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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.


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Journal ArticleDOI
TL;DR: The data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.
Abstract: A new set of multiplexed PCR primers has been applied to the analysis of human skeletal remains to determine their efficacy in analyzing degraded DNA. These primer sets, known as Miniplexes, produce shorter amplicons (50-280base pairs (bp)) than standard short tandem repeat (STR) kits, but still utilize the 13 CODIS STR loci, providing results that are searchable on national DNA databases. In this study, a set of 31 different human remains were exposed to a variety of environmental conditions, extracted, and amplified with commercial and Miniplex DNA typing kits. The amplification efficiency of the Miniplex sets was then compared with the Promega PowerPlex s 16 system. Sixty-four percent of the samples generated full profiles when amplified with the Miniplexes, while only 16% of the samples generated full profiles with the Pow- erplex s 16 kit. Complete profiles were obtained for 11 of the 12 Miniplex loci with amplicon sizes less than 200bp. These data suggest smaller PCR amplicons may provide a useful alternative to mitochondrial DNA for anthropological and forensic analysis of degraded DNA from human skeletal remains.

55 citations

Journal ArticleDOI
TL;DR: The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.
Abstract: Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in Treponema pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR assay for detection of the A2058G mutation, and upon discovery of the A2059G mutation, we modified the assay into a triplex format to simultaneously detect both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 specimens PCR positive for T. pallidum that were obtained from an azithromycin resistance surveillance study conducted in the United States were analyzed. Sixty-six (51.2%) of the 129 samples with the A2058G mutation were identified by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation by the triplex PCR. The proportions of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or the A2059G mutation among the T. pallidum strains were 35.6, 51.2, and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism analyses for the rapid detection of both point mutations associated with macrolide resistance in T. pallidum.

55 citations

Journal ArticleDOI
TL;DR: The assay was 100% sensitive and specific for detecting the expected species and offers a quick and accurate strategy for identifying E. coli, E. albertii, and E. fergusonii in either a single reaction or by in silico PCR with sequence assemblies.

54 citations

Journal ArticleDOI
TL;DR: This multiplex RT-PCR technique demonstrates a higher sensitivity of virus detection than DAS-ELISA, and also could detect viruses in some samples that DAS -ELISA failed to detect.
Abstract: Search for a host RNA molecule appropriate as an internal control for reverse transcription-polymerase chain reaction (RT-PCR) detection of viruses in potato (Solanum tuberosum) was conducted. The 18S ribosomal RNA (rRNA) was compared with the commonly used nad2 mRNA in terms of detection sensitivity and degradation kinetics. Detection of 18S rRNA was 5 magnitudes more sensitive than that of nad2 mRNA. The 18S rRNA also displayed degradation kinetics more similar to that of Potato virus X (PVX). Based on this result, reaction components and cycling parameters were optimized for a multiplex RT-PCR protocol for simultaneous detection of five potato viruses using 18S rRNA as an internal control. The protocol simultaneously amplified cDNAs from Potato virus A, PVX, Potato virus Y, Potato leaf roll virus, Potato virus S, and 18S rRNA. The multiplex RT-PCR protocol was able to detect all viruses in different combinations. The technique was 100-fold greater for detection of PVX than that of commercial double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), and also could detect viruses in some samples that DAS-ELISA failed to detect. This multiplex RT-PCR technique demonstrates a higher sensitivity of virus detection than DAS-ELISA.

54 citations

Journal ArticleDOI
TL;DR: A multiplex polymerase chain reaction (PCR) assay that detects key DEC virulence genes (stx1, stx2, eae, bfpA, ipaH, LT, STh, aaiC, aatA) is developed and proposed to screen for DEC directly in stool or stool broths.

54 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
2023275
2022448
2021172
2020176
2019221
2018220