Rapid genotyping of hepatitis C virus by direct cycle sequencing of PCR-amplified cDNAs and capillary electrophoresis analysis.
There is an increasing demand for the genotyping of hepatitis C virus (HCV), since it has been shown that different HCV genotypes are associated with distinct profiles of pathogenicity and responses to antiviral treatment. Hence, there is a need for a simple and precise genotyping assay for routine diagnosis of HCV types and subtypes. Here we show that direct sequencing, considered as the reference method, can provide an accurate and rapid method for large-scale screening of HCV genotypes. PCR-amplified cDNAs of the HCV 5' non-coding region (5' NCR) were obtained from the widespread "Amplicor" HCV detection system. Semi-purified PCR products were directly cycle-sequenced in a single tube using multicolour dye terminator chemistry. Sample loading, electrophoresis and sequence analysis were automatically achieved by a capillary electrophoresis-based genetic analyser. Out of a total of 500 samples, HCV subtype 1b accounted for the majority of the infections (41%), followed by HCV 3 (31%) and HCV 1a (22%). This procedure failed to identify a genotype in only 3 samples. In addition, several cases of mixed HCV infection were also documented. The combination of direct cycle sequencing of PCR products with capillary electrophoresis provides a simple and rapid method convenient for routine HCV genotyping analysis.
Laboratoire de Virologie, Faculté de Médecine, Nice, France.
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A
Doglio
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C
Laffont
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Thyss
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JC
Lefebvre
D001331:Automation; D018076:DNA, Complementary / Q000097:blood / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005260:Female; D005838:Genotype; D016174:Hepacivirus / Q000145:classification* / Q000235:genetics*; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D008297:Male; D012367:RNA, Viral / Q000097:blood / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic