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Genotyping of hepatitis C virus by melting curve analysis: analytical characteristics and performance.

Knowledge of the hepatitis C virus (HCV) genotype is important in guiding antiviral therapy (1). Viral pharmacogenomic studies have demonstrated that patients infected with genotype-1 HCV respond poorly to interferon-ribavirin therapy and may require a longer course of therapy (1)(2)(3). In the United States, where the most common HCV types are 1a/b, 2a/c, 2b, and 3a, it is particularly important to distinguish patients infected with type 1a/b from those infected with types 2 and 3. To meet the need for this information, we have developed a rapid-cycle, real-time PCR assay with melting-curve analysis for genotyping of HCV (4). This method uses reverse transcription-PCR performed in a block cycler followed by a seminested PCR with product identification using fluorescence resonance energy transfer (FRET) probes and DNA melting curves in a single tube. The FRET probes were designed to identify HCV types 1, 2a/c, 2b, 3a, and 3b/4. Other less common genotypes will likely either not be amplified (types 6b, 7b, and 11a) or will produce a product with a non-type 1 melting temperature ( T m) (4).

Real-time PCR has gained widespread use in clinical analyses since its introduction in 1991 (5), but little has been published on the performance characteristics of such assays over periods longer than a few days or weeks. The objective of the present study was to determine the analytical characteristics of the above HCV genotyping assay and its performance in routine use. The study period covered 23 months with 92 runs performed by six operators on four different LightCycler® instruments, using the exact assay described above (4).

Patient samples were analyzed in groups of 5 to 13. Each analytical …