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.

Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR.

Abstract: Mismatch amplification mutation assay primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, was coupled with primers for the Shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. Analysis of 108 bacteria showed that all Escherichia coli serotype O157:H7 strains were identified simultaneously with the SLT types encoded by these strains.

Multiplex PCR amplification from the CFTR gene using DNA prepared from buccal brushes/swabs

Abstract: Traditionally, DNA used for PCR-based diagnostic analysis has originated from white cells fractionated from whole blood. Although this method yields substantial quantities of DNA, there are some drawbacks to the procedure, including the inconvenience of drawing blood, risk of exposure to blood-borne pathogens, liquid sample handling, and the somewhat involved extraction procedure. Alternatively, DNA for genetic diagnosis has been derived from finger stick blood samples, hair roots, cheek scrapings, and urine samples. Oral saline rinses have also been used extensively as a means of collecting buccal epithelial cells as a DNA source. However, this method still requires liquid sample handling. Herein, we present our results involving the rapid extraction of DNA from buccal cells collected on cytology brushes and swabs for use in PCR reactions, specifically the multiplex amplification of 5 exons within the CFTR gene. The quality of DNA isolated from buccal cells, collected in this manner, has been sufficient to reproducibly support multiplex amplification. Cheek cell samples and the DNA prepared from them as described here are highly stable. The success rate of PCR amplification on DNA prepared from buccal cells is 99%. In a blind study comparing the analysis of 12 mutations responsible for cystic fibrosis in multiplex products amplified with DNA from both blood and buccal cell samples from 464 individuals, there was 100% correlation of results for blood and cheek cell DNA, validating the use of DNA extracted from cheek cells collected on cytology brushes for use in genetic testing.

[A high-throughput SNP typing system for genome-wide association studies].

Abstract: SNPs are useful markers for identifying genes responsible for and/or associated with common diseases, and for directing personalized medical care. Furthermore, because they are so frequent in the genome and can be genotyped quite easily, SNPs can serve as markers for a whole genome association study. However, one of the most difficult issues to be solved for whole-genome association studies using SNPs is reduction of the amount of genomic DNA for genotyping. The presently available technologies require too much genomic DNA to be practical. To overcome this problem, we combined the Invader assay with multiplex PCR performed in the presence of Taq polymerase antibody as well as a novel 384-well card system that reduces the reaction volume. We amplified 96 genomic DNA fragments simultaneously in a single tube, and analyzed each SNP using the Invader assay. Since we used 10-20 nanograms of genomic DNA as a template for multiplex PCR, the amount needed to assay one SNP was only 0.1-0.2 nanograms. Our results strongly indicate the feasibility of undertaking genome-wide association studies using blood samples of only 5-10 milliliters. Using these technologies, which allow us to perform as many as 450,000 typings in one day, our system should let us identify the genes responsible for many diseases and/or pharmacological responsiveness.

Minisequencing: A Specific Tool for DNA Analysis and Diagnostics on Oligonucleotide Arrays

Abstract: We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.

Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory.

Abstract: A multiplex PCR assay for detection of the staphylococcal mecA gene (the structural gene for penicillin-binding protein 2a) was compared with agar dilution and disk diffusion susceptibility test methods for identifying methicillin resistance. The multiplex PCR assay combined two primer sets (mecA and 16S rRNA) in a single reaction. A total of 500 staphylococcal isolates (228 isolates of Staphylococcus aureus and 272 isolates of coagulase-negative staphylococci) from clinical specimens were studied. For S. aureus, 40 of 40 mecA-positive isolates and 4 of 188 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 100 and 98%, respectively). In 3 of 4 discordant isolates, resistance was due to hyperproduction of beta-lactamase. For coagulase-negative staphylococci, 148 of 159 mecA-positive isolates and 0 of 113 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 93 and 100%, respectively). Twenty-six isolates were categorized as indeterminate because of the absence of a detectable 16S rRNA product. Four of these 26 isolates contained mecA when retested. The assay is designed to be incorporated into the work flow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner.