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      Multiplex polymerase chain reaction: A practical approach

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          Abstract

          Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features. J. Clin. Lab. Anal. 16:47–51, 2002. © 2002 Wiley‐Liss, Inc.

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          Author and article information

          Journal
          J Clin Lab Anal
          J. Clin. Lab. Anal
          10.1002/(ISSN)1098-2825
          JCLA
          Journal of Clinical Laboratory Analysis
          John Wiley & Sons, Inc. (New York )
          0887-8013
          1098-2825
          08 January 2002
          2002
          : 16
          : 1 ( doiID: 10.1002/jcla.v16:1 )
          : 47-51
          Affiliations
          [ 1 ]Virology Department, Hellenic Pasteur Institute, Athens, Greece
          [ 2 ]Laboratoire de Biologie Cellulaire et Moléculaire, Université de La Rochelle, La Rochelle, France
          Author notes
          [*] [* ]Virology Laboratory, Hellenic Pasteur Institute, 127 Vasilissis Sofias Ave., Athens 115 21, Greece
          Article
          PMC6808141 PMC6808141 6808141 JCLA2058
          10.1002/jcla.2058
          6808141
          11835531
          1acce6a7-70c8-477a-9bc7-51055ef89d05
          Copyright © 2002 Wiley‐Liss, Inc.
          History
          : 19 April 2001
          : 16 October 2001
          Page count
          Figures: 0, Tables: 0, References: 27, Pages: 5, Words: 3443
          Categories
          Original Article
          Original Articles
          Custom metadata
          2.0
          2002
          Converter:WILEY_ML3GV2_TO_NLMPMC version:5.7.0 mode:remove_FC converted:23.10.2019

          PCR buffer concentration,primer‐to‐template ratio,dNTP/MgCl2 balance,multiplex PCR

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