Multiplex polymerase chain reaction

About: Multiplex polymerase chain reaction is a research topic. Over the lifetime, 6409 publications have been published within this topic receiving 221244 citations.

The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.

Abstract: With current methodology, DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) that can identify blood, saliva, semen and menstrual blood in individual stains or in mixtures of body fluids. Messenger RNA transcripts specific to each type of body fluid have been identified and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) system developed to identify these body fluids along with three housekeeping genes. This multiplex can detect semen and seminal fluid (semen without spermatozoa present). Furthermore, we have targeted the co-isolation of RNA and DNA from the same sample and, with the RT-PCR multiplex, we can determine the type of body fluid present as well as generate a DNA profile(s) from the same stain.

Development of a single-reaction multiplex PCR toxin typing assay for Staphylococcus aureus strains.

Abstract: We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureus that utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes.

Nucleic acid amplification strategies for DNA microarray-based pathogen detection.

Abstract: DNA microarray-based screening and diagnostic technologies have long promised comprehensive testing capabilities. However, the potential of these powerful tools has been limited by front-end target-specific nucleic acid amplification. Despite the sensitivity and specificity associated with PCR amplification, the inherent bias and limited throughput of this approach constrain the principal benefits of downstream microarray-based applications, especially for pathogen detection. To begin addressing alternative approaches, we investigated four front-end amplification strategies: random primed, isothermal Klenow fragment-based, phi29 DNA polymerase-based, and multiplex PCR. The utility of each amplification strategy was assessed by hybridizing amplicons to microarrays consisting of 70-mer oligonucleotide probes specific for enterohemorrhagic Escherichia coli O157:H7 and by quantitating their sensitivities for the detection of O157:H7 in laboratory and environmental samples. Although nearly identical levels of hybridization specificity were achieved for each method, multiplex PCR was at least 3 orders of magnitude more sensitive than any individual random amplification approach. However, the use of Klenow-plus-Klenow and phi29 polymerase-plus-Klenow tandem random amplification strategies provided better sensitivities than multiplex PCR. In addition, amplification biases among the five genetic loci tested were 2- to 20-fold for the random approaches, in contrast to >4 orders of magnitude for multiplex PCR. The same random amplification strategies were also able to detect all five diagnostic targets in a spiked environmental water sample that contained a 63-fold excess of contaminating DNA. The results presented here underscore the feasibility of using random amplification approaches and begin to systematically address the versatility of these approaches for unbiased pathogen detection from environmental sources.

Detection and quantification of Phytophthora ramorum, P. kernoviae, P. citricola and P. quercina in symptomatic leaves by multiplex real-time PCR

Abstract: SUMMARY New species of Phytophthora such as Phytophthora ramorum, P. kernoviae and P. quercina together with P. citricola are plant pathogens which impact on forest health, natural ecosystem stability and international trade. A real-time multiplex PCR approach based on TaqMan PCR was developed to simultaneously identify and detect these four Phytophthora species. Specific primers and probes labelled with FAM (P. ramorum), Yakima Yellow (P. kernoviae), Rox (P. citricola) and Cy5 (P. quercina) were designed in different regions of the ras-related protein (Ypt1) gene. A new set of Black Hole Quenchers (BHQ), which dissipate energy as heat rather than fluorescence, were utilized. The method proved to be highly specific in tests with target DNA from 72 Phytophthora isolates (35 species). For all pathogens, the detection limit was 100 fg of target DNA and was not improved utilizing a nested approach to provide a first round of amplification with Phytophthora spp.-specific primers. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficients ranged from 0.947 to 0.996) and were not affected by the presence of plant extracts, indicating the appropriateness of the method for qualitative and quantitative analyses. Two universal primers and a TaqMan probe were also developed to evaluate the quality and quantity of extracted DNA and to avoid false negatives. The reliability of the entire procedure was assessed using both artificially and naturally infected leaves of a range of plant species. The method, combined with a rapid procedure for DNA extraction, proved to be rapid, reliable, sensitive and cost effective as multiple pathogens were detected within the same plant extract by using different primer/probe combinations.

Multiplex Real-Time PCR Assay for Detection of Influenza and Human Respiratory Syncytial Viruses

Abstract: A multiplex real-time PCR assay was developed with a LightCycler instrument for detection of influenza viruses A and B and the human respiratory syncytial virus (HRSV). Detection of each viral product and of an internal control was based on determination of specific melting temperatures by the LightCycler software. The lower limit of detection in the multiplex PCR assay was found to be 50 copies for each viral target. In an evaluation of nasopharyngeal samples collected from hospitalized children (ages, 0 to 3 years) with acute respiratory tract infections during the winter of 2001 to 2002, a viral pathogen was detected by the multiplex PCR test in 139 (66.8%) of 208 cases, including 45 (21.6%) influenza A virus infections, no (0%) influenza B virus infections, 106 (51%) HRSV infections, and 12 (5.8%) coinfections. The multiplex PCR test was compared to rapid antigen detection assays for influenza viruses A and B (Directigen; Becton Dickinson, Sparks, Md.) and HRSV (RSV TestPack; Abbott Laboratories, Abbott Park, Ill.) in 172 and 204 samples, respectively. After resolution of discrepant test results by use of additional PCR assays targeting other viral genes, the sensitivity (Se) and specificity (Sp) of the multiplex PCR assay for influenza A virus were 100 and 97.7% compared to 43.6 and 98.5% for the antigenic test. Similarly, the Se and Sp of the multiplex PCR assay for HRSV were 94.5 and 98.9% compared to 81.6 and 94.7% for the antigenic test. In conclusion, our multiplex real-time PCR assay combines both rapidity and sensitivity for detecting the most important respiratory viral pathogens in children.