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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: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples is developed.
Abstract: Aims: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. Methods and Results: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC-d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which coamplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 104 CFU ml−1 (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre-enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. Conclusions: The developed mPCR assay provides specific detection of S. Typhi. Significance and Impact of the Study: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.

71 citations

Journal ArticleDOI
TL;DR: In an effort to develop a new class of molecular diagnostic assay that can rapidly assess drug resistance, a multiplexed assay was established using molecular beacons corresponding to the wild-type cyp51A gene and seven mutant alleles encoding either Arg54, Lys54, Val54, Trp54, or Glu54.
Abstract: Aspergillus fumigatus is an important cause of life-threatening invasive fungal disease in patients with compromised immune systems. Resistance to itraconazole in A. fumigatus is closely linked to amino acid substitutions in Cyp51A that replace Gly54. In an effort to develop a new class of molecular diagnostic assay that can rapidly assess drug resistance, a multiplexed assay was established. This assay uses molecular beacons corresponding to the wild-type cyp51A gene and seven mutant alleles encoding either Arg54, Lys54, Val54, Trp54, or Glu54. Molecular beacon structure design and real-time PCR conditions were optimized to increase the assay specificity. The multiplex assay was applied to the analysis of chromosomal DNA samples from a collection of 48 A. fumigatus clinical and laboratory-derived isolates, most with reduced susceptibility to itraconazole. The cyp51A allelic identities for codon 54 were established for all of the strains tested, and mutations altering Gly54 in 23 strains were revealed. These mutations included G54W (n = 1), G54E (n = 12), G54K (n = 3), G54R (n = 3), and G54V (n = 4). Molecular beacon assay results were confirmed by DNA sequencing. Multiplex real-time PCR with molecular beacons is a powerful technique for allele differentiation and analysis of resistance mutations that is dynamic and suitable for rapid high-throughput assessment of drug resistance.

71 citations

Journal ArticleDOI
TL;DR: A qualitative SYBR Green-based real-time multiplex RT-PCR for the simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) genomes in DBS may represent a reliable alternative for the detection of HIV-1/HCV co-infection, in rapid and relatively inexpensive screening programmes.

71 citations

Journal ArticleDOI
TL;DR: This multiplex PCR assay was used to identify 31 Leuconostoc strains isolated from kimchi, a fermented-cabbage product, and the results showed perfect correlation with the results of a polyphasic method, including 16S rDNA sequencing and DNA-DNA hybridization.
Abstract: A multiplex polymerase chain reaction (PCR) assay has been developed for rapid and reliable identification of Leuconostoc species, by using species-specific primers targeted to the genes encoding 16S rRNA. This assay can detect and differentiate Leuconostoc species from mixed populations in natural sources as well as from pure cultures, within 3 h. This assay system consists of a total of 10 primers, two primers from each target species, and comprises two multiplex PCR reactions: one reaction for Leuconostoc carnosum, Leuconostoc citreum and Leuconostoc mesenteroides, and another reaction for Leuconostoc gelidum and Leuconostoc lactis. This multiplex PCR assay was used to identify 31 Leuconostoc strains isolated from kimchi, a fermented-cabbage product, and the results showed perfect correlation with the results of a polyphasic method, including 16S rDNA sequencing and DNA–DNA hybridization. In addition, this assay enables simultaneous detection of the above-mentioned Leuconostoc species when chromosomal DNA from these Leuconostoc species was mixed. Thus, these results suggest that this multiplex PCR is a rapid and reliable method for identification of Leuconostoc species in pure cultures or in mixed populations.

71 citations

Book ChapterDOI
TL;DR: This chapter describes the different steps necessary for establishing a molecular beacon real-time PCR assay: target design, primer design, optimization of the amplification reaction conditions using SYBR Green, molecular beacon design, and molecular beacon synthesis and characterization.
Abstract: During the last few years, several innovative technologies have become available for performing sensitive and accurate genetic analyses. These techniques use fluorescent detection strategies in combination with nucleic acid amplification protocols. Most commonly used is the real-time polymerase chain reaction (PCR). To achieve the maximum potential of a real-time PCR assay, several parameters must be evaluated and optimized independently. This chapter describes the different steps necessary for establishing a molecular beacon real-time PCR assay: (1) target design, (2) primer design, (3) optimization of the amplification reaction conditions using SYBR Green, (4) molecular beacon design, and (5) molecular beacon synthesis and characterization. The last section provides an example of a multiplex quantitative real-time PCR.

71 citations


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