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

Abstract

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 genome and a 142-bp HIV-1 quantitation standard fragment, a 244-bp fragment from 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 primers; (ii) on the capacity of these four biotinylated PCR products to hybridize to their specific oligonucleotide probe-coated microspheres; and (iii) on the ability of the flow cytometer to discriminate between distinct fluorescent-microsphere categories. Absence of cross-hybridization between the unrelated oligonucleotide probes and PCR products generated by the multiplex reverse transcription-PCR (RT-PCR) and the highly sensitive detection method allowed us to assess unambiguously the HIV-1 viral load and the infectious status of 35 serologically well-established clinical samples and 20 seronegative blood donor plasma samples tested. The results indicate that multiplex RT-PCR and flow cytometer microsphere-based hybridization assays, when combined, provide a rapid, sensitive, and specific method for the quantitation and detection of the major viral agents of infectious diseases in a single plasma sample.

FIG. 1

Typical dot plots of green fluorescence versus red fluorescence for the mixture of the four oligonucleotide-coated microsphere populations for the simultaneous detection of RT-PCR products from HIV-1, HCV, and HBV nucleic acids.

FIG. 2

Typical dot plots of green fluorescence versus red fluorescence for virus-free or seropositive plasma samples. (A) Virus-free plasma sample; (B) HIV-1-seropositive plasma sample; (C) HIV-1- and HBV-seropositive plasma sample; (D) HCV-seropositive plasma sample.

FIG. 3

Comparison of HIV-1 RNA viral loads (copies per milliliter from 21 specimens after HIV-1 RT-PCR (x axis) and multiplex RT-PCR (y axis). Linear regression was calculated as follows: log (HIV-1 RT-PCR) = 1.07 log (multiplex RT-PCR) − 0.37; r = 0.956.