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.

Development of a Multiplex PCR Technique for Detection and Epidemiological Typing of Salmonella in Human Clinical Samples

Abstract: We have developed a multiplex PCR assay for Salmonella detection and epidemiological typing. Six sets of primers were designed to detect the major Salmonella serotypes and phage types in Spain. An internal amplification control was designed in order to detect PCR inhibition. The different amplification profiles obtained allowed us to detect Salmonella bacteria and to distinguish the clinically prevalent Salmonella enterica serotypes Enteritidis, Typhimurium and subspecies I serotype 4,5,12:i:−. Using this method, we could detect a specific band for DT104 and U302 phage types in Salmonella serotype Typhimurium. Salmonella enterica serotype Hadar and other C2 serogroup strains showed two specific band profiles. In the validation stage, the assay was reproducible for all serotypes studied, apart from some C2 serogroup strains. When the technique was applied to clinical stool specimens, the prevalent serotypes Enteritidis and Typhimurium were detected with a sensitivity of 93%, specificity of 100%, and efficiency of 98%. Also, a low PCR inhibition rate (8%) was obtained. The overall agreement of the multiplex PCR with conventional culture-based techniques was 95% for Salmonella typing using Cohen's kappa index.

Detection and differentiation of Listeria spp. by a single reaction based on multiplex PCR

Abstract: The iap gene encodes the protein p60, which is common to all Listeria species. A previous comparison of the DNA sequences indicated conserved and species-specific gene portions. Based on these comparisons, a combination consisting of only five different primers that allows the specific detection and differentiation of Listeria species with a single multiplex PCR and subsequent gel analysis was selected. One primer was derived from the conserved 3' end and is specific for all Listeria species; the other four primers are specific for Listeria monocytogenes, L. innocua, L. grayi, or the three grouped species L. ivanovii, L. seeligeri, and L. welshimeri, respectively. The PCR method, which also enables the simultaneous detection of L. monocytogenes and L. innocua, was evaluated against conventional biotyping with 200 food hygiene-relevant Listeria strains. The results indicated the superiority of this technique. Thus, this novel type of multiplex PCR may be useful for rapid Listeria species confirmation and for identification of Listeria species for strains isolated from different sources.

DNA differential diagnosis of taeniasis and cysticercosis by multiplex PCR

Abstract: Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome c oxidase subunit 1 gene yielded evident differential products unique for Taenia saginata and Taenia asiatica and for American/African and Asian genotypes of Taenia solium with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9 T. solium carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a T. solium carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to T. solium carriers, 827-bp markers were detected by multiplex PCR in all T. saginata carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections.

Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique.

Abstract: We have developed a rapid procedure for the detection of serum hepatitis B virus (HBV) DNA using the polymerase chain reaction (PCR) technique. HBV DNA is released from virions by incubating serum with 0.1 M NaOH for 60 min at 37 degrees C. The mixture is brought to neutral pH with HCl, and the HBV DNA sequences are detected by agarose gel electrophoresis and ethidium bromide staining after PCR amplification with two successive sets of primer pairs. The detection limit of this method (i.e., 10(-5) pg of HBV DNA) is equivalent to that previously determined by one round of PCR amplification and Southern blot hybridization analysis. The advantages are that the assay can be completed in 1 day, is very sensitive, and does not require the use of radiolabeled reagents. Images