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Journal of Clinical Microbiology, November 2006, p. 4149-4156, Vol. 44, No. 11
0095-1137/06/$08.00+0     doi:10.1128/JCM.01230-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Multiplex Real-Time Reverse Transcription-PCR Assay for Determination of Hepatitis C Virus Genotypes

Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington 98195,¹ Program in Infectious Diseases, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109²

Received 15 June 2006/ Returned for modification 29 August 2006/ Accepted 6 September 2006

A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n = 110) and a subset that were incorrectly called 2a by the RFLP method (n = 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications.

Published ahead of print on 20 September 2006.

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