viruses, bacteria, and parasites. A multiplex PCR assay to simultaneously identify human Astrovirus (HAstV), Calicivirus (HuCVs), Entamoeba histolytica (E. histolytica), and enteroinvasive Escherichia coli (EIEC) in stool samples is described. A total of 103 samples were individually analyzed by ELISA (enzyme-linked immunosorbent assays) and RT-PCR/PCR. HAstV and HuCVs were detected in four out of 103 samples (3.8 %) by RT-PCR, but ELISAs found only one sample as positive for HuCVs (2.5 %). E. histolytica was identified in two out of 19 samples (10.5 %) and EIEC in 13 out of 20 samples (70 %) by PCR, and all PCR products were sequenced to verify their identities. Our multiplex PCR results demonstrate the simultaneous amplification of different pathogens such as HAstV, EIEC, and E. histolytica in the same reaction, though the HuCVs signal was weak in every replicate. Regardless, this multiplex PCR protocol represents a novel tool for the identification of distinct pathogens and may provide support for the diagnosis of AGE in children. PMID:22711331

  18. Predicting Salmonella enterica subsp. enterica Serotypes by Repetitive Extragenic Palindromic Sequence-Based PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The DiversiLabTM System, which employs repetitive extragenic palindromic sequence-based PCR (rep-PCR) to genotype microorganisms, was evaluated as a method to predict the serotype of Salmonella isolates. Two hundred and thirty-three Salmonella isolates belonging to 14 frequently isolated serotypes f...

  19. Sequence polymorphism can produce serious artifacts in real-time PCR assays: lessons from Pacific oysters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Since it was first described in the mid-1990s, quantitative real time PCR (Q-PCR) has been widely used in many fields of biomedical research and molecular diagnostics. This method is routinely used to validate whole transcriptome analyses such as DNA microarrays, suppressive subtractive hybridizati...

  20. A High Malaria Prevalence Identified by PCR among Patients with Acute Undifferentiated Fever in India

    PubMed Central

    Haanshuus, Christel Gill; Chandy, Sara; Manoharan, Anand; Vivek, Rosario; Mathai, Dilip; Xena, Deepika; Singh, Ashita; Langeland, Nina; Blomberg, Bjørn; Vasanthan, George; Sitaram, Usha; Appasamy, Jonathan; Nesaraj, Joel; Henry, Anil; Patil, Suvarna; Alvarez-Uria, Gerardo; Armstrong, Lois; Mørch, Kristine

    2016-01-01

    Background Approximately one million malaria cases were reported in India in 2015, based on microscopy. This study aims to assess the malaria prevalence among hospitalised fever patients in India identified by PCR, and to evaluate the performance of routine diagnostic methods. Methods During June 2011-December 2012, patients admitted with acute undifferentiated fever to seven secondary level community hospitals in Assam (Tezpur), Bihar (Raxaul), Chhattisgarh (Mungeli), Maharashtra (Ratnagiri), Andhra Pradesh (Anantapur) and Tamil Nadu (Oddanchatram and Ambur) were included. The malaria prevalence was assessed by polymerase chain reaction (PCR), routine microscopy, and a rapid diagnostic test (RDT) with PCR as a reference method. Results The malaria prevalence by PCR was 19% (268/1412) ranging from 6% (Oddanchatram, South India) to 35% (Ratnagiri, West India). Among malaria positive patients P. falciparum single infection was detected in 46%, while 38% had P. vivax, 11% mixed infections with P. falciparum and P. vivax, and 5% P. malariae. Compared to PCR, microscopy had sensitivity of 29% and specificity of 98%, while the RDT had sensitivity of 24% and specificity of 99%. Conclusions High malaria prevalence was identified by PCR in this cohort. Routine diagnostic methods had low sensitivity compared to PCR. The results suggest that malaria is underdiagnosed in rural India. However, low parasitaemia controlled by immunity may constitute a proportion of PCR positive cases, which calls for awareness of the fact that other pathogens could be responsible for the febrile disease in submicroscopic malaria. PMID:27389396

  1. Comparison of PCR and Plaque Assay for Detection and Enumeration of Coliphage in Polluted Marine Waters

    PubMed Central

    Rose, J. B.; Zhou, X.; Griffin, D. W.; Paul, J. H.

    1997-01-01

    A total of 68 marine samples from various sites impacted by sewage and storm waters were analyzed by both the plaque assay and a reverse transcriptase (RT) PCR technique for F(sup+)-specific coliphage. The coliphage levels detected by the plaque assay averaged 1.90 x 10(sup4) PFU/100.0 ml. Using a most probable number (MPN) PCR approach, the levels averaged 2.40 x 10(sup6) MPN-PCR units/100.0 ml. Two samples were positive by RT-PCR but negative by plaque assay, and 12 samples were positive by plaque assay but negative by RT-PCR (levels lower than 11.00 PFU/100.0 ml). The host system used for the plaque assay may detect somatic coliphage in addition to the F(sup+)-specific coliphage. When it is used as an indicator of pollution, contamination may be missed with more restrictive systems. The difference in results may be due to the sensitivity, specificity, or inhibition of RT-PCR in marine samples. This study provides information on quantifying PCR results by an MPN method and insights into interpretation of PCR data for detection of viruses in marine environments. PMID:16535737

  2. 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...

  3. LONG-PCR AS A TOOL FOR DETECTING REARRANGEMENTS OF ARTHROPOD MTDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rearrangements of the mitochondrial DNA gene order have been used to help define the pattern of evolutionary divergence in arthropod taxa. We have employed a combination of highly conserved insect-based PCR primers with long-PCR to survey fourteen non-insect arthropods for mitochondrial gene rearra...

  4. 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...

  5. Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism of gap Gene

    PubMed Central

    Yugueros, Javier; Temprano, Alejandro; Sánchez, María; Luengo, José María; Naharro, Germán

    2001-01-01

    Oligonucleotide primers specific for the Staphylococcus aureus gap gene were previously designed to identify 12 Staphylococcus spp. by PCR. In the present study, AluI digestion of PCR-generated products rendered distinctive restriction fragment length polymorphism patterns that allowed 24 Staphylococcus spp. to be identified with high specificity. PMID:11574593

  6. Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

    PubMed Central

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

    2005-01-01

    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 × 102 CFU/cm2. PMID:15812058

  7. Hypervirulent Clostridium difficile PCR-Ribotypes Exhibit Resistance to Widely Used Disinfectants

    PubMed Central

    Dawson, Lisa F.; Valiente, Esmeralda; Donahue, Elizabeth H.; Birchenough, George; Wren, Brendan W.

    2011-01-01

    The increased prevalence of Clostridium difficile infection (CDI) has coincided with enhanced transmissibility and severity of disease, which is often linked to two distinct clonal lineages designated PCR-ribotype 027 and 017 responsible for CDI outbreaks in the USA, Europe and Asia. We assessed sporulation and susceptibility of three PCR-ribotypes; 012, 017 and 027 to four classes of disinfectants; chlorine releasing agents (CRAs), peroxygens, quaternary ammonium compounds (QAC) and biguanides. The 017 PCR-ribotype, showed the highest sporulation frequency under these test conditions. The oxidizing biocides and CRAs were the most efficacious in decontamination of C. difficile vegetative cells and spores, the efficacy of the CRAs were concentration dependent irrespective of PCR-ribotype. However, there were differences observed in the susceptibility of the PCR-ribotypes, independent of the concentrations tested for Virkon®, Newgenn®, Proceine 40® and Hibiscrub®. Whereas, for Steri7® and Biocleanse® the difference observed between the disinfectants were dependent on both PCR-ribotype and concentration. The oxidizing agent Perasafe® was consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five Log10 reduction in spore titre. The PCR-ribotype and concentration dependent differences in the efficacy of the disinfectants in this study indicate that disinfectant choice is a factor for llimiting the survival and transmission of C. difficile spores in healthcare settings. PMID:22039420

  8. Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments

    PubMed Central

    2012-01-01

    Background Multiplex PCR has been successfully applied in many areas since it was first reported in 1988; however, it suffers from poor universality. Results A novel method called Universal Multiplex PCR (UM-PCR) was created, which simultaneously amplifies multiple target fragments from genomic DNA. The method has two steps. First, the universal adapter-F and universal adapter-R are connected to the forward primers and the reverse primers, respectively. Hairpin structures and cross dimers of five pairs of adapter-primers are detected. Second, UM-PCR amplification is implemented using a novel PCR procedure termed “Two Rounds Mode” (three and 28–32 cycles). The first round (the first three cycles) is named the “One by One Annealing Round”. The second round (28–32 cycles) combines annealing with extension. In the first two cycles of the first round, primers only amplify the specific templates; there are no templates for the universal adapters. The templates of universal adapters begin to be synthesized from the second cycle of the first round, and universal adapters and primers commence full amplification from the third cycle of the first round. Conclusions UM-PCR greatly improves the universality of multiplex PCR. UM-PCR could rapidly detect the genetic purity of maize seeds. In addition, it could be applied in other areas, such as analysis of polymorphisms, quantitative assays and identifications of species. PMID:22894545

  9. Examining Interactions Between Legumes and Aphamonyces euteiches with Real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time PCR has many applications in the study of host-pathogen interactions including improving the resolution of disease screening programs in selecting highly resistant plants. Identifying the earliest time in the disease process that real-time PCR can be used to reliably identify resistant gen...

  10. An improved multiplex IC-RT-PCR assay distinguishes nine strains of Potato virus Y

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex RT-PCR assay was previously developed to identify a group of PVY isolates with unusual recombinant structures, e.g. PVYNTN-NW and SYR-III, and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended cons...

  11. 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.

  12. Evaluation of reference genes for quantitative RT-PCR in Lolium perenne

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Quantitative real-time RT-PCR provides an important tool for analyzing gene expression if proper internal standards are used. The aim of this study was to identify and evaluate reference genes for use in real-time quantitative RT-PCR in perennial ryegrass (Lolium perenne L.) during plant developmen...

  13. PCR-based Detection of Spiroplasma citri Associated with Citrus Stubborn Disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR detection of Spiroplasma citri, the causal agent of citrus stubborn disease, was improved using primers based on sequences of the P89 adhesin gene and the P58 putative adhesin multigene of S. citri. PCR was compared with isolation by culturing for detection of S. citri in two 20 A citrus orchar...

  14. A Web-Based Adaptive Tutor to Teach PCR Primer Design

    ERIC Educational Resources Information Center

    van Seters, Janneke R.; Wellink, Joan; Tramper, Johannes; Goedhart, Martin J.; Ossevoort, Miriam A.

    2012-01-01

    When students have varying prior knowledge, personalized instruction is desirable. One way to personalize instruction is by using adaptive e-learning to offer training of varying complexity. In this study, we developed a web-based adaptive tutor to teach PCR primer design: the PCR Tutor. We used part of the Taxonomy of Educational Objectives (the…

  15. High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

    PubMed Central

    2011-01-01

    Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ∼2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100 000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics. PMID:22035192

  16. Sequence characterisation of deletion breakpoints in the dystrophin gene by PCR

    SciTech Connect

    Abbs, S.; Sandhu, S.; Bobrow, M.

    1994-09-01

    Partial deletions of the dystrophin gene account for 65% of cases of Duchenne muscular dystrophy. A high proportion of these structural changes are generated by new mutational events, and lie predominantly within two `hotspot` regions, yet the underlying reasons for this are not known. We are characterizing and sequencing the regions surrounding deletion breakpoints in order to: (i) investigate the mechanisms of deletion mutation, and (ii) enable the design of PCR assays to specifically amplify mutant and normal sequences, allowing us to search for the presence of somatic mosaicism in appropriate family members. Using this approach we have been able to demonstrate the presence of somatic mosaicism in a maternal grandfather of a DMD-affected male, deleted for exons 49-50. Three deletions, namely of exons 48-49, 49-50, and 50, have been characterized using a PCR approach that avoids any cloning procedures. Breakpoints were initially localized to within regions of a few kilobases using Southern blot restriction analyses with exon-specific probes and PCR amplification of exonic and intronic loci. Sequencing was performed directly on PCR products: (i) mutant sequences were obtained from long-range or inverse-PCR across the deletion junction fragments, and (ii) normal sequences were obtained from the products of standard PCR, vectorette PCR, or inverse-PCR performed on YACs. Further characterization of intronic sequences will allow us to amplify and sequence across other deletion breakpoints and increase our knowledge of the mechanisms of mutation in the dystophin gene.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  18. Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding

    PubMed Central

    Best, Katharine; Oakes, Theres; Heather, James M.; Shawe-Taylor, John; Chain, Benny

    2015-01-01

    The polymerase chain reaction (PCR) is one of the most widely used techniques in molecular biology. In combination with High Throughput Sequencing (HTS), PCR is widely used to quantify transcript abundance for RNA-seq, and in the context of analysis of T and B cell receptor repertoires. In this study, we combine DNA barcoding with HTS to quantify PCR output from individual target molecules. We develop computational tools that simulate both the PCR branching process itself, and the subsequent subsampling which typically occurs during HTS sequencing. We explore the influence of different types of heterogeneity on sequencing output, and compare them to experimental results where the efficiency of amplification is measured by barcodes uniquely identifying each molecule of starting template. Our results demonstrate that the PCR process introduces substantial amplification heterogeneity, independent of primer sequence and bulk experimental conditions. This heterogeneity can be attributed both to inherited differences between different template DNA molecules, and the inherent stochasticity of the PCR process. The results demonstrate that PCR heterogeneity arises even when reaction and substrate conditions are kept as constant as possible, and therefore single molecule barcoding is essential in order to derive reproducible quantitative results from any protocol combining PCR with HTS. PMID:26459131

  19. Use PCR and a Single Hair To Produce a "DNA Fingerprint."

    ERIC Educational Resources Information Center

    Campbell, A. Malcolm; And Others

    1997-01-01

    Presents a laboratory procedure that involves students extracting their own DNA from a single hair follicle, using the polymerase chain reaction (PCR) to amplify a polymorphic locus, performing electrophoresis on the PCR products on an agarose gel, and visualizing the alleles to generate a "DNA fingerprint." Discusses theoretical background,…

  20. Isolation of Listeria monocytogenes from challenged turkeys using real time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have hypothesized that stress-induced subclinical infection of turkeys with L. monocytogenes (Lm) may be a source of processing plant contamination. The objective of this work was to compare conventional culture methods and Taqman® real time PCR (RTi PCR) for isolation of Lm from joints of challe...

  1. New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

    PubMed Central

    Gueimonde, Miguel; Tölkkö, Satu; Korpimäki, Teemu; Salminen, Seppo

    2004-01-01

    The application of a real-time quantitative PCR method (5′ nuclease assay), based on the use of a probe labeled at its 5′ end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 × 104 cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces. PMID:15240297

  2. Identification of Erwinia species isolated from apples and pears by differential PCR.

    PubMed

    Gehring, I; Geider, K

    2012-04-01

    Many pathogenic and epiphytic bacteria isolated from apples and pears belong to the genus Erwinia; these include the species E. amylovora, E. pyrifoliae, E. billingiae, E. persicina, E. rhapontici and E. tasmaniensis. Identification and classification of freshly isolated bacterial species often requires tedious taxonomic procedures. To facilitate routine identification of Erwinia species, we have developed a PCR method based on species-specific oligonucleotides (SSOs) from the sequences of the housekeeping genes recA and gpd. Using species-specific primers that we report here, differentiation was done with conventional PCR (cPCR) and quantitative PCR (qPCR) applying two consecutive primer annealing temperatures. The specificity of the primers depends on terminal Single Nucleotide Polymorphisms (SNPs) that are characteristic for the target species. These PCR assays enabled us to distinguish eight Erwinia species, as well as to identify new Erwinia isolates from plant surfaces. When performed with mixed bacterial cultures, they only detected a single target species. This method is a novel approach to classify strains within the genus Erwinia by PCR and it can be used to confirm other diagnostic data, especially when specific PCR detection methods are not already available. The method may be applied to classify species within other bacterial genera. PMID:22330936

  3. Assessing the Validity of Diagnostic Quantitative PCR Assays for Phakopsora pachyrhizi and P. meibomiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There are 123 confirmed species in the genus Phakopsora worldwide, with 19 species reported in the continental United States. In 2002, a quantitative PCR (qPCR) diagnostic assay was developed by Frederick et al. that has been used for detecting Phakopsora pachyrhizi in spore trapping studies. Based ...

  4. High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites.

    PubMed

    Taniuchi, Mami; Verweij, Jaco J; Noor, Zannatun; Sobuz, Shihab U; Lieshout, Lisette van; Petri, William A; Haque, Rashidul; Houpt, Eric R

    2011-02-01

    Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites-Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis-were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites. PMID:21292910

  5. 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. PMID:24957236

  6. 16S rRNA region based PCR protocol for identification and subtyping of Parvimonas micra

    PubMed Central

    Ota-Tsuzuki, C.; Brunheira, A.T.P.; Mayer, M.P.A.

    2008-01-01

    The present study established a PCR protocol in order to identify Parvimonas micra and to evaluate the intra-species diversity by PCR-RFLP of 16S rRNA partial sequence. The data indicated that the protocol was able to identify this species which could be clustered in five genotypes. PMID:24031274

  7. Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

    PubMed Central

    Luo, Xiao-Feng; Jiao, Jian-Hua; Zhang, Wen-Yue; Pu, Han-Ming; Qu, Bao-Jin; Yang, Bing-Ya; Hou, Min; Ji, Min-Jun

    2016-01-01

    AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher

  8. A multiplex PCR for the simultaneous detection of Tenacibaculum maritimum and Edwardsiella tarda in aquaculture.

    PubMed

    Castro, Nuria; Toranzo, Alicia E; Magariños, Beatriz

    2014-06-01

    A specific and sensitive multiplex PCR (mPCR) method was developed as a useful tool for the simultaneous detection of two important flatfish pathogens in marine aquaculture, Tenacibaculum maritimum and Edwardsiella tarda. In fish tissues, the average detection limit for these mPCR-amplified organisms was 2 × 10 ⁵ ± 0.2 CFU/g and 4 × 10 ⁵ ± 0.3 CFU/g, respectively. These values are similar or even lower than those previously obtained using the corresponding single PCR. Moreover, mPCR did not produce any nonspecific amplification products when tested against 36 taxonomically related and unrelated strains belonging to 33 different bacterial species. Large amounts of DNA from one of the target bacterial species in the presence of low amounts from the other did not have a significant effect on the amplification sensitivity of the latter. PMID:26418855

  9. Species-specific PCR for the identification of goat cashmere and sheep wool.

    PubMed

    Geng, Rong-Qing

    2015-02-01

    In order to establish rapid and species-specific method of goat cashmere and sheep wool identification, a polymerase chain reaction using specific primer pairs targeting mitochondrial D-loop was developed. The goat specific primers yielded a 294 bp PCR fragment and the sheep specific primers yielded three PCR fragments of which only the 404 bp fragment was found highly diagnostic. The specificity and reliability of the developed species-specific PCR assay was validated by considering as many as 500 cashmere and wool samples. The developed species-specific PCR was found effective in detecting mixed samples of cashmere and wool precisely with the relative content over 9.09%. The species-specific PCR method proved to be low cost, fast, easy and reliable alternative to determine the addition of sheep wool in goat cashmere. PMID:25444936

  10. PCR for detection of Clostridium botulinum type C in avian and environmental samples.

    PubMed

    Franciosa, G; Fenicia, L; Caldiani, C; Aureli, P

    1996-04-01

    A PCR was developed and applied for the detection of Clostridium botulinum type C in 18 avian and environmental samples collected during an outbreak of avian botulism, and the results were compared with those obtained by conventional methodologies based on the mouse bioassay. PCR and mouse bioassay results compared well (100%) after the enrichment of samples, but PCR results directly indicated the presence of this microorganism in six samples, while only one of these contained the type C botulinal neurotoxin before enrichment. The PCR assay was sensitive (limit of detection between 15 and 15 x 10(3) spores per PCR), specific (no amplification products were obtained with other clostridia), and rapid, since sonicated and heated samples provided enough template for amplification without any DNA purification. Eleven isolates of C. botulinum type C were recovered from mallards (Anas platyrhynchos), grey herons (Ardea cinerea), and mud during investigation of this outbreak. PMID:8815101

  11. PCR for detection of Clostridium botulinum type C in avian and environmental samples.

    PubMed Central

    Franciosa, G; Fenicia, L; Caldiani, C; Aureli, P

    1996-01-01

    A PCR was developed and applied for the detection of Clostridium botulinum type C in 18 avian and environmental samples collected during an outbreak of avian botulism, and the results were compared with those obtained by conventional methodologies based on the mouse bioassay. PCR and mouse bioassay results compared well (100%) after the enrichment of samples, but PCR results directly indicated the presence of this microorganism in six samples, while only one of these contained the type C botulinal neurotoxin before enrichment. The PCR assay was sensitive (limit of detection between 15 and 15 x 10(3) spores per PCR), specific (no amplification products were obtained with other clostridia), and rapid, since sonicated and heated samples provided enough template for amplification without any DNA purification. Eleven isolates of C. botulinum type C were recovered from mallards (Anas platyrhynchos), grey herons (Ardea cinerea), and mud during investigation of this outbreak. PMID:8815101

  12. Dielectrophoresis chips improve PCR detection of the food-spoiling yeast Zygosaccharomyces rouxii in apple juice.

    PubMed

    del Carmen Jaramillo, Maria; Huttener, Mario; Alvarez, Juan Manuel; Homs-Corbera, Antoni; Samitier, Josep; Torrents, Eduard; Juárez, Antonio

    2015-07-01

    Dielectrophoretic (DEP) manipulation of cells present in real samples is challenging. We show in this work that an interdigitated DEP chip can be used to trap and wash a population of the food-spoiling yeast Zygosaccharomyces rouxii that contaminates a sample of apple juice. By previously calibrating the chip, the yeast population loaded is efficiently trapped, washed, and recovered in a small-volume fraction that, in turn, can be used for efficient PCR detection of this yeast. DEP washing of yeast cells gets rid of PCR inhibitors present in apple juice and facilitates PCR analysis. This and previous works on the use of DEP chips to improve PCR analysis show that a potential use of DEP is to be used as a treatment of real samples prior to PCR. PMID:25808673

  13. Detecting Newcastle disease virus in combination of RT-PCR with red blood cell absorption.

    PubMed

    Yi, Jianzhong; Liu, Chengqian

    2011-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) has limited sensitivity when treating complicated samples, such as feces, waste-water in farms, and nucleic acids, protein rich tissue samples, all the factors may interfere with the sensitivity of PCR test or generate false results. In this study, we developed a sensitive RT-PCR, combination of red blood cell adsorption, for detecting Newcastle disease virus (NDV). One pair of primers which was highly homologous to three NDV pathotypes was designed according to the consensus nucleocapsid protein (NP) gene sequence. To eliminate the interfere of microbes and toxic substances, we concentrated and purified NDV from varied samples utilizing the ability of NDV binding red blood cells (RBCs). The RT-PCR coupled with red blood cell adsorption was much more sensitive in comparison with regular RT-PCR. The approach could also be used to detect other viruses with the property of hemagglutination, such as influenza viruses. PMID:21535888

  14. 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. PMID:27371921

  15. Coagulase gene polymorphisms detected by PCR in Staphylococcus aureus isolated from subclinical bovine mastitis in Turkey.

    PubMed

    Karahan, Murat; Cetinkaya, Burhan

    2007-09-01

    The genetic relatedness of coagulase (coa) positive Staphylococcus aureus isolated from cows with subclinical mastitis in Turkey was investigated by polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) analysis. Among 700 milk samples positive in the California Mastitis Test (CMT), species specific PCR identified 200 (28.6%) isolates as S. aureus and 161 (80.5%) of these isolates were positive for the 3' end of the coa gene by PCR. Most isolates (n=135, 83.9%) produced a single band on coa PCR, with molecular sizes ranging from 500 to 1400bp, whereas a small number of isolates (n=26, 16.1%) yielded two amplification products. Coa RFLP analysis using AluI and Hin6I revealed 23 and 22 band patterns, respectively. The detection of double bands by coa PCR, previously reported in human isolates, suggests that milking personnel can play a role in the transmission of S. aureus. PMID:16901735

  16. Rapid and Inexpensive Screening of Genomic Copy Number Variations Using a Novel Quantitative Fluorescent PCR Method

    PubMed Central

    Han, Joan C.; Elsea, Sarah H.; Pena, Heloísa B.; Pena, Sérgio Danilo Junho

    2013-01-01

    Detection of human microdeletion and microduplication syndromes poses significant burden on public healthcare systems in developing countries. With genome-wide diagnostic assays frequently inaccessible, targeted low-cost PCR-based approaches are preferred. However, their reproducibility depends on equally efficient amplification using a number of target and control primers. To address this, the recently described technique called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR) was shown to reliably detect four human syndromes by quantifying DNA amplification in an internally controlled PCR reaction. Here, we confirm its utility in the detection of eight human microdeletion syndromes, including the more common WAGR, Smith-Magenis, and Potocki-Lupski syndromes with 100% sensitivity and 100% specificity. We present selection, design, and performance evaluation of detection primers using variety of approaches. We conclude that MQF-PCR is an easily adaptable method for detection of human pathological chromosomal aberrations. PMID:24288428

  17. Acoustic detection of DNA conformation in genetic assays combined with PCR.

    PubMed

    Papadakis, G; Tsortos, A; Kordas, A; Tiniakou, I; Morou, E; Vontas, J; Kardassis, D; Gizeli, E

    2013-01-01

    Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost. PMID:23778520

  18. Acoustic detection of DNA conformation in genetic assays combined with PCR

    PubMed Central

    Papadakis, G.; Tsortos, A.; Kordas, A.; Tiniakou, I.; Morou, E.; Vontas, J.; Kardassis, D.; Gizeli, E.

    2013-01-01

    Application of PCR to multiplexing assays is not trivial; it requires multiple fluorescent labels for amplicon detection and sophisticated software for data interpretation. Alternative PCR-free methods exploiting new concepts in nanotechnology exhibit high sensitivities but require multiple labeling and/or amplification steps. Here, we propose to simplify the problem of simultaneous analysis of multiple targets in genetic assays by detecting directly the conformation, rather than mass, of target amplicons produced in the same PCR reaction. The new methodology exploits acoustic wave devices which are shown to be able to characterize in a fully quantitative manner multiple double stranded DNAs of various lengths. The generic nature of the combined acoustic/PCR platform is shown using real samples and, specifically, during the detection of SNP genotyping in Anopheles gambiae and gene expression quantification in treated mice. The method possesses significant advantages to TaqMan assay and real-time PCR regarding multiplexing capability, speed, simplicity and cost. PMID:23778520

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

    PubMed

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

    2015-01-01

    We detected Legionella species in 111 bath water samples and 95 cooling tower water samples by using a combination of conventional plate culture, quantitative polymerase chain reaction (qPCR) and qPCR combined with ethidium monoazide treatment (EMA-qPCR) methods. In the case of bath water samples, Legionella spp. were detected in 30 samples by plate culture, in 85 samples by qPCR, and in 49 samples by EMA-qPCR. Of 81 samples determined to be Legionella-negative by plate culture, 56 and 23 samples were positive by qPCR and EMA-qPCR, respectively. Therefore, EMA treatment decreased the number of Legionella-positive bath water samples detected by qPCR. In contrast, EMA treatment had no effect on cooling tower water samples. We therefore expect that EMA-qPCR is a useful method for the rapid detection of viable Legionella spp. from bath water samples. PMID:25817816

  20. Immunomagnetic bead-based recovery and real time quantitative PCR (RT iq-PCR) for sensitive quantification of aflatoxin B(1).

    PubMed

    Babu, Dinesh; Muriana, Peter M

    2011-08-01

    Aflatoxin B(1) is an unavoidable natural mycotoxin that enters the food chain by contamination of food grains and feedstuffs, potentially posing carcinogenic risks to animal and human health. Immuno-PCR methods have the potential to address the need of meeting the regulatory limits by detecting trace levels of toxins present in food and animal feeds. This paper describes a real-time immuno-quantitative PCR (RT-iqPCR) assay for quantification of aflatoxin B(1) suspended in methanol:water solution that can also serve as an extraction solvent. Immuno-PCR approaches were examined including direct vs. indirect sandwich assays using monoclonal vs. polyclonal antibodies. Our best approach was obtained using monoclonal antibodies to capture aflatoxin in solution prior to immobilizing the F(c) portion of the capture antibodies onto to protein G magnetic beads. This was followed by the addition of a polyclonal 'signal antibody' tethered with an oligonucleotide template for a subsequent PCR assay. The RT-iqPCR assay described herein leads to the sensitive detection and quantification of aflatoxin B(1) from 10ppb down to 0.1ppb with high correlation (r(2)=0.97) and efficiency (99.5%). The approach also detected the high-dose 'hook effect' phenomenon (excess antigen) which was overcome by the use of dilution protocols to eliminate false negatives that may occur at levels above quantification limits of the assay. The RT-iqPCR approach discussed here is presented as a model system that could easily be adapted for aflatoxin detection in a variety of food or animal feed samples using a simple methanol:water solution as an extraction solvent. PMID:21596071