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.

Detection and Characterization of Atypical Mycobacteria by the Polymerase Chain Reaction

Abstract: The purpose of this study was to develop a simple protocol of nested reamplification polymerase chain reaction (PCR) to detect and characterize diverse mycobacterial species. DNA extracted from 126 pure mycobacterial cultures isolated from clinical specimens was amplified by nested PCR with use of a novel set of oligonucleotide primers specific for the 65-kDa antigen gene of mycobacteria. The PCR products were each digested with three restriction enzymes and electrophoresed on an agarose gel. The observed DNA fragment sizes of the different species with each enzyme were compiled into a simple algorithm. This method can rapidly detect and characterize a wide variety of mycobacterial species, including the most common pathogens Mycobacterium tuberculosis, Mycobacterium avium-intracellulare, and Mycobacterium kansasii, without hybridization to labeled probes. The application of this method to surgical pathology was demonstrated by amplification and identification of atypical mycobacteria, including M. kansasii and Mycobacterium leprae, in formalin-fixed paraffin-embedded tissue. This protocol broadens the diagnostic potential of PCR for rapidly diagnosing mycobacterial infection in clinical samples, particularly in paraffin-embedded tissue sections.

Duplications and de novo deletions of the SMNt gene demonstrated by fluorescence-based carrier testing for spinal muscular atrophy.

Abstract: Approximately 95% of individuals with spinal muscular atrophy (SMA) lack both copies of the SMNt gene at 5q13. The presence of a nearly identical centromeric homolog of the SMNt gene, SMNc, necessitates a quantitative polymerase chain reaction approach to direct carrier testing. Adapting a radioactivity-based method described previously, multiplex polymerase chain reaction was performed using fluorescently labeled primers followed by analysis on an ABI 373a DNA sequencer. The SMNt copy number was calculated from ratios of peak areas using both internal and genomic standards. Samples from 60 presumed carriers (50 parents of affected individuals and 10 relatives implicated by linkage analysis) and 40 normal control individuals were tested. Normalized results (to the mean of five or more control samples harboring two copies of the SMNt gene) were consistently within the ranges of 0.4 to 0.6 for carriers (one copy) and 0.8 to 1.2 for normal controls (two copies), without overlap. Combining linkage analyses with direct carrier test results demonstrated de novo deletions associated with crossovers, unaffected individuals carrying two SMNt gene copies on one chromosome and zero SMNt gene copies on the other chromosome, and unaffected individuals with three copies of the SMNt gene. This report demonstrates that fluorescence-based carrier testing for SMA is accurate, reproducible, and useful for genetic risk assessment, and that carrier testing may need to be combined with linkage analysis in certain circumstances.

Development and evaluation of a multiplex PCR for rapid detection and differentiation of Mycobacterium tuberculosis complex members from non-tuberculous mycobacteria.

Abstract: Most mycobacterial infections are still caused by Mycobacterium tuberculosis complex (MTC) strains; however, infections by non-tuberculous mycobacteria (NTM) are increasing, particularly among immunocompromised patients. Conventional species-specific identification and proper patient management are delayed due to the slow-growing nature of mycobacteria. We have developed a multiplex PCR (mPCR) targeting the oxyR-ahpC intergenic region and rpoB gene for direct detection and differentiation of clinical isolates as MTC or NTM in primary culture. Two amplicons of 473 bp and 235 bp from MTC members and a single amplicon of 136 bp from NTM are expected. The mPCR was developed using several mycobacterial species and was evaluated by testing extracted DNA from liquid cultures, flagged as positive for bacterial growth, of 100 consecutive mycobacterial isolates. The results were validated by DNA sequencing of the species-specific 16S-23S internal transcribed spacer (ITS) region. The mPCR with template DNA from reference Mycobacterium spp. yielded the expected amplicons. When 100 consecutive clinical isolates of Mycobacterium spp. were tested, 92 strains yielded MTC member-specific amplicons, and DNA sequences from 10 randomly selected isolates matched completely with the ITS sequence from M. tuberculosis. Eight isolates were identified as NTM, and DNA sequencing of the ITS region confirmed the NTM status of each of these isolates. The mPCR developed in this study allowed rapid detection and differentiation of primary cultures as MTC or NTM, thus helping in timely institution of specific therapy.