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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. PCR und Real-Time PCR

    NASA Astrophysics Data System (ADS)

    Konrad, Regina; Busch, Ulrich

    Die vielfältigen Anwendungsmöglichkeiten der Polymerasekettenreaktion (polymerase chain reaction, PCR) machen sie zu einer der wichtigsten und am häufigsten eingesetzten Methoden in der molekularbiologischen Forschung und Diagnostik. Für diese Technologie wurde der Erfinder der Methode, Kary Mullis, 1993 mit dem Nobelpreis ausgezeichnet. Die PCR erlaubt einen hochsensitiven und spezifischen in-vitro-Nachweis von Desoxyribonukleinsäuren (DNA), da im Zuge der Reaktion Sequenzabschnitte gezielt vermehrt werden. Innerhalb weniger Stunden können aus einem einzigen Zielmolekül 1012 identische Moleküle entstehen [1].

  7. 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...

  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. Pcr by Thermal Convection

    NASA Astrophysics Data System (ADS)

    Braun, Dieter

    The Polymerase Chain Reaction (PCR) allows for highly sensitive and specific amplification of DNA. It is the backbone of many genetic experiments and tests. Recently, three labs independently uncovered a novel and simple way to perform a PCR reaction. Instead of repetitive heating and cooling, a temperature gradient across the reaction vessel drives thermal convection. By convection, the reaction liquid circulates between hot and cold regions of the chamber. The convection triggers DNA amplification as the DNA melts into two single strands in the hot region and replicates into twice the amount in the cold region. The amplification progresses exponentially as the convection moves on. We review the characteristics of the different approaches and show the benefits and prospects of the method.

  10. PCR in forensic genetics.

    PubMed

    Morling, Niels

    2009-04-01

    Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more and more advanced, special investigations in cases concerning crime, paternity, relationship, disaster victim identification etc. The present review gives an update on the use of DNA investigations in forensic genetics.

  11. 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...

  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.

  13. Applications of Digital PCR for Clinical Microbiology.

    PubMed

    Kuypers, Jane; Jerome, Keith R

    2017-03-15

    Digital PCR (dPCR) is an important new tool for use in the clinical microbiology laboratory. Its advantages over quantitative PCR (qPCR), including absolute quantification without a standard curve, improved precision, improved accuracy in the presence of inhibitors, and more accurate quantitation when amplification efficiency is low, make dPCR the assay of choice for several specimen testing applications. This mini-review will discuss the advantages and disadvantages of dPCR compared to qPCR, its applications in clinical microbiology and the considerations for implementation of the method in a clinical laboratory.

  14. PCR hot-start using duplex primers.

    PubMed

    Kong, Deming; Shen, Hanxi; Huang, Yanping; Mi, Huaifeng

    2004-02-01

    A new technique of PCR hot-start using duplex primers has been developed which can decrease the undesirable products arising throughout PCR amplification thereby giving better results than a manual hot-start method.

  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 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.

  17. Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples.

    PubMed

    Yang, Rongchang; Paparini, Andrea; Monis, Paul; Ryan, Una

    2014-12-01

    Clinical microbiology laboratories rely on quantitative PCR for its speed, sensitivity, specificity and ease-of-use. However, quantitative PCR quantitation requires the use of a standard curve or normalisation to reference genes. Droplet digital PCR provides absolute quantitation without the need for calibration curves. A comparison between droplet digital PCR and quantitative PCR-based analyses was conducted for the enteric parasite Cryptosporidium, which is an important cause of gastritis in both humans and animals. Two loci were analysed (18S rRNA and actin) using a range of Cryptosporidium DNA templates, including recombinant plasmids, purified haemocytometer-counted oocysts, commercial flow cytometry-counted oocysts and faecal DNA samples from sheep, cattle and humans. Each method was evaluated for linearity, precision, limit of detection and cost. Across the same range of detection, both methods showed a high degree of linearity and positive correlation for standards (R(2)⩾0.999) and faecal samples (R(2)⩾0.9750). The precision of droplet digital PCR, as measured by mean Relative Standard Deviation (RSD;%), was consistently better compared with quantitative PCR, particularly for the 18S rRNA locus, but was poorer as DNA concentration decreased. The quantitative detection of quantitative PCR was unaffected by DNA concentration, but droplet digital PCR quantitative PCR was less affected by the presence of inhibitors, compared with quantitative PCR. For most templates analysed including Cryptosporidium-positive faecal DNA, the template copy numbers, as determined by droplet digital PCR, were consistently lower than by quantitative PCR. However, the quantitations obtained by quantitative PCR are dependent on the accuracy of the standard curve and when the quantitative PCR data were corrected for pipetting and DNA losses (as determined by droplet digital PCR), then the sensitivity of both methods was comparable. A cost analysis based on 96 samples revealed that

  18. 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…

  19. Direct in situ rt-PCR.

    PubMed

    Lossi, Laura; Gambino, Graziana; Salio, Chiara; Merighi, Adalberto

    2011-01-01

    In situ polymerase chain reaction (PCR) is a histological technique that exploits the advantages of PCR for detection of mRNA directly in tissue sections. It somehow conjugates together PCR and in situ hybridization that is more traditionally employed for mRNA localization in cell organelles, intact cells, or tissue sections. This chapter describes the application of in situ PCR for neuropeptide mRNA localization. We provide here a detailed protocol for direct in situ reverse transcription (RT) PCR (RT-PCR) with nonradioactive probes after fixation and paraffin embedding or cryosectioning. Digoxigenin-labeled nucleotides (digoxigenin-11-dUTP) are incorporated in the PCR product after RT and subsequently detected with an anti-digoxigenin antibody conjugated with alkaline phosphatase. The procedure can be modified for use with fluorescent probes and employed in combination with enzyme/fluorescence immunocytochemical labeling.

  20. Multiplexed Primer Prediction for PCR

    SciTech Connect

    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 sequences 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.

  1. Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR.

    PubMed

    Garofalo, Cristiana; Bancalari, Elena; Milanović, Vesna; Cardinali, Federica; Osimani, Andrea; Sardaro, Maria Luisa Savo; Bottari, Benedetta; Bernini, Valentina; Aquilanti, Lucia; Clementi, Francesca; Neviani, Erasmo; Gatti, Monica

    2017-02-02

    The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loads<5.0 Logcfug(-1). Furthermore, LH-PCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products.

  2. Pitfalls in PCR troubleshooting: Expect the unexpected?

    PubMed Central

    Schrick, Livia; Nitsche, Andreas

    2015-01-01

    PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings. PMID:27077041

  3. 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.

  4. Absolute quantification by droplet digital PCR versus analog real-time PCR

    PubMed Central

    Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh

    2014-01-01

    Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

  5. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

    PubMed

    Farrell, D J

    1999-02-01

    Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

  6. [Detection of cytomegalovirus by real-time PCR in HIV-positive plasm].

    PubMed

    Ruiz-Tachiquín, Martha Eugenia; Gómez-Delgado, Alejandro; Valdez-Salazar, Hilda Alicia; Aguilera, Penélope

    2014-01-01

    INTRODUCCIÓN: el citomegalovirus es responsable de infecciones persistentes, generalmente asintomáticas en personas sanas pero que en ausencia de una respuesta inmune efectiva puede causar enfermedad severa, por ello es muy importante su detección temprana en los individuos con trastornos de la inmunidad. El objetivo de esta investigación fue hacer un análisis del límite de detección, sensibilidad y concordancia de la reacción en cadena de la polimerasa (PCR) en punto final con los obtenidos con la PCR en tiempo real. MÉTODOS: se realizó un estudio transversal con 43 muestras de plasma humano positivas al virus de la inmunodeficiencia humano, provenientes de individuos de 18 o más años de edad, de uno u otro sexo. Todas las muestras tuvieron una carga viral-VIH mayor a 100 000 copias/mL. Para la PCR en punto final se empleó un método comercial para identificar UL54 (gen viral blanco) y para la PCR en tiempo real se amplificaron fragmentos de los genes UL54 (gen temprano) y UL83 (gen tardío) del citomegalovirus humano.

  7. 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...

  8. 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…

  9. Real-time PCR and PCR-tandem Mass Spectrometry for Biodetection

    DTIC Science & Technology

    2005-10-01

    Real - time PCR and PCR- tandem mass spectrometry for biodetection Alvin Fox, University of South Carolina, School of Medicine Report Documentation...TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR and PCRtandem mass spectrometry for biodetection 5a. CONTRACT NUMBER 5b...interspace region Bacillus subtilis W23 standard Blank Barn dust House dust Cycle Real - time PCR (16s rRNA) - environmental samples Real - time

  10. Pre-PCR processing: strategies to generate PCR-compatible samples.

    PubMed

    Rådström, Peter; Knutsson, Rickard; Wolffs, Petra; Lövenklev, Maria; Löfström, Charlotta

    2004-02-01

    Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

  11. A new PCR method: one primer amplification of PCR-CTPP products.

    PubMed

    Yin, Guang; Mitsuda, Yoko; Ezaki, Takayuki; Hamajima, Nobuyuki

    2012-10-01

    Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.

  12. Real-Time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei

    DTIC Science & Technology

    2005-10-01

    1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions

  13. Quantitative real-time PCR eliminates false-positives in colony screening PCR.

    PubMed

    Skarratt, Kristen K; Fuller, Stephen J

    2014-01-01

    We report an alternative approach to colony screening using real-time PCR (qPCR) which can be used instead of the traditional end-point PCR to eliminate false-positives and reduce processing times. False-positive transformants can easily be distinguished from true-positives by comparing Ct values derived from qPCR amplification curves. In addition, the use of qPCR allows for more efficient processing since a gel electrophoresis step is not required and the screening process is no longer limited by the capacity of the gel apparatus.

  14. PCR for capsular typing of Haemophilus influenzae.

    PubMed Central

    Falla, T J; Crook, D W; Brophy, L N; Maskell, D; Kroll, J S; Moxon, E R

    1994-01-01

    A PCR method for the unequivocal assignment of Haemophilus influenzae capsular type (types a to f) was developed. PCR primers were designed from capsule type-specific DNA sequences cloned from the capsular gene cluster of each of the six capsular types. PCR product was amplified only from the capsular type for which the primers were designed. Product was confirmed by using either an internal oligonucleotide or restriction endonuclease digestion. A total of 172 H. influenzae strains of known capsular type (determined genetically) comprising all capsular types and noncapsulate strains were tested by PCR capsular typing. In all cases the PCR capsular type corresponded to the capsular genotype determined by restriction fragment length polymorphism analysis of the cap region. When used in conjunction with PCR primers derived from the capsular gene bexA, capsulate, noncapsulate, and capsule-deficient type b mutant strains could be differentiated. PCR capsular typing overcomes the problems of cross-reaction and autoagglutination associated with the serotyping of H. influenzae strains. The rapid and unequivocal capsular typing method that is described will be particularly important for typing invasive H. influenzae strains isolated from recipients of H. influenzae type b vaccine. Images PMID:7814470

  15. Quantitative DNA Analysis Using Droplet Digital PCR.

    PubMed

    Vossen, Rolf H A M; White, Stefan J

    2017-01-01

    Droplet digital PCR (ddPCR) is based on the isolated amplification of thousands of individual DNA molecules simultaneously, with each molecule compartmentalized in a droplet. The presence of amplified product in each droplet is indicated by a fluorescent signal, and the proportion of positive droplets allows the precise quantification of a given sequence. In this chapter we briefly outline the basis of ddPCR, and describe two different applications using the Bio-Rad QX200 system: genotyping copy number variation and quantification of Illumina sequencing libraries.

  16. The potential advantages of digital PCR for clinical virology diagnostics.

    PubMed

    Hall Sedlak, Ruth; Jerome, Keith R

    2014-05-01

    Digital PCR (dPCR), a new nucleic acid amplification technology, offers several potential advantages over real-time or quantitative PCR (qPCR), the current workhorse of clinical molecular virology diagnostics. Several studies have demonstrated dPCR assays for human cytomegalovirus or HIV, which give more precise and reproducible results than qPCR assays without sacrificing sensitivity. Here we review the literature comparing dPCR and qPCR performance in viral molecular diagnostic assays and offer perspective on the future of dPCR in clinical virology diagnostics.

  17. Recent advances in quantitative PCR (qPCR) applications in food microbiology.

    PubMed

    Postollec, Florence; Falentin, Hélène; Pavan, Sonia; Combrisson, Jérôme; Sohier, Danièle

    2011-08-01

    Molecular methods are being increasingly applied to detect, quantify and study microbial populations in food or during food processes. Among these methods, PCR-based techniques have been the subject of considerable focus and ISO guidelines have been established for the detection of food-borne pathogens. More particularly, real-time quantitative PCR (qPCR) is considered as a method of choice for the detection and quantification of microorganisms. One of its major advantages is to be faster than conventional culture-based methods. It is also highly sensitive, specific and enables simultaneous detection of different microorganisms. Application of reverse-transcription-qPCR (RT-qPCR) to study population dynamics and activities through quantification of gene expression in food, by contrast with the use of qPCR, is just beginning. Provided that appropriate controls are included in the analyses, qPCR and RT-qPCR appear to be highly accurate and reliable for quantification of genes and gene expression. This review addresses some important technical aspects to be considered when using these techniques. Recent applications of qPCR and RT-qPCR in food microbiology are given. Some interesting applications such as risk analysis or studying the influence of industrial processes on gene expression and microbial activity are reported.

  18. Sensitive and accurate quantification of human malaria parasites using droplet digital PCR (ddPCR)

    PubMed Central

    Koepfli, Cristian; Nguitragool, Wang; Hofmann, Natalie E.; Robinson, Leanne J.; Ome-Kaius, Maria; Sattabongkot, Jetsumon; Felger, Ingrid; Mueller, Ivo

    2016-01-01

    Accurate quantification of parasite density in the human host is essential for understanding the biology and pathology of malaria. Semi-quantitative molecular methods are widely applied, but the need for an external standard curve makes it difficult to compare parasite density estimates across studies. Droplet digital PCR (ddPCR) allows direct quantification without the need for a standard curve. ddPCR was used to diagnose and quantify P. falciparum and P. vivax in clinical patients as well as in asymptomatic samples. ddPCR yielded highly reproducible measurements across the range of parasite densities observed in humans, and showed higher sensitivity than qPCR to diagnose P. falciparum, and equal sensitivity for P. vivax. Correspondence in quantification was very high (>0.95) between qPCR and ddPCR. Quantification between technical replicates by ddPCR differed 1.5–1.7-fold, compared to 2.4–6.2-fold by qPCR. ddPCR facilitates parasite quantification for studies where absolute densities are required, and will increase comparability of results reported from different laboratories. PMID:27982132

  19. Transgene detection by digital droplet PCR.

    PubMed

    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.

  20. 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.

  1. 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.

  2. Evaluation of Altona Diagnostics RealStar Zika Virus RT-PCR Test Kit for Zika virus PCR testing.

    PubMed

    L'Huillier, Arnaud G; Lombos, Ernesto; Tang, Elaine; Perusini, Stephen; Eshaghi, Alireza; Nagra, Sandeep; Frantz, Christine; Olsha, Romy; Kristjanson, Erik; Dimitrova, Kristina; Safronetz, David; Drebot, Mike; Gubbay, Jonathan B

    2017-03-15

    Background: With the emerging ZIKA virus (ZIKV) epidemic, accessible real-time reverse-transcription PCR (rRT-PCR) assays are needed to streamline testing. The commercial Altona Diagnostics RealStar ZIKV rRT-PCR Test Kit has been approved for Emergency Use Authorization by the FDA. Our aim was to verify Altona-PCR, by comparing it to the CDC-designed dual target ZIKV virus rRT-PCR reference assay (Reference-PCR), and describe demographics of patients tested for ZIKV by rRT-PCR in Ontario, Canada.Methods: A large set of clinical specimens were tested for ZIKV by Altona-PCR and Reference-PCR. Positive or equivocal specimens underwent PCR and Sanger sequencing targeting ZIKV NS5 gene.Results: 671 serum specimens were tested by Reference-PCR: 58 (8.6%) were positive, 193 (28.8%) equivocal and 420 (62.6%) negative. Ninety percent of Reference-PCR positive patients were tested in the first 5 days after symptom onset. Altona-PCR was performed on 284/671 tested specimens by Reference-PCR. Altona-PCR was positive in 53/58 (91%) Reference-PCR positive and 16/193 (8%) Reference-PCR equivocal specimens; ZIKV NS5 PCR was positive in all 68 Altona-PCR positive specimens, and negative in all 181 Altona-PCR negative specimens that underwent NS5 PCR.Conclusion: Most ZIKV PCR positive cases are detected in the first five days of illness. Altona-PCR has very good sensitivity (91%) and specificity (97%) compared to Reference-PCR. Altona-PCR can be used for ZIKV diagnostic testing, with less extensive verification requirements compared to a laboratory developed test.

  3. [Identification of Mycobacterium avium-intracellulare complex by PCR of AIDS and disseminated mycobacteriosis].

    PubMed

    García-Elorriaga, Guadalupe; Degollado-Estrada, Edgar; Villagómez-Ruiz, Alfredo; Cortés-Torres, Nancy; Arreguín-Reséndiz, Lilián; Del Rey-Pineda, Guillermo; González-Bonilla, César

    2016-01-01

    Introducción: el objetivo de este artículo es Identificar y diferenciar el complejo MAC por PCR en pacientes con SIDA y micobacteriosis diseminada. Métodos: se llevó a cabo un estudio transversal para identificar MAC por biología molecular. Se sintetizaron dos conjuntos de iniciadores: MAV y MIN, para M. avium y M. intracellulare, respectivamente. El ADN total de células obtenidas de 29 aislados clínicos y muestras de suero de otros 24 pacientes con SIDA e infección micobacteriana diseminada fue extraído y se amplificó por PCR con los iniciadores MAV y MIN. Cada uno de los iniciadores MAV y MIN amplificó un segmento altamente específico de 1.3 kb del ADN homólogo, respectivamente. Resultados: veintinueve ADN de los aislados clínicos de MAC identificadas por Gen-Probe AccuProbes se amplificaron con los iniciadores MAV (M. avium). De las 24 muestras clínicas, 3 fueron positivas para M. avium y 6 para M. tuberculosis. Conclusiones: nuestros resultados demostraron que la técnica de PCR se puede aplicar para la diferenciación de M. avium y M. intracellulare por iniciadores específicos 16S rRNA. En pacientes con estadio avanzado de SIDA y en quienes se sospecha micobacteriosis diseminada, la presencia de anemia (incluso con cultivos negativos) fosfatasa alcalina elevada y una mediana de CD4 de 15.9/ml, se debe considerar seriamente el diagnóstico de infección por MAC; sugerimos que, de acuerdo con nuestros resultados, se justifica una estratificación más precisa de los pacientes en términos de sus recuentos de células T CD4.

  4. ParaDIS_lib

    SciTech Connect

    Cook, Richard D.

    2016-05-25

    The ParaDIS_lib software is a project that is funded by the DOE ASC Program. Its purpose is to provide visualization and analysis capabilities for the existing ParaDIS parallel dislocation dynamics simulation code.

  5. 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.

  6. pcrEfficiency: a Web tool for PCR amplification efficiency prediction

    PubMed Central

    2011-01-01

    Background Relative calculation of differential gene expression in quantitative PCR reactions requires comparison between amplification experiments that include reference genes and genes under study. Ignoring the differences between their efficiencies may lead to miscalculation of gene expression even with the same starting amount of template. Although there are several tools performing PCR primer design, there is no tool available that predicts PCR efficiency for a given amplicon and primer pair. Results We have used a statistical approach based on 90 primer pair combinations amplifying templates from bacteria, yeast, plants and humans, ranging in size between 74 and 907 bp to identify the parameters that affect PCR efficiency. We developed a generalized additive model fitting the data and constructed an open source Web interface that allows the obtention of oligonucleotides optimized for PCR with predicted amplification efficiencies starting from a given sequence. Conclusions pcrEfficiency provides an easy-to-use web interface allowing the prediction of PCR efficiencies prior to web lab experiments thus easing quantitative real-time PCR set-up. A web-based service as well the source code are provided freely at http://srvgen.upct.es/efficiency.html under the GPL v2 license. PMID:22014212

  7. Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR.

    PubMed

    de Morais, Rayana Carla Silva; Gonçalves, Suênia da Cunha; Costa, Pietra Lemos; da Silva, Kamila Gaudêncio; da Silva, Fernando José; Silva, Rômulo Pessoa E; de Brito, Maria Edileuza Felinto; Brandão-Filho, Sinval Pinto; Dantas-Torres, Filipe; de Paiva-Cavalcanti, Milena

    2013-04-01

    Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.

  8. 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...

  9. 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 ...

  10. PCR Techniques in Next-Generation Sequencing.

    PubMed

    Goswami, Rashmi S

    2016-01-01

    With the advent of next-generation sequencing and its prolific use in the clinical realm, it would appear that techniques such as PCR would not be in high demand. This is not the case however, as PCR techniques play an important role in the success of NGS technology. Although NGS has rapidly become an important part of clinical molecular diagnostics, whole genome sequencing is still difficult to implement in a clinical laboratory due to high costs of sequencing, as well as issues surrounding data processing, analysis, and data storage, which can reduce efficiency and increase turnaround times. As a result, targeted sequencing is often used in clinical diagnostics, due to its increased efficiency. PCR techniques play an integral role in targeted NGS sequencing, allowing for the generation of multiple NGS libraries and the sequencing of multiple targeted regions simultaneously. We will outline the methods we employ in PCR amplification of targeted genomic regions for cancer mutation hotspots using the Ampliseq Cancer Hotspot v2 panel (Life Technologies, Carlsbad, CA).

  11. Manufacturing DNA microarrays from unpurified PCR products

    PubMed Central

    Diehl, Frank; Beckmann, Boris; Kellner, Nadine; Hauser, Nicole C.; Diehl, Susanne; Hoheisel, Jörg D.

    2002-01-01

    For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(l-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products. PMID:12177307

  12. Miniaturized technology for DNA typing: cassette PCR.

    PubMed

    Manage, Dammika P; Pilarski, Linda M

    2015-01-01

    With the smaller size, low cost, and rapid testing capabilities, miniaturized lab-on-a-chip devices can change the way medical diagnostics are currently performed in the health-care system. We have demonstrated such a device that is self-contained, simple, disposable, and inexpensive. It is capable of performing DNA amplification on an inexpensive instrument suitable for near point of care settings. This technology will enable on the spot evaluation of patients in the clinic for faster medical decision-making and more informed therapeutic choices. Our device, a gel capillary cassette, termed cassette PCR, contains capillary reaction units each holding a defined primer set, with arrays of capillary reaction units for simultaneously detecting multiple targets. With the exception of the sample to be tested, each capillary reaction unit holds all the reagents needed for PCR in a desiccated form that can be stored at room temperature for up to 3 months and even longer in colder conditions. It relies on capillary forces for sample delivery of microliter volumes through capillaries, hence avoiding the need for pumps or valves. In the assembled cassette, the wax architecture supporting the capillaries melts during the PCR and acts as a vapor barrier as well as segregating capillaries with different primer sets. No other chip sealing techniques are required. Cassette PCR accepts raw samples such as urine, genital swabs, and blood. The cassette is made with off-the-shelf components and contains integrated positive and negative controls.

  13. 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…

  14. New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

    PubMed Central

    Herthnek, David; Bölske, Göran

    2006-01-01

    Background Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. Results Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. Conclusion We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used. PMID:17020599

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed

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

    2017-01-01

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

  17. 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.

  18. 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.

  19. How Many Microorganisms Are Present? Quantitative Reverse Transcription PCR (qRT-PCR)

    NASA Astrophysics Data System (ADS)

    Price, Andy; Álvarez, Laura Acuña; Whitby, Corinne; Larsen, Jan

    Quantitative reverse transcription PCR (qRT-PCR) is a variation of conventional quantitative or real-time PCR, whereby mRNA is first converted into the complementary DNA (cDNA) by reverse transcription, the cDNA is then subsequently quantified by qPCR. The use of mRNA as the initial template allows the quantification of gene transcripts, rather than gene copy numbers. mRNA is only produced by actively metabolising cells and is produced by its corresponding gene to provide a 'blueprint' in order for a cell to manufacture a specific protein. Conventional qPCR detects not only DNA present in actively metabolising cells but also inactive and dead cells. qRT-PCR has the advantage that only actively metabolising cells are detected, hence provides a more reliable measure of microbial activity in oilfield samples. When qRT-PCR is combined with primers and probes for specific genes, the activity of microbial processes important in the oilfield, such as sulphate reduction, methanogenesis and nitrate reduction can be monitored.

  20. A survey of tools for the analysis of quantitative PCR (qPCR) data.

    PubMed

    Pabinger, Stephan; Rödiger, Stefan; Kriegner, Albert; Vierlinger, Klemens; Weinhäusel, Andreas

    2014-09-01

    Real-time quantitative polymerase-chain-reaction (qPCR) is a standard technique in most laboratories used for various applications in basic research. Analysis of qPCR data is a crucial part of the entire experiment, which has led to the development of a plethora of methods. The released tools either cover specific parts of the workflow or provide complete analysis solutions. Here, we surveyed 27 open-access software packages and tools for the analysis of qPCR data. The survey includes 8 Microsoft Windows, 5 web-based, 9 R-based and 5 tools from other platforms. Reviewed packages and tools support the analysis of different qPCR applications, such as RNA quantification, DNA methylation, genotyping, identification of copy number variations, and digital PCR. We report an overview of the functionality, features and specific requirements of the individual software tools, such as data exchange formats, availability of a graphical user interface, included procedures for graphical data presentation, and offered statistical methods. In addition, we provide an overview about quantification strategies, and report various applications of qPCR. Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

  1. 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 ...

  2. Rapid PCR thermocycling using microscale thermal convection.

    PubMed

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

    2011-03-05

    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

  3. 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%.

  4. 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

  5. A nanofluidic system for massively parallel PCR

    NASA Astrophysics Data System (ADS)

    Brenan, Colin; Morrison, Tom; Roberts, Douglas; Hurley, James

    2008-02-01

    Massively parallel nanofluidic systems are lab-on-a-chip devices where solution phase biochemical and biological analyses are implemented in high density arrays of nanoliter holes micro-machined in a thin platen. Polymer coatings make the interior surfaces of the holes hydrophilic and the exterior surface of the platen hydrophobic for precise and accurate self-metered loading of liquids into each hole without cross-contamination. We have created a "nanoplate" based on this concept, equivalent in performance to standard microtiter plates, having 3072 thirty-three nanoliter holes in a stainless steel platen the dimensions of a microscope slide. We report on the performance of this device for PCR-based single nucleotide polymorphism (SNP) genotyping or quantitative measurement of gene expression by real-time PCR in applications ranging from plant and animal diagnostics, agricultural genetics and human disease research.

  6. PCR (Polymerase Chain Reaction) Testing for Leishmaniasis

    DTIC Science & Technology

    1993-10-20

    reactions. Prior to temperature cycling, this moderately heat stable enzyme selectively excises uracil residues which have been incorporated into DNA... temperature . Despite these precautions, some non-specific annealing of PCR primers does occur, even with single copy gene detection, and more so with...Mallinckrodt) in slightly hypotonic saline and allowed to remain at ambient temperature for 5 minutes, during which time RBC’s are lysed. Centrifugation

  7. 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.

  8. 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

  9. Microfluidic gradient PCR (MG-PCR): a new method for microfluidic DNA amplification.

    PubMed

    Zhang, Chunsun; Xing, Da

    2010-02-01

    This study develops a new microfluidic DNA amplification strategy for executing parallel DNA amplification in the microfluidic gradient polymerase chain reaction (MG-PCR) device. The developed temperature gradient microfluidic system is generated by using an innovative fin design. The device mainly consists of modular thermally conductive copper flake which is attached onto a finned aluminum heat sink with a small fan. In our microfluidic temperature gradient prototype, a non-linear temperature gradient is produced along the gradient direction. On the copper flake of length 45 mm, width 40 mm and thickness 4 mm, the temperature gradient easily spans the range from 97 to 52 degrees Celsius. By making full use of the hot (90-97 degrees Celsius) and cold (60-70 degrees Celsius) regions on the temperature gradient device, the parallel, two-temperature MG-PCR amplification is feasible. As a demonstration, the MG-PCR from three parallel reactions of 112-bp Escherichia coli DNA fragment is performed in a continuous-flow format, in which the flow of the PCR reagent in the closed loop is induced by the buoyancy-driven nature convection. Although the prototype is not optimized, the MG-PCR amplification can be completed in less than 45 min. However, the MG-PCR thermocycler presented herein can be further scaled-down, and thus the amplification times and reagent consumption can be further reduced. In addition, the currently developed temperature gradient technology can be applied onto other continuous-flow MG-PCR systems or used for other analytical purposes such as parallel and combination measurements, and fluorescent melting curve analysis.

  10. Overcoming inhibition in real-time diagnostic PCR.

    PubMed

    Hedman, Johannes; Rådström, Peter

    2013-01-01

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

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

    PubMed

    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.

  12. 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

  13. 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

  14. Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

    PubMed

    Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

    2016-10-29

    Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally.

  15. Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

    PubMed

    Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

    2016-12-01

    Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally.

  16. 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.

  17. Miniaturized detection system for handheld PCR assays

    NASA Astrophysics Data System (ADS)

    Richards, James B.; Benett, William J.; Stratton, Paul; Hadley, Dean R.; Nasarabadi, Shanavaz L.; Milanovich, Fred P.

    2000-12-01

    We have developed and delivered a four chamber, battery powered, handheld instrument referred to as the HANAA which monitors the polymerase chain reaction (PCR) process using a TaqMan based fluorescence assay. The detection system differs form standard configurations in two essential ways. First, the size is miniaturized, with a combined cycling and optics plug-in module for a duplex assay begin about the size of a small box of matches. Second, the detection/analysis system is designed to call a positive sample in real time.

  18. 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 ...

  19. Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

    PubMed

    Greenspoon, S A; Sykes, K L V; Ban, J D; Pollard, A; Baisden, M; Farr, M; Graham, N; Collins, B L; Green, M M; Christenson, C C

    2006-12-20

    Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the

  20. Rapid direct PCR for ABO blood typing.

    PubMed

    Lee, Hwan Young; Park, Myung Jin; Kim, Na Young; Yang, Woo Ick; Shin, Kyoung-Jin

    2011-01-01

    Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.

  1. A Guide to Using STITCHER for Overlapping Assembly PCR Applications.

    PubMed

    O'Halloran, Damien M

    2017-01-01

    Overlapping PCR is commonly used in many molecular applications that include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping assembly PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online.

  2. Accurate quantification of supercoiled DNA by digital PCR.

    PubMed

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

    2016-04-11

    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.

  3. 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

  4. Droplet digital PCR for absolute quantification of pathogens.

    PubMed

    Gutiérrez-Aguirre, Ion; Rački, Nejc; Dreo, Tanja; Ravnikar, Maja

    2015-01-01

    The recent advent of different digital PCR (dPCR) platforms is enabling the expansion of this technology for research and diagnostic applications worldwide. The main principle of dPCR, as in other PCR-based methods including quantitative PCR (qPCR), is the specific amplification of a nucleic acid target. The distinctive feature of dPCR is the separation of the reaction mixture into thousands to millions of partitions which is followed by a real time or end point detection of the amplification. The distribution of target sequences into partitions is described by the Poisson distribution, thus allowing accurate and absolute quantification of the target from the ratio of positive against all partitions at the end of the reaction. This omits the need to use reference materials with known target concentrations and increases the accuracy of quantification at low target concentrations compared to qPCR. dPCR has also shown higher resilience to inhibitors in a number of different types of samples. In this chapter we describe the droplet digital PCR (ddPCR) workflow for the detection and quantification of pathogens using the droplet digital Bio-Rad platform QX100. We present as an example the quantification of the quarantine plant pathogenic bacterium, Erwinia amylovora.

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

    PubMed

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

    2009-03-01

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

  6. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    PubMed

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

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

    PubMed

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

    2012-09-01

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

  8. 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.

  9. 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.

  10. Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA.

    PubMed

    Tang, Hui; Cai, Qingchun; Li, Hu; Hu, Peng

    2016-06-16

    Quantitative real-time PCR (qPCR) has been widely implemented for clinical hepatitis B viral load testing, but a lack of standardization and relatively poor precision hinder its usefulness. Droplet digital PCR (ddPCR) is a promising tool that offers high precision and direct quantification. In this study, we compared the ddPCR QX100 platform by Bio-Rad with the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA) to detect serial plasmid DNA dilutions of known concentrations as well as HBV DNA extracted from patient serum samples. Both methods showed a high degree of linearity and quantitative correlation. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays. Patient sample quantifications by ddPCR and qPCR were highly agreeable based on the Bland-Altman analysis. Collectively, our findings demonstrate that ddPCR offers improved analytical sensitivity and specificity for HBV measurements and is suitable for clinical HBV detection.

  11. 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.

  12. 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.

  13. COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

    PubMed Central

    GÖKMEN, Tülin GÜVEN; SOYAL, Ayben; KALAYCI, Yıldız; ÖNLEN, Cansu; KÖKSAL, Fatih

    2016-01-01

    SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM), serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT) and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP) of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively). The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01%) than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis. PMID:27680169

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

    PubMed

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

    2017-04-01

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

  15. Comparison of the immunofluorescence assay with RT-PCR and nested PCR in the diagnosis of canine distemper.

    PubMed

    Jóźwik, A; Frymus, T

    2005-05-01

    Two pairs of primers were prepared, both localized within the sequences of the nucleoprotein gene (NP) of canine distemper virus (CDV). A number of experiments were done to optimize the conditions of RT-PCR and nested PCR methods. The nucleic acids of the Onderstepoort, Rockborn, Snyder Hill and Lederle strains of CDV could be detected with these primers. However, they did not react with the sequences of the Edmonston strain of the measles virus. The detection limit for RT-PCR was 10 TCID50 and for nested PCR 0.1 TCID50 of CDV. The RT-PCR was able to demonstrate the nucleic acid of CDV in the blood of all seven puppies vaccinated with a modified live virus. Blood samples of 23 dogs clinically suspected of distemper were examined by RT-PCR combined with nested PCR, and the results were compared with the detection of the CDV antigen in the smears from the mucous membranes by the direct immunofluorescence (IF) test. Of the 23 dogs, 12 were positive in nested PCR, six in the IF assay, and only two in single RT-PCR. It is concluded that nested PCR seems to be the most sensitive method for ante-mortem diagnosis of canine distemper, especially in its subacute or chronic forms.

  16. Microfluidics-Based PCR for Fusion Transcript Detection.

    PubMed

    Chen, Hui

    2016-01-01

    The microfluidic technology allows the production of network of submillimeter-size fluidic channels and reservoirs in a variety of material systems. The microfluidic-based polymerase chain reaction (PCR) allows automated multiplexing of multiple samples and multiple assays simultaneously within a network of microfluidic channels and chambers that are co-ordinated in controlled fashion by the valves. The individual PCR reaction is performed in nanoliter volume, which allows testing on samples with limited DNA and RNA. The microfluidics devices are used in various types of PCR such as digital PCR and single molecular emulsion PCR for genotyping, gene expression, and miRNA expression. In this chapter, the use of a microfluidics-based PCR for simultaneous screening of 14 known fusion transcripts in patients with leukemia is described.

  17. Misuse of PCR assay for diagnosis of mollusc protistan infections.

    PubMed

    Burreson, Eugene M

    2008-06-19

    Polymerase chain reaction (PCR) assays are useful tools for pathogen surveillance, but they are only proxy indications of pathogen presence in that they detect a DNA sequence. To be useful for detection of actual infections, PCR assays must be thoroughly tested for sensitivity and specificity, and ultimately validated against a technique, typically histology, which allows visualization of the parasite in host tissues. There is growing use of PCR assays for pathogen surveillance, but too often the assumption is made that a positive PCR result verifies an infection in a tested host. This assumption is valid only if the assay has been properly validated for the geographic area and for the hosts examined. Researchers should interpret unvalidated PCR assay results with caution, and editors and reviewers should insist that robust validations support all assertions that PCR results confirm infections.

  18. Combined Overlap Extension PCR Method for Improved Site Directed Mutagenesis

    PubMed Central

    Chong, Nikson Fatt-Ming

    2016-01-01

    The combined overlap extension PCR (COE-PCR) method developed in this work combines the strengths of the overlap extension PCR (OE-PCR) method with the speed and ease of the asymmetrical overlap extension (AOE-PCR) method. This combined method allows up to 6 base pairs to be mutated at a time and requires a total of 40–45 PCR cycles. A total of eight mutagenesis experiments were successfully carried out, with each experiment mutating between two to six base pairs. Up to four adjacent codons were changed in a single experiment. This method is especially useful for codon optimization, where doublet or triplet rare codons can be changed using a single mutagenic primer set, in a single experiment. PMID:27995143

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

    PubMed

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

    2016-01-06

    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.

  20. Forensic implications of PCR inhibition--A review.

    PubMed

    Alaeddini, Reza

    2012-05-01

    Polymerase chain reaction (PCR) is currently the method of choice for the identification of human remains in forensic coursework. DNA samples from crime scenes often contain co-purified impurities which inhibit PCR. PCR inhibition is the most common cause of PCR failure when adequate copies of DNA are present. Inhibitors have been routinely reported in forensic investigations of DNA extracted from a variety of templates. Humic compounds, a series of substances produced during decay process have been considered as the materials contaminating DNA in soil, natural waters and recent sediments. Those compounds have been frequently assigned as PCR inhibitors. The current report reviews the characteristics of PCR inhibition, including the proposed mechanisms of inhibition, detection methods and the available technologies to remove or overcome the inhibitory activities.

  1. Blood grouping based on PCR methods and agarose gel electrophoresis.

    PubMed

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  2. Direct PCR Improves the Recovery of DNA from Various Substrates.

    PubMed

    Templeton, Jennifer E L; Taylor, Duncan; Handt, Oliva; Skuza, Pawel; Linacre, Adrian

    2015-11-01

    This study reports on the comparison of a standard extraction process with the direct PCR approach of processing low-level DNA swabs typical in forensic investigations. Varying concentrations of control DNA were deposited onto three commonly encountered substrates, brass, plastic, and glass, left to dry, and swabbed using premoistened DNA-free nylon FLOQswabs(™) . Swabs (n = 90) were either processed using the DNA IQ(™) kit or, for direct PCR, swab fibers (~2 mm(2) ) were added directly to the PCR with no prior extraction. A significant increase in the height of the alleles (p < 0.005) was observed when using the direct PCR approach over the extraction methodology when controlling for surface type and mass of DNA deposited. The findings indicate the potential use of direct PCR for increasing the PCR product obtained from low-template DNA samples in addition to minimizing contamination and saving resources.

  3. Specific detection of chikungunya virus using a RT-PCR/nested PCR combination.

    PubMed

    Pfeffer, M; Linssen, B; Parke, M D; Kinney, R M

    2002-02-01

    Chikungunya (CHIK) virus is enzootic in many countries in Asia and throughout tropical Africa. In Asia the virus is transmitted from primates to humans almost exclusively by Aedes aegypti, while various aedine mosquito species are responsible for human infections in Africa. The clinical picture is characterized by a sudden onset of fever, rash and severe pain in the joints which may persist in a small proportion of cases. Although not listed as a haemorrhagic fever virus, illness caused by CHIK virus can be confused with diseases such as dengue or yellow fever, based on the similarity of the symptoms. Thus, laboratory confirmation of suspected cases is required to launch control measures during an epidemic. CHIK virus diagnosis based on virus isolation is very sensitive, yet requires at least a week in conjunction with virus identification using monovalent sera. We developed a reverse transcription-polymerase chain reaction (RT-PCR) assay which amplifies a 427-bp fragment of the E2 gene. Specificity was confirmed by testing representative strains of all known alphavirus species. To verify further the viral origin of the amplicon and to enhance sensitivity, a nested PCR was performed subsequently. This RT-PCR/nested PCR combination was able to amplify a CHIK virus-specific 172-bp amplicon from a sample containing as few as 10 genome equivalents. This assay was successfully applied to four CHIK virus isolates from Asia and Africa as well as to a vaccine strain developed by USAMRIID. Our method can be completed in less than two working days and may serve as a sensitive alternative in CHIK virus diagnosis.

  4. 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

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

    2014-10-01

    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.

  5. 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.

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

    PubMed

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

    2014-10-01

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

  7. The inhibitory effect of pentobarbitone on reverse transcription-PCR.

    PubMed

    Hyun, Changbaig; Filippich, Lucio John; Hughes, Ian

    2005-01-31

    Pentobarbitone sodium (Sodium 5-ethyl-5[1-methylbutyl]-pentobarbitone) is a short-acting barbiturate that is commonly used to euthanase animals. As part of our studies into the molecular genetics of copper toxicosis in Bedlington terrier dogs, reverse-transcription (RT)-PCR was noted to always fail on RNA samples collected from livers of dogs sacrificed by pentobarbitone injection. When samples were collected without pentobarbitone, however, RT-PCR was always successful. We suspected the possible inhibition by pentobarbitone sodium of either reverse transcriptase or Taq polymerase. In vitro studies showed that pentobarbitone interference of PCR occurred at >4 microg/microl. To identify if pentobarbitone produced competitive inhibition, each components (Taq polymerase, MgCl(2), dNTP, etc.) of the PCR was individually altered. However, inhibition still persisted, suggesting that multiple PCR components may be affected. Also it was shown that pentobarbitone interference was not dependent on the PCR product size. Simple dilution of pentobarbitone contaminated DNA solutions, and the addition of bovine serum albumin (BSA) to the PCR mix overcame pentobarbitone interference. In vivo, PCR by pentobarbitone was found to be compounded by high DNA concentration and pentobarbitone contamination. In addition, both high DNA concentration and pentobarbitone contamination could be overcome through dilution and the addition of BSA. Further work is required to quantify pentobarbitone concentration in the liver-extracted DNA and RNA samples before this inhibition effect on PCR can be fully elucidated.

  8. 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.

  9. Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing.

    PubMed

    Zhang, Rui; Li, Xin; Ramaswami, Gokul; Smith, Kevin S; Turecki, Gustavo; Montgomery, Stephen B; Li, Jin Billy

    2014-01-01

    We developed a targeted RNA sequencing method that couples microfluidics-based multiplex PCR and deep sequencing (mmPCR-seq) to uniformly and simultaneously amplify up to 960 loci in 48 samples independently of their gene expression levels and to accurately and cost-effectively measure allelic ratios even for low-quantity or low-quality RNA samples. We applied mmPCR-seq to RNA editing and allele-specific expression studies. mmPCR-seq complements RNA-seq for studying allelic variations in the transcriptome.

  10. 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.

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

    PubMed

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

    2012-03-01

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

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

    PubMed

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

    2016-12-01

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

  13. Hypervariable spacer regions are good sites for developing specific PCR-RFLP markers and PCR primers for screening actinorhizal symbionts.

    PubMed

    Varehese, Rajani; Chauhan, Vineeta S; Misra, Arvind K

    2003-06-01

    While the ribosomal RNA like highly conserved genes are good molecular chronometers for establishing phylogenetic relationships, they can also be useful in securing the amplification of adjoining hyper-variable regions. These regions can then be used for developing specific PCR primers or PCR-RFL profiles to be used as molecular markers. We report here the use of ITS region of rrn operon of Frankia for developing PCR-RFL profiles capable of discriminating between closely related frankiae. We have also made use of the ITS1 region of the nuclear rrn operon of Alnus nepalensis (D Don) for designing a PCR primer for specific amplification of nuclear DNA of this tree.

  14. Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project.

    PubMed

    Hein, Ingeborg; Flekna, Gabriele; Krassnig, Martina; Wagner, Martin

    2006-09-01

    A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).

  15. 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

  16. Applications of competitor RNA in diagnostic reverse transcription-PCR.

    PubMed

    Kleiboeker, Steven B

    2003-05-01

    Detection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.

  17. 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...

  18. Detection of Salmonella spp. in oysters by PCR.

    PubMed Central

    Bej, A K; Mahbubani, M H; Boyce, M J; Atlas, R M

    1994-01-01

    PCR DNA amplification of a region of the himA gene of Salmonella typhimurium specifically detected Salmonella spp. In oysters, 1 to 10 cells of Salmonella spp. were rapidly detected by the PCR following a pre-enrichment step to increase sensitivity and to ensure that detection was based on the presence of viable Salmonella spp. Images PMID:8117091

  19. 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...

  20. 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

  1. Evaluation of Aspergillus PCR protocols for testing serum specimens.

    PubMed

    White, P Lewis; Mengoli, Carlo; Bretagne, Stéphane; Cuenca-Estrella, Manuel; Finnstrom, Niklas; Klingspor, Lena; Melchers, Willem J G; McCulloch, Elaine; Barnes, Rosemary A; Donnelly, J Peter; Loeffler, Juergen

    2011-11-01

    A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 μl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

  2. Comparison between ICT and PCR for diagnosis of Chlamydia trachomatis.

    PubMed

    Khan, E R; Hossain, M A; Paul, S K; Mahmud, C; Hasan, M M; Rahman, M M; Nahar, K; Kubayashi, N

    2012-04-01

    Chlamydia trachomatis is an obligate intracellular gram-negative bacterium which is the most prevalent cause of bacterial sexually transmitted infections (STI). The present study was carried to diagnose genital Chlamydia trachomatis infection among women of reproductive age, attending Mymensingh Medical College Hospital, during July 2009 to June 2010 by Immunochromatographic test (ICT) and Polymerase chain reaction (PCR). A total of 70 females were included in this study. Out of 70 cases 56 were symptomatic and 14 asymptomatic. Endocervical swabs were collected from each of the cases and examined by Immunochromatographic test (ICT) for antigen detection and Polymerase chain reaction (PCR) for detection of endogenous plasmid-based nucleic acid. A total 29(41.4%) of the cases were found positive for C. trachomatis either by ICT or PCR. Of the 56 symptomatic cases, 19(33.9%) were found ICT positive and 17(30.4%) were PCR positive. Among 14 asymptomatic females, 2(14.3%) were ICT positive and none were PCR positive. Though PCR is highly sensitive but a total of twelve cases were found ICT positive but PCR negative. It may be due to presence of plasmid deficient strain of C trachomatis which could be amplified by ompA based (Chromosomal gene) multiplex PCR.

  3. Comparison of two DNA extractions and nested PCR, real-time PCR, a new commercial PCR assay, and bacterial culture for detection of Mycobacterium avium subsp. paratuberculosis in bovine feces.

    PubMed

    Christopher-Hennings, Jane; Dammen, Matthew A; Weeks, Shelleen R; Epperson, William B; Singh, Shri N; Steinlicht, Gina L; Fang, Ying; Skaare, Jessica L; Larsen, Jill L; Payeur, Janet B; Nelson, Eric A

    2003-03-01

    In this study, 5 combinations of 2 DNA extractions and 3 polymerase chain reaction (PCR) techniques were compared with culture for the detection of Mycobacterium paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southern blot technique, nested PCR (nPCR), or real-time PCR, and a university-developed extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93% and 100% and specificity between 95% and 100%, when compared with culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southern blot, nPCR, or real-time PCR, or a university-developed extraction with real-time PCR would result in similar sensitivities to culture for the identification of M. paratuberculosis from bovine feces and are valid alternatives to culture.

  4. Detection of episomal banana streak badnavirus by IC-PCR.

    PubMed

    Harper, G; Dahal, G; Thottappilly, G; Hull, R

    1999-04-01

    A polymerase chain reaction (PCR) based strategy to detect episomal banana streak badnavirus (BSV) in banana and plantain plants that carry integrated BSV sequences was developed. Antisera used in immuno-capture polymerase chain reaction (IC-PCR) are capable of binding a large number of BSV serotypes. The primers used for PCR are capable of annealing to and amplifying across the aspartic protease-reverse transcriptase domain boundaries of both episomal and integrated BSV sequences and result in similar or identical sequence size fragments from either template. However, we show that under the conditions selected for IC-PCR, nuclear, mitochondrial or chloroplast genomic sequences are not amplified and thus only captured episomal BSV is amplified. IC-PCR is suitable for the large-scale screening of Musa for episomal BSV which is necessary for germplasm movement.

  5. 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

  6. Rapid diagnosis of poliovirus infection by PCR amplification.

    PubMed Central

    Chezzi, C

    1996-01-01

    A single-tube, single-primer-set reverse transcription-PCR assay was developed for the rapid detection of polioviruses in infected tissue culture fluids and clinical materials. The poliovirus-specific PCR primers are located in the VP1-2A region of the poliovirus genome. They generate a 290-bp product and can be used in duplex reactions with general enterovirus primers. The primers span the region used for genotype determination, so that genotype analysis of wild-type polioviruses can be performed by direct sequencing of the PCR products. Of 125 virus isolates typed as polioviruses by neutralization assays, 125 (100%) were also positive by PCR, and of 38 isolates typed as non-polio enteroviruses by conventional techniques, 38 (100%) were also negative by PCR. The assay described here is rapid, highly sensitive, and specific and has clinical applicability in the diagnosis of poliovirus infections. PMID:8784577

  7. Teaching PCR Through Inquiry in an Undergraduate Biology Laboratory Course

    NASA Astrophysics Data System (ADS)

    Dorighi, K. M.; Betancourt, J.; Sapp, J.; Quan, T. K.; Lee, J.

    2010-12-01

    In this paper, we describe the design and implementation of an inquiry-based laboratory unit on the Polymerase Chain Reaction (PCR). This unit was designed and taught for the undergraduate Eukaryotic Genetics Laboratory class (Bio105L) at the University of California, Santa Cruz. Our activity utilizes an authentic molecular biology research question to teach the underlying molecular mechanisms and experimental technique of PCR, as well as fundamental scientific process skills such as planning experiments, making predictions and interpreting data. In particular, the activity prompts students to use PCR to determine which gene has been deleted in a region of the Drosophila genome. During this activity, students also gained technical experience in common molecular biology techniques, learned about additional applications of PCR and used a hands-on approach to model each step of PCR.

  8. Transcript quantitation in total yeast cellular RNA using kinetic PCR

    PubMed Central

    Kang, John J.; Watson, Robert M.; Fisher, Mary E.; Higuchi, Russell; Gelfand, David H.; Holland, Michael J.

    2000-01-01

    Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT–PCR, kRT–PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT–PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of ±20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators. PMID:10606670

  9. Multiplex PCR identification of Taenia spp. in rodents and carnivores.

    PubMed

    Al-Sabi, Mohammad N S; Kapel, Christian M O

    2011-11-01

    The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.

  10. PCR thermal management in an integrated Lab on Chip

    NASA Astrophysics Data System (ADS)

    Singh, Janak; Ekaputri, Mayang

    2006-04-01

    Thermal management modelling and simulations of a polymerase chain reaction (PCR) device to be integrated on a lab on chip (LOC) have been carried out and presented. A typical MEMS PCR in symmetrical configuration is the base model for this study. When the PCR device is integrated on a fluidic chip with many other bio-analysis components such as DNA extraction, RNA extraction, electro-chemical sensor, flow through components and channels etc., thermal symmetry required for uniform temperature across the PCR chamber is normally lost. In this paper, ANSYS 8.0 simulations in varying conditions and corresponding physical basis have been investigated and presented. Model optimizations are carried out when PCR chamber is placed, one, in the centre (symmetry) and two, in the corner (asymmetry) of the integrated chip. In both cases, temperature uniformity within ±0.5 °C variation is obtained.

  11. [Detection of influenza virus (RT-PCR assay and others)].

    PubMed

    Matsuzaki, Yoko

    2003-11-01

    Viral isolation is the conventional method for influenza virus diagnosis but it is less useful for immediate patient management. RT-PCR is the sensitive and rapid assay for the detection of respiratory viruses. Single step and multiplex RT-PCR is able to detect several viruses simultaneously in a single reaction. Real time PCR(TaqMan method) is able to detect the amplicon directly by release of a fluorescent reporter of the probe during the amplification reactions. This procedure can save time since it eliminates post-PCR processing steps. These RT-PCR methods should be useful for the accurate and rapid diagnosis of influenza virus infection, especially severe cases such as pneumonia and encephalopathy.

  12. PCR detection of Babesia ovata from questing ticks in Japan.

    PubMed

    Sivakumar, Thillaiampalam; Tattiyapong, Muncharee; Okubo, Kazuhiro; Suganuma, Keisuke; Hayashida, Kyoko; Igarashi, Ikuo; Zakimi, Satoshi; Matsumoto, Kotaro; Inokuma, Hisashi; Yokoyama, Naoaki

    2014-04-01

    Babesia ovata is a tick-transmitted hemoprotozoan parasite of cattle. In the present study, we analyzed tick DNA samples (n=1459) prepared from questing ticks collected from various cattle pastures in Hokkaido (Shibecha, Taiki, Otofuke, Memuro, and Shin-Hidaka districts) and Okinawa (Yonaguni Island) prefectures of Japan for B. ovata. When all the tick DNA samples were screened by a previously described B. ovata-specific apical membrane antigen-1 (AMA-1) gene-based polymerase chain reaction (PCR) assay, none of the DNA samples was positive. Therefore, we developed a PCR assay based on the protozoan beta-tubulin (β-tubulin) gene to detect B. ovata from ticks in Japan. In the specificity test, the PCR assay amplified the expected 444-bp target gene fragment from B. ovata DNA. No PCR products were amplified from DNA samples from other blood pathogens, bovine blood, or ticks. In addition, the PCR assay detected 100 fg of B. ovata-genomic DNA extracted from an in vitro culture of the parasites. Subsequently, when all the tick DNA samples were screened by this new PCR assay, 18 were positive for B. ovata. Positive samples were found only in the Yonaguni and Memuro areas. In Okinawa, where all the ticks were identified as Haemaphysalis longicornis, 9.7% of the samples were PCR-positive, while a single tick (Ixodes ovatus) from Memuro was infected with B. ovata. When the nucleotide sequences of the PCR amplicons were phylogenetically analyzed, they formed a separate clade containing a previously described β-tubulin gene sequence from B. ovata (Miyake strain), confirming that the PCR assay had detected only B. ovata from the tick DNA samples. This is the first report that describes the PCR detection of B. ovata in ticks. The findings warrant transmission experiments to evaluate I. ovatus as a potential vector of B. ovata.

  13. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

    PubMed Central

    2011-01-01

    Background The polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. Results Pyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. Conclusion rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection. PMID:21831278

  14. An investigation of PCR inhibition using Plexor(®) -based quantitative PCR and short tandem repeat amplification.

    PubMed

    Thompson, Robyn E; Duncan, George; McCord, Bruce R

    2014-11-01

    A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor(®) real-time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and CT takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex(®) 16 HS System. The overall results demonstrate that real-time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.

  15. 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.

  16. 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

  17. 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.

  18. Real time PCR in childhood tuberculosis: a valuable diagnostic tool.

    PubMed

    Dayal, Rajeshwar; Kashyap, Haripal; Pounikar, Gajanand; Kamal, Raj; Yadav, Neeraj Kumar; Singh, Manoj Kumar; Chauhan, Devendra Singh; Goyal, Ankur

    2015-02-01

    The present study was conducted to detect and quantitate Mycobacterium tuberculosis from various body fluid specimens of cases of tuberculosis by real time PCR technique and compare results with conventional PCR technique and culture. One hundred fifteen children (<18 y) with tuberculosis (diagnosed as per IAP guidelines) and 32 disease matched controls from the Department of Pediatrics, S.N. Medical College, Agra, were included in the study. Different body fluids (CSF, gastric aspirate, pleural fluid, ascitic fluid and lymph node aspirate) were subjected to culture, conventional PCR targeting insertion sequence 1S6110 and Real time PCR targeting 16srRNA of Mycobacterium tuberculosis. Real time PCR showed significantly better results than culture in all body fluids (p < 0.05). It was superior to conventional PCR in CSF (p < 0.05) but showed comparable results in gastric aspirate, pleural fluid, ascitic fluid and lymph node aspirate (p > 0.05). Hence, real time PCR is a promising diagnostic tool for childhood tuberculosis, particularly tubercular meningitis.

  19. 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.

  20. Multiplex PCR to detect four different tomato-infecting pathogens.

    PubMed

    Quintero-Vásquez, Gabriela Alejandra; Bazán-Tejeda, María Luisa; Martínez-Peñafiel, Eva; Kameyama-Kawabe, Luis; Bermúdez-Cruz, Rosa María

    2013-07-01

    This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200-800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.

  1. Molecular characterization of Corynebacterium pseudotuberculosis isolates using ERIC-PCR.

    PubMed

    Guimarães, Alessandro de Sá; Dorneles, Elaine Maria Seles; Andrade, Giovanna Ivo; Lage, Andrey Pereira; Miyoshi, Anderson; Azevedo, Vasco; Gouveia, Aurora Maria Guimarães; Heinemann, Marcos Bryan

    2011-12-15

    Caseous lymphadenitis is an infectious sheep and goats disease caused by Corynebacterium pseudotuberculosis and characterized by abscesses in superficial and visceral lymph nodes. C. pseudotuberculosis strains isolated from these hosts have been shown to be very difficult to type by the existing methods. The aim of this study is evaluating the potential of the Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR) as a tool for molecular typing of C. pseudotuberculosis strains isolated in sheep. One hundred and twenty seven isolates of C. pseudotuberculosis were isolated from lesions suspected to have had caseous lymphadenitis collected from sheep at the slaughterhouse. Animals were from 24 flocks in 13 municipalities of the Minas Gerais State, Brazil. Species identification of the isolates was performed by routine biochemical tests and mPCR. Fingerprint was performed by RAPD using ERIC-1R, ERIC-2 and ERIC-1R+ERIC-2 primers. Seventeen different genotypes were generated by ERIC 1-PCR, 21 genotypes by ERIC 2-PCR and 21 genotypes by ERIC 1+2-PCR. Hunter-Gaston Discrimination Index (HGDI) found for ERIC 1, ERIC 2, ERIC 1+2 PCR were 0.69, 0.87, and 0.84, respectively. For most herds evaluated observed at most three different genotypes among isolates from animals of these property, in all ERIC-PCR assays. However a few flocks observed between four and nine genotypes per flock. The W Kendall value found for correlation among the three techniques of ERIC-PCR was 0.91 (P<5.0 x 10(-6)). The results show that ERIC-PCR has good discriminatory power and advantages over other DNA-based typing methods, making it a useful tool to discriminate C. pseudotuberculosis isolates.

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

    PubMed

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

    2015-05-05

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

  3. 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.

  4. PCR diagnosis of Pneumocystis pneumonia: a bivariate meta-analysis.

    PubMed

    Lu, Yuan; Ling, Guoya; Qiang, Chenyi; Ming, Qinshou; Wu, Cong; Wang, Ke; Ying, Zouxiao

    2011-12-01

    We undertook a bivariate meta-analysis to assess the overall accuracy of respiratory specimen PCR assays for diagnosing Pneumocystis pneumonia. The summary sensitivity and specificity were 0.99 (95% confidence interval, 0.96 to 1.00) and 0.90 (0.87 to 0.93). Subgroup analyses showed that quantitative PCR analysis and the major surface glycoprotein gene target had the highest specificity value (0.93). Respiratory specimen PCR results are sufficient to confirm or exclude the disease for at-risk patients suspected of having Pneumocystis pneumonia.

  5. 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.

  6. 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

  7. Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR): a simple method for suppressing PCR amplification of specific DNA sequences using ORNs.

    PubMed

    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.

  8. COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids

    PubMed Central

    2016-01-01

    Avoiding complementarity between primers when designing a PCR assay constitutes a central rule strongly anchored in the mind of the molecular scientist. 3’-complementarity will extend the primers during PCR elongation using one another as template, consequently disabling further possible involvement in traditional target amplification. However, a 5’-complementarity will leave the primers unchanged during PCR cycles, albeit sequestered to one another, therefore also suppressing target amplification. We show that 5’-complementarity between primers may be exploited in a new PCR method called COMplementary-Primer-Asymmetric (COMPAS)-PCR, using asymmetric primer concentrations to achieve target PCR amplification. Moreover, such a design may paradoxically reduce spurious non-target amplification by actively sequestering the limiting primer. The general principles were demonstrated using 5S rDNA direct repeats as target sequences to design a species-specific assay for identifying Salmo salar and Salmo trutta using almost fully complementary primers overlapping the same target sequence. Specificity was enhanced by using 3’-penultimate point mutations and the assay was further developed to enable identification of S. salar x S. trutta hybrids by High Resolution Melt analysis in a 35 min one-tube assay. This small paradigm shift, using highly complementary primers for PCR, should help develop robust assays that previously would not be considered. PMID:27783658

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

    PubMed

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

    2016-02-01

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

  10. COMplementary Primer ASymmetric PCR (COMPAS-PCR) Applied to the Identification of Salmo salar, Salmo trutta and Their Hybrids.

    PubMed

    Anglès d'Auriac, Marc B

    2016-01-01

    Avoiding complementarity between primers when designing a PCR assay constitutes a central rule strongly anchored in the mind of the molecular scientist. 3'-complementarity will extend the primers during PCR elongation using one another as template, consequently disabling further possible involvement in traditional target amplification. However, a 5'-complementarity will leave the primers unchanged during PCR cycles, albeit sequestered to one another, therefore also suppressing target amplification. We show that 5'-complementarity between primers may be exploited in a new PCR method called COMplementary-Primer-Asymmetric (COMPAS)-PCR, using asymmetric primer concentrations to achieve target PCR amplification. Moreover, such a design may paradoxically reduce spurious non-target amplification by actively sequestering the limiting primer. The general principles were demonstrated using 5S rDNA direct repeats as target sequences to design a species-specific assay for identifying Salmo salar and Salmo trutta using almost fully complementary primers overlapping the same target sequence. Specificity was enhanced by using 3'-penultimate point mutations and the assay was further developed to enable identification of S. salar x S. trutta hybrids by High Resolution Melt analysis in a 35 min one-tube assay. This small paradigm shift, using highly complementary primers for PCR, should help develop robust assays that previously would not be considered.

  11. Specific detection of viable Legionella cells by combined use of photoactivated ethidium monoazide and PCR/real-time PCR.

    PubMed

    Chang, Bin; Sugiyama, Kanji; Taguri, Toshitsugu; Amemura-Maekawa, Junko; Kura, Fumiaki; Watanabe, Haruo

    2009-01-01

    Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10(4)- to 10(5)-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.

  12. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex-PCR and of HPV by single PCR.

    PubMed

    Gimenes, Fabrícia; Medina, Fabiana Soares; Abreu, André Luelsdorf Pimenta de; 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.

  13. 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

  14. A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis.

    PubMed

    Boonyayatra, S; Fox, L K; Besser, T E; Sawant, A; Gay, J M; Raviv, Z

    2012-01-01

    The focus of the current research was to develop real-time PCR assays with improved sensitivity and the capacity to simultaneously speciate the 3 most common mycoplasma mastitis agents: Mycoplasma bovis, Mycoplasma californicum, and Mycoplasma bovigenitalium. Real-time PCR was chosen because it provides rapid results. Partial 16S rRNA gene sequencing was used as the gold standard for evaluating candidate real-time PCR assays. To ascertain the real-time PCR assay specificity, reference strains of Mycoplasma species, Acholeplasma axanthum, and common gram-positive and gram-negative mastitis pathogens were tested. No cross-reactions were observed. Mycoplasma spp. isolated from bovine milk samples (n=228) and other organ sites (n=40) were tested by the real-time PCR assays and the partial 16S rRNA gene sequencing assay. Overall accuracy of this novel real-time PCR was 98.51%; 4 of 228 isolates identified as M. bovis by the partial 16S rRNA gene sequencing assay were identified as both M. bovis and M. californicum by real-time PCR. Subsequent amplicon sequencing suggested the presence of both M. bovis and M. californicum in these 4 samples. Using a cycle threshold of 37, the detection limits for real-time PCR were 10 copies of DNA template for both M. bovis and M. bovigenitalium, and 1 copy for M. californicum. This real-time PCR assay is a diagnostic technique that may be used as a screening tool or as a confirmation test for mycoplasma mastitis.

  15. Clinical Performance of Aspergillus PCR for Testing Serum and Plasma: a Study by the European Aspergillus PCR Initiative.

    PubMed

    White, P Lewis; Barnes, Rosemary A; Springer, Jan; Klingspor, Lena; Cuenca-Estrella, Manuel; Morton, C Oliver; Lagrou, Katrien; Bretagne, Stéphane; Melchers, Willem J G; Mengoli, Carlo; Donnelly, J Peter; Heinz, Werner J; Loeffler, Juergen

    2015-09-01

    Aspergillus PCR testing of serum provides technical simplicity but with potentially reduced sensitivity compared to whole-blood testing. With diseases for which screening to exclude disease represents an optimal strategy, sensitivity is paramount. The associated analytical study confirmed that DNA concentrations were greater in plasma than those in serum. The aim of the current investigation was to confirm analytical findings by comparing the performance of Aspergillus PCR testing of plasma and serum in the clinical setting. Standardized Aspergillus PCR was performed on plasma and serum samples concurrently obtained from hematology patients in a multicenter retrospective anonymous case-control study, with cases diagnosed according to European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) consensus definitions (19 proven/probable cases and 42 controls). Clinical performance and clinical utility (time to positivity) were calculated for both kinds of samples. The sensitivity and specificity for Aspergillus PCR when testing serum were 68.4% and 76.2%, respectively, and for plasma, they were 94.7% and 83.3%, respectively. Eighty-five percent of serum and plasma PCR results were concordant. On average, plasma PCR was positive 16.8 days before diagnosis and was the earliest indicator of infection in 13 cases, combined with other biomarkers in five cases. On average, serum PCR was positive 10.8 days before diagnosis and was the earliest indicator of infection in six cases, combined with other biomarkers in three cases. These results confirm the analytical finding that the sensitivity of Aspergillus PCR using plasma is superior to that using serum. PCR positivity occurs earlier when testing plasma and provides sufficient sensitivity for the screening of invasive aspergillosis while maintaining methodological simplicity.

  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. 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-...

  18. Identification of Alocasia odora (Kuwazuimo in Japanese) Using PCR Method.

    PubMed

    Hagino, Kayo; Nakano, Hisako; Shimizu, Motomu; Terai, Akiko; Ogai, Mami; Aragane, Masako; Abe, Tomohiro; Sasamoto, Takeo

    2017-01-01

    Kuwazuimo (Alocasia odora) and shimakuwazuimo (Alocasia cucullata) are evergreen perennial plants that originated in East Asia. Although inedible, they are occasionally eaten by mistake because they resemble satoimo (Colocasia esculenta), and this has caused food poisoning in Japan. It is not easy to determine the cause of a food poisoning outbreak from the shape or chemical composition when the available sample is small. Therefore, we developed a new primer pair for PCR to identify kuwazuimo and shimakuwazuimo in small samples, based on the internal transcribed spacer (ITS) region of ribosomal DNA. Using PCR with the developed primer pair, we detected all samples of kuwazuimo obtained from the market, while excluding 17 other kinds of crops. The samples were identified as shimakuwazuimo by DNA sequencing of the PCR products. The present PCR method showed high specificity and was confirmed to be applicable to the identification of kuwazuimo and shimakuwazuimo from various crops.

  19. Single-molecule emulsion PCR in microfluidic droplets.

    PubMed

    Zhu, Zhi; Jenkins, Gareth; Zhang, Wenhua; Zhang, Mingxia; Guan, Zhichao; Yang, Chaoyong James

    2012-06-01

    The application of microfluidic droplet PCR for single-molecule amplification and analysis has recently been extensively studied. Microfluidic droplet technology has the advantages of compartmentalizing reactions into discrete volumes, performing highly parallel reactions in monodisperse droplets, reducing cross-contamination between droplets, eliminating PCR bias and nonspecific amplification, as well as enabling fast amplification with rapid thermocycling. Here, we have reviewed the important technical breakthroughs of microfluidic droplet PCR in the past five years and their applications to single-molecule amplification and analysis, such as high-throughput screening, next generation DNA sequencing, and quantitative detection of rare mutations. Although the utilization of microfluidic droplet single-molecule PCR is still in the early stages, its great potential has already been demonstrated and will provide novel solutions to today's biomedical engineering challenges in single-molecule amplification and analysis.

  20. Rapid PCR amplification of DNA utilizing Coriolis effects.

    PubMed

    Mårtensson, Gustaf; Skote, Martin; Malmqvist, Mats; Falk, Mats; Asp, Allan; Svanvik, Nicke; Johansson, Arne

    2006-08-01

    A novel polymerase chain reaction (PCR) method is presented that utilizes Coriolis and centrifugal effects, produced by rotation of the sample disc, in order to increase internal circulatory rates, and with them temperature homogenization and mixing speeds. A proof of concept has been presented by testing a rapid 45-cycle PCR DNA amplification protocol. During the repeated heating and cooling that constitutes a PCR process, the 100 microL samples were rotated at a speed equivalent to an effective acceleration of gravity of 7,000 g. A cycle time of 20.5 s gave a total process time of 15 min to complete the 45 cycles. A theoretical and numerical analysis of the resulting flow, which describes the increased mixing and temperature homogenization, is presented. The device gives excellent reaction speed efficiency, which is beneficial for rapid PCR.

  1. 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

  2. DNA microarray-based PCR ribotyping of Clostridium difficile.

    PubMed

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

    2015-02-01

    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.

  3. [Fut1 gene mutation for para-bombay blood type individual in Fujian Province of China].

    PubMed

    Huang, Hao-Bou; Fan, Li-Ping; Wai, Shi-Jin; Zeng, Feng; Lin, Hai-Yan; Zhang, Rong

    2010-10-01

    This study was aimed to investigate the molecular mechanisms for para-Bombay blood type individual in Fujian Province of China. The para-Bombay blood type of this individual was identified by routine serological techniques. The full coding region of alpha (1,2) fucosyltransferase (FUT1) gene of this individual was amplified by polymerase chain reaction (PCR), then the PCR product was cloned into T vector. The mutation in coding region of fut1 gene was identified by TA cloning, so as to explore the molecular mechanisms for para-Bombay blood type individual. The results indicated that the full coding region of fut1 gene was successfully amplified by PCR. AG deletion at position 547-552 on 2 homologous chromosomes was detected by TA cloning method, leading to a reading frame shift and a premature stop codon. It is concluded that genetic mutation of fut1 gene in this para-bombay blood type individual was h1h1 homozygotic type.

  4. 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

  5. PCR test for detecting Taenia solium cysticercosis in pig carcasses.

    PubMed

    Sreedevi, Chennuru; Hafeez, Mohammad; Kumar, Putcha Anand; Rayulu, Vukka Chengalva; Subramanyam, Kothapalli Venkata; Sudhakar, Krovvidi

    2012-01-01

    Polymerase chain reaction (PCR) test was employed to detect Taenia solium DNA in muscle lesions for validation of the meat inspection results of slaughtered pigs. Two sets of oligonucleotide primers, one targeted against the large subunit rRNA gene (TBR primers) and the other targeted against cytochrome c oxidase subunit 1 gene (Cox1 primers) of T. solium were used in this study. On reactivity in PCR test, the TBR primers and the Cox1 primers yielded products of 286 and 984 bp, respectively, in cysticercosis positive cases. Both the sets of primers were found to be highly specific, since they did not yield any PCR product in negative controls. A total of 225 pig carcasses were screened for cysticercosis by meat inspection, out of which 25 carcasses with visible cysts (16 viable and 9 degenerated cysts) were also confirmed to be positive for cysticercosis in PCR test. However, out of the 35 carcasses with suspected lesions on meat inspection, only two were found to be positive for cysticercosis in PCR test. The detection limits for both the primer sets were analyzed. The TBR primer set could detect up to 10 pg of cysticercus DNA, whereas the Cox1 primer set could detect only up to 1 ng. It is evident from the study that PCR test is an efficient tool for validation of meat inspection results and also to rule out ambiguity in carcass judgment of suspected cases of porcine cysticercosis.

  6. Improved PCR Amplification of Broad Spectrum GC DNA Templates.

    PubMed

    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.

  7. PCR performance of a thermostable heterodimeric archaeal DNA polymerase.

    PubMed

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  8. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    PubMed Central

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

  9. 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.

  10. Specific PCR product primer design using memetic algorithm.

    PubMed

    Yang, Cheng-Hong; Cheng, Yu-Huei; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2009-01-01

    To provide feasible primer sets for performing a polymerase chain reaction (PCR) experiment, many primer design methods have been proposed. However, the majority of these methods require a relatively long time to obtain an optimal solution since large quantities of template DNA need to be analyzed. Furthermore, the designed primer sets usually do not provide a specific PCR product size. In recent years, evolutionary computation has been applied to PCR primer design and yielded promising results. In this article, a memetic algorithm (MA) is proposed to solve primer design problems associated with providing a specific product size for PCR experiments. The MA is compared with a genetic algorithm (GA) using an accuracy formula to estimate the quality of the primer design and test the running time. Overall, 50 accession nucleotide sequences were sampled for the comparison of the accuracy of the GA and MA for primer design. Five hundred runs of the GA and MA primer design were performed with PCR product lengths of 150-300 bps and 500-800 bps, and two different methods of calculating T(m) for each accession nucleotide sequence were tested. A comparison of the accuracy results for the GA and MA primer design showed that the MA primer design yielded better results than the GA primer design. The results further indicate that the proposed method finds optimal or near-optimal primer sets and effective PCR products in a dry dock experiment. Related materials are available online at http://bio.kuas.edu.tw/ma-pd/.

  11. 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

    2016-05-18

    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.

  12. 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

  13. 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.

  14. Detection of Leishmania infantum in 4 different dog samples by real-time PCR and ITS-1 nested PCR.

    PubMed

    Carvalho Ferreira, Aline Leandra; Carregal, Virgínia Mendes; de Almeida Ferreira, Sidney; Leite, Rodrigo Souza; de Andrade, Antero Silva Ribeiro

    2014-04-01

    The canine visceral leishmaniasis (CVL) diagnosis is an important step of visceral leishmaniasis control program in Brazil, which involves the elimination of infected dogs, the main animal reservoir host of the disease. The aim of the present study was to evaluate a sensitive real-time PCR method for Leishmania infantum detection in 4 different clinical samples of dogs, including the noninvasive conjunctival swab (CS) sample. The results of real-time PCR were compared with those obtained using internal transcribed spacer 1 nested PCR. Animals were divided into 2 groups based on the absence or presence of CVL clinical sings. The CS associated with real-time PCR, using primers addressed to kinetoplast DNA minicircles, was able to detect L. infantum infection in 96.7% of dogs without clinical signs and in 100% of the symptomatic animals, demonstrating the importance of these procedures for diagnosing CVL.

  15. Assessing UV Inactivation of Adenovirus 41 Using Integrated Cell Culture Real-Time qPCR/RT-qPCR.

    PubMed

    Ding, Ning; Craik, Stephen A; Pang, Xiaoli; Lee, Bonita; Neumann, Norman F

    2017-04-01

      Enteric adenoviruses are among most UV-resistant viruses in water. Cytopathic effects (CPE)-based cell culture TCID50 assay as a conventional virus assessment approach has major drawbacks for enteric adenovirus since it is selective on cell lines and takes longer time to show CPE. Integrated cell culture real-time quantitative PCR (ICC-qPCR) and reverse transcriptase (RT)-qPCR were applied in this study, in comparison with TCID50, to assess UV inactivation of adenovirus type 41 (Ad41) in water. Adenovirus type 41 was exposed to UV doses of 40, 80, 160, and 320 mJ/cm2 using a collimated beam apparatus. There was no significant difference of inactivation at conducted UV doses between measurements using TCID50 assay and ICC-RT-qPCR. Both assays fitted the Chick-Watson model at 95% confidence level. The inactivation measured by ICC-qPCR did not fit the Chick-Watson model. In summary, ICC-RT-qPCR is the most appropriate alternate to CPE-based assay for assessing UV inactivation of enteric adenoviruses.

  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.

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

    PubMed

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

    1994-12-01

    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.

  18. 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.

  19. Pneumocystis sp. in bats evaluated by qPCR.

    PubMed

    Cavallini Sanches, E M; Ferreiro, L; Andrade, C P; Pacheco, S M; Almeida, L L; Spanamberg, A; Wissmann, G

    2013-03-01

    Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals. Accurate quantification of Pneumocystis sp. is essential for the correct interpretation of many research experiments investigating this organism. The objectives of this study were to detect the presence of Pneumocystis sp. in bats by qPCR, and to distinguish colonization from infection. Probes and primers for real time PCR (qPCR) were designed based on the gene of major surface glycoprotein (MSG) of Pneumocystis sp., in order to analyze 195 lung tissue samples from bats captured (2007-2009). All samples were also analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit rRNA (mtSSU rRNA) to confirm the results. The qPCR assay was standardized using a standard curve made with the DNA extracted from bronchoalveolar lavage positive for Pneumocystis jirovecii. The average Ct was found to be between 13 and 14 (calibration curve) for the detection of infection with Pneumocystis sp. and above these values for colonization. It was considered as negative samples the ones that had Ct values equal to 50. Out of the total 195 samples, 47 (24.1%) bat lung DNA samples were positive for Pneumocystis sp. by qPCR. The most common bat species found were: Tadarida brasiliensis (23.4%), Histiotus velatus (17.0%), Desmodus rotundus (14.9%) and Molossus molossus (8.5%). The average cycle threshold of the positive samples (bats) was 25.8 and standard deviation was 1.7. The DNA samples with Ct values greater than 14 suggest that these animals might be colonized by Pneumocystis sp. Results obtained in this study demonstrated the usefulness of the qPCR procedure for identification of Pneumocystis sp. and for distinction between its colonizing or infectious status in bats.

  20. Development of internal controls for PCR detection of Bacillus anthracis.

    PubMed

    Brightwell, G; Pearce, M; Leslie, D

    1998-12-01

    This work describes the development and evaluation of a multiplex polymerase chain reaction (PCR) for the detection of Bacillus anthracis strains harbouring plasmid pX02. The multiplex also incorporated an internal control (IC) to avoid false negative reactions. Internal controls consisted of plasmids containing modified PCR target sequences, corresponding to the capC and BA813 genes of B. anthracis, which were then co-amplified with the original target sequences using the same set of amplimers. The initial IC construct comprised of an internally deleted form of the genomic target sequence cloned into pUC19. A series of nested DNA fragments corresponding to the 23S rRNA sequences of Bacillus cereus were then subcloned into the point of deletion, producing a number of IC constructs with similar sequences but increasing product size on PCR amplification. Neither the presence of IC DNA template or IC PCR product size affected the specificity or non-specific cross-reactivity of the original PCR assay. The concentration of IC was critical, too much IC DNA template would out compete the genomic DNA template, thus giving a false negative result. However, when the concentration of IC was optimal assay sensitivity was not compromised.

  1. 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

  2. High-throughput PCR in silicon based microchamber array.

    PubMed

    Nagai, H; Murakami, Y; Yokoyama, K; Tamiya, E

    2001-12-01

    Highly integrated hybridization assay and capillary electrophoresis have improved the throughput of DNA analysis. The shift to high throughput analysis requires a high speed DNA amplification system, and several rapid PCR systems have been developed. In these thermal cyclers, the temperature was controlled by effective methodology instead of a large heating/cooling block preventing rapid thermal cycling. In our research, high speed PCR was performed using a silicon-based microchamber array and three heat blocks. The highly integrated microchamber array was fabricated by semiconductor microfabrication techniques. The temperature of the PCR microchamber was controlled by alternating between three heat blocks of different temperature. In general, silicon has excellent thermal conductivity, and the heat capacity is small in the miniaturized sample volume. Hence, the heating/cooling rate was rapid, approximately 16 degrees C/s. The rapid PCR was therefore completed in 18 min for 40 cycles. The thermal cycle time was reduced to 1/10 of a commercial PCR instrument (Model 9600, PE Applied Biosystems-3 h).

  3. 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.

  4. 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

  5. Optimizing methods for PCR-based analysis of predation.

    PubMed

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

    2011-09-01

    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.

  6. Electrochemistry-based real-time PCR on a microchip.

    PubMed

    Yeung, Stephen S W; Lee, Thomas M H; Hsing, I-Ming

    2008-01-15

    The development of handheld instruments for point-of-care DNA analysis can potentially contribute to the medical diagnostics and environmental monitoring for decentralized applications. In this work, we demonstrate the implementation of a recently developed electrochemical real-time polymerase chain reaction (ERT-PCR) technique on a silicon-glass microchip for simultaneous DNA amplification and detection. This on-chip ERT-PCR process requires the extension of an oligonucleotide in both solution and at solid phases and intermittent electrochemical signal measurement in the presence of all the PCR reagents. Several important parameters, related to the surface passivation and electrochemical scanning of working electrodes, were investigated. It was found that the ERT-PCR's onset thermal cycle ( approximately 3-5), where the analytical signal begins to be distinguishable from the background, is much lower than that of the fluorescence-based counterparts for high template DNA situations (3 x 10(6) copies/microL). By carefully controlling the concentrations of the immobilized probe and the enzyme polymerase, improvements have been made in obtaining a meaningful electrochemical signal using a lower initial template concentration. This ERT-PCR technique on a microchip platform holds significant promise for rapid DNA detection for point-of-care testing applications.

  7. 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.

  8. Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR (PMA-qPCR).

    PubMed

    Li, Dan; Tong, Tiezheng; Zeng, Siyu; Lin, Yiwen; Wu, Shuxu; He, Miao

    2014-02-01

    The detection of viable bacteria in wastewater treatment plants (WWTPs) is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide (PMA) combined with the quantitative polymerase chain reaction (PMA-qPCR) to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 micromol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.

  9. [Use of nested PCR in detection of the plague pathogen].

    PubMed

    Glukhov, A I; Gordeev, S A; Al'tshuler, M L; Zykova, I E; Severin, S E

    2003-07-01

    Causative agents of plague, i.e. bacterium Yersina pestis (in the subcutaneous tissues of rodents) and their cutaneous parasites need to be isolated to enable plague prevention. A comparatively new method of polymerase chain reaction (PCR) opens up new possibilities of determining Y. pestis just within several hours and without any cultivation. The article contains a description of the PCR-method, which makes it possible to distinguish the culture of Y. pestis from cultures of other microorganism, including speci of Yersina. The method is of the cluster-type, i.e. it is made up of subsequent PC reactions with the substrate for the second reaction being the product of the first one. The cluster nature of the method preconditions a higher sensitivity and specificity versus the ordinary PCR.

  10. Heat properties of an integrated micro PCR vessel

    NASA Astrophysics Data System (ADS)

    Zhao, Zhan; Cui, Dafu; Yu, Zhongyao; Wang, Li; Xia, Shanhong; Cui, Zheng

    2001-09-01

    The PCR amplification is based on multiple temperature cycles of DNA synthesis; each includes denaturation of the template, annealing of the primers to complementary sites in the template and primer extension. The key technique of PCR amplification is the heating control in design and fabrication of its chip form. The specifications of the chip are heat properties. In this paper the heat properties of a micro PCR vessel integration heater and temperature sensor was introduced. The temperature distribution of the vessel was simulated with software tool IntelliSuite. The temperatures cycles were measured and the time response of the chip was discussed. It is found that the integrate micro vessel is a very useful tool not only for DNA synthesis but also as a biochemical reactor for many other biological and chemical analyses.

  11. Noninvasive genotyping of common marmoset (Callithrix jacchus) by fingernail PCR.

    PubMed

    Takabayashi, Shuji; Katoh, Hideki

    2015-07-01

    The common marmoset (Callithrix jacchus) is a New World primate that is a useful model for medical studies. In this study, we report a convenient, reliable, and noninvasive procedure to genotype a living common marmoset by using fingernails. This method was used to successfully genotype DNA by restriction fragment length polymorphism (RFLP) PCR without prior purification, by using the KOD FX PCR enzyme kit. Additionally, there is no sample contamination from hematopoietic chimera derived from fused placenta in utero. We compared chimeric levels between various tissues in females with male littermates using quantitative fluorescent (QF)-PCR to prepare a reliable DNA source for genetic analyses, such as genotyping, gene mapping, or genomic sequencing. The chimerism detected appeared to be restricted to lymphatic tissues, such as bone marrow, thymus, spleen, lymph nodes and blood cells. As a result, DNA from fingernails with the quick is the best DNA source for genetic research in living marmosets.

  12. Detection of alcohol-tolerant hiochi bacteria by PCR.

    PubMed Central

    Nakagawa, T; Shimada, M; Mukai, H; Asada, K; Kato, I; Fujino, K; Sato, T

    1994-01-01

    We report a sensitive and rapid method for detection of hiochi bacteria by PCR. This method involves the electrophoresis of amplified DNA. Nucleotide sequences of the spacer region between 16S and 23S rRNA genes of 11 Lactobacillus strains were identified by analysis of PCR products. Five primers were designed by analysis of similarities among these sequences. A single cell of Lactobacillus casei subsp. casei could be detected when purified genomic DNA was used as the template. When various cell concentrations of L. casei subsp. casei were added to 50 ml of pasteurized sake and the cells were recovered, the detection limit was about one cell. No discrete band was observed in electrophoresis after PCR when human, Escherichia coli, mycoplasma, Acholeplasma, yeast, or mold DNA was used as the template. Images PMID:7510942

  13. Multiplex PCR for identification of herpes virus infections in adolescents.

    PubMed

    Durzyńska, Julia; Pacholska-Bogalska, Joanna; Kaczmarek, Maria; Hanć, Tomasz; Durda, Magdalena; Skrzypczak, Magdalena; Goździcka-Józefiak, Anna

    2011-02-01

    The aim of the study was to develop a multiplex PCR (mPCR) for a rapid and simultaneous detection of herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), and human cytomegalovirus (HCMV) DNA in squamous oral cells obtained from adolescents. Accuracy of the method was tested in a group of 513 adolescents, almost 11% of subjects were positive for infection with herpes viruses. Correlations with gender, age, and place of residence were sought. A similar incidence of HSV-2 and HCMV was found (4.3% and 5.4%, respectively) and the incidence of HSV-1 was the lowest (1%) in the study group. Conversely to HSV-2, HCMV was detected mostly in the youngest individuals. The same occurrence of all viruses was observed in boys and girls. The mPCR method described is suggested as a useful tool for epidemiologic studies of active herpes infections.

  14. 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.

  15. Design and evaluation of consensus PCR assays for henipaviruses.

    PubMed

    Feldman, K S; Foord, A; Heine, H G; Smith, I L; Boyd, V; Marsh, G A; Wood, J L N; Cunningham, A A; Wang, L-F

    2009-10-01

    Henipaviruses were first discovered in the 1990s, and their potential threat to public health is of increasing concern with increasing knowledge. Old-world fruit bats are the reservoir hosts for these viruses, and spill-over events cause lethal infections in a wide range of mammalian species, including humans. In anticipation of these spill-over events, and to investigate further the geographical range of these genetically diverse viruses, assays for detection of known and potentially novel strains of henipaviruses are required. The development of multiple consensus PCR assays for the detection of henipaviruses, including both SYBR Green and TaqMan real-time PCRs and a conventional heminested PCR is described. The assays are highly sensitive and have defined specificity. In addition to being useful tools for detection of known and novel henipaviruses, evaluation of assay efficiency and sensitivity across both biological and synthetic templates has provided valuable insight into consensus PCR design and use.

  16. [Application of rapid PCR to authenticate medicinal snakes].

    PubMed

    Chen, Kang; Jiang, Chao; Yuan, Yuan; Huang, Lu-Qi; Li, Man

    2014-10-01

    To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.

  17. Authentication of medicinal plants by SNP-based multiplex PCR.

    PubMed

    Lee, Ok Ran; Kim, Min-Kyeoung; Yang, Deok-Chun

    2012-01-01

    Highly variable intergenic spacer and intron regions from nuclear and cytoplasmic DNA have been used for species identification. Noncoding internal transcribed spacers (ITSs) located in 18S-5.8S-26S, and 5S ribosomal RNA genes (rDNAs) represent suitable region for medicinal plant authentication. Noncoding regions from two cytoplasmic DNA, chloroplast DNA (trnT-F intergenic spacer region), and mitochondrial DNA (fourth intron region of nad7 gene) are also successfully applied for the proper identification of medicinal plants. Single-nucleotide polymorphism (SNP) sites obtained from the amplification of intergenic spacer and intron regions are properly utilized for the verification of medicinal plants in species level using multiplex PCR. Multiplex PCR as a variant of PCR technique used to amplify more than two loci simultaneously.

  18. 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

  19. Rapid PCR of STR markers: Applications to human identification.

    PubMed

    Romsos, Erica L; Vallone, Peter M

    2015-09-01

    Multiplex PCR with fluorescently labeled primers has been an essential method for the amplification of short tandem repeats used in human identify testing. Within the STR workflow of extraction, quantitation, amplification, separation, and detection, multiplex PCR is commonly identified as the bottleneck in the process. The time requirement of up to three hours to complete 28-30 cycles of multiplex PCR for STR genotyping is the greatest amount of time required for a single step within the process. The historical use of commercially available thermal cyclers and heat stable polymerases may have given the impression that large multiplex will always require long PCR cycling times to ensure that all of the varying sized targets (typically 100-400bp) can be amplified in a balanced manner throughout the multiplex. However, with the advent of improved polymerases and faster thermal cyclers the time required for the amplification of large STR multiplexes is no longer on the order of three hours, but as little as 14min. Faster amplification times can be performed while retaining the balance and integrity of large multiplex PCRs by implementation of alternate polymerases and thermal cyclers. With the reduction in PCR cycling times there has also been an impact on the development of integrated and microfluidics devices which employ the use of reduced or rapid thermal cycling protocols as part of their integration. Similarly, PCR inhibitor resistant polymerases can also reduce overall STR processing times for reference samples by eliminating the need for DNA extraction and purification that is additionally implemented within the development of integrated DNA typing devices.

  20. Monitoring of geosmin producing Anabaena circinalis using quantitative PCR.

    PubMed

    Tsao, Hsiang-Wei; Michinaka, Atsuko; Yen, Hung-Kai; Giglio, Steven; Hobson, Peter; Monis, Paul; Lin, Tsair-Fuh

    2014-02-01

    Geosmin is one of the most commonly detected off-flavor chemicals present in reservoirs and drinking water systems. Quantitative real-time PCR (qPCR) is useful for quantifying geosmin-producers by focusing on the gene encoding geosmin synthase, which is responsible for geosmin synthesis. In this study, several primers and probes were designed and evaluated to detect the geosmin synthase gene in cyanobacteria. The specificity of primer and probe sets was tested using 21 strains of laboratory cultured cyanobacteria isolated from surface waters in Australia (18) and Taiwan (2), including 6 strains with geosmin producing ability. The results showed that the primers designed in this study could successfully detect all geosmin producing strains tested. The selected primers were used in a qPCR assay, and the calibration curves were linear from 5 × 10(1) to 5 × 10(5) copies mL(-1), with a high correlation coefficient (R(2) = 0.999). This method was then applied to analyze samples taken from Myponga Reservoir, South Australia, during a cyanobacterial bloom event. The results showed good correlations between qPCR techniques and traditional methods, including cell counts determined by microscopy and geosmin concentration measured using gas chromatography (GC) coupled with a mass selective detector (MSD). Results demonstrate that qPCR could be used for tracking geosmin-producing cyanobacteria in drinking water reservoirs. The qPCR assay may provide water utilities with the ability to properly characterize a taste and odor episode and choose appropriate management and treatment options.

  1. 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

  2. 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

  3. [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.

  4. Novel PCR assay for determining the genetic sex of mice.

    PubMed

    McFarlane, L; Truong, V; Palmer, J S; Wilhelm, D

    2013-01-01

    A number of studies require the determination of the genetic sex of mouse embryos before sexual differentiation and/or of mutant mice that display partial or complete sex reversal. The majority of current methods for sexing by PCR involve multiplexing of 2 primer pairs. We have developed a novel sexing PCR using a single primer pair that amplifies fragments from the X and the Y chromosome with a clear size difference between the respective amplicons. This assay provides a rapid and reliable method to identify the genetic sex of mice across different mouse strains.

  5. Sheep poxvirus identification by PCR in cell cultures.

    PubMed

    Mangana-Vougiouka, O; Markoulatos, P; Koptopoulos, G; Nomikou, K; Bakandritsos, N; Papadopoulos, O

    1999-01-01

    A simple, rapid and specific diagnostic polymerase chain reaction (PCR) method was developed for sheep poxvirus identification. The primers used were from the sequenced genomes of the capripox viruses KS-1 and InS-1. Six different sheep pox isolates were tested against two orf (parapox) and three animal herpesviruses as controls. Material from uninfected cell cultures was also used as control. The sensitivity of the PCR was approximately equivalent with each of the two primers and for the six sheep pox isolates. All the negative control virus DNAs were negative and differed clearly from those of the sheep pox strains.

  6. 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.

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

    PubMed

    Rodriguez-Lazaro, David; Hernandez, Marta

    2013-01-01

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

  8. [Basics of PCR and related techniques applied in veterinary parasitology].

    PubMed

    Ben Abderrazak, S

    2004-01-01

    We attempte through the following overall review pertaining to the basics of PCR techniques (Polymerase Chain Reaction), to introduce the main applications used in veterinary parasitology. A major problem restricting the application possibilities of molecular biology techniques is of quantitative nature. Amplification techniques represent a real revolution, for it makes possible the production of tens, even hundreds of nanogrammes of sequences when starting from very small quantities. The PCR technique has dramatically transformed the strategies used so far in molecular biology and subsequently research and medical diagnosis.

  9. Microsatellite amplification in plants: optimization procedure of major PCR components.

    PubMed

    Ghaffari, Sana; Hasnaoui, Nejib

    2013-01-01

    Microsatellites (SSRs) are the most informative and popular class of molecular markers used for diverse purposes, particularly in plants: genetic diversity study, marker assisted selection, breeding, mapping, phylogenetics and phylogeography, systematics, etc. They have become a routine technique practically in each laboratory for studying molecular plant genetics. Despite their wide utilization, however, setup and optimization of various conditions involved in PCR amplification is a prerequisite for reliable inference of results. In this chapter, we describe optimization of SSR-PCR conditions and give ranges of concentrations for different parameters. The protocol provided here is inspired from bench work on the use of microsatellite to study diversity of Vitis vinifera germplasm.

  10. Multiplex PCR for Detection and Typing of Porcine Circoviruses

    PubMed Central

    Ouardani, M.; Wilson, L.; Jetté, R.; Montpetit, C.; Dea, S.

    1999-01-01

    Sets of oligonucleotide primers were designed according to the sequences of the open reading frames (ORFs) ORF1 and ORF2 of the prototype nonpathogenic PK-15 strain of porcine circovirus (PCV) type 1 (PCV-1). By the PCR performed with the various primer sets, genomic DNA or RNA from other bacterial or viral pathogens of the respiratory tracts of pigs could not be amplified. A positive amplification reaction could be visualized with DNA extracted from a viral suspension containing as few as 10 viral particles per ml. No DNA fragment could be amplified from lysates of continuous porcine cell lines (PT, ST, and PFT cells) known to be negative for PCV. When tested with clinical samples from pigs, the results of the single PCR method showed nearly 93% (13 of 14 samples) correlation with histopathological and immunohistochemical findings. Interestingly, subclinical PCV infections could be detected by single PCR with clinical samples that have been submitted from animals with irrelevant cases of respiratory and/or enteric problems. On the basis of the nucleotide sequences of PCV strains (PCV-2) recently associated with outbreaks of postweaning multisystemic wasting syndrome (PWMS) in Quebec, Canada, pig farms, other primers were designed from the PCV-1 genome, and these primers failed to amplify genomic fragments specific to the ORF1 or ORF2 genes of clinical isolates associated with PWMS but amplified DNA from the PCV-1 strain. Two rapid multiplex PCR (mPCR) methods have been developed to distinguish between both genotypes of PCV. By those two mPCR methods, (i) species-specific primer pairs were used to amplify a DNA fragment of 488 bp specific for the ORF2 genes of both genotypes, whereas a 375-bp fragment was amplified from the ORF1 gene of the PCV-1 strain only, or (ii) species-specific primer pairs were used to amplify a DNA fragment of 646 bp specific for the ORF1 genes of both genotypes, whereas a 425-bp fragment was amplified from the ORF2 gene of the PCV-1 strain

  11. High-throughput quantitative real-time PCR.

    PubMed

    Arany, Zoltan P

    2008-07-01

    Recent technical advances in quantitative real-time PCR (qRT-PCR) have allowed for extensive miniaturization, thereby rendering the technique amenable to high-throughput assays. Large numbers of different nucleic acids can now rapidly be measured quantitatively. Many investigations can benefit from this approach, including determination of gene expression in hundreds of samples, determination of hundreds of genes in a few samples, or even quantification of nucleic acids other than mRNA. A simple technique is described here to quantify 1880 transcripts of choice from any number of starting RNA samples.

  12. 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.

  13. Critical appraisal of quantitative PCR results in colorectal cancer research: can we rely on published qPCR results?

    PubMed

    Dijkstra, J R; van Kempen, L C; Nagtegaal, I D; Bustin, S A

    2014-06-01

    The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published. But how reliable are the published results? Since the validity of gene expression data is greatly dependent on appropriate normalisation to compensate for sample-to-sample and run-to-run variation, we have evaluated the adequacy of normalisation procedures in qPCR-based experiments. Consequently, we assessed all colorectal cancer publications that made use of qPCR from 2006 until August 2013 for the number of reference genes used and whether they had been validated. Using even these minimal evaluation criteria, the validity of only three percent (6/179) of the publications can be adequately assessed. We describe common errors, and conclude that the current state of reporting on qPCR in colorectal cancer research is disquieting. Extrapolated to the study of cancer in general, it is clear that the majority of studies using qPCR cannot be reliably assessed and that at best, the results of these studies may or may not be valid and at worst, pervasive incorrect normalisation is resulting in the wholesale publication of incorrect conclusions. This survey demonstrates that the existence of guidelines, such as MIQE, is necessary but not sufficient to address this problem and suggests that the scientific community should examine its responsibility and be aware of the implications of these findings for current and future research.

  14. Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

    PubMed

    Yamada, Kazuhiro; Ibata, Ami; Suzuki, Masahiro; Matsumoto, Masakado; Yamashita, Teruo; Minagawa, Hiroko; Kurane, Ryuichiro

    2015-01-01

    Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future.

  15. qPCR assays to quantify genes and gene expression associated with microbial perchlorate reduction.

    PubMed

    De Long, Susan K; Kinney, Kerry A; Kirisits, Mary Jo

    2010-11-01

    Quantitative PCR (qPCR) assays targeting cld (developed in this work) and pcrA (previously described) were used to quantify these perchlorate-related genes in a perchlorate-reducing enrichment culture. Transcript copies were quantified in perchlorate-reducing Rhodocyclaceae strain JDS4. Oxygen and nitrate inhibited expression of cld and pcrA.

  16. 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

  17. Electrochemical genosensor based on peptide nucleic acid-mediated PCR and asymmetric PCR techniques: Electrostatic interactions with a metal cation.

    PubMed

    Kerman, Kagan; Vestergaard, Mun'delanji; Nagatani, Naoki; Takamura, Yuzuru; Tamiya, Eiichi

    2006-04-01

    The unique structure of peptide nucleic acids (PNAs), linking the N-(2-aminoethyl)glycine units that create a neutral backbone, and prevent it from acting as a primer for DNA polymerase, has been utilized in an electrochemical biosensor scheme for simple and sensitive detection of hybridization. When the PNA is targeted against a single-nucleotide polymorphism (SNP) or wild-type site on the gene, PNA-mediated polymerase chain reaction (PCR) clamping method effectively blocks the formation of a PCR product. In our report, PNA probe for PCR clamping was targeted against the wild-type site of alcohol dehydrogenase. The electrostatic interactions between the negatively charged DNA and neutral PNA molecules with redox-active metal cation cobalt(III)hexamine ([Co(NH3)6]3+) were monitored using differential pulse voltammetry. The electrostatic binding of [Co(NH3)6]3+ to DNA provided the basis for the discrimination against PNA/PNA, PNA/DNA, and DNA/DNA hybrid molecules. We have optimized the experimental conditions, such as probe concentration, [Co(NH3)6]3+ concentration, accumulation time for [Co(NH3)6]3+, and target concentration. A new pretreatment method has also been employed to allow fast and simple detection of hybridization reaction between the PCR amplicon and the probe on glassy carbon electrode (GCE) surface. This method was based on the application of a high-temperature treatment (95 degrees C, 5 min), followed by a 1-min incubation in the presence of DNA primers. The excess concentration of DNA primers prevented the rehybridization of the denatured strands, while enabling the target gene sequence to bind with the immobilized probe. Additionally, asymmetric PCR was employed to detect the presence of genetically modified organism in standard Roundup Ready soybean samples. The amplicons of asymmetric PCR, which were predominantly single-stranded DNA as a result of unequal primer concentration, hybridized with the DNA probe on the sensor surface efficiently. The

  18. Deletion-targeted multiplex PCR (DTM-PCR) for identification of Beijing/W genotypes of Mycobacterium tuberculosis.

    PubMed

    Chen, Jing; Tsolaki, Anthony G; Shen, Xin; Jiang, Xi; Mei, Jian; Gao, Qian

    2007-09-01

    Beijing/W strains of Mycobacterium tuberculosis cause the vast majority of tuberculosis cases in Shanghai, China. Such highly prevalent strains are considered as hypervirulent and are often associated with multi-drug resistance, treatment failure and HIV status. We present a reliable and fast detection method to identify these Beijing/W strains, which can be applied to screening large numbers of samples at low cost. Using this Deletion-Targeted Multiplex PCR (DTM-PCR) method for detecting these strains, we obtained 100% sensitivity and specificity.

  19. A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed.

    PubMed

    Rudi, Knut; Rud, Ida; Holck, Askild

    2003-06-01

    We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5' 'HEAD' region and a 3' region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1-2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5' nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The

  20. 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.

  1. 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,...

  2. Enumeration of Mycobacterium leprae Using Real-Time PCR

    PubMed Central

    Truman, Richard W.; Andrews, P. Kyle; Robbins, Naoko Y.; Adams, Linda B.; Krahenbuhl, James L.; Gillis, Thomas P.

    2008-01-01

    Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae–infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories. PMID:18982056

  3. 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.

  4. 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...

  5. 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 ...

  6. Real-Time PCR Quantification of Methanobrevibacter oralis in Periodontitis

    PubMed Central

    Bringuier, Amélie; Khelaifia, Saber; Richet, Hervé; Aboudharam, Gérard

    2013-01-01

    A real-time PCR assay developed to quantify Methanobrevibacter oralis indicated that its inoculum significantly correlated with periodontitis severity (P = 0.003), despite a nonsignificant difference in prevalence between controls (3/10) and patients (12/22) (P = 0.2, Fisher test). The M. oralis load can be used as a biomarker for periodontitis. PMID:23254133

  7. Fuel utilization in a progressive conversion reactor (PCR)

    SciTech Connect

    Leyse, C.F.; Judd, J.L.

    1981-05-01

    Preliminary studies indicate that for once-through fuel cycles, the PCR offers potential improvements over current LWRs in the following major areas: improved uranium utilization (reduced uranium demand), degraded plutonium product in spent fuel, reduced plutonium content of spent fuel, reduced amount of spent fuel, reduced fissile content of spent fuel, and reduced separative work.

  8. Identification of Staphylococcus spp. using (GTG)₅-PCR fingerprinting.

    PubMed

    Svec, Pavel; Pantůček, Roman; Petráš, Petr; Sedláček, Ivo; Nováková, Dana

    2010-12-01

    A group of 212 type and reference strains deposited in the Czech Collection of Microorganisms (Brno, Czech Republic) and covering 41 Staphylococcus species comprising 21 subspecies was characterised using rep-PCR fingerprinting with the (GTG)₅ primer in order to evaluate this method for identification of staphylococci. All strains were typeable using the (GTG)₅ primer and generated PCR products ranging from 200 to 4500 bp. Numerical analysis of the obtained fingerprints revealed (sub)species-specific clustering corresponding with the taxonomic position of analysed strains. Taxonomic position of selected strains representing the (sub)species that were distributed over multiple rep-PCR clusters was verified and confirmed by the partial rpoB gene sequencing. Staphylococcus caprae, Staphylococcus equorum, Staphylococcus sciuri, Staphylococcus piscifermentans, Staphylococcus xylosus, and Staphylococcus saprophyticus revealed heterogeneous fingerprints and each (sub)species was distributed over several clusters. However, representatives of the remaining Staphylococcus spp. were clearly separated in single (sub)species-specific clusters. These results showed rep-PCR with the (GTG)₅ primer as a fast and reliable method applicable for differentiation and straightforward identification of majority of Staphylococcus spp.

  9. 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.

  10. PCR Detection of Helicobacter pylori in Clinical Samples

    PubMed Central

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

    2014-01-01

    Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etio-logically 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

  11. 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.

  12. 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,…

  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. 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

  15. Application of nested PCR for diagnosis of histoplasmosis.

    PubMed

    Ohno, Hideaki; Tanabe, Koichi; Umeyama, Takashi; Kaneko, Yukihiro; Yamagoe, Satoshi; Miyazaki, Yoshitsugu

    2013-10-01

    Histoplasmosis is a fungal infection that, although not endemic in Japan, has seen a rise in the number of Japanese cases since the mid-1980s. Diagnosis of the disease is not straightforward, and the main method of detection, fungal culture (which has biosafety-related issues), is of low sensitivity in general. Alternative methods that depend on antibody or antigen detection have had limited use. We have developed a histoplasmosis detection method based on PCR amplification of the Histoplasma capsulatum M antigen gene. We compared this method with fungal culture and serological diagnostic techniques. Among five cases that were finally diagnosed as histoplasmosis, the fungal culture method was only successful in identifying one such case. Although the presence of anti-H. capsulatum antibodies was confirmed in three cases, our PCR method identified four of five cases of histoplasmosis. The performance of our PCR method could not be compared with the antigen detection method, which is used in the United States but is not routinely used in Japan. However, the PCR method was shown to have high sensitivity and specificity for H. capsulatum. Although the number of histoplasmosis cases examined in this study was small, our data suggest that the molecular diagnosis technique has potential for increasing the reliability of histoplasmosis diagnosis when used in combination with established methods.

  16. 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

  17. DNA extraction protocol for rapid PCR detection of pathogenic bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virtually all current assays for foodborne pathogens, including PCR assays, are conducted after lengthy cultural enrichment of the sample to increase the concentration of the target organism. This delays detection by many hours, prevents quantitation, and limits the ability to detect multiple organ...

  18. 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...

  19. 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) ...

  20. 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.

  1. Primer design for PCR reactions in forensic biology.

    PubMed

    Elkins, Kelly M

    2015-01-01

    The polymerase chain reaction (PCR) is a popular method to copy DNA in vitro. Its invention revolutionized fields ranging from clinical medicine to anthropology, molecular biology, and forensic biology. The method employs one of many available heat-stable DNA polymerases in a reaction that is repeated many times in situ. The DNA polymerase reads a template DNA strand and using the components of the reaction mix, catalyzes the addition of free 2'-deoxynucleotide triphosphate nitrogenous bases to short segment of DNA that forms a complement with the template via Watson-Crick base pairing. This short segment of DNA is referred to as a PCR primer and it is essential to the success of the reaction. The most widely used application of PCR in forensic labs is the amplification of short tandem repeat (STR) loci used in DNA typing. The STRs are routinely evaluated in concert with 16 or more reactions, a multiplex, run in one test tube simultaneously. In a multiplex, it is essential that the primers work specifically and accurately on the intended reactions without hindering the other reactions. The primers, which are very specific, also can be used to probe single nucleotide polymorphisms (SNPs) in a DNA sequence of interest by single base extension. Primers are often designed using one of many available automated software packages. Here the process of manually designing PCR primers for forensic biology using no-cost software is described.

  2. 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.

  3. A novel nested PCR for the diagnosis of calicivirus infections in the cat.

    PubMed

    Marsilio, Fulvio; Di Martino, Barbara; Decaro, Nicola; Buonavoglia, Canio

    2005-01-05

    A novel nested PCR (nPCR) assay is reported on the diagnosis of the feline calicivirus (FCV) infection. The test was performed on 47 ocular and 40 pharyngeal swabs collected from 47 cats with respiratory syndrome; among the 87 samples examined, 18 ocular and 23 pharyngeal swabs were positive in nPCR. The nPCR sensitivity was compared to other diagnostic techniques such as virus isolation on cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR). The nPCR was more sensitive than the virus isolation and RT-PCR; therefore, it can be used for calicivirosis diagnosis in cats.

  4. Two-temperature LATE-PCR endpoint genotyping

    PubMed Central

    Sanchez, J Aquiles; Abramowitz, Jessica D; Salk, Jesse J; Reis, Arthur H; Rice, John E; Pierce, Kenneth E; Wangh, Lawrence J

    2006-01-01

    Background In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. Results Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the

  5. Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications.

    PubMed

    Cao, Lei; Cui, Xingye; Hu, Jie; Li, Zedong; Choi, Jane Ru; Yang, Qingzhen; Lin, Min; Ying Hui, Li; Xu, Feng

    2017-04-15

    Since the invention of polymerase chain reaction (PCR) in 1985, PCR has played a significant role in molecular diagnostics for genetic diseases, pathogens, oncogenes and forensic identification. In the past three decades, PCR has evolved from end-point PCR, through real-time PCR, to its current version, which is the absolute quantitive digital PCR (dPCR). In this review, we first discuss the principles of all key steps of dPCR, i.e., sample dispersion, amplification, and quantification, covering commercialized apparatuses and other devices still under lab development. We highlight the advantages and disadvantages of different technologies based on these steps, and discuss the emerging biomedical applications of dPCR. Finally, we provide a glimpse of the existing challenges and future perspectives for dPCR.

  6. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything

    PubMed Central

    Kralik, Petr; Ricchi, Matteo

    2017-01-01

    Real time PCR (quantitative PCR, qPCR) is now a well-established method for the detection, quantification, and typing of different microbial agents in the areas of clinical and veterinary diagnostics and food safety. Although the concept of PCR is relatively simple, there are specific issues in qPCR that developers and users of this technology must bear in mind. These include the use of correct terminology and definitions, understanding of the principle of PCR, difficulties with interpretation and presentation of data, the limitations of qPCR in different areas of microbial diagnostics and parameters important for the description of qPCR performance. It is not our intention in this review to describe every single aspect of qPCR design, optimization, and validation; however, it is our hope that this basic guide will help to orient beginners and users of qPCR in the use of this powerful technique. PMID:28210243

  7. 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.

  8. STITCHER: A web resource for high-throughput design of primers for overlapping PCR applications.

    PubMed

    O'Halloran, Damien M

    2015-06-01

    Overlapping PCR is routinely used in a wide number of molecular applications. These include stitching PCR fragments together, generating fluorescent transcriptional and translational fusions, inserting mutations, making deletions, and PCR cloning. Overlapping PCR is also used for genotyping by traditional PCR techniques and in detection experiments using techniques such as loop-mediated isothermal amplification (LAMP). STITCHER is a web tool providing a central resource for researchers conducting all types of overlapping PCR experiments with an intuitive interface for automated primer design that's fast, easy to use, and freely available online (http://ohalloranlab.net/STITCHER.html). STITCHER can handle both single sequence and multi-sequence input, and specific features facilitate numerous other PCR applications, including assembly PCR, adapter PCR, and primer walking. Field PCR, and in particular, LAMP, offers promise as an on site tool for pathogen detection in underdeveloped areas, and STITCHER includes off-target detection features for pathogens commonly targeted using LAMP technology.

  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

    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.

  10. 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

  11. Detection of glycopeptide resistance genes in enterococci by multiplex PCR

    PubMed Central

    Bhatt, Puneet; Sahni, A.K.; Praharaj, A.K.; Grover, Naveen; Kumar, Mahadevan; Chaudhari, C.N.; Khajuria, Atul

    2014-01-01

    Background Vancomycin Resistant Enterococci (VRE) are a major cause of nosocomial infections. There are various phenotypic and genotypic methods of detection of glycopeptide resistance in enterococci. This study utilizes multiplex PCR for reliable detection of various glycopeptides resistance genes in VRE. Method This study was conducted to detect and to assess the prevalence of vancomycin resistance among enterococci isolates. From October 2011 to June 2013, a total of 96 non-repetitive isolates of enterococci from various clinical samples were analyzed. VRE were identified by Kirby Bauer disc diffusion method with Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) of all isolates for vancomycin and teicoplanin was determined by E-test. Multiplex PCR was carried out for all enterococci isolates using six sets of primers. Results Out of 96 isolates, 14 (14.6%) were found to be resistant to vancomycin by vancomycin E-test method (MIC ≥32 μg/ml). Out of these 14 isolates, 13 were also resistant to teicoplanin (MIC ≥16 μg/ml). VanA gene was detected in all the 14 isolates by Multiplex PCR. One of the PCR amplicons was sent for sequencing and the sequence received was submitted in the GenBank (GenBank accession no. KF181100). Conclusion Prevalence of VRE in this study was 14.6%. Multiplex PCR is a robust, sensitive and specific technique, which can be used for rapid detection of various glycopeptide resistance genes. Rapid identification of patients infected or colonized with VRE is essential for implementation of appropriate control measures to prevent their spread. PMID:25609863

  12. 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.

  13. Discrimination between Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capricolum using PCR-RFLP and PCR.

    PubMed

    Cillara, Grazia; Manca, Maria Giovanna; Longheu, Carla; Tola, Sebastiana

    2015-09-01

    In this study, the dihydrolipoyl dehydrogenase (lpdA) gene was used to distinguish Mycoplasma mycoides subsp. capri (Mmc) from Mycoplasma capricolum subsp. capricolum (Mcc), two of four Mycoplasma species that cause contagious agalactia in sheep and goats. After alignment of nucleotide sequences of both species, specific primer sets were designed from unchanging and variable gene segments. The first primer set LPD-C1-F/LPD-C1-R was used to amplify a 911 bp fragment that was subsequently co-digested with FastDigest PstI, SspI, EcoRI and ClaI enzymes. The PCR-RFLP profiles differentiated the two mycoplasma species. The second primer set was used to distinguish Mmc from Mcc by single tube PCR. Both methods were further applied to identify 54 isolates collected from dairy herds from different provinces in Sardinia. The results of this study showed that PCR-RFLP and PCR could be used in routine diagnosis for rapid and specific simultaneous discrimination of Mmc and Mcc.

  14. 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.

  15. [Primers design and optimization of PCR and nested-PCR assays for the specific detection of Tritrichomonas foetus].

    PubMed

    Fernandes, Paula Rogério; Da Silva, Andréa Caetano; Gambarini, Maria Lúcia; Linhares, Guido Fontgalland C

    2008-01-01

    Tritrichomonas foetus is a pathogenic protozoan that causes a venereal disease in cattle known as bovine genital tricomonosis. In spite of the efficacy to recognize the target genomic DNA, the protocols so far developed for the diagnosis of this organism by PCR promote some inespecific amplifications or they are unable to discriminate T. foetus against other species within the genus. The objective of this study was to assess and optimize PCR and nested-PCR assays for the specific diagnosis of T. foetus, using novel primers selected from the alignment of sequences of the genes 18S rRNA, 5.8S rRNA, 28S rRNA and of the internal transcribed spacers of the rDNA (ITS1 and ITS2). A pair of primers was constructed for the genus-specific amplification of a 648 bp fragment and two others to amplify T. foetus species-specific fragments of 343 and 429 bp. No cross amplification was observed against Bos taurus genomic DNA neither against the DNA of usual bovine genital pathogens. Both, single and nested-PCR assays, presented analytical sensitivity to detect at least two T. foetus organisms.

  16. 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...

  17. 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.

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. False-Positive Viability PCR Results: An Association with Microtubes.

    PubMed

    Agustí, Gemma; Fittipaldi, Mariana; Codony, Francesc

    2017-03-01

    Currently, one of the most challenged points to expand the use of viability PCR technique is achieving the complete exclusion of dead cells amplification signals, thus avoiding the overestimation of live cells population. Considering that, and based on the hypothesis that DNA may be retained by microtube walls, the impact of the microtube was addressed on signals from live and heat-killed cells. A double-dye reagent, PEMAX™, which comprises a mix of photo-reactive azide forms of phenanthridium, was used in this work. We found that if both the incubation and the photoactivation steps are carried out in different microtubes, the dead cell signal is greatly reduced than when those steps are done in the same tube. Therefore, the strategy depicted in this study presents a simple and efficient step in minimizing false-positive signal when employing viability PCR.

  5. Tuberculosis diagnosed by PCR analysis of synovial fluid.

    PubMed

    Fujimoto, Nobukazu; Gemba, Kenichi; Yao, Atsushi; Ozaki, Shinji; Ono, Katsuichiro; Wada, Sae; Fujii, Yasuhiro; Namba, Yoshifumi; Kishimoto, Takumi

    2010-02-01

    Tuberculosis is a leading cause of mortality due to an infectious agent worldwide. It often affects multiple organs by hematogenous spread of Mycobacterium tuberculosis, but knee-joint involvement is extremely rare, comprising approximately 0.1% of all forms of tuberculosis. We present a case of tuberculous pleuritis with knee-joint involvement. Cytological and biochemical analysis of the pleural fluid and a biopsy specimen of the cervical lymph node indicated tuberculosis, but a definitive diagnosis was not given. A confirmed diagnosis was finally obtained through PCR analysis of the synovial fluid. Tuberculosis should be included in the differential diagnosis in patients with persistent pain and swelling of the knee. PCR analysis of the synovial fluid is a quick and useful method for the diagnosis.

  6. Development of a PCR for identification of Bordetella hinzii.

    PubMed

    Register, Karen B

    2013-06-01

    Bordetella hinzii infects primarily poultry and immunocompromised humans. It is closely related to the etiologic agent of turkey coryza, Bordetella avium. Distinguishing between B. avium and B. hinzii is difficult, and there is no method for identification of B. hinzii suitable for use by diagnostic laboratories. This report details the development of a B. hinzii-specific PCR targeting the ompA gene. Assay sensitivity is 100% based on analysis of 48 B. hinzii isolates from diverse geographic locations representing all known ribotypes. Evaluation of 71 isolates of B. avium and 20 other bacterial isolates from poultry, comprising gram-negative and gram-positive commensals and pathogens of nine genera, demonstrated an assay specificity of 100%. The ompA PCR is a rapid, reliable, and accurate method for identification of B. hinzii and provides a valuable new tool for veterinary diagnostic laboratories investigating poultry respiratory disease outbreaks.

  7. DPPrimer – A Degenerate PCR Primer Design Tool

    PubMed Central

    Gahoi, Shachi; Arya, L; Anil, Rai; Marla, ss

    2013-01-01

    Designed degenerate primers unlike conventional primers are superior in matching and amplification of large number of genes, from related gene families. DPPrimer tool was designed to predict primers for PCR amplification of homologous gene from related or diverse plant species. The key features of this tool include platform independence and user friendliness in primer design. Embedded features such as search for functional domains, similarity score selection and phylogebetic tree further enhance the user friendliness of DPPrimer tool. Performance of DPPrimer tool was evaluated by successful PCR amplification of ADP-glucose phosphorylase genes from wheat, barley and rice. Availability DPPrimer is freely accessible at http://202.141.12.147/DGEN_tool/index.html PMID:24307773

  8. Zip nucleic acids are potent hydrolysis probes for quantitative PCR

    PubMed Central

    Paris, Clément; Moreau, Valérie; Deglane, Gaëlle; Voirin, Emilie; Erbacher, Patrick; Lenne-Samuel, Nathalie

    2010-01-01

    Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3′ end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes. PMID:20071749

  9. 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.

  10. Chemical surface management for micro PCR in silicon chip thermocyclers

    NASA Astrophysics Data System (ADS)

    Felbel, Jana; Bieber, Ivonne; Koehler, Johann M.

    2002-11-01

    Silicon, silicon dioxide, glass and other key materials of micro system technology show an inhibiting effect on PCR. This negative influence becomes seriously, if devices are miniaturized, particularly in case of flow-through devices due to their high surface to volume ratio. In contrast, alkyl-substituted surfaces do not inhibit the reaction. Although the silanization improves the compatibility, the suppression of inhibition by wall surface treatment was not stable over longer time intervals. Therefore, the stability of chemical surface modifications was studied in dependence of silanization, material, pH, temperature and buffer composition. The efficiency of surface covering by molecular substitution was characterized by wetting experiments as well as by PCR test runs. The results show that the surface treatment can be optimized by the choice of silanization agents and the concentration of surface active additives.

  11. DNA probe and PCR-specific reaction for Lactobacillus plantarum.

    PubMed

    Quere, F; Deschamps, A; Urdaci, M C

    1997-06-01

    A 300 bp DNA fragment of Lactobacillus plantarum isolated by randomly amplified polymorphic DNA (RAPD) analysis was cloned and sequenced. This fragment was tested using a dot-blot DNA hybridization to technique for its ability to identify Lact. plantarum strains. This probe hybridized with all Lact. plantarum strains tested and with some strains of Lact. pentosus, albeit more weakly. Two internal primers of this probe were selected (LbP11 and LbP12) and polymerase chain reaction (PCR) was carried out. All Lact. plantarum strains tested amplified a 250 bp fragment contrary to the other LAB species tested. This specific PCR for Lact. plantarum was also performed from colonies grown on MRS medium with similar results. These methods enabled the rapid and specific detection and identification of Lact. plantarum.

  12. Targeted resequencing and variant validation using pxlence PCR assays.

    PubMed

    Coppieters, Frauke; Verniers, Kimberly; De Leeneer, Kim; Vandesompele, Jo; Lefever, Steve

    2016-01-01

    The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage. In addition, data indicates a positive correlation between the predictive in silico specificity score and the amount of assay non-specific coverage.

  13. Targeted resequencing and variant validation using pxlence PCR assays

    PubMed Central

    Coppieters, Frauke; Verniers, Kimberly; De Leeneer, Kim; Vandesompele, Jo; Lefever, Steve

    2015-01-01

    The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage. In addition, data indicates a positive correlation between the predictive in silico specificity score and the amount of assay non-specific coverage. PMID:27077044

  14. SNP genotyping using single-tube fluorescent bidirectional PCR.

    PubMed

    Waterfall, Christy M; Cobb, Benjamin D

    2002-07-01

    SNP genotyping is a well-populatedfield with a large number of assay formats offering accurate allelic discrimination. However, there remains a discord between the ultimate goal of rapid, inexpensive assays that do not require complex design considerations and involved optimization strategies. We describe the first integration of bidirectional allele-specific amplification, SYBR Green I, and rapid-cycle PCR to provide a homogeneous SNP-typing assay. Wild-type, mutant, and heterozygous alleles were easily discriminated in a single tube using melt curve profiling of PCR products alone. We demonstrate the effectiveness and reliability of this assay with a blinded trial using clinical samples from individuals with sickle cell anemia, sickle cell trait, or unaffected individuals. The tests were completed in less than 30 min without expensive fluorogenic probes, prohibiting design rules, or lengthy downstream processing for product analysis.

  15. 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

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

    DOEpatents

    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.

  17. Thermosiphon-based PCR reactor: experiment and modeling.

    PubMed

    Chen, Zongyuan; Qian, Shizhi; Abrams, William R; Malamud, Daniel; Bau, Haim H

    2004-07-01

    A self-actuated, flow-cycling polymerase chain reaction (PCR) reactor that takes advantage of buoyancy forces to continuously circulate reagents in a closed loop through various thermal zones has been constructed, tested, and modeled. The heating required for the PCR is advantageously used to induce fluid motion without the need for a pump. Flow velocities on the order of millimeters per second are readily attainable. In our preliminary prototype, we measured a cross-sectionally averaged velocity of 2.5 mm/s and a cycle time of 104 s. The flow velocity is nearly independent of the loop's length, making the device readily scalable. Successful amplifications of 700- and 305-bp fragments of Bacillus cereus genomic DNA have been demonstrated. Since the device does not require any moving parts, it is particularly suitable for miniature systems.

  18. Culture independent PCR: an alternative enzyme discovery strategy.

    PubMed

    Jacobsen, Jonas; Lydolph, Magnus; Lange, Lene

    2005-01-01

    Degenerate primers were designed for use in a culture-independent PCR screening of DNA from composite fungal communities, inhabiting residues of corn stovers and leaves. According to similarity searches and alignments amplified clone sequences affiliated with glycosyl hydrolase family 7 and glycosyl hydrolase family 45 though significant sequence divergence was observed. Glycosyl hydrolases from families 7 and 45 play a crucial role in biomass conversion to fuel ethanol. Research in this renewable energy source has two objectives: (i) To contribute to development of a renewable alternative to world's limited crude fossil oil reserves and (ii) to reduce air pollution. Amplification with 18S rDNA-specific primers revealed species within the ascomycetous orders Sordariales and Hypocreales as well as basidiomycetous order Agaricales to be present in these communities. Our study documents the value of culture-independent PCR in microbial diversity studies and could add to development of a new enzyme screening technology.

  19. Specific detection by PCR of Streptococcus agalactiae in milk.

    PubMed

    Martinez, G; Harel, J; Gottschalk, M

    2001-01-01

    The aim of this study was to develop a simple and specific method for direct detection of Streptococcus agalactiae from cow's milk. The method was based on polymerase chain reaction (PCR) using species-specific and universal primers derived from the 16S rRNA gene. The amplification product was verified by restriction endonuclease digest and sequencing. Specific identification was proven on a collection of 147 S. agalactiae isolates of bovine and human origin. In addition, 17 strains belonging to different bacterial species that potentially can be found in milk samples also tested negative. The PCR developed was used for direct detection of S. agalactiae in milk, using for the first time with gram-positive bacteria the nucleic acid-binding properties of diatomaceous earth. The test, which has high specificity, high sensitivity (100 cfu/mL), and can be carried out in less than 24 h, represents an innovative diagnostic tool for the detection of S. agalactiae in milk.

  20. 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.

  1. Evaluation of PCR Systems for Field Screening of Bacillus anthracis

    PubMed Central

    Ozanich, Richard M.; Colburn, Heather A.; Victry, Kristin D.; Bartholomew, Rachel A.; Arce, Jennifer S.; Heredia-Langner, Alejandro; Jarman, Kristin; Kreuzer, Helen W.

    2017-01-01

    There is little published data on the performance of hand-portable polymerase chain reaction (PCR) systems that can be used by first responders to determine if a suspicious powder contains a potential biothreat agent. We evaluated 5 commercially available hand-portable PCR instruments for detection of Bacillus anthracis. We used a cost-effective, statistically based test plan to evaluate systems at performance levels ranging from 0.85-0.95 lower confidence bound (LCB) of the probability of detection (POD) at confidence levels of 80% to 95%. We assessed specificity using purified genomic DNA from 13 B. anthracis strains and 18 Bacillus near neighbors, potential interference with 22 suspicious powders that are commonly encountered in the field by first responders during suspected biothreat incidents, and the potential for PCR inhibition when B. anthracis spores were spiked into these powders. Our results indicate that 3 of the 5 systems achieved 0.95 LCB of the probability of detection with 95% confidence levels at test concentrations of 2,000 genome equivalents/mL (GE/mL), which is comparable to 2,000 spores/mL. This is more than sufficient sensitivity for screening visible suspicious powders. These systems exhibited no false-positive results or PCR inhibition with common suspicious powders and reliably detected B. anthracis spores spiked into these powders, though some issues with assay controls were observed. Our testing approach enables efficient performance testing using a statistically rigorous and cost-effective test plan to generate performance data that allow users to make informed decisions regarding the purchase and use of field biodetection equipment. PMID:28192050

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

    EPA Pesticide Factsheets

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

  3. 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

  4. PCR detection of aflatoxin producing fungi and its limitations.

    PubMed

    Levin, Robert E

    2012-05-01

    Unlike bacterial toxins that are primarily peptides and are therefore encoded by a single gene, fungal toxins such as the aflatoxins are multi-ring structures and therefore require a sequence of structural genes for their biological synthesis. There is therefore no specific PCR for any one of the four biologically produced aflatoxins. Unfortunately, the structural genes presently in use for PCR detection of aflatoxin producing fungi are also involved in the synthesis of other fungal toxins such as sterigmatocystin by Aspergillus versicolor and Aspergillus nidulans and therefore lack absolute specificity for aflatoxin producing fungi (Table 1). In addition, the genomic presence of several structural genes involved in aflatoxin biosynthesis does not guarantee the production of aflatoxin by all isolates of Aspergillus flavus and Aspergillus parasiticus. The most widely used DNA target regions for discriminating Aspergillus species are those of the rDNA complex, mainly the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) and the variable regions in the 5'-end of the 28S rRNA gene. Since these sequence regions are unrelated to the structural genes involved in aflatoxin biosynthesis there successful amplification can be used for species identification but do not confirm aflatoxin production. This review therefore presents the various approaches and limitations in the use of the PCR in attempting to detect aflatoxin producing fungi.

  5. STITCHER 2.0: primer design for overlapping PCR applications.

    PubMed

    O'Halloran, Damien M; Uriagereka-Herburger, Isabel; Bode, Katrin

    2017-03-30

    Overlapping polymerase chain reaction (PCR) is a common technique used by researchers in very diverse fields that enables the user to 'stitch' individual pieces of DNA together. Previously, we have reported a web based tool called STITCHER that provides a platform for researchers to automate the design of primers for overlapping PCR applications. Here we present STITCHER 2.0, which represents a substantial update to STITCHER. STITCHER 2.0 is a newly designed web tool that automates the design of primers for overlapping PCR. Unlike STITCHER, STITCHER 2.0 considers diverse algorithmic parameters, and returns multiple result files that include a facility for the user to draw their own primers as well as comprehensive visual guides to the user's input, output, and designed primers. These result files provide greater control and insight during experimental design and troubleshooting. STITCHER 2.0 is freely available to all users without signup or login requirements and can be accessed at the following webpage: www.ohalloranlab.net/STITCHER2.html.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. Identification of Candida spp. by phenotypic tests and PCR

    PubMed Central

    Marinho, Sandra Aparecida; Teixeira, Alice Becker; Santos, Otávio Silveira; Cazanova, Ricardo Flores; Ferreira, Carlos Alexandre Sanchez; Cherubini, Karen; de Oliveira, Sílvia Dias

    2010-01-01

    The correct identification of Candida species is of great importance, as it presents prognostic and therapeutical significance, allowing an early and appropriate antifungical therapy. The purpose of this study was to identify isolates of Candida spp. from oral mucosa of 38 patients with oral candidosis evaluated in 2004 by phenotypic methods and PCR, discriminating C. albicans from the other Candida species. The tests used for phenotypic analysis were germ-tube and chlamydoconidia production, culture in CHROMAgar™ Candida, carbohydrate assimilation test, growth at 45ºC and culture in Tween 80 agar. Genotypic confirmation was performed by PCR. Phenotypic tests showed that 63.2% strains formed germ-tubes, 73.7% produced chlamydoconidia, and 63.2% showed green colonies in chromogenic medium, presumptively indicating C. albicans or C. dubliniensis. The carbohydrate assimilation test confirmed these results. A total of 21% strains were identified as C. krusei and 13.2% were indicative of C. tropicalis. Of these later strains, three produced chlamydoconidia. The association of other phenotypic tests with culture in Tween 80 agar identified 95.8% of strains as C. albicans and 4.2% as C. dubliniensis. All 24 strains indicative of C. albicans and C. dubliniensis were confirmed by PCR as C. albicans. PMID:24031493

  11. [PCR-based diagnosis of mucormycosis in tissue samples].

    PubMed

    Bialek, R; Zelck, U E

    2013-11-01

    Mucormycosis is characterized by a rapid, often fatal progression. Early diagnosis of invasive mucormycosis is the key for timely therapeutic intervention and improved survival. Contrary to the more prevalent aspergillosis, effective antifungal therapy of mucormycosis is mainly limited to amphotericin B. Given the importance to guide the timely initiation of amphotericin B and possible surgical intervention, rapid and specific identification of fungal hyphae is essential. Conventional histopathology depends on abundance and morphology of the fungi as well as on the skills of the personnel, and usually shows an accuracy of 80 %. PCR assays targeting fungal ribosomal genes to identify Mucorales at least at genus level increase sensitivity, allow a rapid identification as well as detection of double mold infections. Thus, PCR assays are beneficial to complement existing approaches. They are recommended to rapidly specify tissue diagnosis and accurate identification of fungi. This will help to guide effective therapy and thereby, survival will increase. Retrospective analyses of mucormycosis by PCR help to evaluate therapeutic interventions and will optimize treatment options.

  12. 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.

  13. 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

  14. STITCHER 2.0: primer design for overlapping PCR applications

    PubMed Central

    O’Halloran, Damien M.; Uriagereka-Herburger, Isabel; Bode, Katrin

    2017-01-01

    Overlapping polymerase chain reaction (PCR) is a common technique used by researchers in very diverse fields that enables the user to ‘stitch’ individual pieces of DNA together. Previously, we have reported a web based tool called STITCHER that provides a platform for researchers to automate the design of primers for overlapping PCR applications. Here we present STITCHER 2.0, which represents a substantial update to STITCHER. STITCHER 2.0 is a newly designed web tool that automates the design of primers for overlapping PCR. Unlike STITCHER, STITCHER 2.0 considers diverse algorithmic parameters, and returns multiple result files that include a facility for the user to draw their own primers as well as comprehensive visual guides to the user’s input, output, and designed primers. These result files provide greater control and insight during experimental design and troubleshooting. STITCHER 2.0 is freely available to all users without signup or login requirements and can be accessed at the following webpage: www.ohalloranlab.net/STITCHER2.html. PMID:28358011

  15. A PCR-based linkage map of human chromosome 1

    SciTech Connect

    Engelstein, M.; Hudson, T.J.; Lane, J.M.; Lee, M.K.; Dracopoli, C. ); Leverone, B.; Landes, G.M. ); Peltonen, L. ); Weber, J.L. )

    1993-02-01

    A genetic linkage map of human chromosome 1 based entirely on PCR-typable markers has been developed using 38 simple sequence repeat (SSR) polymorphisms. These SSRs include 36 dinucleotide repeats and 2 tetranucleotide repeats. The average heterozygosity at these markers was 0.73 and ranged form 0.52 to 0.95. Multipoint linkage analysis was used to develop a map of these 38 markers in which the relative placement of each locus is supported by likelihood odds > 1000:1. This PCR-based map was anchored at the centromere by the D1Z5 [alpha]-satellite polymorphism, and the ends of the map were defined by D1Z2 and D1S68, which are the most distal loci in the CEPH consortium map of chromosome 1. The sex-averaged, male, and female maps extend for 328, 273, and 409 cM, respectively. The average distance between markers on the sex-averaged map is 8 cM, and the largest interval is 32 cM. This map of highly informative PCR-based markers will provide a rapid means of screening human chromosome 1 for the presence of disease genes. 36 refs., 4 figs., 4 tabs.

  16. 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.

  17. Colorimetric Integrated PCR Protocol for Rapid Detection of Vibrio parahaemolyticus

    PubMed Central

    Cheng, Kewen; Pan, Daodong; Teng, Jun; Yao, Li; Ye, Yingwang; Xue, Feng; Xia, Fan; Chen, Wei

    2016-01-01

    Rapid detection of pathogens is of great significance for food safety and disease diagnosis. A new colorimetric method for rapid and easy detection of Vibrio parahaemolyticus (V. parahaemolyticus or Vp) has been developed in this research. A specific sequence was designed and integrated with the forward primer for molecular detection of Vp. This specific sequence was tested and treated as the horseradish peroxidase (HRP)-mimicking DNAzyme and could be amplified during the polymerase chain reaction (PCR) process. The products of PCR including the sequence of HRP-mimicking DNAzyme could produce the distinguished color in the presence of catalysis substrates. The optical signal of the catalysis reaction, which is in a linear relationship with the initial template of Vp, could be determined with the naked eye or measured with Ultraviolet-visible (UV-vis) for qualitative and quantitative detections, respectively. Based on the optical signal intensity, rapid and easy detection of Vp was successfully achieved with satisfied sensitivity and specificity. Furthermore, the detection of tdh, trh, tlh and toxR virulence genes of two Vp species (Vp 33847 and Vp 17802) were all performed successfully with this developed colorimetric integrated PCR protocol, which demonstrated potential applicability for the rapid detection of other bacteria. PMID:27690041

  18. Monitoring infection: from blood culture to polymerase chain reaction (PCR).

    PubMed

    Book, Malte; Lehmann, Lutz Eric; Zhang, XiangHong; Stüber, Frank

    2013-06-01

    In patients with sepsis, diagnosis of blood stream infection (BSI) is a key concern to the therapist. Direct verification of pathogens in the blood stream executed by blood cultures (BC) still is regarded as the gold standard up to date. The quickest possible initiation of an appropriate antimicrobial therapy is a cornerstone of an effective therapy. Moreover, in this view BC can also serve to identify antimicrobial agents to target the pathogen. However, when employing BC the time needed until microbiological results are available ranges from 24 up to 72 h. Moreover, infections caused by multiple pathogens often remain undetected and concurrent antibiotic therapy may lower the overall sensitivity. Alternative pathogen characterization can be performed by polymerase chain reaction (PCR) based amplification methods. Results using PCR can be obtained within 6-8 h. Therefore, the time delay until an appropriate therapy can be reduced enormously. Moreover, these methods have the potential to enhance the sensitivity in the diagnosis of blood stream infections. Therefore, PCR based methods might be a valuable adjunct to present procedures of diagnosing bacteraemia.

  19. 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

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

    PubMed

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

    2008-05-01

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

  1. 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.

  2. Enzymological considerations for a theoretical description of the quantitative competitive polymerase chain reaction (QC-PCR).

    PubMed

    Schnell, S; Mendoza, C

    1997-02-21

    The enzymological principles of the polymerase chain reaction (PCR) and of the quantitative competitive PCR (QC-PCR) are developed, proposing a theoretical framework that will facilitate quantification in experimental methodologies. It is demonstrated that the specificity of the QC-PCR, i.e. the ratio of the target initial velocity to that of the competitor template, remains constant not only during a particular amplification but also for increasing initial competitor concentrations. Linear fitting procedures are thus recommended that will enable a quantitative estimate of the initial target concentration. Finally, expressions for the efficiency of the PCR and QC-PCR are derived that are in agreement with previous experimental inferences.

  3. Nuclear DNA PCR-RFLPs that distinguish African and European honey bee groups of subspecies. II: Conversion of long PCR markers to standard PCR.

    PubMed

    Suazo, Alonso; Hall, H Glenn

    2002-08-01

    Nuclear DNA PCR-RFLPs previously found in amplifications of three long (> 5 kbp) anonymous regions of DNA were made analyzable using standard PCR procedures. RFLP analyses were simplified by restricting the amplifications to sections, within each locus, that contained most of the informative polymorphic sites. AluI digests of locus L-1 section 2 (L-1S2) revealed three suballeles of which one was African-specific (Apis mellifera scutellata Lepeletier) and one was east European-predominant (A. m. ligustica Spinola, A. m. carnica Pollman, and A. m. caucasica Gorbachev). Alleles found originally at locus L-2 with AvaI were determined in RFLP analysis of two sections, L-2S1int and L-2S2, resulting in two African-specific and two east European-predominant suballeles. Suballele identity was determined by the combination of banding patterns from both fragments. Polymorphisms revealed by HaeIII in locus L-2 were analyzed in amplifications and digests of L-2SM1int. an 830 bpfragment within L-2S1. Seven suballeles were found of which two were African-specific and three were east European-specific or predominant, including one suballele specific to the east European subspecies A. m. caucasica. In locus L-5, RFLPs were detected with HaeIII, DdeI, and SpeI. HaeIII polymorphisms were analyzed by amplification and digestion offragments L-5S1xt and L-5S1ter: Five suballeles were found of which three were African-specific and one east European-predominant. For DdeI, all five alleles originally found with long PCR could be identified in RFLP analyses of three sections. Two African-specific, one east European-specific, and one west European-predominant (A. m. mellifera L. and A. m. iberica Goetze) suballeles were found. A west European-predominant suballele was also found in RFLP analysis of L-5S3 with SpeI. Allele frequency data from Old World and US. populations are presented.

  4. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    PubMed Central

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-01-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

  5. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

    PubMed

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-10-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

  6. 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.

  7. Quantitative real-time PCR (qPCR) for the detection and quantification of dactylogyrid parasites infecting Lutjanus guttatus.

    PubMed

    Soler-Jiménez, L C; García-Gasca, A; Fajer-Ávila, E J

    2017-03-07

    Severe infections of the spotted rose snapper Lutjanus guttatus resulting from dactylogyrid monogeneans present a risk to aquaculture. Currently, the diagnosis of this infection requires the morphological identification and manual quantification of parasites. Based on the characterization of the 28S rRNA gene of dactylogyrid species present in L. guttatus, specific primers were designed for real-time polymerase chain reaction (qPCR) using EvaGreen® chemistry. The standard curve method estimated the number of dactylogyrids accurately. A total of 85 gill samples from cage-cultured fish infected with dactylogyrids were analysed. The estimated number of dactylogyrids using this molecular method was very similar to the manual count that was performed initially. The standardized qPCR approach will be helpful as a complementary method for the early routine monitoring of dactylogyrid infections and for epidemiological studies in which a high number of fish must be studied.

  8. Biofunctionalization of Polyoxometalates with DNA Primers, Their Use in the Polymerase Chain Reaction (PCR) and Electrochemical Detection of PCR Products.

    PubMed

    Debela, Ahmed M; Ortiz, Mayreli; Beni, Valerio; Thorimbert, Serge; Lesage, Denis; Cole, Richard B; O'Sullivan, Ciara K; Hasenknopf, Bernold

    2015-12-01

    The bioconjugation of polyoxometalates (POMs), which are inorganic metal oxido clusters, to DNA strands to obtain functional labeled DNA primers and their potential use in electrochemical detection have been investigated. Activated monooxoacylated polyoxotungstates [SiW11 O39 {Sn(CH2 )2 CO}](8-) and [P2 W17 O61 {Sn(CH2 )2 CO}](6-) have been used to link to a 5'-NH2 terminated 21-mer DNA forward primer through amide coupling. The functionalized primer was characterized by using a battery of techniques, including electrophoresis, mass spectrometry, as well as IR and Raman spectroscopy. The functionality of the POM-labeled primers was demonstrated through hybridization with a surface-immobilized probe. Finally, the labeled primers were successfully used in the polymerase chain reaction (PCR) and the PCR products were characterized by using electrophoresis.

  9. Simplified diagnosis of malaria infection: GFM/PCR/ELISA a simplified nucleic acid amplification technique by PCR/ELISA.

    PubMed

    Machado, R L; Garret, D O; Adagu, I S; Warhurst, D C; Póvoa, M M

    1998-01-01

    We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

  10. 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.

  11. Efficacy comparison of adjuvants in PcrV vaccine against Pseudomonas aeruginosa pneumonia.

    PubMed

    Hamaoka, Saeko; Naito, Yoshifumi; Katoh, Hideya; Shimizu, Masaru; Kinoshita, Mao; Akiyama, Koichi; Kainuma, Atsushi; Moriyama, Kiyoshi; Ishii, Ken J; Sawa, Teiji

    2017-02-01

    Vaccination against the type III secretion system of P. aeruginosa is a potential prophylactic strategy for reducing the incidence and improving the poor prognosis of P. aeruginosa pneumonia. In this study, the efficacies of three different adjuvants, Freund's adjuvant (FA), aluminum hydroxide (alum) and CpG oligodeoxynucleotide (ODN), were examined from the viewpoint of inducing PcrV-specific immunity against virulent P. aeruginosa. Mice that had been immunized intraperitoneally with recombinant PcrV formulated with one of the above adjuvants were challenged intratracheally with a lethal dose of P. aeruginosa. The PcrV-FA immunized group attained a survival rate of 91%, whereas the survival rates of the PcrV-alum and PcrV-CpG groups were 73% and 64%, respectively. In terms of hypothermia recovery after bacterial instillation, PcrV-alum was the most protective, followed by PcrV-FA and PcrV-CpG. The lung edema index was lower in the PcrV-CpG vaccination group than in the other groups. PcrV-alum immunization was associated with the greatest decrease in myeloperoxidase in infected lungs, and also decreased the number of lung bacteria to a similar number as in the PcrV-FA group. There was less neutrophil recruitment in the lungs of mice vaccinated with PcrV-alum or PcrV-CpG than in those of mice vaccinated with PcrV-FA or PcrV alone. Overall, in terms of mouse survival the PcrV-CpG vaccine, which could be a relatively safe next-generation vaccine, showed a comparable effect to the PcrV-alum vaccine.

  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. Broad-range real time PCR and DNA sequencing for the diagnosis of bacterial meningitis.

    PubMed

    Deutch, Susanna; Pedersen, Lisbeth N; Pødenphant, Lone; Olesen, Rikke; Schmidt, Michael B; Møller, Jens K; Ostergaard, Lars

    2006-01-01

    Rapid aetiological diagnosis of bacterial meningitis is crucial for the early targeting of antimicrobial and adjuvant therapy. Broad-range polymerase chain reaction (PCR) targeting the 16S rRNA gene allows aetiological diagnosis of bacterial meningitis when applied to cerebrospinal fluid (CSF). We assessed the additional diagnostic effect of applying a novel broad-range real time PCR and subsequent DNA sequencing to culture, microscopy, and broad-range conventional PCR on CSF in patients with suspected bacterial meningitis. Broad-range conventional PCR and broad-range real time PCR with subsequent DNA sequencing were applied to 206 CSF specimens collected consecutively from 203 patients aged 6 d to 86 y. Patients' charts were reviewed for clinical information. 17 pathogens were identified by PCR and DNA sequencing or culture. Three specimens were negative by culture but positive by broad-range real time PCR. Three specimens were positive by culture but negative by broad-range real time PCR. Compared with culture, the sensitivity of broad-range real time PCR was 86%, and the specificity 98%. Conventional PCR resulted in a sensitivity of 64% and specificity of 98%. Broad-range real time PCR was generally comparable to culture of CSF and may be a useful supplement, particularly when antimicrobial therapy has been administered. Broad-range real time PCR was more sensitive than broad-range conventional PCR and microscopy.

  14. Comparison of standard, quantitative and digital PCR in the detection of enterotoxigenic Bacteroides fragilis.

    PubMed

    Purcell, Rachel V; Pearson, John; Frizelle, Frank A; Keenan, Jacqueline I

    2016-09-30

    Gut colonization with enterotoxigenic Bacteroides fragilis (ETBF) appears to be associated with the development of colorectal cancer. However, differences in carriage rates are seen with various testing methods and sampling sites. We compared standard PCR, SYBR green and TaqMan quantitative PCR (qPCR) and digital PCR (dPCR) in detecting the B. fragilis toxin (bft) gene from cultured ETBF, and from matched luminal and faecal stool samples from 19 colorectal cancer patients. Bland-Altman analysis found that all three quantitative methods performed comparably in detecting bft from purified bacterial DNA, with the same limits of detection (<1 copy/μl). However, SYBR qPCR under-performed compared to TaqMan qPCR and dPCR in detecting bft in clinical stool samples; 13/38 samples were reported positive by SYBR, compared to 35 and 36 samples by TaqMan and dPCR, respectively. TaqMan qPCR and dPCR gave bft copy numbers that were 48-fold and 75-fold higher for the same samples than SYBR qPCR, respectively (p < 0.001). For samples that were bft-positive in both fecal and luminal stools, there was no difference in relative abundance between the sites, by any method tested. From our findings, we recommend the use of TaqMan qPCR as the preferred method to detect ETBF from clinical stool samples.

  15. Development of an RNA extraction protocol for detection of waterborne viruses by reverse transcriptase quantitative PCR (RT-qPCR).

    PubMed

    Jothikumar, N; Sobsey, M D; Cromeans, T L

    2010-10-01

    RNA extraction from environmental samples yields frequently an RNA preparation containing inhibitors of molecular reactions. Commercial RNA extraction kits commonly permit extraction of only 0.1-0.2 ml sample volume. An RNA extraction buffer (RNAX buffer) was formulated for the extraction of viral RNA from 4.0 ml using a silica column based protocol. To evaluate the RNAX buffer based protocol, we used hepatitis A virus (HAV) and coxsackievirus B3 (CVB3) to monitor the RNA extraction efficiency from environmental samples. For evaluation of viral RNA recovery from water concentrates which were prepared from river and pond water by PEG concentration, serial ten fold dilutions of two waterborne viruses were added to the water concentrates for evaluation by quantitative detection. Quantitative recovery of HAV and CVB3 was determined by reverse transcriptase quantitative real-time PCR (RT-qPCR). The extracted RNA was compatible with RT-qPCR and sensitivity of detection of 0.8PFU per reaction was found with RNAX buffer and the developed protocol. This level of sensitivity was obtained using viral RNA extracted from 4.0 ml of an inoculated water sample concentrate. The RNAX buffer developed in this study could be applicable to the detection of other pathogens in water and food.

  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.

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

    PubMed Central

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

    2016-01-01

    Real-time quantitative reverse transcription PCR (qRT-PCR) offers a robust method for measurement of gene expression levels. Selection of reliable reference gene(s) for gene expression study is conducive to reduce variations derived from different amounts of RNA and cDNA, the efficiency of the reverse transcriptase or polymerase enzymes. Until now reference genes identified for other members of the family Pasteurellaceae have not been validated for Avibacterium paragallinarum. The aim of this study was to validate nine reference genes of serovars A, B, and C strains of A. paragallinarum in different growth phase by qRT-PCR. Three of the most widely used statistical algorithms, geNorm, NormFinder and ΔCT method were used to evaluate the expression stability of reference genes. Data analyzed by overall rankings showed that in exponential and stationary phase of serovar A, the most stable reference genes were gyrA and atpD respectively; in exponential and stationary phase of serovar B, the most stable reference genes were atpD and recN respectively; in exponential and stationary phase of serovar C, the most stable reference genes were rpoB and recN respectively. This study provides recommendations for stable endogenous control genes for use in further studies involving measurement of gene expression levels. PMID:27942007

  18. 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.

  19. 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...

  20. Data Analysis of Transcriptomic Sequences and qPCR Validations for Microbial Communities during Algal Blooms

    EPA Pesticide Factsheets

    A training opportunity is open to a highly microbial-research-motivated student to conduct sequence analysis, explore novel genes and metabolic pathways, validate resultant findings using qPCR/RT-qPCR and summarize the findings

  1. Rapid detection and biovar differentiation of Ureaplasma urealyticum in clinical specimens by PCR.

    PubMed

    Teng, L J; Ho, S W; Ho, H N; Liaw, S J; Lai, H C; Luh, K T

    1995-07-01

    On the basis of the nucleotide sequence of the multiple-banded (MB) antigen genes of Ureaplasma urealyticum, a polymerase chain reaction (PCR) technique was developed for rapid detection and biovar differentiation of U. urealyticum in a total of 100 urogenital specimens from 50 female patients. Positive PCR UM-1 amplification was found in 28 cervical swabs and 31 urine samples. Overall agreement between PCR and culture was 95%. Members of the two biovars of U. urealyticum could be distinguished by the size of the PCR UM-1 amplification products. Biovar differentiation was also demonstrated by two additional sets of PCRs: PCR UM-2 and UM-3. The PCR UM-2 was used to amplify biovar 1, while PCR UM-3 amplified biovar 2 specifically. The results indicated that use of the MB antigen gene as a target for PCR amplification could provide rapid and specific detection and biotyping of ureaplasma DNA in urogenital samples.

  2. 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.

  3. Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis.

    PubMed

    Keid, L B; Soares, R M; Vasconcellos, S A; Salgado, V R; Megid, J; Richtzenhain, L J

    2010-07-17

    The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

  4. 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

  5. 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...

  6. Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat.

    PubMed

    Brusa, Victoria; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Lirón, Juan P; Leotta, Gerardo A

    2015-12-01

    Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation.

  7. Development of TaqMan probe-based insulated isothermal PCR (iiPCR) for sensitive and specific on-site pathogen detection.

    PubMed

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

    2012-01-01

    Insulated isothermal PCR (iiPCR), established on the basis of Ralyeigh-Bénard convection, is a rapid and low-cost platform for nucleic acid amplification. However, the method used for signal detection, namely gel electrophoresis, has limited the application of iiPCR. In this study, TaqMan probe-based iiPCR system was developed to obviate the need of post-amplification processing. This system includes an optical detection module, which was designed and integrated into the iiPCR device to detect fluorescent signals generated by the probe. TaqMan probe-iiPCR assays targeting white spot syndrome virus (WSSV) and infectious myonecrosis virus were developed for preliminary evaluation of this system. Significant elevation of fluorescent signals was detected consistently among positive iiPCR reactions in both assays, correlating with amplicon detection by gel electrophoresis analysis. After condition optimization, a threshold value of S/N (fluorescent intensity(after)/fluorescent intensity(before)) for positive reactions was defined for WSSV TaqMan probe-iiPCR on the basis of 20 blank reactions. WSSV TaqMan probe-iiPCR generated positive S/Ns from as low as 10(1) copies of standard DNA and lightly infected Litopenaeus vannamei. Compared with an OIE-certified nested PCR, WSSV TaqMan probe-iiPCR showed a sensitivity of 100% and a specificity of 96.67% in 120 WSSV-free or lightly infected shrimp samples. Generating positive signals specifically and sensitively, TaqMan probe-iiPCR system has a potential as a low-cost and rapid on-site diagnostics method.

  8. 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

  9. 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

  10. Comparative PCR-based fingerprinting of Vibrio cholerae isolated in Malaysia.

    PubMed

    Shuan Ju Teh, Cindy; Thong, Kwai Lin; Osawa, Ro; Heng Chua, Kek

    2011-01-01

    Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.

  11. A specific real-time PCR assay for the detection of Bordetella pertussis.

    PubMed

    Vincart, Benoit; De Mendonça, Ricardo; Rottiers, Sylvianne; Vermeulen, Françoise; Struelens, Marc J; Denis, Olivier

    2007-07-01

    A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.

  12. Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR.

    PubMed Central

    Leys, E J; Griffen, A L; Strong, S J; Fuerst, P A

    1994-01-01

    By using PCR, Actinobacillus actinomycetemcomitans strains were identified directly from plaque samples without the need to isolate or culture bacteria. DNA fragments were generated by a nested, two-step PCR amplification of the ribosomal spacer region between the 16S and 23S rRNA genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a second amplification with primers specific to A. actinomycetemcomitans. The ribosomal DNA spacer region was amplified from as few as 10 bacterial cells within a total population of 10(8) cells (0.00001%), and cross-reactivity between species was not observed. DNA fragments specific for Porphyromonas gingivalis were generated from the same samples by using a P. gingivalis-specific primer, and equivalent sensitivity and specificity were observed. A. actinomycetemcomitans was detected in 60% and P. gingivalis was detected in 79% of 52 subjects tested. Sequence analysis of the spacer region DNA fragment for A. actinomycetemcomitans gave precise strain identification, producing unique sequences for seven reference strains and identification of nine plaque-derived isolates. A phylogenetic tree based on quantitative sequence relationships was constructed. Two-step PCR amplification directly from plaque samples combined with sequence analysis of the ribosomal DNA spacer region provides a sensitive assay for detection and strain identification of multiple species directly from a single plaque sample. This simplified approach provides a practical method for large-scale studies on the transmission and pathogenicity of periodontitis-associated bacteria. Images PMID:8051258

  13. Multiplex PCR testing for nine different sexually transmitted infections.

    PubMed

    Kriesel, John D; Bhatia, Amiteshwar S; Barrus, Cammie; Vaughn, Mike; Gardner, Jordan; Crisp, Robert J

    2016-12-01

    Current sexually transmitted infection (STI) testing is not optimal due to delays in reporting or missed diagnoses due to a lack of comprehensive testing. The FilmArray® (BioFire Diagnostics, LLC, Salt Lake City, Utah) is a user-friendly, fully automated, multiplex PCR system that is being developed for rapid point-of-care use. A research-use-only STI panel including multiple PCR primer sets for each organism was designed to detect Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum, Trichomonas vaginalis, Mycoplasma genitalium, Ureaplasma urealyticum, Haemophilus ducreyi, and herpes simplex virus (HSV) types 1 and 2. Standard clinical testing included Gram stain, nucleic acid amplification, wet mount examination, herpes simplex virus culture, and syphilis IgG. Standard clinical tests were not available for all the organisms tested by the FilmArray STI panel. Two hundred and ninety-five clinical specimens from 190 subjects were directly compared to standard testing. Urine (n = 146), urethral/cervical swabs (31), oral swabs (60), rectal swabs (43), and ulcer swabs (15) were tested. Among the tested samples, FilmArray detected C. trachomatis in 39 (13%), N. gonorrhoeae in 20 (7%), T. vaginalis in nine (3%), HSV 1 in five (2%), HSV 2 in five (2%), U. urealyticum in 36 (12%), M. genitalium in eight (3%), and T. pallidum in 11 (4%). Concordance between the FilmArray STI panel and standard nucleic acid amplification testing for C. trachomatis was 98% and for N. gonorrhoeae was 97%. Multiplex PCR STI testing has the potential to improve public health by providing rapid, sensitive, and reliable results within the clinic or nearby laboratory.

  14. 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

  15. 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.

  16. Comparison of multilocus sequence typing (MLST) and repetitive sequence-based PCR (rep-PCR) fingerprinting for differentiation of Campylobacter jejuni isolated from broiler in Chiang Mai, Thailand.

    PubMed

    Patchanee, Prapas; Chokboonmongkol, Chomporn; Zessin, Karl-Hans; Alter, Thomas; Pornaem, Sarinya; Chokesajjawatee, Nipa

    2012-11-01

    We compared rapid fingerprinting using repetitive sequence-based PCR (rep-PCR) for subtyping Campylobacter jejuni isolates to the widely used multilocus sequence typing (MLST). Representative C. jejuni isolates (n = 16) from broilers were analyzed using MLST and rep-PCR. Both techniques demonstrated an equal discriminatory power of 0.8917, and 9 subgroups were identified. Clonal identification of all 16 isolates was identical for both techniques. The rep-PCR as described in this study may be used as a rapid and cost-effective alternative for subtyping of C. jejuni isolates, or as an effective screening tool in large epidemiological studies.

  17. Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis

    PubMed Central

    Sanchez, J. Aquiles; Pierce, Kenneth E.; Rice, John E.; Wangh, Lawrence J.

    2004-01-01

    Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)–PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80–250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CFΔ508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells. PMID:14769930

  18. 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).

  19. [Differentiation of geographic biovariants of smallpox virus by PCR].

    PubMed

    Babkin, I V; Babkina, I N

    2010-01-01

    Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the geographic origin. This is ORF O1L of VARV. The primers were calculated for synthesis of this ORF fragment by PCR, which makes it possible to distinguish South America-Western Africa genotype from other VARV strains. Subsequent RFLP analysis reliably differentiated Asian strains from African strains (except Western Africa isolates). This method has been tested using 16 VARV strains from various geographic regions. The developed approach is simple, fast and reliable.

  20. PCR-based typing of IncC plasmids.

    PubMed

    Harmer, Christopher J; Hall, Ruth M

    IncC (A/C2) plasmids are known to play an important role in the spread of multiple antibiotic resistance determinants, including extended-spectrum β-lactamases and carbapenamases, amongst Gram negative bacterial populations. The ability to identify and track these plasmids is valuable in epidemiological and clinical studies. A recent comparative analysis of the backbones of sequenced IncC plasmids identified two distinct lineages, type 1 and type 2, with different evolutionary histories. Here, a simple PCR method to rapidly assign plasmids to one of these lineages by detecting variable regions in the backbone was developed. This PCR scheme uses two primer pairs to assign the plasmid to a lineage, and an additional two PCRs can be used to detect the i1 and i2 insertions, which are only found in type 2. PCRs were also developed to detect the presence or absence of the sul2-containing ARI-B island, which is found in some plasmids belonging to both type 1 and type 2, and the ARI-A island found in most type 1 plasmids. The PCR strategy was validated using sequenced type 1 plasmids pRMH760 and pDGO100, and the type 2 plasmid pSRC119-A/C, and a collection of non-IncC plasmids in Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae backgrounds. An IncC plasmid detected in an antibiotic susceptible commensal E. coli isolate was examined and found to be a type 1, lacking any antibiotic resistance islands and missing a large backbone segment. Examination of pIP40a, an IncC plasmid isolated in Paris in 1969, by PCR revealed that it belongs to type 1 but lacks ARI-A. However, it includes both ends of the integrative element GIsul2, whereas only remnants of one end of this element are found in more recently isolated IncC plasmids. The sequence of pIP40a was determined and confirmed the assignment to type 1 and revealed the presence of a complete copy of GIsul2.

  1. Amelogenin sex determination by pyrosequencing of short PCR products.

    PubMed

    Tschentscher, Frank; Frey, Ulrich H; Bajanowski, Thomas

    2008-07-01

    We developed an assay, which allows the sex determination of human DNA samples by pyrosequencing of short PCR products. A 48/45-bp stretch including primers of the amelogenin gene with a 3-bp insertion on the Y chromosome was chosen for analysis. In an initial study, we correctly typed 50 male and 50 female DNA samples from unrelated donors. First experiments with forensic samples, which failed in conventional analyses, indicate that this approach might be an advantage when dealing with degraded DNA.

  2. [Frequency of intestinal microsporidian infections in HIV-positive patients, as diagnosis by quick hot Gram chromotrope staining and PCR].

    PubMed

    Botero, Jorge H; Montoya, Martha Nelly; Vanegas, Adriana Lucía; Díaz, Abel; Navarro-i-Martínez, Luis; Bornay, Fernando Jorge; Izquierdo, Fernando; del Aguila, Carmen; Agudelo, Sonia del Pilar

    2004-12-01

    Microsporidia are intracellular obligate parasites, today mainly associated with diarrhea in AIDS patients. Microsporidia prevalence ranges from 8% to 52% in different countries, as evaluated by several diagnostic methods, such as the stain test and PCR. In Medellín, Colombia, its frequency is unknown, and hence, a study was undertaken to determine the frequency of intestinal microsporidiosis in HIV patients, by means of the quick-hot Gram chromotrope test and the PCR. A prospective and descriptive study of an intentional population of all HIV-positive patients was sent to the Grupo Interdisciplinario para el Estudio de las Parasitosis Intestinales laboratory by institutions treating the HIV-positive patients of Medellín between August 2001 and September 2002. The clinical-epidemiological survey included a serial stool test with direct concentration and special stains for coccidiae and intestinal microsporidia. In addition, counts of lymphocytes TCD4+ and viral load were requested. One hundred and three patients with ages ranging from 2-74 years were evaluated. Seventy percent presented with diarrhea--mostly in men (83.5%). The overall frequency of intestinal microsporidiosis was 3.9% and that of other intestinal parasitic infections was 39.8%. Three of the four patients positive for microsporida were infected with Enterocytozoon bieneusi and one with Encephalitozoon intestinalis. The microsporidiosis frequency was relatively low with 3 of the 4 cases associated with protracted diarrhea, counts of LTCD4+ below 100 cel/microl and viral loads up to 100,000 copies.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. 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...

  5. Avian influenza virus detection and quantitation by real-time RT-PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of rRT-PCR are: high sensitivity, high specificity, rapid time-to-result, scalability, cost, and its inherentl...

  6. 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.

  7. 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...

  8. 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...

  9. 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...

  10. Cytochrome b gene quantitative PCR for diagnosing Plasmodium falciparum infection in travelers.

    PubMed

    Farrugia, Cécile; Cabaret, Odile; Botterel, Françoise; Bories, Christian; Foulet, Françoise; Costa, Jean-Marc; Bretagne, Stéphane

    2011-06-01

    A cytochrome b (cytb) gene quantitative PCR (qPCR) assay was developed to diagnose malaria in travelers. First, manual and automated DNA extractions were compared and automated DNA extraction of 400 μl of blood was found to be more efficient. Sensitivity was estimated using the WHO international standard for Plasmodium falciparum DNA and compared to that of a previously published qPCR targeting the 18S rRNA coding gene (18S qPCR). The limit of detection of the cytb qPCR assay was 20 DNA copies (i.e., 1 parasite equivalent) per 400 μl of extracted whole blood and was comparable for the two qPCR assays. Both qPCR assays were used on blood samples from 265 consecutive patients seen for suspicion of malaria. There were no microscopy-positive and qPCR-negative samples. Positive cytb qPCR results were observed for 51 samples, and all but 1 were also 18S qPCR positive. Eight (16%) of these 51 samples were negative by microscopic examination. The 8 cytb qPCR-positive and microscopy-negative samples were from African patients, 3 of whom had received antimalarial drugs. Three non-P. falciparum infections were correctly identified using an additional qPCR assay. The absence of PCR inhibitors was tested for by the use of an internal control of mouse DNA to allow reliable quantification of circulating DNA. The high analytical sensitivity of both qPCR assays combined with automated DNA extraction supports its use as a laboratory tool for diagnosis and parasitemia determination in emergencies. Whether to treat qPCR-positive and microscopy-negative patients remains to be determined.

  11. Insulated Isothermal Reverse Transcriptase PCR (iiRT-PCR) for Rapid and Sensitive Detection of Classical Swine Fever Virus.

    PubMed

    Lung, O; Pasick, J; Fisher, M; Buchanan, C; Erickson, A; Ambagala, A

    2016-10-01

    Classical swine fever (CSF) is an OIE-listed disease that can have a severe impact on the swine industry. User-friendly, sensitive, rapid diagnostic tests that utilize low-cost field-deployable instruments for CSF diagnosis can be useful for disease surveillance and outbreak monitoring. In this study, we describe validation of a new probe-based insulated isothermal reverse transcriptase PCR (iiRT-PCR) assay for rapid detection of classical swine fever virus (CSFV) on a compact, user-friendly device (POCKIT(™) Nucleic Acid Analyzer) that does not need data interpretation by the user. The assay accurately detected CSFV RNA from a diverse panel of 33 CSFV strains representing all three genotypes plus an additional in vitro-transcribed RNA from cloned sequences representing a vaccine strain. No cross-reactivity was observed with a panel of 18 viruses associated with livestock including eight other pestivirus strains (bovine viral diarrhoea virus type 1 and type 2, border disease virus, HoBi atypical pestivirus), African swine fever virus, swine vesicular disease virus, swine influenza virus, porcine respiratory and reproductive syndrome virus, porcine circovirus 1, porcine circovirus 2, porcine respiratory coronavirus, vesicular exanthema of swine virus, bovine herpes virus type 1 and vesicular stomatitis virus. The iiRT-PCR assay accurately detected CSFV as early as 2 days post-inoculation in RNA extracted from serum samples of experimentally infected pigs, before appearance of clinical signs. The limit of detection (LOD95% ) calculated by probit regression analysis was 23 copies per reaction. The assay has a sample to answer turnaround time of less than an hour using extracted RNA or diluted or low volume of neat serum. The user-friendly, compact device that automatically analyses and displays results could potentially be a useful tool for surveillance and monitoring of CSF in a disease outbreak.

  12. Digital PCR on an integrated self-priming compartmentalization chip.

    PubMed

    Zhu, Qiangyuan; Qiu, Lin; Yu, Bingwen; Xu, Yanan; Gao, Yibo; Pan, Tingting; Tian, Qingchang; Song, Qi; Jin, Wei; Jin, Qinhan; Mu, Ying

    2014-03-21

    An integrated on-chip valve-free and power-free microfluidic digital PCR device is for the first time developed by making use of a novel self-priming compartmentalization and simple dehydration control to realize 'divide and conquer' for single DNA molecule detection. The high gas solubility of PDMS is exploited to provide the built-in power of self-priming so that the sample and oil are sequentially sucked into the device to realize sample self-compartmentalization based on surface tension. The lifespan of its self-priming capability was about two weeks tested using an air-tight packaging bottle sealed with a small amount of petroleum jelly, which is significant for a practical platform. The SPC chip contains 5120 independent 5 nL microchambers, allowing the samples to be compartmentalized completely. Using this platform, three different abundances of lung cancer related genes are detected to demonstrate the feasibility and flexibility of the microchip for amplifying a single nucleic acid molecule. For maximal accuracy, within less than 5% of the measurement deviation, the optimal number of positive chambers is between 400 and 1250 evaluated by the Poisson distribution, which means one panel can detect an average of 480 to 4804 template molecules. This device without world-to-chip connections eliminates the constraint of the complex pipeline control, and is an integrated on-chip platform, which would be a significant improvement to digital PCR automation and more user-friendly.

  13. PCR-free digital minisatellite tandem repeat genotyping.

    PubMed

    Chen, Yuchao; Seo, Tae Seok

    2011-06-01

    We demonstrated a proof-of-concept for novel minisatellite tandem repeat typing, called PCR-free digital VNTR (variable number tandem repeat) typing, which is composed of three steps: a ligation reaction instead of PCR thermal cycling, magnetic bead-based solid-phase capture for purification, and an elongated sample stacking microcapillary electrophoresis (μCE) for sensitive digital coding of repeat number. We designed a 16-bp fluorescently labeled ligation probe which is complementary to a repeat unit of a biotinylated synthetic template mimicking the human D1S80 VNTR locus and is randomly hybridized with the minisatellite tandem repeats. A quick isothermal ligation reaction was followed to link the adjacent ligation probes on the DNA templates, and then the ligated products were purified by streptavidin-coated magnetic beads. After a denaturing step, a large amount of ligated products whose size difference was equivalent to the repeat unit were released and recovered. Through the elongated sample stacking μCE separation on a microdevice, the fluorescence signal of the ligated products was generated in the electropherogram and the peak number was directly counted which was exactly matched with the repeat number of VNTR locus. We could successfully identify the minisatellite tandem repeat number with only 5 fmol of DNA template in 30 min.

  14. Enzymatic amplification of DNA by PCR: standard procedures and optimization.

    PubMed

    Kramer, M F; Coen, D M

    2001-05-01

    This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR) and for optimizing this reaction for the sequence and primer set of interest. Important variables that can influence the outcome of PCR include the MgCl(2) concentration and the cycling temperatures. Additives that promote polymerase stability and processivity or increase hybridization stringency, and strategies that reduce nonspecific primer-template interactions, especially prior to the critical first cycle, can greatly improve sensitivity, specificity, and yield. This protocol is designed to optimize the reaction components and conditions in one or two stages. The first stage determines the optimal MgCl(2) concentration and screens several enhancing additives. To further improve specificity, sensitivity and yield, the second stage compares methods for optimizing initial specific hybridization to prevent polymerization of misprimed sequences prior to thermal cycling. For initial inhibition of polymerase activity, temperature (i.e., cooling reagents), physical separation ("hot start" method), and reversible antibody binding are compared.

  15. 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.

  16. Nested PCR for Specific Diagnosis of Taenia solium Taeniasis▿

    PubMed Central

    Mayta, Holger; Gilman, Robert H.; Prendergast, Emily; Castillo, Janeth P.; Tinoco, Yeny O.; Garcia, Hector H.; Gonzalez, Armando E.; Sterling, Charles R.

    2008-01-01

    Taeniasis due to Taenia solium is a disease with important public health consequences, since the larval stage is not exclusive to the animal intermediate, the pig, but also infects humans, causing neurocysticercosis. Early diagnosis and treatment of T. solium tapeworm carriers is important to prevent human cysticercosis. Current diagnosis based on microscopic observation of eggs lacks both sensitivity and specificity. In the present study, a nested-PCR assay targeting the Tso31 gene was developed for the specific diagnosis of taeniasis due to T. solium. Initial specificity and sensitivity testing was performed using stored known T. solium-positive and -negative samples. The assay was further analyzed under field conditions by conducting a case-control study of pretreatment stool samples collected from a population in an area of endemicity. Using the archived samples, the assay showed 97% (31/32) sensitivity and 100% (123/123) specificity. Under field conditions, the assay had 100% sensitivity and specificity using microscopy/enzyme-linked immunosorbent assay coproantigen testing as the gold standards. The Tso31 nested PCR described here might be a useful tool for the early diagnosis and prevention of taeniasis/cysticercosis. PMID:17989190

  17. 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

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

    PubMed

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

    2015-12-01

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

  19. Sex determination in 6 bovid species by duplex PCR.

    PubMed

    Prashant; Gour, Digpal S; Dubey, Prem P; Jain, Anubhav; Gupta, Subhash C; Joshi, Balwinder K; Kumar, Dinesh

    2008-01-01

    Sex determination in domestic animals is of potential value to livestock breeding programs. The aim of this study was to develop a simple and accurate PCR-based sex determination protocol, which can be applicable to 6 major domesticated species of the family Bovidae, viz. Bos frontalis, B. grunniens, B. indicus, Bubalus bubalis, Capra hircus, and Ovis aries. In silico analysis was done to identify conserved DNA sequence in the HMG box region of the sex-determining region of the Y-chromosome (SRY gene) across the bovids. Duplex PCR assay, including the SRY gene and the GAPDH housekeeping gene, was optimized by using genomic DNA extracted from blood samples of known sex. It was possible to identify the sex of animals by amplifying both gender-specific (SRY) and autosomal (GAPDH) genes simultaneously in the duplex reaction, with the male yielding two bands and the female one band. The protocol was subjected to a blind test that showed a 100 percent specificity and accuracy, thus it can be used in sex determination in livestock breeding programs.

  20. Triplet repeat primed PCR simplifies testing for Huntington disease.

    PubMed

    Jama, Mohamed; Millson, Alison; Miller, Christine E; Lyon, Elaine

    2013-03-01

    Diagnostic and predictive testing for Huntington disease (HD) requires an accurate determination of the number of CAG repeats in the Huntingtin (HHT) gene. Currently, when a sample appears to be homozygous for a normal allele, additional testing is required to confirm amplification from both alleles. If the sample still appears homozygous, Southern blot analysis is performed to rule out an undetected expanded HTT allele. Southern blot analysis is expensive, time-consuming, and labor intensive and requires high concentrations of DNA. We have developed a chimeric PCR process to help streamline workflow; true homozygous alleles are easily distinguished by this simplified method, and only very large expanded alleles still require Southern blot analysis. Two hundred forty-six HD samples, previously run with a different fragment analysis method, were analyzed with our new method. All samples were correctly genotyped, resulting in 100% concordance between the methods. The chimeric PCR assay was able to identify expanded alleles up to >150 CAG repeats. This method offers a simple strategy to differentiate normal from expanded CAG alleles, thereby reducing the number of samples reflexed to Southern blot analysis. It also provides assurance that expanded alleles are not routinely missed because of allele dropout.

  1. Analysis of electrophoretic patterns of arbitrarily primed PCR profiling.

    PubMed

    Trifunović, Dragana; Radović, Milan; Ristić, Zoran; Guzvić, Miodrag; Dimitrijević, Bogomir

    2005-11-01

    We present a mathematical algorithm for the analysis of electrophoretic patterns resulting from arbitrarily primed PCR profiling. The algorithm is based on the established mathematical procedures applied to the analysis of digital images of gel patterns. The algorithm includes (a) transformation of the image into a matrix form, (b) identification of every electrophoretic lane as a set of matrix columns that are further mathematically processed, (c) averaging of matrix columns corresponding to electrophoretic lanes that define lane representatives, (d) elimination of "smiling" bands, (e) solving the problem of a lane offset, and (f) removal of the background. Representation of individual electrophoretic lanes in the form of functions allows interlane comparisons and further mathematical analysis. Direct comparison of selected lanes was obtained by employing correlation analysis. Gel images were those obtained after arbitrarily primed PCR analysis of DNA that underwent damage induced by gamma radiation from a (60)Co source. The applied method proved to be useful for elimination of subjectivity of visual inspection. It offers the possibility to avoid overlooking important differences in case of suboptimal electrophoretic resolution. In addition, higher precision is achieved in the assessment of quantitative differences due to better insight into experimental artifacts. These simple mathematical methods offer an open-type algorithm, i.e., this algorithm enables easy implementation of different parameters that may be useful for other analytical needs.

  2. 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.

  3. Nested PCR for specific diagnosis of Taenia solium taeniasis.

    PubMed

    Mayta, Holger; Gilman, Robert H; Prendergast, Emily; Castillo, Janeth P; Tinoco, Yeny O; Garcia, Hector H; Gonzalez, Armando E; Sterling, Charles R

    2008-01-01

    Taeniasis due to Taenia solium is a disease with important public health consequences, since the larval stage is not exclusive to the animal intermediate, the pig, but also infects humans, causing neurocysticercosis. Early diagnosis and treatment of T. solium tapeworm carriers is important to prevent human cysticercosis. Current diagnosis based on microscopic observation of eggs lacks both sensitivity and specificity. In the present study, a nested-PCR assay targeting the Tso31 gene was developed for the specific diagnosis of taeniasis due to T. solium. Initial specificity and sensitivity testing was performed using stored known T. solium-positive and -negative samples. The assay was further analyzed under field conditions by conducting a case-control study of pretreatment stool samples collected from a population in an area of endemicity. Using the archived samples, the assay showed 97% (31/32) sensitivity and 100% (123/123) specificity. Under field conditions, the assay had 100% sensitivity and specificity using microscopy/enzyme-linked immunosorbent assay coproantigen testing as the gold standards. The Tso31 nested PCR described here might be a useful tool for the early diagnosis and prevention of taeniasis/cysticercosis.

  4. Real-Time PCR Method for Detection of Zygomycetes ▿

    PubMed Central

    Hata, D. Jane; Buckwalter, Seanne P.; Pritt, Bobbi S.; Roberts, Glenn D.; Wengenack, Nancy L.

    2008-01-01

    Zygomycete infections can be devastating in immunocompromised hosts. Difficulties in the histopathologic differentiation of this class from other filamentous fungi (e.g., Aspergillus spp., Fusarium spp.) may lead to delays in diagnosis and initiation of appropriate treatment, thereby significantly affecting patient outcome. A real-time PCR assay was developed to detect species of the zygomycete genera Absidia, Apophysomyces, Cunninghamella, Mucor, Rhizopus, and Saksenaea in culture and tissue samples. Primers and fluorescence resonance energy transfer hybridization probes were designed to detect a 167-bp conserved region of the multicopy zygomycete cytochrome b gene. A plasmid containing target sequence from Mucor racemosus was constructed as a positive control. The analytical sensitivity of the assay is 10 targets/μl, and a specificity panel consisting of other filamentous fungi, yeasts (Candida spp.), and bacteria demonstrated no cross-reactivity in the assay. The clinical sensitivity and specificity of the assay from culture isolates were 100% (39/39) and 92% (59/64), respectively. Sensitivity and specificity determined using a limited number of fresh tissue specimens were both 100% (2/2). The sensitivity seen with formalin-fixed, paraffin-embedded tissues was 56% (35/62), and the specificity was 100% (19/19). The speed, sensitivity, and specificity of the PCR assay indicate that it is useful for the rapid and accurate detection of zygomycetes. PMID:18480229

  5. Population diversity of ammonium oxidizers investigated by specific PCR amplification

    USGS Publications Warehouse

    Ward, B.B.; Voytek, M.A.; Witzel, K.-P.

    1997-01-01

    The species composition of ammonia-oxidizing bacteria in aquatic environments was investigated using PCR primers for 16S rRNA genes to amplify specific subsets of the total ammonia-oxidizer population. The specificity of the amplification reactions was determined using total genomic DNA from known nitrifying strains and non-nitrifying strains identified as having similar rDNA sequences. Specificity of amplification was determined both for direct amplification, using the nitrifier specific primers, and with nested amplification, in which the nitrifier primers were used to reamplify a fragment obtained from direct amplification with Eubacterial universal primers. The present level of specificity allows the distinction between Nitrosomonas europaea, Nitrosomonas sp. (marine) and the other known ammonia-oxidizers in the beta subclass of the Proteobacteria. Using total DNA extracted from natural samples, we used direct amplification to determine presence/absence of different species groups. Species composition was found to differ among depths in vertical profiles of lake samples and among samples and enrichments from various other aquatic environments. Nested PCR yielded several more positive reactions, which implies that nitrifier DNA was present in most samples, but often at very low levels.

  6. New Approaches on Quantification of Campylobacter jejuni in Poultry Samples: The Use of Digital PCR and Real-time PCR against the ISO Standard Plate Count Method

    PubMed Central

    Papić, Bojan; Pate, Mateja; Henigman, Urška; Zajc, Urška; Gruntar, Igor; Biasizzo, Majda; Ocepek, Matjaž; Kušar, Darja

    2017-01-01

    Campylobacteriosis is the most frequently reported bacterial food-borne illness in the European Union and contaminated broiler meat is considered the most important source of infection in humans. The aim of the present study was to evaluate real-time PCR (qPCR) and digital PCR (dPCR) for quantification of Campylobacter jejuni in 75 broiler neck-skin samples collected from a poultry slaughterhouse, and to compare them with the ISO 10272-2 standard plate count method. For qPCR standard curve, C. jejuni-negative neck-skin samples were spiked with C. jejuni suspension with a known number of bacterial cells. The observed CFU/g values by qPCR correlated greatly with the expected values and qPCR showed good performance with the reliable limit of detection (rLOD) and limit of quantification (LOQ) of three and 31 target copies per reaction, respectively. However, both rLOD (1219 CFU/g) and LOQ (12,523 CFU/g) were beyond the EFSA-proposed critical limit of 500–1,000 CFU/g of neck skin. Although C. jejuni cell counts were ≤1,000 CFU/g in only 7/75 samples by plate counting, they were ≤LOQ in 60/75 and ≤rLOD in 26/75 (≤1,000 CFU/g in 24/75) samples by qPCR. A strong and statistically significant correlation was observed between qPCR and dPCR. Both PCR-based methods correlated significantly with the plate count method; however, the correlation was moderate. Using the Bland–Altman analysis, an average agreement was noted between all three methods, although with a large standard deviation. A significant bias toward overestimation in dPCR was observed, probably due to the relatively high number of false positive calls. The linear dynamic range was comparable in both PCR-based methods; however, qPCR proved to be more suitable for routine use. In the future, the establishment of a reliable molecular quantification of C. jejuni in poultry samples showing a wide range of contamination levels down to the proposed critical limit is needed to enable time- and cost

  7. Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers.

    PubMed

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

    2012-01-01

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

  8. Molecular diagnosis of canine visceral leishmaniasis: identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR.

    PubMed

    Quaresma, Patrícia Flávia; Murta, Silvane Maria Fonseca; Ferreira, Eduardo de Castro; da Rocha-Lima, Ana Cristina Vianna Mariano; Xavier, Ana Amélia Prates; Gontijo, Célia Maria Ferreira

    2009-09-01

    The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dye-based system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmania infantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples.

  9. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-03-01

    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.

  12. [Application of the multiplex PCR and PCR-RFLP method in the identification of the Bacillus anthracis].

    PubMed

    Szymajda, Urszula; Bartoszcze, Michał

    2005-01-01

    The aim of this study was to apply the multiplex PCR and PCR-RFLP method for the identification of the B. anthracis strains and to distinguish those bacteria from other members of the Bacillus cereus group. The multiplex PCR method enables to detect the virulence factors, i.e. the toxin and the capsule in B. anthracis strains. To do that, the authors have used 5 primer pairs specific for the fragments of lef, cya, pag genes which are present in the pXO1 plasmid and encode the toxin, the cap gene, which is present in the pXO2 plasmid and encodes the capsule, and the Ba813 chromosomal sequence. Among the four B. anthracis strains examined, three contained two plasmids and the Ba813 chromosomal sequence, while the fourth one contained the pXO1 plasmid only, together and the Ba813 chromosomal sequence. Other bacterial species, belonging to the B. cereus group, were also examined: 6 strains of B. cereus, 4 strains of B. thuringiensis and one strain of B. mycoides. The presence of Ba813 chromosomal sequence has been detected in two B. cereus strains. Neither plasmids nor Ba813 chromosomal sequence have been discovered in other B. cereus, B. thuringiensis and B. mycoides strains. The results of the survey indicate that the Ba813 chromosomal sequence does not occur solely in B. anthracis strains. The PCR-RFLP method with the use of SG-749f and SG-749r primers enabled to demonstrate the presence of DNA sequence (SG-749) in B. anthracis, B. cereus, B. thuringiensis and B. mycoides strains. Restriction analysis with enzyme AluI of the SG-749 sequence, has shown the presence of two DNA fragments at the size of about 90 and 660 bp in all B. anthracis strains. The restriction profile obtained was characteristic for B. anthracis strains and it did not occur in other investigated bacterial species belonging to the B. cereus group. It was not observed even in such B. cereus strains in which the presence of Ba813 sequence was discovered and it enabled to differentiate between B

  13. ddpcr: an R package and web application for analysis of droplet digital PCR data

    PubMed Central

    Attali, Dean; Bidshahri, Roza; Haynes, Charles; Bryan, Jennifer

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel platform for exact quantification of DNA which holds great promise in clinical diagnostics. It is increasingly popular due to its digital nature, which provides more accurate quantification and higher sensitivity than traditional real-time PCR. However, clinical adoption has been slowed in part by the lack of software tools available for analyzing ddPCR data. Here, we present ddpcr – a new R package for ddPCR visualization and analysis. In addition, ddpcr includes a web application (powered by the Shiny R package) that allows users to analyze ddPCR data using an interactive graphical interface. PMID:27703666

  14. ddpcr: an R package and web application for analysis of droplet digital PCR data.

    PubMed

    Attali, Dean; Bidshahri, Roza; Haynes, Charles; Bryan, Jennifer

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel platform for exact quantification of DNA which holds great promise in clinical diagnostics. It is increasingly popular due to its digital nature, which provides more accurate quantification and higher sensitivity than traditional real-time PCR. However, clinical adoption has been slowed in part by the lack of software tools available for analyzing ddPCR data. Here, we present ddpcr - a new R package for ddPCR visualization and analysis. In addition, ddpcr includes a web application (powered by the Shiny R package) that allows users to analyze ddPCR data using an interactive graphical interface.

  15. Single-primer PCR correction: a strategy for false-positive exclusion.

    PubMed

    Ma, J; Wang, P W; Yao, D; Wang, Y P; Yan, W; Guan, S C

    2011-02-01

    Polymerase chain reaction (PCR) technology plays an important role in molecular biology research, but false-positive and nonspecific PCR amplification have plagued many researchers. Currently, research on the optimization of the PCR system focuses on double-primer-based PCR products. This research has shown that PCR amplification based on single-primer binding to the DNA template is an important contributing factor to obtaining false-positive results, fragment impurity, and nonspecific fragment amplification, when the PCR conditions are highly restricted during PCR-based target gene cloning, detection of transgenic plants, simple-sequence repeat marker-assisted selection, and mRNA differential display. Here, we compared single- and double-primer amplification and proposed "single-primer PCR correction"; improvements in PCR that eliminate interference caused by single-primer-based nonspecific PCR amplification were demonstrated and the precision and success rates of experiments were increased. Although for some kinds of experiments, the improvement effect of single-primer PCR correction was variable, the precision and success rate could be elevated at 12-50% in our experiment by this way.

  16. Design of primers and probes for quantitative real-time PCR methods.

    PubMed

    Rodríguez, Alicia; Rodríguez, Mar; Córdoba, Juan J; Andrade, María J

    2015-01-01

    Design of primers and probes is one of the most crucial factors affecting the success and quality of quantitative real-time PCR (qPCR) analyses, since an accurate and reliable quantification depends on using efficient primers and probes. Design of primers and probes should meet several criteria to find potential primers and probes for specific qPCR assays. The formation of primer-dimers and other non-specific products should be avoided or reduced. This factor is especially important when designing primers for SYBR(®) Green protocols but also in designing probes to ensure specificity of the developed qPCR protocol. To design primers and probes for qPCR, multiple software programs and websites are available being numerous of them free. These tools often consider the default requirements for primers and probes, although new research advances in primer and probe design should be progressively added to different algorithm programs. After a proper design, a precise validation of the primers and probes is necessary. Specific consideration should be taken into account when designing primers and probes for multiplex qPCR and reverse transcription qPCR (RT-qPCR). This chapter provides guidelines for the design of suitable primers and probes and their subsequent validation through the development of singlex qPCR, multiplex qPCR, and RT-qPCR protocols.

  17. Dry-reagent-based PCR as a novel tool for the rapid detection of Clostridium spp.

    PubMed

    Seise, Barbara; Pollok, Sibyll; Seyboldt, Christian; Weber, Karina

    2013-10-01

    Improved conventional PCR techniques are required for the rapid on-site detection of human and animal diseases. In this context, a PCR method using dry-stored reagents intended for the detection of Clostridium spp. is presented. Basic PCR reagents (BSA, PCR buffer, MgCl₂ and primers), which were dried on polyolefin matrices, showed stability at ambient temperatures for up to 10 months without any loss of functionality. An outstanding advantage of our amelioration is the elimination of PCR process errors caused by the improper storage and handling of liquid reagents. Moreover, our PCR-based amplification can be performed in less than 30 min, saving time compared with conventional detection methods. Thus, dry-reagent-based PCR is implementable in a suitcase-like modular device for the rapid on-site detection of microbial pathogens such as blackleg of ruminants caused by Clostridium chauvoei.

  18. Optimized PCR assay for detection of white spot syndrome virus (WSSV).

    PubMed

    Nunan, Linda M; Lightner, Donald V

    2011-01-01

    A rapid PCR assay for detection of white spot syndrome virus (WSSV) was developed based on the nested PCR procedure described by Lo et al. (1996) and outlined as the recommended PCR diagnostic assay in the Manual of Diagnostic Tests for Aquatic Animals published by the Office of International Epizootics (OIE, 2009). The optimized procedure incorporated the second step primers used in the nested WSSV PCR. By adjusting the annealing temperature and shortening the cycling times, this modified assay is substantially faster and as sensitive as the recommended OIE protocol. The modified PCR test was compared directly to the two-step nested PCR protocol and a modified nested procedure. The sensitivity of the published assay was determined by template dilutions of semi-purified WSSV virions that had been quantitated using real-time PCR for detection of WSSV. Various isolates were tested using the modified procedure, to ensure that the assay was able to detect WSSV from different geographical locations.

  19. Molecular identification of Mucorales in human tissues: contribution of PCR electrospray-ionization mass spectrometry.

    PubMed

    Alanio, A; Garcia-Hermoso, D; Mercier-Delarue, S; Lanternier, F; Gits-Muselli, M; Menotti, J; Denis, B; Bergeron, A; Legrand, M; Lortholary, O; Bretagne, S

    2015-06-01

    Molecular methods are crucial for mucormycosis diagnosis because cultures are frequently negative, even if microscopy suggests the presence of hyphae in tissues. We assessed PCR/electrospray-ionization mass spectrometry (PCR/ESI-MS) for Mucorales identification in 19 unfixed tissue samples from 13 patients with proven or probable mucormycosis and compared the results with culture, quantitative real-time PCR, 16S-23S rRNA gene internal transcribed spacer region (ITS PCR) and 18S PCR sequencing. Concordance with culture identification to both genus and species levels was higher for PCR/ESI-MS than for the other techniques. Thus, PCR/ESI-MS is suitable for Mucorales identification, within 6 hours, for tissue samples for which microscopy results suggest the presence of hyphae.

  20. High-throughput STR analysis for DNA database using direct PCR.

    PubMed

    Sim, Jeong Eun; Park, Su Jeong; Lee, Han Chul; Kim, Se-Yong; Kim, Jong Yeol; Lee, Seung Hwan

    2013-07-01

    Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high-throughput and cost-effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter-loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time- and cost-saving manner.

  1. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    PubMed Central

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  2. Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

    PubMed

    Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

    2011-01-01

    In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

  3. [Evaluation of pathogen disinfection efficacy by chlorine and monochloramine disinfection based on quantitative PCR combined with propidium monoazide (PMA-qPCR)].

    PubMed

    Tong, Tie-Zheng; Wu, Shu-Xu; Li, Dan; He, Miao; Yang, Tian; Shi, Han-Chang

    2011-04-01

    A novel detection method of quantitative PCR combined with a DNA intercalating dye propidium monoazide (PMA-qPCR) was developed and then applied to analyze inactivation efficacy of chlorine and monochloramine on E. coli as a representative organism. The results shows that PMA removed 99.94% and 99.99% DNA from non-viable E. coli and Salmonella cells respectively and PMA-qPCR could effectively differentiate viable bacteria from non-viable bacteria; According to the first-order kinetic model, the inactivation coefficients on E. coli obtained by PMA-qPCR were 2.24 L x (mg x min)-1 and 0.0175 L x (mg x min)-1 for chlorine and monochloramine respectively, both of which were lower than those obtained by traditional plating counting method. In order to inactivate 99% of E. coli, the ct values by PMA-qPCR were 0.9 mg L(-1) min and more than 100 mg x L(-1) x min for chlorine and monochloramine while those by plating counting method were only 0.6 mg x L(-1) x min and 20 mg x L(-1) min, respectively; E. coli concentration detected by conventional qPCR kept almost the same when ct value increased, indicating that conventional qPCR was unable to evaluate inactivation efficacy of both chlorine and monochloramine disinfection. In summary, PMA-qPCR shows to be a promising method for evaluating disinfection efficacy by chlorine and monochloramine more accurately.

  4. 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...

  5. 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

  6. 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.

  7. 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.

  8. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    PubMed

    Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V

    2012-03-01

    In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA.

  9. Transmission trials, ITS2-PCR and RAPD-PCR show identity of Toxocara canis isolates from red fox and dog.

    PubMed

    Epe, C; Meuwissen, M; Stoye, M; Schnieder, T

    1999-07-01

    Toxocara canis isolates from dog and from red fox were compared in transmission trials and with molecular analysis using RAPD-PCR technique and comparison of the ITS2 sequence. After oral infection of bitches with 20,000 embryonated T. canis eggs of vulpine and canine origin, the vertical transmission to pup's was examined. All animals of both groups developed typical clinical symptoms of toxocarosis. The haematological, serological, parasitological and post mortem results showed no differences between both isolates except for the infectivity of T. canis stages in mice where the fox isolate showed a significant higher infectivity than the dog isolate. The RAPD-PCR showed a similarity coefficient of 0.95, similar to the range of intraspecific variation in Toxocara cati and Toxascaris leonina specimens as outgroups. The ITS2 comparison showed a 100% identity between both isolates with no intraspecific variations. Therefore, the study shows that the fox and the dog isolate of T. canis were identical in infectivity, transmission and molecular structure; a host adaptation could not be found and the fox has to be seen as a reservoir for T. canis infections in dogs. Considering the increasing number of foxes in urban areas the importance of helminth control in dogs is stressed.

  10. In planta distribution of 'Candidatus Liberibacter asiaticus' as revealed by polymerase chain reaction (PCR) and real-time PCR.

    PubMed

    Tatineni, Satyanarayana; Sagaram, Uma Shankar; Gowda, Siddarame; Robertson, Cecile J; Dawson, William O; Iwanami, Toru; Wang, Nian

    2008-05-01

    Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide, and is caused by a phloem-limited fastidious prokaryotic alpha-proteobacterium that is yet to be cultured. In this study, a combination of traditional polymerase chain reaction (PCR) and real-time PCR targeting the putative DNA polymerase and 16S rDNA sequence of 'Candidatus Liberibacter asiaticus,' respectively, were used to examine the distribution and movement of the HLB pathogen in the infected citrus tree. We found that 'Ca. Liberibacter asiaticus' was distributed in bark tissue, leaf midrib, roots, and different floral and fruit parts, but not in endosperm and embryo, of infected citrus trees. Quantification analysis of the HLB bacterium indicated that it was distributed unevenly in planta and ranged from 14 to 137,031 cells/mug of total DNA in different tissues. A relatively high concentration of 'Ca. Liberibacter asiaticus' was observed in fruit peduncles. Our data from greenhouse-infected plants also indicated that 'Ca. Liberibacter asiaticus' was transmitted systemically from infection site to different parts of the plant. Understanding the distribution and movement of the HLB bacterium inside an individual citrus tree is critical for discerning its virulence mechanism and to develop management strategies for HLB.

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

    PubMed

    Cao, Heping; Cao, Fangping; Roussel, Anne-Marie; Anderson, Richard A

    2013-12-01

    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 false-positive signals and that the length of the amplicon affects the intensity of the amplification. Previous results demonstrate that TaqMan assay is more sensitive but generates lower calculated expression levels than SYBR Green assay in quantifying seven mRNAs in tung tree tissues. The objective of this study is to expand the analysis using animal cells. We compared both qPCR assays for quantifying 24 mRNAs including those coding for glucose transporter (Glut) and mRNA-binding protein tristetraprolin (TTP) in mouse 3T3-L1 adipocytes and RAW264.7 macrophages. The results showed that SYBR Green and TaqMan qPCR were reliable for quantitative gene expression in animal cells. This result was supported by validation analysis of Glut and TTP family gene expression. However, SYBR Green qPCR overestimated the expression levels in most of the genes tested. Finally, both qPCR instruments (Bio-Rad's CFX96 real-time system and Applied Biosystems' Prism 7700 real-time PCR instrument) generated similar gene expression profiles in the mouse cells. These results support the conclusion that both qPCR assays (TaqMan and SYBR Green qPCR) and both qPCR instruments (Bio-Rad's CFX96 real-time system and Applied Biosystems' Prism 7700 real-time PCR instrument) are reliable for quantitative gene expression analyses in animal cells but SYBR Green qPCR generally overestimates gene expression levels than TaqMan qPCR.

  12. 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

  13. 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.

  14. 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.

  15. Microfluidic digital PCR enables multigene analysis of individual environmental bacteria.

    PubMed

    Ottesen, Elizabeth A; Hong, Jong Wook; Quake, Stephen R; Leadbetter, Jared R

    2006-12-01

    Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA-based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment.

  16. 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.

  17. DNA methylation testing and marker validation using PCR: diagnostic applications.

    PubMed

    Egger, Gerda; Wielscher, Matthias; Pulverer, Walter; Kriegner, Albert; Weinhäusel, Andreas

    2012-01-01

    DNA methylation provides a fundamental epigenetic mechanism to establish and promote cell-specific gene-expression patterns, which are inherited by subsequent cell generations. Thus, the epigenome determines the differentiation into a cell lineage but can also program cells to become abnormal or malignant. In humans, different germline and somatic diseases have been linked to faulty DNA methylation. In this article, we will discuss the available PCR-based technologies to assess differences in DNA methylation levels mainly affecting 5-methylcytosine in the CpG dinucleotide context in hereditary syndromal and somatic pathological conditions. We will discuss some of the current diagnostic applications and provide an outlook on how DNA methylation-based biomarkers might provide novel tools for diagnosis, prognosis or patient stratification for diseases such as cancer.

  18. 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.

  19. 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.

  20. An expanded nuclear phylogenomic PCR toolkit for Sapindales1

    PubMed Central

    Collins, Elizabeth S.; Gostel, Morgan R.; Weeks, Andrea

    2016-01-01

    Premise of the study: We tested PCR amplification of 91 low-copy nuclear gene loci in taxa from Sapindales using primers developed for Bursera simaruba (Burseraceae). Methods and Results: Cross-amplification of these markers among 10 taxa tested was related to their phylogenetic distance from B. simaruba. On average, each Sapindalean taxon yielded product for 53 gene regions (range: 16–90). Arabidopsis thaliana (Brassicales), by contrast, yielded product for two. Single representatives of Anacardiaceae and Rutacaeae yielded 34 and 26 products, respectively. Twenty-six primer pairs worked for all Burseraceae species tested if highly divergent Aucoumea klaineana is excluded, and eight of these amplified product in every Sapindalean taxon. Conclusions: Our study demonstrates that customized primers for Bursera can amplify product in a range of Sapindalean taxa. This collection of primer pairs, therefore, is a valuable addition to the toolkit for nuclear phylogenomic analyses of Sapindales and warrants further investigation. PMID:28101434

  1. [PCR detection of transgenic elements in feed raw material].

    PubMed

    Wang, Ying; Yu, Dao-Jian; Kang, Lin; Zhang, Gui-Ming; Jin, Xian-Zhong; Yang, Wei-Dong; Huang, Pei-Qing; Wu, Qiong; Chen, Zhi-Nan; Chu, Cheng-Cai; Cheng, Ying-Hui

    2002-05-01

    Based on the heterogeneous genes usually used in transgenic crops, the PCR technique was performed with primers derived from CaMV 35S promoter (35S-promoter,originated from cauliflower mosaic virus), NOS terminator (nopaline synthase-terminator,derived from Agrobacterium tumefaciens), EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene, and CryIA(b) (delta-endotoxin,evolved from Bacillus thuringiensis subsp. kurstaki) gene to detect transgenic agents from feed raw materials of soybean dregs and corn gluten meal, respectively. Endogenous corn Zein (a protein extracted from corn gluten) gene, soybean Lectin (chitin-binding protein) gene and negative, positive control were applied for avoiding false results. The method established here has been successfully applied in detecting transgenic elements in imported feed raw material.

  2. Detection of genetically modified organisms by electrochemiluminescence PCR method.

    PubMed

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

    2004-10-15

    With the development of biotechnology, more and more genetically modified organisms (GMOs) have entered commercial market. Because of the safety concerns, detection and characterization of GMOs have attracted much attention recently. In this study, electrochemiluminescence polymerase chain reaction (ECL-PCR) combined with hybridization technique was applied to detect the GMOs in genetically modified (GM) soybeans and papayas for the first time. Whether the soybeans and the papayas contain GM components was discriminated by detecting the Cauliflower mosaic virus 35S (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 soybeans and papayas. The technique may provide a new means in GMOs detection due to its simplicity and high efficiency.

  3. Identification of canine papillomavirus by PCR in Greyhound dogs

    PubMed Central

    Anis, Eman A.; Frank, Linda A.; Francisco, Raquel

    2016-01-01

    Background Corns are hard protuberances that occur on the digital footpads of Greyhound dogs. The cause of these lesions is unknown and there is little information about them in the veterinary literature. We received anecdotal examples of dog to dog spread of corns suggesting an infectious cause. The aim of this study was to determine if papillomavirus (PV) is associated with Greyhound corns. Methods We examined four corns from two unrelated adult Greyhound dogs that resided in Florida and Washington, respectively, for PV by PCR. The samples were obtained by owner coring of two lesions from one dog and laser removal of two lesions from the other dog. Total nucleic acid was extracted and DNA was amplified using two PCR primer sets that have been shown to amplify a broad range of PVs from humans and animals: FAP59/ FAP64 and MY11/ MY09. The DNA sequences were compared with all sequences in GenBank. Formalin-fixed, paraffin-embedded tissue from the footpads of four dogs with other inflammatory dermatoses were also examined. Results PV DNA was amplified from all four corn lesions, while no PV DNA was amplified from other tissues. Comparison of the 444-bp sequences amplified by the MY11/ MY09 primers identified two different PVs. One showed 96% nucleotide sequence similarity with the L1 gene of canine PV type 12. The other showed 78% similarity to canine PV type 16 and, therefore, represents a novel PV. In one of the corns, infection by two of the identified PVs was found. Discussion These results suggest PV infection could be involved in the pathogenesis of corns in Greyhound dogs. PMID:27957392

  4. 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

  5. Modified COLD-PCR for detection of minor microorganisms in wine samples during the fermentation.

    PubMed

    Takahashi, Masayuki; Masaki, Kazuo; Mizuno, Akihiro; Goto-Yamamoto, Nami

    2014-05-01

    The detection of low-abundant microorganism is difficult when in a sample in which a specific microorganism represents an overwhelming majority using polymerase chain reaction (PCR)-based methods. A modified CO-amplification at Lower Denaturation temperature PCR (mCOLD-PCR) method was developed to detect low-abundant microorganisms using a double-strand RNA probe to inhibit the amplification of the sequence of a major microorganism. Combining the mCOLD-PCR and downstream application (e.g., denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS)), low-abundant microorganisms were detected more efficiently, even when a specific microorganism represents an overwhelming majority of the sample. We demonstrated that mCOLD-PCR-DGGE enabled us to detect Schizosaccharomyces pombe in a model sample coexisting with 10,000 times as many Saccharomyces cerevisiae. When mCOLD-PCR-DGGE was applied in the microbiota analysis of a fermenting white wine, Candida sp. and Cladosporium sp., which were not detected by conventional PCR, were detected. According to the NGS analysis after mCOLD-PCR of a fermenting red wine, the detection ratio of Saccharomyces was decreased dramatically, and the detection ratios of other microorganisms and the numbers of genera detected were increased compared with the conventional PCR. Thus, the application of mCOLD-PCR will reveal comprehensive microbiota of fermented foods, beverages, and so on.

  6. Development of a novel PCR-RFLP assay for improved detection and typing of bovine papillomaviruses.

    PubMed

    Kawauchi, Kyoko; Takahashi, Chiaki; Ishihara, Ryoko; Hatama, Shinichi

    2015-06-15

    A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed to detect and type bovine papillomaviruses (BPVs) from tumors in cattle. Two degenerate primer sets targeting the BPV L1 gene, subAup/subAdw and subBup/subBdw, and one restriction enzyme RsaI were used in this assay. In silico analyses of the restriction enzyme sites in the PCR fragments of 13 BPV sequences (BPV-1 to -13) revealed that all known BPVs are differentiated by the PCR-RFLP assay. Analyses of 63 previously typed clinical samples, that included teat papillomas and both esophageal and urinary bladder cancer biopsies, show that the assay clearly differentiates between eight clinically important BPV types (BPV-1 to -6, -9, -10), and discriminates between single and multiple infections. To further assess the reliability of the PCR-RFLP method amplified fragments were sequenced. A high correlation (95%) was observed when the results of the PCR-RFLP method were compared with PCR-sequencing. Differences in typing occurred for 3 of 63 specimens; PCR-RFLP identified additional BPV types in these specimens, while the PCR-sequencing identified only one. These results indicate that the PCR-RFLP method reported here is simpler and more reliable in the detection and typing of BPVs from bovine tumor samples than PCR-sequencing.

  7. 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.

  8. [Application study of droplet digital PCR to detect maternal cell contamination in prenatal diagnosis].

    PubMed

    Geng, J; Liu, C; Zhou, X C; Ma, J; Du, L; Lu, J; Zhou, W N; Hu, T T; Lyu, L J; Yin, A H

    2017-02-25

    Objective: To develop a new method based on droplet digital PCR (DD-PCR) for detection and quantification of maternal cell contamination in prenatal diagnosis. Methods: Invasive prenatal samples from 40 couples of β(IVS-Ⅱ-654)/β(N) thalassemia gene carriers who accepted prenatal diagnosis in Affiliated Women and Children's Hospital of Guangzhou Medical University from October 2015 to December 2016 were analyzed retrospectively. Specific primers and probes were designed. The concentration gradient were 50%, 25%, 12.5%, 6.25%, 3.125%, 1.562 5%. There were 40 groups of prenatal diagnostic samples. Comparing DD-PCR with quantitative fluorescent-PCR (QF-PCR) based on the short tandem repeats for assement of the sensitivity and accuracy of maternal cell contamination, respectively. Results: DD-PCR could quantify the maternal cell contamination as low as 1.562 5%. The result was proportional to the dilution titers. In the 40 prenatal samples, 6 cases (15%, 6/40) of maternal cell contamination were detected by DD-PCR, while the QF-PCR based on short tandem repeat showed 3 cases (7.5%, 3/40) with maternal cell contamination, DD-PCR was more accurate (P=0.002) . Conclusion: DD-PCR is a precise and sensitive method in the detection of maternal cell contamintation. It could be useful in clinical application.

  9. Development of a novel immuno-PCR for detection of avian leukosis virus.

    PubMed

    Xie, Quan; Zhang, Jianjun; Shao, Hongxia; Wan, Zhimin; Tian, Xiaoyan; Yang, Jialiang; Pang, Mayun; Qian, Kun; Gao, Wei; Wang, Chengming; Qin, Aijian; Ye, Jianqiang

    2016-10-01

    Avian leukosis virus (ALV) is an important pathogen for various neoplasms, including lymphoid, myeloid, and erythroid neoplasms, and it causes significant economic loss in the poultry industry. Several efficient methods for the detection of ALV have been reported. However, these previously developed approaches are based on either PCR or immunoassays. Here, we used a proximity ligation technique and combined PCR with the immunoassay to develop a novel immuno-PCR (Im-PCR) approach for the detection of ALV. Our data showed that the Im-PCR had high specificity and sensitivity to ALV. The Im-PCR method selectively reacted to ALV but not to the other avian viruses tested. The limit of detection of Im-PCR could reach 0.5 TCID50. Moreover, the results of Im-PCR were in agreement with results from commercial ELISA when the clinical cloaca samples were used for ALV detection. The present results demonstrate that the novel Im-PCR method can be efficiently applied to detect ALV in a clinical setting. Our data also highlight that Im-PCR may have promising applications in the diagnosis of pathogens.

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

    PubMed

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

    2016-01-01

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

  11. Integration of nanoparticle cell lysis and microchip PCR for one-step rapid detection of bacteria.

    PubMed

    Wan, Weijie; Yeow, John T W

    2012-04-01

    This paper describes an integrated microchip system as an efficient and cost-effective solution involving Nanotechnology and Lab-on-a-Chip technology for the rapid detection of bacteria. The system is based on using surface-modified gold nanoparticles for efficient cell lysis followed by microchip PCR without having to remove the nanoparticles from the PCR solution. Poly(quaternary ammonium) modified gold nanoparticles are used to provide a novel and efficient cell lysis method without the need to go through time-consuming, expensive and complicated microfabrication processes as most of current cell lysis methods for Lab-on-a-Chip applications do. It also facilitates the integration of cell lysis and PCR by sharing the same reaction chamber as PCR uses. It is integrated with a prototype microchip PCR system consisting of a physical microchip PCR device and an automated temperature control mechanism. The research work explores solutions for the problem of PCR inhibition caused by gold nanoparticles as well as for the problem of non-specific PCR amplification in the integrated microchip system. It also explores the possibility of greatly reducing PCR cycling time to achieve the same result compared to the protocol for a regular PCR machine. The simplicity of the setup makes it easy to be integrated with other Lab-on-a-Chip functional modules to create customized solutions for target applications.

  12. Evaluation of a PCR/ESI-MS platform to identify respiratory viruses from nasopharyngeal aspirates.

    PubMed

    Lin, Yong; Fu, Yongfeng; Xu, Menghua; Su, Liyun; Cao, Lingfeng; Xu, Jin; Cheng, Xunjia

    2015-11-01

    Acute respiratory tract infection is a major cause of morbidity and mortality worldwide, particularly in infants and young children. High-throughput, accurate, broad-range tools for etiologic diagnosis are critical for effective epidemic control. In this study, the diagnostic capacities of an Ibis platform based on the PCR/ESI-MS assay were evaluated using clinical samples. Nasopharyngeal aspirates (NPAs) were collected from 120 children (<5 years old) who were hospitalized with lower respiratory tract infections between November 2010 and October 2011. The respiratory virus detection assay was performed using the PCR/ESI-MS assay and the DFA. The discordant PCR/ESI-MS and DFA results were resolved with RT-PCR plus sequencing. The overall agreement for PCR/ESI-MS and DFA was 98.3% (118/120). Compared with the results from DFA, the sensitivity and specificity of the PCR/ESI-MS assay were 100% and 97.5%, respectively. The PCR/ESI-MS assay also detected more multiple virus infections and revealed more detailed subtype information than DFA. Among the 12 original specimens with discordant results between PCR/ESI-MS and DFA, 11 had confirmed PCR/ESI-MS results. Thus, the PCR/ESI-MS assay is a high-throughput, sensitive, specific and promising method to detect and subtype conventional viruses in respiratory tract infections and allows rapid identification of mixed pathogens.

  13. 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

  14. 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.

  15. Determination of Bifidobacterium and Lactobacillus in breast milk of healthy women by digital PCR.

    PubMed

    Qian, L; Song, H; Cai, W

    2016-09-01

    Breast milk is one of the most important sources of postnatal microbes. Quantitative real-time polymerase chain reaction (qRT-PCR) is currently used for the quantitative analysis of bacterial 16S rRNA genes in breast milk. However, this method relies on the use of standard curves and is imprecise when quantitating target DNA of low abundance. In contrast, droplet digital PCR (DD-PCR) provides an absolute quantitation without the need for calibration curves. A comparison between DD-PCR and qRT-PCR was conducted for the quantitation of Bifidobacterium and Lactobacillus 16S RNA genes in human breast milk, and the impacts of selected maternal factors were studied on the composition of these two bacteria in breast milk. From this study, DD-PCR reported between 0-34,460 16S rRNA gene copies of Bifidobacterium genera and between 1,108-634,000 16S rRNA gene copies of Lactobacillus genera in 1 ml breast milk. The 16S rRNA gene copy number of Lactobacillus genera was much greater than that of Bifidobacterium genera in breast milk. DD-PCR showed a 10-fold lower limit of quantitation as compared to qRT-PCR. A higher correlation and agreement was observed between qRT-PCR and DD-PCR in Lactobacillus quantitation as compared to Bifidobacterium quantitation. Based on our DD-PCR quantitation, a low abundance of Bifidobacterium bacteria in breast milk was correlated to higher pre-pregnancy body mass index (BMI). However, no significant difference was observed for these two bacteria in breast milk between mothers who had vaginal deliveries and caesarean deliveries. This study suggests that DD-PCR is a better tool to quantitate the bacterial load of breast milk compared to the conventional qRT-PCR method. The number of breast milk Bifidobacterium bacteria is influenced by maternal pre-pregnancy BMI.

  16. 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.

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

    PubMed

    Taylor, Sean C; Mrkusich, Eli M

    2014-01-01

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

  18. Comparison of the quantification of KRAS mutations by digital PCR and E-ice-COLD-PCR in circulating-cell-free DNA from metastatic colorectal cancer patients.

    PubMed

    Sefrioui, David; Mauger, Florence; Leclere, Laurence; Beaussire, Ludivine; Di Fiore, Frédéric; Deleuze, Jean-François; Sarafan-Vasseur, Nasrin; Tost, Jörg

    2017-02-01

    Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy.

  19. Application of a spotting sample preparation technique for the detection of pathogens in woody plants by RT-PCR and real-time PCR (TaqMan).

    PubMed

    Osman, Fatima; Rowhani, Adib

    2006-05-01

    An extraction technique for reverse transcription-PCR (RT-PCR) detection of plant pathogens including viruses, bacteria and phytoplasma is described. The total nucleic acid of these plant pathogens was obtained by direct spotting of crude sap derived from infected leaf, petiole or cambial tissue onto two different types of membranes, positively charged Hybond N(+) Nylon and FTA membranes, and processed for use in PCR. Thirteen different plant viruses, Xylella fastidiosa (causal agent of Pierce's disease) and phytoplasmas were included in the experiment. A thermal treatment (95 degrees C for 10 min) of the Hybond N(+) Nylon discs in a buffered solution improved the detection, but for FTA membrane discs the thermal treatment was not required and the discs were directly placed in the PCR reaction cocktail. Specific amplification of genomic or ribosomal RNA fragments of these pathogens was obtained by one-step RT-PCR except for X. fastidiosa in which a fragment of the genomic DNA was used for amplification. The same sample preparation methods also worked well for real-time RT-PCR (TaqMan). The sample preparation techniques reported here could be used to store samples for future PCR test or for long distance shipment to a detection laboratory.

  20. 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

  1. Fusion primer and nested integrated PCR (FPNI-PCR): a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

    PubMed Central

    2011-01-01

    Background The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow. Results Here, we report a simple and effective fusion primer and nested integrated PCR method (FPNI-PCR) for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs). These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis. Conclusions The successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, FPNI-PCR represents a more sensitive, rapid and accurate technique than the established TAIL-PCR and hiTAIL-PCR procedures. PMID:22093809

  2. Molecular typing of native Bacillus thuringiensis isolates from diverse habitats in India using REP-PCR and ERIC-PCR analysis.

    PubMed

    Katara, Jawahar; Deshmukh, Rupesh; K Singh, Nagendra; Kaur, Sarvjeet

    2012-01-01

    Bacillus thuringiensis is a bacterium of great agronomic and scientific interest. The subspecies of this bacterium colonize and kill a large variety of host insects and even nematodes, but each strain does so with a high degree of specificity. Therefore molecular typing and diversity analysis of B. thuringiensis has enormous importance for discrimination of strains isolated from different sources. In this study, 113 native B. thuringiensis isolates collected from diverse habitats and locations in India and 27 B. thuringiensis type strains obtained from the Bacillus Genetic Stock Centre (BGSC), Ohio State University, USA and used as reference, were analyzed for molecular typing. Genotypic data of 140 B. thuringiensis isolates and type strains was generated by using REP-PCR and ERIC-PCR primers and unweighted pair group method with arithmetic mean (UPGMA) analysis using NTSYSpc2.2 and grouped into 4 main clusters. All the groups have isolates from diverse origins. No group was found to represent any specific origin or location. The observed patterns of REP-PCR and ERIC-PCR pattern were discriminatory enough to reveal differences in the B. thuringiensis isolates and reference strains. The resolution power and marker index of the ERIC-PCR (RP 9.39, MI 6.34) was found to be higher than that of the REP-PCR (RP 6.20, MI 4.48). The REP-PCR and ERIC-PCR markers have been found to be useful for discrimination of B. thuringiensis isolates and reference strains. ERIC-PCR was the more informative of the two techniques. This study showed that the B. thuringiensis isolates collected from diverse habitats in India had a high degree of genetic diversity.

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

    PubMed

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

    2012-09-01

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

  4. Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR.

    PubMed

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas; Hung, Tran Quang; Wolff, Anders; Bang, Dang Duong

    2017-04-01

    Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to increase the yield of the solid-phase amplification, we studied various parameters including the length, the density, as well as the annealing position of the solid support primer. A dramatic increase in the signal-to-noise (S/N) ratio was observed when increasing the length of solid support primers from 45 to 80 bp. The density of the primer on the surface was found to be important for the S/N ratio of the SP-PCR, and the optimal S/N was obtained with a density of 1.49 × 10(11) molecules/mm(2). In addition, the use of solid support primers with a short overhang at the 5' end would help improve the S/N ratio of the SP-PCR. With optimized conditions, SP-PCR can achieve amplification efficiency comparable to conventional PCR, with a limit of detection of 1.5 copies/μl (37.5 copies/reaction). These improvements will pave the way for wider applications of SP-PCR in various fields such as clinical diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR.

  5. Diagnosis of Kyasanur forest disease by nested RT-PCR, real-time RT-PCR and IgM capture ELISA.

    PubMed

    Mourya, Devendra T; Yadav, Pragya D; Mehla, Rajeev; Barde, Pradip V; Yergolkar, Prasanna N; Kumar, Sandeep R P; Thakare, Jyotsna P; Mishra, Akhilesh C

    2012-12-01

    Kyasanur forest disease (KFD) is a zoonotic viral disease caused by infection by a Flavivirus, a member of the family Flaviviridae. KFD is a public health concern in the Karnataka State in southern India. Available conventional diagnostic tests such as virus isolation and serological tests, such as haemagglutination inhibition and complement fixation tests are time consuming. This study reports the development of a nested RT-PCR [nRT-PCR] and a TaqMan-based real-time RT-PCR and IgM antibodies capture ELISA [MAC-ELISA] for rapid and accurate diagnosis of suspected KFD cases. The nRT-PCR and the TaqMan-based real-time RT-PCR assays were developed using gene sequences of the NS-5/non-coding region. Both the assays detected KFD viral RNA in acute phase human serum samples and can provide early diagnosis of infection. Real-time RT-PCR was found to be more sensitive than nRT-PCR, which could detect 38 copies of KFDV RNA. MAC-ELISA was developed for the detection of recent infections. Although real-time RT-PCR and nRT-PCR require expensive reagents, expensive equipment and trained personnel, the developed MAC-ELISA can be used easily in the affected areas. These tests add to the existing diagnosis arsenal against haemorrhagic viruses that are prevalent in India. These assays will also help to extend our knowledge of the pathology of KFD virus and its associated clinical features, by measuring the viral titre during infection and at the time of seroconversion. Information, which is not available currently because of the lack of appropriate diagnostic methods. In addition, early laboratory diagnosis of KFDV infection will help in the application of appropriate control measures and management of KFD cases.

  6. Comparison of real-time multiplex human papillomavirus (HPV) PCR assays with the linear array HPV genotyping PCR assay and influence of DNA extraction method on HPV detection.

    PubMed

    Roberts, Christine C; Swoyer, Ryan; Bryan, Janine T; Taddeo, Frank J

    2011-05-01

    Real-time human papillomavirus (HPV) type-specific multiplex PCR assays were developed to detect HPV DNA in specimens collected for the efficacy determination of the quadrivalent HPV (type 6, 11, 16, and 18) L1 virus-like particle (VLP) vaccine (Gardasil). We evaluated the concordance between type-specific multiplex HPV PCR and the widely used, commercially available Roche Linear Array genotyping PCR assay. Female genital swab specimens were tested for the presence of L1, E6, and E7 sequences of HPV type 6 (HPV6), HPV11, HPV16, HPV18, HPV31, HPV45, HPV52, and HPV58 and E6 and E7 sequences of HPV33, HPV35, HPV39, HPV51, HPV56, and HPV59 in type- and gene-specific real-time multiplex PCR assays. Specimens were also tested for the presence of L1 sequences using two versions of the Roche Linear Array genotyping assay. Measures of concordance of a modified version of the Linear Array and the standard Linear Array PCR assay were evaluated. With specimen DNA extraction using the Qiagen Spin blood kit held as the constant, multiplex PCR assays detect more HPV-positive specimens for the 14 HPV types common to both than either version of the Linear Array HPV genotyping assay. Type-specific agreements between the assays were good, at least 0.838, but were often driven by negative agreement in HPV types with low prevalence, as evidenced by reduced proportions of positive agreement. Overall HPV status agreements ranged from 0.615 for multiplex PCR and standard Linear Array to 0.881 for multiplex PCR and modified Linear Array. An alternate DNA extraction technique, that used by the Qiagen MinElute kit, impacted subsequent HPV detection in both the multiplex PCR and Linear Array assays.

  7. 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.

  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 competitive-nested PCR and real-time PCR in detecting BCR-ABL fusion transcripts in chronic myeloid leukemia patients.

    PubMed

    Guo, J Q; Lin, H; Kantarjian, H; Talpaz, M; Champlin, R; Andreeff, M; Glassman, A; Arlinghaus, R B

    2002-12-01

    Real-time RT-PCR has great advantages for estimating transcript levels in a variety of situations. These include relative rapid assay times (hours), reliability and ease of performing replicate analyses. In contrast, competitive PCR is a very labor-intensive procedure requiring a few days to generate useful data. We compared the same samples from CML patients by both methods. Importantly, we used the Bcr-Abl junction plasmid DNA, which is used as a competitor in the manual competitive PCR assay, to generate a standard curve for the real-time assay. This permitted reporting the real-time data as the number of BCR-ABL transcripts per microg of total RNA, which is the same format used for the competitive PCR assay. In this study, a total of 435 peripheral blood and marrow samples from 285 CML patients were analyzed by RT-PCR; these patients were undergoing therapy by STI-571, interferon, and bone marrow transplantation treatment. Most samples also had assay values for the Philadelphia chromosome (Ph), FISH and Western blotting for the Bcr-Abl oncoprotein. Our findings indicated that the real-time assay was less sensitive than the manual competitive RT-PCR assay (t = 5.118; P < 0.001). Of interest, the transcript levels in cell line mixtures with various ratios of K562/KG-1 (BCR-ABL positive/negative) cells were also significantly higher with the competitive RT-PCR assays than real-time RT-PCR, except for levels of BCR-ABL below 200 transcripts per microg of RNA. In both patient and cell line experiments, dividing the BCR-ABL transcripts by the total ABL transcripts virtually eliminated the difference between real-time BCR-ABL transcript values and quantitative competitive BCR-ABL transcript values, indicating that both BCR-ABL and ABL transcripts were underestimated by the real-time assay. In addition, the increased sensitivity of the nested, competitive RT-PCR was readily apparent in patients with minimal residual disease, which by the real-time were negative in the

  10. Group-specific PCR-RFLP and real-time PCR methods for detection and tentative discrimination of strictly anaerobic beer-spoilage bacteria of the class Clostridia.

    PubMed

    Juvonen, Riikka; Koivula, Teija; Haikara, Auli

    2008-07-15

    The strictly anaerobic brewery contaminants of the genera Pectinatus, Megasphaera, Selenomonas and Zymophilus in the class Clostridia constitute an important group of spoilage bacteria of unpasteurised, packaged beers. The aim of this study was to develop and evaluate group-specific PCR methods to detect and differentiate these bacteria in beer. A group-specific primer pair targeting a 342-bp variable region of the 16S rRNA gene was designed and evaluated in end-point PCR with gel electrophoresis and in real-time PCR with SYBR Green I dye. Significant cross-reactions with DNAs from any of the forty-two brewery-related, non-target microbes or from real brewery samples were not detected in either PCR system. The group-specific end-point and real-time PCR products could be differentiated according to species/genus and spoilage potential using restriction fragment length polymorphism (KpnI, XmnI, BssHII, ScaI) and melting point curve analysis, respectively. In combination with a rapid DNA extraction method, the PCR reactions detected ca 10(0)-10(3) CFU per 25 ml of beer depending on the strain and on the PCR system. The end-point and real-time PCR analysis took 6-7 h and 2-3 h, respectively. Pre-PCR enrichment of beer samples for 1-3 days ensured the detection of even a single cultivable cell. The PCR and cultivation results of real brewery samples were mostly congruent but the PCR methods were occasionally more sensitive. The PCR methods developed allow the detection of all the nine beer-spoilage Pectinatus, Megasphaera, Selenomonas and Zymophilus species in a single reaction and their differentiation below group level and reduce the analysis time for testing of their presence in beer samples by 1-2 days. The methods can be applied for brewery routine quality control and for studying occurrence, diversity and numbers of the strictly anaerobic beer spoilers in the brewing process.

  11. 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

  12. Multi-primer target PCR for rapid identification of bovine DRB3 alleles.

    PubMed

    Ledwidge, S A; Mallard, B A; Gibson, J P; Jansen, G B; Jiang, Z H

    2001-08-01

    Multi-primer target polymerase chain reaction (MPT-PCR) is a rapid method for the identification of specific BoLA-DRB3 alleles. In a single PCR reaction, the presence of two alleles associated with increased risk, DRB3.2*23 (DRB3*2701-2703, 2705-2707) and decreased risk, DRB3.2*16 (DRB3*1501, 1502), of mastitis in Canadian Holstein can be detected. Two outer primers amplify exon 2 of DRB3. Simultaneously, two inner, allele-specific primers amplify individual alleles. Initially, 40 cows previously typed by PCR-restriction fragment length polymorphism (PCR-RFLP) were genotyped using the multi-primer approach. An additional 30 cows were first genotyped by multi-primer target PCR, then by PCR-RFLP. All animals were correctly identified and there were no false positives. This technique can readily be modified to identify other BoLA alleles of interest.

  13. Fast detection of genetic information by an optimized PCR in an interchangeable chip.

    PubMed

    Wu, Jinbo; Kodzius, Rimantas; Xiao, Kang; Qin, Jianhua; Wen, Weijia

    2012-02-01

    In this paper, we report the construction of a polymerase chain reaction (PCR) device for fast amplification and detection of DNA. This device consists of an interchangeable PCR chamber, a temperature control component as well as an optical detection system. The DNA amplification happens on an interchangeable chip with the volumes as low as 1.25 μl, while the heating and cooling rate was as fast as 12.7°C/second ensuring that the total time needed of only 25 min to complete the 35 cycle PCR amplification. An optimized PCR with two-temperature approach for denaturing and annealing (Td and Ta) of DNA was also formulated with the PCR chip, with which the amplification of male-specific sex determining region Y (SRY) gene marker by utilizing raw saliva was successfully achieved and the genetic identification was in-situ detected right after PCR by the optical detection system.

  14. International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

    PubMed

    Whale, Alexandra S; Devonshire, Alison S; Karlin-Neumann, George; Regan, Jack; Javier, Leanne; Cowen, Simon; Fernandez-Gonzalez, Ana; Jones, Gerwyn M; Redshaw, Nicholas; Beck, Julia; Berger, Andreas W; Combaret, Valérie; Dahl Kjersgaard, Nina; Davis, Lisa; Fina, Frederic; Forshew, Tim; Fredslund Andersen, Rikke; Galbiati, Silvia; González Hernández, Álvaro; Haynes, Charles A; Janku, Filip; Lacave, Roger; Lee, Justin; Mistry, Vilas; Pender, Alexandra; Pradines, Anne; Proudhon, Charlotte; Saal, Lao H; Stieglitz, Elliot; Ulrich, Bryan; Foy, Carole A; Parkes, Helen; Tzonev, Svilen; Huggett, Jim F

    2017-02-07

    This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

  15. Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

    PubMed

    Waterfall, Christy M; Eisenthal, Robert; Cobb, Benjamin D

    2002-12-20

    A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.

  16. Quantitative Reverse Transcription-qPCR-Based Gene Expression Analysis in Plants.

    PubMed

    Abdallah, Heithem Ben; Bauer, Petra

    2016-01-01

    The investigation of gene expression is an initial and essential step to understand the function of a gene in a physiological context. Reverse transcription-quantitative real-time PCR (RT-qPCR) assays are reproducible, quantitative, and fast. They can be adapted to study model and non-model plant species without the need to have whole genome or transcriptome sequence data available. Here, we provide a protocol for a reliable RT-qPCR assay, which can be easily adapted to any plant species of interest. We describe the design of the qPCR strategy and primer design, considerations for plant material generation, RNA preparation and cDNA synthesis, qPCR setup and run, and qPCR data analysis, interpretation, and final presentation.

  17. QUANTITATIVE VS. CONVENTIONAL PCR FOR DETECTION OF HUMAN ADENOVIRUSES IN WATER AND SEDIMENT SAMPLES

    PubMed Central

    STAGGEMEIER, Rodrigo; BORTOLUZZI, Marina; HECK, Tatiana Moraes da Silva; SPILKI, Fernando Rosado; ALMEIDA, Sabrina Esteves de Matos

    2015-01-01

    SUMMARY Human Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively. PMID:26422153

  18. QUANTITATIVE VS. CONVENTIONAL PCR FOR DETECTION OF HUMAN ADENOVIRUSES IN WATER AND SEDIMENT SAMPLES.

    PubMed

    Staggemeier, Rodrigo; Bortoluzzi, Marina; Heck, Tatiana Moraes da Silva; Spilki, Fernando Rosado; Almeida, Sabrina Esteves de Matos

    2015-01-01

    Human Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively.

  19. Evaluation of different enrichment methods for pathogenic Yersinia species detection by real time PCR

    PubMed Central

    2014-01-01

    Background Yersiniosis is a zoonotic disease reported worldwide. Culture and PCR based protocols are the most common used methods for detection of pathogenic Yersinia species in animal samples. PCR sensitivity could be increased by an initial enrichment step. This step is particularly useful in surveillance programs, where PCR is applied to samples from asymptomatic animals. The aim of this study was to evaluate the improvement in pathogenic Yersinia species detection using a suitable enrichment method prior to the real time PCR (rtPCR). Nine different enrichment protocols were evaluated including six different broth mediums (CASO, ITC, PSB, PBS, PBSMSB and PBSSSB). Results The analysis of variance showed significant differences in Yersinia detection by rtPCR according to the enrichment protocol used. These differences were higher for Y. pseudotuberculosis than for Y. enterocolitica. In general, samples incubated at lower temperatures yielded the highest detection rates. The best results were obtained with PBSMSB and PBS2. Application of PBSMSB protocol to free-ranging wild board samples improved the detection of Y. enterocolitica by 21.2% when compared with direct rtPCR. Y. pseudotuberculosis detection was improved by 10.6% when results obtained by direct rtPCR and by PBSMSB enrichment before rtPCR were analyzed in combination. Conclusions The data obtained in the present study indicate a difference in Yersinia detection by rtPCR related to the enrichment protocol used, being PBSMSB enrichment during 15 days at 4°C and PBS during 7 days at 4°C the most efficient. The use of direct rtPCR in combination with PBSMSB enrichment prior to rtPCR resulted in an improvement in the detection rates of pathogenic Yersinia in wild boar and could be useful for application in other animal samples. PMID:25168886

  20. Evidence for a functional dimeric form of the PcrA helicase in DNA unwinding.

    PubMed

    Yang, Ye; Dou, Shuo-Xing; Ren, Hua; Wang, Peng-Ye; Zhang, Xing-Dong; Qian, Min; Pan, Bing-Yi; Xi, Xu Guang

    2008-04-01

    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 approximately 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 approximately 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.

  1. Development of an updated PCR assay for detection of African swine fever virus.

    PubMed

    Luo, Yuzi; Atim, Stella A; Shao, Lina; Ayebazibwe, Chrisostom; Sun, Yuan; Liu, Yan; Ji, Shengwei; Meng, Xing-Yu; Li, Su; Li, Yongfeng; Masembe, Charles; Ståhl, Karl; Widén, Frederik; Liu, Lihong; Qiu, Hua-Ji

    2017-01-01

    Due to the current unavailability of vaccines or treatments for African swine fever (ASF), which is caused by African swine fever virus (ASFV), rapid and reliable detection of the virus is essential for timely implementation of emergency control measures and differentiation of ASF from other swine diseases with similar clinical presentations. Here, an improved PCR assay was developed and evaluated for sensitive and universal detection of ASFV. Primers specific for ASFV were designed based on the highly conserved region of the vp72 gene sequences of all ASFV strains available in GenBank, and the PCR assay was established and compared with two OIE-validated PCR tests. The analytic detection limit of the PCR assay was 60 DNA copies per reaction. No amplification signal was observed for several other porcine viruses. The novel PCR assay was more sensitive than two OIE-validated PCR assays when testing 14 strains of ASFV representing four genotypes (I, V, VIII and IX) from diverse geographical areas. A total of 62 clinical swine blood samples collected from Uganda were examined by the novel PCR, giving a high agreement (59/62) with a superior sensitive universal probe library-based real-time PCR. Eight out of 62 samples tested positive, and three samples with higher Ct values (39.15, 38.39 and 37.41) in the real-time PCR were negative for ASFV in the novel PCR. In contrast, one (with a Ct value of 29.75 by the real-time PCR) and two (with Ct values of 29.75 and 33.12) ASFV-positive samples were not identified by the two OIE-validated PCR assays, respectively. Taken together, these data show that the novel PCR assay is specific, sensitive, and applicable for molecular diagnosis and surveillance of ASF.

  2. Detection of Babesia microti parasites by highly sensitive 18S rRNA reverse transcription PCR.

    PubMed

    Hanron, Amelia E; Billman, Zachary P; Seilie, Annette M; Chang, Ming; Murphy, Sean C

    2017-03-01

    Babesia are increasingly appreciated as a cause of transfusion-transmitted infection. Sensitive methods are needed to screen blood products. We report herein that B. microti 18S rRNA is over 1,000-fold more abundant than its coding genes, making reverse transcription PCR (RT-PCR) much more sensitive than PCR. Babesia 18S rRNA may be useful for screening the blood supply.

  3. 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

  4. Quantitative PCR analysis of salivary pathogen burden in periodontitis.

    PubMed

    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 T

  5. Simultaneous detection and differentiation of Campylobacter jejuni, C. coli, and C. lari in chickens by multiplex real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A multiplex real-time PCR (qPCR) assay was developed to detect and differentiate the three most commonly found and harmful species of Campylobacter in a single PCR reaction. The qPCR primers and TaqMan probes were designed to amplify the unique DNA sequences of hipO, cdtA, and pepT genes which are s...

  6. 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

  7. 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.

  8. Competitor calibration and analysis of competitive amplified PCR products by high-performance liquid chromatography (HPLC).

    PubMed

    Köhler, T

    1999-01-01

    To assay gene expression or virus genomes in tissues or body fluids, competitive polymerase chain reaction (cPCR) is now performed in many laboratories. cPCR is a quantitative adaption of the PCR method in which a known number of copies of a synthetic RNA (1) or DNA (2-4) is coamplified with the target sample and therefore compete for the common primers and reagents in the same reaction tube. After coamplification, both products are distinguished by characteristic features, e.g., size, mostly by electrophoretic methods, or by probe-specific hybridization, e.g., PCR-ELISA (4,5).

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

    PubMed

    Deepak, Sa; Kottapalli, Kr; Rakwal, R; Oros, G; Rangappa, Ks; Iwahashi, H; Masuo, Y; Agrawal, Gk

    2007-06-01

    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 21(st) 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.

  10. Rapid detection of Vibrio cholerae O139 Bengal from stool specimens by PCR.

    PubMed Central

    Albert, M J; Islam, D; Nahar, S; Qadri, F; Falklind, S; Weintraub, A

    1997-01-01

    In a previous study using pure bacterial cultures in a PCR assay, a primer pair corresponding to a unique chromosomal region of Vibrio cholerae O139 Bengal generated an amplicon from only V. cholerae O139 Bengal. PCR with the same primer pair was used to screen 180 diarrheal stool specimens. All the 67 V. cholerae O139 culture-positive stool specimens were positive by PCR, and the remaining specimens, which contained either other recognized enteric pathogens or no pathogens, were all negative by PCR. PMID:9163504

  11. 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.

  12. Regulation of PCR efficiency with magnetic nanoparticles in a rotating magnetic field

    NASA Astrophysics Data System (ADS)

    Higashi, Toshiaki; Nagaoka, Yutaka; Minegishi, Hiroaki; Echigo, Akinobu; Usami, Ron; Maekawa, Toru; Hanajiri, Tatsuro

    2011-04-01

    The polymerase chain reaction (PCR) method is widely used for the reproduction and amplification of specific DNA segments in vitro, and a novel PCR method using nanomaterials such as gold nanoparticles has recently been reported. This paper reports on the regulation of PCR efficiency with superparamagnetic nanoparticles in a rotating magnetic field. The level of efficiency was successfully regulated in a rotating magnetic field by the authors, and decreased with increasing frequency of the field. The results obtained show that simply controlling the structure and dynamics of magnetic nanoparticle clusters in a rotating magnetic field can regulate PCR efficiency.

  13. 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

  14. Calibration-free assays on standard real-time PCR devices

    PubMed Central

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

    2017-01-01

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

  15. Linear amplification mediated PCR--localization of genetic elements and characterization of unknown flanking DNA.

    PubMed

    Gabriel, Richard; Kutschera, Ina; Bartholomae, Cynthia C; von Kalle, Christof; Schmidt, Manfred

    2014-06-25

    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.

  16. Calibration-free assays on standard real-time PCR devices

    NASA Astrophysics Data System (ADS)

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

    2017-03-01

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

  17. Using Multiplex PCR for Assessing the Quality of Whole Genome Amplified DNA.

    PubMed

    El-Heliebi, Amin; Chen, Shukun; Kroneis, Thomas

    2015-01-01

    This chapter describes a simple and inexpensive multiplex PCR-based method to assess the quality of whole genome amplification (WGA) products generated from heat-induced random fragmented DNA. A set of four primer pairs is used to amplify DNA sequences of WGA products in and downstream of GAPDH gene in yielding 100, 200, 300, and 400 bp fragments. PCR products are analyzed by agarose gel electrophoresis and the respective WGA quality is classified according to the number of obtained PCR bands. WGA products that yield three or four PCR bands are considered to be of high quality and yield good results when analyzed by means of array comparative genome hybridization (CGH).

  18. 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

  19. [Clarification of a break-in theft crime by multiplex PCR analysis of cigarette butts].

    PubMed

    Hochmeister, M; Haberl, J; Borer, V; Rudin, O; Dirnhofer, R

    1995-01-01

    This paper describes the first use of multiplex PCR amplification kits for the analysis of DNA extracted from cigarette butts in a criminal case. Two suspects could be excluded as potential contributors to the samples, whereas the multi locus PCR-based DNa profile derived from the cigarette butts was consistent with a DNA profile derived from a third suspect. For identity testing in criminal cases where cigarette butts are involved, commercially available PCR amplification kits provide currently the most powerful tool. Furthermore this PCR-based analysis can be implemented into most application orientated laboratories.

  20. Quantitative analysis of somatic mitochondrial DNA mutations by single-cell single-molecule PCR.

    PubMed

    Kraytsberg, Yevgenya; Bodyak, Natalya; Myerow, Susan; Nicholas, Alexander; Ebralidze, Konstantin; Khrapko, Konstantin

    2009-01-01

    Mitochondrial genome integrity is an important issue in somatic mitochondrial genetics. Development of quantitative methods is indispensable to somatic mitochondrial genetics as quantitative studies are required to characterize heteroplasmy and mutation processes, as well as their effects on phenotypic developments. Quantitative studies include the identification and measurement of the load of pathogenic and non-pathogenic clonal mutations, screening mitochondrial genomes for mutations in order to determine the mutation spectra and characterize an ongoing mutation process. Single-molecule PCR (smPCR) has been shown to be an effective method that can be applied to all areas of quantitative studies. It has distinct advantages over conventional vector-based cloning techniques avoiding the well-known PCR-related artifacts such as the introduction of artificial mutations, preferential allelic amplifications, and "jumping" PCR. smPCR is a straightforward and robust method, which can be effectively used for molecule-by-molecule mutational analysis, even when mitochondrial whole genome (mtWG) analysis is involved. This chapter describes the key features of the smPCR method and provides three examples of its applications in single-cell analysis: di-plex smPCR for deletion quantification, smPCR cloning for clonal point mutation quantification, and smPCR cloning for whole genome sequencing (mtWGS).

  1. Comparison of Simplexa Flu A/B & RSV PCR with cytospin-immunofluorescence and laboratory-developed TaqMan PCR in predominantly adult hospitalized patients.

    PubMed

    Landry, Marie L; Ferguson, David

    2014-08-01

    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).

  2. Characterization of Theileria species by PCR using specific target sequences.

    PubMed

    Maxia, L; Reale, S; Loria, G R; Greco, A; Vitale, F; Glorioso, N S; Sparagano, O

    1999-09-01

    Theileriosis is an infectious disease in tropical countries and in the Mediterranean area. It is caused by Theileria, a haemoprotozoan, transmitted by vectors belonging to the Ixodidae. In Southern Italy and in Sicily the infection is due mainly to T. annulata, but in some cases other species are involved in the disease. The authors describe a method to identify theileriosis in cattle blood samples, using PCR and hybridization techniques. Different primer sets were used to amplify different DNA target sequences, both genus and species specific. Blood samples from cattle were collected in Sicily. The DNA extracted from blood samples was employed first to detect the presence of the 18S ribosomal subunit gene specific for Theileria genus. Successively the positive samples were analysed to identify the species, T. annulata or T. buffeli/orientalis, using as target sequences for amplification respectively a fragment of the TAMS-1 and p33/34 antigens gene. Here the authors describe for the first time the presence of T. buffeli/orientalis infection in Sicilian herds. In fact 66% of positive blood samples were T. buffeli/orientalis infected.

  3. Generation of competitor DNA fragments for quantitative PCR.

    PubMed

    Uberla, K; Platzer, C; Diamantstein, T; Blankenstein, T

    1991-11-01

    A convenient and generally applicable method for the generation of competitor DNA fragments for quantitative PCR is described. Using mouse-specific primers, fragments are amplified from DNA of an evolutionarily distantly related species under low-stringency annealing conditions. Because these artificially created fragments contain the mouse primer specific ends, they can be used for the quantification of the mouse DNA amplified by these primers. Competitor DNA fragments that differ in size from the corresponding mouse DNA are selected to distinguish both fragments visually by gel electrophoresis. Competitor DNA fragments were generated for mouse beta-actin, interleukin-1, and tumor necrosis factor (TNF). Co-amplification of beta-actin cDNA for adjustment of equal amounts of input cDNA and subsequently TNF cDNA from lipopolysaccharide (LPS)-activated and nonactivated spleen cells with serial dilutions of the respective competitor DNA fragments allowed a semiquantitative comparison of the ratio of TNF mRNA present in both cDNA samples. Under certain conditions, the competitor DNA fragments can be used to determine the approximate molar concentration of a mRNA.

  4. A PCR survey for posterior Hox genes in amphibians.

    PubMed

    Mannaert, An; Roelants, Kim; Bossuyt, Franky; Leyns, Luc

    2006-02-01

    Hox genes encode transcription factors that play a key role in specifying the body plan in metazoans and are therefore essential in explaining patterns of evolutionary diversity. As an ancient tetrapod group with diverse limb types, amphibians are important for understanding the origin and diversification of limbs in land vertebrates. We conducted a PCR survey in two species of each amphibian order to identify Hox-9 to Hox-13, known to function in limb development. Fifteen distinct posterior Hox genes and one retro-pseudogene were identified, and the former confirm the existence of four Hox clusters in each amphibian order. Some genes expected to occur in all tetrapods, based on the posterior Hox complement of mammals, fishes and coelacanth, were not recovered from our survey, and may have been lost. Hoxd-12 is absent in frogs and possibly other amphibians. Considering its function in autopodial development, the loss of this gene may be related to the absence of the fifth finger in frogs and salamanders.

  5. Host Immunity to Clostridium difficile PCR Ribotype 017 Strains

    PubMed Central

    Jafari, Nazila V.; Songane, Mario; Stabler, Richard A.; Elawad, Mamoun; Wren, Brendan W.; Allan, Elaine

    2014-01-01

    Clostridium difficile is an important nosocomial pathogen and the leading cause of antibiotic-associated diarrhea. Multilocus sequence typing indicates that C. difficile strains belong to five distinct genetic clades encompassing several PCR ribotypes (RT). Since their emergence in 2003, hypervirulent RT027 strains have been a major focus of research; in contrast, our current understanding of RT017-mediated disease pathogenesis lags far behind. In this study, we aimed to characterize host immunity to CF5 and M68, two genetically well-defined RT017 strains. Both strains engaged with host Toll-like receptor 2/6 (TLR2/6), TLR2-CD14, and TLR5 to similar extents in a model cell line. Despite this, CF5 mediated significantly greater dendritic cell (DC) interleukin-12 (IL-12), IL-27, and IL-10 immunity than M68. Both strains elicited similar IL-1β mRNA levels, and yet only M68 caused a marked increase in secretory IL-1β. A CF5 cocultured-DC cytokine milieu drove an equipotent Th1 and Th17 response, while M68 promoted greater Th17 immunity. Human gastrointestinal ex vivo cytokine responses to both strains were characterized. Taken together, our data suggest that C. difficile strains mediate overlapping and yet distinct mucosal and DC/T cell immunity. Finally, toxin-driven IL-1β release supports the hypothesis that this cytokine axis is a likely target for therapeutic intervention for C. difficile infection. PMID:25225246

  6. A simplified universal genomic DNA extraction protocol suitable for PCR.

    PubMed

    Wang, T Y; Wang, L; Zhang, J H; Dong, W H

    2011-03-29

    Conventional genomic DNA extraction protocols need expensive and hazardous reagents for decontamination of phenolic compounds from the extracts and are only suited for certain types of tissue. We developed a simple, time-saving and cost-efficient method for genomic DNA extraction from various types of organisms, using relatively innocuous reagents. The protocol employs a single purification step to remove contaminating compounds, using a silica column and a non-hazardous buffer, and a chaotropic-detergent lysing solution that hydrolyzes RNA and allows the selective precipitation of DNA from cell lysates. We used this system to extract genomic DNA from different tissues of various organisms, including algae (Dunaliella salina), human peripheral blood, mouse liver, Escherichia coli, and Chinese hamster ovary cells. Mean DNA yields were 20-30 μg/cm(3) from fresh tissues (comparable to yields given by commercial extraction kits), and the 260/280 nm absorbance ratio was 1.8-2.0, demonstrating a good degree of purity. The extracted DNA was successfully used in PCR, restriction enzyme digestion and for recombinant selection studies.

  7. [Simultaneous screening method for Bordetella species by conventional PCR assay].

    PubMed

    Akiyama, Yumi; Saito, Etsuko; Enomoto, Miki; Tsuji, Hidetaka; Chikahira, Masatsugu; Yoshida, Masashi

    2013-11-01

    A simultaneous screening method using conventional PCR was developed for the detection and discrimination of Bordetella pertussis, Bordetella parapertussis, and Bordetella holmesii. A formulated multiprex method employing 4 kinds of paired primers on amplification of 4 corresponding different insertion sequences (IS481, IS1001, IS1002 and hIS1001) enabled rapid screening and identification. The detection limits of each DNA extracted from 3 kinds of Bordetella species were 5fg/microL for each. Obscure existences of B. pertussis and B. holmesii at low levels were confirmed with the LAMP method. This multiplex assay was applied to the clinical specimens obtained from patients with pertussis-like symptoms at sentinel clinics under the epidemiological surveillance of infectious diseases of Hyogo prefecture in FY2012. Among 42 nasopharyngeal swabs, B. pertussis was detected from 12 samples including 8 samples collected at outbreak in nursery school. The use of this method for the surveillance of infectious agents enabled us to search for 3 kinds of Bordetella species at once with low costs.

  8. 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.

  9. 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.

  10. Development of Cross-Assembly Phage PCR-Based Methods ...

    EPA Pesticide Factsheets

    Technologies that can characterize human fecal pollution in environmental waters offer many advantages over traditional general indicator approaches. However, many human-associated methods cross-react with non-human animal sources and lack suitable sensitivity for fecal source identification applications. The genome of a newly discovered bacteriophage (~97 kbp), the Cross-Assembly phage or “crAssphage”, assembled from a human gut metagenome DNA sequence library is predicted to be both highly abundant and predominately occur in human feces suggesting that this double stranded DNA virus may be an ideal human fecal pollution indicator. We report the development of two human-associated crAssphage endpoint PCR methods (crAss056 and crAss064). A shotgun strategy was employed where 384 candidate primers were designed to cover ~41 kbp of the crAssphage genome deemed favorable for method development based on a series of bioinformatics analyses. Candidate primers were subjected to three rounds of testing to evaluate assay optimization, specificity, limit of detection (LOD95), geographic variability, and performance in environmental water samples. The top two performing candidate primer sets exhibited 100% specificity (n = 70 individual samples from 8 different animal species), >90% sensitivity (n = 10 raw sewage samples from different geographic locations), LOD95 of 0.01 ng/µL of total DNA per reaction, and successfully detected human fecal pollution in impaired envi

  11. PCR-Based Identification of Oral Streptococcal Species

    PubMed Central

    Zhu, Min; Dawson, Deborah V.; Cao, Huojun; Levy, Steven M.

    2016-01-01

    The microbial etiology of dental caries is still debated. Among the hypothesized contributors are the “low pH streptococci,” a designation given to unusually acid proficient strains among the primary plaque colonizers S. oralis, S. mitis, S. gordonii, and S. anginosus. However, accurate assignment of species is difficult among the oral streptococci. Our objective was to develop a streamlined method for identifying strains of S. oralis and S. mitis from plaque samples so that they could be analyzed in a separate study devoted to low pH streptococci and caries. Two independent PCR amplifications of a locus highly conserved among streptococci were used for presumptive species identification. Multilocus sequence analysis (MLSA) was used to measure accuracy. Sensitivity was 100% for selecting S. oralis and S. mitis among the clones sampled. Specificity was good except for the most closely related species that could not be reliably distinguished even by MLSA. The results with S. oralis and S. mitis were used to identify new primer sets that expanded the utility of the approach to other oral streptococcal species. These novel primer sets offer a convenient means of presumptive identification that will have utility in many studies where large scale, in-depth genomic analyses are not practical. PMID:27703479

  12. PCR analysis for Wolbachia in human and canine Demodex mites.

    PubMed

    Borgo, Sibylle N; Sattler, Elke C; Hogardt, Michael; Adler, Kristin; Plewig, Gerd

    2009-10-01

    In many skin diseases such as Demodex folliculitis, rosacea- or steroid-induced rosacea Demodex mites are present in abundance and are at least partially held responsible for causing these disorders. Although it is known that these diseases respond well to tetracyclines, it is unclear if this is due to the antiinflammatory effects of the antibiotics or to an antibacterial effect on so far unknown bacteria within the Demodex mites. As in filariasis, where the response to doxycycline can be explained by the presence of Wolbachia within the filarial nematodes, this study was performed to see whether Wolbachia also use Demodex mites as their hosts. Human and canine Demodex mite samples were taken by skin scrapings and tested by PCR for the presence of Wolbachia DNA. Wolbachia pipientis DNA was used as positive control. In none of the DNA extracts, Wolbachia were detected showing no evidence for the presence of these bacteria in Demodex mites. The response of Demodex aggravated or Demodex caused diseases to tetracyclines seems not to be due to the presence of Wolbachia in Demodex mites in contrast to the results seen in filariasis.

  13. 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.

  14. Real-Time PCR Identification of Unique Bacillus anthracis Sequences.

    PubMed

    Cieślik, P; Knap, J; Kolodziej, M; Mirski, T; Joniec, J; Graniak, G; Zakowska, D; Winnicka, I; Bielawska-Drózd, A

    2015-01-01

    Bacillus anthracis is a spore-forming, Gram-positive microorganism. It is a causative agent of anthrax, a highly infectious disease. It belongs to the "Bacillus cereus group", which contains other closely related species, including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus weihenstephanensis, and Bacillus pseudomycoides. B. anthracis naturally occurs in soil environments. The BA5345 genetic marker was used for highly specific detection of B. anthracis with TaqMan probes. The detection limit of a real-time PCR assay was estimated at the level of 16.9 copies (CI95% - 37.4 to 37.86, SD = 0.2; SE = 0.118). Oligonucleotides designed for the targeted sequences (within the tested locus) revealed 100 % homology to B. anthracis strain reference sequences deposited in the database (NCBI) and high specificity to all tested B. anthracis strains. Additional in silico analysis of plasmid markers pag and cap genes with B. anthracis strains included in the database was carried out. Our study clearly indicates that the BA5345 marker can be used with success as a chromosomal marker in routine identification of B. anthracis; moreover, detection of plasmid markers indicates virulence of the examined strains.

  15. ABO genotyping by PCR-RFLP and cloning and sequencing.

    PubMed

    Haak, Wolfgang; Burger, Joachim; Alt, Kurt Werner

    2004-12-01

    A refined PCR-RFLP based method was established to genotype ABO blood groups. The main objective of this study was to make the techniques also suitable for working with degraded DNA. Specific primer design was carried out to choose fragments shorter than 200 bp as necessary in forensic and archaeological applications. Four fragments of exon 6 and 7 of the ABO gene were amplified and digested by in total 7 restriction endonucleases. Particular attention was paid to the base changes at nucleotide positions 261(delG), 297, 526, 703, 721, 771, 796 and 1060(delC) in order to distinguish the six common alleles A101, A201, B, O01, O02 and O03. Furthermore, this method also enables determination of seven of the less frequent alleles: A104, A204, Ax02, Ax03, O05, O06 and O07. The method was applied successfully to a series of blood samples with known phenotypes and genotypes as well as DNA extracted from a thirty year old blood stain and an ancient tooth sample. However, working with ancient DNA requires additional cloning and sequencing of the RFLP-typing results due to DNA post mortem damages such as deaminations, which could lead to false typing results.

  16. 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.

  17. Rapid detection of equine influenza virus H3N8 subtype by insulated isothermal RT-PCR (iiRT-PCR) assay using the POCKIT™ Nucleic Acid Analyzer.

    PubMed

    Balasuriya, Udeni B R; Lee, Pei-Yu Alison; Tiwari, Ashish; Skillman, Ashley; Nam, Bora; Chambers, Thomas M; Tsai, Yun-Long; Ma, Li-Juan; Yang, Pai-Chun; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

    2014-10-01

    Equine influenza (EI) is an acute, highly contagious viral respiratory disease of equids. Currently, equine influenza virus (EIV) subtype H3N8 continues to be the most important respiratory pathogen of horses in many countries around the world. The need to achieve a rapid diagnosis and to implement effective quarantine and movement restrictions is critical in controlling the spread of EIV. In this study, a novel, inexpensive and user-friendly assay based on an insulated isothermal RT-PCR (iiRT-PCR) method on the POCKIT™, a field-deployable device, was described and validated for point-of-need detection of EIV-H3N8 in clinical samples. The newly established iiRT-PCR assay targeting the EIV HA3 gene was evaluated for its sensitivity using in vitro transcribed (IVT) RNA, as well as ten-fold serial dilutions of RNA extracted from the prototype H3N8 strain A/equine/Miami/1/63. Inclusivity and exclusivity panels were tested for specificity evaluation. Published real-time RT-PCR (rRT-PCR) assays targeting the NP and HA3 genes were used as the reference standards for comparison of RNA extracted from field strains and from nasal swab samples collected from experimentally infected horses, respectively. Limit of detection with a 95% probability (LoD95%) was estimated to be 11copies of IVT RNA. Clinical sensitivity analysis using RNA prepared from serial dilutions of a prototype EIV (Miami 1/63/H3N8) showed that the iiRT-PCR assay was about 100-fold more sensitive than the rRT-PCR assay targeting the NP gene of EIV subtype H3N8. The iiRT-PCR assay identified accurately fifteen EIV H3N8 strains and two canine influenza virus (CIV) H3N8 strains, and did not cross-react with H6N2, H7N7, H1N1 subtypes or any other equine respiratory viral pathogens. Finally, 100% agreement was found between the iiRT-PCR assay and the universal influenza virus type A rRT-PCR assay in detecting the EIV A/equine/Kentucky/7/07 strain in 56 nasal swab samples collected from experimentally inoculated

  18. Profound inhibition of the PCR step of CF V3 multiplex PCR/OLA assay by the use of UV-irradiated plastic reaction tubes.

    PubMed

    Fox, David H; Huang, Chih-Kang; Du, Juan; Chang, Tylis Y; Pan, Qiulu

    2007-06-01

    Supplies, such as bags of plastic reaction tubes, are sometimes left in the laminar flow hoods unintentionally while the ultraviolet (UV) lamp is illuminated overnight. In addition, UV irradiation is used for sterilization and amplicon inactivation to avoid contamination. The oligonucleotide ligation assay (OLA) is a unique approach to mutation detection of point mutations, small deletions, and small insertions. Recently, we encountered problems with this assay and peak heights were much lower or disappeared. After going through systemic trouble-shooting, we found that profound inhibition of the polymerase chain reaction (PCR) step of CF V3 multiplex PCR/OLA assay by the use of UV-irradiated plastic reaction tubes. When UV-irradiated tubes used throughout the assay, tubes exposed for 8 weeks at 0.7 m from the UV source gave a reduction of 60% and 67% in the assay products on the basis of sum of peak heights. Tubes exposed for 3 weeks at 0.1 m from the UV source totally eliminated assay product yielding no peaks. Further experiments showed that the inhibition happened mostly in the PCR step. Burgess and Hall had reported that inhibition of PCR of human glyceraldehydes-3-phosphate dehydrogenase transcripts after UV irradiating the tubes. This showed that the inhibition was not assay-specific. The reason that the inhibition of PCR was more profound could be due to a multiplex PCR assay and small reaction volume. The mechanism of PCR inhibition by UV irradiation is not clear. In conclusion, plastic reaction tubes intended for PCR/OLA assays should not be exposed to UV.

  19. 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.

  20. Standard Addition Quantitative Real-Time PCR (SAQPCR): A Novel Approach for Determination of Transgene Copy Number Avoiding PCR Efficiency Estimation

    PubMed Central

    Zhu, Changqing; Wang, Weiwei; Grierson, Donald; Xu, Changjie; Chen, Kunsong

    2013-01-01

    Quantitative real-time polymerase chain reaction (qPCR) has been previously applied to estimate transgene copy number in transgenic plants. However, the results can be erroneous owing to inaccurate estimation of PCR efficiency. Here, a novel qPCR approach, named standard addition qPCR (SAQPCR), was devised to accurately determine transgene copy number without the necessity of obtaining PCR efficiency data. The procedures and the mathematical basis for the approach are described. A recombinant plasmid harboring both the internal reference gene and the integrated target gene was constructed to serve as the standard DNA. It was found that addition of suitable amounts of standard DNA to test samples did not affect PCR efficiency, and the guidance for selection of suitable cycle numbers for analysis was established. Samples from six individual T0 tomato (Solanum lycopersicum) plants were analyzed by SAQPCR, and the results confirmed by Southern blot analysis. The approach produced accurate results and required only small amounts of plant tissue. It can be generally applied to analysis of different plants and transgenes. In addition, it can also be applied to zygosity analysis. PMID:23308234

  1. Thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR.

    PubMed

    Le, Yilin; Chen, Huayou; Zagursky, Robert; Wu, J H David; Shao, Weilan

    2013-08-01

    Polymerase chain reaction (PCR) is a powerful method to produce linear DNA fragments. Here we describe the Tma thermostable DNA ligase-mediated PCR production of circular plasmid (PPCP) and its application in directed evolution via in situ error-prone PCR. In this thermostable DNA ligase-mediated whole-plasmid amplification method, the resultant DNA nick between the 5' end of the PCR primer and the extended newly synthesized DNA 3' end of each PCR cycle is ligated by Tma DNA ligase, resulting in circular plasmid DNA product that can be directly transformed. The template plasmid DNA is eliminated by 'selection marker swapping' upon transformation. When performed under an error-prone condition with Taq DNA polymerase, PPCP allows one-step construction of mutagenesis libraries based on in situ error-prone PCR so that random mutations are introduced into the target gene without altering the expression vector plasmid. A significant difference between PPCP and previously published methods is that PPCP allows exponential amplification of circular DNA. We used this method to create random mutagenesis libraries of a xylanase gene and two cellulase genes. Screening of these libraries resulted in mutant proteins with desired properties, demonstrating the usefulness of in situ error-prone PPCP for creating random mutagenesis libraries for directed evolution.

  2. Trypanosoma rangeli: RAPD-PCR and LSSP-PCR analyses of isolates from southeast Brazil and Colombia and their relation with KPI minicircles.

    PubMed

    Marquez, D S; Ramírez, L E; Moreno, J; Pedrosa, A L; Lages-Silva, E

    2007-09-01

    This study presents the first genetic characterization of five Trypanosoma rangeli isolates from Minas Gerais, in the southeast of Brazil and their comparison with Colombian populations by minicircle classification, RAPD-PCR and LSSP-PCR analyses. Our results demonstrated a homogenous T. rangeli population circulating among Didelphis albiventris as reservoir host in Brazil while heterogeneous populations were found in different regions of Colombia. KP1(+) minicircles were found in 100% isolates from Brazil and in 36.4% of the Colombian samples, whereas the KP2 and KP3 minicircles were detected in both groups. RAPD-PCR and LSSP-PCR profiles revealed a polymorphism within KP1(+) and KP1(-) T. rangeli populations and allowed the division of T. rangeli in two branches. The Brazilian KP1(+) isolates were more homogenous than the KP1(+) isolates from Colombia. The RAPD-PCR were entirely consistent with the distribution of KP1 minicircles while those obtained by LSSP-PCR were associated in 88.9% and 71.4% with KP1(+) and KP1(-) populations, respectively.

  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.

  4. 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.

  5. 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.

  6. 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.

  7. Standardization and validation of simple PCR, duplex PCR and RAPD in comparison to blood smear examination for diagnosing bovine tropical theileriosis.

    PubMed

    Sudan, Vikrant; Shanker, Daya; Jaiswal, Amit; Singh, Amit; Pandey, Vijay

    2017-03-01

    Bovine Tropical Theileriosis (BTT) is an important vector-borne protozoan disease that imposing serious constraints on the health and productivity of domestic cattle. It is matter of common fact that following recovery from primary infection, cattle become persistent carriers and act as reservoirs of infection thereby, playing a critical role in disease epidemiology. The present study describes the comparative diagnostic efficiency of simplex PCR, duplex PCR and RAPD assays for detection of Theileria annulata in cattle. An optimized simple PCR and duplex PCR assay were established using TAMS F/R as primer sets encoding for 721 bp amplicon alongside a RAPD with arbitrary primer coding for 963 bp product of T. annulata. The simple PCR and duplex PCR detected pathogen with almost same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA of another pathogen without nonspecific amplifications. RAPD failed to give comparable results and suffered from limitations of sensitivity as well as specificity. The developed assays may be seen as a good tool for epidemiological studies aiming at assessing the burden of chronic infections and improving control of the associated diseases in endemic regions.

  8. 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

  9. DNA and RNA isolated from tissues processed by microwave-accelerated apparatus MFX-800-3 are suitable for subsequent PCR and Q-RT-PCR amplification.

    PubMed

    Bödör, Csaba; Schmidt, Otto; Csernus, Balázs; Rajnai, Hajnalka; Szende, Béla

    2007-01-01

    Over the past decade, methods of molecular biology have appeared in diagnostic pathology and are routinely applied on formalin-fixed, paraffin-embedded histological samples, processed via conventional embedding methods. Due to its reagent- and cost-effectiveness, embedding techniques that utilize microwave acceleration in one or more steps of histoprocessing are increasingly used by numerous laboratories. The demand arises that tissues processed this way should also be suitable for the requirements of molecular pathology. In this study, both conventionally embedded and MFX-800-3 machine-processed tissue samples from the same source were used for isolation of DNA and RNA and for performing PCR and real-time PCR. PCR amplification of the beta-globin gene, as well as the real-time PCR amplification of the ABL mRNA was successful in all cases. Our conclusion is that samples processed by the vacuum assisted automatic microwave histoprocessor MFX-800-3 are perfectly applicable for DNA and RNA isolation and provide appropriate templates for further PCR and realtime PCR studies.

  10. Quantitative multiplex real-time PCR assay for shrimp allergen: comparison of commercial master mixes and PCR platforms in rapid cycling.

    PubMed

    Eischeid, Anne C; Kasko, Sasha M

    2015-01-01

    Real-time PCR has been used widely in numerous fields. In food safety, it has been applied to detection of microbes and other contaminants, including food allergens. Interest in rapid (fast) cycling real-time PCR has grown because it yields results in less time than does conventional cycling. However, fast cycling can adversely affect assay performance. Here we report on tests of commercial master mixes specifically designed for fast real-time PCR using a shrimp allergen assay we previously developed and validated. The objective of this work was to determine whether specialized commercial master mixes lead to improved assay performance in rapid cycling. Real-time PCR assays were carried out using four different master mixes and two different rapid cycling protocols. Results indicated that specialized master mixes did yield quality results. In many cases, linear ranges spanned up to 7 orders of magnitude, R(2) values were at least 0.95, and reaction efficiencies were within or near the optimal range of 90 to 110%. In the faster of the two rapid cycling protocols tested, assay performance and PCR amplification were markedly better for the shorter PCR product. In conclusion, specialized commercial master mixes were effective as part of rapid cycling protocols, but conventional cycling as used in our previous work is more reliable for the shrimp assay tested.

  11. PCR diagnosis of visceral leishmaniasis in asymptomatic dogs using conjunctival swab samples.

    PubMed

    Leite, Rodrigo Souza; Ferreira, Sidney de Almeida; Ituassu, Leonardo Trindade; de Melo, Maria Norma; de Andrade, Antero Silva Ribeiro

    2010-06-24

    The efficacy of conjunctival swab (CS) as a sampling method for visceral leishmaniasis (VL) diagnosis by PCR of asymptomatic dogs was evaluated. The CS was compared to blood samples (B) and skin biopsies (SB), two less invasive samples potentially useful for massive screening of dogs. Thirty asymptomatic dogs, with serological and parasitological positive tests, were used. The samples were analyzed by two PCR methods: kDNA PCR-hybridization and ITS-1 nPCR. The DNA sample volume used was of 1.0 microL and 10.0 microL respectively. Using CS samples the kDNA PCR-hybridization was able to detected parasite DNA in 24/30 dogs (80%) using the right conjunctiva (RC) and 23/30 dogs (76.6%) with the left conjunctiva (LC), 17/30 dogs (56.7%) by means of SB and 4/30 dogs (13.3%) with B. The CS positivity obtained combining RC and LC results was of 90% (27/30 dogs). The assay of CS samples by ITS-1 nPCR revealed that 25/30 dogs (83.3%) were positive when using RC and 20/30 dogs (66.6%) were positive when using LC. Via the same method 15/30 dogs (50.0%) were positive by SB and 17/30 dogs (56.7%) with B. The CS positivity obtained by ITS-1 nPCR combining RC and LC was of 83.3%. The CS positivities for RC and LC were significantly higher (p<0.05) than SB and B for kDNA PCR-hybridization method. Statistical difference in relation to SB and B was verified by ITS-1 nPCR only for RC. The kDNA PCR-hybridization and ITS-1 nPCR methods showed similar sensitivities for CS and SB samples. On the other hand, for blood samples, the positivity of ITS-1 nPCR was significantly higher than the one obtained by the kDNA PCR-hybridization, indicating that sensitivity of PCR methods can vary according to the biological sample examined. Our results showed that CS was suitable to detect Leishmania DNA in asymptomatic animals when comparing to other low-invasive samples. The CS sensitivities obtained in this study were similar to the ones observed in other studies for VL diagnosis in symptomatic dogs

  12. [Detection of wheat as an allergenic substance in food by a nested PCR method].

    PubMed

    Hashimoto, Hiroyuki; Makabe, Yuhki; Hasegawa, Yasuyuki; Sajiki, Junko; Miyamoto, Fumio

    2008-02-01

    A nested PCR method was developed for the detection of DNAs extracted from allergenic substances (here, wheat) in food. Because of DNA fragmentation, detection of wheat-specific DNA extracted from food, such as retort pouch food, is very difficult. Therefore, to improve the sensitivity of detection, a nested PCR primer pair (Wtr01NE2-5' and Wtr10NE5-3': amplicon size 97 bp) was newly designed within the region of the PCR products amplified by the official Japanese primer pair (Wtr01-5' and Wtr10-3'; amplicon size 141 bp) for wheat. Genomic DNAs of seven kinds of commercial processed foods containing wheat, wheat flour and three kinds of wheat flours pressure-heated at 100, 121 and 131 degrees C were extracted with a commercial ion-exchange type kit by modifying the Japanese official method. The nested PCR method involved two PCR procedures. First, PCR was performed by varying both the PCR reagents and cycling conditions of the Japanese official method. Second, PCR was performed using the first PCR products diluted 200-fold with TE buffer. The Japanese official method enabled detection of only four of the seven kinds of foods and three of the four kinds of flours (one sample was just a trace), while the nested PCR method detected all seven foods and all four flours. Investigation of the detectability of the four kinds of wheat flours depending on the size of the amplified fragment using five primer pairs showed that its size must be kept to less than approximately 100 bp. The nested PCR method significantly improved the sensitivity of detection of wheat-specific DNA.

  13. 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

  14. 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.

  15. Impact of multiplex PCR on antimicrobial treatment in febrile neutropenia: a randomized controlled study.

    PubMed

    Idelevich, Evgeny A; Silling, Gerda; Niederbracht, Yvonne; Penner, Hanna; Sauerland, Maria Cristina; Tafelski, Sascha; Nachtigall, Irit; Berdel, Wolfgang E; Peters, Georg; Becker, Karsten

    2015-10-01

    Multiplex PCR (mPCR) directly from blood has been suggested as a promising method for rapid identification of pathogens causing sepsis. This study aimed to investigate whether mPCR has any impact on antimicrobial treatment. Hematological patients with febrile neutropenia were randomized into two groups. In the study group, mPCR was performed as an addition to standard diagnostics, and PCR finding was immediately communicated to the clinicians, thus being available for decision making. In the control group, clinicians were not aware of PCR result. PCR samples were collected simultaneously with clinically indicated blood culture specimens from peripheral vein and/or central venous catheter at fever onset and once again if fever persisted up to 72 h. Overall, 74 patients of the study group and 76 patients of the control group were enrolled and 253 samples collected. Therapy was changed to targeted antimicrobial therapy (AMT) in 12 patients (16.2%) in the study group and in 12 patients (15.8%) in the control group. For patients with changes, the median time to change to the targeted AMT was 21.4 h in the study group and 47.5 h in the control group (p = 0.018). In the study group, 57.1% (8/14) of changes to targeted AMT was due to PCR finding. PCR led to AMT change in 9.5% (7/74) of study group patients, i.e., in 33.3% (7/21) of patients who had positive PCR finding. There were no significant differences in patient outcomes (secondary endpoints). In conclusion, PCR method accelerates change to the targeted AMT in febrile neutropenic patients.

  16. Identification and characterization of dermatophyte species and strains with PCR amplification.

    PubMed

    Liu, Guofang; He, Chenghua; Zhang, Haibin

    2014-08-01

    The aim of the present study was to use two polymerase chain reaction (PCR) methods, with (GACA)4 and non-transcribed spacer (NTS) as primers, to identify and characterize dermatophyte isolates from dogs and cats to a species and strain level. A total of 45 isolates from nine dermatophyte species were collected from pet dogs and cats and subjected to PCR amplification with the microsatellite primer (GACA)4. Dermatophyte strains of three of the same species collected from four cities were subjected to PCR amplification with the NTS