Detection of hepatitis C virus by a user-developed reverse transcriptase-PCR and use of amplification products for subsequent genotyping.
BACKGROUND
Detection, quantitation and genotyping of hepatitis C virus (HCV) are important in selecting appropriate therapy. Current commercially available HCV genotyping kits, including sequencing-based TRUGENE HCV 5'NC and hybridization-based INNO-LiPA HCV II assays, rely on amplification products (amplicons) generated by HCV reverse-transcriptase (RT)-PCR methods such as the Roche AMPLICOR HCV test.


METHODS
We developed a one-step RT-PCR assay to amplify and detect HCV RNA, and the resulting amplicons were used for HCV genotyping (TRUGENE). A total of 142 clinical samples were used to compare results from the RT-PCR/TRUGENE assay and those generated by the COBAS AMPLICOR and INNO-LiPA tests.


RESULTS
Eighty-seven of 108 plasma specimens which were positive by AMPLICOR were also positive by the user-developed RT-PCR, giving a sensitivity of 100.0%. The RT-PCR detected 2 of 21 AMPLICOR-negative specimens and none of 34 HCV-EIA-negative serum specimens, giving a specificity of 96.4%. The 87 amplicons from the RT-PCR yielded HCV genotypes. HCV genotype results from both TRUGENE and INNO-LiPA were all in agreement. The TRUGENE and INNO-LiPA assays identified 69 (79.3%) and 47 (54.0%) specimens, respectively at the subtype level. HCV subtype information agreed by both assays in 34 of 36 (99.4%) specimens. One specimen with HCV genotypes 2 and 4 by INNO-LiPA was classified as a single genotype 2 by TRUGENE.


CONCLUSIONS
Our data showed that the user-developed RT-PCR has comparable sensitivity and specificity for the detection of HCV in clinical specimens. The amplicons generated by the RT-PCR can be used for HCV genotyping by the sequencing-based TRUGENE assay.
Tang
Yi-Wei
YW
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA. yiwei.tang@vanderbilt.edu
Li
Haijing
H
Roberto
Ann
A
Warner
Diane
D
Yen-Lieberman
Belinda
B
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification*; D006526:Hepatitis C / Q000175:diagnosis / Q000821:virology; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014773:Virology / Q000379:methods
D004279:DNA, Viral