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CLINICAL MICROBIOLOGY REVIEWS, Oct. 2000, p. 559–570 Vol. 13, No. 4 0893-8512/00/$04.00 0 Copyright © 2000, American Society for Microbiology. All Rights Reserved. Multiplex PCR: Optimization and Application in Diagnostic Virology ELFATH M. ELNIFRO,1 AHMED M. ASHSHI,1 ROBERT J. COOPER,1 AND PAUL E. KLAPPER1,2* School of Medicine, The University of Manchester,1 and Clinical Virology, Central Manchester Healthcare Trust,2 Manchester, United Kingdom INTRODUCTION 559 PRINCIPLE AND DEVELOPMENT OF MULTIPLEX PCR559 Primers and Multiplex PCR Efficiency559 Other PCR Components 560 Variations in Methodology To Improve Sensitivity and Specificity561 General Considerations for Multiplex PCR Development561 APPLICATION OF MULTIPLEX PCR IN DIAGNOSTIC VIROLOGY 561 Neurotropic Viruses561 Respiratory Viruses 563 Genito-Urinary Infections564 Ocular Infections 565 Immunocompromised Patients 565 Other Applications566 CONCLUSION AND PERSPECTIVES567 REFERENCES 568 INTRODUCTION and discuss the application and potential of this technique in the field of diagnostic virology. During the past decade, advances in PCR technology and other DNA signal and target amplification techniques have resulted in these molecular diagnostics becoming