Simultaneous detection of multiplex-amplified human immunodeficiency virustype 1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA using a flow cytometer microsphere-based hybridization assay

The feasibility of performing a multiplex assay for the detection of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) RNAs and hepatitis B virus (HBV) DNA is demonstrated. This assay is based (i) on the coamplification of a 142-bp fragment from the gag region of the HIV-1 geno me and a 142-bp HIV-1 quantitation standard fragment, a 244-bp fragment fro m the 5' noncoding region of the HCV genome, and a 104-bp fragment from the pre-C and C gene regions of the HBV genome, using three sets of specific p rimers; (ii) on the capacity of these four biotinylated PCR products to hyb ridize; to their specific oligonucleotide probe-coated microspheres; and (i ii) on the ability of the flow cytometer to discriminate between distinct f luorescent-microsphere categories. Absence of cross-hybridization between t he unrelated oligonucleotide probes and PCR products generated by the multi plex reverse transcription-PCR (RT-PCR) and the highly sensitive detection method allowed us to assess unambiguously the HIV-1 viral load and the infe ctious status of 35 serologically well-established clinical samples and 20 seronegative blood donor plasma samples tested. The results indicate that m ultiplex RT-PCR and flow cytometer microsphere-based hybridization assays, when combined, provide a rapid, sensitive, and specific method for the quan titation and detection of the major viral agents of infectious diseases in a single plasma sample.