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.

Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB)—a practical epidemiological and diagnostic tool

Abstract: Combining multiplex PCR, sequentially, with reverse line blot hybridization (mPCR/RLB) is a convenient, objective way to identify up to 43 targets in 43 individual specimens simultaneously (using a 45-lane membrane format). It is more flexible and less expensive than DNA microarray. The number of targets is adequate for epidemiological and most clinical diagnostic applications; based on the same target (43) and specimen numbers (43), it is much more practical than conventional uniplex PCR (uPCR) and mPCR. We have used the protocol to identify and subtype bacteria, viruses and fungi and identify pathogens in clinical specimens; potentially, it could be used for many other applications, such as detection of mutations in, or identification of alleles of, eukaryotic genes. Development of each assay involves (i) careful primer and probe design, based on literature and sequence database searches, which are critical to success of the assay; and (ii) bench-top evaluation, using known samples, controls and dilution series, to confirm sensitivity, specificity and reproducibility. The assay takes about one and half working days to complete; about 4 h for the mPCR and 6 h for the RLB, including a total of 4 h 'hands-on' time.

Multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis.

Abstract: Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are strongly associated with periodontitis. However, little is known about their distribution in periodontally healthy individuals, because culturing techniques are not sufficiently sensitive. A modified multiplex PCR was developed to address that question. Our method uses two species-specific forward primers in combination with a single reverse primer. These primers target variable and conserved regions of the 16S rRNA gene. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans and P. gingivalis cells. Primer specificity was tested against (i) six A. actinomycetemcomitans strains and four P. gingivalis strains, (ii) seven different species of oral bacteria, and (iii) supra- and subgingival plaque from 20 subjects. The multiplex PCR had a lower limit of detection of 2 A. actinomycetemcomitans and 30 P. gingivalis cells. Species-specific amplicons were obtained for all A. actinomycetemcomitans and P. gingivalis strains tested and did not occur with seven other bacterial species unless A. actinomycetemcomitans and P. gingivalis were added. Neither target species was detected in supragingival plaque; A. actinomycetemcomitans was detected in one subgingival specimen, and P. gingivalis was detected in another. When plaque samples were spiked with 10 A. actinomycetemcomitans cells and 100 P. gingivalis cells, species-specific amplicons were detected. These findings show our multiplex PCR to be highly sensitive and specific while allowing simultaneous detection of A. actinomycetemcomitans and P. gingivalis. This assay has potential applications in epidemiological studies, diagnosis, treatment planning, and monitoring of periodontal pathogens.

Performing quantitative reverse-transcribed polymerase chain reaction experiments.

Abstract: Quantitative polymerase chain reaction (PCR) is as old as PCR, but it has had to wait for the introduction of real-time PCR instruments to become widely used. These instruments allow monitoring of the PCR reaction on line; they involve the use of a fluorescent probe that allows quantification of the amplified DNA. Different fluorescent formats and different applications have been developed for quantitative PCR, but this chapter focuses on the use of the SYBR Green label for the quantification of specific cDNAs in reverse transcription mixes: RT-PCR. We propose optimal reaction conditions for the reactions to be performed on the different available instruments and discuss the important parameters for setting up experiments: specificity, efficiency, and reproducibility. We also introduce the reader to the problems of relative quantification.

Multiplex polymerase chain reaction for subgenus‐specific detection of human adenoviruses in clinical samples

Abstract: The polymerase chain reaction (PCR) has been used previously for the detection and typing of adenoviruses directly in clinical samples. Since under clinical conditions subgenus-specific identification is often sufficient, we extended the genus- and type-specific PCR by a subgenus-specific PCR. By sequencing several loop I4 gene regions of the hexon (major adenovirus coat protein) and comparing them to published sequences, subgenus-specific sequences were identified in this region. By using primers targeted to this region and to a conserved hexon gene region, a multiplex, nonnested PCR for the detection and subgenus-specific identification of adenoviruses could be established. The six subgenus-specific amplimers are distinguishable by agarose gel electrophoresis, and subsequent restriction analysis is not necessary. The specificity of the subgenus-specific primer pairs was tested on 23 adenovirus prototypes, representing all six subgenera, on 9 subgenus B and D intermediate strains, and on 16 subgenus C genome types. Furthermore, multiplex, subgenus-specific PCR was performed directly with 100 clinical specimens, including stool samples, ocular swabs, and throat swabs. Adenoviruses of all subgenera could be detected. Especially for clinical application, the rapid, one-step differentiation between subgenus D adenoviruses, causing the severe and highly contagious epidemic keratoconjunctivitis, and subgenus B and E adenoviruses, causing relative harmless ocular infections, is of great importance. The subgenus-specific PCR could also facilitate the primary classification of unknown virus isolates.