Multiplex real-time quantitative RT-PCR assay for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1.
A multiplex real-time quantitative reverse transcription (RT)-PCR assay was developed for simultaneous detection, identification and quantification of hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) in plasma or serum samples. Genomic amplification of one virus was unaffected by the simultaneous amplification of the other two. Competition between HCV and HIV-1 amplifications slightly affected the yield of HIV-1 amplification. However, quantitation was possible when a single virus was present. The 95% detection limits were 30, 167 and 680IU/ml for HBV DNA, HCV RNA and HIV-1 RNA, respectively. The multiplex assay detected with similar efficiency strains of HBV genotypes A-F, HCV genotypes 1-6, and HIV-1 subtypes A-G. Applied to 267 pools of 10 plasmas from blood donors, multiplex screening indicated that the assay was reproducible, sensitive, and specific. This assay has the potential to be used for large-scale nucleic acid testing (NAT) of blood donations.
Candotti
Daniel
D
National Blood Service Cambridge, Long Road, Cambridge CB2 2PT, UK. dc241@cam.ac.uk
Temple
Jillian
J
Owusu-Ofori
Shirley
S
Allain
Jean-Pierre
JP
D001483:Base Sequence; D001782:Blood Donors; D004279:DNA, Viral / Q000097:blood / Q000235:genetics; D005838:Genotype; D005869:Ghana; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D016174:Hepacivirus / Q000235:genetics* / Q000302:isolation & purification*; D006515:Hepatitis B virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D012367:RNA, Viral / Q000097:blood / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014773:Virology / Q000379:methods
D004279:DNA, Viral; D012367:RNA, Viral