Topic
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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TL;DR: A rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species, which will permit more accurate assessment of the role of various B. fragilIS group members in infections and of the degree of antimicrobial resistance in each of the group members.
Abstract: We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.
107 citations
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TL;DR: A novel method for the fast identification of genetic material utilizing a micro-DNA amplification and analysis device (micro-DAAD) consisting of multiple PCR microreactors with integrated DNA microarrays was developed and successful amplification of the genetic material and the consecutive analysis of the fluorescent-labeled amplicons by the integrated oligonucleotide probes were demonstrated.
Abstract: A novel method for the fast identification of genetic material utilizing a micro-DNA amplification and analysis device (μ-DAAD) consisting of multiple PCR microreactors with integrated DNA microarrays was developed. The device was fabricated in Si-technology and used for the genotyping of Chinese medicinal plants on the basis of differences in the noncoding region of the 5S-rRNA gene. Successful amplification of the genetic material and the consecutive analysis of the fluorescent-labeled amplicons in the μ-DAAD by the integrated oligonucleotide probes were demonstrated. Parallel analysis was performed by loading the four PCR reactors of the μ-DAAD with different samples of 3-μL volume. Temperature sensors and heating elements of the μ-DAAD enable precise temperature control and fast cycling, allowing the rapid completion of a combined amplification and analysis (hybridization) experiment.
107 citations
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TL;DR: A polymerase chain reaction (PCR) based approach to the detection and identification of pathogenic fungi which has potential for the diagnosis of systemic mycoses and species-specific primers which only amplified homologous DNA are described.
Abstract: We describe a polymerase chain reaction (PCR) based approach to the detection and identification of pathogenic fungi which has potential for the diagnosis of systemic mycoses. Primers to sequences of the large subunit ribosomal DNA genes, which are universally conserved within the fungal kingdom, were capable of amplifying DNA from 43 strains representing 20 species (12 genera) of medically important fungi. Sequence analysis of the products obtained from Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans allowed us to design species-specific primers which only amplified homologous DNA. The use of these two PCRs in tandem allows the detection (universal PCR) and identification (species-specific PCR) of a fungal pathogen within 8 h from simulated clinical specimens.
107 citations
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TL;DR: A species-specific 760-base pair (bp) BamHI to EcoRI DNA fragment (fMG-2) was isolated from a Mycoplasma gallisepticum genomic library constructed in plasmid pUC8, and a pair of 25 base primers, designated amplification (Amp) left (L) and right (R) primers was synthesized.
Abstract: A species-specific 760-base pair (bp) BamHI to EcoRI DNA fragment (fMG-2) was isolated from a Mycoplasma gallisepticum (MG) genomic library constructed in plasmid pUC8. Based on the DNA sequence data of fMG-2, a pair of 25 base primers, designated amplification (Amp) left (L) and right (R) primers, was synthesized. When used in the polymerase chain reaction (PCR), the Amp L and R primers directed amplification of DNA of 16 MG strains yielding an expected 732-bp product, but did not amplify DNA of Escherichia coli, calf thymus, lambda phage, pUC8 plasmid, or 16 other species of avian mycoplasmas. As low as 10(-6) picogram of MG DNA, a fraction of the total chromosomal content of one cell, was detected following amplification by PCR. PCR amplification products were visualized by either ethidium bromide/ultraviolet exposure or hybridization with a 481-bp probe (fMG-3) prepared from the central region of fMG-2.
107 citations
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TL;DR: The multiplex assay had a tendency to detect the species of highest mtDNA concentration only, and better detection of all three species in a combination of human, bovine, and swine effluents was accomplished by running each real-time PCR primer/ probe set singly.
Abstract: Multiplex real-time PCR amplifying fecal mitochondrial DNA (mtDNA) combined with rapid, crude DNA preparations are promising additions to surface water source tracking methods. Amplification of eukaryotic mitochondrial DNA identifies the fecal source directly and can be used in conjunction with other intestinal microbial methods to characterize effluents. Species-specific primers and dual-labeled probes for human, swine, and bovine NADH dehydrogenase subunit 5 (ND5) genes were created for multiplex real-time PCR in feces and effluent slurries. The linear range of the multiplex assay was 10(2)-10(7) mtDNA copies for human, bovine, and swine effluent in combination (equal volumes). PCR amplification efficiencies for bovine, human, and swine mtDNA when assayed in combination were 93, 107, and 92% respectively. Linear regression correlation coefficients (r2) were 0.99 for all standard curves except for human mtDNA in combination (r2 = 0.95). Multiplex amplification of bovine, human, and swine mtDNA (ND5) exhibited no cross-reactions between the effluents from three species of interest. Also, no cross-reactions were observed with effluents of other vertebrates: sheep, goat, horse, dog, cat, Canada goose, broiler, layer, turkey, and tilapia. Performed as a blind test, the PCR operator was able to correctly identify all but two effluent challenge samples (10/12 or 83% correct) with no false positives (22/22 or 100% correct). The multiplex assay had a tendency to detect the species of highest mtDNA concentration only. Better detection of all three species in a combination of human, bovine, and swine effluents was accomplished by running each real-time PCR primer/ probe set singly. Real-time PCR detection limit was calculated as 2.0 x 10(6) mitochondrial copies or 0.2 g of human feces per 100 mL effluent. Some carry-over mtDNA PCR signal from consumed beef, but not pork, was found in feces of human volunteers.
107 citations