Multiplex Polymerase Chain Reaction

James B. Mahony and Max A. Chernesky

McMaster University Regional Virology and Chlamydiology Laboratory

St. Joseph's Hospital

Hamilton, Ontario, Canada L8N 4A6

I. Introduction

II. Methodology

A. Principle

B. Applications

C. Strengths and Weaknesses

III. Developing a Multiplex PCR Assay

A. Primers

B. Optimizing Conditions

C. Amplicon Analysis

D. Evaluating Performance

E. Troubleshooting

IV. Quality Assurance

A. Anticontamination Measures

B. Controls

C. Limitations

D. Interpretation References

I. INTRODUCTION

In a few short years, the polymerase chain reaction (PCR) has had a major impact on how clinical laboratories diagnose infectious diseases. PCR has been used for the diagnosis of infections caused by several noncultivatable viruses including human papillomavirus (HPV), parvovirus B19, and BK/ JC papovaviruses. Arguably, the greatest impact of PCR has been in the field of retro virology, where it has been used to detect infections in seronegative

MOLECULAR METHODS FOR VIRUS DETECTION

Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

individuals. Quantitative PCR (see Chapter 9), is also proving useful in evaluating the efficacy of new antiviral agents in clinical trials.

Multiplex PCR (M-PCR) is the simultaneous detection of more than one target sequence. The first reports appearing in the literature that used M-PCR involved the diagnosis of inherited genetic diseases. The majority of M-PCR studies have involved the detection of mutations in the dystrophin gene and the cystic fibrosis transmembrane regulatory gene (Chamberlain et al., 1988,1990; Fortina et al., 1992; Kilimann et al., 1992; Ko et al., 1992; Morrall and Estivill, 1992; Picci et al., 1992; Richards et al., 1993). A computer search of the medical literature (Medline: Mesh Headings, PCR and multiplex, September 1993) turned up 112 papers on M-PCR of which only 11 addressed the detection of infectious agents. The development of multitarget co-amplification, or M-PCR, has not yet had an impact on clinical laboratories, but as more virologists explore the tremendous potential of this technique, M-PCR will assume a major role in the diagnostic virology laboratory.

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