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.
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.
Defoort
J P
JP
Immunoanalysis Department, Immunotech, a Beckman-Coulter Company, 13276 Marseille Cedex 9, France. defoort@immunotech.fr
Martin
M
M
Casano
B
B
Prato
S
S
Camilla
C
C
Fert
V
V
D000163:Acquired Immunodeficiency Syndrome / Q000097:blood / Q000150:complications / Q000175:diagnosis*; D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral / Q000097:blood*; D005240:Feasibility Studies; D005434:Flow Cytometry / Q000379:methods; D015750:Genes, gag; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification*; D006509:Hepatitis B / Q000097:blood / Q000150:complications / Q000175:diagnosis*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification*; D006526:Hepatitis C / Q000097:blood / Q000150:complications / Q000175:diagnosis*; D006801:Humans; D008863:Microspheres; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral; D012367:RNA, Viral