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Sample records for techniques pcr amplification

  1. SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

    Directory of Open Access Journals (Sweden)

    Ricardo Luiz Dantas MACHADO

    1998-09-01

    Full Text Available 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.Relatamos a adaptação de uma técnica para coleta de amostras (MFV e outra para extração, amplificação de DNA de parasitas da malária para diagnóstico por PCR/ELISA. O método de coleta de amostras requer menos habilidade e economisa tempo e dinheiro, assim reduzindo a mais da metade o custo. O material é também adequado para análise genética em especimens frescos ou estocados, preparados por este método.

  2. Preventing PCR amplification carryover contamination in a clinical laboratory.

    Science.gov (United States)

    Aslanzadeh, Jaber

    2004-01-01

    During the past two decades PCR and several other DNA/RNA amplification techniques have become important diagnostic tools in clinical laboratories. Amplification products contamination has been the main impediment to using these techniques routinely in diagnostic laboratories. Over the years, several creative pre- and post-amplification methods have been developed that prevent amplicon carryover contamination. These procedures, coupled with automated systems that employ real-time amplification and simultaneous detection in a closed system, have substantially reduced the possibility of false positive results due to amplification products carryover contamination.

  3. Development of a loop-mediated isothermal amplification technique and comparison with quantitative real-time PCR for the rapid visual detection of canine neosporosis.

    Science.gov (United States)

    Mahittikorn, Aongart; Thammasonthijarern, Nipa; Roobthaisong, Amonrattana; Udonsom, Ruenruetai; Popruk, Supaluk; Siri, Sukhontha; Mori, Hirotake; Sukthana, Yaowalark

    2017-08-23

    Dogs are the definitive hosts of Neospora caninum and play an important role in the transmission of the parasite. Despite the high sensitivity of existing molecular tools such as quantitative real-time PCR (qPCR), these techniques are not suitable for use in many countries because of equipment costs and difficulties in implementing them for field diagnostics. Therefore, we developed a simplified technique, loop-mediated isothermal amplification (LAMP), for the rapid visual detection of N. caninum. LAMP specificity was evaluated using a panel containing DNA from a range of different organisms. Sensitivity was evaluated by preparing 10-fold serial dilutions of N. caninum tachyzoites and comparing the results with those obtained using qPCR. Assessment of the LAMP results was determined by recognition of a colour change after amplification. The usefulness of the LAMP assay in the field was tested on 396 blood and 115 faecal samples from dogs, and one placenta from a heifer collected in Lopburi, Nakhon Pathom, Sa Kaeo, and Ratchaburi provinces, Thailand. Specificity of the LAMP technique was shown by its inability to amplify DNA from non-target pathogens or healthy dogs. The detection limit was the equivalent of one genome for both LAMP and qPCR. LAMP and qPCR detected positive N. caninum infection in 15 of 396 (3.8%) blood samples; LAMP detected 9/115 (7.8%) positive faecal samples, while qPCR detected 5/115 (4.3%) positive faecal samples. The placental tissue was shown to be positive by both techniques. Agreement between LAMP and qPCR was perfect in blood samples (kappa value, 1.00) and substantial in faecal samples (kappa value, 0.697). This is the first known LAMP assay developed for the amplification of N. caninum. The technique effectively and rapidly detected the parasite with high sensitivity and specificity and was cost-effective. This assay could be used in the field to confirm the diagnosis of canine or bovine neosporosis.

  4. (PCR) and loop-mediated isothermal amplification

    African Journals Online (AJOL)

    SAM

    2014-03-26

    Mar 26, 2014 ... better alternative for PCR, even in low technology laboratories. In addition, these findings revealed that the possibility of fatal fusariosis due to F. solani is not so rare in HIV positive patients. Key words: Fusarium solani, loop-mediated isothermal amplification (LAMP), HIV, polymerase chain reaction. (PCR).

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

    Science.gov (United States)

    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.

  6. A DNA-Based Encryption Method Based on Two Biological Axioms of DNA Chip and Polymerase Chain Reaction (PCR) Amplification Techniques.

    Science.gov (United States)

    Zhang, Yunpeng; Wang, Zhiwen; Wang, Zhenzhen; Liu, Xin; Yuan, Xiaojing

    2017-09-27

    Researchers have gained a deeper understanding of DNA-based encryption and its effectiveness in enhancing information security in recent years. However, there are many theoretical and technical issues about DNA-based encryption that need to be addressed before it can be effectively used in the field of security. Currently, the most popular DNA-based encryption schemes are based on traditional cryptography and the integration of existing DNA technology. These schemes are not completely based on DNA computing and biotechnology. Herein, as inspired by nature, encryption based on DNA has been developed, which is, in turn, based on two fundamental biological axioms about DNA sequencing: 1) DNA sequencing is difficult under the conditions of not knowing the correct sequencing primers and probes, and 2) without knowing the correct probe, it is difficult to decipher precisely and sequence the information of unknown and mixed DNA/peptide nucleic acid (PNA) probes, which only differ in nucleotide sequence, arranged on DNA chips (microarrays). In essence, when creating DNA-based encryption by means of biological technologies, such as DNA chips and polymerase chain reaction (PCR) amplification, the encryption method discussed herein cannot be decrypted, unless the DNA/PNA probe or PCR amplification is known. The biological analysis, mathematical analysis, and simulation results demonstrate the feasibility of the method, which provides much stronger security and reliability than that of traditional encryption methods. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. SISH/CISH or qPCR as alternative techniques to FISH for determination of HER2 amplification status on breast tumors core needle biopsies: a multicenter experience based on 840 cases.

    Science.gov (United States)

    Jacquemier, Jocelyne; Spyratos, Frédérique; Esterni, Benjamin; Mozziconacci, Marie-Joëlle; Antoine, Martine; Arnould, Laurent; Lizard, Sarab; Bertheau, Philippe; Lehmann-Che, Jacqueline; Fournier, Cécile Blanc; Krieger, Sophie; Bibeau, Frédéric; Lamy, Pierre-Jean; Chenard, Marie Pierre; Legrain, Michèle; Guinebretière, Jean-Marc; Loussouarn, Delphine; Macgrogan, Gaëtan; Hostein, Isabelle; Mathieu, Marie Christine; Lacroix, Ludovic; Valent, Alexander; Robin, Yves Marie; Revillion, Françoise; Triki, Magali Lacroix; Seaume, Aline; Salomon, Anne Vincent; de Cremoux, Patricia; Portefaix, Geneviève; Xerri, Luc; Vacher, Sophie; Bièche, Ivan; Penault-Llorca, Frédérique

    2013-07-22

    Until now, FISH has been the gold standard technique to identify HER2 amplification status in ambiguous cases of breast cancer. Alternative techniques have been developed to increase the capacities of investigating HER2 amplification status. The aims of this multicenter study in a large series of breast cancer patients were to prospectively compare the level of performance of CISH, SISH, and qPCR alternative techniques on paraffin-embedded core biopsies with "gold standard FISH" for evaluation of HER2 amplification status. This study was performed on 840 cases scored by immunohistochemistry (IHC): 0=317 (38%), 1+=183 (22%), 2+=109 (13%), 3+=231 (27%). Each of the 15 French centers participating in the study analyzed 56 breast carcinoma cases diagnosed on fixed paraffin-embedded core biopsies. HER2 amplification status was determined by commercially available FISH used as the reference technique with determination of the HER2/CEN17 ratio or HER2 copy number status. The alternative techniques performed on the same cases were commercially available SISH or CISH and a common qPCR method especially designed for the study including a set of 10 primer pairs: 2 for HER2 (exons 8 and 26), 5 to evaluate chromosome 17 polysomy TAOK1, UTP6, MRM1, MKS1, SSTR2 and 3 for diploidy control TSN, LAP3 and ADAMTS16. The concordance between IHC and FISH was 96% to 95% based on the HER2/CEN17 ratio (n=766) or HER2 copy number (n=840), respectively. The concordance of the alternative techniques with FISH was excellent: 97% and 98% for SISH (498 and 587 cases), 98% and 75% for CISH (108 and 204 cases) and 95% and 93% (699 and 773 cases) for qPCR based on the HER2/CEN17 ratio or HER2 copy number, respectively. Similarly, sensitivity ranged from 99% to 95% for SISH, 100% to 99% for CISH and 89% to 80% for qPCR. The concordance with FISH (ratio) in the 2+ cases was 89% for SISH, 100% for CISH and 93% for qPCR. These alternative techniques showed an excellent concordance with FISH in core

  8. PCR amplification on microarrays of gel immobilized oligonucleotides

    Science.gov (United States)

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

    2003-11-04

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

  9. Principles and technical aspects of PCR amplification

    National Research Council Canada - National Science Library

    Pelt-Verkuil, Elizabeth van; Belkum, Alex van; Hays, John P

    2008-01-01

    ... to illustrate any particularly important concepts or comments. Indeed, all commercial PCR biotechnology companies offer information about their products on internet sites and in online technical manuals. These online resources will be invaluable for any readers requiring more detailed PCR protocols. The authors have provided references for many PCR co...

  10. Reduction of heteroduplex formation in PCR amplification

    Czech Academy of Sciences Publication Activity Database

    Michu, Elleni; Mráčková, Martina; Vyskot, Boris; Žlůvová, Jitka

    2010-01-01

    Roč. 54, č. 1 (2010), s. 173-176 ISSN 0006-3134 R&D Projects: GA AV ČR(CZ) KJB600040801; GA ČR(CZ) GD204/09/H002; GA AV ČR(CZ) IAA600040801; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : polymerase chain reaction * reconditioning PCR * mixed-template PCR Subject RIV: BO - Biophysics Impact factor: 1.582, year: 2010

  11. Quality control for quantitative PCR based on amplification compatibility test

    Czech Academy of Sciences Publication Activity Database

    Tichopád, Aleš; Bar, T.; Pecen, Ladislav; Kitchen, R.R.; Kubista, Mikael; Pfaffl, M.W.

    2010-01-01

    Roč. 50, č. 4 (2010), s. 308-312 ISSN 1046-2023 R&D Projects: GA AV ČR IAA500520809; GA AV ČR IAA500970904 Institutional research plan: CEZ:AV0Z50520701 Keywords : Quantitative PCR * Quality control * Amplification efficiency Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.527, year: 2010

  12. Enhancing PCR Amplification of DNA from Recalcitrant Plant Specimens Using a Trehalose-Based Additive

    Directory of Open Access Journals (Sweden)

    Tharangamala Samarakoon

    2013-01-01

    Full Text Available Premise of the study: PCR amplification of DNA extracted from plants is sometimes difficult due to the presence of inhibitory compounds. An effective method to overcome the inhibitory effect of compounds that contaminate DNA from difficult plant specimens is needed. Methods and Results: The effectiveness of a PCR additive reagent containing trehalose, bovine serum albumin (BSA, and polysorbate-20 (Tween-20 (TBT-PAR was tested. PCR of DNA extracted from fresh, silica-dried, and herbarium leaf material of species of Achariaceae, Asteraceae, Lacistemataceae, and Samydaceae that failed using standard techniques were successful with the addition of TBT-PAR. Conclusions: The addition of TBT-PAR during routine PCR is an effective method to improve amplification of DNA extracted from herbarium specimens or plants that are known to contain PCR inhibitors.

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

    Directory of Open Access Journals (Sweden)

    Yeates C

    1998-01-01

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

  14. Whole Genome Amplification of Day 3 or Day 5 Human Embryos Biopsies Provides a Suitable DNA Template for PCR-Based Techniques for Genotyping, a Complement of Preimplantation Genetic Testing

    Directory of Open Access Journals (Sweden)

    Elizabeth Schaeffer

    2017-01-01

    Full Text Available Our objective was to determine if whole genome amplification (WGA provides suitable DNA for qPCR-based genotyping for human embryos. Single blastomeres (Day 3 or trophoblastic cells (Day 5 were isolated from 342 embryos for WGA. Comparative Genomic Hybridization determined embryo sex as well as Trisomy 18 or Trisomy 21. To determine the embryo’s sex, qPCR melting curve analysis for SRY and DYS14 was used. Logistic regression indicated a 4.4%, 57.1%, or 98.8% probability of a male embryo when neither gene, SRY only, or both genes were detected, respectively (accuracy = 94.1%, kappa = 0.882, and p<0.001. Fluorescent Capillary Electrophoresis for the amelogenin genes (AMEL was also used to determine sex. AMELY peak’s height was higher and this peak’s presence was highly predictive of male embryos (AUC = 0.93, accuracy = 81.7%, kappa = 0.974, and p<0.001. Trisomy 18 and Trisomy 21 were determined using the threshold cycle difference for RPL17 and TTC3, respectively, which were significantly lower in the corresponding embryos. The Ct difference for TTC3 specifically determined Trisomy 21 (AUC = 0.89 and RPL17 for Trisomy 18 (AUC = 0.94. Here, WGA provides adequate DNA for PCR-based techniques for preimplantation genotyping.

  15. Improved DOP-PCR (iDOP-PCR: A robust and simple WGA method for efficient amplification of low copy number genomic DNA.

    Directory of Open Access Journals (Sweden)

    Konstantin A Blagodatskikh

    Full Text Available Whole-genome amplification (WGA techniques are used for non-specific amplification of low-copy number DNA, and especially for single-cell genome and transcriptome amplification. There are a number of WGA methods that have been developed over the years. One example is degenerate oligonucleotide-primed PCR (DOP-PCR, which is a very simple, fast and inexpensive WGA technique. Although DOP-PCR has been regarded as one of the pioneering methods for WGA, it only provides low genome coverage and a high allele dropout rate when compared to more modern techniques. Here we describe an improved DOP-PCR (iDOP-PCR. We have modified the classic DOP-PCR by using a new thermostable DNA polymerase (SD polymerase with a strong strand-displacement activity and by adjustments in primers design. We compared iDOP-PCR, classic DOP-PCR and the well-established PicoPlex technique for whole genome amplification of both high- and low-copy number human genomic DNA. The amplified DNA libraries were evaluated by analysis of short tandem repeat genotypes and NGS data. In summary, iDOP-PCR provided a better quality of the amplified DNA libraries compared to the other WGA methods tested, especially when low amounts of genomic DNA were used as an input material.

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

    OpenAIRE

    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 b...

  17. Direct PCR amplification of forensic touch and other challenging DNA samples: A review.

    Science.gov (United States)

    Cavanaugh, Sarah E; Bathrick, Abigail S

    2018-01-01

    DNA evidence sample processing typically involves DNA extraction, quantification, and STR amplification; however, DNA loss can occur at both the DNA extraction and quantification steps, which is not ideal for forensic evidence containing low levels of DNA. Direct PCR amplification of forensic unknown samples has been suggested as a means to circumvent extraction and quantification, thereby retaining the DNA typically lost during those procedures. Direct PCR amplification is a method in which a sample is added directly to an amplification reaction without being subjected to prior DNA extraction, purification, or quantification. It allows for maximum quantities of DNA to be targeted, minimizes opportunities for error and contamination, and reduces the time and monetary resources required to process samples, although data analysis may take longer as the increased DNA detection sensitivity of direct PCR may lead to more instances of complex mixtures. ISO 17025 accredited laboratories have successfully implemented direct PCR for limited purposes (e.g., high-throughput databanking analysis), and recent studies indicate that direct PCR can be an effective method for processing low-yield evidence samples. Despite its benefits, direct PCR has yet to be widely implemented across laboratories for the processing of evidentiary items. While forensic DNA laboratories are always interested in new methods that will maximize the quantity and quality of genetic information obtained from evidentiary items, there is often a lag between the advent of useful methodologies and their integration into laboratories. Delayed implementation of direct PCR of evidentiary items can be attributed to a variety of factors, including regulatory guidelines that prevent laboratories from omitting the quantification step when processing forensic unknown samples, as is the case in the United States, and, more broadly, a reluctance to validate a technique that is not widely used for evidence samples. The

  18. Human minisatellite alleles detectable only after PCR amplification.

    Science.gov (United States)

    Armour, J A; Crosier, M; Jeffreys, A J

    1992-01-01

    We present evidence that a proportion of alleles at two human minisatellite loci is undetected by standard Southern blot hybridization. In each case the missing allele(s) can be identified after PCR amplification and correspond to tandem arrays too short to detect by hybridization. At one locus, there is only one undetected allele (population frequency 0.3), which contains just three repeat units. At the second locus, there are at least five undetected alleles (total population frequency 0.9) containing 60-120 repeats; they are not detected because these tandem repeats give very poor signals when used as a probe in standard Southern blot hybridization, and also cross-hybridize with other sequences in the genome. Under these circumstances only signals from the longest tandemly repeated alleles are detectable above the nonspecific background. The structures of these loci have been compared in human and primate DNA, and at one locus the short human allele containing three repeat units is shown to be an intermediate state in the expansion of a monomeric precursor allele in primates to high copy number in the longer human arrays. We discuss the implications of such loci for studies of human populations, minisatellite isolation by cloning, and the evolution of highly variable tandem arrays.

  19. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection ofCylindrocladium scopariumon Eucalyptus.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Tian-Min Qiao

    2016-10-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  2. A stochastic model of the processes in PCR based amplification of STR DNA in forensic applications.

    Science.gov (United States)

    Weusten, Jos; Herbergs, Jos

    2012-01-01

    In forensic DNA profiling use is made of the well-known technique of PCR. When the amount of DNA is high, generally unambiguous profiles can be obtained, but for low copy number DNA stochastic effects can play a major role. In order to shed light on these stochastic effects, we present a simple model for the amplification process. According to the model, three possible things can happen to an individual single DNA strand in each complete cycle: successful amplification, no amplification, or amplification with the introduction of stutter. The model is developed in mathematical terms using a recursive approach: given the numbers of chains at a given cycle, the numbers in the next can be described using a multinomial probability distribution. A full set of recursive relations is derived for the expectations and (co)variances of the number of amplicon chains with no, 1 or 2 stutters. The exact mathematical solutions of this set are given, revealing the development of the expectations and (co)variances as function of the cycle number. The equations reveal that the expected number of amplicon chains without stutter grows exponentially with the cycle number, but for the chains with stutter the relation is more complex. The relative standard deviation on the numbers of chains (coefficient of variation) is inversely proportional to the square root of the expected number of DNA strands entering the amplification. As such, for high copy number DNA the stochastic effects can be ignored, but they play an important role at low concentrations. For the allelic peak, the coefficient of variation rapidly stabilizes after a few cycles, but for the chains with stutter the decrease is more slowly. Further, the ratio of the expected intensity of the stutter peak over that of the allelic peak increases linearly with the number of cycles. Stochastic models, like the one developed in the current paper, can be important in further developing interpretation rules in a Bayesian context

  3. The sensitivity of real-time PCR amplification targeting invasive Salmonella serovars in biological specimens

    Directory of Open Access Journals (Sweden)

    Chau Tran

    2010-05-01

    Full Text Available Abstract Background PCR amplification for the detection of pathogens in biological material is generally considered a rapid and informative diagnostic technique. Invasive Salmonella serovars, which cause enteric fever, can be commonly cultured from the blood of infected patients. Yet, the isolation of invasive Salmonella serovars from blood is protracted and potentially insensitive. Methods We developed and optimised a novel multiplex three colour real-time PCR assay to detect specific target sequences in the genomes of Salmonella serovars Typhi and Paratyphi A. We performed the assay on DNA extracted from blood and bone marrow samples from culture positive and negative enteric fever patients. Results The assay was validated and demonstrated a high level of specificity and reproducibility under experimental conditions. All bone marrow samples tested positive for Salmonella, however, the sensitivity on blood samples was limited. The assay demonstrated an overall specificity of 100% (75/75 and sensitivity of 53.9% (69/128 on all biological samples. We then tested the PCR detection limit by performing bacterial counts after inoculation into blood culture bottles. Conclusions Our findings corroborate previous clinical findings, whereby the bacterial load of S. Typhi in peripheral blood is low, often below detection by culture and, consequently, below detection by PCR. Whilst the assay may be utilised for environmental sampling or on differing biological samples, our data suggest that PCR performed directly on blood samples may be an unsuitable methodology and a potentially unachievable target for the routine diagnosis of enteric fever.

  4. Colony-PCR Is a Rapid Method for DNA Amplification of Hyphomycetes

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    Georg Walch

    2016-04-01

    Full Text Available Fungal pure cultures identified with both classical morphological methods and through barcoding sequences are a basic requirement for reliable reference sequences in public databases. Improved techniques for an accelerated DNA barcode reference library construction will result in considerably improved sequence databases covering a wider taxonomic range. Fast, cheap, and reliable methods for obtaining DNA sequences from fungal isolates are, therefore, a valuable tool for the scientific community. Direct colony PCR was already successfully established for yeasts, but has not been evaluated for a wide range of anamorphic soil fungi up to now, and a direct amplification protocol for hyphomycetes without tissue pre-treatment has not been published so far. Here, we present a colony PCR technique directly from fungal hyphae without previous DNA extraction or other prior manipulation. Seven hundred eighty-eight fungal strains from 48 genera were tested with a success rate of 86%. PCR success varied considerably: DNA of fungi belonging to the genera Cladosporium, Geomyces, Fusarium, and Mortierella could be amplified with high success. DNA of soil-borne yeasts was always successfully amplified. Absidia, Mucor, Trichoderma, and Penicillium isolates had noticeably lower PCR success.

  5. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

    DEFF Research Database (Denmark)

    Hummelshoj, Lone; Ryder, Lars P; Madsen, Hans O

    2005-01-01

    in intronic DNA, the aim was to inhibit the amplification of genomic DNA without affecting the amplification of reverse-transcribed spliced mRNA. LNA was designed to bind within intron 5 in the x-box binding protein 1 (XBP1) gene. An irrelevant LNA oligonucleotide served as a negative control. In both PCR...... and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More...

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

    Science.gov (United States)

    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. External and semi-internal controls for PCR amplification of homologous sequences in mixed templates

    DEFF Research Database (Denmark)

    Kalle, Elena; Gulevich, Alexander; Rensing, Christopher Günther T

    2013-01-01

    . The complexity of the external and semi-internal amplification controls must be comparable with the assumed complexity of the samples. We also emphasize that amplification controls should be applied in multi-template PCR regardless of the post-assay method used to analyze products.......In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur...... in a single template assay. Yet, multi-template PCR has been used without appropriate attention to quality control and assay validation, in spite of the fact that such practice diminishes the reliability of results. External and internal amplification controls became obligatory elements of good laboratory...

  8. Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content.

    Science.gov (United States)

    Ishihara, Satoru; Kotomura, Naoe; Yamamoto, Naoki; Ochiai, Hiroshi

    2017-08-15

    Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. QCM-based rapid detection of PCR amplification products of Ehrlichia canis.

    Science.gov (United States)

    Bunroddith, Kespunyavee; Viseshakul, Nareerat; Chansiri, Kosum; Lieberzeit, Peter

    2018-02-25

    Ehrlichia canis is an intracellular parasitic bacterium and arthropod-borne pathogen that receives growing attention, because it leads to increasing morbidity and mortality in animals. It does so by causing canine monocytotropic ehrlichiosis (CME). Infected canines may lack obvious clinical signs and stay in chronic stage. Herein we report a rapid screening method based on PCR assay combined with quartz crystal microbalance (QCM) to design a DNA sensor for detecting E. canis in early stages of infection. The test relies on DNA amplification of target nucleotide sequences via PCR followed by detecting DNA-DNA hybridization using QCM. The approach did not result in any cross-hybridization toward other blood bacteria or parasites in dogs, such as Anaplasma platys, Babesia canis and Trypanosoma spp, but turned out selective for the target species. The limit of detection of QCM was as low as 4.1 × 10 9 molecules/μl of 289 bp E. canis PCR products corresponding to 22 copy numbers/μl of E. canis. Furthermore, the technique is also simple, does not require complicated equipment and can in principle be reused. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Novel degenerate PCR method for whole genome amplification applied to Peru Margin (ODP Leg 201 subsurface samples

    Directory of Open Access Journals (Sweden)

    Amanda eMartino

    2012-01-01

    Full Text Available A degenerate PCR-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. The method, which we have called Random Amplification Metagenomic PCR (RAMP, involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3’ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10X. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin, and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa show that community analysis can be sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low biomass samples.

  11. PCR amplification of microsatellites from single cells of Karenia brevis preserved in Lugol's iodine solution.

    Science.gov (United States)

    Henrichs, D W; Renshaw, M A; Santamaria, C A; Richardson, B; Gold, J R; Campbell, L

    2008-01-01

    A simple and effective protocol is described for multiplex polymerase chain reaction (PCR) amplification of single cells of Karenia brevis. The protocol requires minimum processing, avoids additions that might dilute target DNA template, and can be used on cells preserved in Lugol's iodine preservative. Destaining of Lugol's-preserved cells with sodium thiosulfate allowed successful amplification of single-copy, nuclear-encoded microsatellites in single cells of K. brevis that have been preserved for up to 6 years.

  12. A new real-time PCR method to overcome significant quantitative inaccuracy due to slight amplification inhibition

    Directory of Open Access Journals (Sweden)

    Stocchi Laura

    2008-07-01

    Full Text Available Abstract Background Real-time PCR analysis is a sensitive DNA quantification technique that has recently gained considerable attention in biotechnology, microbiology and molecular diagnostics. Although, the cycle-threshold (Ct method is the present "gold standard", it is far from being a standard assay. Uniform reaction efficiency among samples is the most important assumption of this method. Nevertheless, some authors have reported that it may not be correct and a slight PCR efficiency decrease of about 4% could result in an error of up to 400% using the Ct method. This reaction efficiency decrease may be caused by inhibiting agents used during nucleic acid extraction or copurified from the biological sample. We propose a new method (Cy0 that does not require the assumption of equal reaction efficiency between unknowns and standard curve. Results The Cy0 method is based on the fit of Richards' equation to real-time PCR data by nonlinear regression in order to obtain the best fit estimators of reaction parameters. Subsequently, these parameters were used to calculate the Cy0 value that minimizes the dependence of its value on PCR kinetic. The Ct, second derivative (Cp, sigmoidal curve fitting method (SCF and Cy0 methods were compared using two criteria: precision and accuracy. Our results demonstrated that, in optimal amplification conditions, these four methods are equally precise and accurate. However, when PCR efficiency was slightly decreased, diluting amplification mix quantity or adding a biological inhibitor such as IgG, the SCF, Ct and Cp methods were markedly impaired while the Cy0 method gave significantly more accurate and precise results. Conclusion Our results demonstrate that Cy0 represents a significant improvement over the standard methods for obtaining a reliable and precise nucleic acid quantification even in sub-optimal amplification conditions overcoming the underestimation caused by the presence of some PCR inhibitors.

  13. Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR

    Directory of Open Access Journals (Sweden)

    Seiki Kuramitsu

    2013-03-01

    Full Text Available Polymerase chain reaction (PCR-related technologies are hampered mainly by two types of error: nonspecific amplification and DNA polymerase-generated mutations. Here, we report that both errors can be suppressed by the addition of a DNA mismatch-recognizing protein, MutS, from a thermophilic bacterium. Although it had been expected that MutS has a potential to suppress polymerase-generated mutations, we unexpectedly found that it also reduced nonspecific amplification. On the basis of this finding, we propose that MutS binds a mismatched primer-template complex, thereby preventing the approach of DNA polymerase to the 3' end of the primer. Our simple methodology improves the efficiency and accuracy of DNA amplification and should therefore benefit various PCR-based applications, ranging from basic biological research to applied medical science.

  14. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

    DEFF Research Database (Denmark)

    Hummelshoj, Lone; Ryder, Lars P; Madsen, Hans O

    2005-01-01

    and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More...

  15. Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR)

    NARCIS (Netherlands)

    Ruiz-Villalba, Adrián; van Pelt-Verkuil, Elizabeth; Gunst, Quinn D.; Ruijter, Jan M.; van den Hoff, Maurice J. B.

    2017-01-01

    Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated

  16. PCR amplification of repetitive sequences as a possible approach in relative species quantification

    DEFF Research Database (Denmark)

    Ballin, Nicolai Zederkopff; Vogensen, Finn Kvist; Karlsson, Anders H

    2012-01-01

    Abstract Both relative and absolute quantifications are possible in species quantification when single copy genomic DNA is used. However, amplification of single copy genomic DNA does not allow a limit of detection as low as one obtained from amplification of repetitive sequences. Amplification...... of repetitive sequences is therefore frequently used in absolute quantification but problems occur in relative quantification as the number of repetitive sequences is unknown. A promising approach was developed where data from amplification of repetitive sequences were used in relative quantification of species...... to relatively quantify the amount of chicken DNA in a binary mixture of chicken DNA and pig DNA. However, the designed PCR primers lack the specificity required for regulatory species control....

  17. Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

    DEFF Research Database (Denmark)

    Boessenkool, Sanne; Epp, Laura S.; Haile, James Seymour

    2012-01-01

    , or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA, we first optimized the design and concentration of blocking primer in the PCR......Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results because of competition....... Subsequently, 454 pyrosequencing of ancient permafrost samples amplified with and without the addition of blocking primer revealed that DNA sequences from a diversity of mammalian representatives of the Beringian megafauna were retrieved only when the blocking primer was added to the PCR. Notably, we observe...

  18. Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

    Science.gov (United States)

    Girard, Laurie D; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G

    2015-02-07

    The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design

  19. A Novel Low Temperature PCR Assured High-Fidelity DNA Amplification

    Directory of Open Access Journals (Sweden)

    Shaoxia Zhou

    2013-06-01

    Full Text Available As previously reported, a novel low temperature (LoTemp polymerase chain reaction (PCR catalyzed by a moderately heat-resistant (MHR DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94–96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV type 52 (HPV-52 as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3'–5' exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories.

  20. PCR amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophthora.

    Science.gov (United States)

    Ristaino, J B; Madritch, M; Trout, C L; Parra, G

    1998-03-01

    We have developed a PCR procedure to amplify DNA for quick identification of the economically important species from each of the six taxonomic groups in the plant pathogen genus Phytophthora. This procedure involves amplification of the 5.8S ribosomal DNA gene and internal transcribed spacers (ITS) with the ITS primers ITS 5 and ITS 4. Restriction digests of the amplified DNA products were conducted with the restriction enzymes RsaI, MspI, and HaeIII. Restriction fragment patterns were similar after digestions with RsaI for the following species: P. capsici and P. citricola; P. infestans, P. cactorum, and P. mirabilis; P. fragariae, P. cinnamomi, and P. megasperma from peach; P. palmivora, P. citrophthora, P. erythroseptica, and P. cryptogea; and P. megasperma from raspberry and P. sojae. Restriction digests with MspI separated P. capsici from P. citricola and separated P. cactorum from P. infestans and P. mirabilis. Restriction digests with HaeIII separated P. citrophthora from P. cryptogea, P. cinnamomi from P. fragariae and P. megasperma on peach, P. palmivora from P. citrophthora, and P. megasperma on raspberry from P. sojae. P. infestans and P. mirabilis digests were identical and P. cryptogea and P. erythroseptica digests were identical with all restriction enzymes tested. A unique DNA sequence from the ITS region I in P. capsici was used to develop a primer called PCAP. The PCAP primer was used in PCRs with ITS 1 and amplified only isolates of P. capsici, P. citricola, and P. citrophthora and not 13 other species in the genus. Restriction digests with MspI separated P. capsici from the other two species. PCR was superior to traditional isolation methods for detection of P. capsici in infected bell pepper tissue in field samples. The techniques described will provide a powerful tool for identification of the major species in the genus Phytophthora.

  1. Dynamic solid phase DNA extraction and PCR amplification in polyester-toner based microchip.

    Science.gov (United States)

    Duarte, Gabriela R M; Price, Carol W; Augustine, Brian H; Carrilho, Emanuel; Landers, James P

    2011-07-01

    A variety of substrates have been used for fabrication of microchips for DNA extraction, PCR amplification, and DNA fragment separation, including the more conventional glass and silicon as well as alternative polymer-based materials. Polyester represents one such polymer, and the laser-printing of toner onto polyester films has been shown to be effective for generating polyester-toner (PeT) microfluidic devices with channel depths on the order of tens of micrometers. Here, we describe a novel and simple process that allows for the production of multilayer, high aspect-ratio PeT microdevices with substantially larger channel depths. This innovative process utilizes a CO(2) laser to create the microchannel in polyester sheets containing a uniform layer of printed toner, and multilayer devices can easily be constructed by sandwiching the channel layer between uncoated cover sheets of polyester containing precut access holes. The process allows the fabrication of deep channels, with ~270 μm, and we demonstrate the effectiveness of multilayer PeT microchips for dynamic solid phase extraction (dSPE) and PCR amplification. With the former, we found that (i) more than 65% of DNA from 0.6 μL of blood was recovered, (ii) the resultant DNA was concentrated to greater than 3 ng/μL (which was better than other chip-based extraction methods), and (iii) the DNA recovered was compatible with downstream microchip-based PCR amplification. Illustrative of the compatibility of PeT microchips with the PCR process, the successful amplification of a 520 bp fragment of λ-phage DNA in a conventional thermocycler is shown. The ability to handle the diverse chemistries associated with DNA purification and extraction is a testimony to the potential utility of PeT microchips beyond separations and presents a promising new disposable platform for genetic analysis that is low cost and easy to fabricate.

  2. PCR amplification and species determination of microsporidia in formalin-fixed feces after immunomagnetic separation.

    Science.gov (United States)

    Dowd, S E; Gerba, C P; Enriquez, F J; Pepper, I L

    1998-01-01

    The term microsporidia is used to describe several species of opportunistic protozoan parasites. Encephalitozoon intestinalis and Enterocytozoon bieneusi have been found in stools of more than 40% of AIDS patients with diarrhea. Diagnosis of infection with these small protozoans has been difficult, and until recently their occurrence has not been well documented. Formalin is widely used to preserve clinical specimens, but due to the nature of the fixation process, subsequent analysis, especially analysis by the PCR, is difficult. This study evaluated methods used to prepare formalin-fixed fecal specimens for PCR amplification of microsporidial DNA. Two methods were devised to allow PCR detection and subsequent identification of microsporidia in formalin-fixed fecal specimens to the species level. One method involved immunomagnetic separation to concentrate microsporidial spores from fecal specimens. In the second method Chelex resin (Bio-Rad, Hercules, Calif.) was used to remove inhibitory substances, followed by a DNA concentration step. Both methods resulted in reproducible, confirmed detection of microsporidia in formalinized fecal specimens and subsequent species determination by PCR sequencing. The detection sensitivity was two in vitro culture-derived spores (Encephalitozoon intestinalis) for the direct PCR. The reproducible detection sensitivity for DNA amplification from formalin-fixed fecal samples was 200 spores for either the Chelex method or the immunomagnetic bead separation method. Thus, we developed two methods for rapid, inexpensive detection of microsporidial spores in formalin-fixed fecal specimens.

  3. LOMA: A fast method to generate efficient tagged-random primers despite amplification bias of random PCR on pathogens

    Directory of Open Access Journals (Sweden)

    Lee Wah

    2008-09-01

    Full Text Available Abstract Background Pathogen detection using DNA microarrays has the potential to become a fast and comprehensive diagnostics tool. However, since pathogen detection chips currently utilize random primers rather than specific primers for the RT-PCR step, bias inherent in random PCR amplification becomes a serious problem that causes large inaccuracies in hybridization signals. Results In this paper, we study how the efficiency of random PCR amplification affects hybridization signals. We describe a model that predicts the amplification efficiency of a given random primer on a target viral genome. The prediction allows us to filter false-negative probes of the genome that lie in regions of poor random PCR amplification and improves the accuracy of pathogen detection. Subsequently, we propose LOMA, an algorithm to generate random primers that have good amplification efficiency. Wet-lab validation showed that the generated random primers improve the amplification efficiency significantly. Conclusion The blind use of a random primer with attached universal tag (random-tagged primer in a PCR reaction on a pathogen sample may not lead to a successful amplification. Thus, the design of random-tagged primers is an important consideration when performing PCR.

  4. Study On Application Of Molecular Techniques (RAPD-PCR And RAMP-PCR) To Detect Mutation In Rice Breeding

    International Nuclear Information System (INIS)

    Hoang Thi My Linh; Phan, D. T. Son; Nguyen Thi Vang; Nguyen, T. T. Hien; Le XuanTham

    2007-01-01

    The project was carried out in 2007 with the purpose of consideration for using the two simple and inexpensive molecular techniques to estimate changes in DNA of rice mutant after gamma irradiation. Three rice cultivars: Basmati370, Tam Thom (TT1), IR64 and three gamma irradiated mutants BDS, TDS and VND 95-20 respectively, were used. Suitable DNA extraction procedure was obtained. PCR optimization was conducted on three important factors including: amount of MgCl 2 , DNA concentration and annealing temperature. 2.5 mM of MgCl 2 for RAPD-PCR and 3.75 mM for RAMP-PCR were found the best. 40 ng DNA provided a good amplification for RAMP-PCR; this figure was 50 ng for RAPD-PCR. Annealing temperatures were determined at 36 o C for RAPD primer and at 55±3 o C for Microsatellite primer. Final results showed that, both RAPD-PCR and RAMP-PCR could detect changes in DNA of rice mutants after gamma irradiation compared to their parents. Percentage of DNA changes determined by RAPD-PCR and RAMP-PCR on Basmati370 and its mutant BDS were 11.49% and 21.2% respectively; These on TT1 and TDS were 8.98% and 15.4%; and on IR64 and VND 95-20 were 3.45% and 4.95%. (author)

  5. Single cell PCR amplification of diatoms using fresh and preserved samples

    Science.gov (United States)

    Hamilton, Paul B.; Lefebvre, Keely E.; Bull, Roger D.

    2015-01-01

    Single cell Chelex® DNA extraction and nested PCR amplification were used to examine partial gene sequences from natural diatom populations for taxonomic and phylogenetic studies at and above the level of species. DNA was extracted from cells that were either fresh collected or stored in RNAlater. Extractions from Lugol's fixation were also attempted with limited success. Three partial gene sequences (rbcL, 18S, and psbA) were recovered using existing and new primers with a nested or double nested PCR approach with amplification and success rates between 70 and 96%. An rbcL consensus tree grouped morphologically similar specimens and was consistent across the two primary sample treatments: fresh and RNAlater. This tool will greatly enhance the number of microscopic diatom taxa (and potentially other microbes) available for barcoding and phylogenetic studies. The near-term increase in sequence data for diatoms generated via routine single cell extractions and PCR will act as a multiproxy validation of longer-term next generation genomics. PMID:26528252

  6. Application of Isothermal Amplification Techniques for Identification of Madurella mycetomatis, the Prevalent Agent of Human Mycetoma.

    Science.gov (United States)

    Ahmed, Sarah A; van de Sande, Wendy W J; Desnos-Ollivier, Marie; Fahal, Ahmed H; Mhmoud, Najwa A; de Hoog, G S

    2015-10-01

    Appropriate diagnosis and treatment of eumycetoma may vary significantly depending on the causative agent. To date, the most common fungus causing mycetoma worldwide is Madurella mycetomatis. This species fails to express any recognizable morphological characteristics, and reliable identification can therefore only be achieved with the application of molecular techniques. Recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) are proposed as alternatives to phenotypic methods. Species-specific primers were developed to target the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of M. mycetomatis. Both isothermal amplification techniques showed high specificity and sufficient sensitivity to amplify fungal DNA and proved to be appropriate for detection of M. mycetomatis. Diagnostic performance of the techniques was assessed in comparison to conventional PCR using biopsy specimens from eumycetoma patients. RPA is reliable and easy to operate and has the potential to be implemented in areas where mycetoma is endemic. The techniques may be expanded to detect fungal DNA from environmental samples. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  7. Pre-amplification in the context of high-throughput qPCR gene expression experiment

    Czech Academy of Sciences Publication Activity Database

    Korenková, Vlasta; Scott, J.; Novosadová, Vendula; Jindřichová, Marie; Langerová, Lucie; Švec, David; Šídová, Monika; Sjoback, R.

    2015-01-01

    Roč. 16, č. 5 (2015) ISSN 1471-2199 R&D Projects: GA ČR(CZ) GAP304/12/1585; GA ČR(CZ) GA15-08239S; GA ČR GA13-02154S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : High-throughput qPCR * Gene expression * Exponential pre-amplification Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.500, year: 2015

  8. Identification of periodontopathogen microorganisms by PCR technique

    Directory of Open Access Journals (Sweden)

    Milićević Radovan

    2008-01-01

    Full Text Available INTRODUCTION Periodontitis is an inflammatory disease of the supporting tissues of teeth and is a major cause of tooth loss in adults. The onset and progression of periodontal disease is attributed to the presence of elevated levels of a consortium of pathogenic bacteria. Gram negative bacteria, mainly strict anaerobes, play the major role. OBJECTIVE The present study aimed to assess the presence of the main types of microorganisms involved in the aetiopathogenesis of periodontal disease: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Treponema denticola, Tanerella forsythia and Prevotella intermedia in different samples collected from the oral cavity of 90 patients diagnosed with periodontitis. METHOD Bacterial DNA detection was performed in diverse biological materials, namely in dental plaque, gingival tissue and saliva, by means of multiplex PCR, a technique that allows simultaneous identification of two different bacterial genomes. RESULTS In the dental plaque of the periodontitis patients, Treponema denticola dominated. In the gingival tissue, Tannerella forsythia and Treponema denticola were the microbiota most frequently detected, whilst in saliva Treponema denticola and Eikenella corrodens were found with the highest percentage. CONCLUSION The identification of microorganisms by multiplex PCR is specific and sensitive. Rapid and precise assessment of different types of periodontopathogens is extremely important for early detection of the infection and consequently for the prevention and treatment of periodontal disease. In everyday clinical practice, for routine bacterial evaluation in patients with periodontal disease, the dental plaque is the most suitable biological material, because it is the richest in periodontal bacteria.

  9. Validation of the AmpFlSTR« SEfiler Plus(TM) PCR Amplification kit for forensic STR analysis

    DEFF Research Database (Denmark)

    Fredslund, Stine Frisk; Mogensen, Helle Smidt; Morling, Niels

    2009-01-01

    Validation of the AmpFlSTR« SEfiler Plus(TM) PCR Amplification kit with 29 and 30 PCR cycles for forensic STR analysis demonstrated that the kit had fewer artefacts than the AmpFlSTR« SGM Plus(TM) kit (28 PCR cycles). The SEfiler Plus kit was more sensitive and devoid of colour artefacts, but sho......Validation of the AmpFlSTR« SEfiler Plus(TM) PCR Amplification kit with 29 and 30 PCR cycles for forensic STR analysis demonstrated that the kit had fewer artefacts than the AmpFlSTR« SGM Plus(TM) kit (28 PCR cycles). The SEfiler Plus kit was more sensitive and devoid of colour artefacts...

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

    DEFF Research Database (Denmark)

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas

    2017-01-01

    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...... 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 × 1011 molecules/mm2. 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...

  11. Validation of the AmpFlSTR« SEfiler Plus(TM) PCR Amplification kit for forensic STR analysis

    DEFF Research Database (Denmark)

    Fredslund, Stine Frisk; Mogensen, Helle Smidt; Morling, Niels

    2009-01-01

    Validation of the AmpFlSTR« SEfiler Plus(TM) PCR Amplification kit with 29 and 30 PCR cycles for forensic STR analysis demonstrated that the kit had fewer artefacts than the AmpFlSTR« SGM Plus(TM) kit (28 PCR cycles). The SEfiler Plus kit was more sensitive and devoid of colour artefacts, but sho......, but showed more stutters, drop-ins, drop-outs and allelic imbalances...

  12. HER-2 and INT-2 amplification estimated by quantitative PCR in paraffin-embedded ovarian cancer tissue samples.

    Science.gov (United States)

    Hruza, C; Dobianer, K; Beck, A; Czerwenka, K; Hanak, H; Klein, M; Leodolter, S; Medl, M; Müllauer-Ertl, S; Preiser, J

    1993-01-01

    Competitive polymerase chain reaction (PCR) systems were developed for rapid and quantitative estimation of HER-2 (c-erbB-2) and INT-2 oncogene amplification in paraffin-embedded ovarian cancer tissue samples. The beta-globin gene was used as reference and DNA from paraffin-embedded placenta tissue as single copy control. Reliability of the PCR method could be demonstrated by comparing dot blot data with PCR data of identical tumour samples. The PCR method was used to determine HER-2 and INT-2 copy numbers in 196 ovarian cancer samples. HER-2 and INT-2 were found to be amplified in 40 and 19%, respectively. In 8% HER-2 copy numbers were greater than five, but no high INT-2 copies were noted. Kaplan-Meier estimates did not reveal significant association with overall survival. Indirect correlation between HER-2 and INT-2 amplification was observed. The present PCR system is a valuable method for prospective and retrospective studies.

  13. [Identification and diagnosis of Taylorella equigenitalis by a DNA amplification method (PCR)].

    Science.gov (United States)

    Miserez, R; Frey, J; Krawinkler, M; Nicolet, J

    1996-01-01

    A polymerase chain reaction (PCR) for identification of Taylorella equigenitalis was developed. The oligonucleotide primers are based on the DNA sequence of the rrs gene of T. equigenitalis, encoding for the 16S ribosomal RNA. Analysis of 21 strains of T. equigenitalis from England, USA and Switzerland showed an amplification product of 410 bp with identical Sau3A restriction profile. The sensitivity of the PCR-Assay was estimated to detect 50 to 500 bacteria of T. equigenitalis in a mixture with frequently found contaminants. Further analysis of culture from 60 genital swabs, taken in the course of the control of the contagious equine metritis in horses and donkeys, of experimental assays as well as of two positive cases from the diagnostic showed that this PCR-assay can be used to identify and to detect strains of T. equigenitalis. In addition, preliminary results indicate that the method is also applicable for direct in vitro establishment of the presence of T. equigenitalis in clinical samples.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. COBAS AMPLICOR: fully automated RNA and DNA amplification and detection system for routine diagnostic PCR.

    Science.gov (United States)

    DiDomenico, N; Link, H; Knobel, R; Caratsch, T; Weschler, W; Loewy, Z G; Rosenstraus, M

    1996-12-01

    The COBAS AMPLICOR system automates amplification and detection of target nucleic acids, making diagnostic PCR routine for a variety of infectious diseases. The system contains a single thermal cycler with two independently regulated heating/cooling blocks, an incubator, a magnetic particle washer, a pipettor, and a photometer. Amplified products are captured on oligonucleotide-coated paramagnetic microparticles and detected with use of an avidin-horseradish peroxidase (HRP) conjugate. Concentrated solutions of amplicon or HRP were pipetted without detectable carryover. Amplified DNA was detected with an intraassay CV of < 4.5%; the combined intraassay CV for amplification and detection was < 15%. No cross-reactivity was observed when three different target nucleic acids were amplified in a single reaction and detected with three target-specific capture probes. The initial COBAS AMPLICOR menu includes qualitative tests for diagnosing infections with Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and hepatitis C virus. All tests include an optional Internal Control to provide assurance that specimens are successfully amplified and detected.

  16. Direct Identification of Enteroviruses in Cerebrospinal Fluid of Patients with Suspected Meningitis by Nested PCR Amplification

    Directory of Open Access Journals (Sweden)

    Alexandr Krasota

    2016-01-01

    Full Text Available Enteroviruses, the most common human viral pathogens worldwide, have been associated with serous meningitis, encephalitis, syndrome of acute flaccid paralysis, myocarditis and the onset of diabetes type 1. In the future, the rapid identification of the etiological agent would allow to adjust the therapy promptly and thereby improve the course of the disease and prognosis. We developed RT-nested PCR amplification of the genomic region coding viral structural protein VP1 for direct identification of enteroviruses in clinical specimens and compared it with the existing analogs. One-hundred-fifty-nine cerebrospinal fluids (CSF from patients with suspected meningitis were studied. The amplification of VP1 genomic region using the new method was achieved for 86 (54.1% patients compared with 75 (47.2%, 53 (33.3% and 31 (19.5% achieved with previously published methods. We identified 11 serotypes of the Enterovirus species B in 2012, including relatively rare echovirus 14 (E-14, E-15 and E-32, and eight serotypes of species B and 5 enteroviruses A71 (EV-A71 in 2013. The developed method can be useful for direct identification of enteroviruses in clinical material with the low virus loads such as CSF.

  17. Detection of Flavobacterium psychrophilum from fish tissue and water samples by PCR amplification

    DEFF Research Database (Denmark)

    Wiklund, T.; Madsen, Lone; Bruun, Morten Sichlau

    2000-01-01

    Rainbow trout fry syndrome and cold-water disease, caused by Flavobacterium psychrophilum, are important diseases in farmed salmonids. Some of the presently available techniques for the detection of Fl. psychrophilum are either time consuming or lack sufficient sensitivity. In the present...... investigation, the possible detection of Fl. psychrophilum from fish tissue and water samples was examined using nested PCR with DNA probes against a sequence of the 16S rRNA genes. The DNA was extracted using Chelex(R) 100 chelating resin. The primers, which were tested against strains isolated from diseased...... to be more sensitive than agar cultivation of tissue samples from the brain of rainbow trout injected with Fl. psychrophilum. In non-sterile fresh water seeded with Fl. psychrophilum the detection limit of the PCR- assay was 1.7 cfu in the PCR tube, corresponding to 110 cfu ml(-1) water. The PCR...

  18. Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their gpd (glyceraldehyde-3-phosphate dehydrogenase) genes.

    Science.gov (United States)

    Kreuzinger, N; Podeu, R; Gruber, F; Göbl, F; Kubicek, C P

    1996-01-01

    Degenerated oligonucleotide primers designed to flank an approximately 1.2-kb fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) from ascomycetes and basidiomycetes were used to amplify the corresponding gpd fragments from several species of the ectomycorrhizal fungal taxa Boletus, Amanita, and Lactarius. Those from B. edulis, A. muscaria, and L. deterrimus were cloned and sequenced. The respective nucleotide sequences of these gene fragments showed a moderate degree of similarity (72 to 76%) in the protein-encoding regions and only a low degree of similarity in the introns (56 to 66%). Introns, where present, occurred at conserved positions, but the respective positions and numbers of introns in a given taxon varied. The amplified fragment from a given taxon could be distinguished from that of others by both restriction nuclease cleavage analysis and Southern hybridization. A procedure for labeling DNA probes with fluorescein-12-dUTP by PCR was developed. These probes were used in a nonradioactive hybridization assay, with which the gene could be detected in 2 ng of chromosomal DNA of L. deterrimus on slot blots. Taxon-specific amplification was achieved by the design of specific oligonucleotide primers. The application of the gpd gene for the identification of mycorrhizal fungi under field conditions was demonstrated, with Picea abies (spruce) mycorrhizal roots harvested from a northern alpine forest area as well as from a plant-breeding nursery. The interference by inhibitory substances, which sometimes occurred in the DNA extracted from the root-fungus mixture, could be overcome by using very diluted concentrations of template DNA for a first round of PCR amplification followed by a second round with nested oligonucleotide primers. We conclude that gpd can be used to detect ectomycorrhizal fungi during symbiotic interaction. PMID:8795234

  19. Fibroblast growth factor receptor 1 amplification in non-small cell lung cancer by quantitative real-time PCR.

    Directory of Open Access Journals (Sweden)

    Shirish M Gadgeel

    Full Text Available Amplification of the fibroblast growth factor receptor 1 (FGFR1 gene has been described in tumors of non-small-cell lung cancer (NSCLC patients. Prior reports showed conflicting rates of amplification frequency and clinical relevance.We developed a reliable real-time quantitative PCR assay to assess the frequency of FGFR1 amplification and assessed the optimal cutoff level of amplification for clinical application.In a training cohort of 203 NSCLCs, we established that a 3.5-fold amplification optimally divided patients into groups with different survival rates with a clear threshold level. Those with FGFR1 amplification levels above 3.5-fold had an inferior survival. These data were confirmed in a validation cohort of 142 NSCLC. After adjusting for age, sex, performance status, stage, and histology, patients with FGFR1 amplification levels above 3.5 fold had a hazard ratio of 2.91 (95% CI- 1.14, 7.41; pvalue-0.025 for death in the validation cohort. The rates of FGFR1 amplification using the cutoff level of 3.5 were 5.1% in squamous cell and 4.1% in adenocarcinomas. There was a non-significant trend towards higher amplifications rates in heavy smokers (> 15 pack-years of cigarette consumption as compared to light smokers.Our data suggest that a 3.5-fold amplification of FGFR1 is of clinical importance in NSCLC. Our cutpoint analysis showed a clear threshold effect for the impact of FGFR1 amplification on patients' survival, which can be used as an initial guide for patient selection in trials assessing efficacy of novel FGFR inhibitors.

  20. Rapid detection of Puccinia triticina causing leaf rust of wheat by PCR and loop mediated isothermal amplification.

    Science.gov (United States)

    Manjunatha, C; Sharma, Sapna; Kulshreshtha, Deepika; Gupta, Sangeeta; Singh, Kartar; Bhardwaj, Subhash C; Aggarwal, Rashmi

    2018-01-01

    Leaf rust of wheat caused by Puccinia triticina has significant impact on wheat production worldwide. Effective and quick detection methodologies are required to mitigate yield loss and time constraints associated with monitoring and management of leaf rust of wheat. In the present study, detection of P. triticina has been simplified by developing a rapid, reliable, efficient and visual colorimetric method i.e., loop mediated isothermal amplification of DNA (LAMP). Based on in silico analysis of P. triticina genome, PTS68, a simple sequence repeat was found highly specific to leaf rust fungus. A marker (PtRA68) was developed and its specificity was validated through PCR technique which gave a unique and sharp band of 919 bp in P. triticina pathotypes only. A novel gene amplification method LAMP which enables visual detection of pathogen by naked eye was developed for leaf rust pathogen. A set of six primers was designed from specific region of P. triticina and conditions were optimised to complete the observation process in 60 minutes at 65o C. The assay developed in the study could detect presence of P. triticina on wheat at 24 hpi (pre-symptomatic stage) which was much earlier than PCR without requiring thermal cycler. Sensitivity of LAMP assay developed in the study was 100 fg which was more sensitive than conventional PCR (50 pg) and equivalent to qPCR (100 fg). The protocol developed in the study was utilized for detection of leaf rust infected samples collected from different wheat fields. LAMP based colorimetric detection assay showed sky blue color in positive reaction and violet color in negative reaction after addition of 120 μM hydroxyl napthol blue (HNB) solution to reaction mixture. Similarly, 0.6 mg Ethidium bromide/ml was added to LAMP products, placed on transilluminator to witness full brightness in positive reaction and no such brightness could be seen in negative reaction mixture. Further, LAMP products spread in a ladder like banding pattern in

  1. Rapid genome detection of Schmallenberg virus and bovine viral diarrhea virus by use of isothermal amplification methods and high-speed real-time reverse transcriptase PCR.

    Science.gov (United States)

    Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2014-06-01

    Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application-specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  2. Temperature Switch PCR (TSP: Robust assay design for reliable amplification and genotyping of SNPs

    Directory of Open Access Journals (Sweden)

    Mather Diane E

    2009-12-01

    Full Text Available Abstract Background Many research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs. Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP, a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays. Results We demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method. Conclusion Theoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.

  3. Simple and reliable procedure for PCR amplification of genomic DNA from yeast cells using short sequencing primers

    DEFF Research Database (Denmark)

    Haaning, J; Oxvig, C; Overgaard, Michael Toft

    1997-01-01

    by means of PCR without any prior DNA purification steps. This method involves a simple boiling step of whole yeast cells in the presence of detergent, and subsequent amplification of genomic DNA using short sequencing primers in a polymerase chain reaction assay with a decreasing annealing temperature...

  4. Gene amplification by PCR and subcloning into a GFP-fusion plasmid expression vector as a molecular biology laboratory course*.

    Science.gov (United States)

    Bornhorst, Joshua A; Deibel, Michael A; Mulnix, Amy B

    2004-05-01

    A novel experimental sequence for the advanced undergraduate laboratory course has been developed at Earlham College. Utilizing recent improvements in molecular techniques for a time-sensitive environment, undergraduates were able to create a chimera of a selected gene and green fluorescent protein (GFP) in a bacterial expression plasmid over the course of a single semester in a weekly 3-h laboratory period. Students designed PCR primers for amplification of the selected gene using computational DNA sequence analysis tools. During the experimental portion of the course, students amplified and ligated the target DNA into a commercially available GFP expression vector. Following transformation of the ligation product, plasmids were harvested from the resulting bacterial colonies and were analyzed by restriction digestion to confirm the creation of the chimeric GFP-DNA. This course gave students valuable experience with commonly used molecular techniques in an authentic research project. In addition, students gained experience with experimental design and execution. The techniques presented here are flexible and can be generalized for use with almost any DNA sequence and expression vector. This series also serves as an example of how faculty can adapt their ongoing research projects to the undergraduate laboratory. Copyright © 2004 International Union of Biochemistry and Molecular Biology, Inc.

  5. Molecular Technique to Reduce PCR Bias for Deeper Understanding of Microbial Diversity

    Science.gov (United States)

    Vaishampayan, Parag A.; Venkateswaran, Kasthuri J.

    2012-01-01

    Current planetary protection policies require that spacecraft targeted to sensitive solar system bodies be assembled and readied for launch in controlled cleanroom environments. A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination. However, despite a growing understanding of the diverse microbial populations present in cleanrooms, less abundant microbial populations are probably not adequately taken into account due to technological limitations. This novel approach encompasses a wide spectrum of microbial species and will represent the true picture of spacecraft cleanroom-associated microbial diversity. All of the current microbial diversity assessment techniques are based on an initial PCR amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of a minor template appears to be suppressed by the amplification of a more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck overlooks the presence of the less abundant minority population and may underestimate their role in the ecosystem maintenance. DNA intercalating agents such as propidium monoazide (PMA) covalently bind with DNA molecules upon photolysis using visible light, and make it unavailable for DNA polymerase enzyme during polymerase chain reaction (PCR). Environmental DNA samples will be treated with suboptimum PMA concentration, enough to intercalate with 90 99% of the total DNA. The probability of PMA binding with DNA from abundant bacterial species will be much higher than binding with DNA from less abundant species. This will increase the relative DNA concentration of

  6. Detection of Escherichia coli Serogroups O26 and O113 by PCR Amplification of the wzx and wzy Genes

    OpenAIRE

    DebRoy, Chitrita; Roberts, Elisabeth; Kundrat, James; Davis, Michael A.; Briggs, Connie E.; Fratamico, Pina M.

    2004-01-01

    PCR-based assays for detecting enterohemorrhagic Escherichia coli serogroups O26 and O113 were developed by targeting the wzx (O-antigen flippase) and the wzy (O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113 E. coli serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple...

  7. Detection of Escherichia coli serogroups O26 and O113 by PCR amplification of the wzx and wzy genes.

    Science.gov (United States)

    DebRoy, Chitrita; Roberts, Elisabeth; Kundrat, James; Davis, Michael A; Briggs, Connie E; Fratamico, Pina M

    2004-03-01

    PCR-based assays for detecting enterohemorrhagic Escherichia coli serogroups O26 and O113 were developed by targeting the wzx (O-antigen flippase) and the wzy (O-antigen polymerase) genes found in the O-antigen gene cluster of each organism. The PCR assays were specific for the respective serogroups, as there was no amplification of DNA from non-O26 and non-O113 E. coli serogroups or from other bacterial genera tested. Using the PCR assays, we were able to detect the organisms in seeded apple juice inoculated at concentration levels as low as replacing antigen-based serotyping.

  8. A general method for nested RT-PCR amplification and sequencing the complete HCV genotype 1 open reading frame

    Directory of Open Access Journals (Sweden)

    Tavis John E

    2005-12-01

    Full Text Available Abstract Background Hepatitis C virus (HCV is a pathogenic hepatic flavivirus with a single stranded RNA genome. It has a high genetic variability and is classified into six major genotypes. Genotype 1a and 1b cause the majority of infections in the USA. Viral genomic sequence information is needed to correlate viral variation with pathology or response to therapy. However, reverse transcription-polymerase chain reaction (RT-PCR of the HCV genome must overcome low template concentration and high target sequence diversity. Amplification conditions must hence have both high sensitivity and specificity yet recognize a heterogeneous target population to permit general amplification with minimal bias. This places divergent demands of the amplification conditions that can be very difficult to reconcile. Results RT and nested PCR conditions were optimized independently and systematically for amplifying the complete open reading frame (ORF from HCV genotype 1a and 1b using several overlapping amplicons. For each amplicon, multiple pairs of nested PCR primers were optimized. Using these primers, the success rate (defined as the rate of production of sufficient DNA for sequencing with any one of the primer pairs for a given amplicon for amplification of 72 genotype 1a and 1b patient plasma samples averaged over 95% for all amplicons. In addition, two sets of sequencing primers were optimized for each genotype 1a and 1b. Viral consensus sequences were determined by directly sequencing the amplicons. HCV ORFs from 72 patients have been sequenced using these primers. Sequencing errors were negligible because sequencing depth was over 4-fold and both strands were sequenced. Primer bias was controlled and monitored through careful primer design and control experiments. Conclusion Optimized RT-PCR and sequencing conditions are useful for rapid and reliable amplification and sequencing of HCV genotype 1a and 1b ORFs.

  9. Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

    Directory of Open Access Journals (Sweden)

    Maria Cristina Carvalho do Espírito-Santo

    2012-10-01

    Full Text Available Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg of feces. Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

  10. Detection of Hepatitis B Virus (HBV) in Blood Serum By Means of PCR (Polymerase Chain Reaction) Technique

    International Nuclear Information System (INIS)

    Lina, M.; Dadang, S.; Suhadi, F.

    2002-01-01

    Research for detecting the presence of HBV DNA in serum with PCR technique by using two pairs of oligonucleotide primers, has been carried out. Ten serum consisted of 5 HBsAg positive serum, I HBsAg weak positive serum, 3 HBsAg negative serum, and I sampel with negative HBV DNA as a previous PCR product trom another laboratory, were used to purify and to extract the DNA of virus, the sample pretreatment was done with Boom method. The two pairs of primers used for the- PCR process, were PC1 and PC2 and P1 and P2. The amplification process by means of PC1 and PC2 primer was carried out with two treatments, l.a. and l.b treatments of 5 HBsAg positive serum samples, 3 were positive for HBV DNA by PCR test with l.a. treatment. The PCR test by means of either the same primer but different treaunent (l.b treatment) or different pair of primer (pI and P2 pimer), revealed the presence of HBV DNA in all of HBsAg serum mentioned above of HBsAg negative Seruln, I serum was positive for HBV DNA and it was an amplification product of PCR test by using PI and P2 primer. The amplification products of PCR processwith either l.b treatment or PI and P2 primer, showed the positive results for I HBV positive serum as a previous PCR product trom another laboratory. All of the PCR test in this research provided the negative HBV DNA result in the HBsAg weak positive serum. The DNA amplification process by means of PI and P2 primer was more sensitive compared with PC I and PC2 primer

  11. Ligation-mediated PCR amplification of specific fragments from a class-II restriction endonuclease total digest.

    OpenAIRE

    Guilfoyle, R A; Leeck, C L; Kroening, K D; Smith, L M; Guo, Z

    1997-01-01

    A method is described which permits the ligation- mediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest. Feasibility was tested using Bcl I and phage lambda DNA as a model enzyme and amplicon system, respectively. Bcl I is one of many widely used restriction enzymes which cleave at palindromic recognition sequences and leave 5'-protruding ends of defined sequence. Using a single pair of universal primers, a given fragment can be specifically am...

  12. PCR amplification of Bartonella koehlerae from human blood and enrichment blood cultures

    Directory of Open Access Journals (Sweden)

    Breitschwerdt Edward B

    2010-08-01

    Full Text Available Abstract Background Cats appear to be the primary reservoir host for Bartonella koehlerae, an alpha Proteobacteria that is most likely transmitted among cat populations by fleas (Ctenocephalides felis. Bartonella koehlerae has caused endocarditis in a dog and in one human patient from Israel, but other clinically relevant reports involving this bacterium are lacking. Despite publication of numerous, worldwide epidemiological studies designed to determine the prevalence of Bartonella spp. bacteremia in cats, B. koehlerae has never been isolated using conventional blood agar plates. To date, successful isolation of B. koehlerae from cats and from the one human endocarditis patient has consistently required the use of chocolate agar plates. Results In this study, Bartonella koehlerae bacteremia was documented in eight immunocompetent patients by PCR amplification and DNA sequencing, either prior to or after enrichment blood culture using Bartonella alpha Proteobacteria growth medium. Presenting symptoms most often included fatigue, insomnia, joint pain, headache, memory loss, and muscle pain. Four patients were also infected with Bartonella vinsonii subsp. berkhoffii genotype II. After molecular documentation of B. koehlerae infection in these patients, a serological test was developed and serum samples were tested retrospectively. Bartonella koehlerae antibodies were not detected (titers B. koehlerae antibody titers of 1:64 or greater. Conclusions Although biased by a study population consisting of individuals with extensive arthropod and animal exposure, the results of this study suggest that B. koehlerae bacteremia is more common in immunocompetent people than has been previously suspected. Future studies should more thoroughly define modes of transmission and risk factors for acquiring infection with B. koehlerae. In addition, studies are needed to determine if B. koehlerae is a cause or cofactor in the development of arthritis, peripheral

  13. Forensic and population genetic analyses of Danes, Greenlanders and Somalis typed with the Yfiler® Plus PCR amplification kit

    DEFF Research Database (Denmark)

    Olofsson, Jill Katharina; Mogensen, Helle Smidt; Buchard, Anders

    2015-01-01

    Recently, the Yfiler(®) Plus PCR Amplification Kit (Yfiler(®) Plus, Thermo Fisher Scientific, Waltham, MA, USA) was introduced. Yfiler(®) Plus amplifies 27 Y-chromosomal short tandem repeat loci (Y-STRs) and adds ten new Y-STRs to those analysed with the commonly used AmpFlSTR(®) Yfiler(®) PCR Am...... in crime case investigations. However, the inclusion of seven RM Y-STRs in Yfiler(®) Plus makes it less attractive for relationship testing because of the relatively high combined mutation rate, approximately 15%....

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

    Directory of Open Access Journals (Sweden)

    Kirill V Sergueev

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

  15. Quantitative PCR Detection of c-erbB-2 Gene Amplification.

    Science.gov (United States)

    Dobianer, K; Medl, M; Spona, J

    2001-01-01

    The association of c-erbB-2 oncogene amplification and prognostic factors was intensively studied in human gynecological carcinomas, especially in mammary carcinoma (1). Positive lymph nodes, estrogen and progesterone receptor negative tumors, and short survival time correlate with c-erbB-2 amplification.

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

    Science.gov (United States)

    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. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. MALS: an efficient strategy for multiple site-directed mutagenesis employing a combination of DNA amplification, ligation and suppression PCR

    Directory of Open Access Journals (Sweden)

    Drayna Dennis T

    2009-09-01

    Full Text Available Abstract Background Multiple approaches for the site-directed mutagenesis (SDM have been developed. However, only several of them are designed for simultaneous introduction of multiple nucleotide alterations, and these are time consuming. In addition, many of the existing multiple SDM methods have technical limitations associated with type and number of mutations that can be introduced, or are technically demanding and require special chemical reagents. Results In this study we developed a quick and efficient strategy for introduction of multiple complex mutations in a target DNA without intermediate subcloning by using a combination of connecting SDM and suppression PCR. The procedure consists of sequential rounds, with each individual round including PCR amplification of target DNA with two non-overlapping pairs of oligonucleotides. The desired mutation is incorporated at the 5' end of one or both internal oligonucleotides. DNA fragments obtained during amplification are mixed and ligated. The resulting DNA mixture is amplified with external oligonucleotides that act as suppression adapters. Suppression PCR limits amplification to DNA molecules representing full length target DNA, while amplification of other types of molecules formed during ligation is suppressed. To create additional mutations, an aliquot of the ligation mixture is then used directly for the next round of mutagenesis employing internal oligonucleotides specific for another region of target DNA. Conclusion A wide variety of complex multiple mutations can be generated in a short period of time. The procedure is rapid, highly efficient and does not require special chemical reagents. Thus, MALS represents a powerful alternative to the existing methods for multiple SDM.

  18. Comparison of the Becton Dickinson strand displacement amplification and Cobas Amplicor Roche PCR for the detection of Chlamydia trachomatis: pooling versus individual tests

    DEFF Research Database (Denmark)

    Bang, D; Angelsø, Lene; Schirakow, Bente

    2003-01-01

    The objective of the study was to examine the influence of pooling Chlamydia trachomatis specimens. We compared Becton Dickinson ProbeTec strand displacement amplification (SDA) with Cobas Amplicor Roche (PCR). With PCR as the standard, SDA performed equally well in single-sample testing. For poo......The objective of the study was to examine the influence of pooling Chlamydia trachomatis specimens. We compared Becton Dickinson ProbeTec strand displacement amplification (SDA) with Cobas Amplicor Roche (PCR). With PCR as the standard, SDA performed equally well in single-sample testing...

  19. An improved colony-PCR method for filamentous fungi for amplification of pcr-fragments of several kilobases

    NARCIS (Netherlands)

    Zeijl, C.M.J. van; Kamp, E.H.M. van de; Punt, P.J.; Selten, G.C.M.; Hauer, B.; Gorcom, R.F.M. van; Hondel, C.A.M.J.J. van den

    1998-01-01

    A method was developed to perform PCR directly on mycelial pellets or colonies treated with NOVOzym 234. The method allows rapid screening of large numbers of transformants of both sporulating and non-sporulating fungi for the presence of (co)transforming plasmid copies or for specific genetic

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

    Science.gov (United States)

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

    2009-07-01

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

  1. Amplification of marine methanotrophic enrichment DNA with 16S rDNA PCR primers for type II alpha proteobacteria methanotrophs.

    Science.gov (United States)

    Rockne, Karl J; Strand, Stuart E

    2003-09-01

    Type II alpha proteobacteria methanotrophs are capable of a wide range of cometabolic transformations of chlorinated solvents and polycyclic aromatic hydrocarbons (PAHs), and this activity has been exploited in many terrestrial bioremediation systems. However, at present, all known obligately marine methanotrophic isolates are Type I gamma proteobacteria which do not have this activity to the extent of Type II methanotrophs. In previous work in our laboratory, determining the presence of Type II alpha proteobacteria methanotrophs in marine enrichment cultures that co-metabolized PAHs required a more sensitive assay. 16S rDNA PCR primers were designed based on oligonucleotide probes for serine pathway methanotrophs and serine pathway methylotrophs with an approximate amplification fragment size of 870 base pairs. Comparison of the primers using double primer BLAST searches in established nucleotide databases showed potential amplification with all Methylocystis and Methylosinus spp., as well as potential amplification with Methylocella palustrus. DNA from Methylosinus trichosporium OB3b, a Type II methanotroph, amplified with the primers with a fragment size of approximately 850 base pairs, whereas DNA extracted from Methylomonas methanica, a Type I methanotroph, did not. The primers were used to amplify DNA extracted from two marine methanotrophic enrichment cultures: a low nitrogen/low copper enrichment to select for Type II methanotrophs and a high nitrogen/high copper enrichment to select for Type I methanotrophs. Although DNA from both cultures amplified with the PCR primers, amplification was stronger in cultures that were specifically enriched for Type II methanotrophs, suggesting the presence of higher numbers of Type II methanotrophs. These results provide further evidence for the existence of Type II marine methanotrophs, suggesting the possibility of exploiting cometabolic activity in marine systems.

  2. Amplification of Mycoplasma haemofelis DNA by a PCR for point-of-care use.

    Science.gov (United States)

    Hawley, Jennifer; Yaaran, Tal; Maurice, Sarah; Lappin, Michael R

    2018-01-01

    We compared a qualitative in-clinic (IC)-PCR for the detection of Mycoplasma haemofelis DNA with the results of a commercial qualitative laboratory-based, conventional (c)PCR. In order to determine the specificity of both tests, Bartonella spp. samples were included. Forty-three previously tested blood samples with known PCR results for hemoplasmas and Bartonella spp. were selected. The samples were split between 2 laboratories. At the first laboratory, DNA was purified and run on 2 cPCR assays for the detection of hemoplasmas and Bartonella spp. At the second laboratory, DNA was purified using 2 purification protocols and both run in the IC-PCR assay. The cPCR results confirmed that 18 samples were positive for M. haemofelis, 5 for ' Candidatus M. haemominutum', 8 for Bartonella henselae, 2 for Bartonella clarridgeiae, and 10 were negative for both genera. No mixed infections were observed. The IC-PCR assay for the detection of M. haemofelis had a sensitivity of 94.4% and specificity of 96%, when using the same DNA purification method as the first laboratory. Using the second purification method, the sensitivity of the IC-PCR assay was 77.8% and specificity was 96%. Bartonella species were not detected by the IC-PCR M. haemofelis assay. The IC-PCR assay decreased the amount of time to final result compared to a cPCR assay.

  3. Comparative evaluation of DNA extraction methods for amplification by qPCR of superficial vs intracellular DNA from Bacillus spores.

    Science.gov (United States)

    Brauge, Thomas; Faille, Christine; Inglebert, Gaëlle; Dubois, Thomas; Morieux, Paul; Slomianny, Christian; Midelet-Bourdin, Graziella

    2018-02-02

    This study was designed to assess the efficiency of eight extraction methods regarding their ability to release superficial (exogenous) and intracellular (endogenous) DNA from B. cereus spores for subsequent analysis by quantitative PCR (qPCR). B. cereus spore suspensions were subjected to both commercial DNA extraction kits and mechanical DNA extraction methods. The spores were observed by transmission electron microscopy to evaluate any damage caused during extraction. The efficiency of both extraction and purification were assessed using a qPCR assay targeting the bclA gene. Most of the extraction methods assessed, except the passage through the French press or the use of the QIAamp DNA Blood Mini kit without 95°C treatment, allowed the amplification of significant amounts of DNA. By using propidium monoazide, which is a photoreactive DNA-binding dye, the presence of non-negligible amounts of amplifiable DNA at the spore surface was highlighted. A further set of extraction assays was then performed on spores previously treated with PMA. The results of this study show that both superficial and intracellular spore DNA can be released by extraction methods to a greater or lesser extent and then further amplified by qPCR. The Precellys extraction allowed the detection of both intracellular and superficial DNA, the DNeasy Blood & Tissue kit the specific detection of intracellular DNA, while the Instagene kit detected only superficial DNA. Of the methods tested in this study, the Precellys extraction was the most efficient in terms of further DNA detection. In order to verify the presence or absence of B. cereus spores in food or on surfaces in the food environment, the use of an efficient extraction method is required, followed by a qPCR analysis on the DNA released. Conversely, in order to quantify the population of Bacillus spores, any superficial DNA must be blocked, e.g. with PMA, prior to intracellular DNA extraction and further amplification. Copyright © 2018

  4. Highly sensitive detection of gluten-containing cereals in food samples by real-time Loop-mediated isothermal AMPlification (qLAMP) and real-time polymerase chain reaction (qPCR).

    Science.gov (United States)

    Garrido-Maestu, Alejandro; Azinheiro, Sarah; Fuciños, Pablo; Carvalho, Joana; Prado, Marta

    2018-04-25

    The treatment of gluten-related disorders is based on a lifelong, and strict, gluten-free diet. Thus, reliable and sensitive methods are required to detect the presence of gluten contamination. Traditional techniques rely on the detection of these proteins based on specific antibodies, but recent approaches go for an indirect route detecting the DNA that indicates the presence of cereals with gluten content. In the current study two different DNA amplification techniques, real-time PCR (qPCR) and real-time Loop-mediated isothermal AMPlification (qLAMP), were evaluated for their capability to detect and quantify gluten. Different detection strategies, based on these DNA amplification techniques, were tested. Even though good specificity results were obtained with the different approaches, overall qPCR proved more sensitive than qLAMP. This is the first study reporting a qLAMP based-method for the detection of gluten-containing cereals, along with its evaluation in comparison with qPCR. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Whole Blood PCR Amplification with Pfu DNA Polymerase and Its Application in Single-Nucleotide Polymorphism Analysis.

    Science.gov (United States)

    Liu, Er-Ping; Wang, Yan; He, Xiao-Hui; Guan, Jun-Jie; Wang, Jin; Qin, Zheng-Hong; Sun, Wan-Ping

    2015-11-01

    Point-of-care genetic analysis may require polymerase chain reaction (PCR) to be carried out on whole blood. However, human blood contains natural inhibitors of PCR such as hemoglobin, immunoglobulin G, lactoferrin, and proteases, as well as anticoagulant agents, including EDTA and heparin that can reduce whole blood PCR efficiency. Our purpose was to develop a highly specific, direct whole blood single-nucleotide polymorphism (SNP) analysis method based on allele-specific (AS) PCR that is mediated by Pfu DNA polymerase and phosphorothioate-modified AS primers. At high Mg(2+) concentrations, Pfu DNA polymerase efficiently amplified genomic DNA in a reaction solution containing up to 14% whole blood. Among the three anticoagulants tested, Pfu DNA polymerase showed the highest activity with sodium citrate. Meanwhile, Triton X-100 and betaine inhibited Pfu DNA polymerase activity in whole blood PCR, whereas trehalose had virtually no effect. These findings provided for the development of a low-cost, simple, and fast direct whole blood genotyping method that uses Pfu DNA polymerase combined with phosphorothioate AS primers for CYP2C9*3 and VKORC1(-1639) loci. With its high DNA amplification efficiency and tolerance of various blood conditions, Pfu DNA polymerase can be used in clinical laboratories to analyze SNPs in whole blood samples.

  6. Factors affecting success of PCR amplification of microsatellite loci from otter faeces

    Czech Academy of Sciences Publication Activity Database

    Hájková, Petra; Zemanová, Barbora; Bryja, Josef; Hájek, B.; Roche, K.; Tkadlec, Emil; Zima, Jan

    2006-01-01

    Roč. 6, č. 2 (2006), s. 559-562 ISSN 1471-8278 R&D Projects: GA ČR(CZ) GA206/03/0757 Institutional research plan: CEZ:AV0Z60930519 Keywords : amplification success * Lutra * faecal DNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.220, year: 2006

  7. By-Product Formation in Repetitive PCR Amplification of DNA Libraries during SELEX

    DEFF Research Database (Denmark)

    Tolle, Fabian; Wilke, Julian; Wengel, Jesper

    2014-01-01

    The selection of nucleic acid aptamers is an increasingly important approach to generate specific ligands binding to virtually any molecule of choice. However, selection-inherent amplification procedures are prone to artificial by-product formation that prohibits the enrichment of target-recogniz...

  8. Enzymological description of multitemplate PCR-Shrinking amplification bias by optimizing the polymerase-template ratio

    Czech Academy of Sciences Publication Activity Database

    Ingr, M.; Dostál, Jiří; Majerová, Taťána

    2015-01-01

    Roč. 382, Oct 7 (2015), s. 178-186 ISSN 0022-5193 R&D Projects: GA MŠk LO1302; GA ČR GBP208/12/G016 Institutional support: RVO:61388963 Keywords : amplification efficiency * amplicons pooling * species diversity * library preparation * processivity Subject RIV: CE - Biochemistry Impact factor: 2.049, year: 2015

  9. Recent advances in cell-free PrPSc amplification technique.

    Science.gov (United States)

    Atarashi, Ryuichiro

    2009-01-01

    The development of amplification technology for abnormal forms of prion protein in vitro has had a great impact on the field of prion research. This novel technology has generated new possibilities for understanding the molecular basis of prions and for developing an early diagnostic test for prion diseases. This review provides an overview of recent progress in cell-free PrPSc amplification techniques.

  10. Comparison of DNA probe, PCR amplification, ELISA and culture methods for the rapid detection of Salmonella in poultry

    International Nuclear Information System (INIS)

    Qasem, J.A.; Al-Mouqati, S.; Rajkumar, G.

    2005-01-01

    The identification of Salmonella spp. from poultry meat was studied by comparing bacterial detection using the Gene-Trak colorimetric hybridization method, a PCR amplification kit and an Enzyme Linked Immunosorbent Assay (ELISA), and these methods were compared with the conventional methodology proposed by the United States Food and Drug Administration (US FDA) for detection of Salmonella in food samples. Forty positive and negative samples were studied. The three methods yielded similar results with levels of Salmonella greater than 10 CFU per sample, even when the samples were highly contaminated with competing bacteria. In contrast, 20 CFU of seed inoculum per sample was the lowest level of Salmonella detectable with all three methods and the standard culture method. The detection limits of the PCR and ELISA assays were 5 CFU/g after enrichment at 37 deg. C for 6 and 9 hours, respectively. Compared with conventional bacteriology, all three methods here demonstrated high sensitivity and specificity for Salmonella. (author)

  11. Isolation by the 5 anchored PCR technique and characterization of ...

    Indian Academy of Sciences (India)

    Isolation by the 5 anchored PCR technique and characterization of eighteen microsatellite loci in horseshoe crab (Tachypleus gigas). LIEW PUI LING, ABU BAKAR ADIBAH, SOON GUAN TAN. ∗. , ANNIE CHRISTIANUS and QAMARUZ ZAMAN FARIDAH. Universiti Putra Malaysia, UPM Serdang, Selangor, 43400, Malaysia.

  12. PCR-based techniques for leprosy diagnosis: from the laboratory to the clinic.

    Directory of Open Access Journals (Sweden)

    Alejandra Nóbrega Martinez

    2014-04-01

    Full Text Available In leprosy, classic diagnostic tools based on bacillary counts and histopathology have been facing hurdles, especially in distinguishing latent infection from active disease and diagnosing paucibacillary clinical forms. Serological tests and IFN-gamma releasing assays (IGRA that employ humoral and cellular immune parameters, respectively, are also being used, but recent results indicate that quantitative PCR (qPCR is a key technique due to its higher sensitivity and specificity. In fact, advances concerning the structure and function of the Mycobacterium leprae genome led to the development of specific PCR-based gene amplification assays for leprosy diagnosis and monitoring of household contacts. Also, based on the validation of point-of-care technologies for M. tuberculosis DNA detection, it is clear that the same advantages of rapid DNA detection could be observed in respect to leprosy. So far, PCR has proven useful in the determination of transmission routes, M. leprae viability, and drug resistance in leprosy. However, PCR has been ascertained to be especially valuable in diagnosing difficult cases like pure neural leprosy (PNL, paucibacillary (PB, and patients with atypical clinical presentation and histopathological features compatible with leprosy. Also, the detection of M. leprae DNA in different samples of the household contacts of leprosy patients is very promising. Although a positive PCR result is not sufficient to establish a causal relationship with disease outcome, quantitation provided by qPCR is clearly capable of indicating increased risk of developing the disease and could alert clinicians to follow these contacts more closely or even define rules for chemoprophylaxis.

  13. PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

    Science.gov (United States)

    Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

    1992-06-01

    Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

  14. Comparison of loop-mediated isothermal amplification (LAMP) and nested-PCR assay targeting the RE and B1 gene for detection of Toxoplasma gondii in blood samples of children with leukaemia.

    Science.gov (United States)

    Fallahi, Shirzad; Seyyed Tabaei, Seyyed Javad; Pournia, Yadollah; Zebardast, Nozhat; Kazemi, Bahram

    2014-07-01

    Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this paper, a newly described DNA amplification technique, loop-mediated isothermal amplification (LAMP), and nested-PCR targeting the repeated element (RE) and B1 gene, were compared to each other for the detection of Toxoplasma gondii DNA in blood samples of children with leukaemia. One hundred ten blood samples from these patients were analyzed by LAMP and nested-PCR. Out of 50 seropositive samples (IgM+, IgG+), positive results were obtained with 92% and 86% on RE, B1-LAMP and 82% and 68% on RE, B1-nested PCR analyses, respectively. Of the 50 seronegative samples, three, two and one samples were detected positive by RE-LAMP, B1-LAMP and RE-nested PCR assays, respectively, while none were detected positive by B1-nested PCR. None of the 10 IgM-, IgG+ samples was detected positive after testing LAMP and nested-PCR assays in duplicate. This is the first report of a study in which the LAMP method was applied with high sensitivity and efficacy for the diagnosis of T. gonii in blood samples of children with leukaemia. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Detection of bond formations by DNA-programmed chemical reactions and PCR amplification.

    Science.gov (United States)

    Li, Yizhou; Zhang, Mingda; Zhang, Chi; Li, Xiaoyu

    2012-10-04

    A system capable of performing both DNA-templated chemical reactions and detection of bond formations is reported. Photocleavable DNA templates direct reactions. Products from bond-forming events re-ligate original templates, amplifiable by PCR, therefore distinguishing bond formation from background. This system provides a novel approach for discovering potential new chemical reactions.

  16. Signal Amplification by Glyco-qPCR for Ultrasensitive Detection of Carbohydrates: Applications in Glycobiology**

    Science.gov (United States)

    Kwon, Seok Joon; Lee, Kyung Bok; Solakyildirim, Kemal; Masuko, Sayaka; Ly, Mellisa; Zhang, Fuming; Li, Lingyun; Dordick, Jonathan S.; Linhardt, Robert J.

    2012-01-01

    Tiny amounts of carbohydrates (ca. 1 zmol) can be detected quantitatively by a real-time method based on the conjugation of carbohydrates with DNA markers (see picture). The proposed method (glyco-qPCR) provides uniform, ultrasensitive detection of carbohydrates, which can be applied to glycobiology, as well as carbohydrate-based drug discovery. PMID:23073897

  17. Image data analysis in qPCR: A method for smart analysis of DNA amplification

    Directory of Open Access Journals (Sweden)

    Massimo Orazio Spata

    2015-12-01

    Full Text Available In this paper, a method for the direct quantitative analysis of amplified DNA via q-Polymerase Chain Reaction (qPCR in miniaturised silicon-based chip system is presented. The designed tool presented here allows the automatic extraction of meaningful information from input fluorescent images by means of digital image processing algorithm. In particular, a smart mathematical model, optimizing the integration of all the analysis steps of the fluorescence data from on chip multiple real time PCR, is described. Such a tool is able to load the digital input images, select and smartly detect the region of interest for fluorescence, elaborate the data input, calculate the average fluorescence values and finally, plot the fitted curve as output, giving also, for each well, both the Cycle Threshold (CT and Slope parameters.

  18. Histopathologic Effects of Dirofilaria Immitis Microfilaria on Internal Organs of Dog Confirming by PCR Technique

    Directory of Open Access Journals (Sweden)

    S Simsek

    2012-06-01

    Full Text Available Background: The heartworm disease is an infectious disease of dogs with Dirofilaria immitis combined with cardiovascular and circulatory abnormalities. The heartworm disease can become a serious health risk when associated with a severe infection. In this study, a male, 8 year-old dog that died suddenly was necropsied and all tissues were examined grossly.Methods: Major organs including heart, lungs, liver, spleen, kidneys, brain, eyes, and testis were fixed in 10% neutral formalin, embedded in paraffin, sectioned at 5-µm thickness, stained with hematoxylin and eosin, and examined with a light microscope. For each examined organ, paraffin-embedded tissues were cut and placed in eppendorf tubes for genomic DNA extraction. PCR was performed using two sets of primers for amplification of a 302 bp ITS-2 gene fragment and a 203 bp cytochrome oxidase subunit 1 (CO1 gene fragment of D. immitis.Results: During the necropsy examination, 46 adult D. immitis were found in the portal vein, right ventricle, and atrium of the heart and pulmonary trunk. Microscopically, microfilarias were found throughout the vessels of different organs including lungs, kidneys, liver, heart, brain, and spleen. All tissues examined by PCR were positive for D. immitis ITS-2 and CO1.Conclusion: PCR technique now represents an effective method for identification of D. immitis from formalin-fixed samples.

  19. Identification of Tilletia species using rep-PCR fingerprinting technique

    Directory of Open Access Journals (Sweden)

    Župunski Vesna

    2011-01-01

    Full Text Available Analyzing 167 non-processed seed samples of wheat, it was found that 145 samples (86.8 % were contaminated with Tilletia species, while 22 (13.2 % samples were not contaminated. By using rep-PCR fingerprinting technique, it was found that DNA isolates of T. tritici originated from Serbian wheat samples had 80 % similarity with positive control for T. tritici. One isolate shared similarity of 60% with T. tritici, T. controversa and T. laevis. It was supposed that this isolate belongs to T. bromi. Isolate of T. laevis shared a similarity of 70 % with isolates of T. tritici and T. controversa, while T. walkeri was more than 10 % similar with T. tritici, T. controversa and T. laevis. Although T. controversa and T. tritici had high percent of genetic similarity, they were clustered separately. Our results suggest that rep-PCR fingerprinting could be a useful tool for monitoring presence of morphologically similar Tilletia species in wheat production areas.

  20. Design factors that influence PCR amplification success of cross-species primers among 1147 mammalian primer pairs

    Directory of Open Access Journals (Sweden)

    Beckett Stephanie E

    2006-10-01

    of successful amplifications are the number of index species mismatches, GC-richness of the target amplimer, and the relatedness of the target species to the index species, at least under the single PCR condition used. The 1147 cross-species primer pairs can be used in a high throughput manner to generate data for studies on the genetics and genomics of non-sequenced mammalian genomes.

  1. PCR-based amplification and heterologous expression of Pseudomonas alcohol dehydrogenase genes from the soil metagenome for biocatalysis.

    Science.gov (United States)

    Itoh, Nobuya; Isotani, Kentaro; Makino, Yoshihide; Kato, Masaki; Kitayama, Kouta; Ishimota, Tuyoshi

    2014-02-05

    The amplification of useful genes from metagenomes offers great biotechnological potential. We employed this approach to isolate alcohol dehydrogenase (adh) genes from Pseudomonas to aid in the synthesis of optically pure alcohols from various ketones. A PCR primer combination synthesized by reference to the adh sequences of known Pseudomonas genes was used to amplify full-length adh genes directly from 17 samples of DNA extracted from soil. Three such adh preparations were used to construct Escherichia coli plasmid libraries. Of the approximately 2800 colonies obtained, 240 putative adh-positive clones were identified by colony-PCR. Next, 23 functional adh genes named using the descriptors HBadh and HPadh were analyzed. The adh genes obtained via this metagenomic approach varied in their DNA and amino acid sequences. Expression of the gene products in E. coli indicated varying substrate specificity. Two representative genes, HBadh-1 and HPadh-24, expressed in E. coli and Pseudomonas putida, respectively, were purified and characterized in detail. The enzyme products of these genes were confirmed to be useful for producing anti-Prelog chiral alcohols. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Chum-RNA allows preparation of a high-quality cDNA library from a single-cell quantity of mRNA without PCR amplification.

    Science.gov (United States)

    Tougan, Takahiro; Okuzaki, Daisuke; Nojima, Hiroshi

    2008-09-01

    Linear RNA amplification using T7 RNA polymerase is useful in genome-wide analysis of gene expression using DNA microarrays, but exponential amplification using polymerase chain reaction (PCR) is still required for cDNA library preparation from single-cell quantities of RNA. We have designed a small RNA molecule called chum-RNA that has enabled us to prepare a single-cell cDNA library after four rounds of T7-based linear amplification, without using PCR amplification. Chum-RNA drove cDNA synthesis from only 0.49 femtograms of mRNA (730 mRNA molecules) as a substrate, a quantity that corresponds to a minor population of mRNA molecules in a single mammalian cell. Analysis of the independent cDNA clone of this library (6.6 x 10(5) cfu) suggests that 30-fold RNA amplification occurred in each round of the amplification process. The size distribution and representation of mRNAs in the resulting one-cell cDNA library retained its similarity to that of the million-cell cDNA library. The use of chum-RNA might also facilitate reactions involving other DNA/RNA modifying enzymes whose Michaelis constant (K(m)) values are around 1 mM, allowing them to be activated in the presence of only small quantities of substrate.

  3. Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples

    NARCIS (Netherlands)

    Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; Doorn, van Joop; Pham, Khanh; Cambra, Miguel A.; Berruete, Isabel M.; Pothier, Joël F.; Duffy, Brion; Olmos, Antonio; López, María M.

    2015-01-01

    Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample

  4. Accurate quantification of microorganisms in PCR-inhibiting environmental DNA extracts by a novel Internal Amplification Control approach using Biotrove OpenArrays

    NARCIS (Netherlands)

    Van Doorn, R.; Klerks, M.; van Gent-Pelzer, M.; Speksnijder, A.G.C.L.; Kowalchuk, G.A.; Schoen, C.D.

    2009-01-01

    PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally

  5. Detection of the food allergen celery via loop-mediated isothermal amplification technique.

    Science.gov (United States)

    Zahradnik, Celine; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

    2014-11-01

    Since 2005, celery and celery products have to be labeled according to Directive 2003/89/EC due to their allergenic potential. In order to provide a DNA-based, rapid and simple detection method suitable for high-throughput analysis, a loop-mediated isothermal amplification (LAMP) assay for the detection of celery (Apium graveolens) was developed. The assay was tested for specificity for celery since closely related species also hold food relevance. The limit of detection (LOD) for spiked food samples was found to be as low as 7.8 mg of dry celery powder per kilogram. An evaluation of different amplification and detection platforms was performed to show reliable detection independent from the instrument used for amplification (thermal cycler or heating block) and detection mechanisms (real-time fluorescence detection, agarose gel electrophoresis or nucleic acid staining). The analysis of 10 commercial food samples representing diverse and complex food matrices, and a false-negative rate of 0% for approximately 24 target copies or 0.08 ng celery DNA for three selected food matrices show that LAMP has the potential to be used as an alternative strategy for the detection of allergenic celery. The performance of the developed LAMP assay turned out to be equal or superior to the best available PCR assay for the detection of celery in food products.

  6. Multiplex RT-PCR amplification of HIV genes to create a completely autologous DC-based immunotherapy for the treatment of HIV infection.

    Directory of Open Access Journals (Sweden)

    Irina Tcherepanova

    Full Text Available BACKGROUND: Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. METHODOLOGY/PRINCIPAL FINDINGS: The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. CONCLUSION/SIGNIFICANCE: This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce

  7. Multiplex RT-PCR Amplification of HIV Genes to Create a Completely Autologous DC-Based Immunotherapy for the Treatment of HIV Infection

    Science.gov (United States)

    Tcherepanova, Irina; Harris, Jason; Starr, Aijing; Cleveland, Jaclyn; Ketteringham, Helen; Calderhead, David; Horvatinovich, Joe; Healey, Don; Nicolette, Charles A.

    2008-01-01

    Background Effective therapy for HIV-infected individuals remains an unmet medical need. Promising clinical trials with dendritic cell (DC)-based immunotherapy consisting of autologous DC loaded with autologous virus have been reported, however, these approaches depend on large numbers of HIV virions to generate sufficient doses for even limited treatment regimens. Methodology/Principal Findings The present study describes a novel approach for RT-PCR amplification of HIV antigens. Previously, RT-PCR amplification of autologous viral sequences has been confounded by the high mutation rate of the virus which results in unreliable primer-template binding. To resolve this problem we developed a multiplex RT-PCR strategy that allows reliable strain-independent amplification of highly polymorphic target antigens from any patient and requires neither viral sequence data nor custom-designed PCR primers for each individual. We demonstrate the application of our RT-PCR process to amplify translationally-competent RNA encoding regions of Gag, Vpr, Rev and Nef. The products amplified using this method represent a complex mixture of autologous antigens encoded by viral quasispecies. We further demonstrate that DCs electroporated with in vitro-transcribed HIV RNAs are capable of stimulating poly-antigen-specific CD8+ T cell responses in vitro. Conclusion/Significance This study describes a strategy to overcome patient to patient viral diversity enabling strain-independent RT-PCR amplification of RNAs encoding sequence divergent quasispecies of Gag, Vpr, Rev and Nef from small volumes of infectious plasma. The approach allows creation of a completely autologous therapy that does not require advance knowledge of the HIV genomic sequences, does not have yield limitations and has no intact virus in the final product. The simultaneous use of autologous viral antigens and DCs may provoke broad patient-specific immune responses that could potentially induce effective control of viral

  8. Restriction Cascade Exponential Amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR.

    Science.gov (United States)

    Ghindilis, Andrey L; Smith, Maria W; Simon, Holly M; Seoudi, Ihab A; Yazvenko, Nina S; Murray, Iain A; Fu, Xiaoqing; Smith, Kenneth; Jen-Jacobson, Linda; Xu, Shuang-Yong

    2015-01-13

    An alternative to qPCR was developed for nucleic acid assays, involving signal rather than target amplification. The new technology, Restriction Cascade Exponential Amplification (RCEA), relies on specific cleavage of probe-target hybrids by restriction endonucleases (REase). Two mutant REases for amplification (Ramp), S17C BamHI and K249C EcoRI, were conjugated to oligonucleotides, and immobilized on a solid surface. The signal generation was based on: (i) hybridization of a target DNA to a Ramp-oligonucleotide probe conjugate, followed by (ii) specific cleavage of the probe-target hybrid using a non-immobilized recognition REase. The amount of Ramp released into solution upon cleavage was proportionate to the DNA target amount. Signal amplification was achieved through catalysis, by the free Ramp, of a restriction cascade containing additional oligonucleotide-conjugated Ramp and horseradish peroxidase (HRP). Colorimetric quantification of free HRP indicated that the RCEA achieved a detection limit of 10 aM (10(-17) M) target concentration, or approximately 200 molecules, comparable to the sensitivity of qPCR-based assays. The RCEA assay had high specificity, it was insensitive to non-specific binding, and detected target sequences in the presence of foreign DNA. RCEA is an inexpensive isothermal assay that allows coupling of the restriction cascade signal amplification with any DNA target of interest.

  9. The cost effectiveness of Nucleic Acid Amplification Techniques for the diagnosis of tuberculosis.

    Science.gov (United States)

    Hughes, Ralph; Wonderling, David; Li, Bernadette; Higgins, Bernard

    2012-02-01

    There is wide variation in the techniques deployed to diagnose tuberculosis in the UK, with little agreement on which tools or strategies are cost effective. This analysis therefore comprehensively evaluated the cost effectiveness of currently available diagnostic strategies for routine diagnosis of TB in the NHS. The analysis compared strategies consisting of Nucleic Acid Amplification Techniques, culture and microscopy. A decision tree was used to estimate costs and Quality-Adjusted Life Years (QALYs) from a UK health service perspective. The sensitivity and specificity of each test determined the true and false positive and negative results in patients suspected of having active tuberculosis. These results led to either early, correct diagnosis or delayed diagnosis and the associated costs and QALYs. The presence of active tuberculosis combined with the side effects of treatment was associated with reduction in quality of life. Costs included were test costs, drug costs and the management of tuberculosis. Drug costs were based on generic UK list prices. Uncertainty in the model was explored through probabilistic and deterministic sensitivity analyses. The cost effective strategy at threshold of £20,000 per QALY was a strategy using only sputum microscopy and culture routinely, meaning Nucleic Acid Amplification Techniques are not cost effective at baseline. When the prevalence of tuberculosis was increased, however, nucleic acid amplification became cost effective at the same threshold. Aside from the prevalence, the results were shown to be robust. At low tuberculosis prevalence, Nucleic Acid Amplification Techniques may not be cost effective but their potential in higher prevalence situations is considerable. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. The potential role of incorporating real-time PCR and DNA sequencing for amplification and detection of 16S rRNA gene signatures in neonatal sepsis.

    Science.gov (United States)

    Midan, Dina A; Abo El Fotoh, Wafaa Moustafa M; El Shalakany, Abeer H

    2017-06-01

    This study aimed to explore whether 16S rRNA gene amplification by real time PCR and sequencing could serve as genetic-based methods in rapid and accurate diagnosis of neonatal sepsis. This case control study was conducted on 40 neonates suffering from sepsis like manifestations recruited from the neonatal intensive care unit of Menoufia university hospital over a period of 6 months. Their blood samples were used for paired analysis of bacterial growth using BACTEC 9050 instrument and real time PCR assay with subsequent DNA sequencing for bacterial species identification. The detection rate of culture proven sepsis was 70%. By using real time 16S r RNA PCR amplification method, the detection of bacteria was improved to 80%. Real time PCR revealed sensitivity, specificity, positive predictive value and negative predictive value of [100%, 66.7%, 87.5% and 100%] respectively. Compared to culture, the 16S rRNA real time PCR demonstrated a high negative value for ruling out neonatal sepsis. There was significant statistical difference between the PCR positive and negative cases as regards the hematological sepsis score. The results demonstrated the ability of DNA sequencing to recognize 4 pathogens which were negative by blood culture. The time consumed to detect sepsis using blood culture was up to 5 days while it took up to 16 h only by PCR and sequencing methods. 16S rRNA gene amplification by real time PCR and sequence analysis could be served as ideal and reliable genetic-based methods to diagnose and rule out sepsis with provision of additional data that cannot be obtained by routine laboratory tests with a shorter turnaround time than those with culture-based protocols.

  11. Rolling circle amplification of metazoan mitochondrialgenomes

    Energy Technology Data Exchange (ETDEWEB)

    Simison, W. Brian; Lindberg, D.R.; Boore, J.L.

    2005-07-31

    Here we report the successful use of rolling circle amplification (RCA) for the amplification of complete metazoan mt genomes to make a product that is amenable to high-throughput genome sequencing techniques. The benefits of RCA over PCR are many and with further development and refinement of RCA, the sequencing of organellar genomics will require far less time and effort than current long PCR approaches.

  12. Development and evaluation of internal amplification controls for use in a real-time duplex PCR assay for detection of Campylobacter coli and Campylobacter jejuni.

    Science.gov (United States)

    Randall, Luke; Lemma, Fabrizio; Rodgers, John; Vidal, Ana; Clifton-Hadley, Felicity

    2010-02-01

    A common problem of both conventional and real-time PCR assays is failure of DNA amplification due to the presence of inhibitory substances in samples. In view of this, our aim was to develop and evaluate internal amplification controls (IACs) for use with an existing duplex real-time PCR assay for Campylobacter coli and Campylobacter jejuni. Both competitive and non-competitive IACs were developed and evaluated. The competitive approach involved a DNA fragment of the coding region of the fish viral haemorrhagic septicaemia virus, flanked by the mapA PCR primers, whilst the non-competitive approach utilized an extra set of universal 16S rDNA primers. Both IAC-PCR assay types were evaluated using cultures of Campylobacter and chicken caecal content samples. Both IACs were sensitive to caecal inhibitors, making them suitable for detecting inhibition which could lead to false-negatives. Results showed that both IACs at optimum concentrations worked well without reducing the overall sensitivity of the PCR assay. Compared to culture, the optimized competitive IAC-PCR assay detected 45/47 positives (sensitivity 93.6 %, specificity 80.1 %); however, it had the advantage over culture in that it could detect mixed infections of C. coli and C. jejuni and was capable of giving a result for a sample within a day.

  13. Development of rapid and simple method for DNA extraction from cannabis resin based on the evaluation of relative PCR amplification ability.

    Science.gov (United States)

    Yamamuro, Tadashi; Iwata, Yuko T; Segawa, Hiroki; Kuwayama, Kenji; Tsujikawa, Kenji; Kanamori, Tatsuyuki; Inoue, Hiroyuki

    2018-04-04

    In recent years, analysis of cannabis DNA has been increasingly used in forensic drug tests. However, in the case of cannabis resin, a processed marijuana product, complicated procedures are required for the extraction of clean DNA, as the presence of various impurities inhibits PCR amplification. Therefore, in this study, we attempted to identify the factors that would allow quick and simple DNA extraction from cannabis resin with a commercially available kit. We also constructed a simple assay system for comparing relative amplification efficiencies by end-point PCR and used it to evaluate the purity of the obtained DNA solutions. For extraction with a kit that contains a silica column, reducing the starting amount of resin, using the residue remaining after methanol extraction, dilution of the final solution, extraction with an equal amount of powdered activated carbon or an excess amount of polyvinylpolypyrrolidone, and the addition of an appropriate amount of polyvinylpyrrolidone to the solution after extraction were effective measures that improved amplification efficiency. Furthermore, the use of the most rapid alkaline extraction kit combined with the addition of powdered activated carbon allowed obtaining DNA solutions with sufficient amplification efficiency in about 10min. These findings should be useful for routine DNA analysis of cannabis resin during forensic examination. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Analysis of antimicrobial effect of MTAD solution in infected canal system using PCR technique

    Directory of Open Access Journals (Sweden)

    Mitić Aleksandar

    2013-01-01

    Full Text Available Introduction. Clinically acceptable antiseptic should possess organolithic-mineralolithic properties and antimicrobial efficacy, and should be non-toxic. Objective. The aim of the paper was to assess the presence of genomes of the most common microorganisms (Porphyromonas gingivalis, Agregatibacter actinomycetemcomitans, Tanerella forsythensis, Prevotella intermedia, Treponema denticola and Enterococcus faecalis in infected tooth root canals before and after rinsing with solution of doxycycline, citric acid and detergent Tween-80 (MTAD in patients with clinically diagnosed primary apex periodontitis. Methods. The content of primarily infected canals before and after using the MTAD solution was used as a biological material in which the presence of microorganisms DNA was proved. For the detection of bacterial genome the multiplex PCR technique was applied. Results. The percentage of positive samples before canal treatment was 100%. In infected root canals E. faecalis was most dominant (37%. In a relatively high percentage we detected P. intermedia (25%, A. actinomycetemcomitans (20%, T. denticola (17%, T. forsythensis (15% and P. gingivalis (10%. After rinsing the canal system using MTAD solution, there was a statistically significant decrease in E. faecalis (12%, P. intermedia (0%, T. forsythensis (0% and P. gingivalis (0%. The presence of other bacteria was also diminished but not statistically significantly. Conclusion. With the application of multiplex PCR technique which provided a simultaneous amplification of various genomic sequences, using several pairs of primers, the most dominant in infected root canals were E. faecalis. P. intermedia, A. actinomycetemcomitans, T. denticola, T. forsythensis and P. gingivalis. After mechanic treatment and irrigation of root canals with MTAD solution, P. intermedia, P. gingivalis and T. forsythensis were not found. The presence of E. faecalis, A. actinomycetemcomitans and T. denticola was diminished

  15. Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR for diagnosis of natural infection with canine distemper virus

    Directory of Open Access Journals (Sweden)

    Wong Min-Liang

    2010-06-01

    Full Text Available Abstract Background Canine distemper virus (CDV is present worldwide and produces a lethal systemic infection of wild and domestic Canidae. Pre-existing antibodies acquired from vaccination or previous CDV infection might interfere the interpretation of a serologic diagnosis method. In addition, due to the high similarity of nucleic acid sequences between wild-type CDV and the new vaccine strain, current PCR derived methods cannot be applied for the definite confirmation of CD infection. Hence, it is worthy of developing a simple and rapid nucleotide-based assay for differentiation of wild-type CDV which is a cause of disease from attenuated CDVs after vaccination. High frequency variations have been found in the region spanning from the 3'-untranslated region (UTR of the matrix (M gene to the fusion (F gene (designated M-F UTR in a few CDV strains. To establish a differential diagnosis assay, an amplification refractory mutation analysis was established based on the highly variable region on M-F UTR and F regions. Results Sequences of frequent polymorphisms were found scattered throughout the M-F UTR region; the identity of nucleic acid between local strains and vaccine strains ranged from 82.5% to 93.8%. A track of AAA residue located 35 nucleotides downstream from F gene start codon highly conserved in three vaccine strains were replaced with TGC in the local strains; that severed as target sequences for deign of discrimination primers. The method established in the present study successfully differentiated seven Taiwanese CDV field isolates, all belonging to the Asia-1 lineage, from vaccine strains. Conclusions The method described herein would be useful for several clinical applications, such as confirmation of nature CDV infection, evaluation of vaccination status and verification of the circulating viral genotypes.

  16. Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites

    NARCIS (Netherlands)

    van der Meide, Wendy; Guerra, Jorge; Schoone, Gerard; Farenhorst, Marit; Coelho, Leila; Faber, William; Peekel, Inge; Schallig, Henk

    2008-01-01

    DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays

  17. Combined amplification and hybridization techniques for genome scanning in vegetatively propagated crops

    International Nuclear Information System (INIS)

    Kahl, G.; Ramser, J.; Terauchi, R.; Lopez-Peralta, C.; Asemota, H.N.; Weising, K.

    1998-01-01

    A combination of PCR- and hybridization-based genome scanning techniques and sequence comparisons between non-coding chloroplast DNA flanking tRNA genes has been employed to screen Dioscorea species for intra- and interspecific genetic diversity. This methodology detected extensive polymorphisms within Dioscorea bulbifera L., and revealed taxonomic and phylogenetic relationships among cultivated Guinea yams varieties and their potential wild progenitors. Finally, screening of yam germplasm grown in Jamaica permitted reliable discrimination between all major cultivars. Genome scanning by micro satellite-primed PCR (MP-PCR) and random amplified polymorphic DNA (RAPD) analysis in combination with the novel random amplified micro satellite polymorphisms (RAMPO) hybridization technique has shown high potential for the genetic analysis of yams, and holds promise for other vegetatively propagated orphan crops. (author)

  18. Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

    Science.gov (United States)

    de Souza, Marcela; Matsuzawa, Tetsuhiro; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Busso-Lopes, Ariane Fidelis; Levin, Anna Sara Shafferman; Schreiber, Angélica Zaninelli; Mikami, Yuzuru; Gonoi, Tohoru; Kamei, Katsuhiko; Moretti, Maria Luiza; Trabasso, Plínio

    2017-08-01

    The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

  19. DNA Amplification Techniques for the Detection of Toxoplasma gondii Tissue Cysts in Meat Producing Animals: A Narrative Review Article

    Directory of Open Access Journals (Sweden)

    Farooq RIAZ

    2016-12-01

    Full Text Available Background: Toxoplasma gondii is an intracellular parasite, which infects one-third population of world. Humans and animals acquire infection by ingesting oocytes from feces of cats or by meat of other animals having cysts that may lead to congenital, ocular or cephalic toxoplasmosis. Either it is important to detect T. gondii from meat of food animals from retail shops or directly at slaughterhouses, which is meant for export.Methods: The current research was done without time limitation using such terms as follows: “Toxoplasma gondii”, “Meat”, “Tissue cyst”, “PCR”, “LAMP”, “Screening” and “Immunological assay” alone or in combination, in English language. The used electronic databases for searching included as follows: PubMed, Scopus, Google Scholar, Web of Science and Science Direct. The searches were limited to the published papers to English language.Results: Sensitivity of different molecular techniques for diagnosis of Toxoplasma is real-time PCR > LAMP > conventional PCR. In addition to these DNA analysis tools, bioassay in mice and cats is considered as “gold standard” to detect T. gondii. Conclusion: This review article will help the readers for grasping advantages and limitations of different diagnostic tools for screening meat samples for T. gondii. This review also makes bibliography about the type of meat sample to be processed for diagnosis and different primers or sequences to be targeted for T. gondii by number of researches for its detection from meat or tissue sample using DNA amplification techniques.

  20. Detection of Treponema pallidum Sp. Pallidum DNA in Cerebrospinal Fluid (CSF) by Two PCR Techniques.

    Science.gov (United States)

    Castro, Rita; Águas, Maria João; Batista, Teresa; Araújo, Carlos; Mansinho, Kamal; Pereira, Filomena da Luz Martins

    2016-09-01

    Laboratory diagnosis of neurosyphilis is complicated especially when it is asymptomatic, no single laboratory test result being appropriate to diagnose central nervous system infectivity caused by Treponema pallidum. Our objective was to evaluate two polymerase chain reaction (PCR) techniques for the detection of T. pallidum DNA in the cerebrospinal fluid (CSF) of patients with syphilis. One hundred twenty-four CSF samples from patients with reactive blood tests for syphilis were obtained. Two PCR techniques (47-PCR, polA-PCR) were used to detect T. pallidum DNA. The laboratory criteria used for the diagnosis of neurosyphilis to which the PCR techniques were compared were those recommended by the IUSTI: 2008 European guidelines on the management of syphilis. Treponema pallidum DNA was detected amplified in 37 of 124 (29.8%) and 30 of 124 (24.2%) samples with the 47-PCR and polA-PCR, respectively. Sensitivities were 75.8% and 69.7% and specificities 86.8% and 92.3%, respectively, for 47-PCR and polA-PCR techniques, respectively. The three CSF samples of patients with primary syphilis did not fulfill the criteria of neurosyphilis and DNA was only detected in one by the 47-PCR. In samples from secondary syphilis and neurosyphilis, three of nine and nine of nine respectively, results were coincident for the two PCR techniques and neurosyphilis criteria. Major discrepancies between the two PCR techniques and neurosyphilis diagnostic criteria were observed in latent syphilis. Beyond some limitations of the study, which are discussed here, both PCR techniques seem to be useful for the diagnosis of neurosyphilis, although 47-PCR presents a higher sensitivity and polA-PCR a higher specificity. © 2016 Wiley Periodicals, Inc.

  1. Rapid detection of Salmonella in raw chicken breast using real-time PCR combined with immunomagnetic separation and whole genome amplification.

    Science.gov (United States)

    Hyeon, Ji-Yeon; Deng, Xiangyu

    2017-05-01

    We presented the first attempt to combine immunomagnetic separation (IMS), whole genome amplification by multiple displacement amplification (MDA) and real-time PCR for detecting a bacterial pathogen in a food sample. This method was effective in enabling real-time PCR detection of low levels of Salmonella enterica Serotype Enteritidis (SE) (∼10 CFU/g) in raw chicken breast without culture enrichment. In addition, it was able to detect refrigeration-stressed SE cells at lower concentrations (∼0.1 CFU/g) in raw chicken breast after a 4-h culture enrichment, shortening the detection process from days to hours and displaying no statistical difference in detection rate in comparison with a culture-based detection method. By substantially improving performance in SE detection over conventional real-time PCR, we demonstrated the potential of IMS-MDA real-time PCR as a rapid, sensitive and affordable method for detecting Salmonella in food. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. An evaluation of the PCR-RFLP technique to aid molecular-based monitoring of felids and canids in India

    Directory of Open Access Journals (Sweden)

    Home Chandrima

    2010-06-01

    Full Text Available Abstract Background The order Carnivora is well represented in India, with 58 of the 250 species found globally, occurring here. However, small carnivores figure very poorly in research and conservation policies in India. This is mainly due to the dearth of tested and standardized techniques that are both cost effective and conducive to small carnivore studies in the field. In this paper we present a non-invasive genetic technique standardized for the study of Indian felids and canids with the use of PCR amplification and restriction enzyme digestion of scat collected in the field. Findings Using existing sequences of felids and canids from GenBank, we designed primers from the 16S rRNA region of the mitochondrial genome and tested these on ten species of felids and five canids. We selected restriction enzymes that would cut the selected region differentially for various species within each family. We produced a restriction digestion profile for the potential differentiation of species based on fragment patterns. To test our technique, we used felid PCR primers on scats collected from various habitats in India, representing varied environmental conditions. Amplification success with field collected scats was 52%, while 86% of the products used for restriction digestion could be accurately assigned to species. We verified this through sequencing. A comparison of costs across the various techniques currently used for scat assignment showed that this technique was the most practical and cost effective. Conclusions The species-specific key developed in this paper provides a means for detailed investigations in the future that focus on elusive carnivores in India and this approach provides a model for other studies in areas of Asia where many small carnivores co-occur.

  3. The principle and application of new PCR Technologies

    Science.gov (United States)

    Yu, Miao; Cao, Yue; Ji, Yubin

    2017-12-01

    Polymerase chain reaction (PCR) is essentially a selective DNA amplification technique commonlyapplied for genetic testing and molecular diagnosis because of its high specificity and sensitivity.PCR technologies as the key of molecular biology, has realized that the qualitative detection of absolute quantitative has been changed. It has produced a variety of new PCR technologies, such as extreme PCR, photonic PCR, o-amplification at lower denaturation temperature PCR, nanoparticle PCR and so on. In this paper, the principle and application of PCR technologies are reviewed, and its development is prospected too.

  4. [Detection of epidermal growth factor receptor gene mutations in different types of non-small cell lung cancer by droplet digital PCR and amplification refractory mutation system].

    Science.gov (United States)

    Li, R; Ye, S B; He, Y; Wang, X; Wu, N; Xia, Q Y; Shen, Q; Shi, S S

    2017-11-08

    Objective: To compare amplification refractory mutation system(ARMS) and droplet digital PCR (ddPCR) in the detection of epidermal growth factor receptor (EGFR) gene mutations in patients with non-small cell lung cancer (NSCLC), and to investigate the clinical value of ddPCR. Methods: A total of 79 specimens of NSCLC, including 22 cases of cell block, 18 cases of surgical specimens, 12 cases of biopsy specimens and 27 cases of plasma samples, were analyzed for the mutation status of EGFR gene by ARMS and droplet digital PCR method. Results: In 18 cases of surgical specimens and 12 cases of biopsy specimens, the detection results by the two methods were identical with positive rates of 9/18 and 5/12, respectively. In 22 cases of effusion cell blocks, ARMS detected 19-del and L858R of EGFR gene in two cases, in which droplet digital PCR detected 19-del+ T790M mutations in one case and L858R+ T790M mutation in another. L858R mutation was detected by droplet digital PCR in one case but ARMS assay was negative. The remaining 19 cases were consistent by the two methods. In blood samples, the positive rate was 33.3%(9/27) by ARMS and 37.0%(10/27) by droplet digital PCR. Two cases showed L858R and 19-del+ T790M mutation by droplet digital PCR but ARMS assay detected only 19-del. The remaining 25 cases were consistent by the two methods. Conclusion: Droplet digital PCR method is more sensitive and accurate than ARMS for the detection of EGFR mutations in pleural fluid and blood samples, can be used in clinical test.

  5. Evaluation of amplification refractory mutation system (ARMS technique for quick and accurate prenatal gene diagnosis of CHM variant in choroideremia

    Directory of Open Access Journals (Sweden)

    Yang L

    2017-12-01

    Full Text Available Lisha Yang,1 Iqra Ijaz,1 Jingliang Cheng,1,2 Chunli Wei,1,3 Xiaojun Tan,4 Md Asaduzzaman Khan,1 Xiaodong Fu,5 Junjiang Fu1–3 1Key Laboratory of Epigenetics and Oncology, the Research Center for Precision Medicine, Southwest Medical University, Luzhou, 2Department of Pathology, Hunan Normal University College of Medicine, Changsha, 3State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Macau (SAR, 4Reproductive and Genetic Center, the Central Hospital of Xiangtan City, Xiangtan, 5Department of Obstetrics and Gynecology, First Affiliated Hospital of Southwest Medical University, Luzhou, People’s Republic of China Abstract: Choroideremia is a rare X-linked recessive inherited disorder that causes chorioretinal dystrophy leading to visual impairment in its early stages which finally causes total blindness in the affected person. It is caused due to mutations in the CHM gene. In this study, we have recruited a pedigree with choroideremia and detected a nonsense variant (c.C799T:p.R267X in CHM of the proband (I:1. Different primer sets for amplification refractory mutation system (ARMS were designed and PCR conditions were optimized. Then, we evaluated the sequence variant in the patient, carrier, and a fetus by using ARMS technique to identify if they inherited the pathogenic gene from parental generation; we used amniotic fluid DNA for the diagnosis of the gene in the fetus. The primer pairs, WT2+C and MT+C, amplified high specific products in different DNAs which were verified by Sanger sequencing. Based on our results, ARMS technique is fast, accurate, and reliable prenatal gene diagnostic tool to assess CHM variants. Taken together, our study indicates that ARMS technique can be used as a potential molecular tool in the diagnosis of prenatal mutation for choroideremia as well as other genetic diseases in undeveloped and developing countries, where there might be shortage of medical resources

  6. Prospective comparison of a PCR assay and a microbiological culture technique for identification of pathogens from blood and non-blood samples in septic patients.

    Science.gov (United States)

    Plettig, Runa; Nowak, Andreas; Balau, Veronika; Hahnenkamp, Klaus; Usichenko, Taras

    2015-01-01

    Molecular amplification techniques are suggested to be a useful adjunct in early detection of pathogens in septic patients. The aim was to study the feasibility of a polymerase chain reaction (PCR) assay compared to the standard microbiological culture (MC) technique in identification of pathogenic microorganisms from blood and non-blood samples in septic patients. Samples for pathogen identification were taken during febrile septic episodes (SE) in 54 patients with sepsis and analyzed using both MC and PCR. Semi-automated multiplex PCR, provided by Philips Medical Systems, was able to detect nine different pathogens. The accuracy of pathogen identification using PCR vs. MC as well as the time-saving effect of PCR on the potential decision-making process for antimicrobial therapy was evaluated. In a total of 258 samples taken during 87 SE, both methods yielded more pathogens from the non-blood than blood samples (87 % vs. 45 %; p = 0.002). PCR identified more pathogens than MC in the blood samples (98 vs. 21; p technique. In the non-blood samples, PCR was comparable to that of MC.

  7. Sex Determination in Ostrich (Struthio camelus Using PCR Technique

    Directory of Open Access Journals (Sweden)

    M Alipanah

    2011-12-01

    Full Text Available The important of ostrich breeding have been increased in the Iran as other country around the world. Lack of sexual dimorphism in many species of birds including Ostrich (Struthio camelus, making it difficult to differentiate between males and females, especially in young birds. This can be problematic for breeding programs. Blood samples from randomly chicks in the first days were collected and then DNA was extracted from blood. For sex typing used PCR by OSFES primer that produced a 423 fragment. Our results shown that sex determination using PCR method for chicks in the first days is useful, easy and inexpensive.

  8. Identification of endotrypanum species from a sloth, a squirrel and Lutzomyia sandflies in ecuador by PCR amplification and sequencing of the mini-exon gene.

    Science.gov (United States)

    Katakura, Ken; Mimori, Tatsuyuki; Furuya, Masato; Uezato, Hiroshi; Nonaka, Shigeo; Okamoto, Munehiro; Gomez L, Eduardo A; Hashiguchi, Yoshihisa

    2003-05-01

    PCR amplification and nucleotide sequencing of the mini-exon gene revealed that four strains isolated from a sloth (Choloepus hoffmanni), a squirrel (Sciurus granatensis) and two sandflies (Lutzomyia hartmanni) in Ecuador were indistinguishable from Endotrypanum monterogeii. Another strain isolated from Lu. hartmanni showed the high sequence similarity to E. schaudinni. Since three of these strains have been previously identified as Leishmania (Viannia) equatorensis, the results demonstrate that L. (V.) equatorensis is genetically closely related to the genus Endotrypanum. The present study also indicates that Endotrypanum species are distributed in arboreal animals and sandflies in Ecuador, and that mini-exon gene amplification is useful for epidemiological studies of Leishmania and Endotrypanum in the New World.

  9. Comparison of Nested-PCR technique and culture method in ...

    African Journals Online (AJOL)

    USER

    2010-04-05

    Apr 5, 2010 ... tuberculosis (GUTB) compared with acid fast staining and culture method. In total ... tuberculosis; PCR, polymerase chain reaction; MTB,. Mycobacterium tuberculosis;. EPTB, extrapulmonary tuberculosis; AFB, acid-fast bacilli; SDS, sodium ..... Dingtoumda B, Diallo B, Defer MC, Sombié I, Zanetti S, Sechi LA.

  10. Evaluation of most-probable-number-PCR method with internal amplification control for the counting of total and pathogenic Vibrio parahaemolyticus in frozen shrimps.

    Science.gov (United States)

    Copin, S; Robert-Pillot, A; Malle, P; Quilici, M L; Gay, M

    2012-01-01

    The most-probable-number (MPN) method is often time-consuming for the isolation, detection, and quantification of Vibrio parahaemolyticus from natural sources. MPN counting of V. parahaemolyticus bacteria usually involves the isolation of typical V. parahaemolyticus colonies on selective medium, with subsequent confirmation by biochemical identification. In this study, we evaluated the use of a PCR on MPN enrichment cultures (MPN-PCR) for the direct detection of total and pathogenic V. parahaemolyticus cells in frozen shrimp. This reaction targeted the R72H, tdh, and trh sequences. An internal amplification control was added to the samples before R72H amplification. There was an excellent correlation between the results of the two methods for artificially inoculated and natural shrimp samples. Of 36 natural samples, 28 tested positive for the presence of V. parahaemolyticus, with an MPN value of 2 × 10(-1) to 9.2 × 10(1) per g. No pathogenic V. parahaemolyticus cells were detected. The test had a detection limit of one V. parahaemolyticus organism per g and was completed within two working days. These results support the use of the combination of PCR with MPN for the detection of total or potentially pathogenic V. parahaemolyticus cells in frozen shrimp.

  11. Development of Multiplex-Mismatch Amplification Mutation-PCR Assay for Simultaneous Detection of Campylobacter jejuni and Mutation in gyrA Gene Related to Fluoroquinolone Resistance.

    Science.gov (United States)

    Cui, Mingquan; Wu, Chenbin; Zhang, Peng; Wu, Congming

    2016-11-01

    Campylobacter jejuni, a foodborne pathogen, is the major cause of enteritis in humans worldwide, however, its increasing resistance to fluoroquinolones reported recently is of a major concern. In the present study, multiplex-mismatch amplification mutation assay-polymerase chain reaction (MMAMA-PCR) was developed for the first time with the aim to quickly identify C. jejuni and to detect the single nucleotide mutation (C-257 to T) frequently observed in gyrA gene, associated with the acquisition of resistance to fluoroquinolones. In this assay, mismatch amplification mutation primers for the detection of gyrA mutation in C. jejuni were coupled with primers for the hip gene encoding for hippuricase and 16S rRNA gene of C. jejuni, respectively, in the multiplex PCR assay. The specificity and accuracy of this method were analyzed by the use of 78 C. jejuni strains with previously confirmed resistance phenotypes and the mutation (C-257 to T) in gyrA gene, as well as 107 clinical isolates of various bacterial species, including 29 C. jejuni isolates. This study indicates that MMAMA-PCR is a promising assay for the rapid identification of C. jejuni with a specific mutation in gyrA gene, responsible for the resistance to fluoroquinolones.

  12. Quantification of Campylobacter spp. in pig feces by direct real-time PCR with an internal control of extraction and amplification.

    Science.gov (United States)

    Leblanc-Maridor, Mily; Garénaux, Amélie; Beaudeau, François; Chidaine, Bérangère; Seegers, Henri; Denis, Martine; Belloc, Catherine

    2011-04-01

    The rapid and direct quantification of Campylobacter spp. in complex substrates like feces or environmental samples is crucial to facilitate epidemiological studies on Campylobacter in pig production systems. We developed a real-time PCR assay for detecting and quantifying Campylobacter spp. directly in pig feces with the use of an internal control. Campylobacter spp. and Yersinia ruckeri primers-probes sets were designed and checked for specificity with diverse Campylobacter, related organisms, and other bacterial pathogens before being used in field samples. The quantification of Campylobacter spp. by the real-time PCR then was realized on 531 fecal samples obtained from experimentally and naturally infected pigs; the numeration of Campylobacter on Karmali plate was done in parallel. Yersinia ruckeri, used as bacterial internal control, was added to the samples before DNA extraction to control DNA-extraction and PCR-amplification. The sensitivity of the PCR assay was 10 genome copies. The established Campylobacter real-time PCR assay showed a 7-log-wide linear dynamic range of quantification (R²=0.99) with a detection limit of 200 Colony Forming Units of Campylobacter per gram of feces. A high correlation was found between the results obtained by real-time PCR and those by culture at both qualitative and quantitative levels. Moreover, DNA extraction followed by real-time PCR reduced the time needed for analysis to a few hours (within a working day). In conclusion, the real-time PCR developed in this study provides new tools for further epidemiological surveys to investigate the carriage and excretion of Campylobacter by pigs. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples.

    Science.gov (United States)

    Palacio-Bielsa, Ana; López-Soriano, Pablo; Bühlmann, Andreas; van Doorn, Joop; Pham, Khanh; Cambra, Miguel A; Berruete, Isabel M; Pothier, Joël F; Duffy, Brion; Olmos, Antonio; López, María M

    2015-05-01

    Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample preparation were analyzed. Kappa coefficient, sensitivity, specificity, likelihood ratios and post-test probability of detection were estimated to manage the risk associated with the use of the two methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. PCR

    African Journals Online (AJOL)

    Jane

    2011-07-11

    Jul 11, 2011 ... then in age group 31 to 40 years, 2.25% (2/89) CMV DNA were detected by PCR and 0% was recorded in age group of above 40 years. The overall prevalence of human cytomegalovirus (HCMV) infection in 16 ..... genome revisited: Comparison with the chimpanzee cytomegalovirus genome. J. Gen. Virol.

  15. Sex Determination in Ostrich (Struthio camelus) Using PCR Technique

    OpenAIRE

    M Alipanah; .H Taghavi; A Torkamanzehi; M Yosefelahi

    2011-01-01

    The important of ostrich breeding have been increased in the Iran as other country around the world. Lack of sexual dimorphism in many species of birds including Ostrich (Struthio camelus), making it difficult to differentiate between males and females, especially in young birds. This can be problematic for breeding programs. Blood samples from randomly chicks in the first days were collected and then DNA was extracted from blood. For sex typing used PCR by OSFES primer that produced a 423 fr...

  16. Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques

    Directory of Open Access Journals (Sweden)

    PAULO Z. PASSAMANI

    2017-12-01

    Full Text Available ABSTRACT Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS, micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR. Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

  17. Protocol for chromosome-specific probe construction using PRINS, micromanipulation and DOP-PCR techniques.

    Science.gov (United States)

    Passamani, Paulo Z; Carvalho, Carlos R; Soares, Fernanda A F

    2018-01-01

    Chromosome-specific probes have been widely used in molecular cytogenetics, being obtained with different methods. In this study, a reproducible protocol for construction of chromosome-specific probes is proposed which associates in situ amplification (PRINS), micromanipulation and degenerate oligonucleotide-primed PCR (DOP-PCR). Human lymphocyte cultures were used to obtain metaphases from male and female individuals. The chromosomes were amplified via PRINS, and subcentromeric fragments of the X chromosome were microdissected using microneedles coupled to a phase contrast microscope. The fragments were amplified by DOP-PCR and labeled with tetramethyl-rhodamine-5-dUTP. The probes were used in fluorescent in situ hybridization (FISH) procedure to highlight these specific regions in the metaphases. The results show one fluorescent red spot in male and two in female X chromosomes and interphase nuclei.

  18. Allele dropout caused by a non-primer-site SNV affecting PCR amplification--a call for next-generation primer design algorithm.

    Science.gov (United States)

    Lam, Ching-wan; Mak, Chloe Miu

    2013-06-05

    PCR-based technology is indispensable for genetic diagnosis. On the other hand, allele dropout is one significant cause of genotyping errors. Most allele dropout mechanisms are related to annealing failure caused by single nucleotide variant (SNV) situated inside the primer sequences. Here, we demonstrate a novel allele dropout mechanism caused by a non-primer-binding-site SNV. We demonstrate that the apparent homozygosity of NM_000137.1(FAH):c.1035_1037del was caused by allele dropout. The non-primer-binding-site SNV causes a strong secondary hairpin structure formation of the PCR products and leads to amplification failure. SNV check of the primer sequences per se during primer design is not adequate to avoid allele dropout. The next-generation primer design software should analyze the secondary structure of primers and template sequence taking SNV in both sequences into account in order to avoid genotyping errors. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman, Johan Frederik; Pierik, A.

    2012-01-01

    Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the

  20. Identification and differentiation of the twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

    Directory of Open Access Journals (Sweden)

    Narender S Maan

    Full Text Available Bluetongue (BT is an arthropod-borne viral disease, which primarily affects ruminants in tropical and temperate regions of the world. Twenty six bluetongue virus (BTV serotypes have been recognised worldwide, including nine from Europe and fifteen in the United States. Identification of BTV serotype is important for vaccination programmes and for BTV epidemiology studies. Traditional typing methods (virus isolation and serum or virus neutralisation tests (SNT or VNT are slow (taking weeks, depend on availability of reference virus-strains or antisera and can be inconclusive. Nucleotide sequence analyses and phylogenetic comparisons of genome segment 2 (Seg-2 encoding BTV outer-capsid protein VP2 (the primary determinant of virus serotype were completed for reference strains of BTV-1 to 26, as well as multiple additional isolates from different geographic and temporal origins. The resulting Seg-2 database has been used to develop rapid (within 24 h and reliable RT-PCR-based typing assays for each BTV type. Multiple primer-pairs (at least three designed for each serotype were widely tested, providing an initial identification of serotype by amplification of a cDNA product of the expected size. Serotype was confirmed by sequencing of the cDNA amplicons and phylogenetic comparisons to previously characterised reference strains. The results from RT-PCR and sequencing were in perfect agreement with VNT for reference strains of all 26 BTV serotypes, as well as the field isolates tested. The serotype-specific primers showed no cross-amplification with reference strains of the remaining 25 serotypes, or multiple other isolates of the more closely related heterologous BTV types. The primers and RT-PCR assays developed in this study provide a rapid, sensitive and reliable method for the identification and differentiation of the twenty-six BTV serotypes, and will be updated periodically to maintain their relevance to current BTV distribution and

  1. DNA Amplification Techniques for the Detection ofToxoplasma gondiiTissue Cysts in Meat Producing Animals: A Narrative Review Article.

    Science.gov (United States)

    Riaz, Farooq; Rashid, Imran; Akbar, Haroon; Shehzad, Wasim; Islam, Saher; Bajwa, Amna Arshad; Saeed, Khalid; Ashraf, Kamran

    2016-01-01

    Toxoplasma gondii is an intracellular parasite, which infects one-third population of world. Humans and animals acquire infection by ingesting oocytes from feces of cats or by meat of other animals having cysts that may lead to congenital, ocular or cephalic toxoplasmosis. Either it is important to detect T. gondii from meat of food animals from retail shops or directly at slaughterhouses, which is meant for export. The current research was done without time limitation using such terms as follows: " Toxoplasma gondii ", "Meat", "Tissue cyst", "PCR", "LAMP", "Screening" and "Immunological assay" alone or in combination, in English language. The used electronic databases for searching included as follows: Pub-Med, Scopus, Google Scholar, Web of Science and Science Direct. The searches were limited to the published papers to English language. Sensitivity of different molecular techniques for diagnosis of Toxoplasma is real-time PCR > LAMP > conventional PCR. In addition to these DNA analysis tools, bioassay in mice and cats is considered as "gold standard" to detect T. gondii . This review article will help the readers for grasping advantages and limitations of different diagnostic tools for screening meat samples for T. gondii . This review also makes bibliography about the type of meat sample to be processed for diagnosis and different primers or sequences to be targeted for T. gondii by number of researches for its detection from meat or tissue sample using DNA amplification techniques.

  2. Isolation of epithelial cells from acrylic removable dentures and gender identification by amplification of SRY gene using real time PCR.

    Science.gov (United States)

    George, Renjith; Sriram, G; Saraswathi, Tr; Sivapathasundharam, B

    2010-01-01

    This study evaluates the usefulness of acrylic dentures as the source of DNA for forensic analysis. Thirty-eight samples (21 males and 17 females) were collected and stored for different time periods. The epithelial cells adhered to the dentures were retrieved and the genomic DNA was extracted. All the samples yielded sufficient amount of DNA for analysis irrespective of the storage time. Gender determination was done by amplification of the sex determining region on the Y chromosome (SRY) using real-time polymerase chain reaction with 100% accuracy, within minimal time. With this study, we conclude that saliva-stained acrylic dentures can act as a source of forensic DNA and co-amplification of SRY gene with other routine sex typing markers will give unambiguous gender identification.

  3. Modified Polyadenylation-Based RT-qPCR Increases Selectivity of Amplification of 3′-MicroRNA Isoforms

    Directory of Open Access Journals (Sweden)

    Charlotte Nejad

    2018-01-01

    Full Text Available MicroRNA (miRNA detection by reverse transcription (RT quantitative real-time PCR (RT-qPCR is the most popular method currently used to measure miRNA expression. Although the majority of miRNA families are constituted of several 3′-end length variants (“isomiRs”, little attention has been paid to their differential detection by RT-qPCR. However, recent evidence indicates that 3′-end miRNA isoforms can exhibit 3′-length specific regulatory functions, underlining the need to develop strategies to differentiate 3′-isomiRs by RT-qPCR approaches. We demonstrate here that polyadenylation-based RT-qPCR strategies targeted to 20–21 nt isoforms amplify entire miRNA families, but that primers targeted to >22 nt isoforms were specific to >21 nt isoforms. Based on this observation, we developed a simple method to increase selectivity of polyadenylation-based RT-qPCR assays toward shorter isoforms, and demonstrate its capacity to help distinguish short RNAs from longer ones, using synthetic RNAs and biological samples with altered isomiR stoichiometry. Our approach can be adapted to many polyadenylation-based RT-qPCR technologies already exiting, providing a convenient way to distinguish long and short 3′-isomiRs.

  4. Development of an internal amplification control system for a real-time PCR assay for detection of Neisseria meningitidis in CSF and EDTA blood.

    Science.gov (United States)

    McIver, Christopher J; Bell, Sydney M; Er, Noel

    2014-06-01

    The aim of this study was to assemble and assess a non-competitive internal amplification control (IAC) system targeting the Escherichia coli alanine racemase (alr) gene to include in a real-time polymerase chain reaction (PCR) assay for Neisseria meningitidis. Primers and hybridisation probes specific for the IAC were designed and assessed for specificity. Amplification efficiency and limit of detection for the assembled assay was extrapolated using standard curves constructed with serial dilutions of N. meningitidis in saline, pooled cerebrospinal fluid (CSF) and EDTA blood. The 95% confidence limits (CI) were calculated for IAC crossing-points recorded for assays for N. meningitidis ctrA in saline (negative blank), and N. meningitides-negative samples of CSF and EDTA blood. These limits served as a reference range against which the IAC crossing-points recorded for prospective assays are compared to detect sample inhibition. This system was used in testing consecutive EDTA blood samples from two cases of meningococcal disease. The IAC system is specific for Escherichia coli and Shigella species. The amplification efficiency of the assembled assay for N. meningitidis and ability to detect low target DNA levels was not compromised with the inclusion of the IAC system. The IAC crossing-points varied in clinical samples of CSF and EDTA blood. The elucidated reference range for EDTA blood was used to detect sample inhibition in one of the two clinical cases investigated.The IAC system monitors the performance of all processes in the assembled assay for N. meningitidis. Measuring IAC crossing-points serves as an indicator of sample stability and inhibitory properties when testing single or multiple samples from the same patient. Specificity for E. coli and Shigella species enables inclusion in assays of different targets within the same laboratory. Reporting PCR assay results in the context of the IAC crossing-points and reference ranges validates against sample

  5. A prospective study of two isothermal amplification assays compared with real-time PCR, CCNA and toxigenic culture for the diagnosis of Clostridium difficile infection.

    Science.gov (United States)

    Neuendorf, Martina; Guadarrama-Gonzalez, Raquel; Lamik, Birgit; MacKenzie, Colin R

    2016-02-12

    New molecular methods of detecting Clostridium difficile infection (CDI) provide the routine lab with a sensitive random access method to produce results that are available in a shorter time than traditional methods. In this prospective study a total of 989 stool specimens were tested over a period of 16 months in parallel using two isothermal amplification assays, AmpliVue® (Quidel) and Illumigene® (Meridian) and the results compared to those from toxigenic culture. In addition all specimens were tested using a cytotoxic cell neutralisation assay (CCNA) and three different Real-time PCR targeting a C. difficile-specific 16S rDNA sequence or the toxin genes tcdA, tcdB/tcdB027 or cdtB. AmpliVue® was positive in 242 (24.5%) and Illumigene® in 228 (23.1%) specimens. 167 (16.9%) specimens were positive in toxigenic culture. Real-time-tcdA and -tcdB PCR was positive in 211 (21.3%) specimens, Real-time-cdtB PCR was positive in 101 (10.2%) specimens and C. difficile-PCR (16S rDNA) in 267 (27.0%) specimens. The respective sensitivity, specificity, positive predictive value and negative predictive value compared to toxigenic culture were 91, 89, 62 and 98% for AmpliVue® and 91, 91, 67 and 98% for Illumigene®.

  6. Isolation of epithelial cells from acrylic removable dentures and gender identification by amplification of SRY gene using real time PCR

    OpenAIRE

    Renjith George; G Sriram; T R Saraswathi; B Sivapathasundharam

    2010-01-01

    This study evaluates the usefulness of acrylic dentures as the source of DNA for forensic analysis. Thirty-eight samples (21 males and 17 females) were collected and stored for different time periods. The epithelial cells adhered to the dentures were retrieved and the genomic DNA was extracted. All the samples yielded sufficient amount of DNA for analysis irrespective of the storage time. Gender determination was done by amplification of the sex determining region on the Y chromosome (SRY) us...

  7. Development of real-time PCR and loop-mediated isothermal amplification (LAMP assays for the differential detection of digital dermatitis associated treponemes.

    Directory of Open Access Journals (Sweden)

    Kelly Anklam

    Full Text Available Bovine digital dermatitis (DD is a severe infectious cause of lameness in cattle worldwide, with important economic and welfare consequences. There are three treponeme phylogroups (T. pedis, T. phagedenis, and T. medium that are implicated in playing an important causative role in DD. This study was conducted to develop real-time PCR and loop-mediated isothermal amplification (LAMP assays for the detection and differentiation of the three treponeme phylogroups associated with DD. The real-time PCR treponeme phylogroup assays targeted the 16S-23S rDNA intergenic space (ITS for T. pedis and T. phagedenis, and the flagellin gene (flaB2 for T. medium. The 3 treponeme phylogroup LAMP assays targeted the flagellin gene (flaB2 and the 16S rRNA was targeted for the Treponeme ssp. LAMP assay. The real-time PCR and LAMP assays correctly detected the target sequence of all control strains examined, and no cross-reactions were observed, representing 100% specificity. The limit of detection for each of the three treponeme phylogroup real-time PCR and LAMP assays was ≤ 70 fg/μl. The detection limit for the Treponema spp. LAMP assay ranged from 7-690 fg/μl depending on phylogroup. Treponemes were isolated from 40 DD lesion biopsies using an immunomagnetic separation culture method. The treponeme isolation samples were then subjected to the real-time PCR and LAMP assays for analysis. The treponeme phylogroup real-time PCR and LAMP assay results had 100% agreement, matching on all isolation samples. These results indicate that the developed assays are a sensitive and specific test for the detection and differentiation of the three main treponeme phylogroups implicated in DD.

  8. Development of loop-mediated isothermal amplification and PCR assays for rapid and simple detection of Campylobacter fetus subsp. venerealis.

    Science.gov (United States)

    Yamazaki, Wataru; Taguchi, Masumi; Misawa, Naoaki

    2010-07-01

    Campylobacter fetus is divided into CFV and CFF. Because CFV causes bovine genital campylobacteriosis, differentiation of the two subspecies is essential to the implementation of efficient CFV control and eradication programs. We have developed LAMP and duplex PCR assays for rapid and simple detection of CFV. The LAMP assay correctly detected 7 CFV strains and did not detect 53 CFF, 35 non-fetus Campylobacter and 25 non-Campylobacter strains. The PCR assay successfully differentiated the two subspecies. The LAMP and PCR assays were faster than conventional biochemical assays, requiring for detection less than 50 min and less than 4 hr, respectively, from the beginning of DNA extraction from a single colony on blood agar to final determination. Our LAMP and PCR assays are rapid and practical tools for detection of CFV.

  9. DNA polymorphism of butyrophilin gene by PCR-RFLP technique ...

    African Journals Online (AJOL)

    We used the polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) technique to screen for DNA polymorphism in 109 cattle. In all cattle, we amplified an 863 fragment consisting of part of exon 8. The amplified fragment digested with HaeIII restriction endonuclease and subjected to electrophoretic ...

  10. Direct PCR amplification of DNA from human bloodstains, saliva, and touch samples collected with microFLOQ® swabs.

    Science.gov (United States)

    Ambers, Angie; Wiley, Rachel; Novroski, Nicole; Budowle, Bruce

    2018-01-01

    Previous studies have shown that nylon flocked swabs outperform traditional fiber swabs in DNA recovery due to their innovative design and lack of internal absorbent core to entrap cellular materials. The microFLOQ ® Direct swab, a miniaturized version of the 4N6 FLOQSwab ® , has a small swab head that is treated with a lysing agent which allows for direct amplification and DNA profiling from sample collection to final result in less than two hours. Additionally, the microFLOQ ® system subsamples only a minute portion of a stain and preserves the vast majority of the sample for subsequent testing or re-analysis, if desired. The efficacy of direct amplification of DNA from dilute bloodstains, saliva stains, and touch samples was evaluated using microFLOQ ® Direct swabs and the GlobalFiler™ Express system. Comparisons were made to traditional methods to assess the robustness of this alternate workflow. Controlled studies with 1:19 and 1:99 dilutions of bloodstains and saliva stains consistently yielded higher STR peak heights than standard methods with 1ng input DNA from the same samples. Touch samples from common items yielded single source and mixed profiles that were consistent with primary users of the objects. With this novel methodology/workflow, no sample loss occurs and therefore more template DNA is available during amplification. This approach may have important implications for analysis of low quantity and/or degraded samples that plague forensic casework. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Concordance between nucleic acid amplification technique and culture for the diagnosis of gonorrhoea.

    Science.gov (United States)

    Creighton, S; Revell, B; Barrow, A

    2009-05-01

    The objective of the study was to evaluate the concordance between nucleic acid amplification technique (NAAT) and culture for the diagnosis of Neisseria gonorrhoeae among attendees at a genitourinary medicine clinic in East London. All patients testing positive for N. gonorrhoeae on NAAT and/or culture between 1 April 2007 and 31 August 2008 at the Department of Sexual Health at Homerton University Hospital were included. Male patients had a first void urine sample for NAAT and urethral culture; female patients had a self-taken vulval swab or endocervical sample sent for NAAT and an endocervical culture sample. After interim analysis, discrepant results had both NAAT and culture repeated prior to treatment. Of 159 male patients with a positive NAAT, 22 (13%) had a negative culture. Among 135 female patients with a positive NAAT, 36 (27%) had a negative culture. Three men had a positive culture and negative NAAT. Nineteen of the discrepant samples were retested prior to treatment and 12 (63%) had spontaneously revered to negative. In conclusion, there was concordance in 84% of male and 67% of female samples. In two-thirds of the discrepant cases, the previously positive NAAT had become negative prior to treatment. This study highlights the importance of consideration of the clinical picture when assessing results.

  12. The detection of Yersinia enterocolitica in surface water by quantitative PCR amplification of the ail and yadA genes.

    Science.gov (United States)

    Cheyne, Bo M; Van Dyke, Michele I; Anderson, William B; Huck, Peter M

    2010-09-01

    Yersinia enterocolitica has been detected in surface water, and drinking untreated water is a risk factor for infection. PCR-based methods have been used to detect Y. enterocolitica in various sample types, but quantitative studies have not been conducted in water. In this study, quantitative PCR (qPCR)-based methods targeting the Yersinia virulence genes ail and yadA were used to survey the Grand River watershed in southern Ontario, Canada. Initial testing of reference strains showed that ail and yadA PCR assays were specific for pathogenic biotypes of Y. enterocolitica; however the genes were also detected in one clinical Yersinia intermedia isolate. A survey of surface water from the Grand River watershed showed that both genes were detected at five sampling locations, with the ail and yadA genes detected in 38 and 21% of samples, respectively. Both genes were detected more frequently at colder water temperatures. A screening of Yersinia strains isolated from the watershed showed that the ail gene was detected in three Y. enterocolitica 1A/O:5 isolates. Results of this study show that Yersinia virulence genes were commonly detected in a watershed used as a source of drinking water, and that the occurrence of these genes was seasonal.

  13. Specific and sensitive detection of Ralstonia solanacearum in soil on the basis of PCR amplification of fliC fragments

    NARCIS (Netherlands)

    Schonfeld, J.; Heuer, H.; Elsas, van J.D.; Smalla, K.

    2003-01-01

    Ralstonia solanacearum is the causative agent of bacterial wilt in many important crops. A specific and sensitive PCR detection method that uses primers targeting the gene coding for the flagella subunit, fliC, was established. Based on the first fliC gene sequence of R. solanacearum strain K60

  14. Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification: A Novel Analytically Rapid, Sensitive, Multiplex Loop-Mediated Isothermal Amplification Detection Technique.

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Lan, Ruiting; Xu, Huaqing; Ma, Aijing; Li, Dongxun; Dai, Hang; Yuan, Xuejiao; Xu, Jianguo; Ye, Changyun

    2015-07-01

    Loop-mediated isothermal amplification (LAMP) is restricted to detecting a single target, limiting the usefulness of this method. To achieve multiplex LAMP-based detection, we developed a novel approach we called the multiple endonuclease restriction real-time-LAMP assay. In this system, the LAMP forward or backward inner primers contain 5' end short sequences that are recognized by the restriction endonuclease Nb.BsrDI, and the new forward or backward inner primers were modified at the 5' end with a fluorophore and in the middle with a dark quencher. Nb.BsrDI digests the newly synthesized double-stranded terminal sequences (5' end short sequences and their complementary sequences), which releases the quenching, resulting in a gain of signal. The assay permitted real-time detection of single or multiple target sequences in a single tube, and the positive results can be obtained in as short as 12 minutes. The novel methodology is highly efficient and specific, detecting down to 250 fg of DNA per reaction of Listeria DNA tested, and was successful in evaluating raw meat samples. The multiple endonuclease restriction real-time-LAMP technology, which is an extension of LAMP to accommodate robust, target-specific, and multiplex detection, provides a molecular diagnostic tool with less detection time and high sensitivity and specificity compared with those of LAMP and quantitative real-time PCR. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  15. Underestimation of ammonia-oxidizing bacteria abundance by amplification bias in amoA-targeted qPCR

    DEFF Research Database (Denmark)

    Dechesne, Arnaud; Musovic, Sanin; Palomo, Alejandro

    2016-01-01

    Molecular methods to investigate functional groups in microbial communities rely on the specificity and selectivity of the primer set towards the target. Here, using rapid sand filters for drinking water production as model environment, we investigated the consistency of two commonly used quantit......RNA. In contrast, both approaches performed very similarly at waterworks with high Cluster 7 prevalence. Our results highlight that caution is warranted when comparing AOB abundances obtained using different qPCR primer sets....

  16. Risk Minimization Measures for Blood Screening HIV-1 Nucleic Acid Amplification Technique Assays in Germany

    Science.gov (United States)

    Chudy, Michael; Kress, Julia; Halbauer, Jochen; Heiden, Margarethe; Funk, Markus B.; Nübling, C. Micha

    2014-01-01

    Summary Background Several publications describe HIV-1 RNA false-negative results or viral load underquantitation associated with Communauté Européenne(CE)-marked qualitative or quantitative nucleic acid amplification technique (NAT) assays. 6 cases occurred during blood screening in Germany, with 2 of them causing HIV-1 transmissions to recipients of blood components. The implicated NAT assays were mono-target assays amplifying in different viral genome regions (gag or long terminal repeat). Methods Specimens characterized by HIV-1 NAT underquantitation or false-negative NAT results were comparatively investigated in CE-marked HIV-1 NAT systems of different design to identify potential reasons. The target regions of the viral nucleic acids were sequenced and these sequences compared to primers and probes of the assays. Potential risk minimization measures were considered for quantitative and blood-screening HIV-1 NAT systems. Results Nucleotide sequencing of the viral target region in cases of HIV-1 RNA underquantitation or false-negative test results revealed new HIV-1 variants that were mismatched with primers and probes used in some mono-target assays. So far, dualtarget NAT assays have not been associated with mismatch-based false-negative test results. From 2015, the Paul Ehrlich Institute will request HIV-1 NAT assays of dual-target design or an analogous solution for further reducing the risk in blood screening. Conclusion HIV differs from other blood-borne viruses with regard to its fast evolution of new viral variants. The evolution of new sequences is hardly predictable; therefore, NAT assays with only 1 target region appear to be more vulnerable to sequence variations than dual-target assays. The associated risk may be higher for HIV-1 NAT assays used for blood screening compared to quantitative assays used for monitoring HIV-1-infected patients. In HIV-1 screening NAT assays of dual-target design may adequately address the risk imposed by new HIV-1

  17. Absolute quantification of Bovine Viral Diarrhea Virus (BVDV) RNA by the digital PCR technique

    International Nuclear Information System (INIS)

    Flatschart, R B; Almeida, D O; Medeiros, M N; Granjeiro, J M; Folgueras-Flatschart, A V; Heinemann, M B

    2015-01-01

    The quality control of cell lines used in research and industry is critical to ensure confidence in experimental results and to guarantee the safety of biopharmaceuticals to consumers. The BVDV is a common adventitious agent in many cell lines. We preliminarly evaluate the use of Digital Droplet PCR (ddPCR) for the detection and enumeration of genome copies of BVDV in cell culture and on FBS. The application of a commercial Real-Time PCR kit with the ddPCR technique was successful on different matrices. The technique allowed the absolute quantification of the genome without the use of calibration standards, suggesting its promising application on the development of reference materials for quantification of nucleic acids

  18. Absolute quantification of Bovine Viral Diarrhea Virus (BVDV) RNA by the digital PCR technique

    Science.gov (United States)

    Flatschart, R. B.; Almeida, D. O.; Heinemann, M. B.; Medeiros, M. N.; Granjeiro, J. M.; Folgueras-Flatschart, A. V.

    2015-01-01

    The quality control of cell lines used in research and industry is critical to ensure confidence in experimental results and to guarantee the safety of biopharmaceuticals to consumers. The BVDV is a common adventitious agent in many cell lines. We preliminarly evaluate the use of Digital Droplet PCR (ddPCR) for the detection and enumeration of genome copies of BVDV in cell culture and on FBS. The application of a commercial Real-Time PCR kit with the ddPCR technique was successful on different matrices. The technique allowed the absolute quantification of the genome without the use of calibration standards, suggesting its promising application on the development of reference materials for quantification of nucleic acids.

  19. Strategy for Extracting DNA from Clay Soil and Detecting a Specific Target Sequence via Selective Enrichment and Real-Time (Quantitative) PCR Amplification

    Science.gov (United States)

    Yankson, Kweku K.; Steck, Todd R.

    2009-01-01

    We present a simple strategy for isolating and accurately enumerating target DNA from high-clay-content soils: desorption with buffers, an optional magnetic capture hybridization step, and quantitation via real-time PCR. With the developed technique, μg quantities of DNA were extracted from mg samples of pure kaolinite and a field clay soil. PMID:19633108

  20. Quantification of alpha-tubulin isotypes by sandwich ELISA with signal amplification through biotinyl-tyramide or immuno-PCR

    Czech Academy of Sciences Publication Activity Database

    Dráberová, Eduarda; Stegurová, Lucie; Sulimenko, Vadym; Hájková, Zuzana; Dráber, Petr; Dráber, Pavel

    2013-01-01

    Roč. 395, 1-2 (2013), s. 63-70 ISSN 0022-1759 R&D Projects: GA AV ČR KAN200520701; GA ČR GAP302/12/1673; GA ČR GPP302/11/P709; GA ČR GAP302/10/1759; GA ČR GA301/09/1826; GA MŠk(CZ) LD13015; GA MŠk LD12073 Institutional support: RVO:68378050 Keywords : alpha-tubulin isotypes * biotinyl-tyramide * ELISA * immuno-PCR * mast cells Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.005, year: 2013

  1. PCR amplification, cloning, and construction of HIV-1 infectious molecular clones from virtually full-length HIV-1 genomes.

    Science.gov (United States)

    Ehrenberg, Philip K; Michael, Nelson L

    2005-01-01

    The development of mixtures of highly processive and high-fidelity thermostable DNA polymerases has enabled the routine recovery of DNA sequences in excess of 25 kb generated by polymerase chain reaction. This powerful tool has been instrumental in the ability to recover virtually full-length HIV-1 proviral DNA as a single, contiguous fragment. Such fragments allow for the clean interpretation of the genomic organization of HIV-1 provirus, as they are not confounded by molecular mosaicism that accrues to overlapping subgenomic amplification strategies. We detail here a robust procedure to produce virtually full-length, single contiguous 9.2-kb HIV-1 amplimers whose full-length infectious potential is reconstituted upon cloning into long terminal repeat-replacement vectors. Large numbers of HIV-1 proviral clones can now be quickly generated and screened to identify the fraction of the viral quasispecies with the highest capacity for viral replication. The methods used to construct long terminal repeat-replacement vectors, amplify HIV-1 provirus, reconstitute full-length provirus, and recover viral stocks will be illustrated using a circulating recombinant form 1 (CRF01_AE, formerly known as subtype E) primary isolate.

  2. Multiplex PMA-qPCR Assay with Internal Amplification Control for Simultaneous Detection of Viable Legionella pneumophila, Salmonella typhimurium, and Staphylococcus aureus in Environmental Waters.

    Science.gov (United States)

    Li, Haiyan; Xin, Hongyi; Li, Sam Fong Yau

    2015-12-15

    Pathogenic microorganisms are responsible for many infectious diseases, and pathogen monitoring is important and necessary for water quality control. This study for the first time explored a multiplex quantitative real-time PCR (qPCR) technique combined with propidium monoazide (PMA) to simultaneously detect viable Legionella pneumophila, Salmonella typhimurium, and Staphylococcus aureus in one reaction from water samples. Sodium lauroyl sarcosinate (sarkosyl) was applied to enhance the dead bacterial permeability of PMA. The sensitivity of the multiplex PMA-qPCR assay achieved two colony-forming units (CFU) per reaction for L. pneumophila and three CFU per reaction for S. typhimurium and S. aureus. No PCR products were amplified from all nontarget control samples. Significantly, with comparable specificity and sensitivity, this newly invented multiplex PMA-qPCR assay took a much shorter time than did conventional culture assays when testing water samples with spiked bacteria and simulated environmental water treatment. The viable multiplex PMA-qPCR assay was further successfully applied to pathogen detection from rivers, canals, and tap water samples after simple water pretreatment.

  3. A study on the determination of risk factors associated with babesiosis and prevalence of Babesia sp., by PCR amplification, in small ruminants from Southern Punjab (Pakistan

    Directory of Open Access Journals (Sweden)

    Iqbal F.

    2011-08-01

    Full Text Available Babesiosis is a parasitic infection due to the multiplication of tick borne parasite, Babesia sp., in erythrocytes of host, which includes a wide variety of vertebrates including small ruminants causing decreased livestock output and hence economic losses. The objective of the present study was to establish a PCR based method for the detection of Babesia sp. in small ruminant population in Southern Punjab and to determine the risk factors involve in the spread of babesiosis. A total of 107 blood samples were collected from 40 sheep and 67 goats in seven districts of Southern Punjab from randomly selected herds. Data on the characteristics of the animals and the herd were collected through questionnaires. 36 blood samples (34% of total produced the DNA fragment specific for 18S rRNA gene of Babesia sp., by PCR amplification, of which 20 were sheep and 16 were goats. Samples from all seven district contained Babesia positive samples and prevalence varied between 18 to 68%. It was observed that male animals (P = 0.009 and young animals under one year of age (P = 0.01 were more prone to the parasite. It was observed that herds consist of more than 15 animals (P = 0.007, composed of mixed species of small ruminants (P = 0.022, associated with dogs (P = 0.003 and dogs having ticks on their bodies (P = 0.011 were among the major risk factors for the spread of babesiosis in small ruminants.

  4. A study on the determination of risk factors associated with babesiosis and prevalence of Babesia sp., by PCR amplification, in small ruminants from Southern Punjab (Pakistan).

    Science.gov (United States)

    Iqbal, F; Fatima, M; Shahnawaz, S; Naeem, M; Shaikh, Rs; Ali, M; Shaikh, As; Aktas, M; Ali, M

    2011-08-01

    Babesiosis is a parasitic infection due to the multiplication of tick borne parasite, Babesia sp., in erythrocytes of host, which includes a wide variety of vertebrates including small ruminants causing decreased livestock output and hence economic losses. The objective of the present study was to establish a PCR based method for the detection of Babesia sp. in small ruminant population in Southern Punjab and to determine the risk factors involve in the spread of babesiosis. A total of 107 blood samples were collected from 40 sheep and 67 goats in seven districts of Southern Punjab from randomly selected herds. Data on the characteristics of the animals and the herd were collected through questionnaires. 36 blood samples (34% of total) produced the DNA fragment specific for 18S rRNA gene of Babesia sp., by PCR amplification, of which 20 were sheep and 16 were goats. Samples from all seven district contained Babesia positive samples and prevalence varied between 18 to 68%. It was observed that male animals (P = 0.009) and young animals under one year of age (P = 0.01) were more prone to the parasite. It was observed that herds consist of more than 15 animals (P = 0.007), composed of mixed species of small ruminants (P = 0.022), associated with dogs (P = 0.003) and dogs having ticks on their bodies (P = 0.011) were among the major risk factors for the spread of babesiosis in small ruminants.

  5. Immuno-PCR, a new technique for the serodiagnosis of tuberculosis.

    Science.gov (United States)

    Mehta, Promod K; Dahiya, Bhawna; Sharma, Suman; Singh, Netrapal; Dharra, Renu; Thakur, Zoozeal; Mehta, Neeru; Gupta, Krishna B; Gupta, Mahesh C; Chaudhary, Dhruva

    2017-08-01

    Rapid and accurate diagnosis of tuberculosis (TB) is essential to control the disease. The conventional microbiological tests have limitations and there is an urgent need to devise a simple, rapid and reliable point-of-care (POC) test. The failure of TB diagnostic tests based on antibody detection due to inconsistent and imprecise results has stimulated renewed interest in the development of rapid antigen detection methods. However, the World Health Organization (WHO) has emphasized to continue research for designing new antibody-based detection tests with improved accuracy. Immuno-polymerase chain reaction (I-PCR) combines the simplicity and versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification capacity and sensitivity of PCR thus leading to several-fold increase in sensitivity in comparison to analogous ELISA. In this review, we have described the serodiagnostic potential of I-PCR assays for an early diagnosis of TB based on the detection of potential mycobacterial antigens and circulating antibodies in body fluids of TB patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Multiplex PCR-based simultaneous amplification of selectable marker and reporter genes for the screening of genetically modified crops.

    Science.gov (United States)

    Randhawa, Gurinder Jit; Chhabra, Rashmi; Singh, Monika

    2009-06-24

    The development and commercialization of genetically modified (GM) crops with enhanced insect and herbicide resistance, abiotic stress tolerance, and improved nutritional quality has expanded dramatically. Notwithstanding the huge potential benefits of GM crops, the perceived environmental risks associated with these crops need to be addressed in proper perspective. One critical concern is the adventitious presence or unintentional mixing of GM seed in non-GM seed lots, which can seriously affect the global seed market. It would therefore be necessary though a challenging task to develop reliable, efficient, and economical assays for GM detection, identification, and quantification in non-GM seed lots. This can be systematically undertaken by preliminary screening for control elements and selectable or scorable (reporter) marker genes. In this study, simplex and multiplex polymerase chain reaction (PCR) assays individually as well as simultaneously amplifying the commonly used selectable marker genes, i.e., aadA, bar, hpt, nptII, pat encoding, respectively, for aminoglycoside-3'-adenyltransferase, Streptococcus viridochromogenes phosphinothricin-N-acetyltransferase, hygromycin phosphotransferase, neomycin phosphotransferase, Streptococcus hygroscopicus phosphinothricin-N-acetyltransferase, and a reporter gene uidA encoding beta-d-glucuronidase, were developed as a reliable tool for qualitative screening of GM crops. The efficiency of the assays was also standardized in the test samples prepared by artificial mixing of transgenic seed samples in different proportions. The developed multiplex PCR assays will be useful in verifying the GM status of a sample irrespective of the crop and GM trait.

  7. HPV in oral squamous cell carcinomas of a Brazilian population: amplification by PCR HPV em carcinoma epidermóide de boca em população brasileira: amplificação por PCR

    Directory of Open Access Journals (Sweden)

    Elena Riet Correa Rivero

    2006-03-01

    Full Text Available Human Papilomaviruses (HPV are a group of viruses associated with benign and malignant lesions of cutaneous and mucosal epithelia. Some "high risk" HPV types, especially HPV 16 and 18, are strongly correlated with cervical and anogenital cancers and are also related to the genesis of oral squamous cell carcinomas (OSCC. The aim of this work was to investigate the incidence of HPV infection in 40 paraffin-embedded or fresh specimens of OSCC, using PCR amplification of the viral DNA. Literature based primers (GP5+/GP6+ were used in order to amplify HPV DNA from the L1 gene, present in more than 22 types of HPV. A condyloma case with HPV 16 and 18 detected by in situ hybridization was used as a positive control. Amplification of HPV was observed only in the positive control. No squamous cell carcinoma cases showed DNA viral amplification. Absence of HPV DNA amplification by PCR in the analyzed specimens of OSCCs suggests that this virus not always plays a role in the carcinogenesis process. Discrepancy with some studies found in the literature may be related to methodology or population differences.O papilomavírus humano (HPV constitui um grupo de vírus associados com lesões benignas e malignas do epitélio da pele e das mucosas. Alguns tipos de HPV, ditos oncogênicos, especialmente os HPVs 16 e 18, estão fortemente correlacionados com os cânceres cervical e anogenital, e também têm sido implicados na gênese do carcinoma epidermóide de boca. O objetivo deste estudo foi investigar a incidência de infecção pelo HPV, em 40 casos de carcinoma epidermóide de boca emblocados ou não em parafina, utilizando-se para isso a amplificação do DNA viral por PCR. Foram utilizados os iniciadores GP5+/GP6+, que amplificam parte da região L1 do vírus, comum a mais de 22 tipos de HPVs. Como controle positivo para as reações, foi utilizado um caso de condiloma, o qual foi positivo para os HPVs 16 e 18 na hibridização in situ. A amplifica

  8. An Undergraduate Laboratory Experiment for Upper-Level Forensic Science, Biochemistry, or Molecular Biology Courses: Human DNA Amplification Using STR Single Locus Primers by Real-Time PCR with SYBR Green Detection

    Science.gov (United States)

    Elkins, Kelly M.; Kadunc, Raelynn E.

    2012-01-01

    In this laboratory experiment, real-time polymerase chain reaction (real-time PCR) was conducted using published human TPOX single-locus DNA primers for validation and various student-designed short tandem repeat (STR) primers for Combined DNA Index System (CODIS) loci. SYBR Green was used to detect the amplification of the expected amplicons. The…

  9. Assessing HER2 amplification by IHC, FISH, and real-time polymerase chain reaction analysis (real-time PCR) following LCM in formalin-fixed paraffin embedded tissue from 40 women with ovarian cancer

    DEFF Research Database (Denmark)

    Hillig, Thore; Thode, Jørgen; Breinholt, Ellen Marie

    2012-01-01

    We compare HER2 receptor amplification analysis by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and real-time polymerase chain reaction (real-time PCR) DNA copy-number assay following laser capture microdissection (LCM) in formalin-fixed paraffin embedded tissue from 40...

  10. 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.

    Science.gov (United States)

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

    2012-09-01

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

  11. Gene Amplification by PCR and Subcloning into a GFP-Fusion Plasmid Expression Vector as a Molecular Biology Laboratory Course

    Science.gov (United States)

    Bornhorst, Joshua A.; Deibel, Michael A.; Mulnix, Amy B.

    2004-01-01

    A novel experimental sequence for the advanced undergraduate laboratory course has been developed at Earlham College. Utilizing recent improvements in molecular techniques for a time-sensitive environment, undergraduates were able to create a chimera of a selected gene and green fluorescent protein (GFP) in a bacterial expression plasmid over the…

  12. Method for signal conditioning and data acquisition system, based on variable amplification and feedback technique

    International Nuclear Information System (INIS)

    Conti, Livio; Sgrigna, Vittorio; Zilpimiani, David; Assante, Dario

    2014-01-01

    An original method of signal conditioning and adaptive amplification is proposed for data acquisition systems of analog signals, conceived to obtain a high resolution spectrum of any input signal. The procedure is based on a feedback scheme of the signal amplification with aim at maximizing the dynamic range and resolution of the data acquisition system. The paper describes the signal conditioning, digitization, and data processing procedures applied to an a priori unknown signal in order to enucleate its amplitude and frequency content for applications in different environments: on the ground, in space, or in the laboratory. An electronic board of the conditioning module has also been constructed and described. In the paper are also discussed the main fields of application and advantages of the method with respect to those known today

  13. The evaluation of histo-blood group ABO typing by flow cytometric and PCR-amplification of specific alleles analyses and their application in clinical laboratories.

    Science.gov (United States)

    Aki, Kensaku; Izumi, Azusa; Hosoi, Eiji

    2012-01-01

    ABO antigens are oligosaccharide antigens, and are widely distributed on red blood and tissue cells as well as in saliva and body fluid. Therefore, these antigens are important not only for blood transfusion, but also for tissue cell and organ transplantations. Also, blood, hair, and seminal fluid are important sources of evidence at crime scenes, and these antigens are some of the most important markers for personal identification in forensic investigations. Here, we describe the development and use of quantitative analysis of A, B, and H antigens on red blood cells by employing flow cytometric analysis and the ABO genotyping method based on PCR-amplification of specific alleles (PASA) within DNA, especially from blood and saliva. In this study, flow cytometric analysis could be used to compare the differences between the expression of A and/or B and H antigens on red blood cells with various phenotypes, and the PASA method was able to determine the genotype of the type cisA(2)B(3) pedigree using only DNA extracted from saliva. These analysis methods are simple and useful for judging the ABO blood group system and genotyping, and are used widely throughout research and clinical laboratories and forensic fields.

  14. Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification

    Directory of Open Access Journals (Sweden)

    van Rotterdam Bart J

    2010-12-01

    Full Text Available Abstract Background Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. Results Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. Conclusions The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum

  15. Influence of selected stool concentration techniques on the effectiveness of PCR examination in Giardia intestinalis diagnostics.

    Science.gov (United States)

    Stojecki, K; Sroka, J; Karamon, J; Kusyk, P; Cencek, T

    2014-01-01

    Giardia intestinalis is a widespread parasitic protozoa which has great significance as a public health threat. Molecular diagnostics of stool sample can be unreliable because of the presence of inhibitors of enzymatic reactions. The aim of this study was to determine the effectiveness of selected pre-treatment methods of fecal samples for further PCR-based diagnostics of G. intestinalis, and the effect of each component of pre-treatment solutions on PCR reactions. Seven stool concentration techniques were compared. The results showed that the most efficient concentration method for stool sample preparation for detection of G. intestinalis by PCR is centrifugal flotation with Percoll (with saturated NaNO3 as the flotation solution). This method is relatively inexpensive, less labor-intensive, and suitable for epidemiological monitoring and clinical investigations.

  16. Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

    Science.gov (United States)

    Gille, Johan J. P.; Floor, Karijn; Kerkhoven, Lianne; Ameziane, Najim; Joenje, Hans; de Winter, Johan P.

    2012-01-01

    Fanconi anemia (FA) is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD) and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of FANCA, FANCB, FANCC, FANCE, FANCF, and FANCG and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed. PMID:22778927

  17. Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

    Directory of Open Access Journals (Sweden)

    Johan J. P. Gille

    2012-01-01

    Full Text Available Fanconi anemia (FA is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of FANCA, FANCB, FANCC, FANCE, FANCF, and FANCG and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed.

  18. Trypanosomosis in The Gambia: prevalence in working horses and donkeys detected by whole genome amplification and PCR, and evidence for interactions between trypanosome species

    Directory of Open Access Journals (Sweden)

    Jallow Jibril

    2008-02-01

    Full Text Available Abstract Background The Gambia has an increasing population of equidae largely used for agriculture and transportation. A review of cases at The Gambian Horse and Donkey Trust (GHDT indicated that a common reason for presentation is a poorly defined medical condition often attributed to trypanosomosis. There are few reports describing the prevalence or the range of clinical signs associated with infection with different species of trypanosomes in horses and donkeys, but given the importance of these animals, the role of trypanosomosis requires investigation. Results In total 241 animals from the Central River Division in The Gambia (183 horses and 58 donkeys were screened using Whole Genome Amplification (WGA followed by trypanosome species identification using polymerase chain reaction (PCR. The results indicated overall trypanosome prevalence of 91%; with an infection rate of 31% for Trypanosoma congolense Savannah, 87% for Trypanosoma vivax and 18% for Trypanosoma brucei sp. Multiple species were present in 43% of infections. Microscopy had a good specificity (100% and positive predictive value (100% for trypanosome detection, but the sensitivity (20% and negative predictive value (10.5% were low relative to PCR-based diagnosis. Infection with T congolense showed the greatest negative effect on packed cell volume (PCV, while infection with T. brucei sp also had a significant, although lesser, negative effect on PCV. In addition, cases positive by microscopy were associated with significantly lower PCV. However, concurrent infection with T. vivax appeared to cause less effect on PCV, compared to animals infected with T. congolense alone. Conclusion The prevalence of Trypanosomosis was high in both horses and donkeys. Infection with T. congolense appeared to have the greatest clinical significance, while T. vivax infection may be of limited clinical significance in this population. Indeed, there is evidence of T. vivax co-infection ameliorating

  19. Multiplex Ligation-Dependent Probe Amplification Technique for Copy Number Analysis on Small Amounts of DNA Material

    DEFF Research Database (Denmark)

    Sørensen, Karina; Andersen, Paal; Larsen, Lars

    2008-01-01

    The multiplex ligation-dependent probe amplification (MLPA) technique is a sensitive technique for relative quantification of up to 50 different nucleic acid sequences in a single reaction, and the technique is routinely used for copy number analysis in various syndromes and diseases. The aim...... of the study was to exploit the potential of MLPA when the DNA material is limited. The DNA concentration required in standard MLPA analysis is not attainable from dried blood spot samples (DBSS) often used in neonatal screening programs. A novel design of MLPA probes has been developed to permit for MLPA...... analysis on small amounts of DNA. Six patients with congenital adrenal hyperplasia (CAH) were used in this study. DNA was extracted from both whole blood and DBSS and subjected to MLPA analysis using normal and modified probes. Results were analyzed using GeneMarker and manual Excel analysis. A total...

  20. Comparison of whole genome amplification techniques for human single cell exome sequencing.

    Science.gov (United States)

    Borgström, Erik; Paterlini, Marta; Mold, Jeff E; Frisen, Jonas; Lundeberg, Joakim

    2017-01-01

    Whole genome amplification (WGA) is currently a prerequisite for single cell whole genome or exome sequencing. Depending on the method used the rate of artifact formation, allelic dropout and sequence coverage over the genome may differ significantly. The largest difference between the evaluated protocols was observed when analyzing the target coverage and read depth distribution. These differences also had impact on the downstream variant calling. Conclusively, the products from the AMPLI1 and MALBAC kits were shown to be most similar to the bulk samples and are therefore recommended for WGA of single cells. In this study four commercial kits for WGA (AMPLI1, MALBAC, Repli-G and PicoPlex) were used to amplify human single cells. The WGA products were exome sequenced together with non-amplified bulk samples from the same source. The resulting data was evaluated in terms of genomic coverage, allelic dropout and SNP calling.

  1. Detection of Pneumocystis in lungs of bats from Brazil by PCR amplification Detecção de Pneumocystis em pulmões de morcegos no Brasil por Nested-PCR

    Directory of Open Access Journals (Sweden)

    Edna Maria Cavallini Sanches

    2009-06-01

    Full Text Available Pneumocystis has been isolated from a wide range of unrelated mammalian hosts, including humans, domestic and wild animals. It has been demonstrated that the genome of Pneumocystis of one host differs markedly from that of other hosts. Also, variation in the chromosome and DNA sequence of Pneumocystis within a single host species has been observed. Since information about the occurrence and nature of infections in wild animals is still limited, the objective of this work was to detect the presence of Pneumocystis sp. in lungs of bats from two states from Brazil by Nested-PCR amplification. The bats, captured in caves and in urban areas, were obtained from the Program of Rabies Control of two States in Brazil, Mato Grosso and Rio Grande do Sul, located in the Mid-Western and Southern regions of the country, respectively. DNAs were extracted from 102 lung tissues and screened for Pneumocystis by nested PCR at the mtLSU rRNA gene and small subunit of mitochondrial ribosomal RNA (mtSSU rRNA. Gene amplification was performed using the mtLSU rRNA, the primer set pAZ102H - pAZ102E and pAZ102X - pAZY, and the mtSSU rRNA primer set pAZ102 10FRI - pAZ102 10R-RI and pAZ102 13RI - pAZ102 14RI. The most frequent bats were Tadarida brasiliensis (25, Desmodus rotundus (20, and Nyctinomops laticaudatus (19. Pneumocystis was more prevalent in the species Nyctinomops laticaudatus (26.3% = 5/19, Tadarida brasiliensis (24% = 6/25, and Desmodus rotundus (20% = 4/20. Besides these species, Pneumocystis also was detected in lungs from Molossus molossus (1/11, 9.1%, Artibeus fimbriatus (1/1, 100%, Sturnira lilium (1/3, 33.3%, Myotis levis (2/3, 66.7%and Diphylla ecaudata (1/2, 50%. PCR products which could indicate the presence of Pneumocystis (21.56% were identified in DNA samples obtained from 8 out of 16 classified species from both states (5 bats were not identified. This is the first report of detection of Pneumocystis in bats from Brazil.Pneumocystis tem sido

  2. Sampling methods for rumen microbial counts by Real-Time PCR techniques

    Directory of Open Access Journals (Sweden)

    S. Puppo

    2010-02-01

    Full Text Available Fresh rumen samples were withdrawn from 4 cannulated buffalo females fed a fibrous diets in order to quantify bacteria concentration in the rumen by Real-Time PCR techniques. To obtain DNA of a good quality from whole rumen fluid, eight (M1-M8 different pre-filtration methods (cheese cloths, glass-fibre and nylon filter in combination with various centrifugation speeds (1000, 5000 and 14,000 rpm were tested. Genomic DNA extraction was performed either on fresh or frozen samples (-20°C. The quantitative bacteria analysis was realized according to Real-Time PCR procedure for Butyrivibrio fibrisolvens reported in literature. M5 resulted the best sampling procedure allowing to obtain a suitable genomic DNA. No differences were revealed between fresh and frozen samples.

  3. Typing some of lactic acid bacteria in Syria using PCR and FT-IR techniques

    International Nuclear Information System (INIS)

    Al-Mariri, A.; Sharabi, N. D.

    2008-11-01

    Lactic Acid Bacteria (LAB) are considered to be the most useful microorganisms. They are beneficial in flavoring foods, inhibiting pathogenic as well as spoilage bacteria in food products. The isolates of LAB were obtained from traditional Syrian dairy products (white cheese and curdled yogurt) obtained from different regions in Syria. The isolates were subjected to phenotypic characterization analyses. The PCR technique of bacterial DNA was evaluated as an advanced tool for the identification of LAB. It was found that strains: E. faecium, E. faecalis and S. thermophilus dominate in white cheese and in yogurt. Our results demonstrated that we could identify LAB using Fourier transform infrared spectroscopy (FT-IR) patterns. (Authors)

  4. Typing some of lactic acid bacteria in Syria using PCR and FT-IR techniques

    International Nuclear Information System (INIS)

    Al-Mariri, A.; Sharabi, N. D.

    2010-01-01

    Lactic Acid Bacteria (LAB) are considered to be the most useful microorganisms. They are beneficial in flavoring foods, inhibiting pathogenic as well as spoilage bacteria in food products. The isolates of LAB were obtained from traditional Syrian dairy products (white cheese and curdled yogurt) obtained from different regions in Syria. The isolates were subjected to phenotypic characterization analyses. The PCR technique of bacterial DNA was evaluated as an advanced tool for the identification of LAB. It was found that strains: E. faecium, E. faecalis and S. thermophilus dominate in white cheese and in yogurt. Our results demonstrated that we could identify LAB using Fourier transform infrared spectroscopy (FT-IR) patterns. (author)

  5. Standardization of the PCR technique for the detection of delta toxin in Staphylococcus spp.

    Directory of Open Access Journals (Sweden)

    C. Marconi

    2005-06-01

    Full Text Available Coagulase-negative staphylococci (CNS, components of the normal flora of neonates, have emerged as important opportunistic pathogens of nosocomial infections that occur in neonatal intensive care units. Some authors have reported the ability of some CNS strains, particularly Staphylococcus epidermidis, to produce a toxin similar to S. aureus delta toxin. This toxin is an exoprotein that has a detergent action on the membranes of various cell types resulting in rapid cell lysis. The objectives of the present study were to standardize the Polymerase Chain Reaction (PCR technique for the detection of the gene responsible for the production of delta toxin (hld gene in staphylococcal species isolated from catheters and blood cultures obtained from neonates, and to compare the results to those obtained with the phenotypic synergistic hemolysis method. Detection of delta toxin by the phenotypic and genotypic method yielded similar results for the S. aureus isolates. However, in S. epidermidis, a higher positivity was observed for PCR (97.4% compared to the synergistic hemolysis method (86.8%. Among CNS, S. epidermidis was the most frequent isolate and was a delta toxin producer. Staphylococcus simulans and S. warneri tested positive by the phenotypic method, but their positivity was not confirmed by PCR for the hld gene detection. These results indicate that different genes might be responsible for the production of this toxin in different CNS species, requiring highly specific primers for their detection. PCR was found to be a rapid and reliable method for the detection of the hld gene in S. aureus and S. epidermidis.

  6. Application of reverse transcription-PCR and real-time PCR in nanotoxicity research.

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2012-01-01

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

  7. Strategies for the inclusion of an internal amplification control in conventional and real time PCR detection of Campylobacter spp. in chicken fecal samples

    DEFF Research Database (Denmark)

    Lund, Marianne; Madsen, Mogens

    2006-01-01

    To illustrate important issues in optimization of a PCR assay with an internal control four different primer combinations for conventional PCR, two non-competitive and two competitive set-ups for real time PCR were used for detection of Campylobacter spp. in chicken faecal samples. In the convent......To illustrate important issues in optimization of a PCR assay with an internal control four different primer combinations for conventional PCR, two non-competitive and two competitive set-ups for real time PCR were used for detection of Campylobacter spp. in chicken faecal samples....... In the conventional PCR assays the internal control was genomic DNA from Yersinia ruckeri, which is not found in chicken faeces. This internal control was also used in one of the set LIPS in real time PCR. In the three other set-ups different DNA fragments of 109 bp length prepared from two oligos of each 66 bp...

  8. Detection of EnteroinvasiveEscherichia coli by PCR technique in Children with Diarrhea

    Directory of Open Access Journals (Sweden)

    v Aein

    2014-11-01

    Full Text Available Background & aim: EnteroinvasiveEscherichia coliis one of the important agents of invasion to intestinal epithelial cells, damage and cell death which due to dysentery. The aim of this study wastoDetection of EnteroinvasiveEscherichia coli by PCR technique from Children’s Diarrheain yasuj. Methods:This cross-sectional study was performed on 200 stool samples taken from children with diarrhea in Yasuj. After initial identification of E.coli strains by culture and biochemical tests, EIEC gene such as ipaH detected by PCR technicque and antibiotic susceptibility of isolates was evaluated by using disc diffusion (CLSI method. Results: Out of all examined samples, 16(8% EIEC were separated. Antibiotic susceptibility test showed that the most susceptible antibiotic is ciprofloxacin for EIEC and also most resistant antibiotic is ceftizoxime. Conclusion: Results showed that EIEC strains have a moderate prevalence than other studies in our study area. Therefore, for importance of this strain to producing dysentery, hospital-wide surveillance using molecular techniques hase been proposed in other regions of country.

  9. A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp

    Energy Technology Data Exchange (ETDEWEB)

    Jothikumar, N., E-mail: jin2@cdc.gov; Hill, Vincent R.

    2013-06-28

    Highlights: •Uses a single-labeled fluorescent primer for real-time PCR. •The detection sensitivity of PET PCR was comparable to TaqMan PCR. •Melt curve analysis can be performed to confirm target amplicon production. •Conventional PCR primers can be converted to PET PCR primers. -- Abstract: We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3′-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5′-end forms a hairpin structure. A fluorescent dye is attached to 5′-end of either the forward or reverse primer. The presence of guanosine residues at the first and second position of the 3′ dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000–0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource

  10. Cloning and sequencing of an alkaline protease gene from Bacillus lentus and amplification of the gene on the B. lentus chromosome by an improved technique.

    Science.gov (United States)

    Jørgensen, P L; Tangney, M; Pedersen, P E; Hastrup, S; Diderichsen, B; Jørgensen, S T

    2000-02-01

    A gene encoding an alkaline protease was cloned from an alkalophilic bacillus, and its nucleotide sequence was determined. The cloned gene was used to increase the copy number of the protease gene on the chromosome by an improved gene amplification technique.

  11. Establishment of a Pcr Technique for Determination of Htlv-1 Infection in Paraffin-Embedded Tissues

    Directory of Open Access Journals (Sweden)

    M Rastin

    2007-04-01

    Full Text Available Introduction: HTLV-1 , the first known human retrovirus belongs to oncovirus subfamily of retroviruses. The major characteristic of HTLV-1 is its highly restricted geographic prevalence. Northern part of Khorasan is an endemic region of HTLV-1 infection. Epidemiological studies can help in designing preventive programs for HTLV-1 infection. The aim of this study was the establishment of a PCR technique for determination of HTLV-1 infection in paraffin-embedded tissues. Methods: In this experimental laboratory study for establishment of a technique, PCR was initially optimized using Beta-actin primers on various formalin fixed paraffin-embedded tissues from liver, spleen, skin and lymph nodes. The optimized concentration of Mgcl2 was 2mm, primer was 8 pmol. Optimized concentration of DNA was different according to the kind of tissue. HTLV-1 infection was determined by applying tax, pol, env and LTR primers on 50 paraffin-embedded lymph node tissues . The reporoducibility of this technique was shown for skin and lymph node tissues infected with HTLV-1. Resuls: In 50 lymph node tissues, one case with pathologic diagnosis of NHL was positive with all 5 sets of primers (tax, Pol, env and LTR primers and the other case was positive with only two sets of tax primers but was negative with pol, env and LTR primers. The prevalence of infection was 2% among lymph node specimens. (1 of 50 specimens and if the second case is considered, the prevalence would be 4%. Conclusion: Comparison of the results of this study with another study on blood specimens (seroprevalence2.3% was not statistically significant thus confirming the results of one another. (P=0.883

  12. Shape based kinetic outlier detection in real-time PCR

    Directory of Open Access Journals (Sweden)

    D'Atri Mario

    2010-04-01

    Full Text Available Abstract Background Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD. Results Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific. Conclusion Our results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification.

  13. Enrichment of bacteria samples by centrifugation improves the diagnosis of orthopaedics-related infections via real-time PCR amplification of the bacterial methicillin-resistance gene.

    Science.gov (United States)

    Tsuru, Arisa; Setoguchi, Takao; Kawabata, Naoya; Hirotsu, Masataka; Yamamoto, Takuya; Nagano, Satoshi; Yokouchi, Masahiro; Kakoi, Hironori; Kawamura, Hideki; Ishidou, Yasuhiro; Tanimoto, Akihide; Komiya, Setsuro

    2015-07-03

    To effectively treat orthopaedic infections by methicillin-resistant strains, an early diagnosis is necessary. Bacterial cultures and real-time polymerase chain reaction (PCR) have been used to define methicillin-resistant staphylococci. However, even when patients display clinical signs of infections, bacterial culture and real-time PCR often cannot confirm infection. The aim of this study was to prospectively compare the utility of real-time PCR for the mecA gene detection following centrifugation of human samples with suspected orthopaedic infections. In addition to the conventional real-time PCR method, we performed real-time PCR following centrifugation of the sample at 4,830×g for 10 min in a modified real-time PCR (M-PCR) method. We suspended cultured methicillin-resistant Staphylococcus aureus and generated standard dilution series for in vitro experiments. The in vitro detection sensitivity of the M-PCR method was approximately 5.06 times higher than that of the conventional real-time PCR method. We performed bacterial culture, pathological examination, real-time PCR, and M-PCR to examine the infectious fluids and tissues obtained from 36 surgical patients at our hospital. Of these, 20 patients who had undergone primary total hip arthroplasty were enrolled as negative controls. In addition, 15 patients were examined who were clinically confirmed to have an infection, including periprosthetic joint infection (eight patients), pyogenic spondylitis (two patients), infectious pseudoarthrosis (two patients), and after spine surgery (three patients). In one sample from a patient who developed infectious pseudoarthrosis and two samples from surgical site infections after spine surgery, the mecA gene was detected only by the M-PCR method. In one patient with infectious pseudoarthrosis, one patient with infection after arthroplasty, and two patients with purulent spondylitis, the detection sensitivity of the M-PCR method was increased compared with PCR (clinical

  14. Detection of congenital cytomegalovirus in newborns using nucleic acid amplification techniques and its public health implications.

    Science.gov (United States)

    Liu, Guoyu; Hai, Rong; Liu, Fenyong

    2017-10-01

    Human cytomegalovirus (HCMV), a herpesvirus, is an important human pathogen that causes asymptomatic infections in healthy or immunocompetent individuals but can lead to severe and potentially life-threatening complications in immune-immature individuals such as neonates or immune-compromised patients such as organ-transplant recipients and HIV-positive individuals. Congenital HCMV infection represents a significant public health issue and poses substantial healthcare and economic burden to society. This virus causes the most common viral congenital infection worldwide, and is the leading non-genetic cause of sensorineural hearing loss in children in developed countries. Congenital HCMV infection is believed to fulfill the criteria of the American College of Medical Genetics to be considered as a condition targeted for a newborn screening program. This is because congenital HCMV infection can be identified during a time (within 2 days after birth) at which it would not ordinarily be detected clinically, and there are demonstrated benefits of early detection, timely intervention, and efficacious treatment of the condition. Recent progresses in developing polymerase chain reaction-based approaches to detect HCMV in samples obtained from newborns have generated much excitement in the field. In this review, we highlight the recent progress in diagnostic techniques that could potentially be used for the detection of HCMV infection in neonates and its direct implications in public health settings for diagnosing congenital HCMV infection.

  15. Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis.

    Science.gov (United States)

    Waggoner, Jesse J; Balassiano, Ilana; Abeynayake, Janaki; Sahoo, Malaya K; Mohamed-Hadley, Alisha; Liu, Yuanyuan; Vital-Brazil, Juliana Magalhães; Pinsky, Benjamin A

    2014-01-01

    Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species. For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (pLeptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay. The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.

  16. Scope and Limitations of the Nicking Enzyme Amplification Reaction for the Synthesis of Base-Modified Oligonucleotides and Primers for PCR

    Czech Academy of Sciences Publication Activity Database

    Ménová, Petra; Raindlová, Veronika; Hocek, Michal

    2013-01-01

    Roč. 24, č. 6 (2013), s. 1081-1093 ISSN 1043-1802 R&D Projects: GA ČR GA203/09/0317 Institutional support: RVO:61388963 Keywords : isothermal DNA amplification * cross-coupling reactions * nucleoside triphosphates * polymerase incorporation * functionalized DNA * nucleic-acids Subject RIV: CC - Organic Chemistry Impact factor: 4.821, year: 2013

  17. Detection of Staphylococcus aureus enterotoxins in sheep cheese and dairy desserts by multiplex PCR technique.

    Science.gov (United States)

    Ertas, Nurhan; Gonulalan, Zafer; Yildirim, Yeliz; Kum, Erhan

    2010-08-15

    The aim of this study was to investigate the presence of Staphylococcus aureus (S. aureus) and staphylococcal enterotoxins (SEs) genes in sheep cheese and dairy dessert samples by multiplex PCR (mPCR) technique. A total of 150 samples were analyzed consisting of 50 dairy dessert samples and 100 sheep cheese. Coagulase positive staphylococci (CPS) were found in 86 (57.3%) out of 150 analyzed samples. S. aureus were isolated from 60 (60%), 26 (52%) of sheep cheese and from of dairy desserts, respectively. Five suspected colonies were tested from each sheep cheese and dairy dessert samples for phenotypic and genotypic characterizations. A total of 430 isolates from the 86 positive samples were investigated in this study. Eighty (18.6%) isolates were characterized as S. aureus. The enterotoxin genes (sea, seb, sec, sed) were found in 13 (3.02%) out of 80 isolates. From cheese isolates, sea, seb and sed were detected in 5 (1.6%), 2 (0.6%), 1 (0.3%), respectively. From dairy dessert isolates, sea, sec and sed were detected in 3 (2.3%), 1 (0.76%), 1 (0.76%), respectively. The presence of SEs was identified in 12 (2.8%) out of 80 isolates by using ELISA technique. It was determined that these SEs had a distribution of 7 (1.6%) SEA, 2 (0.46%) SEB, 1 (0.23%) SEC, and 2 (0.46%) SED. SEs were found in 7 (2.3%) cheese and 5 (3.8%) dairy dessert isolates. In conclusion, S.aureus and their SEs were found to be present in sheep cheese and dairy desserts in this study. It is emphasized that the presence of S. aureus and their SEs genes in sheep cheese and dairy desserts may be regarded as a potential risk for human health. Copyright 2010 Elsevier B.V. All rights reserved.

  18. Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

    OpenAIRE

    Ram, S.; Vimalin, J.M.; Jambulingam, M.; Tiru, V.; Gopalakrishnan, R.K.; Madhavan, H.N.

    2012-01-01

    Aim: Isolation, dark field detection and microscopic agglutination test (MAT) are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR) - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septi...

  19. New, Improved Version of the mCOP-PCR Screening System for Detection of Spinal Muscular Atrophy Gene (SMN1) Deletion.

    Science.gov (United States)

    Shinohara, Masakazu; Ar Rochmah, Mawaddah; Nakanishi, Kenta; Harahap, Nur Imma Fatimah; Niba, Emma Tabe Eko; Saito, Toshio; Saito, Kayoko; Takeuchi, Atsuko; Bouike, Yoshihiro; Nishio, Hisahide

    2017-09-07

    Spinal muscular atrophy (SMA) is a frequent autosomal recessive disorder, characterized by lower motor neuron loss in the spinal cord. More than 95% of SMA patients show homozygous survival motor neuron 1 (SMN1) deletion. We previously developed a screening system for SMN1 deletion based on a modified competitive oligonucleotide priming-PCR (mCOP-PCR) technique. However, non-specific amplification products were observed with mCOP-PCR, which might lead to erroneous interpretation of the screening results. To establish an improved version of the mCOP-PCR screening system without non-specific amplification. DNA samples were assayed using a new version of the mCOP-PCR screening system. DNA samples had already been genotyped by PCR-restriction fragment length polymorphism (PCR-RFLP), showing the presence or absence of SMN1 exon 7. The new mCOP-PCR method contained a targeted pre-amplification step of the region, including an SMN1-specific nucleotide, prior to the mCOP-PCR step. mCOP-PCR products were electrophoresed on agarose gels. No non-specific amplification products were detected in electrophoresis gels with the new mCOP-PCR screening system. An additional targeted pre-amplification step eliminated non-specific amplification from mCOP-PCR screening.

  20. Molecular Technique to Understand Deep Microbial Diversity

    Science.gov (United States)

    Vaishampayan, Parag A.; Venkateswaran, Kasthuri J.

    2012-01-01

    Current sequencing-based and DNA microarray techniques to study microbial diversity are based on an initial PCR (polymerase chain reaction) amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of the minor template appears to be suppressed by the exponential amplification of the more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck has overlooked the presence of the less abundant minority population, and underestimated their role in the ecosystem maintenance. To generate PCR amplicons for subsequent diversity analysis, bacterial l6S rRNA genes are amplified by PCR using universal primers. Two distinct PCR regimes are employed in parallel: one using normal and the other using biotinlabeled universal primers. PCR products obtained with biotin-labeled primers are mixed with streptavidin-labeled magnetic beads and selectively captured in the presence of a magnetic field. Less-abundant DNA templates that fail to amplify in this first round of PCR amplification are subjected to a second round of PCR using normal universal primers. These PCR products are then subjected to downstream diversity analyses such as conventional cloning and sequencing. A second round of PCR amplified the minority population and completed the deep diversity picture of the environmental sample.

  1. Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques.

    Science.gov (United States)

    Neumaier, M; Braun, A; Wagener, C

    1998-01-01

    The increasing interest in molecular biology diagnostics is a result of the tremendous gain of scientific knowledge in genetics, made possible especially since the introduction of amplification techniques. High expectations have been placed on genetic testing, and the number of laboratories now using the relevant technology is rapidly increasing--resulting in an obvious need for standardization and definition of laboratory organization. This communication is an effort towards that end. We address aspects that should be considered when structuring a new molecular diagnostic laboratory, and we discuss individual preanalytical and analytical procedures, from sampling to evaluation of assay results. In addition, different means of controlling contamination are discussed. Because the methodology is in constant change, no general standards can be defined. Accordingly, this publication is intended to serve as a recommendation for good laboratory practice and internal quality control and as a guide to troubleshooting, primarily in amplification techniques.

  2. Safety evaluation and bacterial community of kung-som using PCR-DGGE technique

    Directory of Open Access Journals (Sweden)

    Sutanate Saelao

    2016-08-01

    Full Text Available This study evaluates the safety of kung-som which was distributed in local markets and using PCR-DGGE technique to identify microflora in kung-som. Lactic acid bacteria (LAB were found at counts of more than 7 log CFU g-1 in all samples and the total viable counts were about 5-8 log CFU g-1 . Bacillus cereus and yeasts were detected at around 2 log CFU g-1 and 5-6log CFU g-1, respectively. For DGGE analysis, LAB and coagulase negative staphylococci (CNS bacteria dominated over other microorganisms. The sequencing of the DNA bands from DGGE gels corresponding to kung-som samples showed the presence of LAB as the major microflora in the products, namely: Lactobacillus farciminis, Lactobacillus plantarum, Lactococcus garvieae, Tetragenococcus halophilus and Weissella thailandensis. In addition, Staphylococcus carnosus was detected in kung-som as minor microflora. These dominant strains would allow the development of defined starter cultures for improving the quality of kung-som.

  3. Application of a Simple In-House PCR-SSP Technique for HLA-B* 27 Typing in Spondyloarthritis Patients

    Directory of Open Access Journals (Sweden)

    Devraj J. Parasannanavar

    2013-01-01

    Full Text Available Background. Microlymphocytotoxicity (MLCT and flowcytometry (FC are the conventional serological methods to detect HLA-B* 27. Due to some disadvantages in these methods, most of the HLA laboratories have now switched over to molecular methods. Molecular techniques based on commercial kits are expensive; as such many laboratories with limited funds in developing countries cannot afford these techniques. Aims. Our main aim was to standardize a simple inexpensive in-house PCR-SSP technique for HLA-B* 27 typing. Materials and Methods. Sequence Specific primers were designed to amplify all the subtypes of B* 27 using IMGT-HLA sequence database. Accuracy was checked by retyping of 90 PCR-SSOP typed controls. Results. The presence of 149 bp specific band with control band on 2% agarose gel showed B* 27 positivity. No discrepancies were found when compared with PCR-SSOP results. The frequency of HLA-B* 27 was found to be significantly increased (68.75% versus 4.40%, O.R 46.909: P value 6.62E-32 among 700 SpA patients as compared to controls. Clinically, 54% of patients had polyarticular arthritis with SI joints involvement (68% and restricted spine flexion (60%. Conclusion. In-house PCR-SSP technique is very simple and inexpensive technique to detect B* 27 allele, which was strongly associated with SpA patients from Western India.

  4. [Comparison study on polymerase chain reaction (PCR) and standard culture technique in detecting mycobacterium tuberculosis to diagnose of joint tuberculosis].

    Science.gov (United States)

    Sun, Yong-sheng; Wen, Jian-min; Lü, Wei-xin; Lou, Si-quan; Jiao, Chang-geng; Yang, Su-min; Xu, Hai-bin; Duan, Yong-zhuang

    2009-07-01

    To study the role of PCR technique in detection of mycobacterium tuberculosis in the samples from joint tuberculosis, and to evaluate the clinical value of PCR in diagnosis of joint tuberculosis. From June 1993 to August 2001, PCR was used to detect DNA of mycobacterium tuberculosis, and the standard culture was applied to detect mycobacterium tuberculosis. Mycobacterium tuberculosis were respectively blindly by the two techniques in the samples obtained from 95 patients with joint tuberculosis (55 males and 40 females, the age ranging from 2 to 75 years, with an average of 34 years). The positive rate of mycobacterium tuberculosis detection was calculated. In the detection of mycobacterium tuberculosis, positive rate was 82% (78/95) in PCR technique, and 16% (15/95) in standard culture technique. There were statistical differences between the two groups (chi2=67, Ptechnique is a rapid, simple, sensitive and specific method for detection of mycobacterium tuberculosis in the samples of joint tuberculosis, showing more marked advantages than the standard culture technique. It is valuable in the early rapid diagnosis and differential diagnosis of joint tuberculosis.

  5. Rapid RT-PCR amplification of full-length poliovirus genomes allows rapid discrimination between wild-type and recombinant vaccine-derived polioviruses.

    Science.gov (United States)

    Boot, Hein J; Schepp, Rutger M; van Nunen, Femke J H B; Kimman, Tjeerd G

    2004-03-01

    Poliomyelitis outbreaks in areas that were free for a long time of wild-type polioviruses have been reported. Characterization at nucleotide level of the causative agents showed that the isolated viruses were recombinant oral polio vaccine (OPV)-derived polioviruses. To allow rapid identification and detailed analysis of such recombinant polioviruses, a robust full-length reverse transcriptase-PCR (RT-PCR) was developed using SuperScript II (RT) and expand (PCR). Without extensive purification, it was possible to amplify and characterize the full-length genomes of all selected vaccine, wild-type, and recombinant vaccine-derived polioviruses within a week. Endonuclease nuclease analysis (SpeI) of the full-length amplicons allowed easy discrimination between recombinant and non-recombinant polioviruses. Furthermore, sequence analysis of cloned full-length amplicons of a recombinant vaccine-derived poliovirus strain showed that the quasi-species nature of a viral stock is preserved during the RT-PCR procedure. This robust and rapid RT-PCR method will allow rapid characterization of (recombinant) poliovirus strains in case of a local poliomyelitis outbreak, and will help to assess the risk of the appearance of such strains after wild-type poliovirus has been eradicated globally.

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

    Science.gov (United States)

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

  7. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature

    DEFF Research Database (Denmark)

    Guðnason, Haukur; Dufva, Hans Martin; Bang, Dang Duong

    2007-01-01

    The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical applications over the last decade. Many applications utilize SYBR Green I dye to follow the accumulation of amplicons in real time. SYBR Green I has, however, a number of limitations that include...... investigate 15 different intercalating DNA dyes for their inhibitory effects on PCR, effects on DNA melting temperature and possible preferential binding to GC-rich sequences. Our results demonstrated that in contrast to the results of SYBR Green I, two intercalating dyes SYTO-13 and SYTO-82 do not inhibit...

  8. Application of polymerase chain reaction (PCR) for diagnosis of equine herpes virus-1 (EHV-1).

    Science.gov (United States)

    Gupta, A K; Singh, B K; Yadav, M P

    1996-11-01

    Fifty aborted foetus samples were diagnosed for the presence of equine herpes virus-1 (EHV-1) by polymerase chain reaction (PCR) technique. Specific primer pair for amplification of a particular segment of EHV-1 DNA in gc region having 3 Hae III restriction endonuclease sites was used. A 409 base pair segment obtained as PCR amplification product in 9 samples was digested with Hae III to confirm the presence of EHV-1 as the infectious agent in aborted tissues. It was observed that PCR technique was more sensitive, specific and rapid than the conventional virological diagnostic methods.

  9. Identification of root-knot nematode species occurring in South Africa using the SCAR-PCR technique

    NARCIS (Netherlands)

    Fourie, H.; Zijlstra, C.; Mc Donald, A.H.

    2001-01-01

    Species of root-knot nematodes occurring in South Africa (Meloidogyne fallax, M. chitwoodi, M. javanica, M. incognita, M. arenaria and M. hapla) were identified and differentiated by the sequence characterised amplified region - polymerase chain reaction (SCAR-PCR) technique. Multiplex internal

  10. Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR

    DEFF Research Database (Denmark)

    Reid, Robert J D; Lisby, Michael; Rothstein, Rodney

    2002-01-01

    . Furthermore, many of the techniques described here rely on preexisting and commercially available adaptamer sets that can be obtained inexpensively rather than designing new primers for every experiment. Although a cost is incurred when performing multiple PCR amplifications, the increase in recombination...... efficiency is dramatic. Finally, the adaptamer-mediated PCR fusion methodology is versatile and can be applied to varied genome manipulations....

  11. Reference gene selection, primer design and amplification interference of quantitative real-time PCR in tomato and orange subjected to environmental and disease stress

    Science.gov (United States)

    Response of gene expression in the oxylipin pathway to chilling and heating in tomatoes at full ripe stage was investigated. Total RNA was isolated from tomato pericarp tissue, and gene expression of Tomlox A-Tomlox D, HPL and ADH were determined by real-time Polymerase Chain Reaction (PCR) using t...

  12. Genetic screening of spinal muscular atrophy using a real-time modified COP-PCR technique with dried blood-spot DNA.

    Science.gov (United States)

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

    2017-10-01

    Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by mutations in SMN1. More than 95% of SMA patients carry homozygous SMN1 deletion. SMA is the leading genetic cause of infant death, and has been considered an incurable disease. However, a recent clinical trial with an antisense oligonucleotide drug has shown encouraging clinical efficacy. Thus, early and accurate detection of SMN1 deletion may improve prognosis of many infantile SMA patients. A total of 88 DNA samples (37 SMA patients, 12 carriers and 39 controls) from dried blood spots (DBS) on filter paper were analyzed. All participants had previously been screened for SMN genes by PCR restriction fragment length polymorphism (PCR-RFLP) using DNA extracted from freshly collected blood. DNA was extracted from DBS that had been stored at room temperature (20-25°C) for 1week to 5years. To ensure sufficient quality and quantity of DNA samples, target sequences were pre-amplified by conventional PCR. Real-time modified competitive oligonucleotide priming-PCR (mCOP-PCR) with the pre-amplified PCR products was performed for the gene-specific amplification of SMN1 and SMN2 exon 7. Compared with PCR-RFLP using DNA from freshly collected blood, results from real-time mCOP-PCR using DBS-DNA for detection of SMN1 exon 7 deletion showed a sensitivity of 1.00 (CI [0.87, 1.00])] and specificity of 1.00 (CI [0.90, 1.00]), respectively. We combined DNA extraction from DBS on filter paper, pre-amplification of target DNA, and real-time mCOP-PCR to specifically detect SMN1 and SMN2 genes, thereby establishing a rapid, accurate, and high-throughput system for detecting SMN1-deletion with practical applications for newborn screening. Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  13. Direct RNA detection without nucleic acid purification and PCR: Combining sandwich hybridization with signal amplification based on branched hybridization chain reaction.

    Science.gov (United States)

    Xu, Yao; Zheng, Zhi

    2016-05-15

    We have developed a convenient, robust and low-cost RNA detection system suitable for high-throughput applications. This system uses a highly specific sandwich hybridization to capture target RNA directly onto solid support, followed by on-site signal amplification via 2-dimensional, branched hybridizing chain polymerization through toehold-mediated strand displacement reaction. The assay uses SYBR Green to detect targets at concentrations as low as 1 pM, without involving nucleic acid purification or any enzymatic reaction, using ordinary oligonucleotides without modification or labeling. The system was demonstrated in the detection of malaria RNA in blood and GAPDH gene expression in cell lysate. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Identification of species of leishmania isolated from patients with cutaneous leishmaniasis in Kermanshah; using RAPD-PCR technique

    Directory of Open Access Journals (Sweden)

    Yazdan Hamzavi

    2010-09-01

    Full Text Available Annually many numbers of pationts with Cutaneous Leishmaniasis (CL have been reported in Kermanshah province- IRAN. The study aimed to identify species of Leishmania isolated from patients with CT in Kermanshah. Seven isolates of Leishmania obtained from patients with CL, without any travelling to other provinces, were cultured in NNN medium. After mass production of leptomonads in RPMI 1640 medium DNA was purified and the species were diagnosed using RAPD-PCR technique. The study of electrophoretic fingerprints of the product of RAPD-PCR in seven isolates showed that Leshmania major was the causative agent of CL patients in Kermanshah province. More studies in this field recommended.

  15. [Fungal composition in massa medicata fermentata based on culture dependent method and independent PCR-SSCP technique].

    Science.gov (United States)

    Chen, Juan; Jiao, Xiao-lin; Yang, Chun-yong; Song, Mei-fang; Gao, Wei-wei

    2014-11-01

    To analyze the fungal composition in Massa Medicata Fermentata based on culture dependent method and independent PCR-SSCP technique. Fungi were directly isolated from Massa Medicata Fermentata samples. The obtained strains were identified according to morphology and DNA sequence. Meanwhile the total fungal DNA was extracted from Massa Medicata Fermentata samples, the cultural independent PCR-SSCP technique based on β-tubulin gene were used to identify the mycobiota. According to cultural method, Aspergillus flavus and Rhizopus oryzae were present in Massa Medicata Fermentata samples, while A. flavus and A. niger were present in fried Massa Medicata Fermentata samples. In contrast, 5 species were obtained by PCR-SSCP technique, A. flavus was overlapped with fungal taxa derived from culture dependent method; A. ambiguu and A. s ivoriensis were dominant with relative abundance of 57% and 35% respectively, while the relative abundance of A. flavus was as low as 4%. None species was obtained from fried Massa Medicata Fermentata samples. PCR-SSCP based on β-tubulin gene could distinguish fungi into species, culture dependent method combined with culture independent method could better understand the fungal composition associated with Massa Medicata Fermentata fermentation.

  16. Identification of a novel, invasive, not-yet-cultivated Treponema sp in the large intestine of pigs by PCR amplification of the 16S rRNA gene

    DEFF Research Database (Denmark)

    Mølbak, Lars; Schou, Kirstine Klitgaard; Jensen, Tim Kåre

    2006-01-01

    Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantii(T) (90.1%). The spirochete, here...... named "Candidatus Treponema suis," was associated with colitis, including invasion of the surface epithelium as well as superficial parts of the mucosa....

  17. Identification of a Novel, Invasive, Not-Yet-Cultivated Treponema sp. in the Large Intestine of Pigs by PCR Amplification of the 16S rRNA Gene▿

    Science.gov (United States)

    Mølbak, Lars; Klitgaard, Kirstine; Jensen, Tim K.; Fossi, Marja; Boye, Mette

    2006-01-01

    Laser capture microdissection in combination with fluorescent in situ hybridization was used to identify an unknown species of spirochetes from the pig colonic mucosa. The 16S rRNA gene was PCR amplified, and the closest related type strain was Treponema bryantiiT (90.1%). The spirochete, here named “Candidatus Treponema suis,” was associated with colitis, including invasion of the surface epithelium as well as superficial parts of the mucosa. PMID:17005743

  18. Rapid Genome Detection of Schmallenberg Virus and Bovine Viral Diarrhea Virus by Use of Isothermal Amplification Methods and High-Speed Real-Time Reverse Transcriptase PCR

    OpenAIRE

    Aebischer, Andrea; Wernike, Kerstin; Hoffmann, Bernd; Beer, Martin

    2014-01-01

    Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (R...

  19. Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

    Directory of Open Access Journals (Sweden)

    Ram, S.

    2012-01-01

    Full Text Available Aim: Isolation, dark field detection and microscopic agglutination test (MAT are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207 blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21% out of 100 culture positive blood samples, three (2.8% out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p<0.001. A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (<0.001 signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin.

  20. Monitoring transmission routes of Listeria spp. in smoked salmon production with repetitive element sequence-based PCR techniques.

    Science.gov (United States)

    Zunabovic, M; Domig, K J; Pichler, I; Kneifel, W

    2012-03-01

    Various techniques have been used for tracing the transmission routes of Listeria species and for the assessment of hygiene standards in food processing plants. The potential of repetitive element sequence-based PCR (Rep-PCR) methods (GTG₅ and REPI + II) for the typing of Listeria isolates (n = 116), including Listeria monocytogenes (n = 46), was evaluated in a particular situation arising from the relocation of a company producing cold-smoked salmon. Pulsed-field gel electrophoresis (PFGE) using three restriction enzymes (ApaI, AscI, and SmaI) was used for comparison. Identical transmission scenarios among two companies could be identified by cluster analysis of L. monocytogenes isolates that were indistinguishable by both Rep-PCR and PFGE. The calculated diversity index (DI) indicates that Rep-PCR subtyping of Listeria species with primer sets GTG₅ and REPI + II has a lower discrimination power than does PFGE. When concatenated Rep-PCR cluster analysis was used, the DI increased from 0.934 (REPI + II) and 0.923 (GTG₅) to 0.956. The discrimination power of this method was similar to that of PFGE typing based on restriction enzyme Apa I (DI = 0.955). Listeria welshimeri may be useful as an indicator for monitoring smoked salmon processing environments. Rep-PCR meets the expectations of a reasonable, fast, and low-cost molecular subtyping method for the routine monitoring of Listeria species. The discriminatory power as characterized by the DI sufficiently quantifies the probability of unrelated isolates being characterized as different subtypes. Therefore, Rep-PCR typing based on two primer systems (GTG₅ and REPI + II) may be a useful tool for monitoring industrial hygiene.

  1. Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Luo, Lijuan; Liu, Dongxin; Luo, Xia; Xu, Yanmei; Hu, Shoukui; Niu, Lina; Xu, Jianguo; Ye, Changyun

    2015-01-01

    Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples. PMID:26697000

  2. Immunomagnetic separation and PCR for detection of Mycobacterium ulcerans.

    OpenAIRE

    Roberts, B; Hirst, R

    1997-01-01

    We have developed a technique based on the use of monodisperse magnetic beads to isolate Mycobacterium ulcerans from heterogenous mixtures, prior to PCR amplification. Using this method, we were able to detect M. ulcerans in water samples taken from Phillip Island, Australia, the site of several outbreaks of M. ulcerans disease in recent times.

  3. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

    Energy Technology Data Exchange (ETDEWEB)

    Malik, Afshan N., E-mail: afshan.malik@kcl.ac.uk [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom); Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom)

    2011-08-19

    Highlights: {yields} Mitochondrial dysfunction is central to many diseases of oxidative stress. {yields} 95% of the mitochondrial genome is duplicated in the nuclear genome. {yields} Dilution of untreated genomic DNA leads to dilution bias. {yields} Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as {beta}-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  4. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

    International Nuclear Information System (INIS)

    Malik, Afshan N.; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

    2011-01-01

    Highlights: → Mitochondrial dysfunction is central to many diseases of oxidative stress. → 95% of the mitochondrial genome is duplicated in the nuclear genome. → Dilution of untreated genomic DNA leads to dilution bias. → Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  5. DVC-FISH and PMA-qPCR techniques to assess the survival of Helicobacter pylori inside Acanthamoeba castellanii.

    Science.gov (United States)

    Moreno-Mesonero, Laura; Moreno, Yolanda; Alonso, José Luis; Ferrús, M Antonia

    2016-01-01

    Free-living amoebae (FLA) are ubiquitous microorganisms commonly found in water. They can act as Trojan Horses for some amoeba-resistant bacteria (ARB). Helicobacter pylori is a pathogenic bacteria, suggested to be transmitted through water, which could belong to the ARB group. In this work, a co-culture assay of H. pylori and Acanthamoeba castellanii, one of the most common FLA, was carried out to identify the presence and survival of viable and potentially infective forms of the bacteria internalized by the amoeba. Molecular techniques including FISH, DVC-FISH, qPCR and PMA-qPCR were used to detect the presence of internalized and viable H. pylori. After 24 h in co-culture and disinfection treatment to kill extra-amoebic bacteria, viable H. pylori cells were observed inside A. castellanii. When PMA-qPCR was applied to the co-culture samples, only DNA from internalized H. pylori cells was detected, whereas qPCR amplified total DNA from the sample. By the combined DVC-FISH method, the viability of H. pylori cells in A. castellanii was observed. Both specific techniques provided evidence, for the first time, that the pathogen is able to survive chlorination treatment in occurrence with A. castellanii and could be very useful methods for performing further studies in environmental samples. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  6. Validation of the (GTG)(5)-rep-PCR fingerprinting technique for rapid classification and identification of acetic acid bacteria, with a focus on isolates from Ghanaian fermented cocoa beans.

    Science.gov (United States)

    De Vuyst, Luc; Camu, Nicholas; De Winter, Tom; Vandemeulebroecke, Katrien; Van de Perre, Vincent; Vancanneyt, Marc; De Vos, Paul; Cleenwerck, Ilse

    2008-06-30

    Amplification of repetitive bacterial DNA elements through the polymerase chain reaction (rep-PCR fingerprinting) using the (GTG)(5) primer, referred to as (GTG)(5)-PCR fingerprinting, was found a promising genotypic tool for rapid and reliable speciation of acetic acid bacteria (AAB). The method was evaluated with 64 AAB reference strains, including 31 type strains, and 132 isolates from Ghanaian, fermented cocoa beans, and was validated with DNA:DNA hybridization data. Most reference strains, except for example all Acetobacter indonesiensis strains and Gluconacetobacter liquefaciens LMG 1509, grouped according to their species designation, indicating the usefulness of this technique for identification to the species level. Moreover, exclusive patterns were obtained for most strains, suggesting that the technique can also be used for characterization below species level or typing of AAB strains. The (GTG)(5)-PCR fingerprinting allowed us to differentiate four major clusters among the fermented cocoa bean isolates, namely A. pasteurianus (cluster I, 100 isolates), A. syzygii- or A. lovaniensis-like (cluster II, 23 isolates), and A. tropicalis-like (clusters III and IV containing 4 and 5 isolates, respectively). A. syzygii-like and A. tropicalis-like strains from cocoa bean fermentations were reported for the first time. Validation of the method and indications for reclassifications of AAB species and existence of new Acetobacter species were obtained through 16S rRNA sequencing analyses and DNA:DNA hybridizations. Reclassifications refer to A. aceti LMG 1531, Ga. xylinus LMG 1518, and Ga. xylinus subsp. sucrofermentans LMG 18788(T).

  7. Identification of neurofibromatosis 1 (NF1) homologous loci by direct sequencing, fluorescence in situ hybridization, and PCR amplification of somatic cell hybrids

    Energy Technology Data Exchange (ETDEWEB)

    Purandare, S.M.; Neil, S.M.; Brothman, A. [Univ. of Utah, Salt Lake City (United States)]|[Jikei Univ., Tokyo (Japan)] [and others

    1995-12-10

    Using fluorescence in situ hybridization (FISH), we have identified seven NF1-related loci, two separate loci on chromosome 2, at bands 2q21 and 2q33-q34, and one locus each on five other chromosomes at bands 14q11.2, 15q11.2, 18p11.2, 21q11.2-q21, and 22q11.2. Application of PCR using NF1 primer pairs and genomic DNA from somatic cell hybrids confirmed the above loci, identified additional loci on chromosomes 12 and 15, and showed that the various loci do not share homology beyond NF1 exon 27b. Sequenced PCR products representing segments corresponding to NF1 exons from these loci demonstrated greater than 95% sequence identity with the NF1 locus. We used sequence differences between bona fide NF1 and NF1-homologous loci to strategically design primer sets to specifically amplify 30 of 36 exons within the 5{prime} end of the NF1 gene. These developments have facilitated mutation analysis at the NF1 locus using genomic DNA as template. 41 refs., 3 figs., 3 tabs.

  8. Magnetic particle separation technique: a reliable and simple tool for RIA/IRMA and quantitative PCR assay

    International Nuclear Information System (INIS)

    Shen Rongsen; Shen Decun

    1998-01-01

    Five types of magnetic particles without or with aldehyde, amino and carboxyl functional groups, respectively were used to immobilize first or second antibody by three models, i. e. physical adsorption, chemical coupling and immuno-affinity, forming four types of magnetic particle antibodies. The second antibody immobilized on polyacrolein magnetic particles through aldehyde functional groups and the first antibodies immobilized on carboxylic polystyrene magnetic particles through carboxyl functional groups were recommended to apply to RIAs and/or IRMAs. Streptavidin immobilized on commercial magnetic particles through amino functional groups was successfully applied to separating specific PCR product for quantification of human cytomegalovirus. In the paper typical data on reliability of these magnetic particle ligands were reported and simplicity of the magnetic particle separation technique was discussed. The results showed that the technique was a reliable and simple tool for RIA/IRMA and quantitative PCR assay. (author)

  9. Evaluation of Digital PCR as a Technique for Monitoring Acute Rejection in Kidney Transplantation

    Directory of Open Access Journals (Sweden)

    Hyeseon Lee

    2017-03-01

    Full Text Available Early detection and proper management of kidney rejection are crucial for the long-term health of a transplant recipient. Recipients are normally monitored by serum creatinine measurement and sometimes with graft biopsies. Donor-derived cell-free deoxyribonucleic acid (cfDNA in the recipient's plasma and/or urine may be a better indicator of acute rejection. We evaluated digital PCR (dPCR as a system for monitoring graft status using single nucleotide polymorphism (SNP-based detection of donor DNA in plasma or urine. We compared the detection abilities of the QX200, RainDrop, and QuantStudio 3D dPCR systems. The QX200 was the most accurate and sensitive. Plasma and/or urine samples were isolated from 34 kidney recipients at multiple time points after transplantation, and analyzed by dPCR using the QX200. We found that donor DNA was almost undetectable in plasma DNA samples, whereas a high percentage of donor DNA was measured in urine DNA samples, indicating that urine is a good source of cfDNA for patient monitoring. We found that at least 24% of the highly polymorphic SNPs used to identify individuals could also identify donor cfDNA in transplant patient samples. Our results further showed that autosomal, sex-specific, and mitochondrial SNPs were suitable markers for identifying donor cfDNA. Finally, we found that donor-derived cfDNA measurement by dPCR was not sufficient to predict a patient's clinical condition. Our results indicate that donor-derived cfDNA is not an accurate predictor of kidney status in kidney transplant patients.

  10. Comparison of error-amplification and haptic-guidance training techniques for learning of a timing-based motor task by healthy individuals.

    Science.gov (United States)

    Milot, Marie-Hélène; Marchal-Crespo, Laura; Green, Christopher S; Cramer, Steven C; Reinkensmeyer, David J

    2010-03-01

    Performance errors drive motor learning for many tasks. Some researchers have suggested that reducing performance errors with haptic guidance can benefit learning by demonstrating correct movements, while others have suggested that artificially increasing errors will force faster and more complete learning. This study compared the effect of these two techniques--haptic guidance and error amplification--as healthy subjects learned to play a computerized pinball-like game. The game required learning to press a button using wrist movement at the correct time to make a flipper hit a falling ball to a randomly positioned target. Errors were decreased or increased using a robotic device that retarded or accelerated wrist movement, based on sensed movement initiation timing errors. After training with either error amplification or haptic guidance, subjects significantly reduced their timing errors and generalized learning to untrained targets. However, for a subset of more skilled subjects, training with amplified errors produced significantly greater learning than training with the reduced errors associated with haptic guidance, while for a subset of less skilled subjects, training with haptic guidance seemed to benefit learning more. These results suggest that both techniques help enhanced performance of a timing task, but learning is optimized if training subjects with the appropriate technique based on their baseline skill level.

  11. PCR-based specific techniques used for detecting the most important pathogens on strawberry: a systematic review.

    Science.gov (United States)

    Mirmajlessi, Seyed Mahyar; Destefanis, Marialaura; Gottsberger, Richard Alexander; Mänd, Marika; Loit, Evelin

    2015-01-15

    Strawberry diseases are a major limiting factor that severely impact plant agronomic performance. Regarding limitations of traditional techniques for detection of pathogens, researchers have developed specific DNA-based tests as sensitive and specific techniques. The aim of this review is to provide an overview of polymerase chain reaction (PCR)-based methods used for detection or quantification of the most widespread strawberry pathogens, such as Fusarium oxysporum f.sp. fragariae, Phytophthora fragariae, Colletotrichum acutatum, Verticillium dahliae, Botrytis cinerea, Macrophomina phaseolina, and Xanthomonas fragariae. An updated and detailed list of published PCR protocols is presented and discussed, aimed at facilitating access to information that could be particularly useful for diagnostic laboratories in order to develop a rapid, cost-effective, and reliable monitoring technique. The study design was a systematic review of PCR-based techniques used for detection and quantification of strawberry pathogens. Using appropriate subject headings, AGRICOLA, AGRIS, BASE, Biological Abstracts, CAB Abstracts, Google Scholar, Scopus, Web of Knowledge, and SpringerLink databases were searched from their inception up to April 2014. Two assessors independently reviewed the titles, abstracts, and full articles of all identified citations. Selected articles were included if one of the mentioned strawberry pathogens was investigated based on PCR methods, and a summary of pre-analytical requirements for PCR was provided. A total of 259 titles and abstracts were reviewed, of which 22 full texts met all the inclusion criteria. Our systematic review identified ten different protocols for X. fragariae, eight for P. fragariae, four for B. cinerea, six for C. acutatum, three for V. dahlia, and only one for F. oxysporum. The accuracy and sensitivity of PCR diagnostic methods is the focus of most studies included in this review. However, a large proportion of errors in laboratories

  12. Rapid and Specific Detection of Acidovorax avenae subsp. citrulli Using SYBR Green-Based Real-Time PCR Amplification of the YD-Repeat Protein Gene.

    Science.gov (United States)

    Cho, Min Seok; Park, Duck Hwan; Ahn, Tae-Young; Park, Dong Suk

    2015-09-01

    The aim of this study was to develop a SYBR Green-based real-time PCR assay for the rapid, specific, and sensitive detection of Acidovorax avenae subsp. citrulli, which causes bacterial fruit blotch (BFB), a serious disease of cucurbit plants. The molecular and serological methods currently available for the detection of this pathogen are insufficiently sensitive and specific. Thus, a novel SYBR Green-based real-time PCR assay targeting the YD-repeat protein gene of A. avenae subsp. citrulli was developed. The specificity of the primer set was evaluated using DNA purified from 6 isolates of A. avenae subsp. citrulli, 7 other Acidovorax species, and 22 of non-targeted strains, including pathogens and non-pathogens. The AC158F/R primer set amplified a single band of the expected size from genomic DNA obtained from the A. avenae subsp. citrulli strains but not from the genomic DNA of other Acidovorax species, including that of other bacterial genera. Using this assay, it was possible to detect at least one genomeequivalents of the cloned amplified target DNA using 5 × 10(0) fg/μl of purified genomic DNA per reaction or using a calibrated cell suspension, with 6.5 colony-forming units per reaction being employed. In addition, this assay is a highly sensitive and reliable method for identifying and quantifying the target pathogen in infected samples that does not require DNA extraction. Therefore, we suggest that this approach is suitable for the rapid and efficient diagnosis of A. avenae subsp. citrulli contaminations of seed lots and plants.

  13. Detection of restriction enzyme-digested target DNA by PCR amplification using a stem-loop primer: application to the detection of hypomethylated fetal DNA in maternal plasma.

    Science.gov (United States)

    Tong, Yu K; Chiu, Rossa W K; Leung, Tak Y; Ding, Chunming; Lau, Tze K; Leung, Tse N; Lo, Y M Dennis

    2007-11-01

    The discovery of cell-free fetal DNA in maternal plasma has opened up new possibilities for noninvasive prenatal diagnosis and monitoring. Among the fetal markers that have been described, methylation markers are sex and polymorphism independent. Methylation-sensitive restriction endonucleases are commonly used to digest hypomethylated DNA molecules, and the hypermethylated molecules remain intact for detection. The positive detection of the cleaved hypomethylated molecules would be useful for certain targets but has not been reported. The use of a stem-loop primer in microRNA detection has previously been described. In this study, DNA assays were designed and performed on maternal plasma, which contained the hypomethylated placental serpin peptidase inhibitor, clade B (ovalbumin), member 5 (SERPINB5; maspin) gene in an excess background of hypermethylated maternal SERPINB5. Detection of the enzyme-digested placenta-derived hypomethylated SERPINB5 molecules was achieved by performing stem-loop extension followed by real-time PCR on maternal plasma. The placental origin of the stem-loop-extended SERPINB5 molecules was confirmed by genotyping. From the real-time PCR results on maternal plasma, stem-loop-extended SERPINB5 promoter sequences were detectable in all 11 enzyme-digested predelivery maternal plasma samples. Postpartum clearance was demonstrated. In 9 cases in which the fetal and maternal SERPINB5 genotypes were distinguishable, the placental-specific genotypes were detected in all predelivery maternal plasma samples. Detection of restriction enzyme-digested hypomethylated placental DNA molecules in maternal plasma by the use of a stem-loop primer represents a novel approach in fetal epigenetic marker detection. The analytical approach may also be generally applicable to the detection of restriction enzyme-digested nucleic acid fragments.

  14. Nucleic acid sequence-based amplification with oligochromatography for detection of Trypanosoma brucei in clinical samples

    NARCIS (Netherlands)

    Mugasa, Claire M.; Laurent, Thierry; Schoone, Gerard J.; Kager, Piet A.; Lubega, George W.; Schallig, Henk D. F. H.

    2009-01-01

    Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma brucei parasites in blood for the diagnosis of human African trypanosomiasis (HAT). Despite good sensitivity, these techniques are not implemented in HAT control

  15. Evaluation of Palm PCRTM G1-12 System: a portable battery-operated PCR thermal cycler

    Directory of Open Access Journals (Sweden)

    Siti Aminah Ahmed

    2016-08-01

    Full Text Available Polymerase chain reaction (PCR is the basis of recombinant and other molecular biological techniques. Availability of cheap and robust PCR platforms enables the tests to be performed easily, even in resource constrained settings. Herein we compared the efficacy of a portable thermal cycler ( Palm PCRTM G1-12 System for rapid DNA amplification against the standard Peltier-based thermal cycler using plasmid DNA and genomic DNA in single and multiplex PCR experiments. Our study revealed that the Palm PCRTM G1-12 System could be a portable DNA amplification system to conduct various molecular techniques, especially in places where resources are limited.

  16. Application of hydroxylamine hydrochloride for post-PCR sterilization.

    Science.gov (United States)

    Aslanzadeh, J

    1993-04-01

    A major problem in the application of polymerase chain reaction (PCR) in diagnostic laboratories is contamination with exogenous nucleic acid, especially aerosolized amplicon, from previous PCR. Although several pre- and post-PCR sterilization techniques have been proposed, an optimal sterilization technique is not yet available. Hydroxylamine hydrochloride is a mutagenic agent that binds to and chemically modifies DNA. In the present study PCR was performed on DNA extracted from Herpes simplex virus (HSV) and Borrelia burgdorferi with two sets of primers that amplified a 92 bp sequence unique to HSV DNA polymerase gene and a 156 bp sequence unique to B. burgdorferi Osp-A gene (35 cycles). Following the amplification, PCR products were treated with 0-500 mM hydroxylamine hydrochloride and incubated at room temperature for 30 min. One microlitre of each hydroxylamine treated PCR product was reamplified for an additional 35 cycles. Pre- and post-hydroxylamine treated PCR products were separated by electrophoresis in 3% agarose gel. Hydroxylamine, at a concentration of 250 mM or higher, was found to effectively modify PCR products and prevent their amplification in subsequent PCR.

  17. Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing

    Directory of Open Access Journals (Sweden)

    Plant Ramona N

    2006-08-01

    Full Text Available Abstract Background Whole genome amplification is an increasingly common technique through which minute amounts of DNA can be multiplied to generate quantities suitable for genetic testing and analysis. Questions of amplification-induced error and template bias generated by these methods have previously been addressed through either small scale (SNPs or large scale (CGH array, FISH methodologies. Here we utilized whole genome sequencing to assess amplification-induced bias in both coding and non-coding regions of two bacterial genomes. Halobacterium species NRC-1 DNA and Campylobacter jejuni were amplified by several common, commercially available protocols: multiple displacement amplification, primer extension pre-amplification and degenerate oligonucleotide primed PCR. The amplification-induced bias of each method was assessed by sequencing both genomes in their entirety using the 454 Sequencing System technology and comparing the results with those obtained from unamplified controls. Results All amplification methodologies induced statistically significant bias relative to the unamplified control. For the Halobacterium species NRC-1 genome, assessed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 119 times greater than those from unamplified material, 164.0 times greater for Repli-G, 165.0 times greater for PEP-PCR and 252.0 times greater than the unamplified controls for DOP-PCR. For Campylobacter jejuni, also analyzed at 100 base resolution, the D-statistics from GenomiPhi-amplified material were 15 times greater than those from unamplified material, 19.8 times greater for Repli-G, 61.8 times greater for PEP-PCR and 220.5 times greater than the unamplified controls for DOP-PCR. Conclusion Of the amplification methodologies examined in this paper, the multiple displacement amplification products generated the least bias, and produced significantly higher yields of amplified DNA.

  18. Applications of Molecular Diagnostic Techniques for Infectious ...

    African Journals Online (AJOL)

    Molecular diagnostic techniques involve a variety of techniques that explore the use of nucleic acid molecules for the identification of a particular pathogenic organism. These techniques include nucleic acid-based typing system, nucleic acid analysis without amplification, polymerase chain reaction (PCR) and other nucleic ...

  19. Development of 23 individual TaqMan® real-time PCR assays for identifying common foodborne pathogens using a single set of amplification conditions.

    Science.gov (United States)

    Cremonesi, Paola; Pisani, Laura Francesca; Lecchi, Cristina; Ceciliani, Fabrizio; Martino, Pieranna; Bonastre, Armand Sanchez; Karus, Avo; Balzaretti, Claudia; Castiglioni, Bianca

    2014-10-01

    Most of the acute intestinal diseases are caused by foodborne pathogens with infants and elderly people being at major risk. The aim of this study was to develop a procedure to simultaneously detect 20 foodborne pathogens in complex alimentary matrices such as milk, cheese and meat. The list of targets include, among the others, Listeria spp., Salmonella spp., Shigella spp., Escherichia coli spp., Campylobacter spp., Clostridium spp. and Staphylococcus aureus. The accuracy of detection was determined by using ATCC strains as positive and negative controls. The achieved sensitivity of each of assays was 1 pg of genomic DNA, which was equivalent to ∼1 cfu. The working ranges of the TaqMan(®) Real-time PCR assays, when used quantitatively on cheese and meat samples inoculated with serial dilution of Listeria spp., Listeria monocytogenes, S. aureus, Salmonella enterica, Shigella boydii, E. coli O157:H7, Bacillus cereus, Campylobacter coli, Yersinia enterocolitica, Enterobacter sakazakii and Pseudomonas aeruginosa was 10(8) cfu/g to 10(4) cfu/g. No matrix interferences were observed. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2013-05-01

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

  1. Quantum dots for a high-throughput Pfu polymerase based multi-round polymerase chain reaction (PCR).

    Science.gov (United States)

    Sang, Fuming; Zhang, Zhizhou; Yuan, Lin; Liu, Deli

    2018-02-26

    Multi-round PCR is an important technique for obtaining enough target DNA from rare DNA resources, and is commonly used in many fields including forensic science, ancient DNA analysis and cancer research. However, multi-round PCR is often aborted, largely due to the accumulation of non-specific amplification during repeated amplifications. Here, we developed a Pfu polymerase based multi-round PCR technique assisted by quantum dots (QDs). Different PCR assays, DNA polymerases (Pfu and Taq), DNA sizes and GC amounts were compared in this study. In the presence of QDs, PCR specificity could be retained even in the ninth-round amplification. Moreover, the longer and more complex the targets were, the earlier the abortion happened in multi-round PCR. However, no obvious enhancement of specificity was found in multi-round PCR using Taq DNA polymerase. Significantly, the fidelity of Pfu polymerase based multi-round PCR was not sacrificed in the presence of QDs. Besides, pre-incubation at 50 °C for an hour had no impact on multi-round PCR performance, which further authenticated the hot start effect of QDs modulated in multi-round PCR. The findings of this study demonstrated that a cost-effective and promising multi-round PCR technique for large-scale and high-throughput sample analysis could be established with high specificity, sensibility and accuracy.

  2. Evaluation of cost-effective total nucleic acids extraction protocols for cultured Mycobacterium tuberculosis; a comparison by PCR amplification of genes associated with drug resistance

    Directory of Open Access Journals (Sweden)

    Gyamfi Oti K

    2010-02-01

    Full Text Available Abstract Background The emergence of drug resistant strains of Mycobacterium tuberculosis complex has made the management of tuberculosis difficult. Also, Mycobacterium species has a peculiar cell wall, made of an impermeable complex structure rich in mycolate, making the lyses of its cell difficult. In order to apply a radio-labelled-probe based detection of mutations in selected genes leading to drug resistance, we concede that the evaluation and modifications of nucleic acid extraction protocols that are less sophisticated and less prone to contamination would be useful in the management of tuberculosis in a resource-constrained setting. Findings The average amount of nucleic acids was determined for different extraction treatments. High temperature treatment only, yielded the lowest amount of nucleic acids, i.e. 15.7 ± 3.2 μg. The average amount of nucleic acids obtained with the addition of TE and triton-X100, was 133.7 ± 8.9 μg, while that obtained with the addition of TE only, and TE and SDS were 68.4 ± 22.7 μg and 70.4 ± 20.3 μg respectively. Other treatments yielded 28.8 ± 6.7 μg, 32.5 ± 2.4 μg and 36.9 ± 15.5 μg. The average amount of nucleic acids obtained with high temperature treatment in TE, and that obtained by freezing prior to high temperature treatment, successfully amplified for the genes of interest (rpoB, KatG, rrs. Conclusion We strongly recommend the use of 1× TE buffer, and freezing and heating for improved lysis of cultured M. tuberculosis, and therefore, as an effective method for the preparation of M. tuberculosis nucleic acid useful for PCR.

  3. Scientific publications about DNA structure-function and PCR technique in Costa Rica: a historic view (1953-2003).

    Science.gov (United States)

    Albertazzi, Federico J

    2004-09-01

    The spreading of knowledge depends on the access to the information and its immediate use. Models are useful to explain specific phenomena. The scientific community accepts some models in Biology after a period of time, once it has evidence to support it. The model of the structure and function of the DNA proposed by Watson & Crick (1953) was not the exception, since a few years later the DNA model was finally accepted. In Costa Rica, DNA function was first mentioned in 1970, in the magazine Biologia Tropical (Tropical Biology Magazine), more than 15 years after its first publication in a scientific journal. An opposite situation occurs with technical innovations. If the efficiency of a new scientific technique is proved in a compelling way, then the acceptance by the community comes swiftly. This was the case of the polymerase chain reaction, or PCR. The first PCR machine in Costa Rica arrived in 1991, only three years after its publication.

  4. C-MYC amplification and expression in stomach cancer samples in Iranian population using two techniques of CISH and IHC

    Directory of Open Access Journals (Sweden)

    Malihea Khaleghian

    2015-07-01

    Results: Our data revealed that both diffuse and intestinal types of gastric cancer occurred significantly in men more than women. Our results showed an indication of some correlation between grades and CISH results, although the difference was not significant. Our data also showed that CISH+ patients (43.1% were more frequent in comparison with IHC+ patients (14.7%. There was a correlation between CISH and IHC. This result revealed that there was a significant difference between grades and IHC. There was also no statistically significant difference between CISH amplification in diffuse and intestinal types. Conclusion: Our conclusion is that for the treatment, management of stomach cancer, and monitoring of progress and prognosis of the tumor that is almost important for patients and clinicians, CISH test is a better and feasible to IHC test, with regards to sensitivity and specificity.

  5. A low cost technique for synthesis of gold nanoparticles using microwave heating and its application in signal amplification for detecting Escherichia Coli O157:H7 bacteria

    Science.gov (United States)

    Thanh Ngo, Vo Ke; Giang Nguyen, Dang; Phat Huynh, Trong; Lam, Quang Vinh

    2016-09-01

    In the present work a low cost technique for preparation of gold nanoparticles (AuNPs) using microwave heating was developed. The effect of different elements (precursor reagents, irradiation time, and microwave radiation power) on the final morphology of AuNPs obtained through the microwave assisted technique has been investigated. The characterization of the samples has been carried out by transmission electron microscopy, UV-vis absorption spectroscopy, Fourier transform infrared spectroscopy, and powder x-ray diffraction. The results showed that to some extent the above-mentioned characterizations influenced the size of synthetized nanoparticles and application of microwave heating has many advantages such as low cost, rapid preparation and highly uniform particles. As an application in quartz crystal microbalance (QCM) immunosensor, AuNPs are conjugated with the Escherichia coli (E.coli) O157:H7 antibodies for signal amplification to detect E.coli O157:H7 bacteria residual in QCM system.

  6. Application of PCR-mediated DNA typing in the molecular epidemiology of medically important microorganisms

    NARCIS (Netherlands)

    A.F. van Belkum (Alex)

    1996-01-01

    textabstractThis thesis describes the development, application and validation of the newer DNA analysis techniques within the field of microbiological epidemiology. Emphasis is placed on the use of the polymerase chain reaction (PCR), a test-tube technique enabling the amplification of (parts of)

  7. Rapid Methods to Distinguish Heterodera schachtii from Heterodera glycines Using PCR Technique

    Directory of Open Access Journals (Sweden)

    Hyoung Rai Ko

    2017-09-01

    Full Text Available The purpose of this study was to develop rapid methods for distinguishing between Heterodera schachtii and H. glycines detected from chinese cabbage fields of highland in Gangwon, Korea. To do this, we performed PCR-RFLP and PCR with the primers set developed in this study for GC147, GC408 and PM001 population, H. schachtii, and YS224, DA142 and BC115 population, H. glycines. Eight restriction enzymes generated RFLP profiles of mtDNA COI region for populations of H. schachtii and H. glycines, repectively. As a result, treatment of two restriction enzymes, RsaI and HinfI, were allowed to distinguish H. schachtii from H. glycines based on the differences of DNA band patterns. The primer set, #JBS1, #JBG1 and #JB3R, amplified specific fragments with 277 and 339 bp of H. schachtii, 339 bp of H. glycines, respectively, while it did not amplify fragments from three root-knot nematodes and two root-lesion nematodes. Thus, the primer set developed in this study could be a good method, which is used to distinguish between H. schachtii and H. glycines.

  8. Development of pork DNA detection in meat products by PCR technique

    Directory of Open Access Journals (Sweden)

    Intaraphad, U.

    2005-09-01

    Full Text Available The polymerase chain reaction (PCR was used to determine pig DNA fragment in meat mixture using specific oligonucleotide primer having 10-30 bp in size to amplify the DNA fragment. Firstly, the β-actin F and the β-actin R primers were used to identify DNA extraction process in beef, pork and chicken. The 284 bp DNA fragment was obtained from beef and chicken meat and 248 bp was amplified from pork. Secondly, pig F and pig R primers for pork were used as the PCR amplifier which resulted in 531 bp DNA fragment from pork; however, it gave negative results for beef and chicken meat. Finally, the obtained nucleotide sequence was compared with AF535163 in the GenBank database and homology was 98%. This work was also submitted to GenBank and obtained an accession number of AY621117.A comparison between the sensitivity of the commercial kit (QIAamp kit and the RSB lysis buffer for the DNA extraction process was carried out by mixing pork with chicken meat at ratios of 1:1, 1:5, 1:10, 1:50, 1:150 and 1:200. QIAamp kit gave the best results at the smallest ratio of 0.5% (1:200, while the RSB lysis buffer gave good results at the ratio of 2% (1:50. This indicated that the QIAamp kit had a higher sensitivity than the RSB lysis buffer. Lastly, a determination of pork DNA fragment from heated pork at 121ºC for 15 min. using the Pig F and Pig R primers for pork were done. The result was similar to that obtained from the fresh pork at 531 bp DNA fragment.

  9. Use of RT-PCR and elisa techniques for the diagnostic of infectious bronchitis virus in broilers at slaughter

    Directory of Open Access Journals (Sweden)

    Davi de Oliveira Almeida

    2015-03-01

    Full Text Available ABSTRACT. Almeida D.O., Tortely R., Nascimento E.R., Khan M., Pereira V.L.A & Babapoor S. [Use of RT-PCR and elisa techniques for the diagnostic of infectious bronchitis virus in broilers at slaughter.] Uso das técnicas de RT-PCR e elisa no diagnóstico da bronquite infecciosa em frangos de corte ao abate. Revista Brasileira de Medicina Veterinária, 37(1:55-59, 2015. Departamento de Saúde Coletiva Veterinária e Saúde Pública, Universidade Federal Fluminense, Rua Vital Brazil Filho, 64, Santa Rosa, Niterói, RJ 24230-340, Brasil. E-mail: elmiro@vm.uff.br Avian infectious bronchitis (IB is a viral, acute and highly contagious disease caused by infectious bronchitis virus (IBV. The disease affects broilers improvement and performance causing major economic losses in the world poultry industry. Generally, IBV infections can be diagnosed by detection of IBV itself or the specific antibody response. This study aimed to detect IBV in broilers under Sanitary Inspection by RT-PCR and ELISA, comparing the results and relating them with the average weight of the flock. Samples were collected from 40 vaccinated broiler flocks under Sanitary Inspection at slaughter. Ten birds from every flock were randomly selected and blood samples were collected for ELISA as well three birds had their trachea and caecal tonsils collected for RT- -PCR test. From 40 flocks, 30 were IBV positive by ELISA and 26 by RT-PCR, in which 15 were detected simultaneously in trachea and caecal tonsil and 5 in each sample. There was no agreement between ELISA and RT-PCR results regarding IBV diagnosis as well positivity in both tests was not statistically significant with the average weight of the flock. There was an improvement on IBV diagnosis when caecal tonsils and tracheas were used instead of just one of them. Considering the only vaccine serotype allowed by Brazilian government is the Mass serotype and its persistence in broilers would only be detected up to 28 days

  10. Multiple strand displacement amplification of mitochondrial DNA from clinical samples

    Directory of Open Access Journals (Sweden)

    Sidransky David

    2008-02-01

    Full Text Available Abstract Background Whole genome amplification (WGA methods allow diagnostic laboratories to overcome the common problem of insufficient DNA in patient specimens. Further, body fluid samples useful for cancer early detection are often difficult to amplify with traditional PCR methods. In this first application of WGA on the entire human mitochondrial genome, we compared the accuracy of mitochondrial DNA (mtDNA sequence analysis after WGA to that performed without genome amplification. We applied the method to a small group of cancer cases and controls and demonstrated that WGA is capable of increasing the yield of starting DNA material with identical genetic sequence. Methods DNA was isolated from clinical samples and sent to NIST. Samples were amplified by PCR and those with no visible amplification were re-amplified using the Multiple Displacement Amplificaiton technique of whole genome amplification. All samples were analyzed by mitochip for mitochondrial DNA sequence to compare sequence concordance of the WGA samples with respect to native DNA. Real-Time PCR analysis was conducted to determine the level of WGA amplification for both nuclear and mtDNA. Results In total, 19 samples were compared and the concordance rate between WGA and native mtDNA sequences was 99.995%. All of the cancer associated mutations in the native mtDNA were detected in the WGA amplified material and heteroplasmies in the native mtDNA were detected with high fidelity in the WGA material. In addition to the native mtDNA sequence present in the sample, 13 new heteroplasmies were detected in the WGA material. Conclusion Genetic screening of mtDNA amplified by WGA is applicable for the detection of cancer associated mutations. Our results show the feasibility of this method for: 1 increasing the amount of DNA available for analysis, 2 recovering the identical mtDNA sequence, 3 accurately detecting mtDNA point mutations associated with cancer.

  11. HLA-DPB1 typing with polymerase chain reaction and restriction fragment length polymorphism technique in Danes

    DEFF Research Database (Denmark)

    Hviid, T V; Madsen, H O; Morling, N

    1992-01-01

    We have used the polymerase chain reaction (PCR) in combination with the restriction fragment length polymorphism (RFLP) technique for HLA-DBP1 typing. After PCR amplification of the polymorphic second exon of the HLA-DPB1 locus, the PCR product was digested with seven allele-specific restriction...

  12. Two-temperature PCR for Microfluidics

    KAUST Repository

    Kodzius, Rimantas

    2010-05-01

    Since its invention in 1983, polymerase chain reaction (PCR) has been the method of choice for DNA amplification. Successful PCR depends on the optimization of several parameters, which is a cumbersome task due to the many variables (conditions and compon

  13. Pyrosequencing for classification of human FcγRIIIA allotypes: a comparison with PCR-based techniques.

    Science.gov (United States)

    Matlawska-Wasowska, Ksenia; Gale, James M; Nickl, Christian K; Khalili, Parisa; Shirley, Brian; Wilson, Bridget S; Vasef, Mohammad A; Winter, Stuart S

    2014-12-01

    Surface-specific antigens expressed by hematopoietic cells are attractive targets for antibody-mediated immunotherapy. Monoclonal antibodies (mAbs) involve various mechanisms to eliminate target cells, including antibody-dependent cellular cytotoxicity (ADCC)- and phagocytosis (ADCP)-mediated killing through natural killer (NK) and macrophage effector cells bearing FcγRIIIA (CD16). The clinical efficacy of ADCC is particularly impacted by a single nucleotide polymorphism (SNP) found in the gene encoding FcγRIIIA (FCGR3A), which generates a variable distribution of the 158 V/V, F/V or F/F CD16 allotypes (F = phenylalanine, V = valine) in the normal human population. Currently, most patients are not screened for CD16 allotypes, creating the potential to include in their treatment a mAb-based therapy that may have limited benefit. Therefore, it is important to identify CD16 allotypes when considering mAb therapies that require ADCC/ADCP. The objective of this study was to develop a reliable PCR-based assay for classification of human FcγRIIIA allotypes. We studied 42 normal human subjects for the incidence of FcγRIIIA-158 polymorphisms using comparative molecular approaches. The results of our study showed 100% accuracy in genotyping by pyrosequencing. In contrast, nested PCR-based allele-specific restriction assay and quantitative PCR techniques proved to be relatively less sensitive and less specific in distinguishing variant genotypes. Since the efficacy of the mAb-based targeted immunotherapy may be highly dependent upon the CD16 polymorphism in a given individual, we recommend pyrosequencing for CD16 allotype testing.

  14. False-positive results and contamination in nucleic acid amplification assays : Suggestions for a prevent and destroy strategy

    NARCIS (Netherlands)

    Borst, A; Box, ATA; Fluit, AC

    Contamination of samples with DNA is still a major problem in microbiology laboratories, despite the wide acceptance of PCR and other amplification techniques for the detection of frequently low amounts of target DNA. This review focuses on the implications of contamination in the diagnosis and

  15. Detection of enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification.

    Science.gov (United States)

    Wang, D; Wang, X; Geng, Y; An, C

    2014-01-01

    The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop-mediated isothermal amplification (LAMP) technique. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conventional reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT-LAMP. The detection rate for EV71 was 56.89% by RT-LAMP, 41.38% by real-time PCR and 34.48% by RT-PCR. The minimum detection limit of RT-LAMP was 0.01 PFU, both of RT-PCR and real-time PCR was 0.1PFU. Non-cross-reactive amplification with other enteroviruses was detected in the survey reports. The effectiveness of RT-LAMP is higher than RT-PCR and real-time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource-poor countries or in developing countries.

  16. Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    D Wang

    2014-01-01

    Full Text Available Purpose : The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD for an early treatment by using loop-mediated isothermal amplification (LAMP technique. Materials and Methods : A reverse-transcription loop-mediated isothermal amplification (RT-LAMP for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conventional reverse-transcription polymerase chain reaction (RT-PCR and real-time PCR. Results : A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT-LAMP. The detection rate for EV71 was 56.89% by RT-LAMP, 41.38% by real-time PCR and 34.48% by RT-PCR. The minimum detection limit of RT-LAMP was 0.01 PFU, both of RT-PCR and real-time PCR was 0.1PFU. Non-cross-reactive amplification with other enteroviruses was detected in the survey reports. Conclusions : The effectiveness of RT-LAMP is higher than RT-PCR and real-time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource-poor countries or in developing countries.

  17. Rapid and Inexpensive Screening of Genomic Copy Number Variations Using a Novel Quantitative Fluorescent PCR Method

    Directory of Open Access Journals (Sweden)

    Martin Stofanko

    2013-01-01

    Full Text Available Detection of human microdeletion and microduplication syndromes poses significant burden on public healthcare systems in developing countries. With genome-wide diagnostic assays frequently inaccessible, targeted low-cost PCR-based approaches are preferred. However, their reproducibility depends on equally efficient amplification using a number of target and control primers. To address this, the recently described technique called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR was shown to reliably detect four human syndromes by quantifying DNA amplification in an internally controlled PCR reaction. Here, we confirm its utility in the detection of eight human microdeletion syndromes, including the more common WAGR, Smith-Magenis, and Potocki-Lupski syndromes with 100% sensitivity and 100% specificity. We present selection, design, and performance evaluation of detection primers using variety of approaches. We conclude that MQF-PCR is an easily adaptable method for detection of human pathological chromosomal aberrations.

  18. Changes in Growth, Genomic DNA, Protein Profiles in Wheat Plant Using Physiological and RAPD-PCR Techniques

    International Nuclear Information System (INIS)

    El-Tarras, A.

    2002-01-01

    Wheat is the major winter cereal crop in the world. The total cultivated area of this crop in Egypt is about two million feddans. Soil salinity represent a serious problem to agriculture in arid and semi-arid in the world. Mexico wheat (Triticum vulgar var. Ycora rojo) was imported in 1999 for cultivation. Mexico wheat was exposed to gamma rays (cobalt 60) from 10 to 80 Krad The unirradiated and irradiated wheat were cultivated in the presence of 0, 5000,10000 and 20000 mg/L of salt solution and 16 hour light /25 degree C. The previous treatment was repeated in combination with 5, 10 mg/l ABA and 10, 20 mg/l GA3 separately. Different accessed parameters were used for evaluation, these parameters were: germination percentage, length of shoots and roots, pigment contents (chl. a,b and a/b carotenoids and total pigments), total protein patterns and RAPD, PCR techniques. The results showed that both of radiation and salinity reduced the percentage of germination. Soaking grains in GA3 considerably increased the shoot and root lengths. Highest value of carotenoids obtained act as a defense mechanism against harmful salinity action. Also, the seedling exposed to 80 Krad and treated with ABA (5 or 10 mg/l) can survive during the experimental period, while plants treated with 10 and 20 mg/l GA3 and exposed to 80 Krad can not survive. At low radiation doses (10 and 20 Krad) there was no difference in the number and density of bands of the total protein patterns, while in the RAPD, PCR technique in presence and/or absence of DNA band in unirradiated and irradiated wheat seeds were observed

  19. CODEHOP PCR and CODEHOP PCR primer design.

    Science.gov (United States)

    Staheli, Jeannette P; Boyce, Richard; Kovarik, Dina; Rose, Timothy M

    2011-01-01

    While PCR primer design for the amplification of known sequences is usually quite straightforward, the design, and successful application of primers aimed at the detection of as yet unknown genes is often not. The search for genes that are presumed to be distantly related to a known gene sequence, such as homologous genes in different species, paralogs in the same genome, or novel pathogens in diverse hosts, often turns into the proverbial search for the needle in the haystack. PCR-based methods commonly used to address this issue involve the use of either consensus primers or degenerate primers, both of which have significant shortcomings regarding sensitivity and specificity. We have developed a novel primer design approach that diminishes these shortcomings and instead takes advantage of the strengths of both consensus and degenerate primer designs, by combining the two concepts into a Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) approach. CODEHOP PCR primers contain a relatively short degenerate 3' core and a 5' nondegenerate clamp. The 3' degenerate core consists of a pool of primers containing all possible codons for a 3-4 aminoacid motif that is highly conserved in multiply aligned sequences from known members of a protein family. Each primer in the pool also contains a single 5' nondegenerate nucleotide sequence derived from a codon consensus across the aligned aminoacid sequences flanking the conserved motif. During the initial PCR amplification cycles, the degenerate core is responsible for specific binding to sequences encoding the conserved aminoacid motif. The longer consensus clamp region serves to stabilize the primer and allows the participation of all primers in the pool in the efficient amplification of products during later PCR cycles. We have developed an interactive web site and algorithm (iCODEHOP) for designing CODEHOP PCR primers from multiply aligned protein sequences, which is freely available online. Here, we describe the

  20. Real-time quantitative PCR of microdissected paraffin-embedded breast carcinoma

    DEFF Research Database (Denmark)

    Gjerdrum, Lise Mette; Sorensen, Boe Sandahl; Kjeldsen, Eigil

    2004-01-01

    We studied the feasibility of using real-time quantitative PCR to determine HER-2 DNA amplification and mRNA expression in microdissected formalin-fixed, paraffin-embedded breast tumors and compared this with standard immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) methods...... tumors as being amplified. Interestingly, all these scored 2+ with the HercepTest, but were negative using FISH. We believe that real-time quantitative PCR analysis of HER-2 DNA amplification following microdissection represents a useful supplementary or perhaps even an alternative technique...

  1. A review on different types of Real-time PCR methods and its optimization

    Directory of Open Access Journals (Sweden)

    M. Keshavarz

    2017-08-01

    Full Text Available In the recent years, Real-time PCR technique introduced as a choice method for diagnosis of infectious diseases in many laboratories. During each cycle of the PCR reaction, this technique combines the polymerase chain reaction chemistry with the utilization of fluorescent reporter molecules for monitoring the production of amplification products. Therefore, the set of features including the high sensitivity and specificity, repeatable data and low contamination risk has made the Real-time PCR technology as an attractive alternative to conventional PCR. This technique is often used to quantify the level of gene expression. Since the whole Real-time PCR reaction is performed within a closed tube, the risk of contamination is reduced and eventually prevent falsepositive results. The aim of present study was to provide a general overview on different types of Real-time PCR methods, their benefits and applications.

  2. Evaluation of ALK gene rearrangement in central nervous system metastases of non-small-cell lung cancer using two-step RT-PCR technique.

    Science.gov (United States)

    Nicoś, M; Krawczyk, P; Wojas-Krawczyk, K; Bożyk, A; Jarosz, B; Sawicki, M; Trojanowski, T; Milanowski, J

    2017-12-01

    RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.

  3. The use of PCR techniques to detect genetic variations in Cassava (Manihot esculenta L. Crantz): minisatellite and RAPD analysis

    International Nuclear Information System (INIS)

    Pawlicki, N.; Sangwan, R.S.; Sangwan-Norreel, B.; Koffi Konan, N.

    1998-01-01

    Cassava is an important tuber crop grown in the tropical and subtropical regions. Recently, we developed protocols for efficient somatic embryogenesis using zygotic embryos and nodal axillary meristems in order to reduce the genotype effect. Thereafter flow cytophotometry and randomly amplified polymorphic DNA (RAPD) markers were used to assess the ploidy level and the genetic fidelity of cassava plants regenerated by somatic embryogenesis. No change in the ploidy level of the regenerated plants was observed in comparison with the control plants. In the same way, monomorphic profiles of RAPD were obtained for the different cassava plants regenerated by somatic embryogenesis. The genetic analysis of calli showed only a few differences. Using two pairs of heterologous micro satellite primers developed in a wild African grass, a monomorphic pattern was also detected. Moreover, cultivars of different origins were also analysed using these PCR techniques. Our data from RAPD and materialistic analyses suggested that these techniques can be efficiently used to detect genetic variations in cassava. (author)

  4. Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes.

    Science.gov (United States)

    Scheen, Anne-Cathrine; Pfeil, Bernard E; Petri, Anna; Heidari, Nahid; Nylinder, Stephan; Oxelman, Bengt

    2012-01-01

    Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing. © 2011 Blackwell Publishing Ltd.

  5. Detection of peroxyl radicals from polluted air by free radical reaction combined with liquid chromatography signal amplification technique.

    Science.gov (United States)

    Wang, Guoying; Jia, Shiming; Niu, Xiuli; Liu, Yanrong; Tian, Haoqi; Chen, Xuefu; Shi, Gaofeng

    2018-01-22

    Free radicals play an important role in the oxidizing power of polluted air, the development of aging-related diseases, the formation of ozone, and the production of secondary particulate matter. The high variability of peroxyl radical concentration has prevented the detection of possible trends or distributions in the concentration of free radicals. We present a new method, free radical reaction combined with liquid chromatography photodiode array detection, for identifying and quantifying peroxyl radicals in polluted air. Functionalized graphene was used for loading peroxyl radicals and reactive molecules in air sampling system, which can facilitate reaction kinetics (charge transfers) between peroxyl radicals and reaction molecules. Separation was performed with and without a preliminary exposure of the polluted air sample to reactive molecule(s) system. The integral chromatographic peak areas before and after air sampling are used to quantify the atmospheric peroxyl radicals in polluted air. The utility of the new technique was tested with measurements carried out in the field. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

    Science.gov (United States)

    Sang, Fuming; Yang, Yang; Yuan, Lin; Ren, Jicun; Zhang, Zhizhou

    2015-09-01

    Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour preincubation at 50 °C before real time PCR. Moreover, the results obtained by QD-based HS PCR were comparable to a commercial Taq antibody DNA polymerase. However, no obvious HS effect of QDs was found in real time PCR using Taq DNA polymerase. The findings of this study demonstrated that a cost-effective high-throughput real time PCR based on QD triggered HS PCR could be established with high consistency, sensitivity and accuracy.Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour

  7. Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR).

    Science.gov (United States)

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

    2017-01-01

    A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

  8. Clinical utility of multiplex ligation-dependent probe amplification technique in identification of aetiology of unexplained mental retardation: A study in 203 Indian patients

    Directory of Open Access Journals (Sweden)

    Vijay R Boggula

    2014-01-01

    Full Text Available Background & objectives: Developmental delay (DD/mental retardation also described as intellectual disability (ID, is seen in 1-3 per cent of general population. Diagnosis continues to be a challenge at clinical level. With the advancement of new molecular cytogenetic techniques such as cytogenetic microarray (CMA, multiplex ligation-dependent probe amplification (MLPA techniques, many microdeletion/microduplication syndromes with DD/ID are now delineated. MLPA technique can probe 40-50 genomic regions in a single reaction and is being used for evaluation of cases with DD/ID. In this study we evaluated the clinical utility of MLPA techniques with different probe sets to identify the aetiology of unexplained mental retardation in patients with ID/DD. Methods: A total of 203 randomly selected DD/ID cases with/without malformations were studied. MLPA probe sets for subtelomeric regions (P070/P036 and common microdeletions/microduplications (P245-A2 and X-chromosome (P106 were used. Positive cases with MLPA technique were confirmed using either fluorescence in situ hybridization (FISH or follow up confirmatory MLPA probe sets. Results: The overall detection rate was found to be 9.3 per cent (19 out of 203. The detection rates were 6.9 and 7.4 per cent for common microdeletion/microduplication and subtelomeric probe sets, respectively. No abnormality was detected with probe set for X-linked ID. The subtelomeric abnormalities detected included deletions of 1p36.33, 4p, 5p, 9p, 9q, 13q telomeric regions and duplication of 9pter. The deletions/duplications detected in non telomeric regions include regions for Prader Willi/Angelman regions, Williams syndrome, Smith Magenis syndrome and Velocardiofacial syndrome. Interpretation & conclusions: Our results show that the use of P245-A2 and P070/P036-E1 probes gives good diagnostic yield. Though MLPA cannot probe the whole genome like cytogenetic microarray, due to its ease and relative low cost it is an

  9. Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR).

    Science.gov (United States)

    Datta, Sibnarayan; Budhauliya, Raghvendra; Chatterjee, Soumya; Vanlalhmuaka; Veer, Vijay; Chakravarty, Runu

    2016-06-01

    Polymerase chain reaction (PCR) is widely used in biological research and diagnostics because of its high sensitivity and specificity. However, the sensitivity of PCR is strongly influenced by topological characteristics of the template. Supercoiled templates are known to inhibit PCR, whereas linearized forms of the same supercoiled templates facilitate PCR. This study was conducted to compare the PCR efficiency of circular supercoiled DNA templates to their restriction endonuclease (RE)-mediated linearized forms. Additionally, we also evaluated the possibility of RE digestion of the circular supercoiled templates within the complete PCR buffer. Following a systematic approach, we demonstrated that circular supercoiled templates could be efficiently linearized by RE in the complete PCR buffer itself. This allowed linearization of circular supercoiled templates and their subsequent amplification in the PCR buffer in a single-tube format. Using this extremely simple RE-PCR approach, we documented up to tenfold increases in detection efficiency of PCR with two different circular supercoiled templates of clinical origin, including an international calibration standard. This inexpensive and easy approach to increasing PCR sensitivity can be easily adapted to any standard PCR protocol aimed at amplifying circular supercoiled genomes. Apart from its application in the development of sensitive clinical diagnostic PCR assays for a large number of organisms, this method could also prove to be very useful in simplifying the existing protocols for other applications where pre-PCR restriction digestion is required, such as mutation detection, genotyping, and selective template amplification.

  10. Pork detection in binary meat mixtures and some commercial food products using conventional and real-time PCR techniques.

    Science.gov (United States)

    Al-Kahtani, Hassan A; Ismail, Elsayed A; Asif Ahmed, Mohammed

    2017-03-15

    Pork DNA was detected in meat mixtures using both conventional PCR and real-time PCR (RT-PCR). Thirty meat mixtures containing beef, chicken, camel, rabbit, goat and sheep with varying percentage of pork (0%, 1%, 5%, 10%, and 20%) and 75 commercial food products, were analyzed using conventional and RT-PCR to determine the presence of pork DNA. Pork DNA standard curves and cycle threshold (Ct) values were used for quantification. The detection limits for pork DNA in the mixtures were 0.22, 0.047, 0.048, 0.0000037, 0.015ng/μl respectively. Unlike conventional PCR, RT-PCR detected pork DNA in nine processed food samples [chicken sausages (2), chicken luncheon (2), turkey meat loaf, milk chocolate with soft nougat, jelly, cake, and candies] at pork DNA concentrations of 0.0001ng/μl or less. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Description of a PCR-based technique for DNA splicing and mutagenesis by producing 5' overhangs with run through stop DNA synthesis utilizing Ara-C

    Directory of Open Access Journals (Sweden)

    Silverman Mel

    2005-09-01

    Full Text Available Abstract Background Splicing of DNA molecules is an important task in molecular biology that facilitates cloning, mutagenesis and creation of chimeric genes. Mutagenesis and DNA splicing techniques exist, some requiring restriction enzymes, and others utilize staggered reannealing approaches. Results A method for DNA splicing and mutagenesis without restriction enzymes is described. The method is based on mild template-dependent polymerization arrest with two molecules of cytosine arabinose (Ara-C incorporated into PCR primers. Two rounds of PCR are employed: the first PCR produces 5' overhangs that are utilized for DNA splicing. The second PCR is based on polymerization running through the Ara-C molecules to produce the desired final product. To illustrate application of the run through stop mutagenesis and DNA splicing technique, we have carried out splicing of two segments of the human cofilin 1 gene and introduced a mutational deletion into the product. Conclusion We have demonstrated the utility of a new PCR-based method for carrying out DNA splicing and mutagenesis by incorporating Ara-C into the PCR primers.

  12. [Retrospective study of the implementation of the qualitative PCR technique in biological samples for monitoring toxoplasmosis in pediatric patients receiving hematopoietic stem cell transplantation].

    Science.gov (United States)

    Nigro, Mónica G; Figueroa, Carlos; Ledesma, Bibiana A

    2014-01-01

    Toxoplasmosis is an opportunistic infection caused by the parasite Toxoplasma gondii. The infection is severe and difficult to diagnose in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). Twelve patients receiving HSCT were monitored post-transplant, by qualitative PCR at the Children's Hospital S.A.M.I.C. "Prof. Dr. Juan P. Garrahan". The monitoring of these patients was defined by a history of positive serology for toxoplasmosis in the donor or recipient and because their hematologic condition did not allow the use of trimethoprim-sulfamethoxazole for prophylaxis. During the patients' monitoring, two of them with positive PCR results showed signs of illness by T. gondii and were treated with pyrimethamine-clindamycin. In two other patients, toxoplasmosis was the cause of death and an autopsy finding, showing negative PCR results. Four patients without clinical manifestations received treatment for toxoplasmosis because of positive PCR detection. In four patients there were no signs of toxoplasmosis disease and negative PCR results during follow-up. The qualitative PCR technique proved useful for the detection of toxoplasmosis reactivation in HSCT recipients, but has limitations in monitoring and making clinical decisions due to the persistence of positive PCR over time and manifestations of toxicity caused by the treatment. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

  13. Use of PCR and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Techniques for Differentiation of Prevotella intermedia Sensu Stricto and Prevotella nigrescens

    Science.gov (United States)

    Premaraj, Thyagaseely; Kato, Naoki; Fukui, Katsuhito; Kato, Haru; Watanabe, Kunitomo

    1999-01-01

    Primers were designed from 16S rRNA sequences of Prevotella intermedia sensu stricto and Prevotella nigrescens and were used to discriminate these two species by PCR. The results were compared with those from the PCR technique using primers designed from arbitrarily primed PCR products by Guillot and Mouton (E. Guillot and C. Mouton, J. Clin. Microbiol. 35:1876–1882, 1997). The specificities of both assays were studied by using P. intermedia ATCC 25611, P. nigrescens ATCC 33563, 174 clinical isolates of P. intermedia sensu lato, and 59 reference strains and 58 clinical isolates of other Prevotella species and/or common oral flora. In addition, the usefulness and reliability of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the differentiation of the two species were examined by comparing the results with those from PCR assays. The controversial lipase test for distinguishing these species was also carried out. Unambiguous differentiation was made by both PCR assays, and the results matched each other. The SDS-PAGE assay was found to misidentify a few strains tested, compared with the results of PCR assays. The lipase test was positive for both species, including the reference strains of P. intermedia and P. nigrescens. We conclude that both PCR assays are simple, rapid, reliable, and specific methods which could be used in clinical studies and that the lipase test is not valuable in the differentiation. The reliable discrimination of the two species by SDS-PAGE is questionable. PMID:10074526

  14. The Use of Polymerase Chain Reaction Amplification for the Detection of Viruses and Bacteria in Severe Community-Acquired Pneumonia.

    Science.gov (United States)

    Siow, Wen Ting; Koay, Evelyn Siew-Chuan; Lee, Chun Kiat; Lee, Hong Kai; Ong, Venetia; Ngerng, Wang Jee; Lim, Hui Fang; Tan, Adeline; Tang, Julian Wei-Tze; Phua, Jason

    2016-01-01

    Pathogens are often not identified in severe community-acquired pneumonia (CAP), and the few studies using polymerase chain reaction (PCR) techniques for virus detection are from temperate countries. This study assesses if PCR amplification improves virus and bacteria detection, and if viral infection contributes to mortality in severe CAP in a tropical setting, where respiratory pathogens have less well-defined seasonality. In this cohort study of patients with severe CAP in an intensive care unit, endotracheal aspirates for intubated patients and nasopharyngeal swabs for non-intubated patients were sent for PCR amplification for respiratory viruses. Blood, endotracheal aspirates for intubated patients, and sputum for non-intubated patients were analysed using a multiplex PCR system for bacteria. Out of 100 patients, using predominantly cultures, bacteria were identified in 42 patients; PCR amplification increased this number to 55 patients. PCR amplification identified viruses in 32 patients. In total, only bacteria, only viruses, and both bacteria and viruses were found in 37, 14, and 18 patients, respectively. The commonest viruses were influenza A H1N1/2009 and rhinovirus; the commonest bacterium was Streptococcus pneumoniae. Hospital mortality rates for patients with no pathogens, bacterial infection, viral infection, and bacterial-viral co-infection were 16.1, 24.3, 0, and 5.6%, respectively (p = 0.10). On multivariable analysis, virus detection was associated with lower mortality (adjusted odds ratio 0.12, 95% confidence interval 0.2-0.99; p = 0.049). Viruses and bacteria were detected in 7 of 10 patients with severe CAP with the aid of PCR amplification. Viral infection appears to be independently associated with lower mortality. © 2016 S. Karger AG, Basel.

  15. Quantitative (real-time) PCR

    International Nuclear Information System (INIS)

    Denman, S.E.; McSweeney, C.S.

    2005-01-01

    Many nucleic acid-based probe and PCR assays have been developed for the detection tracking of specific microbes within the rumen ecosystem. Conventional PCR assays detect PCR products at the end stage of each PCR reaction, where exponential amplification is no longer being achieved. This approach can result in different end product (amplicon) quantities being generated. In contrast, using quantitative, or real-time PCR, quantification of the amplicon is performed not at the end of the reaction, but rather during exponential amplification, where theoretically each cycle will result in a doubling of product being created. For real-time PCR, the cycle at which fluorescence is deemed to be detectable above the background during the exponential phase is termed the cycle threshold (Ct). The Ct values obtained are then used for quantitation, which will be discussed later

  16. Technical aspects of typing for HLA-DP alleles using allele-specific DNA in vitro amplification and sequence-specific oligonucleotide probes. Detection of single base mismatches

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1990-01-01

    The polymerase chain reaction (PCR) is an effective method for in vitro DNA amplification which combined with probing with synthetic oligonucleotides can be used for, e.g., HLA-typing. We have studied the technical aspects of HLA-DP typing with the technique. DNA from mononuclear nucleated cells...... was extracted with either a simple salting out method or phenol/chloroform. Both DNAs could be readily used for PCR. The MgC2 concentration of the PCR buffer and the annealing temperature of the thermal cycle of the PCR were the two most important variables. The MgCl2 concentration and the temperature must...... be carefully titrated for each primer pair in the PCR. The influence of mismatches between the primer and the DNA template were studied and we found that, by using primers differing only from each other at the 3' end, cross-amplification of closely homologous alleles could be avoided. Thus, single base...

  17. Ligation-dependent RT-PCR: a new specific and low-cost technique to detect ALK, ROS, and RET rearrangements in lung adenocarcinoma.

    Science.gov (United States)

    Piton, Nicolas; Ruminy, Philippe; Gravet, Claire; Marchand, Vinciane; Colasse, Élodie; Lamy, Aude; Naoures Mear, Cécile Le; Bibeau, Fréderic; Marguet, Florent; Guisier, Florian; Salaün, Mathieu; Thiberville, Luc; Jardin, Fabrice; Sabourin, Jean-Christophe

    2018-03-01

    Detection of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 (ROS1), and rearranged during transfection (RET) gene rearrangements in lung adenocarcinoma is usually performed by immunohistochemistry (IHC) screening followed by fluorescence in situ hybridization (FISH), which is an expensive and difficult technique. Ligation-dependent reverse transcription polymerase chain reaction (RT-PCR) multiplex technique can detect gene rearrangements using probes specifically hybridized to either side of the break point. PCR products are then sequenced by pyrosequencing or high throughput sequencing in order to identify the two genes involved. The reagent cost is <15 dollars per patient and results are available in 2 days. We have developed a 47-probe LD-RT-PCR kit especially for lung adenocarcinomas. Thirty-nine lung adenocarcinomas were studied: 24 ALK+, 14 ROS1+, and 1 RET+. ALK+ and ROS1+ were IHC+ (D5F3 Ventana for ALK and D4D6 Cell Signaling Technology for ROS1) and all cases were FISH+ (Vysis ALK Breakapart Probe Abbott for ALK, Zytolight SPEC ROS1 Dualcolor Breakapart Probe for ROS1 and Zytolight SPEC RET Dual Color Breakapart for RET); 14 wild type samples were included as negative controls. Using LD-RT-PCR, 15 rearrangements (63%) were detected in the ALK cases (gene partner: EML4 in all cases), 9 rearrangements (64%) in the ROS1 cases (gene partners: CD74 in 8 cases and SLC34A2 in 1 case) and 1 (100%) in the single RET case (gene partner: KIF5B). No rearrangement was found in the 14 negative control cases. Negative cases using LD-RT-PCR could be explained by the fact that some partner genes were not included in our assay and therefore could not be detected. Because it is an affordable, fast, and very simple technique, we propose using LD-RT-PCR when ALK immunostaining is positive. For LD-RT-PCR-negative cases, samples should then be analyzed by FISH.

  18. Amplified RNA degradation in T7-amplification methods results in biased microarray hybridizations

    Directory of Open Access Journals (Sweden)

    Ivell Richard

    2003-11-01

    Full Text Available Abstract Background The amplification of RNA with the T7-System is a widely used technique for obtaining increased amounts of RNA starting from limited material. The amplified RNA (aRNA can subsequently be used for microarray hybridizations, warranting sufficient signal for image analysis. We describe here an amplification-time dependent degradation of aRNA in prolonged standard T7 amplification protocols, that results in lower average size aRNA and decreased yields. Results A time-dependent degradation of amplified RNA (aRNA could be observed when using the classical "Eberwine" T7-Amplification method. When the amplification was conducted for more than 4 hours, the resulting aRNA showed a significantly smaller size distribution on gel electrophoresis and a concomitant reduction of aRNA yield. The degradation of aRNA could be correlated to the presence of the T7 RNA Polymerase in the amplification cocktail. The aRNA degradation resulted in a strong bias in microarray hybridizations with a high coefficient of variation and a significant reduction of signals of certain transcripts, that seem to be susceptible to this RNA degrading activity. The time-dependent degradation of these transcripts was verified by a real-time PCR approach. Conclusions It is important to perform amplifications not longer than 4 hours as there is a characteristic 'quality vs. yield' situation for longer amplification times. When conducting microarray hybridizations it is important not to compare results obtained with aRNA from different amplification times.

  19. IDENTIFIKASI TIPE HLA KELAS II DENGAN TEKNIK PCR

    Directory of Open Access Journals (Sweden)

    Ervi Salwati

    2012-09-01

    Full Text Available HLA (Human Leukocyte Antigen contains a set of genes located together on the short arm of chromosome 6. These genes control immune responses, graft acceptance or rejection and tumor surveillance. These abilities have close relationship with genetic variation (occur in "many forms" or alleles that bind and present antigens to T lymphocytes. Using advanced technology and molecular biology approaches (PCR technique detection of genetic variation in the HLA region (or HLA typing has been performed based on DNA.. PCR is an in vitro technique to amplify the DNA sequence enzymatically. "Sequence Specific Primers" (SSP are designed for this PCR to obtain amplification of specific alleles or groups of alleles. The PCR products are visualized through agarose gel electrophoresis stained with ethidium bromide. The PCR technique requires small amount of whole blood (0.5 - 1 ml, gives rapid, accurate and complete result. This paper discuss identification of HLA class II typing using PCR-SSP technique and show the examples of the results.   Key words: HLA (Human Leukocyte Antigen class II, PCR (Polymerase Chain Reaction

  20. The role of DNA amplification and cultural growth in complicated acute appendicitis

    Directory of Open Access Journals (Sweden)

    Francesca Tocchioni

    2016-09-01

    Full Text Available Bacterial growth of peritoneal fluid specimens obtained during surgical procedures for acute appendicitis may be useful to optimize further antibiotic therapy in complicated cases. DNA amplification represents a fast technique to detect microbial sequences. We aimed to compare the potential of DNA amplification versus traditional bacterial growth culture highlighting advantages and drawbacks in a surgical setting. Peritoneal fluid specimens were collected during surgery from 36 children who underwent appendectomy between May and December 2012. Real-time polymerase chain reaction (RT-PCR and cultures were performed on each sample. RT-PCR showed an amplification of 16S in 18/36 samples, Escherichia coli (in 7 cases, Pseudomonas aeruginosa (3, Fusobacterium necrophorum (3, Adenovirus (2, E.coli (1, Klebsiella pneumoniae (1, Serratia marcescens/Enterobacter cloacae (1. Bacterial growth was instead observed only in four patients (3 E.coli and 1 P.aeruginosa and Bacteroides ovatus. Preoperative C-reactive protein and inflammation degree, the most reliable indicators of bacterial translocation, were elevated as expected. DNA amplification was a quick and useful method to detect pathogens and it was even more valuable in detecting aggressive pathogens such as anaerobes, difficult to preserve in biological cultures; its drawbacks were the lack of biological growths and of antibiograms. In our pilot study RT-PCR and cultures did not influence the way patients were treated.

  1. Optimization of ultrahigh-speed multiplex PCR for forensic analysis.

    Science.gov (United States)

    Gibson-Daw, Georgiana; Crenshaw, Karin; McCord, Bruce

    2018-01-01

    In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl 2 , and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

  2. Amplification factor variable amplifier

    NARCIS (Netherlands)

    Akitsugu, Oshita; Nauta, Bram

    2007-01-01

    PROBLEM TO BE SOLVED: To provide an amplification factor variable amplifier capable of achieving temperature compensation of an amplification factor over a wide variable amplification factor range. ; SOLUTION: A Gilbert type amplification factor variable amplifier 11 amplifies an input signal and

  3. Amplification factor variable amplifier

    NARCIS (Netherlands)

    Akitsugu, Oshita; Nauta, Bram

    2010-01-01

    PROBLEM TO BE SOLVED: To provide an amplification factor variable amplifier capable of achieving temperature compensation of an amplification factor over a wide variable amplification factor range. ;SOLUTION: A Gilbert type amplification factor variable amplifier 11 amplifies an input signal and can

  4. Avaliação de dois métodos de extração de DNA de material parafinado para amplificação em PCR Evaluation of two methods of DNA extraction from paraffin-embedded material for PCR amplification

    Directory of Open Access Journals (Sweden)

    Luciana Estevam Simonato

    2007-04-01

    molecular diagnostic methods based on the polymerase chain reaction (PCR. OBJECTIVE: Two methods of DNA extraction from paraffin-embedded samples were tested aiming at PCR amplification of genomic DNA. MATERIAL AND METHOD: Thirty-five samples were obtained from patients with squamous cell carcinoma of mouth floor treated at the Oral Oncology Center in Universidade Estadual Paulista. The DNA extraction methods included: 1. proteinase K digestion followed by Chelex 100® (BioRad purification and 2. QIAamp DNA minikit® system (Qiagen. Purified DNA was quantified by spectrophometry and a beta-globin gene fragment was amplified using PCR. RESULTS: The DNA concentration from samples applied to the first method presented an average of 120.62 ng/µl and absorbance ratio 260/280 varying between 0.8 and 1.41. From the samples extracted using the second procedure, the mean DNA concentration was 67.38 ng/µl, with absorbance ratio varying between 1.11 and 2.53. DNA samples were submitted to PCR and from 35 samples extracted with both methods, respectively, 29 and 30 were successfully amplified for the beta-globin gene. CONCLUSION: Both methods used to obtain genomic DNA from formalin-fixed paraffin-embedded tissues presented similar performance, revealing their potential to be included in diagnosis of molecular biology, as well as in retrospective studies using archived paraffin-embedded samples.

  5. Random amplified polymorphic DNA and restriction enzyme analysis of PCR amplified rDNA in taxonomy: Two identification techniques for food-borne yeasts

    NARCIS (Netherlands)

    Baleiras Couto, M.M.; Vogels, J.T.W.E.; Hofstra, H.; Veld, J.H.J. Huis in't; Vossen, J.M.B.M. van der

    1995-01-01

    The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be

  6. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Science.gov (United States)

    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

  7. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Directory of Open Access Journals (Sweden)

    Tom eKillelea

    2014-05-01

    Full Text Available 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.

    Science.gov (United States)

    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.

  9. New PCR test for detection of Candida glabrata based on the molecular target chosen by the RAPD technique.

    Science.gov (United States)

    Olchawa, Anna; Krawczyk, Beata; Brillowska-Dabrowska, Anna

    2013-01-01

    Rapid, reliable diagnosis is a necessary condition for the successful treatment of infections. Such diagnostic assays are continually being developed. The paper presents a method for selecting the molecular target for PCR-based diagnostics based on the comparison of RAPD patterns. A sequence encoding Candida glabrata CBS138 hypothetical protein was selected. The limit of detection for PCR and real-time PCR reactions with DNA extracted from blood samples spiked with Candida glabrata was estimated at 1 CFU/ml. The application of the assays developed in this study would thus seem to be promising as a complementary method in the diagnostics of C. glabrata infections.

  10. Detection of Phakopsora pachyrhizi fungus by Polymerase Chain Reaction technique (PCR) after soy grains treatment by electron beams

    International Nuclear Information System (INIS)

    Fanaro, G.B.; Aquino, S.; Guedes, R.L.; Crede, R.G.; Sabundjian, I.T.; Ruiz, M.O.; Villavicencio, A.L.C.H.

    2005-01-01

    Today Brazil, as the largest soy exporter in the world, has undergone the consequences of the contamination of these crops by the Asian dust fungus, being harmed since the plantation up to the harvest, with losses in its productivity ranging 10-80%. As it is a new disease in the Americas, there are not any resistant species to this fungus attack. The grains contamination harms the exportation for countries which do not want to have their crops contaminated, affecting therefore the international commerce and agro-business relationship with those countries Brazil has trade with. The Asian dust is caused by the fungus Phakopsora pachyrhizi and its dissemination is of difficult control, since occurs through the wind dispersion. The P. pachyrhizi is an Asian fungus and was recently found in South Africa, Paraguay, Argentina and Brazil. As an alternative process to minimize these losses is the process to preserve the grains by radiation, the use of the electron accelerator was indicated, since its advantage for the grains exportation industry is fundamental. Besides the possibility of being disconnected when not in use, this source does not need to be recharged, is easily available and has high dose rate, streamlining the process and reducing logistics costs. The present work aims to identify, by the Polymerase Chain Reaction technique (PCR), the P. pachyrhizi fungus presence in the irradiated soy grains, at doses 1 and 2 kGy, at the IPEN-CNEN electron Accelerator, a Dynamitron Machine (Radiation Dynamics Co. model JOB, New York, USA), with 1.5 MeV power and 2.5 mA electrical current. (author)

  11. Comparison of PCR-DGGE and Nested-PCR-DGGE Approach for Ammonia Oxidizers Monitoring in Membrane Bioreactors’ Activated Sludge

    Directory of Open Access Journals (Sweden)

    Ziembińska-Buczyńska Aleksandra

    2014-12-01

    Full Text Available Nitritation, the first stage of ammonia removal process is known to be limiting for total process performance. Ammonia oxidizing bacteria (AOB which perform this process are obligatory activated sludge habitants, a mixture consisting of Bacteria, Protozoa and Metazoa used for biological wastewater treatment. Due to this fact they are an interesting bacterial group, from both the technological and ecological point of view. AOB changeability and biodiversity analyses both in wastewater treatment plants and lab-scale reactors are performed on the basis of 16S rRNA gene sequences using PCR-DGGE (Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis as a molecular biology tool. AOB researches are usually led with nested PCR. Because the application of nested PCR is laborious and time consuming, we have attempted to check the possibility of using only first PCR round to obtain DGGE fingerprinting of microbial communities. In this work we are comparing the nested and non-nested PCR-DGGE monitoring of an AOB community and presenting advantages and disadvantages of both methods used. The experiment revealed that PCR technique is a very sensitive tool for the amplification of even a minute amount of DNA sample. But in the case of nested-PCR, the sensitivity is higher and the template amount could be even smaller. The nested PCR-DGGE seems to be a better tool for AOB community monitoring and complexity research in activated sludge, despite shorter fragments of DNA amplification which seems to be a disadvantage in the case of bacteria identification. It is recommended that the sort of analysis approach should be chosen according to the aim of the study: nested-PCR-DGGE for community complexity analysis, while PCR-DGGE for identification of the dominant bacteria.

  12. Identification of transgenic mice by PCR analysis of saliva.

    Science.gov (United States)

    Irwin, M H; Moffatt, R J; Pinkert, C A

    1996-09-01

    As an alternative to surgically obtaining samples (e.g., tail or tissue biopsy, toe dock, or blood sampling) from weanling mice to screen for transgene integration or other genetic monitoring procedures, we offer a simpler, nonsurgical method. A small amount of saliva, obtained from weanling mice by oral wash using a plastic pipet tip, contains enough oral epithelial cells and lymphocytes to yield sufficient DNA for nested primer polymerase chain reaction (PCR) analysis. The procedure can be repeated many times with minimal stress to the animal, in contrast to tissue biopsy procedures such as tail cutting. Sample analysis is rapid and straightforward; saliva is applied to sample collection paper and then purified using a solid phase DNA purification system. The paper, containing purified DNA, is added directly to PCR cocktail for the first round of amplification. For weanling mice, in the second round of amplification, a small amount of product from the first round is removed and added to PCR cocktail containing the second set of primers. With adult mice, an adequate volume of saliva may be obtained (dependent upon the sensitivity of the particular reaction) to eliminate the need for second-round amplification with nested primers. This technique is reliable, does not require organic solvents, and is more humane than protocols currently in use. Furthermore, this technique could replace hundreds of thousands of surgical biopsies on rodents annually, which are performed for both transgene determination and genetic monitoring procedures.

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

    Directory of Open Access Journals (Sweden)

    Tülin GÜVEN GÖKMEN

    Full Text Available 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.

  14. Infection of Bacterial Endosymbionts in Insects: A Comparative Study of Two Techniques viz PCR and FISH for Detection and Localization of Symbionts in Whitefly, Bemisia tabaci.

    Directory of Open Access Journals (Sweden)

    Harpreet Singh Raina

    Full Text Available Bacterial endosymbionts have been associated with arthropods and large number of the insect species show interaction with such bacteria. Different approaches have been used to understand such symbiont- host interactions. The whitefly, Bemisia tabaci, a highly invasive agricultural pest, harbors as many as seven different bacterial endosymbionts. These bacterial endosymbionts are known to provide various nutritional, physiological, environmental and evolutionary benefits to its insect host. In this study, we have tried to compare two techniques, Polymerase chain reaction (PCR and Flourescence in situ Hybridisation (FISH commonly used for identification and localization of bacterial endosymbionts in B. tabaci as it harbors one of the highest numbers of endosymbionts which have helped it in becoming a successful global invasive agricultural pest. The amplified PCR products were observed as bands on agarose gel by electrophoresis while the FISH samples were mounted on slides and observed under confocal microscope. Analysis of results obtained by these two techniques revealed the advantages of FISH over PCR. On a short note, performing FISH, using LNA probes proved to be more sensitive and informative for identification as well as localization of bacterial endosymbionts in B. tabaci than relying on PCR. This study would help in designing more efficient experiments based on much reliable detection procedure and studying the role of endosymbionts in insects.

  15. An improved method for characterization of the mutation associated to porcine stress syndrome by PCR amplification followed by restriction analysis Um método melhorado para caracterização da mutação associada à síndrome do estresse suíno por amplificação por PCR seguido de análise de restrição

    Directory of Open Access Journals (Sweden)

    Tessália Diniz Luerce

    2009-08-01

    Full Text Available A mutation in the gene coding for the ryanodine receptor 1 (RYR1, also known as halothane (hal gene or swine stress gene, is associated to the porcine stress syndrome (PSS. Detection of the mutation is normally accomplished by PCR amplification of an 81bp fragment of the hal gene, followed by digestion with the HhaI restriction endonuclease. Wild-type allele (N is cut in two fragments, whereas the mutant allele (n is not digested by the restriction enzyme. Electrophoresis of the digested DNA on agarose gel and ethidium bromide staining allows the reading of the result. The correct interpretation is difficult due to the small size of the DNA fragments. In this study we designed a new set of primers for amplification of a 144bp fragment that facilitates the reading of the result. In addition, we optimized the PCR reaction to allow amplification from a single hair bulb, added directly into the PCR mix without previous treatment. This improved method was used to genotype 165 sows and boars used in a breeding program. Forty-nine percent of the animals had the NN genotype, whereas 50% were Nn and only 1% was nn.Uma mutação no gene que codifica o receptor ryanodine 1 (RYR1, também conhecido como gene do halotano (hal ou gene do estresse suíno, está associada à Síndrome do Estresse Suíno (PSS. A mutação é geralmente detectada por PCR, a partir da amplificação de um fragmento de 81pb do gene hal, seguida por digestão com a endonuclease de restrição HhaI. O alelo normal (N é cortado em dois fragmentos, enquanto que o alelo mutado (n não é digerido pela enzima de restrição. A eletroforese do DNA digerido em gel de agarose corado com brometo de etídio permite a leitura do resultado. A interpretação correta é difícil devido ao pequeno tamanho dos fragmentos. Neste estudo, foi projetado um novo par de iniciadores para a amplificação de um fragmento de 144pb, o que facilita a leitura do resultado. Adicionalmente, foi otimizada a rea

  16. Generation of recombinant pestiviruses using a full genome amplification strategy

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Uttenthal, Åse

    Aim Complete genome amplification of viral RNA provides a new tool for generation of modified pestiviruses. We have recently reported a full genome amplification strategy for direct recovery of infectious pestivirus (Rasmussen et al., 2008). This comprised rescue of BDV strain “Gifhorn” from a full...... pestiviruses. Methods Pestivirus genomes were amplified from either total RNA preparations using long RT-PCR or from infectious cDNA clones using long PCR. Viral RNA was extracted from cell cultures inoculated with pestivirus (e.g. BDV “Gifhorn” or BVDV “CP7”) using a combined Trizol/RNeasy protocol. Total RNA......-length RT-PCR amplicon demonstrating that long RT-PCR can be used for direct generation of an infectious pestivirus. The strategy is not limited to amplification of BDV “Gifhorn”, but can be further utilized for amplification of a diverse selection of pestivirus strains and for the generation of modified...

  17. Comparison of two quantitative PCR techniques for porcine circovirus Type 2 (PCV2) nucleic acid in field samples

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Grau-Roma, Llorenc; Sibila, M.

    PMWS (1). PMWS is associated with a high PCV2 load, and a general threshold of 10 7 copies of PCV2 per ml serum has been suggested for PMWS diagnosis (2,3). The objective of this study was to compare the performance of two different real-time quantitative polymerase chain reaction (qPCR) assays for PCV...

  18. Nucleic acid amplification: Alternative methods of polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Md Fakruddin

    2013-01-01

    Full Text Available Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

  19. In Silico PCR Tools for a Fast Primer, Probe, and Advanced Searching.

    Science.gov (United States)

    Kalendar, Ruslan; Muterko, Alexandr; Shamekova, Malika; Zhambakin, Kabyl

    2017-01-01

    The polymerase chain reaction (PCR) is fundamental to molecular biology and is the most important practical molecular technique for the research laboratory. The principle of this technique has been further used and applied in plenty of other simple or complex nucleic acid amplification technologies (NAAT). In parallel to laboratory "wet bench" experiments for nucleic acid amplification technologies, in silico or virtual (bioinformatics) approaches have been developed, among which in silico PCR analysis. In silico NAAT analysis is a useful and efficient complementary method to ensure the specificity of primers or probes for an extensive range of PCR applications from homology gene discovery, molecular diagnosis, DNA fingerprinting, and repeat searching. Predicting sensitivity and specificity of primers and probes requires a search to determine whether they match a database with an optimal number of mismatches, similarity, and stability. In the development of in silico bioinformatics tools for nucleic acid amplification technologies, the prospects for the development of new NAAT or similar approaches should be taken into account, including forward-looking and comprehensive analysis that is not limited to only one PCR technique variant. The software FastPCR and the online Java web tool are integrated tools for in silico PCR of linear and circular DNA, multiple primer or probe searches in large or small databases and for advanced search. These tools are suitable for processing of batch files that are essential for automation when working with large amounts of data. The FastPCR software is available for download at http://primerdigital.com/fastpcr.html and the online Java version at http://primerdigital.com/tools/pcr.html .

  20. Efficiency of rep-PCR fingerprinting as a useful technique for molecular typing of plant pathogenic fungal species: Botryosphaeriaceae species as a case study.

    Science.gov (United States)

    Abdollahzadeh, Jafar; Zolfaghari, Sajedeh

    2014-12-01

    Progress in molecular biology and the advent of rapid and accurate molecular techniques have contributed to precise and rapid detection and differentiation of microbial pathogens. Identification of the Botryosphaeriaceae species based on morphology has been problematic over time. In this study, we used rep-PCR technique as a molecular tool for typing and differentiation of the Botryosphaeriaceae species, well-known and cosmopolitan fungal pathogens on woody plants. Three primer sets BOX, ERIC and REP were used to differentiate 27 species belong to eight genera. The majority of them were examined in terms of typing and differentiation using molecular methods for the first time. All the primer sets were able to generate species-specific DNA fingerprints from all the tested strains, with two exceptions in the genera Diplodia and Spencermartinsia. Despite the deficiency of each primer sets to separate a few species, cluster analysis of combined data sets indicated the ability of rep-PCR technique to separate 26 out of 27 examined species in highly supported clusters corresponded to the species recognized based on DNA sequence data. Our findings revealed the efficiency of rep-PCR for detection and differentiation of the Botryosphaeriaceae species, especially cryptic species with the same ITS sequences and similar morphology. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  1. Loop‑mediated Isothermal Amplification assay (LAMP) based detection of Pasteurella multocida in cases of haemorrhagic septicaemia and fowl cholera.

    Science.gov (United States)

    Bhimani, Mayurkumar; Bhanderi, Bharat; Roy, Ashish

    2015-01-01

    Twenty two isolates of Pasteurella multocida were obtained from different tissues of dead birds and animals (cattle, buffalo, sheep, and goat) suspected of fowl cholera and haemorrhagic septicaemia. The isolates were confirmed as P. multocida by various biochemical tests and PM PCR. An attempt was made to standardize Loop mediated isothermal amplification (LAMP) using newly designed primer sequences of KMT1 gene. Loop mediated isothermal amplification was conducted using 6 sets of primers at 65°C for 30 minutes and the result was confirmed by visual observation using SYBR green fluorescence dye as marker of positive reaction under UV transilluminator. On electrophoretic analysis of the products on 2% agarose gel, a ladder like pattern was observed, which suggested a positive amplification, whereas no amplification was observed in negative controls. Additionally, product of positive reaction yielded a green fluorescence following addition of SYBR green under UV transilluminator. It was observed that LAMP is a more sensitive test than polymerase chain reaction (PCR), as the former could detect DNA to lower limit of 22.8 pg/µl, while the latter could detect DNA to lower limit of 2.28 ng/ µl, thus LAMP could detect 100 times lesser concentration of DNA in comparison to PCR. Loop mediated isothermal amplification is a rather newer molecular technique, which can be used for rapid detection of infectious agent at field level and which does not require sophisticated instrument, i.e. thermal cycler. Furthermore, unlike the conventional PCR technique, LAMP requires lesser time to perform and result can be read visually.

  2. High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yonghua [Iowa State Univ., Ames, IA (United States)

    2000-01-01

    Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an

  3. Direct detection and amplification of Helicobacter pylori ribosomal 16S gene segments from gastric endoscopic biopsies.

    Science.gov (United States)

    Hoshina, S; Kahn, S M; Jiang, W; Green, P H; Neu, H C; Chin, N; Morotomi, M; LoGerfo, P; Weinstein, I B

    1990-01-01

    Helicobacter pylori is an organism thought to play an important causative role in gastritis and peptic ulcer diseases. We have designed an RNA dot blot assay for the detection of H. pylori, using as probe a synthetic oligonucleotide complementary to its 16S rRNA. We have also used oligonucleotide primers, complementary to conserved sequences within bacterial ribosomal 16S genes, to amplify a H. pylori ribosomal 16S DNA fragment via the polymerase chain reaction (PCR). After determining the DNA sequence of this amplified H. pylori fragment, primers were designed for specific PCR amplification of H. pylori ribosomal 16S DNA sequences. Samples from clinical endoscopic biopsies were PCR amplified with universal 16S ribosomal primers to detect the presence of bacteria and with H. pylori-specific primers to uniquely detect H. pylori. Finally, by comparing the H. pylori-specific PCR assay to commonly used diagnostic tests, we demonstrate that the molecular technique of PCR amplification shows promising applications for the clinical detection of H. pylori.

  4. Molecular identification of peste des petits ruminants virus in wild goat and domestic small ruminants by real-time -PCR technique in Erbil-Iraq

    Directory of Open Access Journals (Sweden)

    E.P. Candlan

    2017-06-01

    Full Text Available In July 2010 outbreak was occurred in wild goat in Barzan, Sherwin mizzen and Mergasur in Kurdistan Region- Iraq. There were over 2700 deaths (both young and adult during the period of July 2010 to October 2011. Based on the clinical signs and post-mortem findings, the involvement of peste des petits ruminants virus (PPRV was suspected. This was confirmed by Real Time PCR technique using TaqMan®probes for the detection of Peste des petits ruminants. The results of Real-Time PCR for the 9 sample taken from 9 Wild goat there are 6 sample positive and 3 sample negative and 76 sample from domestic ruminants (sheep and goat 63 samples was negative for PPR. This result confirms the diagnosis domestic ruminants in the region are routinely vaccinated with an attenuated vaccine based on the ‘Nigeria/75/1’ strain of PPRV.

  5. Seeded free-electron and inverse free-electron laser techniques for radiation amplification and electron microbunching in the terahertz range

    Directory of Open Access Journals (Sweden)

    C. Sung

    2006-12-01

    Full Text Available A comprehensive analysis is presented that describes amplification of a seed THz pulse in a single-pass free-electron laser (FEL driven by a photoinjector. The dynamics of the radiation pulse and the modulated electron beam are modeled using the time-dependent FEL code, GENESIS 1.3. A 10-ps (FWHM electron beam with a peak current of 50–100 A allows amplification of a ∼1  kW seed pulse in the frequency range 0.5–3 THz up to 10–100 MW power in a relatively compact 2-m long planar undulator. The electron beam driving the FEL is strongly modulated, with some inhomogeneity due to the slippage effect. It is shown that THz microbunching of the electron beam is homogeneous over the entire electron pulse when saturated FEL amplification is utilized at the very entrance of an undulator. This requires seeding of a 30-cm long undulator buncher with a 1–3 MW of pump power with radiation at the resonant frequency. A narrow-band seed pulse in the THz range needed for these experiments can be generated by frequency mixing of CO_{2} laser lines in a GaAs nonlinear crystal. Two schemes for producing MW power pulses in seeded FELs are considered in some detail for the beam parameters achievable at the Neptune Laboratory at UCLA: the first uses a waveguide to transport radiation in the 0.5–3 THz range through a 2-m long FEL amplifier and the second employs high-gain third harmonic generation using the FEL process at 3–9 THz.

  6. Microfluidic continuous flow digital loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Rane, Tushar D; Chen, Liben; Zec, Helena C; Wang, Tza-Huei

    2015-02-07

    Digital nucleic acid detection is rapidly becoming a popular technique for ultra-sensitive and quantitative detection of nucleic acid molecules in a wide range of biomedical studies. Digital polymerase chain reaction (PCR) remains the most popular way of conducting digital nucleic acid detection. However, due to the need for thermocycling, digital PCR is difficult to implement in a streamlined manner on a single microfluidic device, leading to complex fragmented workflows and multiple separate devices and instruments. Loop-mediated isothermal amplification (LAMP) is an excellent isothermal alternative to PCR with potentially better specificity than PCR because of the use of multiple primer sets for a nucleic acid target. Here we report a microfluidic droplet device implementing all the steps required for digital nucleic acid detection including droplet generation, incubation and in-line detection for digital LAMP. As compared to microchamber or droplet array-based digital assays, the continuous flow operation of this device eliminates the constraints on the number of total reactions imposed by the footprint of the device and the analysis throughput caused by the time for lengthy incubation and transfer of materials between instruments.

  7. Advantages and Limitations of Direct PCR Amplification of Bacterial 16S-rDNA from Resected Heart Tissue or Swabs Followed by Direct Sequencing for Diagnosing Infective Endocarditis: A Retrospective Analysis in the Routine Clinical Setting

    Directory of Open Access Journals (Sweden)

    Daniela Maneg

    2016-01-01

    Full Text Available Infective endocarditis (IE is a life-threatening disease that is associated with high morbidity and mortality. Its long-term prognosis strongly depends on a timely and optimized antibiotic treatment. Therefore, identification of the causative pathogen is crucial and currently based on blood cultures followed by characterization and susceptibility testing of the isolate. However, antibiotic treatment starting prior to blood sampling or IE caused by fastidious or intracellular microorganisms may cause negative culture results. Here we investigate the additional diagnostic value of broad-range PCR in combination with direct sequencing on resected heart tissue or swabs in patients with tissue or swab culture-negative IE in a routine clinical setting. Sensitivity, specificity, and positive and negative predictive values of broad-range PCR from diagnostic material in our patients were 33.3%, 76.9%, 90.9%, and 14.3%, respectively. We identified a total of 20 patients (21.5% with tissue or culture-negative IE who profited by the additional application of broad-range PCR. We conclude that broad-range PCR on resected heart tissue or swabs is an important complementary diagnostic approach. It should be seen as an indispensable new tool for both the therapeutic and diagnostic management of culture-negative IE and we thus propose its possible inclusion in Duke’s diagnostic classification scheme.

  8. Advantages and Limitations of Direct PCR Amplification of Bacterial 16S-rDNA from Resected Heart Tissue or Swabs Followed by Direct Sequencing for Diagnosing Infective Endocarditis: A Retrospective Analysis in the Routine Clinical Setting.

    Science.gov (United States)

    Maneg, Daniela; Sponsel, Janina; Müller, Iris; Lohr, Benedikt; Penders, John; Madlener, Katharina; Hunfeld, Klaus-Peter

    2016-01-01

    Infective endocarditis (IE) is a life-threatening disease that is associated with high morbidity and mortality. Its long-term prognosis strongly depends on a timely and optimized antibiotic treatment. Therefore, identification of the causative pathogen is crucial and currently based on blood cultures followed by characterization and susceptibility testing of the isolate. However, antibiotic treatment starting prior to blood sampling or IE caused by fastidious or intracellular microorganisms may cause negative culture results. Here we investigate the additional diagnostic value of broad-range PCR in combination with direct sequencing on resected heart tissue or swabs in patients with tissue or swab culture-negative IE in a routine clinical setting. Sensitivity, specificity, and positive and negative predictive values of broad-range PCR from diagnostic material in our patients were 33.3%, 76.9%, 90.9%, and 14.3%, respectively. We identified a total of 20 patients (21.5%) with tissue or culture-negative IE who profited by the additional application of broad-range PCR. We conclude that broad-range PCR on resected heart tissue or swabs is an important complementary diagnostic approach. It should be seen as an indispensable new tool for both the therapeutic and diagnostic management of culture-negative IE and we thus propose its possible inclusion in Duke's diagnostic classification scheme.

  9. Noninvasive detection through REMS-PCR technique of K-ras mutations in stool DNA of patients with colorectal cancer.

    Science.gov (United States)

    Mixich, Francisc; Ioana, Mihai; Voinea, Florea; Săftoiu, Adrian; Ciurea, Tudorel

    2007-03-01

    Tumor exfoliated cells that shed into stool are attractive targets for molecular screening and early detection of colon malignancies. Many studies have suggested that the detection of activated ras may have diagnostic or prognostic importance. The aim of this study was to establish the suitability for use in diagnostic laboratories of the noninvasive screening test of K-ras mutation determination in the stool and its routine prognostic value in colorectal cancer. Paired stool and tissue specimens obtained after polypectomy and colorectal biopsy from 28 patients diagnosed solely by histopathological findings with primary colorectal carcinoma, were prospectively studied for K-ras codon 12 mutations by restriction endonuclease-mediated selective (REMS)-PCR. DNA was obtained in 28 of tissue samples (100%) and 26 of stool samples (92.8%). K-ras codon 12 mutation was seen in 14 (50.0%) paired stool and tissue samples. Mutation detection was possible in 1000-fold excess of wild-type sequence. These results may be important in the design of genetic screening programs, determination of prognosis, early detection and treatment for patients with colon malignancy. The sensitivity and specificity of K-ras determination on stool-derived DNA in patients with colorectal carcinoma, support the opportunity of a large-scale trial to validate its use as a screening test. REMS- PCR is not labor intensive, but a sensitive, rapid, and robust assay for the detection of point mutations, and was introduced by us in a routine diagnostic laboratory.

  10. Detection of a putative novel adenovirus by PCR amplification, sequencing and phylogenetic characterisation of two gene fragments from formalin-fixed paraffin-embedded tissues of a cat diagnosed with disseminated adenovirus disease.

    Science.gov (United States)

    Lakatos, Béla; Hornyák, Ákos; Demeter, Zoltán; Forgách, Petra; Kennedy, Frances; Rusvai, Miklós

    2017-12-01

    Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissue samples of a cat that had suffered from disseminated adenovirus infection. The identity of the amplified products from the hexon and DNA-dependent DNA polymerase genes was confirmed by DNA sequencing. The sequences were clearly distinguishable from corresponding hexon and polymerase sequences of other mastadenoviruses, including human adenoviruses. These results suggest the possible existence of a distinct feline adenovirus.

  11. A simple procedure eliminating multiple optimization steps required in developing multiplex PCR reactions

    Energy Technology Data Exchange (ETDEWEB)

    Grondin, V.; Roskey, M.; Klinger, K.; Shuber, T. [Integrated Genetics, Framingham, MA (United States)

    1994-09-01

    The PCR technique is one of the most powerful tools in modern molecular genetics and has achieved widespread use in the analysis of genetic diseases. Typically, a region of interest is amplified from genomic DNA or cDNA and examined by various methods of analysis for mutations or polymorphisms. In cases of small genes and transcripts, amplification of single, small regions of DNA are sufficient for analysis. However, when analyzing large genes and transcripts, multiple PCRs may be required to identify the specific mutation or polymorphism of interest. Ever since it has been shown that PCR could simultaneously amplify multiple loci in the human dystrophin gene, multiplex PCR has been established as a general technique. The properities of multiplex PCR make it a useful tool and preferable to simultaneous uniplex PCR in many instances. However, the steps for developing a multiplex PCR can be laborious, with significant difficulty in achieving equimolar amounts of several different amplicons. We have developed a simple method of primer design that has enabled us to eliminate a number of the standard optimization steps required in developing a multiplex PCR. Sequence-specific oligonucleotide pairs were synthesized for the simultaneous amplification of multiple exons within the CFTR gene. A common non-complementary 20 nucleotide sequence was attached to each primer, thus creating a mixture of primer pairs all containing a universal primer sequence. Multiplex PCR reactions were carried out containing target DNA, a mixture of several chimeric primer pairs and primers complementary to only the universal portion of the chimeric primers. Following optimization of conditions for the universal primer, limited optimization was needed for successful multiplex PCR. In contrast, significant optimization of the PCR conditions were needed when pairs of sequence specific primers were used together without the universal sequence.

  12. Use of PCR-RFLP and PCR-HWP1 for identification of Candida species isolated from cystic fibrosis patients

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    Peyman Solimani

    2014-08-01

    Full Text Available Background: Due to the predisposing conditions in patients with cystic fibrosis (CF caused by defective mucociliary clearance facilitating colonization and invasion with Candida species has dramatically increased. Traditional methods for identifying problems are imminent and time-consuming. Therefore, molecular techniques utilizing amplification of target DNA provide quick and precise methods for the diagnosis and identification of Candida species. Therefore, the aim of the current study was to identify the most medically common isolated Candida species from the air way of CF patients by PCR-RFLP and amplification of HWP1 gene. Materials and Methods: A total of 42 CF patients presenting symptoms who referred to pediatric respiratory diseases research center were screened for the presence of Candida spp. The isolates initially were phenotypically identified and confirmed by molecular approaches based on restriction fragment length polymorphism ( PCR -RFLP for the discrimination of C. albicans of non-albicans and the amplification of HWP1 gene for the discrimination of C. albicans from C. dubliniensis and C. africana was conducted. Results: The results show that C. albicans was the most frequently isolated species (83.8% followed by non-albicans included C. parapsilosis (7.1%, C. glabrata (3.2%, and C. tropicalis (3.2%. The restriction patterns of each Candida species were perfectly specific. Since MspI could not discriminate between the three morphological related species, C. albicans, C. dubliniensis and C. africana, we used PCR amplification of HWP1 gene, which (7.1% species from C. albicans identified as C. dubliniensis, however C. africana strains were not found. Conclusion: The present study found that C. albicans as predominant species wereisolated from the CF patients. It could be concluded that molecular diagnostic methods are reliable and would be useful for the identification of medically important Candida species in clinical samples

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

    Science.gov (United States)

    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.

  14. Comparison of LCD array and IS6110-PCR with conventional techniques for detection of Mycobacterium bovis isolated from Egyptian cattle and Buffaloes.

    Science.gov (United States)

    Zahran, Rasha Nabil; El Behiry, Ayman; Marzouk, Eman; Askar, Tamer

    2014-09-01

    Bovine tuberculosis is a chronic bacterial and major infectious disease of cattle and buffaloes caused by Mycobacterium bovis. Rapid diagnosis of bovine tuberculosis is considered one of the cornerstones for worldwide control as it permits early epidemiological and therapeutic interventions. Therefore, this study was designed to evaluate conventional techniques (tuberculin test, Ziehl Neelsen staining and culturing) in comparison with proven molecular laboratory techniques (LCD array and IS6110 PCR) for identification of Bovine tuberculosis. A total of 902 Egyptian animals (480 buffaloes and 422 cattle) were examined by tuberculin test, and the positive reactors were slaughtered. Tissue samples were collected for staining as well as culturing. Moreover, LCD array and PCR using IS6110 on DNA extracted from tissue and culture samples were carried out for molecular identification of M. bovis. According to the results, the tuberculin positive cases for cattle and buffaloes were 2.14% (9 cases) and 5.62% (27 cases), respectively. After post-mortem examination, the prevalence of tuberculin positive cases with visible lesions was 88.9% for cattle and 14.8% for buffaloes. Alternatively, these percentages were 11.1% and 85.2% for cattle and buffalo carcasses with non-visible lesions. The percentage of cattle and buffaloes showing positive culture was 88.9% and 62.9%, respectively. This percentage was 69.5% after staining with Ziehl Neelsen. In contrast, LCD array and IS6110 were 100%, confirming the isolation results. In conclusion, LCD array depending on 16S RNA and DNA hybridization with specific probes for detection of M. bovis are rapid, sensitive and labor-saving when combined with IS6110-PCR. Copyright © 2014 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

  15. Determining Microeukaryotic Plankton Community around Xiamen Island, Southeast China, Using Illumina MiSeq and PCR-DGGE Techniques.

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    Lingyu Yu

    Full Text Available Microeukaryotic plankton are important components of aquatic environments and play key roles in marine microbial food webs; however, little is known about their genetic diversity in subtropical offshore areas. Here we examined the community composition and genetic diversity of the microeukaryotic plankton in Xiamen offshore water by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis, clone-based sequencing and Illumina based sequencing. The Illumina MiSeq sequencing revealed a much (approximately two orders of magnitude higher species richness of the microeukaryotic community than DGGE, but there were no significant difference in species richness and diversity among the northern, eastern, southern or western stations based on both methods. In this study, Copepoda, Ciliophora, Chlorophyta, Dinophyceae, Cryptophyta and Bacillariophyta (diatoms were the dominant groups even though diatoms were not detected by DGGE. Our Illumina based results indicated that two northern communities (sites N2 and N3 were significantly different from others in having more protozoa and fewer diatoms. Redundancy analysis (RDA showed that both temperature and salinity were the significant environmental factors influencing dominant species communities, whereas the full microeukaryotic community appeared to be affected by a complex of environmental factors. Our results suggested that extensive sampling combined with more deep sequencing are needed to obtain the complete diversity of the microeukaryotic community, and different diversity patterns for both abundant and rare taxa may be important in evaluating the marine ecosystem health.

  16. Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

    Science.gov (United States)

    Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

    2017-11-26

    Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

  17. Use of Tissue Culture Techniques for Producing Virus-Free Plant in Garlic and Their Identification through Real-Time PCR

    Directory of Open Access Journals (Sweden)

    Hatıra Taşkın

    2013-01-01

    Full Text Available This study was performed for comparison of meristem culture technique with shoot tip culture technique for obtaining virus-free plant, comparison of micropropagation success of two different nutrient media, and determination of effectiveness of real-time PCR assay for the detection of viruses. Two different garlic species (Allium sativum and Allium tuncelianum and two different nutrient media were used in this experiment. Results showed that Medium 2 was more successful compared to Medium 1 for both A. tuncelianum and A. sativum (Kastamonu garlic clone. In vitro plants obtained via meristem and shoot tip cultures were tested for determination of onion yellow dwarf virus (OYDV and leek yellow stripe virus (LYSV through real-time PCR assay. In garlic plants propagated via meristem culture, we could not detect any virus. OYDV and LYSV viruses were detected in plants obtained via shoot tip culture. OYDV virus was observed in amount of 80% and 73% of tested plants for A. tuncelianum and A. sativum, respectively. LYSV virus was found in amount of 67% of tested plants of A. tuncelianum and in amount of 87% of tested plants of A. sativum in this study.

  18. Use of tissue culture techniques for producing virus-free plant in garlic and their identification through real-time PCR.

    Science.gov (United States)

    Taşkın, Hatıra; Baktemur, Gökhan; Kurul, Mehmet; Büyükalaca, Saadet

    2013-01-01

    This study was performed for comparison of meristem culture technique with shoot tip culture technique for obtaining virus-free plant, comparison of micropropagation success of two different nutrient media, and determination of effectiveness of real-time PCR assay for the detection of viruses. Two different garlic species (Allium sativum and Allium tuncelianum) and two different nutrient media were used in this experiment. Results showed that Medium 2 was more successful compared to Medium 1 for both A. tuncelianum and A. sativum (Kastamonu garlic clone). In vitro plants obtained via meristem and shoot tip cultures were tested for determination of onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV) through real-time PCR assay. In garlic plants propagated via meristem culture, we could not detect any virus. OYDV and LYSV viruses were detected in plants obtained via shoot tip culture. OYDV virus was observed in amount of 80% and 73% of tested plants for A. tuncelianum and A. sativum, respectively. LYSV virus was found in amount of 67% of tested plants of A. tuncelianum and in amount of 87% of tested plants of A. sativum in this study.

  19. Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format.

    Science.gov (United States)

    Ramlee, Muhammad Khairul; Wang, Jing; Cheung, Alice M S; Li, Shang

    2017-04-08

    The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

  20. Contaminations Occurring in Fungal PCR Assays

    Science.gov (United States)

    Loeffler, Juergen; Hebart, Holger; Bialek, Ralf; Hagmeyer, Lars; Schmidt, Diethard; Serey, Francois-Prâseth; Hartmann, Matthias; Eucker, Jan; Einsele, Hermann

    1999-01-01

    Successful in vitro amplification of fungal DNA in clinical specimens has been reported recently. In a collaboration among five European centers, the frequency and risk of contamination due to airborne spore inoculation or carryover contamination in fungal PCR were analyzed. The identities of all contaminants were specified by cycle sequencing and GenBank analysis. Twelve of 150 PCR assays that together included over 2,800 samples were found to be contaminated (3.3% of the negative controls were contaminated during the DNA extraction, and 4.7% of the PCR mixtures were contaminated during the amplification process). Contaminants were specified as Aspergillus fumigatus, Saccharomyces cerevisiae, and Acremonium spp. Further analysis showed that commercially available products like zymolyase powder or 10× PCR buffer may contain fungal DNA. In conclusion, the risk of contamination is not higher in fungal PCR assays than in other diagnostic PCR-based assays if general precautions are taken. PMID:10074553

  1. Application of bioinformatics on the detection of pathogens by Pcr

    International Nuclear Information System (INIS)

    Rezig, Slim; Sakhri, Saber

    2007-01-01

    Salmonellas are the main responsible agent for the frequent food-borne gastrointestinal diseases. Their detection using classical methods are laborious and their results take a lot of time to be revealed. In this context, we tried to set up a revealing technique of the invA virulence gene, found in the majority of Salmonella species. After amplification with PCR using specific primers created and verified by bioinformatics programs, two couples of primers were set up and they appeared to be very specific and sensitive for the detection of invA gene. (Author)

  2. Quantification of HEV RNA by Droplet Digital PCR

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    Florence Nicot

    2016-08-01

    Full Text Available 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 (ORF3 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.

  3. HLA-DQA1 allele typing by nonisotopic PCR-LIS-SSCP

    OpenAIRE

    Abba, Martin Carlos; Gómez, María Atilia; Golijow, Carlos Daniel

    2001-01-01

    In the present study we used a simple and reliable method for HLA-DQA1 allele typing based on the single-stranded conformation polymorphism (SSCP) properties of DNA molecules obtained by PCR. The technique consists of PCR amplification of a DNA fragment comprising the second exon of the HLA-DQA1 gene, amplicon denaturation using a low ionic strength solution (LIS), and electrophoresis on a small native polyacrylamide gel, followed by a rapid silver staining procedure. In order to validate the...

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

    Science.gov (United States)

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

    2017-12-18

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

  5. Diagnosis Molekuler Virus Flu Burung-A Subtipe H5 Berdasarkan Amplifikasi Gen M dan H5 dengan Metode Onestep Simplex RT-PCR (MOLECULAR DIAGNOSIS OF AVIAN INFLUENZA VIRUS TYPE A AND SUBTYPE H5 BY AMPLIFICATION OF ITS M AND H5 GENES USING ONE STEP SIMPLEX R

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2014-08-01

    Full Text Available Influenza A viruses which belong to the Family of Orthomyxoviridae are a group of viruses withsegmented ssRNA genome. The viruses can be subgroupped into many subtypes on the basis of theirsurface glycoproteins, hemagglutinin (HA and neuraminidase (NA proteins. Among the HA subtypes, H5and H7 have been found to be the most pathogenic. Conventional diagnosis of the viruses is usuallyperformed by isolation of the viruses in embryonated eggs, and hemagglutination (HA and hemagglutinationinhibition test. Although those methods are sensitive and accurate, they are time consuming and requirelaboratory facilities with high biosafety level. Commercial methods such as emzyme-linked immonosorbentassay (ELISA and immunoflurescense assay also provide a rapid result but less sensitive and specificthan conventional methods. Molecular diagnosis by amplification of M and H5 genes using one strepsimplex reverse transcriptase-polymerase chain eraction (RT-PCR provices a rapid and accurate diagnosisfor the viruses. A study was therefore conducted to evaluate the accurate and rapidity of such the moleculartests for diagnosis of avian influenza A virus, subtype H5. As many as 10 sample of the virus isolateswhich were available at the Animal Desease investigation Center in Wates, Yogyakarta, were uses in thisstudy. The virus isolates were firstly propagated in specific antigen negative (SAN chicken embryos andtested by HA/HI test. The viruses were then subjected for the RT-PCR test with varying annealingtemperatures of 500C and 520C. The result showed that all 10 isolates were type A influenza virus and 8out of 10 were influenza A subtype H5 influenza virus. RT-PCR used in this study appears to be moresensitive, rapid and accurate as compared to those by serological and isolation of the virus in embryonatedeggs.

  6. Application of wearable optical coherence tomography (OCT) and loop-mediated isothermal amplification (LAMP) techniques for in situ real-time field inspection of apple Marssonina blotch disease

    Science.gov (United States)

    Wijesinghe, Ruchire Eranga; Lee, Seung-Yeol; Ravichandran, Naresh Kumar; Shirazi, Muhammad Faizan; Han, Sangyeop; Jeong, Hyosang; Kim, Pilun; Jung, Hee-Young; Jeon, Mansik; Kim, Jeehyun

    2017-04-01

    Here we describe the possible application of optical coherence tomography (OCT) to inspect Marssonina coronaria infected apple blotch disease of in situ apple leaves. To fulfill the in situ field inspection requirement, we developed a compact wearable OCT system. For the confirmation of OCT results, simultaneous experiment was performed in realtime using loop-mediated isothermal amplification (LAMP), which is frequently used in agriculture. LAMP method was developed as an alternative approach for the inspection of disease. We performed field inspection for 30 consecutive days, and all the acquired results from both OCT and lamp were compared to confirm the correlation. A clear identification between healthy specimens, apparently healthy but infected specimens, and infected specimens could be obtained through the real-time OCT images, and the correlation between OCT and lamp results was confirmed through the obtained realtime lamp results. Based on this feasibility study, we conclude that the combination of both these diagnosing modalities can be effective for various novel agricultural discoveries.

  7. Optical Parametric Amplification Techniques for the Generation of High-Energy Few-Optical-Cycles IR Pulses for Strong Field Applications

    Directory of Open Access Journals (Sweden)

    Anna G. Ciriolo

    2017-03-01

    Full Text Available Over the last few decades, the investigation of ultrafast phenomena occurring in atoms, molecules and solid-state systems under a strong-field regime of light-matter interaction has attracted great attention. The increasing request for a suitable optical technology is significantly boosting the development of powerful ultrafast laser sources. In this framework, Optical Parametric Amplification (OPA is currently becoming a leading solution for applications in high-power ultra-broadband light burst generation. The main advantage provided by the OPA scheme consists of the possibility of exploring spectral ranges that are inaccessible by other laser technologies, as the InfraRed (IR window. In this paper, we will give an overview on recent progress in the development of high-power few-optical-cycle parametric amplifiers in the near-IR and in the mid-IR spectral domain. In particular, the design of the most advanced OPA implementations is provided, containing a discussion on the key technical aspects. In addition, a review on their application to the study of strong-field ultrafast physical processes is reported.

  8. Utility and limitations of multiplex ligation-dependent probe amplification technique in the detection of cytogenetic abnormalities in products of conception

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    D Saxena

    2016-01-01

    Full Text Available Background and Introduction: Chromosomal abnormality is found in about half of first-trimester abortions. Karyotype is the gold standard to detect chromosomal abnormalities. Multiplex ligation-dependent probe amplification (MLPA offers advantage over karyotype in terms of lower failure rate, faster turnaround time, and much higher resolution than conventional karyotyping and found to be 98% concordant with conventional karyotype. Aim: We performed this study to look for the utility of MLPA in diagnosing chromosomal abnormalities in first-trimester abortions. Materials and Methods: MLPA using subtelomeric SALSA probe sets (P036 and P070 was used to detect cytogenetic abnormalities in products of conception in missed/spontaneous abortions. Results: A total of ninety abortus samples were analyzed by MLPA. Successful results were provided in (67 74.4% of the cases while no conclusion could be drawn in 25.6% (23 of the cases. Fifty-five (82.1% cases were cytogenetically normal and 17.9% (12 had some abnormality. Aneuploidy was detected in 8 (66.7% cases, 3 (25% had double-segment imbalance, and one (8.3% had partial aneuploidy. Conclusion: We suggest that MLPA is a good substitute to traditional karyotype.

  9. [Progress in digital PCR technology and application].

    Science.gov (United States)

    Lin, Jiaqi; Su, Guocheng; Su, Wenjin; Zhou, Changyi

    2017-02-25

    Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.

  10. Cara Preservasi Fitoplasma dari Jaringan Kacang Tanah Bergejala Sapu untuk Deteksi DNA dengan Teknik PCR

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    Siska Irhamnawati Pulogu

    2017-07-01

    Full Text Available Witches‘ broom of peanut caused by phytoplasma is a common disease found in Indonesia. Phytoplasma is able to be detected using polymerase chain reaction (PCR technique. One of important factor which determine the successful of phytoplasma amplification is the DNA availability from fresh tissues. The research was aimed to evaluate some preservation methods of phytoplasma from infected plant samples. The aspects to be evaluated consisted of time (1, 2, 3, and 4 weeks, temperature (-20 °C, 4 °C, and 25 °C, and preservation medium (1X PGB buffer, 3 M NaCl, CTAB buffer, 70% ethanol, non medium, and FTA-card for storing the fresh phytoplasma infected samples. Good preservation method will optimize the phytoplasma DNA amplification using PCR standard technique followed by nested-PCR. The results showed that preservation of samples at -20 °C, 4 °C, and 25 °C in CTAB buffer was able to maintain the tissue freshness for 4 weeks and was able to provide the DNA of either quality or quantity sufficiently for PCR detection. PCR standard using a primer pair P1/P7 showed that not all of the preserved DNA of phytoplasma were amplified positively. However, standard PCR followed by nested-PCR using primer pair fU5/rU3 was able to increase the DNA detectability. Preserved samples derived from various medium and stored for 4 weeks gave positive results.  This results were in contrary with previous same samples which were detected negatively by standard PCR technique.

  11. Development of Rapid Isothermal Amplification Assays for Detection of Phytophthora spp. in Plant Tissue.

    Science.gov (United States)

    Miles, Timothy D; Martin, Frank N; Coffey, Michael D

    2015-02-01

    Several isothermal amplification techniques recently have been developed that are tolerant of inhibitors present in many plant extracts, which can reduce the need for obtaining purified DNA for running diagnostic assays. One such commercially available technique that has similarities with real-time polymerase chain reaction (PCR) for designing primers and a labeled probe is recombinase polymerase amplification (RPA). This technology was used to develop two simple and rapid approaches for detection of Phytophthora spp.: one genus-specific assay multiplexed with a plant internal control and the other species-specific assays for Phytophthora ramorum and P. kernoviae. All assays were tested for sensitivity (ranging from 3 ng to 1 fg of DNA) and specificity using DNA extracted from more than 136 Phytophthora taxa, 21 Pythium spp., 1 Phytopythium sp., and a wide range of plant species. The lower limit of linear detection using purified DNA was 200 to 300 fg of DNA in all pathogen RPA assays. Six different extraction buffers were tested for use during plant tissue maceration and the assays were validated in the field by collecting 222 symptomatic plant samples from over 50 different hosts. Only 56 samples were culture positive for Phytophthora spp. whereas 91 were positive using the Phytophthora genus-specific RPA test and a TaqMan real-time PCR assay. A technique for the generation of sequencing templates from positive RPA amplifications to confirm species identification was also developed. These RPA assays have added benefits over traditional technologies because they are rapid (results can be obtained in as little as 15 min), do not require DNA extraction or extensive training to complete, use less expensive portable equipment than PCR-based assays, and are significantly more specific than current immunologically based methods. This should provide a rapid, field-deployable capability for pathogen detection that will facilitate point-of-sample collection processing

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

    Science.gov (United States)

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

    2017-10-25

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

  13. Assessment of the sensitivity and specificity of serological (IFAT) and molecular (direct-PCR) techniques for diagnosis of leishmaniasis in lagomorphs using a Bayesian approach.

    Science.gov (United States)

    de la Cruz, María Luisa; Pérez, Andres; Domínguez, Mercedes; Moreno, Inmaculada; García, Nerea; Martínez, Irene; Navarro, Alejandro; Domínguez, Lucas; Álvarez, Julio

    2016-08-01

    Leishmaniasis, caused by Leishmania infantum , is a vector-borne zoonotic disease that is endemic to the Mediterranean basin. The potential of rabbits and hares to serve as competent reservoirs for the disease has recently been demonstrated, although assessment of the importance of their role on disease dynamics is hampered by the absence of quantitative knowledge on the accuracy of diagnostic techniques in these species. A Bayesian latent-class model was used here to estimate the sensitivity and specificity of the Immuno-fluorescence antibody test (IFAT) in serum and a Leishmania -nested PCR (Ln-PCR) in skin for samples collected from 217 rabbits and 70 hares from two different populations in the region of Madrid, Spain. A two-population model, assuming conditional independence between test results and incorporating prior information on the performance of the tests in other animal species obtained from the literature, was used. Two alternative cut-off values were assumed for the interpretation of the IFAT results: 1/50 for conservative and 1/25 for sensitive interpretation. Results suggest that sensitivity and specificity of the IFAT were around 70-80%, whereas the Ln-PCR was highly specific (96%) but had a limited sensitivity (28.9% applying the conservative interpretation and 21.3% with the sensitive one). Prevalence was higher in the rabbit population (50.5% and 72.6%, for the conservative and sensitive interpretation, respectively) than in hares (6.7% and 13.2%). Our results demonstrate that the IFAT may be a useful screening tool for diagnosis of leishmaniasis in rabbits and hares. These results will help to design and implement surveillance programmes in wild species, with the ultimate objective of early detecting and preventing incursions of the disease into domestic and human populations.

  14. Digital PCR for direct quantification of viruses without DNA extraction

    OpenAIRE

    Pav?i?, Jernej; ?el, Jana; Milavec, Mojca

    2015-01-01

    DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration mat...

  15. Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review.

    Science.gov (United States)

    Gryson, Nicolas

    2010-03-01

    The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials.

  16. DNA extraction methods for panbacterial and panfungal PCR detection in intraocular fluids.

    Science.gov (United States)

    Mazoteras, Paloma; Bispo, Paulo José Martins; Höfling-Lima, Ana Luisa; Casaroli-Marano, Ricardo P

    2015-07-01

    Three different methods of DNA extraction from intraocular fluids were compared with subsequent detection for bacterial and fungal DNA by universal PCR amplification. Three DNA extraction methods, from aqueous and vitreous humors, were evaluated to compare their relative efficiency. Bacterial (Gram positive and negative) and fungal strains were used in this study: Escherichia coli, Staphylococcus epidermidis and Candida albicans. The quality, quantification, and detection limit for DNA extraction and PCR amplification were analyzed. Validation procedures for 13 aqueous humor and 14 vitreous samples, from 20 patients with clinically suspected endophthalmitis were carried out. The column-based extraction method was the most time-effective, achieving DNA detection limits ≥10(2) and 10(3 )CFU/100 µL for bacteria and fungi, respectively. PCR amplification detected 100 fg, 1 pg and 10 pg of genomic DNA of E. coli, S. epidermidis and C. albicans respectively. PCR detected 90.0% of the causative agents from 27 intraocular samples collected from 20 patients with clinically suspected endophthalmitis, while standard microbiological techniques could detect only 60.0%. The most frequently found organisms were Streptococcus spp. in 38.9% (n = 7) of patients and Staphylococcus spp. found in 22.2% (n = 4). The column-based extraction method for very small inocula in small volume samples (50-100 µL) of aqueous and/or vitreous humors allowed PCR amplification in all samples with sufficient quality for subsequent sequencing and identification of the microorganism in the majority of them.

  17. Use of species-specific PCR for the identification of 10 sea cucumber species

    Science.gov (United States)

    Wen, Jing; Zeng, Ling

    2014-11-01

    We developed a species-specific PCR method to identify species among dehydrated products of 10 sea cucumber species. Ten reverse species-specific primers designed from the 16S rRNA gene, in combination with one forward universal primer, generated PCR fragments of ca. 270 bp length for each species. The specificity of the PCR assay was tested with DNA of samples of 21 sea cucumber species. Amplification was observed in specific species only. The species-specific PCR method we developed was successfully applied to authenticate species of commercial products of dehydrated sea cucumber, and was proven to be a useful, rapid, and low-cost technique to identify the origin of the sea cucumber product.

  18. Use of LH-PCR as a DNA fingerprint technique to trace sediment-associated microbial communities from various land uses

    Science.gov (United States)

    Joe-Strack, J. A.; Petticrew, E. L.

    2012-04-01

    The search for new techniques to effectively and efficiently trace sediment from its source along catchment pathways continues, with a range of new methods being developed and tested annually. A relatively recent approach marries genetic techniques to sediment analysis in order to characterize and differentiate the bacterial populations associated with soil and/or sediment originating from specific locations. Here we present the preliminary results of DNA fingerprint profiles of soil and sediment-associated bacterial communities in and around two different industrial land uses in the central interior of British Columbia, a feedlot and a copper/gold mining site. We assessed the naturally varying 16S rDNA gene using amplicon length heterogeneity-polymerase chain reaction (LH-PCR). Statistical differences between bacterial community profiles were investigated using a suite of methods of which non-metric multidimensional scaling (NMS) and indicator species analysis (ISA) were the most useful. Stronger statistical results were observed for the feedlot data set with spatial differences observed from the source location and within the adjacent creek. Results from the mine site were more difficult to assess although responses were detected in downstream waterways. While bacterial DNA fingerprinting of soil and sediment appears to be a promising tracing technique issues of scale and transferability may limit its use. Lessons learned from this preliminary study will be presented.

  19. 3C-digital PCR for quantification of chromatin interactions.

    Science.gov (United States)

    Du, Meijun; Wang, Liang

    2016-12-06

    Chromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of 3C is to convert physical chromatin interactions into specific DNA ligation products, which are then detected by quantitative polymerase chain reaction (qPCR). However, 3C-qPCR assays are often complicated by the necessity of normalization controls to correct for amplification biases. In addition, qPCR is often limited to a certain cycle number, making it difficult to detect fragment ligations with low frequency. Recently, digital PCR (dPCR) technology has become available, which allows for highly sensitive nucleic acid quantification. Main advantage of dPCR is its high precision of absolute nucleic acid quantification without requirement of normalization controls. To demonstrate the utility of dPCR in quantifying chromatin interactions, we examined two prostate cancer risk loci at 8q24 and 2p11.2 for their interaction target genes MYC and CAPG in LNCaP cell line. We designed anchor and testing primers at known regulatory element fragments and target gene regions, respectively. dPCR results showed that interaction frequency between the regulatory element and MYC gene promoter was 0.7 (95% CI 0.40-1.10) copies per 1000 genome copies while other regions showed relatively low ligation frequencies. The dPCR results also showed that the ligation frequencies between the regulatory element and two EcoRI fragments containing CAPG gene promoter were 1.9 copies (95% CI 1.41-2.47) and 1.3 copies per 1000 genome copies (95% CI 0.76-1.92), respectively, while the interaction signals were reduced on either side of the promoter region of CAPG gene. Additionally, we observed comparable results from 3C-dPCR and 3C-qPCR at 2p11.2 in another cell line (DU145). Compared to traditional 3C-qPCR, our results show that 3C-dPCR is much simpler and more sensitive to detect weak chromatin interactions. It may eliminate

  20. On-chip isothermal, chemical cycling polymerase chain reaction (ccPCR)

    Science.gov (United States)

    Persat, Alexandre; Santiago, Juan

    2008-11-01

    We demonstrate a novel ccPCR technique for microfluidic DNA amplification where temperature is held constant in space and time. The polymerase chain reaction is a platform of choice for biological assays and typically based on a three-step thermal cycling: DNA denaturation, primers annealing and extension by an enzyme. We here demonstrate a novel technique where high concentration chemical denaturants (solvents) denature DNA. We leverage the high electrophoretic mobility of DNA and the electrical neutrality of denaturants to achieve chemical cycling. We focus DNA with isotachophoresis (ITP); a robust electrophoretic preconcentration technique which generates strong electric field gradients and protects the sample from dispersion. We apply a pressure-driven flow to balance electromigration velocity and keep the DNA sample stationary in a microchannel. We drive the DNA through a series of high denaturant concentration zones. DNA denatures at high denaturant concentration. At low denaturant concentration, the enzyme creates complementary strands. DNA reaction kinetics are slower than buffer reactions involved in ITP. We demonstrate successful ccPCR amplification for detection of E. Coli. The ccPCR has the potential for simpler chemistry than traditional PCR.

  1. PCR and Omics Based Techniques to Study the Diversity, Ecology and Biology of Anaerobic Fungi: Insights, Challenges and Opportunities

    Directory of Open Access Journals (Sweden)

    Joan E. Edwards

    2017-09-01

    analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.

  2. PCR and Omics Based Techniques to Study the Diversity, Ecology and Biology of Anaerobic Fungi: Insights, Challenges and Opportunities

    Science.gov (United States)

    Edwards, Joan E.; Forster, Robert J.; Callaghan, Tony M.; Dollhofer, Veronika; Dagar, Sumit S.; Cheng, Yanfen; Chang, Jongsoo; Kittelmann, Sandra; Fliegerova, Katerina; Puniya, Anil K.; Henske, John K.; Gilmore, Sean P.; O'Malley, Michelle A.; Griffith, Gareth W.; Smidt, Hauke

    2017-01-01

    , is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit. PMID:28993761

  3. 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.

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L.

    Science.gov (United States)

    Fatima, Nigar; Ahmad, Naseem; Ahmad, Iqbal; Anis, Mohammad

    2015-09-01

    An improved and methodical in vitro shoot morphogenic approach through axillary bud multiplication was established in a drug yielding plant, Withania somnifera L. Effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyladenine (2iP), and thidiazuron (TDZ)] either singly or in combination with α-napthalene acetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium were tested. The highest regeneration frequency (90 %) with optimum number of shoots (32 ± 0.00)/explant were obtained on MS medium fortified with 2.5 μM 6-benzyladenine (BA) and 0.5 μM NAA and 30 g/l sucrose at pH 5.8. Among the tried TDZ concentrations, 0.5 μM resulted in maximum number of shoots (20.4 ± 0.40)/explant after 4 weeks of exposure. The proliferating shoot cultures established by repeated subculturing of the mother explants on the hormone-free medium produced the highest shoot number (29.4 ± 0.40) with shoot length (6.80 ± 0.12 cm)/explant at fourth subculture passage, which a decline in shoot proliferation was recorded. Different concentrations of NAA were tested for ex vitro rooting of microshoots. The maximum percentage of rooting 100 % with maximum roots (18.3 ± 0.1) was achieved in soilrite when basal portion of the microshoots were treated with 200 μM (NAA) for 15 min per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. The PCR-based single primer amplification reaction (SPAR) methods which include random amplified polymorphic DNA (RAPD) and direct amplification of minisatellite DNA (DAMD) markers has been used for assessment of genetic stability of micropropagated plantlets. No variation was observed in DNA fingerprinting patterns among the micropropagated and the donor plants illustrating their genetic uniformity.

  5. Detection of Phakopsora pachyrhizi fungus by Polymerase Chain Reaction technique (PCR) after soy grains treatment by electron beams; Deteccao da Phakopsora Pachyrhizi por reacao em cadeia pela polimerase (PCR) depois do tratamento dos graos de soja por feixes de eletrons

    Energy Technology Data Exchange (ETDEWEB)

    Fanaro, G.B.; Aquino, S.; Guedes, R.L.; Crede, R.G.; Sabundjian, I.T.; Ruiz, M.O.; Villavicencio, A.L.C.H. [Instituto de Pesquisas Energeticas e Nucleares(IPEN), Sao Paulo (Brazil). Centro de Tecnologia das Radiacoes]. E-mail: villavic@ipen.br

    2005-07-01

    Today Brazil, as the largest soy exporter in the world, has undergone the consequences of the contamination of these crops by the Asian dust fungus, being harmed since the plantation up to the harvest, with losses in its productivity ranging 10-80%. As it is a new disease in the Americas, there are not any resistant species to this fungus attack. The grains contamination harms the exportation for countries which do not want to have their crops contaminated, affecting therefore the international commerce and agro-business relationship with those countries Brazil has trade with. The Asian dust is caused by the fungus Phakopsora pachyrhizi and its dissemination is of difficult control, since occurs through the wind dispersion. The P. pachyrhizi is an Asian fungus and was recently found in South Africa, Paraguay, Argentina and Brazil. As an alternative process to minimize these losses is the process to preserve the grains by radiation, the use of the electron accelerator was indicated, since its advantage for the grains exportation industry is fundamental. Besides the possibility of being disconnected when not in use, this source does not need to be recharged, is easily available and has high dose rate, streamlining the process and reducing logistics costs. The present work aims to identify, by the Polymerase Chain Reaction technique (PCR), the P. pachyrhizi fungus presence in the irradiated soy grains, at doses 1 and 2 kGy, at the IPEN-CNEN electron Accelerator, a Dynamitron Machine (Radiation Dynamics Co. model JOB, New York, USA), with 1.5 MeV power and 2.5 mA electrical current. (author)

  6. Optimization of a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter.

    Science.gov (United States)

    Yamamoto, Naomichi; Kimura, Minoru; Matsuki, Hideaki; Yanagisawa, Yukio

    2010-01-01

    The present study aimed to optimize a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter. In particular, the study optimized conditions for the DNA extraction and real-time PCR amplification to accurately measure airborne fungal concentrations. First, time of fine bead homogenization to extract the DNA from fungal cells was optimized to maximize the DNA yield and prepare the DNA suitable for sensitive and precise quantification by a subsequent real-time PCR analysis. Second, a condition for the real-time PCR amplification was optimized to successfully amplify and quantitate the extracted fungal DNA. In particular, a dilution ratio of the DNA extracts to be introduced to PCR was optimized to achieve an appropriate balance between mitigating PCR inhibition and securing detection sensitivity. Since concentrations of airborne fungi generally observed in indoor and outdoor environments (i.e., 10(1)-10(4) CFU m(-3)) were found to be near the limit of quantification by the generally-used molecular-based detection technique in conjunction with use of gelatin filters, optimizations of these conditions were found to be crucial. Our preliminary result showed that a culture-based method underestimated concentrations of airborne environmental fungi by 1 to 2 orders of magnitude compared to those characterized by the real-time PCR assay. 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  7. Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

    2000-01-01

    A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...... isolates, including 100 problematic "rough" isolates. An internal positive control was designed to use the same Salmonella primers for amplification of a spiked nonrelevant template (116 bp) in the sample tube. The PCR test correctly identified all the Salmonella strains by resulting in positive end......-point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [Delta Rn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (Delta Rn, less than or equal to 0.5). All 100 rough...

  8. Single universal primer multiplex ligation-dependent probe amplification with sequencing gel electrophoresis analysis.

    Science.gov (United States)

    Shang, Ying; Zhu, Pengyu; Xu, Wentao; Guo, Tianxiao; Tian, Wenying; Luo, Yunbo; Huang, Kunlun

    2013-12-15

    In this study, a novel single universal primer multiplex ligation-dependent probe amplification (SUP-MLPA) technique that uses only one universal primer to perform multiplex polymerase chain reaction (PCR) was developed. Two reversely complementary common sequences were designed on the 5' or 3' end of the ligation probes (LPs), which allowed the ligation products to be amplified through only a single universal primer (SUP). SUP-MLPA products were analyzed on sequencing gel electrophoresis with extraordinary resolution. This method avoided the high expenses associated with capillary electrophoresis, which was the commonly used detection instrument. In comparison with conventional multiplex PCR, which suffers from low sensitivity, nonspecificity, and amplification disparity, SUP-MLPA had higher specificity and sensitivity and a low detection limit of 0.1 ng for detecting single crop species when screening the presence of genetically modified crops. We also studied the effect of different lengths of stuffer sequences on the probes for the first time. Through comparing the results of quantitative PCR, the LPs with different stuffer sequences did not affect the ligation efficiency, which further increased the multiplicity of this assay. The improved SUP-MLPA and sequencing gel electrophoresis method will be useful for food and animal feed identification, bacterial detection, and verification of genetic modification status of crops. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Molecular characterization of the rDNA-ITS sequence and a PCR diagnostic technique for Pileolaria terebinthi, the cause of pistachio rust

    Directory of Open Access Journals (Sweden)

    Hossein ALAEI

    2013-01-01

    Full Text Available Eleven samples of the most important pistachio rust (caused by Pileolaria terebinthi (DC. Cast.,, which causes disease on Beneh (Pistacia atlantica Desf. subsp. mutica (Fisch. & Mey. Rech. F and Kasoor (Pistacia khinjuk Stocks., were collected from herbarium specimens and pistachio fields at the Pistachio Research Institute in Rafsanjan, Iran. The complete sequences of ribosomal DNA internal transcribed spacers ITS1 and ITS2 (rDNA ITS from the samples were determined and analysed. In general, very little rDNA ITS sequence variation was observed between rDNA ITS sequences of P. terebinthi samples. The length of the PCR fragments was 621 bp (for ITS1F-ITS4 and 1177 bp (for ITS1F-rust1, and consisted of 67 bp at the 3 ́ end of 18S rDNA, 93 bp of ITS1 region, 154 bp of 5.8S rDNA, 246 bp of the ITS2 region, 57 bp (for ITS1F-ITS4 and 613 bp (for ITS1F-rust1 at the 5 ́ end of the 28S rDNA. Restriction fragment length polymorphisms (RFLPs of the rDNA-ITS region were used to identify Pileolaria terebinthi. Three strong bands of 105, 134 and 381 bp and five bands of 105, 134, 200, 301 and 437 bp are observed for the fragment of “ITS1F-ITS4” and “ITS1F-rust1”, respectively. A PCR-RFLP diagnostic technique provided effective identification of the species by a unique pattern with the specific restriction enzyme XapI (ApoI.

  10. Gene amplification in carcinogenesis

    Directory of Open Access Journals (Sweden)

    Lucimari Bizari

    2006-01-01

    Full Text Available Gene amplification increases the number of genes in a genome and can give rise to karyotype abnormalities called double minutes (DM and homogeneously staining regions (HSR, both of which have been widely observed in human tumors but are also known to play a major role during embryonic development due to the fact that they are responsible for the programmed increase of gene expression. The etiology of gene amplification during carcinogenesis is not yet completely understood but can be considered a result of genetic instability. Gene amplification leads to an increase in protein expression and provides a selective advantage during cell growth. Oncogenes such as CCND1, c-MET, c-MYC, ERBB2, EGFR and MDM2 are amplified in human tumors and can be associated with increased expression of their respective proteins or not. In general, gene amplification is associated with more aggressive tumors, metastases, resistance to chemotherapy and a decrease in the period during which the patient stays free of the disease. This review discusses the major role of gene amplification in the progression of carcinomas, formation of genetic markers and as possible therapeutic targets for the development of drugs for the treatment of some types of tumors.

  11. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.

    Science.gov (United States)

    Santiago-Felipe, S; Tortajada-Genaro, L A; Puchades, R; Maquieira, A

    2014-02-06

    Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40°C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

    Directory of Open Access Journals (Sweden)

    Pesta David

    2003-06-01

    Full Text Available Abstract Background DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein. We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. Results Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. Conclusions The technique detailed

  13. An advanced uracil DNA glycosylase-supplemented loop-mediated isothermal amplification (UDG-LAMP) technique used in the sensitive and specific detection of Cryptosporidium parvum, Cryptosporidium hominis, and Cryptosporidium meleagridis in AIDS patients.

    Science.gov (United States)

    Fallahi, Shirzad; Moosavi, Seyedeh Fatemeh; Karimi, Azadeh; Chegeni, Ali Sharafi; Saki, Mohammad; Namdari, Parsa; Rashno, Mohammad Menati; Varzi, Ali Mohamad; Tarrahi, Mohammad Javad; Almasian, Mohammad

    2017-12-24

    The rapid and accurate detection of Cryptosporidium spp. is critically important for the prevention and timely treatment of cryptosporidiosis in AIDS patients (APs). This study was conducted to examine a UDG-LAMP technique for the first time to diagnose cryptosporidiosis in APs. After collecting demographic and clinical data, three stool samples were collected from the participants (120 volunteering APs). The microscopic examination of stained smears using the acid-fast method and the UDG-LAMP assay were performed for each sample. 10% of APs were infected with Cryptosporidium spp. The number of detected cryptosporidiosis cases using the acid-fast staining and UDG-LAMP methods were significantly different (P < 0.001). Diarrhea and weight loss were found to be significantly associated with cryptosporidiosis in patients (P < 0.05). The pretreatment of LAMP reagents with UDG successfully eliminated the likelihood of product re-amplification remaining from previous reactions. The UDG-LAMP technique could detect cryptosporidiosis in APs with high sensitivity and rapidity without carryover contamination. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Social amplification of risk

    International Nuclear Information System (INIS)

    Kasperson, R.E.; Renn, O.; Slovic, P.; Kasperson, J.X.; Emani, S.

    1989-01-01

    The risks associated with radioactive and other hazardous waste disposal may be expected to interact with societal processes to enlarge or attenuate the consequences of risks and risk events. This article summarizes a conceptual framework that depicts the social amplification of risk. Using a data base of 128 hazard events that have occurred largely over the past ten years, the authors examine the role of physical consequences, media coverage, and public perceptions of risk in generating social and economic impacts. The analysis concludes that social amplification processes substantially shape the nature and magnitude of those impacts but also that such social amplification appears to be systematically related to characteristics of the risks and risk events

  15. Molecular characterization of the capsular antigens of Pasteurella multocida isolates using multiplex PCR

    Directory of Open Access Journals (Sweden)

    Khalid S. Al-Maary

    2017-02-01

    Full Text Available The use of molecular techniques for detection and characterization of the Pasteurella multocida is very important for rapid and specific detection and characterization of the organism. During the period from 15th February, 2014 to 15th April, 2015, 425 nasopharyngeal swabs and 175 lung and spleen samples were collected and examined by conventional methods, 80 strains (18.82% of P. multocida were isolated from the calves, sheep and goat with respiratory manifestation. Meanwhile, 77 strains (44% were isolated from emergency slaughtered animals. All the recovered strains were positive for specific PCR for detection of P. multocida strains previously identified as P. multocida by standard microbiological techniques. Multiplex PCR for molecular typing of the capsular antigens of the recovered P. multocida revealed positive amplification of 1044 bp fragments specific to the capsular antigen type A with 105 strains (66.88%, and amplification 511 bp fragments of the capsular antigen type E with 52 strain (33.12% and absence of B, D and F antigens. Multiplex PCR for molecular typing of the capsular antigens of P. multocida can be used as a simple, sensitive, rapid, reliable technique instead of the serological techniques for identification of the capsular antigens of P. multocida

  16. A one-step multiplex PCR for acanthamoeba keratitis diagnosis and quality samples control.

    Science.gov (United States)

    Maubon, Danièle; Dubosson, Muriel; Chiquet, Christophe; Yera, Hélène; Brenier-Pinchart, Marie-Pierre; Cornet, Muriel; Savy, Olivier; Renard, Elisabeth; Pelloux, Hervé

    2012-05-14

    As the number of cases of Acanthamoeba spp. keratitis (AK) is constantly growing, new diagnostic tools are needed to confirm and guide ophthalmologists in this clinically problematic diagnosis. Molecular diagnosis is particularly well adapted, although only a few real-time PCR techniques have been described recently. The aim of this study was to develop a new PCR technique for the diagnosis of AK by combining the detection of Acanthamoeba DNA with human DNA, thus allowing an accurate interpretation of the PCR result. Different DNA extraction procedures were compared to ensure an optimized amplification of one Acanthamoeba genome. The analytical parameters of this new multiplex Acanthamoeba beta-globin PCR (MAB-PCR) were evaluated. Fourteen eye drops were tested as potential PCR inhibitors. A prospective series of 28 corneal scrapings was subjected to MAB-PCR. The best extraction procedure associated thermal-shock pretreatment followed by a manual extraction procedure. The MAB-PCR parameters displayed excellent specificity and sensitivity, with a detection of 0.02 genome of Acanthamoeba. No eye drops were total PCR inhibitors. Of 28 corneal scrapings, 18 were considered true negatives. Seven could not be interpreted because of insufficient scraping material. Three were considered true positives: every patient progressed favorably on specific and reliable treatment. The MAB-PCR is a new tool to diagnose AK. It allows rapid diagnosis and prompt treatment of this probably underestimated etiology of infectious keratitis. This optimized real-time PCR outperforms the gold standard for Acanthamoeba keratitis diagnosis and it allows a concomitant evaluation of the quality of the corneal scraping, which is necessary for a precise interpretation of the results.

  17. A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter

    DEFF Research Database (Denmark)

    Perelle, S.; Josefsen, Mathilde Hartmann; Hoorfar, Jeffrey

    2004-01-01

    Cycler real-time PCR assay (LC-PCR), which used fluorescent hybridization probes was developed. The test incorporated an internal amplification control co-amplified with the 16S rRNA gene of Campylobacter to monitor potential PCR inhibitors and ensure successful amplifications. The specificity study involving...

  18. Development of rapid and highly sensitive detection of Bean common mosaic necrosis virus in leguminous crops using loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Lee, Siwon; Kim, Heejung; Lee, Jin-Young; Rho, Jae-Young

    2017-11-01

    Bean common mosaic necrosis virus (BCMNV) is a plant pathogenic virus that can infect leguminous crops such as kidney beans, sunn hemp, red beans, and mung beans. BCMNV has not been reported in Korea and is classified as a quarantine plant virus. Currently, the standard diagnostic method for diagnosis of BCMNV is reverse transcription (RT)-nested PCR system. However a more rapid monitoring system is needed to enable the testing of more samples. The use of highly efficient loop-mediated isothermal amplification (LAMP) assay for its detection has not yet been reported, and development of LAMP for detecting BCMNV in this study. In addition, confirmation of LAMP amplification can be achieved using restriction enzyme Mse I (T/TAA). The developed technique could be used for more rapid, specific and sensitive monitoring of BCMNV in leguminous crops than conventional nested RT-PCR. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Diagnosis of Trichomonas vaginalis infection by PCR using vaginal swab samples.

    Science.gov (United States)

    Madico, G; Quinn, T C; Rompalo, A; McKee, K T; Gaydos, C A

    1998-11-01

    Trichomonas vaginalis infection is the most prevalent nonviral sexually transmitted disease (STD) in the world. A PCR test using vaginal swab samples for the detection of T. vaginalis was developed to add T. vaginalis infection to the growing list of STDs that can be detected by DNA amplification techniques. A primer set, BTUB 9/2, was designed to target a well-conserved region in the beta-tubulin genes of T. vaginalis. All strains (15 of 15) of T. vaginalis tested were successfully detected by PCR giving a single predicted product of 112 bp in gel electrophoresis. No such targeted product was amplified with DNA from Trichomonas tenax, Trichomonas gallinae, Chlamydia trachomatis, Neisseria gonorrhoeae, Giardia lamblia, Chilomastix sulcatus, Dientamoeba fragilis, and Entamoeba histolytica. An optimal analytical sensitivity of one T. vaginalis organism per PCR was achieved. Culture, performed with the Inpouch TV culture system, was examined daily with a light microscope to identify T. vaginalis. Twenty-three of 350 (6.6%) vaginal swab samples from women attending an army medical clinic were culture positive for T. vaginalis. Of these culture positive specimens, PCR detected 22 of 23 (96%) with primer set BTUB 9/2, and wet preparation detected only 12 of 23 (52%). Seventeen specimens were BTUB 9/2-PCR positive and culture negative. Ten of these discordant specimens were determined to be as true positive by PCR using primer sets TVA 5-1/6 and/or AP65 A/B, which target different regions in the T. vaginalis genome, and seven were determined to be false positive. The sensitivity of BTUB 9/2-PCR was 97% and the specificity was 98%. The sensitivities of culture and wet preparation were 70 and 36%, respectively. The diagnosis of T. vaginalis infection by PCR is a sensitive and specific method that could be incorporated into a joint strategy for the screening of multiple STDs by using molecular amplification methods.

  20. First evidence of fish genotoxicity induced by heavy metals from landfill leachates: the advantage of using the RAPD-PCR technique.

    Science.gov (United States)

    Ben Salem, Zohra; Capelli, Nicolas; Grisey, Elise; Baurand, Pierre-Emmanuel; Ayadi, Habib; Aleya, Lotfi

    2014-03-01

    Municipal leachates are loaded with heavy metals that can contaminate surface water before discharge into a receiving body of water. The aim of this study is to evaluate the genotoxic effects of heavy metals generated by domestic waste on the common roach Rutilus rutilus in the last of the four interconnected ponds at the Etueffont landfill. We used random amplified polymorphic DNA (RAPD) since it has been shown to be a powerful means of detecting a broad range of DNA damage due to environmental contaminants. Our results show the ability of RAPD analysis to detect significant genetic alterations in roach DNA, after contamination with a set of metals contained in the landfill leachates in comparison to a roach from a non-polluted reference pond. Analysis of electrophoresis profiles indicates apparent changes such as the appearance of new bands or disappearance of bands as compared to the control. In fact, mixed smearing and laddering of DNA fragments in muscle samples support the genotoxic effects of metal deposits in the roach. This study is the first evidence found via the RAPD-PCR technique in the detection of pollutant impacts on fish exposed to landfill leachates. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Determination of haemolytic and non haemolytic genes profiles of Bacillus cereus strains isolated from food samples by polymerase chain reaction (pcr) technique

    Science.gov (United States)

    Jawad, Nisreen; Ahemd, Asmat; Abdullah, Aminah

    2018-04-01

    The aim of this study was to investigate the presence of Bacillus cereus and detection of enterotoxigenic genes in food samples by utilizing a Polymerase Chain Reaction technique (PCR). In this study the providence of B. cereus was carried out to food samples. The B. cereus isolates were investigated for enterotoxigenic gene. The cooked seafood, and raw milk samples were purchased from several restaurants and market in the area of (Bangi, Kajang, Serdang and UKM) Selangor, Malaysia. A total of 60 samples have been analyzed. B. cereus contamination has been formed between 1.4×105 - 3×105 cfu/mL of cooked seafood and raw milk samples. Five colonies have been detected as B. cereus using biochemical test. All B. cereus isolates named BC1 to BC27, were characterized for haemolytic enterotoxin (HBL) complex encoding genes (hblA), non-haemolytic enterotoxin encoding gene (NheA). 10 isolates have been reported to be positive towards hblA and 12 isolates were positive towards NheA. The presence of B. cereus and their enterotoxigenic genes in cooked seafood and raw milk from to food samples obtained may pose a potential risk for public health.

  2. On soliton amplification

    Science.gov (United States)

    Leibovich, S.; Randall, J. D.

    1979-01-01

    The paper considers a modified Korteweg-de Vries equation that permits wave amplification or damping. A 'terminal similarity' solution is identified for large times in amplified systems. Numerical results are given which confirm that the terminal similarity solution is a valid local approximation for mu t sufficiently large and positive, even though the approximation is not uniformly valid in space.

  3. Biomaterials in light amplification

    Science.gov (United States)

    Mysliwiec, Jaroslaw; Cyprych, Konrad; Sznitko, Lech; Miniewicz, Andrzej

    2017-03-01

    Biologically produced or inspired materials can serve as optical gain media, i.e. they can exhibit the phenomenon of light amplification. Some of these materials, under suitable dye-doping and optical pumping conditions, show lasing phenomena. The emerging branch of research focused on obtaining lasing action in highly disordered and highly light scattering materials, i.e. research on random lasing, is perfectly suited for biological materials. The use of biomaterials in light amplification has been extensively reported in the literature. In this review we attempt to report on progress in the development of biologically derived systems able to show the phenomena of light amplification and random lasing together with the contribution of our group to this field. The rich world of biopolymers modified with molecular aggregates and nanocrystals, and self-organized at the nanoscale, offers a multitude of possibilities for tailoring luminescent and light scattering properties that are not easily replicated in conventional organic or inorganic materials. Of particular importance and interest are light amplification and lasing, or random lasing studies in biological cells and tissues. In this review we will describe nucleic acids and their complexes employed as gain media due to their favorable optical properties and ease of manipulation. We will report on research conducted on various biomaterials showing structural analogy to nucleic acids such as fluorescent proteins, gelatins in which the first distributed feedback laser was realized, and also amyloids or silks, which, due to their dye-doped fiber-like structure, allow for light amplification. Other materials that were investigated in that respect include polysaccharides, like starch exhibiting favorable photostability in comparison to other biomaterials, and chitosan, which forms photonic crystals or cellulose. Light amplification and random lasing was not only observed in processed biomaterials but also in living

  4. [Establishment of beta block matching technique].

    Science.gov (United States)

    Zhu, Fa-Ming; Lü, Qin-Feng; Zhang, Wei; Zhang, Hai-Qin; Fu, Qi-Hua; Yan, Li-Xing

    2005-10-01

    The purpose of this study was to establish beta block matching technique. DNA was extracted from whole blood by salting-out method, beta block matching was performed by PCR and GeneScan technique. The results showed that the length of fragments amplificated in 100 samples was different and the range of them was 91-197 bp. Amplification fragments could be divided into four regions: 91-93, 105-113, 125-139 and 177-197 bp respectively. 91 bp DNA fragments could be found in all of samples. The numbers of DNA fragments with different length have been shown high polymorphism and they focused on the range of seven to twenty four. In conclusion, the beta block matching technique is reliable and applicable to the selection of hematopoietic stem cell transplantation donors.

  5. Droplet-based micro oscillating-flow PCR chip

    Science.gov (United States)

    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.

  6. CDK4 amplification predicts recurrence of well-differentiated liposarcoma of the abdomen.

    Directory of Open Access Journals (Sweden)

    Sanghoon Lee

    Full Text Available The absence of CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD and dedifferentiated (DD liposarcomas.From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR.There were 31 WD and 17 DD liposarcomas. Locoregional recurrence was observed in 11 WD and 3 DD liposarcomas. WD liposarcomas showed better patient survival compared to DD liposarcomas (P<0.05. Q-PCR analysis of the liposarcomas revealed the presence of CDK4 amplification in 44 cases (91.7% and MDM2 amplification in 46 cases (95.8%. WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence (P = 0.041. High level of CDK4 amplification (cases with CDK4 amplification higher than the median 7.54 was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P = 0.012 and multivariate analyses (P = 0.020.Level of CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection.

  7. Propidium monoazide combined with real-time quantitative PCR to quantify viable Alternaria spp. contamination in tomato products.

    Science.gov (United States)

    Crespo-Sempere, Ana; Estiarte, Núria; Marín, Sonia; Sanchis, Vicente; Ramos, Antonio J

    2013-08-01

    Alternaria is a common contaminating genus of fungi in fruits, grains, and vegetables that causes severe economic losses to farmers and the food industry. Furthermore, it is claimed that Alternaria spp. are able to produce phytotoxic metabolites, and mycotoxins that are unsafe for human and animal health. DNA amplification techniques are being increasingly applied to detect, identify, and quantify mycotoxigenic fungi in foodstuffs, but the inability of these methods to distinguish between viable and nonviable cells might lead to an overestimation of mycotoxin-producing living cells. A promising technique to overcome this problem is the pre-treatment of samples with nucleic acid intercalating dyes, such as propidium monoazide (PMA), prior to quantitative PCR (qPCR). PMA selectively penetrates cells with a damaged membrane inhibiting DNA amplification during qPCRs. In our study, a primer pair (Alt4-Alt5) to specifically amplify and quantify Alternaria spp. by qPCR was designed. Quantification data of qPCR achieved a detection limit of 10(2)conidia/g of tomato. Here, we have optimized for the first time a DNA amplification-based PMA sample pre-treatment protocol for detecting viable Alternaria spp. cells. Artificially inoculated tomato samples treated with 65μM of PMA, showed a reduction in the signal by almost 7cycles in qPCR between live and heat-killed Alternaria spp. conidia. The tomato matrix had a protective effect on the cells against PMA toxicity, reducing the efficiency to distinguish between viable and nonviable cells. The results reported here indicate that the PMA-qPCR method is a suitable tool for quantifying viable Alternaria cells, which could be useful for estimating potential risks of mycotoxin contamination. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Efficient cloning of alternatively polyadenylated transcripts via hybridization capture PCR.

    Science.gov (United States)

    Rampias, Theodoros N; Fragoulis, Emmanuel G; Sideris, Diamantis C

    2012-01-01

    Cloning of alternatively polyadenylated transcripts is crucial for studying gene expression and function. Recent transcriptome analysis has mainly focused on large EST clone collections. However, EST sequencing techniques in many cases are incapable of isolating rare transcripts or address transcript variability. In most cases, 3' RACE is applied for the experimental identification of alternatively polyadenylated transcripts. However, its application may result in nonspecific amplification and false positive products due to the usage of a single gene specific primer. Additionally, internal poly(A) stretches primed by oligo(dT) primer in mRNAs with AU-rich 3'UTR may generate truncated cDNAs. To overcome these limitations, we have developed a simple and rapid approach combining SMART technology for the construction of a full length cDNA library and hybrid capture PCR for the selection and amplification of target cDNAs. Our strategy is characterized by enhanced specificity compared to other conventional RT-PCR and 3' RACE procedures.

  9. Method for chemical amplification based on fluid partitioning in an immiscible liquid

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Brian L.; Colston, Bill W.; Elkin, Christopher J.

    2017-02-28

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  10. GMO detection using a bioluminescent real time reporter (BART of loop mediated isothermal amplification (LAMP suitable for field use

    Directory of Open Access Journals (Sweden)

    Kiddle Guy

    2012-04-01

    Full Text Available Abstract Background There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART occurs at a constant temperature and only requires a simple light detection and integration device. Results Loop mediated isothermal amplification (LAMP shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART for determination of genetically modified (GM maize target DNA at low levels of contamination (0.1-5.0% GM using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading

  11. Amplification of real-time high resolution melting analysis PCR ...

    African Journals Online (AJOL)

    PKD1 and PKD2 are the two genes responsible for the development of autosomal dominant polycystic kidney disease (ADPKD). PKD1 gene mutations accounts for ≈85% of all ADPKD cases, while the remaining ≈15% of cases is caused by mutations in the PKD2 gene. Genotyping for PKD1 and PKD2 mutations was ...

  12. Detection of pepper leaf curl virus through PCR amplification and ...

    African Journals Online (AJOL)

    Pepper leaf curl virus (PepLCV) is the most destructive pathogen of pepper and causes substantial economic losses of chilli production worldwide. Curling and puckering of leaves and stunted growth of the plants are typical symptoms of the viral infection. For a reliable detection of PepLCV, coat protein specific primer pairs ...

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

    NARCIS (Netherlands)

    Vos, Michel J.; Kampinga, Harm H.

    2008-01-01

    For analyzing protein function, protein dynamics, or protein-protein interactions, the use of chimeric proteins has become an indispensable tool. The generation of DNA constructs coding for such fused proteins can, however, be a tedious process. Currently used strategies often make use of available

  14. (PCR) amplification of DNA from formalin preserved tissue sampl

    African Journals Online (AJOL)

    root

    snow leopard is restricted to the alpine and sub-alpine ecological zones, and the major threats it faces are rela- ted to degradation and fragmentation of its habitat, a lack of effective implementation and enforcement of laws and poaching of its natural prey for illegal trade across snow leopard range. In addition, snow ...

  15. Detection of pepper leaf curl virus through PCR amplification and ...

    African Journals Online (AJOL)

    Administrator

    2011-04-25

    Apr 25, 2011 ... Harlow E, Lane N (1988). Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory. Khan MS, Raj SK, Singh R (2006). First report of Tomato leaf curl New. Delhi virus infecting chilli in India. Plant Pathol. 55: p. 289. Kumar S, Rai M (2005). Chile in India. Chile Pepper Institute Newslett.

  16. Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules

    DEFF Research Database (Denmark)

    Schlemmer, B O; Sorensen, B S; Overgaard, J

    2004-01-01

    , and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real-time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained...... significant (p DNA quantification kit" produces results with a high level of reproducibility and its ease of use allows rapid screening for amplification of HER2. In this paper useful information is given on how real-time PCR compares with FISH and IHC. The data show...... of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression...

  17. Detection of SEA-type α-thalassemia in embryo biopsies by digital PCR.

    Science.gov (United States)

    Lee, Ta-Hsien; Hsu, Ya-Chiung; Chang, Chia Lin

    2017-08-01

    Accurate and efficient pre-implantation genetic diagnosis (PGD) based on the analysis of single or oligo-cells is needed for timely identification of embryos that are affected by deleterious genetic traits in in vitro fertilization (IVF) clinics. Polymerase chain reaction (PCR) is the backbone of modern genetic diagnoses, and a spectrum of PCR-based techniques have been used to detect various thalassemia mutations in prenatal diagnosis (PND) and PGD. Among thalassemias, SEA-type α-thalassemia is the most common variety found in Asia, and can lead to Bart's hydrops fetalis and serious maternal complications. To formulate an efficient digital PCR for clinical diagnosis of SEA-type α-thalassemia in cultured embryos, we conducted a pilot study to detect the α-globin and SEA-type deletion alleles in blastomere biopsies with a highly sensitive microfluidics-based digital PCR method. Genomic DNA from embryo biopsy samples were extracted, and crude DNA extracts were first amplified by a conventional PCR procedure followed by a nested PCR reaction with primers and probes that are designed for digital PCR amplification. Analysis of microfluidics-based PCR reactions showed that robust signals for normal α-globin and SEA-type deletion alleles, together with an internal control gene, can be routinely generated using crude embryo biopsies after a 10 6 -fold dilution of primary PCR products. The SEA-type deletion in cultured embryos can be sensitively diagnosed with the digital PCR procedure in clinics. The adoption of this robust PGD method could prevent the implantation of IVF embryos that are destined to develop Bart's hydrops fetalis in a timely manner. The results also help inform future development of a standard digital PCR procedure for cost-effective PGD of α-thalassemia in a standard IVF clinic. Copyright © 2017. Published by Elsevier B.V.

  18. Assessment of Duplex PCR for the simultaneous diagnose of Mycobacterium spp. and Brucella spp. in cattle

    Directory of Open Access Journals (Sweden)

    Ariel Escobar

    2013-03-01

    Full Text Available Tuberculosis and brucellosis remain important causes of morbidity and mortality in many countries, for the detection of both diseases requires efficient and sensitive tool for effectuate the diagnosis. This study was aimed to evaluate and compare the duplex PCR versus the nested PCR, for detection of Brucella spp. (BR and Mycobacterium spp. (TB. A total of 100 samples of tissues from tracheo-bronchial lymph nodes, bovine lung and bacterial isolate as positive controls were used. Were evaluated ten combinations of primers which were designed to flank the segment of the 16S rRNA sequence (RB and antigen gen MPB70 (TB, the best result for the Duplex PCR was obtained with the primers Bru-2F/Bru-2R for BR and Tub-1F/Tub-N-R for TB. The amplification of the products was 225 and 230-bp respectively. In order to compare the results of the proposed technique, all samples were initially analyzed and compared between PCR and nested PCR (Kappa, k = 0.85 and the concordance between Duplex PCR and nested PCR (k = 0.88 for the two bacteria was very good.

  19. Detection of genetically modified organisms (GMOs using isothermal amplification of target DNA sequences

    Directory of Open Access Journals (Sweden)

    La Mura Maurizio

    2009-02-01

    Full Text Available Abstract Background The most common method of GMO detection is based upon the amplification of GMO-specific DNA amplicons using the polymerase chain reaction (PCR. Here we have applied the loop-mediated isothermal amplification (LAMP method to amplify GMO-related DNA sequences, 'internal' commonly-used motifs for controlling transgene expression and event-specific (plant-transgene junctions. Results We have tested the specificity and sensitivity of the technique for use in GMO studies. Results show that detection of 0.01% GMO in equivalent background DNA was possible and dilutions of template suggest that detection from single copies of the template may be possible using LAMP. Conclusion This work shows that GMO detection can be carried out using LAMP for routine screening as well as for specific events detection. Moreover, the sensitivity and ability to amplify targets, even with a high background of DNA, here demonstrated, highlights the advantages of this isothermal amplification when applied for GMO detection.

  20. Cross-platform comparison of SYBR® Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC study

    Directory of Open Access Journals (Sweden)

    Dial Stacey L

    2008-07-01

    Full Text Available Abstract Background The MicroArray Quality Control (MAQC project evaluated the inter- and intra-platform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006. The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan® Gene Expression PCR Assay, Standardized (Sta RT-PCR™ and QuantiGene®. The data showed great consistency in gene expression measurements across different microarray platforms, different technologies and test sites. However, SYBR® Green real-time PCR, another common technique utilized by half of all real-time PCR users for gene expression measurement, was not addressed in the MAQC study. In the present study, we compared the performance of SYBR Green PCR with TaqMan PCR, microarrays and other quantitative technologies using the same two Reference RNA samples as the MAQC project. We assessed SYBR Green real-time PCR using commercially available RT2 Profiler™ PCR Arrays from SuperArray, containing primer pairs that have been experimentally validated to ensure gene-specificity and high amplification efficiency. Results The SYBR Green PCR Arrays exhibit good reproducibility among different users, PCR instruments and test sites. In addition, the SYBR Green PCR Arrays have the highest concordance with TaqMan PCR, and a high level of concordance with other quantitative methods and microarrays that were evaluated in this study in terms of fold-change correlation and overlap of lists of differentially expressed genes. Conclusion These data demonstrate that SYBR Green real-time PCR delivers highly comparable results in gene expression measurement with TaqMan PCR and other high-density microarrays.

  1. Development of a recombinase polymerase amplification assay for rapid detection of Francisella noatunensis subsp. orientalis.

    Directory of Open Access Journals (Sweden)

    Khalid Shahin

    Full Text Available Francisella noatunensis subsp. orientalis (Fno is the causative agent of piscine francisellosis in warm water fish including tilapia. The disease induces chronic granulomatous inflammation with high morbidity and can result in high mortality. Early and accurate detection of Fno is crucial to set appropriate outbreak control measures in tilapia farms. Laboratory detection of Fno mainly depends on bacterial culture and molecular techniques. Recombinase polymerase amplification (RPA is a novel isothermal technology that has been widely used for the molecular diagnosis of various infectious diseases. In this study, a recombinase polymerase amplification (RPA assay for rapid detection of Fno was developed and validated. The RPA reaction was performed at a constant temperature of 42°C for 20 min. The RPA assay was performed using a quantitative plasmid standard containing a unique Fno gene sequence. Validation of the assay was performed not only by using DNA from Fno, closely related Francisella species and other common bacterial pathogens in tilapia farms, but also by screening 78 Nile tilapia and 5 water samples. All results were compared with those obtained by previously established real-time qPCR. The developed RPA showed high specificity in detection of Fno with no cross-detection of either the closely related Francisella spp. or the other tested bacteria. The Fno-RPA performance was highly comparable to the published qPCR with detection limits at 15 and 11 DNA molecules detected, respectively. The RPA gave quicker results in approximately 6 min in contrast to the qPCR that needed about 90 min to reach the same detection limit, taking only 2.7-3 min to determine Fno in clinical samples. Moreover, RPA was more tolerant to reaction inhibitors than qPCR when tested with field samples. The fast reaction, simplicity, cost-effectiveness, sensitivity and specificity make the RPA an attractive diagnostic tool that will contribute to controlling the

  2. Diagnosis of Neisseria gonorrhoeae among pregnant women by culture method and PCR on cppB gene

    Directory of Open Access Journals (Sweden)

    Jalal Mardaneh

    2013-11-01

    Full Text Available Background: Neisseria gonorrhoeae is a human obligate pathogen and the etiological agent of gonorrhea. Health irreparable complications resulting from gonorrhea disease occur mainly in pregnant women and neonates. Aim of this study was diagnosis of Neisseria gonorrhoeae among pregnant women with using culture and molecular method by amplification of cppB gene with PCR. Material and Methods: In this cross-sectional study, two endocervical swab specimens were obtained from 1100 pregnant women who referred to Shiraz Hospitals. Culture on nonselective and selective media and nucleic acid amplification test (NAAT were performed for detection of Neisseria gonorrhoeae cppB gene. Results: All endocervical swabs cultures on selective and nonselective media were negative for Neisseria gonorrhoeae. Among examined endocervical swabs, 13samples (1.18% were positive by nucleic acid amplification of Neisseria gonorrgoeae cppB gene. Conclusion: Negative results of culture and positive results of PCR in this study indicate that however culture is gold standard method for detection of Neisseria gonorrhoeae but due to bacterial autolysis, poor sampling techniques and improper specimen storage and transport, its value decline as compared with Nucleic acid amplification test (NAAT.

  3. Multiplex polymerase chain reaction (PCR) on a SU-8 chip

    DEFF Research Database (Denmark)

    Christensen, Troels Balmer; Bang, Dang Duong; Wolff, Anders

    2008-01-01

    the PCR. The chip performs very well with respect to heating and cooling rates with values up to around 40 °C/s and 20 °C/s, respectively, and has low power consumption (0.5–2.5 W depending on temperature). Multiplex DNA amplification by PCR for the detection of Campylobacter at species level...

  4. Species specific polymerase chain reaction (PCR) assay for ...

    African Journals Online (AJOL)

    A highly specific single step polymerase chain reaction (PCR) is described for the detection of pig (Sus domesticus) meat. A PCR assay was successfully optimized for amplification of 629 and 322-bp DNA fragment extracted from pig meat using designed species-specific primer pairs based on mitochondrial D-loop and 12S ...

  5. PCR identification of Fusarium genus based on nuclear ribosomal ...

    African Journals Online (AJOL)

    PCR sensitivity ranged from 100 fg to 10 ng for DNA extracted from Fusarium oxysporum mycelium. No amplification products were detected with PCR of DNA from Rhizoctonia solani and Macrophomina phaseolina isolates using these primers. The assay is useful for rapid identification of Fusarium spp. cultures.

  6. Detection of Tritrichomonas Foetus in Cattle Using a PCR ...

    African Journals Online (AJOL)

    A trichomoniasis polymerase chain reaction (PCR) assay was successfully established at the Central Veterinary Laboratory (CVL). Four preputial and 27 vaginal washings of bulls and cows obtained from communal cattle in Zimbabwe's Buhera and Wedza districts were tested using a PCR amplification assay as well as the ...

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

    KAUST Repository

    Wu, Jinbo

    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.

  8. Apparatus, System and Method for Fast Detection of Genetic Information by PCR in an Interchangeable Chip

    KAUST Repository

    Wen, Weijia

    2011-03-03

    A polymerase chain reaction (PCR) device for fast amplification and detection of DNA includes an interchangeable PCR chamber, a temperature control component, and an optical detection system. The DNA amplification is performed on an interchangeable chip with volumes as small as 1.25 µl, while the heating and cooling rate may be as fast as 12.7 °C/second ensuring that the total time needed of only 25 minutes to complete the 35 cycle PCR amplification. The PCR may be performed according to a two-temperature approach for denaturing and annealing (Td and Ta) of DNA with the PCR chip, with which the amplification of male-specific SRY gene marker by utilizing raw saliva may be achieved. The genetic identification may be in-situ detected after PCR by the optical detection system.

  9. Development and evaluation of loop-mediated isothermal amplification assay for rapid detection of Capripoxvirus

    Directory of Open Access Journals (Sweden)

    Kanisht Batra

    2015-11-01

    Full Text Available Aim: The present study was undertaken to develop a nucleic acid-based diagnostic assay loop-mediated isothermal amplification assay (LAMP targeting highly conserved genomic regions of Capripoxvirus (CaPVs and its comparative evaluation with real-time polymerase chain reaction (PCR. Material and Methods: Lyophilized vaccine strain of sheeppox virus (SPPV was used for optimization of LAMP assay. The LAMP assay was designed using envelope immunogenic protein (P32 coding gene targeting highly conserved genomic regions of CaPV responsible for causing sheep pox, goat pox, and lumpy skin disease in sheep, goat and cattle respectively. Serial tenfold dilution of SPPV recombinant plasmid DNA was used for a calculating limit of detection. Analytical sensitivity and specificity were performed. Results: The test described is quick (30 min, sensitive and specific for detection of CaPVs. The described assay did not show any cross-reactivity to other related viruses that cause apparently similar clinical signs. It was found to be ten times more sensitive than conventional PCR however, 100 times less sensitive than quantitative PCR (qPCR. LAMP assay results were monitored by color change method using picogreen dye and agarose gel electrophoresis. Conclusion: LAMP assay can be a very good alternative for CaPV detection to other molecular techniques requiring sophisticated equipments.

  10. The use of droplet digital PCR in liquid biopsies: A highly sensitive technique for MYD88 p.(L265P) detection in cerebrospinal fluid.

    Science.gov (United States)

    Hiemcke-Jiwa, Laura S; Minnema, Monique C; Radersma-van Loon, Joyce H; Jiwa, N Mehdi; de Boer, Mirthe; Leguit, Roos J; de Weger, Roel A; Huibers, Manon M H

    2018-04-01

    The gold standard for diagnosis of central nervous system lymphomas still regards a stereotactic brain biopsy, with the risk of major complications for the patient. As tumor cells can be detected in cerebrospinal fluid (CSF), CSF analysis can be used as an alternative. In this respect, mutation analysis in CSF can be of added value to other diagnostic parameters such a cytomorphology and clonality analysis. A well-known example of targeted mutation analysis entails MYD88 p.(L265P) detection, which is present in the majority of Bing Neel syndrome and primary central nervous system lymphoma (PCNSL) patients. Unfortunately, tumor yield in CSF can be very low. Therefore, use of the highly sensitive droplet digital PCR (ddPCR) might be a suitable analysis strategy for targeted mutation detection. We analyzed 26 formalin fixed paraffin embedded (FFPE) samples (8 positive and 18 negative for MYD88 p.(L265P) mutation) by ddPCR, of which the results were compared with next generation sequencing (NGS). Subsequently, 32 CSF samples were analyzed by ddPCR. ddPCR and NGS results on FFPE material showed 100% concordance. Among the 32 CSF samples, 9 belonged to patients with lymphoplasmacytic lymphoma (LPL) and clinical suspicion of Bing Neel syndrome, and 3 belonged to patients with PCNSL. Nine of these samples tested positive for MYD88 p.(L265P) (8 LPL and 1 PCNSL). This study shows that sensitive MYD88 mutation analysis by ddPCR in CSF is highly reliable and can be applied even when DNA input is low. Therefore, ddPCR is of added value to current diagnostic parameters, especially when the available amount of DNA is limited. Copyright © 2017 John Wiley & Sons, Ltd.

  11. Bayesian techniques for comparison of the test performance of PCR and culture for the identification of Campylobacter in enriched comminuted chicken samples.

    Science.gov (United States)

    Ebel, E D; Williams, M S; Golden, N J; Tankson, J

    2016-05-01

    Using Bayesian methods that do not require the definition of a gold standard, the diagnostic sensitivity and specificity of a real-time polymerase chain reaction (PCR) assay are compared to those of an enriched culture assay for detection of Campylobacter in enriched comminuted chicken samples. Food Safety and Inspection Service comminuted chicken samples were collected from production facilities across the United States. Enriched samples were examined using a commercial real-time PCR kit and plated for culture. Allowing for conditional dependence between these approaches and defining relatively uninformed prior distributions, the 'no gold standard' Bayesian methods generated estimates of the means (95% credible interval) of the posterior distributions for sensitivity and specificity of the PCR as 93% (79, 100%) and 95% (87, 100%) respectively. The estimated sensitivity implies a mean false negative frequency of 7%. The estimated means of the posterior distributions for sensitivity and specificity of the culture assay were 91% (76, 100%) and 96% (88, 100%) respectively. In this case, the mean false negative frequency is 9%. Graphical comparisons of the posterior distributions with their corresponding prior distributions suggested only subtle differences in the sensitivities of both tests, but the posterior distributions for specificities are substantially more certain than the prior distributions. The study suggests that the commercial real-time PCR assay is a more sensitive screening test that would provide timelier negative test results. The modest 1% reduction in specificity of this PCR assay, as compared to an enriched culture assay, is less of a concern for regulatory testing programs if a culture-based confirmatory assay is applied to all presumptive positive samples. The sensitivity and specificity of a PCR assay and a culture assay for Campylobacter in comminuted poultry produced in the United States were estimated. The PCR assay was shown to be an

  12. Detection of viral sequences by internally calibrated gene amplification.

    Science.gov (United States)

    Cheung, R K; Hui, M F; Dosch, H M; Ewart, T E

    1993-05-01

    Inherent pitfalls of the polymerase chain reaction (PCR) can become serious difficulties when transferring research applications to high-volume routine procedures such as biofermentation process control and clinical diagnostics. Difficulties include 1) the danger of accidental sample contamination with positive control templates; 2) variable amplification due to positional effects in thermocycler blocks and unequal primer efficiency for sense/anti-sense strands; and 3) the need for reliable controls, which provide confidence for reporting negative reactions. Using the PCR detection system for Epstein-Barr virus as a model, we have developed a quick process to generate mutant internal co-amplification templates. These can be used for titration of amplification sensitivity. More importantly, single tube co-amplification without titrations allows determination of the minimum sensitivity achieved in each individual reaction; critical information when reporting negative diagnostic results. Mutant and native fragments are easy to distinguish by size, and sample cross contamination can be readily identified. The system should be easily adaptable to gene amplification procedures, which aim to routinely detect the presence of a given gene fragment in a controlled fashion.

  13. Optimizing direct amplification of forensic commercial kits for STR determination.

    Science.gov (United States)

    Caputo, M; Bobillo, M C; Sala, A; Corach, D

    2017-04-01

    Direct DNA amplification in forensic genotyping reduces analytical time when large sample sets are being analyzed. The amplification success depends mainly upon two factors: on one hand, the PCR chemistry and, on the other, the type of solid substrate where the samples are deposited. We developed a workflow strategy aiming to optimize times and cost when starting from blood samples spotted onto diverse absorbent substrates. A set of 770 blood samples spotted onto Blood cards, Whatman ® 3 MM paper, FTA™ Classic cards, and Whatman ® Grade 1 was analyzed by a unified working strategy including a low-cost pre-treatment, a PCR amplification volume scale-down, and the use of the 3500 Genetic Analyzer as the analytical platform. Samples were analyzed using three different commercial multiplex STR direct amplification kits. The efficiency of the strategy was evidenced by a higher percentage of high-quality profiles obtained (over 94%), a reduced number of re-injections (average 3.2%), and a reduced amplification failure rate (lower than 5%). Average peak height ratio among different commercial kits was 0.91, and the intra-locus balance showed values ranging from 0.92 to 0.94. A comparison with previously reported results was performed demonstrating the efficiency of the proposed modifications. The protocol described herein showed high performance, producing optimal quality profiles, and being both time and cost effective. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  14. Comparison between Conventional and Real-Time PCR Assays for Diagnosis of Visceral Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Mariana R. Pereira

    2014-01-01

    Full Text Available The diagnosis of visceral leishmaniasis (VL is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR has proven to be effective in detecting the genome of Leishmania species in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n=12 presented positive results for serology and 79% (n=15 positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient’s blood.

  15. Typing of Y chromosome SNPs with multiplex PCR methods

    DEFF Research Database (Denmark)

    Sanchez Sanchez, Juan Jose; Børsting, Claus; Morling, Niels

    2005-01-01

    We describe a method for the simultaneous typing of Y-chromosome single nucleotide polymorphism (SNP) markers by means of multiplex polymerase chain reaction (PCR) strategies that allow the detection of 35 Y chromosome SNPs on 25 amplicons from 100 to 200 pg of chromosomal deoxyribonucleic acid...... (DNA). Multiplex PCR amplification of the DNA was performed with slight modifications of standard PCR conditions. Single-base extension (SBE) was performed using the SNaPshot kit containing fluorescently labeled ddNTPs. The extended primers were detected on an ABI 3100 sequencer. The most important...... factors for the creation of larger SNP typing PCR multiplexes include careful selection of primers for the primary amplification and the SBE reaction, use of DNA primers with homogenous composition, and balancing the primer concentrations for both the amplification and the SBE reactions....

  16. Deep Diversity: Novel Approach to Overcoming the PCR Bias Encountered During Environmental Analysis of Microbial Populations for Alpha-Diversity

    Science.gov (United States)

    Ramirez, Gustavo A; Vaishampayan, Parag A.

    2011-01-01

    Alpha-diversity studies are of crucial importance to environmental microbiologists. The polymerase chain reaction (PCR) method has been paramount for studies interrogating microbial environmental samples for taxon richness. Phylogenetic studies using this technique are based on the amplification and comparison of the 16S rRNA coding regions. PCR, due disproportionate distribution of microbial species in the environment, increasingly favors the amplification of the most predominant phylotypes with every subsequent reaction cycle. The genetic and chemical complexity of environmental samples are intrinsic factors that exacerbate an inherit bias in PCR-based quantitative and qualitative studies of microbial communities. We report that treatment of a genetically complex total genomic environmental DNA extract with Propidium Monoazide (PMA), a DNA intercalating molecule capable of forming a covalent cross-linkage to organic moieties upon light exposure, disproportionally inactivates predominant phylotypes and results in the exponential amplification of previously shadowed microbial ?-diversity quantified as a 19.5% increase in OUTs reported via phylogenetic screening using PhyloChip.

  17. Loop-Mediated Isothermal Amplification Procedure (LAMP) for Detection of the Potato Zebra Chip Pathogen "Candidatus Liberibacter solanacearum".

    Science.gov (United States)

    Ravindran, Aravind; Lévy, Julien; Pierson, Elizabeth; Gross, Dennis C

    2015-01-01

    An efficient loop-mediated isothermal amplification procedure (LAMP) for the detection of "Candidatus Liberibacter solanacearum" (Lso), the bacterial causal agent of potato zebra chip (ZC) disease, is described in this chapter. Similar to the polymerase chain reaction (PCR), the LAMP employs a bacterial polymerase to amplify specific DNA sequences. However, the method differs from conventional PCR in that it uses six primers specific to the target region to generate a loop structure and autocycling strand displacement rather than thermocycling for sequence amplification. Moreover, unlike PCR that requires agarose gel electrophoresis for resolution, the positive LAMP results can be visualized directly as a precipitate within the reaction tubes. The 16S rDNA gene of "Ca. Liberibacter solanacearum" was used as the target for the design of the six LAMP primers. The LAMP technique is a reliable, rapid, and cost-effective method of detecting the "Ca. Liberibacter solanacearum" pathogen in the potato/tomato psyllid, Bactericera cockerelli, and in field-grown potato plants and tubers.

  18. Inter-laboratory and inter-assay comparison on two real-time PCR techniques for quantification of PCV2 nucleic acid extracted from field samples

    DEFF Research Database (Denmark)

    Hjulsager, Charlotte Kristiane; Grau-Roma, L.; Sibila, M.

    2009-01-01

    Several real-time PCR assays for quantification of PCV2 DNA (qPCR) have been described in the literature. and different in-house assays are being used by laboratories around the world. A general threshold of it copies of PCV2 per millilitre serum for postweaning multisystemic wasting syndrome (PMWS......) diagnosis has been suggested. However, neither inter-laboratory nor inter-assay comparisons have been published so far. In the present study two different qPCR probe assays Used routinely in two laboratories were compared on DNA extracted From serum, nasal and rectal swabs. Results showed a significant...... association between the amount of PCV2 DNA and the amount of total DNA, neither in nasal (p = 0.86) nor in rectal (p=0.78) swabs, suggesting that normalizing of PCV2 DNA load in swab samples to total DNA concentration is not suitable. The present exploratory study highlights the need for the performance...

  19. Bias in the Cq value observed with hydrolysis probe based quantitative PCR can be corrected with the estimated PCR efficiency value

    NARCIS (Netherlands)

    Tuomi, Jari Michael; Voorbraak, Frans; Jones, Douglas L.; Ruijter, Jan M.

    2010-01-01

    For real-time monitoring of PCR amplification of DNA, quantitative PCR (qPCR) assays use various fluorescent reporters. DNA binding molecules and hybridization reporters (primers and probes) only fluoresce when bound to DNA and result in the non-cumulative increase in observed fluorescence.

  20. Hardness amplification in nondeterministic logspace

    OpenAIRE

    Gupta, Sushmita

    2007-01-01

    A hard problem is one which cannot be easily computed by efficient algorithms. Hardness amplification is a procedure which takes as input a problem of mild hardness and returns a problem of higher hardness. This is closely related to the task of decoding certain error-correcting codes. We show amplification from mild average case hardness to higher average case hardness for nondeterministic logspace and worst-to-average amplification for nondeterministic linspace. Finally we explore possible ...

  1. Pathogen Causing Disease of Diagnosis PCR Tecnology

    OpenAIRE

    SEVİNDİK, Emre; KIR, A. Çağrı; BAŞKEMER, Kadir; UZUN, Veysel

    2013-01-01

    Polimerase chain reaction (PCR) with which, the development of recombinant DNA tecnology, a technique commonly used in field of moleculer biology and genetic. Duplication of the target DNA is provided with this technique without the need for cloning. Some fungus species, bacteria, viruses constitutent an important group of pathogenicity in human, animals and plants. There are routinely applied types of PCR in the detection of pathogens infections diseases. These Nested- PCR, Real- Time PCR, M...

  2. COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

    Directory of Open Access Journals (Sweden)

    Masashi Miura

    2013-12-01

    Full Text Available SUMMARY The use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

  3. Application of Multiplex PCR for Detection and Differentiation of Entamoeba histolytica, Entamoeba dispar and Entamoeba moshkovskii.

    Directory of Open Access Journals (Sweden)

    Nozhat Zebardast

    2014-12-01

    Full Text Available Entamoeba moshkovskii and E. dispar are impossible to differentiate microscopically from the pathogenic species E. histolytica. Multiplex polymerase chain reaction (Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment.For detection and differentiation of the three-microscopy indistinguishable Entamoeba species in human, multiplex PCR assay using different DNA extraction methods was studied. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species.A 166-bp PCR product with E. histolytica DNA, a 580-bp product with E. moshkovskii DNA and a 752-bp product with E. dispar DNA were generated in a single-round and multiplex PCR reaction.We recommend this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys.

  4. DNA-based techniques for authentication of processed food and food supplements.

    Science.gov (United States)

    Lo, Yat-Tung; Shaw, Pang-Chui

    2018-02-01

    Authentication of food or food supplements with medicinal values is important to avoid adverse toxic effects, provide consumer rights, as well as for certification purpose. Compared to morphological and spectrometric techniques, molecular authentication is found to be accurate, sensitive and reliable. However, DNA degradation and inclusion of inhibitors may lead to failure in PCR amplification. This paper reviews on the existing DNA extraction and PCR protocols, and the use of small size DNA markers with sufficient discriminative power for molecular authentication. Various emerging new molecular techniques such as isothermal amplification for on-site diagnosis, next-generation sequencing for high-throughput species identification, high resolution melting analysis for quick species differentiation, DNA array techniques for rapid detection and quantitative determination in food products are also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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

  6. Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

    Directory of Open Access Journals (Sweden)

    L.E. Alvares

    2003-12-01

    Full Text Available The reverse transcription-polymerase chain reaction (RT-PCR is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping ß-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19% was estimated for the different genes analyzed (6 to 9 repetitions. The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

  7. Single-step blood direct PCR: A robust and rapid method to diagnose triplet repeat disorders.

    Science.gov (United States)

    Singh, Inder; Swarup, Vishnu; Shakya, Sunil; Goyal, Vinay; Faruq, Mohammed; Srivastava, Achal Kumar

    2017-08-15

    DNA extraction prior to polymerase chain reaction (PCR) amplification in genetic diagnoses of triplet repeat disorders (TRDs) is tedious and labour-intensive and has the limitations of sample contamination with foreign DNA, including that from preceding samples. Therefore, we aimed to develop a rapid, robust, and cost-effective method for expeditious genetic investigation of TRDs from whole blood as a DNA template. Peripheral blood samples were collected from 70 clinically suspected patients of progressive ataxia. The conventional method using genomic DNA and single-step Blood-Direct PCR (BD-PCR) method with just 2μl of whole blood sample were tested to amplify triplet repeat expansion in genes related to spinocerebellar ataxia (SCA) types 1, 2, 3, 12 and Friedreich's ataxia (FRDA). Post-PCR, the allele sizes were mapped and repeat numbers were calculated using GeneMapper and macros run in Microsoft Excel programmes. Successful amplification of target regions was achieved in all samples by both methods. The frequency of the normal and mutated allele was concordant between both methods, diagnosing 37% positive for a mutation in either of the candidate genes. The BD-PCR resulted in higher intensities of product peaks of normal and pathogenic alleles. The nearly-accurate sizing of the normal and expanded allele was achieved in a shorter time (4-5h), without DNA extraction and any risk of cross contamination, which suggests the BD-PCR to be a reliable, inexpensive, and rapid method to confirm TRDs. This technique can be introduced in routine diagnostic procedures of other tandem repeat disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR.

    Science.gov (United States)

    Wills, Melanie K B; Kirby, Andrea M; Lloyd, Vett K

    2018-02-04

    Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to humans through the bite of infected Ixodes ticks. The primary etiological agent in North America is Borrelia burgdorferi sensu stricto. As geographic risk regions expand, it is prudent to support robust surveillance programs that can measure tick infection rates, and communicate findings to clinicians, veterinarians, and the general public. The molecular technique of nested polymerase chain reaction (nPCR) has long been used for this purpose, and it remains a central, inexpensive, and robust approach in the detection of Borrelia in both ticks and wildlife. This article demonstrates the application of nPCR to tick DNA extracts to identify infected specimens. Two independent B. burgdorferi targets, genes encoding Flagellin B (FlaB) and Outer surface protein A (OspA), have been used extensively with this technique. The protocol involves tick collection, DNA extraction, and then an initial round of PCR to detect each of the two Borrelia-specific loci. Subsequent polymerase chain reaction (PCR) uses the product of the first reaction as a new template to generate smaller, internal amplification fragments. The nested approach improves upon both the specificity and sensitivity of conventional PCR. A tick is considered positive for the pathogen when inner amplicons from both Borrelia genes can be detected by agarose gel electrophoresis.

  9. A modified MS-PCR approach to diagnose patients with Prader-Willi and Angelman syndrome.

    Science.gov (United States)

    Dos Santos, Jéssica Fernandes; Mota, Laís R; Rocha, Pedro Henrique Silva Andrade; Ferreira de Lima, Renata Lúcia L

    2016-11-01

    Prader-Willi (PWS) and Angelman (AS) syndromes are clinically distinct neurodevelopmental genetic diseases with multiple phenotypic manifestations. They are one of the most common genetic syndromes caused by non-Mendelian inheritance in the form of genomic imprinting, and can be attributable to the loss of gene expression due to imprinting within the chromosomal region 15q11-q13. Clinical diagnosis of PWS and AS is challenging, and the use of molecular and cytomolecular studies is recommended to help in determining the diagnosis of these conditions. The methylation analysis is a sensible approach; however there are several techniques for this purpose, such as the methylation-sensitive polymerase chain reaction (MS-PCR). This study aims to optimize the MS-PCR assay for the diagnosis of potential PWS and AS patients using DNA modified by sodium bisulfite. We used the MS-PCR technique of PCR described by Kosaki et al. (1997) adapted with betaine. All different concentrations of betaine used to amplify the methylated and unmethylated chromosomal region 15q11-q13 on the gene SNRPN showed amplification results, which increased proportionally to the concentration of betaine. The methylation analysis is a technically robust and reproducible screening method for PWS and AS. The MS-PCR assures a faster, cheaper and more efficient method for the primary diagnosis of the SNRPN gene in cases with PWS and AS, and may detect all of the three associated genetic abnormalities: deletion, uniparental disomy or imprinting errors.

  10. Loop-mediated isothermal amplification as a reliable assay for Toxocara canis infection in pet dogs.

    Science.gov (United States)

    Khoshakhlagh, Paria; Spotin, Adel; Mahami-Oskouei, Mahmoud; Shahbazi, Abbas; Ozlati, Maryam

    2017-09-01

    Keeping of infected dogs as pet results in the potential transmission risk factors for shedding helminthic infections such as toxocariasis. Lack of accurate identification of Toxocara canis eggs in non-dewormed infected pet dogs remains a diagnostic concern among researchers. In this study, dog owners were asked to fill up a questionnaire regarding their pets and their attitude towards the deworming regimen. One hundred faecal samples were collected from pet dogs (Northwest Iran) and were subsequently identified by the ZnSo4 flotation technique, PCR and loop-mediated isothermal amplification (LAMP) assays. The DNA of the recovered T. canis eggs was then extracted and amplified by LAMP and PCR. Furthermore, ITS2 amplicons were sequenced for appraisal of the phylogenetic analysis. Nine, 5 and 11% of T. canis infections were identified by microscopy, PCR and LAMP, respectively. It was detected that LAMP was 10 times (10 -10 to 10 -13  g/μl) more sensitive than PCR (10 -10 to 10 -12  g/μl). The kappa value between LAMP and PCR indicated a faint concurrence (0.463). The kappa coefficient between LAMP and flotation technique indicated a strong agreement (0.667). The highest infection rate (n = 11) was detected in non-dewormed pet dogs, particularly those less than 3 months old (P canis eggs in infected pet dogs. It was proposed that the dog holder's awareness is insufficient to implement regular deworming schedules. Additionally, regional policymakers should broadly revise anthelmintic treatment guidelines.

  11. Evidence of high-elevation amplification versus Arctic amplification.

    Science.gov (United States)

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2016-01-12

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world's high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction.

  12. Evidence of high-elevation amplification versus Arctic amplification

    Science.gov (United States)

    Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

    2016-01-01

    Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction.

  13. Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition.

    Directory of Open Access Journals (Sweden)

    Dimo Dietrich

    Full Text Available Formalin-fixed and paraffin-embedded (FFPE tissues represent a valuable source for biomarker studies and clinical routine diagnostics. However, they suffer from degradation of nucleic acids due to the fixation process. Since genetic and epigenetic studies usually require PCR amplification, this degradation hampers its use significantly, impairing PCR robustness or necessitating short amplicons. In routine laboratory medicine a highly robust PCR performance is mandatory for the clinical utility of genetic and epigenetic biomarkers. Therefore, methods to improve PCR performance using DNA from FFPE tissue are highly desired and of wider interest. The effect of template DNA derived from FFPE tissues on PCR performance was investigated by means of qPCR and conventional PCR using PCR fragments of different sizes. DNA fragmentation was analyzed via agarose gel electrophoresis. This study showed that poor PCR amplification was partly caused by inhibition of the DNA polymerase by fragmented DNA from FFPE tissue and not only due to the absence of intact template molecules of sufficient integrity. This PCR inhibition was successfully minimized by increasing the polymerase concentration, dNTP concentration and PCR elongation time thereby allowing for the robust amplification of larger amplicons. This was shown for genomic template DNA as well as for bisulfite-converted template DNA required for DNA methylation analyses. In conclusion, PCR using DNA from FFPE tissue suffers from inhibition which can be alleviated by adaptation of the PCR conditions, therefore allowing for a significant improvement of PCR performance with regard to variability and the generation of larger amplicons. The presented solutions to overcome this PCR inhibition are of tremendous value for clinical chemistry and laboratory medicine.

  14. Differences between two real-time PCR-based hepatitis C virus (HCV) assays (RealTime HCV and Cobas AmpliPrep/Cobas TaqMan) and one signal amplification assay (Versant HCV RNA 3.0) for RNA detection and quantification.

    Science.gov (United States)

    Vermehren, Johannes; Kau, Annika; Gärtner, Barbara C; Göbel, Reinhild; Zeuzem, Stefan; Sarrazin, Christoph

    2008-12-01

    Hepatitis C virus (HCV) RNA detection and quantification are the key diagnostic tools for the management of hepatitis C. Commercially available HCV RNA assays are calibrated to the HCV genotype 1 (gt1)-based WHO standard. Significant differences between assays have been reported. However, it is unknown which assay matches the WHO standard best, and little is known about the sensitivity and linear quantification of the assays for non-gt1 specimens. Two real-time reverse transcriptase PCR-based assays (RealTime HCV and Cobas Ampliprep/Cobas TaqMan HCV [CAP/CTM]) and one signal amplification-based assay (the Versant HCV RNA, version 3.0, branched DNA [bDNA] assay) were compared for their abilities to quantify HCV RNA in clinical specimens (n = 65) harboring HCV isolates of gt1 to g5. The mean differences in the amounts detected by RealTime HCV in comparison to those detected by the bDNA assay and CAP/CTM were -0.02 and 0.72 log(10) IU/ml HCV RNA, respectively, for gt1; -0.22 and 0.03 log(10) IU/ml HCV RNA, respectively, for gt2; -0.27 and -0.22 log(10) IU/ml HCV RNA, respectively, for gt3; -0.19 and -1.27 log(10) IU/ml HCV RNA, respectively, for gt4; and -0.03 and 0.09 log(10) IU/ml HCV RNA, respectively, for gt5. The lower limits of detection for RealTime HCV and CAP/CTM were 16.8 and 10.3 IU/ml, respectively, for the WHO standard and in the range of 4.7 to 9.0 and 3.4 to 44.4 IU/ml, respectively, for clinical specimens harboring gt1 to gt6. Direct comparison of the two assays with samples of the WHO standard (code 96/798) with high titers yielded slightly smaller amounts by RealTime HCV (-0.2 log(10) at 1,500 IU/ml and -0.3 log(10) at 25,000 IU/ml) and larger amounts by CAP/CTM (0.3 log(10) at 1,500 IU/ml and 0.2 log(10) at 25,000 IU/ml). Finally, all three tests were linear between 4.0 x 10(3) and 1.0 x 10(6) IU/ml (correlation coefficient, >or=0.99). In conclusion, the real-time PCR based assays sensitively detected all genotypes and showed comparable linearities

  15. Differences between Two Real-Time PCR-Based Hepatitis C Virus (HCV) Assays (RealTime HCV and Cobas AmpliPrep/Cobas TaqMan) and One Signal Amplification Assay (Versant HCV RNA 3.0) for RNA Detection and Quantification▿

    Science.gov (United States)

    Vermehren, Johannes; Kau, Annika; Gärtner, Barbara C.; Göbel, Reinhild; Zeuzem, Stefan; Sarrazin, Christoph

    2008-01-01

    Hepatitis C virus (HCV) RNA detection and quantification are the key diagnostic tools for the management of hepatitis C. Commercially available HCV RNA assays are calibrated to the HCV genotype 1 (gt1)-based WHO standard. Significant differences between assays have been reported. However, it is unknown which assay matches the WHO standard best, and little is known about the sensitivity and linear quantification of the assays for non-gt1 specimens. Two real-time reverse transcriptase PCR-based assays (RealTime HCV and Cobas Ampliprep/Cobas TaqMan HCV [CAP/CTM]) and one signal amplification-based assay (the Versant HCV RNA, version 3.0, branched DNA [bDNA] assay) were compared for their abilities to quantify HCV RNA in clinical specimens (n = 65) harboring HCV isolates of gt1 to g5. The mean differences in the amounts detected by RealTime HCV in comparison to those detected by the bDNA assay and CAP/CTM were −0.02 and 0.72 log10 IU/ml HCV RNA, respectively, for gt1; −0.22 and 0.03 log10 IU/ml HCV RNA, respectively, for gt2; −0.27 and −0.22 log10 IU/ml HCV RNA, respectively, for gt3; −0.19 and −1.27 log10 IU/ml HCV RNA, respectively, for gt4; and −0.03 and 0.09 log10 IU/ml HCV RNA, respectively, for gt5. The lower limits of detection for RealTime HCV and CAP/CTM were 16.8 and 10.3 IU/ml, respectively, for the WHO standard and in the range of 4.7 to 9.0 and 3.4 to 44.4 IU/ml, respectively, for clinical specimens harboring gt1 to gt6. Direct comparison of the two assays with samples of the WHO standard (code 96/798) with high titers yielded slightly smaller amounts by RealTime HCV (−0.2 log10 at 1,500 IU/ml and −0.3 log10 at 25,000 IU/ml) and larger amounts by CAP/CTM (0.3 log10 at 1,500 IU/ml and 0.2 log10 at 25,000 IU/ml). Finally, all three tests were linear between 4.0 × 103 and 1.0 × 106 IU/ml (correlation coefficient, ≥0.99). In conclusion, the real-time PCR based assays sensitively detected all genotypes and showed comparable linearities for

  16. Aplicação da técnica de PCR na detecção de Yersinia enterocolitica em suínos abatidos sem inspeção PCR technique application in Yersinia enterocolitica detection in non-inspected swine

    Directory of Open Access Journals (Sweden)

    V.A.M. Teodoro

    2006-02-01

    Full Text Available Avaliou-se a contaminação de carcaças e tonsilas de suínos por Y. enterocolitica em estabelecimentos de abate não inspecionados, comparando a pesquisa microbiológica convencional com a técnica da reação em cadeia de polimerase (PCR e o tipo de amostra analisada (de tonsila ou de carcaça, como subsídio ao monitoramento microbiano em sistemas de análise de perigos e pontos críticos de controle. Calcularam-se os custos dos dois testes. Não se detectou Y. enterocolitica pela técnica microbiológica convencional, mas, pela técnica de PCR foi possível detectar a bactéria em 40% das carcaças e em 43% das tonsilas, incluindo cepas patogênicas nas tonsilas. Não houve diferença entre os resultados positivos para as amostras de tonsilas e esfregaços de superfície das carcaças. A PCR apresentou-se como uma alternativa na detecção do agente e uma técnica aparentemente mais eficaz, econômica e rápida. Os resultados indicam a PCR como um importante recurso para o controle de qualidade da carne suína.The contamination of swine carcasses and tonsils by Yersinia enterocolitica in slaughterhouses without inspection was evaluated. The conventional microbiological analysis was compared with the PCR technique of carcass or tonsil swabs, as a subsidy to the microbiological evaluation in the HACCP system. The costs of the two techniques were also calculated. Y. enterocolitica was not detected by the conventional microbiological analysis. Using the PCR, it was possible to detect this bacterium in carcass (40% and tonsil (43% samples. There was no difference between the positive results for the carcass and tonsil samples. The PCR showed to be a more effective, fast and economic alternative for the Y. enterocolitica detection, as compared to the conventional microbiological analysis.

  17. ASAP: Amplification, sequencing & annotation of plastomes

    Directory of Open Access Journals (Sweden)

    Folta Kevin M

    2005-12-01

    Full Text Available Abstract Background Availability of DNA sequence information is vital for pursuing structural, functional and comparative genomics studies in plastids. Traditionally, the first step in mining the valuable information within a chloroplast genome requires sequencing a chloroplast plasmid library or BAC clones. These activities involve complicated preparatory procedures like chloroplast DNA isolation or identification of the appropriate BAC clones to be sequenced. Rolling circle amplification (RCA is being used currently to amplify the chloroplast genome from purified chloroplast DNA and the resulting products are sheared and cloned prior to sequencing. Herein we present a universal high-throughput, rapid PCR-based technique to amplify, sequence and assemble plastid genome sequence from diverse species in a short time and at reasonable cost from total plant DNA, using the large inverted repeat region from strawberry and peach as proof of concept. The method exploits the highly conserved coding regions or intergenic regions of plastid genes. Using an informatics approach, chloroplast DNA sequence information from 5 available eudicot plastomes was aligned to identify the most conserved regions. Cognate primer pairs were then designed to generate ~1 – 1.2 kb overlapping amplicons from the inverted repeat region in 14 diverse genera. Results 100% coverage of the inverted repeat region was obtained from Arabidopsis, tobacco, orange, strawberry, peach, lettuce, tomato and Amaranthus. Over 80% coverage was obtained from distant species, including Ginkgo, loblolly pine and Equisetum. Sequence from the inverted repeat region of strawberry and peach plastome was obtained, annotated and analyzed. Additionally, a polymorphic region identified from gel electrophoresis was sequenced from tomato and Amaranthus. Sequence analysis revealed large deletions in these species relative to tobacco plastome thus exhibiting the utility of this method for structural and

  18. Efficient audio power amplification - challenges

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Michael A.E.

    2005-07-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where extensive research and development are needed is covered. (au)

  19. Efficient Audio Power Amplification - Challenges

    DEFF Research Database (Denmark)

    Andersen, Michael Andreas E.

    2005-01-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where...... extensive research and development are needed is covered....

  20. Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia.

    Science.gov (United States)

    Khan, Z U; Mustafa, A S

    2001-01-01

    In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.

  1. Clinical value of IS6110-based loop-mediated isothermal amplification for detection of Mycobacterium tuberculosis complex in respiratory specimens.

    Science.gov (United States)

    Aryan, Ehsan; Makvandi, Manoochehr; Farajzadeh, Ahmad; Huygen, Kris; Alvandi, Amir-Hooshang; Gouya, Mohammad-Mehdi; Sadrizadeh, Ali; Romano, Marta

    2013-06-01

    A fundamental to global tuberculosis (TB) control is timely and accurate diagnosis of infectious cases of the disease. Among various methods, techniques based on nucleic acid amplification are the ones with promising prospects. The present study evaluates the diagnostic value of the recently developed IS6110-based loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis complex (MTBC) in sputum specimens. In this cross-sectional study (2008-2009), IS6110-LAMP was evaluated on 101 sputum specimens from 93 highly suspected TB patients and compared to Amplicor MTB test and in-house IS6110-PCR and -nested PCR assays. Culture results or clinical recovery following anti-TB therapy was considered as a reference to prove the TB cases. The overall sensitivity of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 89.6% (69/77 specimens; 95% confidence interval [CI], 80.5-95.4%), 76.6% (59/77 specimens; CI, 65.6-85.5%), 79.2% (61/77 specimens; CI, 68.5-87.6%) and 59.7% (46/77 specimens; CI, 47.9-70.8%). The specificity and positive predictive value (PPV) were 100% for all the tests, and the negative predictive value (NPV) of IS6110-LAMP, Amplicor, nPCR, and PCR were respectively 75%, 57.1%, 60%, and 43.6%. There was an excellent overall agreement between LAMP and nPCR (k 0.828), and between LAMP and Amplicor (k 0.746), in addition to a better tolerance of IS6110-LAMP to inhibitors present in clinical specimens. The better diagnostic performance of IS6110-LAMP compared to Amplicor (p = 0.009), nPCR (p = 0.013) and PCR (p < 0.0001) besides its rapidity, simplicity, and cost-effectiveness makes it a valuable method for the detection of MTBC in clinical samples, particularly in resource-limited settings. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

  2. Is non-union of tibial shaft fractures due to nonculturable bacterial pathogens? A clinical investigation using PCR and culture techniques

    Directory of Open Access Journals (Sweden)

    Gille Justus

    2012-05-01

    Full Text Available Abstract Background Non-union continues to be one of the orthopedist’s greatest challenges. Despite effective culture methods, the detection of low-grade infection in patients with non-union following tibial fracture still presents a challenge. We investigated whether “aseptic” tibial non-union can be the result of an unrecognized infection. Methods A total of 23 patients with non-union following tibial shaft fractures without clinical signs of infection were investigated. Intraoperative biopsy samples obtained from the non-union site were examined by means of routine culture methods and by polymerase chain reaction (PCR for the detection of 16 S ribosomal RNA (rRNA. Control subjects included 12 patients with tibial shaft fractures. Results 23 patients (8 women and 15 men; mean age: 47.4 years were included into this study. Preoperative C-reactive protein levels (mean: 20.8 mg/l and WBC counts (mean: 8,359/μl in the study group were not significantly higher than in the control group. None of the samples of non-union routine cultures yielded microorganism growth. Bacterial isolates were found by conventional culturing methods in only 1 case of an open fracture from the control group. In this case, PCR yielded negative results. 16 S rRNA was detected in tissue specimens from 2 patients (8.7% with non-union. The analysis of these variable species-specific sequences enabled the identification of specific microorganisms (1x Methylobacterium species, 1x Staphylococcus species. Both PCR-positive patients were culture-negative. Conclusions The combination of microbiological culture and broad-range PCR seems to substantially add to the number of microbiological diagnoses obtained and may improve the clinican’s ability to tailor therapy to the individual patient’s needs.

  3. Occurrence of Fungal DNA Contamination in PCR Reagents: Approaches to Control and Decontamination

    Science.gov (United States)

    Czurda, S.; Smelik, S.; Preuner-Stix, S.; Nogueira, F.

    2015-01-01

    Nucleic acid amplification techniques permitting sensitive and rapid screening in patients at risk for invasive fungal infections are an important addition to conventional fungal diagnostic methods. However, contamination with fungal DNA may be a serious threat to the validity of fungal amplification-based assays. Besides rigorous handling procedures to avoid false-positive test results from exogenous sources, we have implemented protocols for comprehensive assessment of fungal contamination in all materials involved in the analytical process. Traces of fungal DNA were found in different commercially available PCR reagents, including lyophilized primers, TaqMan probes, and master mix solutions. These contaminants resulted in a considerable rate of false-positive tests in panfungal real-time PCR analysis. To address this problem, we have established a decontamination protocol based on the activity of a double-strand specific DNase. Using this approach, we have significantly reduced the frequency of false-positive test results attributable to contaminated reagents. On the basis of our findings, we strongly recommend routine monitoring of all reagents used in fungal PCR assays for the presence of relevant contaminants. As long as fungal-grade reagents are not readily available, pretreatment methods facilitating elimination of fungal DNA are critical for reducing the risk of false-positive results in highly sensitive molecular fungal detection assays. PMID:26560539

  4. Simple and rapid protocol for the isolation of PCR-amplifiable DNA from medicinal plants.

    Science.gov (United States)

    Alatar, A A; Mahmoud, M A; Al-Sohaibani, S A; Abd-Elsalam, K A

    2012-02-13

    Medicinal plant species has a valuable economic importance because of its usage as pharmaceuticals, nutritional, as well as its use in popular medication. For DNA-based techniques, nanogram quantities of the purified DNA are requisite to amplify and yield sufficient amounts of PCR products. SDS-based DNA isolation method was used to extract DNA from 11 species of different aromatic and medicinal plants collected from Saudi Arabia. Three hundred milligrams of fresh shredded plant material was necessary. The DNA purity was further confirmed by agarose gel, restriction endonuclease digestion and microsatellite primed-polymerase chain reaction (MP-PCR). DNA yields ranged from 10-20 μg (in 100-μL elution volumes) from all plant material evaluated. The DNA obtained was free of any contaminating proteins, polysaccharides and colored pigments. The extracted genomic DNA was found suitable for restriction digestion and PCR amplification. Our experimental procedure provides an easy, suitable, non-toxic, cheap, and quick process for the amplification of DNA from medical plant tissue.

  5. Amplification biases: possible differences among deviating gene expressions

    Directory of Open Access Journals (Sweden)

    Piumi Francois

    2008-01-01

    Full Text Available Abstract Background Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT and polymerase chain reaction (PCR, the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. Results Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3 and somatic tissues (n = 2, we proceeded to moderate amplifications starting from 1 μg of total RNA (global PCR or IVT one round. Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70% and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID. However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions and relevant (biologically validated. In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples. Conclusion Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i the sample used: brain, ovary or embryos, (ii

  6. HLA-DQA1 typing in Danes by two polymerase chain reaction (PCR) based methods

    DEFF Research Database (Denmark)

    Cowland, J B; Madsen, H O; Morling, N

    1995-01-01

    A total of 280 persons were HLA-DQA1 typed by two different polymerase chain reaction (PCR) based methods; (i) a reverse dot-blot (RDB) method, which can differentiate between six alleles, and (ii) a combined PCR-restriction fragment length polymorphism (PCR-RFLP) and allele-specific amplification...

  7. Effect of progressive visual error amplification on human motor adaptation.

    Science.gov (United States)

    Sung, Cynthia; O'Malley, Marcia K

    2011-01-01

    Amplification of error has been shown to be an effective technique in increasing the rate and extent of learning for motor tasks and has the potential to accelerate rehabilitation following motor impairment. However, current error amplification methods suffer from reduced effectiveness towards the end of training. In this paper, we propose a new approach, progressive error amplification, in which error gains increase as a trainee's performance improves. We tested this approach against conventional error augmentation in a controlled experiment wherein 30 subjects adapted to a visually distorted environment by performing target-hitting tasks under one of three conditions (control, constant error amplification, progressive error amplification). Our results showed that compared with repeated practice, error amplification does not accelerate learning or result in improved task performance with respect to trajectory error, although progressive error amplification does produce lower trajectory errors when training conditions are in effect. These results indicate a need for further tuning of error augmentation methods in order to determine their true potential as a training method. © 2011 IEEE

  8. Accurate quantification of supercoiled DNA by digital PCR

    Science.gov (United States)

    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

  9. Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters: interaction of enrichment media and pre-PCR treatment on carcass rinse samples

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Lübeck, Peter Stephensen; Hansen, F.

    2004-01-01

    As part of a large EU project for standardisation of polymerase chain reaction (PCR), a systematic evaluation of the interaction of enrichment media, type of DNA polymerase and pre-PCR sample treatment for a PCR detecting thermotolerant campylobacters was carried out. The growth-supporting capacity...... and PCR compatibility of enrichment in Preston, Mueller-Hinton and Bolton broth (blood-containing and blood-free) were evaluated. The effect of resin-based DNA extraction and DNA extraction by boiling on the final PCR assay was investigated. The time-course studies indicated that a 20-h sample enrichment...... in blood-containing Bolton broth, followed by a simple resin-based extraction of DNA and a PCR amplification using Tth polymerase, resulted in strong and clear PCR amplicons for target (287 bp) and internal amplification control (IAC, 124 bp). The enrichment PCR-based method, tested on 68 presumably...

  10. Amplification-refractory mutation system (ARMS) analysis of point mutations.

    Science.gov (United States)

    Little, S

    2001-05-01

    The amplification-refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base changes or small deletions. ARMS is based on the use of sequence-specific PCR primers that allow amplification of test DNA only when the target allele is contained within the sample. Following an ARMS reaction the presence or absence of a PCR product is diagnostic for the presence or absence of the target allele. The protocols detailed here outline methods that can be used to analyze human genomic DNA for one or more mutations. The Basic Protocol describes the development and application of an ARMS test for a single mutation; the Alternate Protocol extends this to multiplex ARMS for the analysis of two or more mutations. The Support Protocol describes a rapid DNA extraction method from blood or mouthwash samples that yields DNA compatible with the type of tests described. The amplification-refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base change The amplification-refractory mutation system (ARMS) is a simple method for detecting any mutation involving single base change.

  11. PCR-free detection of genetically modified organisms using magnetic capture technology and fluorescence cross-correlation spectroscopy.

    Science.gov (United States)

    Zhou, Xiaoming; Xing, Da; Tang, Yonghong; Chen, Wei R

    2009-11-26

    The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 microg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.

  12. PCR-free detection of genetically modified organisms using magnetic capture technology and fluorescence cross-correlation spectroscopy.

    Directory of Open Access Journals (Sweden)

    Xiaoming Zhou

    2009-11-01

    Full Text Available The safety of genetically modified organisms (GMOs has attracted much attention recently. Polymerase chain reaction (PCR amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS. The cauliflower mosaic virus 35S (CaMV35S promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 microg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.

  13. PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

    Science.gov (United States)

    Zhou, Xiaoming; Xing, Da; Tang, Yonghong; Chen, Wei R.

    2009-01-01

    The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids. PMID:19956680

  14. Molecular identification of airborne bacteria associated with aerial spraying of bovine slurry waste employing 16S rRNA gene PCR and gene sequencing techniques.

    Science.gov (United States)

    Murayama, Mayumi; Kakinuma, Yuki; Maeda, Yasunori; Rao, Juluri R; Matsuda, Motoo; Xu, Jiru; Moore, Peter J A; Millar, B Cherie; Rooney, Paul J; Goldsmith, Colin E; Loughrey, Anne; McMahon, M Ann S; McDowell, David A; Moore, John E

    2010-03-01

    Polymerase chain reaction amplification of the universal 16S ribosomal RNA (rRNA) gene was performed on a collection of 38 bacterial isolates, originating from air sampled immediately adjacent to the agricultural spreading of bovine slurry. A total of 16 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 34/38 (89.5%) of total bacterial numbers consisting of 12 genera and included Staphylococcus (most common genus isolated), Arthrobacter (2nd most common genus isolated), Brachybacterium, Exiguobacterium, Lactococcus, Microbacterium and Sporosarcina (next most common genera isolated) and finally, Bacillus, Brevibacterium, Frigoribacterium, Mycoplana and Pseudoclavibacter. Gram-negative organisms accounted for only 4/38 (10.5%) bacterial isolates and included the following genera, Brevundimonas, Lysobacter, Psychrobacter and Rhizobium. No gastrointestinal pathogens were detected. Although this study demonstrated a high diversity of the microorganisms present, only a few have been shown to be opportunistically pathogenic to humans and none of these organisms described have been described previously as having an inhalational route of infection and therefore we do not believe that the species of organisms identified pose a significant health and safety threat for immunocompetant individuals. (c) 2009 Elsevier Inc. All rights reserved.

  15. Development of Multiple Cross Displacement Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Shigella spp.

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Xu, Jianguo; Ye, Changyun

    2016-01-01

    Shigella spp., the etiological agent of shigellosis or “bacillary dysentery,” are responsible for considerable morbidity and mortality in excess of a million deaths globally per year. Although PCR-based techniques (such as PCR-based dipstick biosensors) have been used for the molecular diagnosis of infectious disease, these assays were restricted due to the need for a sophisticated thermal cycling apparatus to denature target templates. To facilitate simple and rapid detection of target pathogens, we successfully devised an inexpensive, reliable and nearly instrument-free molecular technique, which incorporates multiple cross displacement amplification (MCDA) combined with a newly designed lateral flow biosensor (LFB) for visual, sensitive and specific detection of Shigella. The MCDA-LFB assay was conducted at 65°C for only 20 min during the amplification stage, and then products were directly analyzed on the biosensor, alleviating the use of special reagents, electrophoresis equipment and amplicon detection instruments. The entire process, including specimen processing (35 min), amplification (20) and detection (2–5 min), can be finished within 1 h. The MCDA-LFB assay demonstrated high specificity for Shigella detection. The analytical sensitivity of the assay was 10 fg of genomic templates per reaction in pure culture and 5.86 CFU per tube in human fecal samples, which was consistent with MCDA by colorimetric indicator, gel electrophoresis, real time turbidity and fluorescence detection. Hence, the simplicity, rapidity and nearly instrument-free platform of the MCDA-LFB assay make it practical for ‘on-site’ diagnosis, point-of-care testing and more. Moreover, the proof-of-concept approach can be reconfigured to detect a wide variety of target sequences by re-designing the specific MCDA primers. PMID:27917160

  16. Development of multiple cross displacement amplification label-based gold nanoparticles lateral flow biosensor for detection of Shigella spp.

    Directory of Open Access Journals (Sweden)

    Yi Wang

    2016-11-01

    Full Text Available Shigella spp., the etiological agent of shigellosis or bacillary dysentery, are responsible for considerable morbidity and mortality in excess of a million deaths globally per year. Although PCR-based techniques (such as PCR-based dipstick biosensors have been used for the molecular diagnosis of infectious disease, these assays were restricted due to the need for a sophisticated thermal cycling apparatus to denature target templates. To facilitate simple and rapid detection of target pathogens, we successfully devised an inexpensive, reliable and nearly instrument-free molecular technique, which incorporates multiple cross displacement amplification (MCDA combined with a newly designed lateral flow biosensor (LFB for visual, sensitive and specific detection of Shigella. The MCDA-LFB assay was conducted at 65 ˚C for only 20 min during the amplification stage, and then products were directly analyzed on the biosensor, alleviating the use of special reagents, electrophoresis equipment and amplicon detection instruments. The entire process, including specimen processing (35 min, amplification (20 and detection (2-5 min, can be finished within 1 h. The MCDA-LFB assay demonstrated high specificity for Shigella detection. The analytical sensitivity of the assay was 10 fg of genomic templates per reaction in pure culture and 5.86 CFU per tube in human fecal samples, which was consistent with MCDA by colorimetric indicator, gel electrophoresis, real time turbidity and fluorescence detection. Hence, the simplicity, rapidity and nearly instrument-free platform of the MCDA-LFB assay make it practical for ‘on-site’ diagnosis, point-of-care testing and more. Moreover, the proof-of-concept approach can be reconfigured to detect a wide variety of target sequences by re-designing the specific MCDA primers.

  17. Development of Multiple Cross Displacement Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection ofShigellaspp.

    Science.gov (United States)

    Wang, Yi; Wang, Yan; Xu, Jianguo; Ye, Changyun

    2016-01-01

    Shigella spp., the etiological agent of shigellosis or "bacillary dysentery," are responsible for considerable morbidity and mortality in excess of a million deaths globally per year. Although PCR-based techniques (such as PCR-based dipstick biosensors) have been used for the molecular diagnosis of infectious disease, these assays were restricted due to the need for a sophisticated thermal cycling apparatus to denature target templates. To facilitate simple and rapid detection of target pathogens, we successfully devised an inexpensive, reliable and nearly instrument-free molecular technique, which incorporates multiple cross displacement amplification (MCDA) combined with a newly designed lateral flow biosensor (LFB) for visual, sensitive and specific detection of Shigella . The MCDA-LFB assay was conducted at 65°C for only 20 min during the amplification stage, and then products were directly analyzed on the biosensor, alleviating the use of special reagents, electrophoresis equipment and amplicon detection instruments. The entire process, including specimen processing (35 min), amplification (20) and detection (2-5 min), can be finished within 1 h. The MCDA-LFB assay demonstrated high specificity for Shigella detection. The analytical sensitivity of the assay was 10 fg of genomic templates per reaction in pure culture and 5.86 CFU per tube in human fecal samples, which was consistent with MCDA by colorimetric indicator, gel electrophoresis, real time turbidity and fluorescence detection. Hence, the simplicity, rapidity and nearly instrument-free platform of the MCDA-LFB assay make it practical for 'on-site' diagnosis, point-of-care testing and more. Moreover, the proof-of-concept approach can be reconfigured to detect a wide variety of target sequences by re-designing the specific MCDA primers.

  18. Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR

    Directory of Open Access Journals (Sweden)

    Kelly Grace Magalhães

    2004-06-01

    Full Text Available From complete mitochondrial DNA sequence of Fasciola hepatica available in Genbank, specific primers were designed for a conserved and repetitive region of this trematode. A pair of primers was used for diagnosis of infected Lymnaea columella by F. hepatica during the pre-patent period simultaneously with another pair of primers which amplified the internal transcribed spacer (ITS region of rDNA from L. columella in a single Multiplex-PCR. The amplification generated a ladder band profile specific for F. hepatica. This profile was observed in positive molluscs at different times of infection, including adult worms from the trematode. The Multiplex-PCR technique showed to be a fast and safe tool for fascioliasis diagnosis, enabling the detection of F. hepatica miracidia in L. columella during the pre-patent period and identification of transmission areas.

  19. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

    Energy Technology Data Exchange (ETDEWEB)

    Santiago-Felipe, S.; Tortajada-Genaro, L.A.; Puchades, R.; Maquieira, A., E-mail: amaquieira@qim.upv.es

    2014-02-06

    Graphical abstract: -- Highlights: •Recombinase polymerase amplification is a powerful DNA method operating at 40 °C. •The combination RPA–ELISA gives excellent performances for high-throughput analysis. •Screening of food safety threats has been done using standard laboratory equipment. •Allergens, GMOs, bacteria, and fungi have been successfully determined. -- Abstract: Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.

  20. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis

    International Nuclear Information System (INIS)

    Santiago-Felipe, S.; Tortajada-Genaro, L.A.; Puchades, R.; Maquieira, A.

    2014-01-01

    Graphical abstract: -- Highlights: •Recombinase polymerase amplification is a powerful DNA method operating at 40 °C. •The combination RPA–ELISA gives excellent performances for high-throughput analysis. •Screening of food safety threats has been done using standard laboratory equipment. •Allergens, GMOs, bacteria, and fungi have been successfully determined. -- Abstract: Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR–ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA–ELISA combination is proposed for amplification at a low, constant temperature (40 °C) in a short time (40 min), for the hybridisation of labelled products to specific 5′-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA–ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA–ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings

  1. An empirical approach for quantifying loop-mediated isothermal amplification (LAMP) using Escherichia coli as a model system.

    Science.gov (United States)

    Subramanian, Sowmya; Gomez, Romel D

    2014-01-01

    Loop mediated isothermal amplification (LAMP) is a highly efficient, selective and rapid DNA amplification technique for genetic screening of pathogens. However, despite its popularity, there is yet no mathematical model to quantify the outcome and no well-defined metric for comparing results that are available. LAMP is intrinsically complex and involves multiple pathways for gene replication, making fundamental modelling nearly intractable. To circumvent this difficulty, an alternate, empirical model is introduced that will allow one to extract a set of parameters from the concentration versus time curves. A simple recipe to deduce the time to positive, Tp--a parameter analogous to the threshold cycling time in polymerase chain reaction (PCR), is also provided. These parameters can be regarded as objective and unambiguous indicators of LAMP amplification. The model is exemplified on Escherichia coli strains by using the two gene fragments responsible for vero-toxin (VT) production and tested against VT-producing (O157 and O45) and non-VT producing (DH5 alpha) strains. Selective amplification of appropriate target sequences was made using well established LAMP primers and protocols, and the concentrations of the amplicons were measured using a Qubit 2.0 fluorometer at specific intervals of time. The data is fitted to a generalized logistic function. Apart from providing precise screening indicators, representing the data with a small set of numbers offers significant advantages. It facilitates comparisons of LAMP reactions independently of the sampling technique. It also eliminates subjectivity in interpretation, simplifies data analysis, and allows easy data archival, retrieval and statistical analysis for large sample populations. To our knowledge this work represents a first attempt to quantitatively model LAMP and offer a standard method that could pave the way towards high throughput automated screening.

  2. An empirical approach for quantifying loop-mediated isothermal amplification (LAMP using Escherichia coli as a model system.

    Directory of Open Access Journals (Sweden)

    Sowmya Subramanian

    Full Text Available Loop mediated isothermal amplification (LAMP is a highly efficient, selective and rapid DNA amplification technique for genetic screening of pathogens. However, despite its popularity, there is yet no mathematical model to quantify the outcome and no well-defined metric for comparing results that are available. LAMP is intrinsically complex and involves multiple pathways for gene replication, making fundamental modelling nearly intractable. To circumvent this difficulty, an alternate, empirical model is introduced that will allow one to extract a set of parameters from the concentration versus time curves. A simple recipe to deduce the time to positive, Tp--a parameter analogous to the threshold cycling time in polymerase chain reaction (PCR, is also provided. These parameters can be regarded as objective and unambiguous indicators of LAMP amplification. The model is exemplified on Escherichia coli strains by using the two gene fragments responsible for vero-toxin (VT production and tested against VT-producing (O157 and O45 and non-VT producing (DH5 alpha strains. Selective amplification of appropriate target sequences was made using well established LAMP primers and protocols, and the concentrations of the amplicons were measured using a Qubit 2.0 fluorometer at specific intervals of time. The data is fitted to a generalized logistic function. Apart from providing precise screening indicators, representing the data with a small set of numbers offers significant advantages. It facilitates comparisons of LAMP reactions independently of the sampling technique. It also eliminates subjectivity in interpretation, simplifies data analysis, and allows easy data archival, retrieval and statistical analysis for large sample populations. To our knowledge this work represents a first attempt to quantitatively model LAMP and offer a standard method that could pave the way towards high throughput automated screening.

  3. A comparison of antigen dipstick assays with polymerase chain reaction (PCR) technique and blood film examination in the rapid diagnosis of malaria.

    Science.gov (United States)

    Lee, M A; Aw, L T; Singh, M

    1999-07-01

    Early diagnosis of malaria for military personnel training in the field is crucial in providing proper treatment for the infected and in taking appropriate preventive measures for the non-infected. Present preliminary diagnosis of malaria in the field depends on the clinical symptoms of the patients and there is a need for rapid diagnosis of malaria in the field. The presence of drug-resistant strains of the Plasmodium species in the region also increases the urgency of finding a quick and sensitive way of identifying the different strains. This study evaluated current methods available for the diagnosis of Plasmodium falciparum and Plasmodium vivax. The dipstick assays, the ParaSight F test and the OptiMAL malaria rapid test were compared with the methods of microscopic examination of blood film and polymerase chain reaction (PCR). On comparison to the blood film and PCR methods, the ParaSight F test has specificity of 98.6% and sensitivity of 91% for P. falciparum detection. The OptiMAL malaria rapid test has a specificity of 100% and 98.6% and sensitivity of 92.8% and 92.6% for P. vivax and P. falciparum detection respectively. We conclude that both tests are suitable for use for rapid malaria diagnosis in the field but the OptiMAL rapid malaria test, which can detect both vivax and falciparum malaria, would be more useful.

  4. Dembo-PCR technique for the detection of bovine abortion, diarrhea, and respiratory disease complex infectious agents in potential vectors and reservoirs.

    Science.gov (United States)

    Rahpaya, Sayed Samim; Tsuchiaka, Shinobu; Kishimoto, Mai; Oba, Mami; Katayama, Yukie; Nunomura, Yuki; Kokawa, Saki; Kimura, Takashi; Kobayashi, Atsushi; Kirino, Yumi; Okabayashi, Tamaki; Nonaka, Nariaki; Mekata, Hirohisa; Aoki, Hiroshi; Shiokawa, Mai; Umetsu, Moeko; Morita, Tatsushi; Hasebe, Ayako; Otsu, Keiko; Asai, Tetsuo; Yamaguchi, Tomohiro; Makino, Shinji; Murata, Yoshiteru; Abi, Ahmad Jan; Omatsu, Tsutomu; Mizutani, Tetsuya

    2017-12-28

    The bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds and rodents. However, experimental data that supports this possibility are scarce. We collected total 117 samples and screened for 44 bovine abortive, diarrheal and respiratory disease complex pathogens by using Dembo-PCR, based on TaqMan real-time PCR. Fifty-seven samples showed positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, Salmonella enterica ser. Dublin, salmonella enterica ser. Typhimurium, and Neospora caninum ; some samples contained multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus represented the most frequently detected pathogens, especially in flies, suggesting the important role of flies in the transmission of these viruses. Additionally, we detected the N. caninum genome from a cockroach sample for the first time. Our data suggests that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of the abortion, diarrhea, and respiratory infectious agents and that they may transmit more than one pathogen at the same time.

  5. [Comparison of Bacteria ERIC-PCR Fingerprints of Index Fingers and Contactants].

    Science.gov (United States)

    Liu, Y T; Sun, D M; Shi, S P; Yang, X

    2018-02-01

    To explore the bacteria relevance between index fingers and contactant' surfaces (mobile phone touch screen and desktop of personal office table). Bacteria were collected from the index fingers, mobile phone touch screen and desktop of personal office table of 10 volunteers. Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprint was established by PCR amplification technique of metagenome. There were 7 volunteers' ERIC-PCR fingerprints of index fingers matched that took from the mobile phone touch screens, and different from each other. There were 3 volunteers' ERIC-PCR fingerprints of index fingers matched that took from desk top of personal office table, and other 7 volunteers' ERIC-PCR fingerprints did not match perfectly with that took from desk top of personal office table, but had at least one similar band for both. The bacteria on index finger shows individual specificity, which on mobile phone touching screen and personal desktop may be a new biological sample of forensic identification. Copyright© by the Editorial Department of Journal of Forensic Medicine.

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

    Science.gov (United States)

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

    2016-01-01

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

  7. DNAzyme Feedback Amplification: Relaying Molecular Recognition to Exponential DNA Amplification.

    Science.gov (United States)

    Liu, Meng; Yin, Qingxin; McConnell, Erin M; Chang, Yangyang; Brennan, John D; Li, Yingfu

    2018-03-26

    Technologies capable of linking DNA amplification to molecular recognition are very desirable for ultrasensitive biosensing applications. We have developed a simple but powerful isothermal DNA amplification method, termed DNAzyme feedback amplification (DFA), that is capable of relaying molecular recognition to exponential DNA amplification. The method incorporates both an RNA-cleaving DNAzyme (RCD) and rolling circle amplification (RCA) carried out by a special DNA polymerase using a circular DNA template. DFA begins with a stimulus-dependent RCA reaction, producing tandemly linked RCDs in long-chain DNA products. These RCDs cleave an RNA-containing DNA sequence to form additional primers that hybridize to the circular DNA molecule, giving rise to DNA assemblies that act as the new inputs for RCA. The RCA reaction and the cleavage event keep on feeding each other autonomously, resulting in exponential growth of repetitive DNA sequences that can be easily detected. This method can be used for the detection of both nucleic acid based targets and non-nucleic acid analytes. In this article, we discuss the conceptual framework of the feedback amplification approach, the essential features of this method as well as remaining challenges and possible solutions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. HLA-DQA1 allele typing by nonisotopic PCR-LIS-SSCP

    Directory of Open Access Journals (Sweden)

    Abba M.C.

    2001-01-01

    Full Text Available In the present study we used a simple and reliable method for HLA-DQA1 allele typing based on the single-stranded conformation polymorphism (SSCP properties of DNA molecules obtained by PCR. The technique consists of PCR amplification of a DNA fragment comprising the second exon of the HLA-DQA1 gene, amplicon denaturation using a low ionic strength solution (LIS, and electrophoresis on a small native polyacrylamide gel, followed by a rapid silver staining procedure. In order to validate the technique and to obtain the allele patterns for the DQA1 gene, 50 cervical samples were typed using this methodology and the commercial Amplitype® HLA DQA1 Amplification and Typing kit. All the alleles detected with the kit were characterized by the LIS-SSCP approach. This procedure proved to be useful for population screening and typing of the DQA1 gene as well as for detecting new alleles or mutations in the donor-recipient molecular matching of HLA class II genes.

  9. Designing of primers for detection of salmonella typhimirium and enteritidis by heminested PCR

    International Nuclear Information System (INIS)

    Ben salem, Issam

    2007-01-01

    Salmonella are the main responsible agent for the frequent food borne gastrointestinal diseases. In Tunisia, this pathogen is considered one of the most important causes of toxiinfections and its detection using classical methods is laborious and requires a large amount of time for revelation. To solve this problem, we developed a rapid molecular technique for the detection of the invA virulence gene sequence which is found in the majority of Salmonella spp. This technique is a hemi nested PCR amplification using specific primers designed and by bioinformatics tools. The detection method consisted of pre-enrichment of the sample in buffered peptone water (BPW), followed by a total DNA extraction step prior to single tube hemi nested PCR amplification. This method was found highly specific and sensitive to detect low levels of salmonella typhimurium and salmonella enteritidis (1cfu/ 25g) in naturally contaminated spicy sausage (merguez) samples. These results can benefit the public health agencies concerning microbiological and quality aspects of the commercial and traditional merguez meat production in Tunisia. (Author)

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

    Science.gov (United States)

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

    2017-04-30

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

  11. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits.

    Science.gov (United States)

    Demeke, Tigst; Jenkins, G Ronald

    2010-03-01

    Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.

  12. Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

    Directory of Open Access Journals (Sweden)

    Saruda Tiwananthagorn

    Full Text Available BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP of 18S rRNA gene as well as polymerase chain reaction (PCR of minicircle kinetoplast DNA gene (PCR-mkDNA have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1. METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L. donovani, and L. (L. infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L

  13. Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection.

    Science.gov (United States)

    Tiwananthagorn, Saruda; Kato, Hirotomo; Yeewa, Ranchana; Muengpan, Amontip; Polseela, Raxsina; Leelayoova, Saovanee

    2017-02-01

    Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis infection in outbreak areas where several species of Leishmania

  14. Comparison of LAMP and PCR for molecular mass screening of sand flies for Leishmania martiniquensis infection

    Science.gov (United States)

    Tiwananthagorn, Saruda; Kato, Hirotomo; Yeewa, Ranchana; Muengpan, Amontip; Polseela, Raxsina; Leelayoova, Saovanee

    2017-01-01

    BACKGROUND Leishmaniasis caused by Leishmania martiniquensis infection has been reported in human and domestic animals of Martinique Island, Germany, Switzerland, USA, Myanmar and Thailand. The peculiar clinical features of disseminated cutaneous and visceral forms co-existence render the urgent need of specific diagnostic tool to identify the natural sand fly vectors for effective prevention and control strategies. Loop-mediated isothermal amplification (LAMP) of 18S rRNA gene as well as polymerase chain reaction (PCR) of minicircle kinetoplast DNA gene (PCR-mkDNA) have never been applied to detect L. martiniquensis and L. siamensis in sand fly vectors. OBJECTIVE The present study was aimed to validate malachite green-LAMP (MG-LAMP) and PCR-mkDNA techniques to detect L. martiniquensis in sand fly vectors, compared with the conventional PCR of internal transcribed spacer 1 (PCR-ITS1). METHODS We compared the validity of LAMP of 18S rRNA gene and PCR-mkDNA, to PCR-ITS1 in simulation model of L. martiniquensis infection in Sergentomyia gemmea sand flies. Attributable to the sensitivity and specificity, PCR-mkDNA was consecutively applied to detect L. martiniquensis in 380 female sand fly individuals captured in the newly identified affected region of Lamphun Province, Thailand. FINDINGS AND MAIN CONCLUSIONS Results showed that PCR-mkDNA could detect at least one promastigote per sand fly, which was 10-time superior to LAMP and PCR-ITS1. In addition, PCR-mkDNA was more specific, able to differentiate L. martiniquensis from other viscerotropic Leishmania species, such as L. siamensis, L. (L.) donovani, and L. (L.) infantum. Consecutively, mass screening of L. martiniquensis in 380 female sand fly individuals by PCR-mkDNA was implemented in a new affected area of Thailand where a patient with leishmaniasis/HIV co-infection resides; however Leishmania DNA was undetected. In conclusion, PCR-mkDNA is a promising tool for molecular mass screening of L. martiniquensis

  15. First experience with FGF-3 (INT-2) amplification in women with epithelial ovarian cancer.

    OpenAIRE

    Rosen, A.; Sevelda, P.; Klein, M.; Dobianer, K.; Hruza, C.; Czerwenka, K.; Hanak, H.; Vavra, N.; Salzer, H.; Leodolter, S.

    1993-01-01

    Estimation of FGF-3 oncogene amplification in DNA samples extracted from paraffin embedded sections of 136 ovarian cancer samples was carried out by a quantitative PCR method. The aim of this study was to elucidate a possible association of FGF-3 copy numbers with established prognostic factors such as age, histology, FIGO stage, grading, postoperative residual tumour mass, ascites, hormone receptor content and preoperative CA 125 serum levels. In addition, correlation of FGF-3 amplification ...

  16. Next generation Chirped Pulse Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Nees, J.; Biswal, S.; Mourou, G. [Univ. Michigan, Center for Ultrafast Optical Science, Ann Arbor, MI (United States); Nishimura, Akihiko; Takuma, Hiroshi

    1998-03-01

    The limiting factors of Chirped Pulse Amplification (CPA) are discussed and experimental results of CPA in Yb:glass regenerative amplifier are given. Scaling of Yb:glass to the petawatt level is briefly discussed. (author)

  17. Identification of three Iranian species of the genus Rattus (Rodentia, Muridae using a PCR-RFLP technique on mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    safieh Akbary rad

    2009-11-01

    Full Text Available Abstract Three species of the genus Rattus Fisher, 1803 have been reported from Iran: the brown rat (R. norvegicus, the black rat (R. rattus and the Himalayan rat (R. pyctoris. The first two were introduced, whilst R. pyctoris is native and lives in mountainous regions from Pakistan to north-eastern Iran. In this study, the mitochondrial DNA from twenty six rats were analysed using a PCR-RFLP (Polymerase Chain Reaction - Restriction Fragments Length Polymorphism method to investigate inter-specific variation. Part of the 16S rRNA and cytochrome b genes were amplified and digested with three restriction enzymes: AluI, MboI and HinfI. Restriction fragments resulted in four different haplotypes and allowed to distinguish the three Rattus species. Our results suggest that the Himalayan rats are more closely related to R. rattus than to R. norvegicus and provide the basics for further phylogenetic studies. Riassunto Identificazione di tre specie iraniane del genere Rattus (Rodentia, Muridae tramite PCR-RFLP su DNA mitocondriale. Tre specie del genere Rattus risultano diffuse in Iran: il surmolotto (R. norvegicus, il ratto nero (R. rattus e il ratto himalayano (R. pyctoris. Le prime due specie sono state introdotte, mentre R. pyctoris è presente nelle aree montane che si sviluppano dal Pakistan all’Iran nordorientale. In questo studio, il DNA mitocondriale di 26 individui è stato analizzato tramite PCR-RFLP per evidenziare variazioni inter-specifiche. Parte dei geni del rRNA 16S e del citocromo b è stata amplificata e quindi sottoposta a digestione tramite tre diversi enzimi: AluI, MboI e HinfI. I frammenti di restrizione hanno permesso di individuare quattro aplotipi mitocondriali e di distinguere le tre specie. I risultati ottenuti suggeriscono che il ratto

  18. A novel and rapid PCR-based method for genotyping human papillomaviruses in clinical samples.

    Science.gov (United States)

    Nelson, J H; Hawkins, G A; Edlund, K; Evander, M; Kjellberg, L; Wadell, G; Dillner, J; Gerasimova, T; Coker, A L; Pirisi, L; Petereit, D; Lambert, P F

    2000-02-01

    Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specimens. By using the MY09-MY11 and the GP5(+)-GP6(+) consensus primer pairs, HPV sequences were amplified by nested PCR from DNA isolated from cervical smear samples. This led to the production of an approximately 140-bp PCR product from the L1 (major capsid) gene of any of the HPVs present in the sample. PCR was performed with a deoxynucleoside triphosphate mixture which resulted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequency of about once or twice per strand. Following the PCR, the product was treated with an enzyme mix that contains uracil N-glycosylase (UNG) and endonuclease IV. UNG removes the uracil base from the nucleotide, and endonuclease IV cleaves the phosphodiester bond at this newly formed abasic site, producing fragments of various sizes. By having end labeled one of the amplification primers, a DNA ladder which is analogous to a "T-sequencing ladder" was produced upon electrophoresis of the products. By comparing this T-sequencing ladder to the known sequences of HPVs, the genotypes of unknown HPV isolates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.

  19. Simple and fast multiplex PCR method for detection of species origin in meat products.

    Science.gov (United States)

    Izadpanah, Mehrnaz; Mohebali, Nazanin; Elyasi Gorji, Zahra; Farzaneh, Parvaneh; Vakhshiteh, Faezeh; Shahzadeh Fazeli, Seyed Abolhassan

    2018-02-01

    Identification of animal species is one of the major concerns in food regulatory control and quality assurance system. Different approaches have been used for species identification in animal origin of feedstuff. This study aimed to develop a multiplex PCR approach to detect the origin of meat and meat products. Specific primers were designed based on the conserved region of mitochondrial Cytochrome C Oxidase subunit I ( COX1 ) gene. This method could successfully distinguish the origin of the pig, camel, sheep, donkey, goat, cow, and chicken in one single reaction. Since PCR products derived from each species represent unique molecular weight, the amplified products could be identified by electrophoresis and analyzed based on their size. Due to the synchronized amplification of segments within a single PCR reaction, multiplex PCR is considered to be a simple, fast, and inexpensive technique that can be applied for identification of meat products in food industries. Nowadays, this technique has been considered as a practical method to identify the species origin, which could further applied for animal feedstuffs identification.

  20. Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification.

    Science.gov (United States)

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

    2017-08-15

    Canine distemper, caused by Canine distemper virus (CDV), is a highly contagious and fatal systemic disease in free-living and captive carnivores worldwide. Recombinase polymerase amplification (RPA), as an isothermal gene amplification technique, has been explored for the molecular detection of diverse pathogens. A real-time reverse transcription RPA (RT-RPA) assay for the detection of canine distemper virus (CDV) using primers and exo probe targeting the CDV nucleocapsid protein gene was developed. A series of other viruses were tested by the RT-RPA.Thirty-two field samples were further tested by RT-RPA, and the resuts were compared with those obtained by the real-time RT-PCR. The RT-RPA assay was performed successfully at 40 °C, and the results were obtained within 3 min-12 min. The assay could detect CDV, but did not show cross-detection of canine parvovirus-2 (CPV-2), canine coronavirus (CCoV), canine parainfluenza virus (CPIV), pseudorabies virus (PRV) or Newcastle disease virus (NDV), demonstrating high specificity. The analytical sensitivity of RT-RPA was 31.8 copies in vitro transcribed CDV RNA, which is 10 times lower than the real-time RT-PCR. The assay performance was validated by testing 32 field samples and compared to real-time RT-PCR. The results indicated an excellent correlation between RT-RPA and a reference real-time RT-PCR method. Both assays provided the same results, and R 2 value of the positive results was 0.947. The results demonstrated that the RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of CDV both in the laboratory and point-of-care facility, especially in the resource-limited settings.

  1. Evaluation of a Solid Phase DNA Binding Matrix for Downstream PCR Analysis

    National Research Council Canada - National Science Library

    Bader, Douglas E; Fisher, Glen R; Stratilo, Chad W

    2005-01-01

    A commercially available solid-phase DNA binding matrix (FTA cards) was evaluated for its ability to capture and release DNA for downstream gene amplification and detection assays using polymerase chain reaction (PCR...

  2. 1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer

    Directory of Open Access Journals (Sweden)

    Dráberová Lubica

    2011-04-01

    Full Text Available Abstract Background Quantitative real-time PCR (qPCR is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI. In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify. Results We found that several DNA dyes (SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight exhibited optimum qPCR performance in buffers of different salt composition. Fidelity assays demonstrated that the observed differences were not caused by changes in Taq DNA polymerase induced mutation frequencies in PCR mixes of different salt composition or containing different DNA dyes. In search for a PCR mix compatible with all the DNA dyes, and suitable for efficient amplification of difficult-to-amplify DNA templates, such as those in whole blood, of medium size and/or GC-rich, we found excellent performance of a PCR mix supplemented with 1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer. These two additives together decreased DNA melting temperature and efficiently neutralized PCR inhibitors present in blood samples. They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives. Furthermore, amplification in the presence of PT enhancer increased the robustness and performance of routinely used qPCRs with short amplicons. Conclusions The combined data indicate that PCR mixes supplemented with PT enhancer are suitable for DNA amplification in the presence of various DNA dyes and for a variety of templates which otherwise can be amplified with difficulty.

  3. Lateral Flow Loop-Mediated Isothermal Amplification Test with Stem Primers: Detection of Cryptosporidium Species in Kenyan Children Presenting with Diarrhea

    Directory of Open Access Journals (Sweden)

    Timothy S. Mamba

    2018-01-01

    Full Text Available Background. Cryptosporidium is a protozoan parasite and a major cause of diarrhea in children and immunocompromised patients. Current diagnostic methods for cryptosporidiosis such as microscopy have low sensitivity while techniques such as PCR indicate higher sensitivity levels but are seldom used in developing countries due to their associated cost. A loop-mediated isothermal amplification (LAMP technique, a method with shorter time to result and with equal or higher sensitivity compared to PCR, has been developed and applied in the detection of Cryptosporidium species. The test has a detection limit of 10 pg/µl (~100 oocysts/ml indicating a need for more sensitive diagnostic tools. This study developed a more sensitive lateral flow dipstick (LFD LAMP test based on SAM-1 gene and with the addition of a second set of reaction accelerating primers (stem primers. Results. The stem LFD LAMP test showed analytical sensitivity of 10 oocysts/ml compared to 100 oocysts/ml (10 pg/ul for each of the SAM-1 LAMP test and nested PCR. The stem LFD LAMP and nested PCR detected 29/39 and 25/39 positive samples of previously identified C. parvum and C. hominis DNA, respectively. The SAM-1 LAMP detected 27/39. On detection of Cryptosporidium DNA in 67 clinical samples, the stem LFD LAMP detected 16 samples and SAM-2 LAMP 14 and nested PCR identified 11. Preheating the templates increased detection by stem LFD LAMP to 19 samples. Time to results from master mix preparation step took ~80 minutes. The test was specific, and no cross-amplification was recorded with nontarget DNA. Conclusion. The developed stem LFD LAMP test is an appropriate method for the detection of C. hominis, C. parvum, and C. meleagridis DNA in human stool samples. It can be used in algorithm with other diagnostic tests and may offer promise as an effective diagnostic tool in the control of cryptosporidiosis.

  4. Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers.

    Science.gov (United States)

    Takahashi, Hirokazu; Yamamoto, Kimiko; Ohtani, Toshio; Sugiyama, Shigeru

    2009-07-01

    The predominant method for DNA cloning is by propagation in biological hosts, but this method has limitations because certain sequences are difficult to clone using any combination of available hosts or vectors. Recently, multiply-primed rolling circle amplification (MPRCA) has been applied to overcome the problems of the DNA cloning via host cells. However, when MPRCA is used to amplify from minute quantities of DNA template, the products are mostly by-product DNA molecules generated by false priming and primer dimer formation. This study demonstrates that MPRCA using random RNA primers[#x02014]instead of DNA primers[#x02014]blocked the synthesis of by-products and succeeded in amplifying one copy of a circular DNA molecule more than 1012-fold to give microgram quantities of amplification product without using submicroliter reaction volumes. Furthermore, a ligation strategy was elaborated to circularize only the desired DNA sequence and eliminate undesired ligation-products. A combination of these methods was able to amplify and ligate a large construct without undesired DNA sequences and at microgram quantities within one day. Therefore, these methods have the possibility to improve DNA cloning techniques that have been restricted by the limitations of PCR methods or by the host cell.

  5. HLA DQA1'İN PCR ÜRÜNLERİNİN KAPİLLER JEL ELEKTROFOREZİ

    OpenAIRE

    YILMAZ, Sevgi; CENGiZ, Salih

    2014-01-01

    Background.- HLA DQA 1 loci are highly informative polymorphic loci that are gaining popularity for identity testing. HLA DQA 1 system easy to work with, fast and reliable. This loci with PCR technique is a very promising tool for genetic investigations in both paternity and crime cases.Design.- Human genomic DNA was extracted by Chelex(r) 100 procedure. The amplification of HLA DQA 1 locus was performed by single locus PCR reaction in the case of HLA DQA 1 locus according to the manufacturer...

  6. Rapid and visual detection ofMycobacterium aviumsubsp.paratuberculosisby recombinase polymerase amplification combined with a lateral flow dipstick.

    Science.gov (United States)

    Guimin, Zhao; Hongmei, Wang; Peili, Hou; Chengqiang, He; Hongbin, He

    2017-12-28

    Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. Quick diagnosis could facilitate control; however widespread point-of-care testing is infrequently done due to the lack of robust method. Isothermal recombinase polymerase amplification (RPA) technique has emerged as a novel DNA amplify assay for use in rapid diagnosis. Here, an RPA combined with lateral flow dipstick (LFD) assay was developed to estimate DNA from M.paratuberculosis . First, the specificity and sensitivity of RPA-nfo primer and probe sets were assessed. The assay successfully detected M.paratuberculosis DNA in 30 minutes at 39°C, limit of detection up to eight copies per reaction, which was equivalent with the real-time quantitative PCR (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium and five pathogenic enteric bacteria. Then, 612 clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR and ELISA assays respectively, also the established RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR. This is the first report utilizing an RPA-LFD assay to visual and rapid detect M.paratuberculosis . Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource constrained setting.

  7. The application of loop-mediated isothermal amplification (LAMP) in food testing for bacterial pathogens and fungal contaminants.

    Science.gov (United States)

    Niessen, Ludwig; Luo, Jie; Denschlag, Carla; Vogel, Rudi F

    2013-12-01

    Bacterial pathogens and toxicants, parasites as well as mycotoxin producing fungi are the major biotic factors influencing the safety of food. Moreover, viral infections and prions may be present as quasi biotic challenging factors. A vast array of culture dependent analytical methods and protocols for food safety testing has been developed during the past decades. Presently, protocols involving molecular biological techniques such as PCR-based nucleic acid amplification and hybridization have become available for many of the known pathogens with their major advantages being rapidness, high sensitivity and specificity. However, this type of assays is still quite labor- and cost intensive and mostly cannot be operated directly in the field. Recently, loop-mediated isothermal amplification (LAMP) of DNA has emerged as an alternative to the use of PCR-based methods not only in food safety testing but also in a wide array of application. Its advantages over PCR-based techniques are even shorter reaction time, no need for specific equipment, high sensitivity and specificity as well as comparably low susceptibility to inhibitors present in sample materials which enables detection of the pathogens in sample materials even without time consuming sample preparation. The present article presents a critical review of the application of LAMP-based methods and their usefulness in detecting and identifying food borne bacterial pathogens and toxicants as well as mycotoxin producing food borne fungi as compared to other methods. Moreover does it elaborate on new developments in the design and automation of LAMP-based assays and their implications for the future developments of food testing. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Development and application of a multiplex PCR assay for rapid detection of 4 major bacterial pathogens in ducks.

    Science.gov (United States)

    Wei, B; Cha, S-Y; Kang, M; Park, I-J; Moon, O-K; Park, C-K; Jang, H-K

    2013-05-01

    Infections with Pasteurella multocida, Salmonella enterica, Riemerella anatipestifer, and Escherichia coli result in high morbidity and mortality, which cause significant economic loss in the poultry industry. It can be difficult to distinguish these pathogens based on clinical signs because these pathogens can cause similar clinical signs and coinfections can occur. Thus, rapid and sensitive detection of these 4 major bacterial pathogens are important in ducks. The aim of this study was to develop a multiplex PCR (mPCR) assay for simultaneously detecting and identifying these 4 pathogenic bacteria in a single tube reaction. The target genes used were KMT1 of P. multocida, the invasion protein gene of S. enterica, 16S rDNA of R. anatipestifer, and the alkaline phosphatase gene of E. coli. The detection limit of the assay for all bacterial DNA was 10 pg. The mPCR did not produce any nonspecific amplification products when tested against other related pathogens, including Staphylococcus aureus, Streptococcus pyogenes, Clostridium perfringens, Mycoplasma gallinarum, Mycoplasma synoviae, and Mycoplasma gallisepticum, which can also infect ducks. We applied mPCR to field samples, and the results were the same as the single PCR results. These results suggest that mPCR for the 4 bacteria is a useful and rapid technique to apply to field samples.

  9. Comparison of culture and acid-fast bacilli stain to PCR for detection of Mycobacterium tuberculosis in clinical samples.

    Science.gov (United States)

    Aslanzadeh, J; de la Viuda, M; Fille, M; Smith, W B; Namdari, H

    1998-08-01

    The major drawback in effective use of polymerase chain reaction (PCR) for detecting Mycobacterium tuberculosis (MTB) in clinical samples is the presence of PCR inhibitors and unique cell components of the organism that complicate DNA extraction and subsequent PCR amplification. A PCR assay with a unique multistep DNA extraction method that minimizes these problems was compared in a prospective study to acid-fast bacilli stain (AFBS) and culture for detecting MTB in clinical samples. A total of 254 clinical specimens in two separate studies were processed for MTB by these techniques. While PCR and culture were 100% sensitive and specific, culture required up to 8 weeks of incubation and additional time to perform biochemical testing to identify the isolated micro-organism. Acid-fast bacilli stain had a specificity of about 87% and did not differentiate among Mycobacterial species. In contrast, the results from PCR were available within 48 h and did not require additional testing to attain a final result. Polymerase chain reaction was highly reliable for detection and confirmation and interpretation of positive AFBS results. The assay was easy to perform with a turn around time of about 2 days.

  10. DNA Fingerprinting Based on Repetitive Sequences of Iranian Indigenous Lactobacilli Species by (GTG5- REP-PCR

    Directory of Open Access Journals (Sweden)

    Farzaneh Tafvizi

    2012-12-01

    Full Text Available Background and Objective: The use of lactobacilli as probiotics requires the application of accurate and reliable methods for the detection and identification of bacteria at the strain level. Repetitive sequence-based polymerase chain reaction (rep-PCR, a DNA fingerprinting technique, has been successfully used as a powerful molecular typing method to determine taxonomic and phylogenetic relationships among bacteria. The aim of this study was to evaluate and detect the genetic diversity of lactobacilli species isolated from different sources in Iran. Material and Methods: Twenty strains were isolated from Iranian traditional yoghurt, cheese, and Tarkhineh. PCR-mediated amplification was carried out by degenerate primers. Sequencing was performed after purification of the PCR product. The rep-PCR fingerprinting by (GTG 5 oligonucleotide primers was conducted for the discrimination and genotypic grouping of isolates. Results: Isolates were deposited as novel stains of lactobacillus casei, brevis, plantarum, and Entrococcus facium in GenBank. Clustering methods were performed on molecular data by NTSYS software, which was also supported by PCO ordination plot. The rep-PCR profiles showed that the 20 isolates produced different banding patterns. In UPGMA dendrogram, three main clusters were formed. Conclusion: According to our findings, rep-PCR appeared to be a very practical method and highly sensitive in the discrimination of the lactobacillus species. The results of sequencing corresponded to the clustering in dendrogram.

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

    Science.gov (United States)

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

    2011-01-01

    We report the successful application of low-power (~30 mW) laser radiation as an optical heating source for high-speed real-time polymerase chain reaction (PCR) amplification of DNA in nanoliter droplets dispersed in an oil phase. Light provides the heating, temperature measurement, and Taqman real-time readout in nanoliter droplets on a disposable plastic substrate. A selective heating scheme using an infrared laser appears ideal for driving PCR because it heats only the droplet, not the oil or plastic substrate, providing fast heating and completing the 40 cycles of PCR in 370 seconds. No microheaters or microfluidic circuitry were deposited on the substrate, and PCR was performed in one droplet without affecting neighboring droplets. The assay performance was quantitative and its amplification efficiency was comparable to that of a commercial instrument. PMID:19129891

  12. Evaluación de la técnica de MSP-PCR para la caracterización molecular de aislamientos de Rhodotorula mucilaginosa provenientes de la Patagonia noroccidental Assessment of the MSP-PCR technique for the molecular characterization of Rhodotorula mucilaginosa isolates from northwestern Patagonia

    Directory of Open Access Journals (Sweden)

    D. Libkind

    2007-09-01

    from 200 environmental pigmented yeast isolates on the basis of phenotypic criteria. (GTG5, (GAC5 and M13 primers were initially evaluated in representative R. mucilaginosa isolates. (GTG5 allowed a good grouping of these isolates and, at the same time, a good differentiation among closely related species, and thus was selected for subsequent studies. R. mucilaginosa isolates (87% presented similar (> 60% MSP-PCR profiles to those of the reference strain CBS 316T. The MSP-PCR technique was effective, both, for the characterization and identification of a large number of R. mucilaginosa environmental isolates as well as for the detection of polymorphisms within the species.

  13. Effect of gamma radiation on the growth of Aspergillus Flavus aflatoxins producer and on the use of polymerase chain reaction technique (PCR) in samples of maize grains artificially inoculated

    International Nuclear Information System (INIS)

    Aquino, Simone

    2003-01-01

    The aim of this present study was to verify the effects of gamma radiation on the growth of Aspergillus flavus Link aflatoxins producer; to demonstrate the application of Polymerase Chain Reaction (PCR) technique in the diagnostic of A. Flavus, as well to verify the effect of radiation in the profile of DNA bands. Twenty samples of grains maize with 200 g each were individually irradiated with 20 kGy, to eliminate the microbial contamination. In following, the samples were inoculated with an toxigenic A. flavus (1x10 6 spores/ml), incubated for 15 days at 25 deg C with a relative humidity of around 97,5% and irradiated with 0, 2; 5 and 10 kGy. The samples, 5 to each dose of irradiation, were individually analyzed for the number of fungal cells, water activity, viability test (fluorescein diacetate and ethidium bromide), PCR and aflatoxins (AFB) detection. The results showed that the doses used were effective in reducing the number of Colony Forming Units (CFU/g) mainly the doses of 5 and 10 kGy. In addition, the viability test showed a decrease of viable cells with increase of irradiation doses. The reduction of AFB 1 and AFB-2, was more efficient with the use of 2 kGy in comparison with the dose of 5 kGy, while the dose of 10 kGy, degraded the aflatoxins. Thereby, it was observed that AFB2 showed to be more radiosensitive. The use of PCR technique showed the presence of DNA bands, in all samples. (author)

  14. Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

    Science.gov (United States)

    Chen, Qianqian; Chen, Xiaoxiang; Zhang, Sichao; Lan, Ke; Lu, Jian; Zhang, Chiyu

    2015-01-01

    The development of simple, accurate, rapid and cost-effective technologies for mutation detection is crucial to the early diagnosis and prevention of numerous genetic diseases, pharmacogenetics, and drug resistance. Proofreading PCR (PR-PCR) was developed for mutation detection in 1998 but is rarely applied due to its low efficiency in allele discrimination. Here we developed a modified PR-PCR method using a ddNTP-blocked primer and a mixture of DNA polymerases with and without the 3'-5' proofreading function. The ddNTP-blocked primer exhibited the best blocking efficiency to avoid nonspecific primer extension while the mixture of a tiny amount of high-fidelity DNA polymerase with a routine amount of Taq DNA polymerase provided the best discrimination and amplification effects. The modified PR-PCR method is quite capable of detecting various mutation types, including point mutations and insertions/deletions (indels), and allows discrimination amplification when the mismatch is located within the last eight nucleotides from the 3'-end of the ddNTP-blocked primer. The modified PR-PCR has a sensitivity of 1-5 × 102 copies and a selectivity of 5 × 10-5 mutant among 107 copies of wild-type DNA. It showed a 100% accuracy rate in the detection of P72R germ-line mutation in the TP53 gene among 60 clinical blood samples, and a high potential to detect rifampin-resistant mutations at low frequency in Mycobacterium tuberculosis using an adaptor and a fusion-blocked primer. These results suggest that the modified PR-PCR technique is effective in detection of various mutations or polymorphisms as a simple, sensitive and promising approach. PMID:25915410

  15. Material Biocompatibility for PCR Microfluidic Chips

    KAUST Repository

    Kodzius, Rimantas

    2010-04-23

    As part of the current miniaturization trend, biological reactions and processes are being adapted to microfluidics devices. PCR is the primary method employed in DNA amplification, its miniaturization is central to efforts to develop portable devices for diagnostics and testing purposes. A problem is the PCR-inhibitory effect due to interaction between PCR reagents and the surrounding environment, which effect is increased in high-surface-are-to-volume ration microfluidics. In this study, we evaluated the biocompatibility of various common materials employed in the fabrication of microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most of the cases, addition of bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, whereas they did show noticeable interaction with the DNA polymerase. Our test, instead of using microfluidic devices, can be easily conducted in common PCR tubes using a standard bench thermocycler. Our data supports an overview of the means by which the materials most bio-friendly to microfluidics can be selected.

  16. Quantification and viability assays of Toxoplasma gondii in commercial "Serrano" ham samples using magnetic capture real-time qPCR and bioassay techniques.

    Science.gov (United States)

    Gomez-Samblas, M; Vílchez, S; Racero, J C; Fuentes, M V; Osuna, A

    2015-04-01

    "Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. To make Serrano ham, pork undergoes a salting and a subsequent fermentation process known as curing. Certain pigs used for meat production are an important source of Toxoplasma gondii infection in humans. We have developed a method for quantifying and assaying the viability of the T. gondii present in commercial Serrano ham samples. A magnetic capture method for the isolation of T. gondii DNA and a qRT-PCR were used to estimate the T. gondii burden in 475 commercial samples of "Serrano" ham in two presentation formats: ham pieces and sliced ham. The infectivity capacity of T. gondii in positive samples was assayed in mice. The global prevalence of T. gondii was 8.84%, ranging from 32.35% in one of the companies to 0% prevalence in three other companies. The infectivity assays revealed that only 4.84% of the positive samples were infective. To the best of our knowledge this is the first report focussing on the prevalence of T. gondii in commercial "Serrano" ham. The method described here could be useful for producers to guarantee the safety of their products. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. DFNB59 Gene Mutation Screening Using PCR-SSCP/HA Technique in Non-syndromic Genetic Hearing Loss in Bushehr Province

    Directory of Open Access Journals (Sweden)

    Fatemeh Azadegan Dehkordi

    2010-09-01

    Full Text Available Background: Hearing impairment (HI is the most prevalent Neurosensory disorder which is heterogenous and can also occur due to environmental causes. The majority of hearing deficiencies are of genetic origin affecting about 60% of the HI cases. A novel gene DFNB59 encodes pejvakin has been recently shown to cause deafness. This study aims to determine the frequency of DFNB59 gene mutations in coding region the gene in Bushehr province. Methods: In this descriptive experimental study, we investigated the presence of DFNB59 gene mutations in Exons (2-7 of the gene in 80 deaf subjects. DNA was extracted using standard phenol –chloroform method. The screening of gene mutations was performed by PCR-SSCP/HA procedure. Finally, the possible mutations were confirmed by direct sequencing. Results: In all, 9 polymorphisms 793C>G were found in 80 non-syndromic, genetic hearing loss subjects studied. However no DFNB59 gene mutation was identified. Conclusion: We conclude that the association of DFNB59 gene mutations with hearing loss is very low in samples studies

  18. Identification of airborne bacterial and fungal species in the clinical microbiology laboratory of a university teaching hospital employing ribosomal DNA (rDNA) PCR and gene sequencing techniques.

    Science.gov (United States)

    Nagano, Yuriko; Walker, Jim; Loughrey, Anne; Millar, Cherie; Goldsmith, Colin; Rooney, Paul; Elborn, Stuart; Moore, John

    2009-06-01

    Universal or "broad-range" PCR-based ribosomal DNA (rDNA) was performed on a collection of 58 isolates (n = 30 bacteria + 28 fungi), originating from environmental air from several locations within a busy clinical microbiology laboratory, supporting a university teaching hospital. A total of 10 bacterial genera were identified including both Gram-positive and Gram-negative genera. Gram-positive organisms accounted for 27/30 (90%) of total bacterial species, consisting of seven genera and included (in descending order of frequency) Staphylococcus, Micrococcus, Corynebacterium, Paenibacillus, Arthrobacter, Janibacter and Rothia. Gram-negative organisms were less frequently isolated 3/30 (10%) and comprised three genera, including Moraxella, Psychrobacter and Haloanella. Eight fungal genera were identified among the 28 fungal organisms isolated, including (in descending order of frequency) Cladosporium, Penicillium, Aspergillus, Thanatephorus, Absidia, Eurotium, Paraphaeosphaeria and Tritirachium, with Cladosporium accounting for 10/28 (35.7%) of the total fungal isolates. In conclusion, this study identified the presence of 10 bacterial and eight fungal genera in the air within the laboratory sampled. Although this reflected diversity of the microorganisms present, none of these organisms have been described previously as having an inhalational route of laboratory-acquired infection. Therefore, we believe that the species of organisms identified and the concentration levels of these airborne contaminants determined, do not pose a significant health and safety threat for immunocompotent laboratory personnel and visitors.

  19. Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

    Directory of Open Access Journals (Sweden)

    Reis Patricia P

    2010-06-01

    Full Text Available Abstract Background MicroRNAs (miRs are non-coding RNA molecules involved in post-transcriptional regulation, with diverse functions in tissue development, differentiation, cell proliferation and apoptosis. miRs may be less prone to degradation during formalin fixation, facilitating miR expression studies in formalin-fixed paraffin-embedded (FFPE tissue. Results Our study demonstrates that the TaqMan Human MicroRNA Array v1.0 (Early Access platform is suitable for miR expression analysis in FFPE tissue with a high reproducibility (correlation coefficients of 0.95 between duplicates, p 35, we show that reproducibility between technical replicates, equivalent dilutions, and FFPE vs. frozen samples is best in the high abundance stratum. We also demonstrate that the miR expression profiles of FFPE samples are comparable to those of fresh-frozen samples, with a correlation of up to 0.87 (p Conclusion Our study thus demonstrates the utility, reproducibility, and optimization steps needed in miR expression studies using FFPE samples on a high-throughput quantitative PCR-based miR platform, opening up a realm of research possibilities for retrospective studies.

  20. Diagnosis of Giardia infections by PCR-based methods in children of an endemic area

    Directory of Open Access Journals (Sweden)

    EB David

    2011-01-01