Characterization of real-time microarrays for simultaneous detection of HIV-1, HIV-2, and hepatitis viruses

Real-time PCR assays for nucleic acid testing (NAT) of hepatitis viruses A-E and for HIV-1 and HIV-2 have been developed; however, a multiplex assay that can simultaneously detect all of these agents is not yet available. Standardized TaqMan assays for detection of hepatitis viruses A-E have been described and applied to TaqMan Array Cards (TAC) which are capable of multiple pathogen detection using a single set of optimized PCR conditions. Assays for three gene regions of HIV-1 (long-terminal repeat (LTR), gag, and polymerase) and HIV-2 (overlap of LTR and gag, protease and integrase) were designed using the hepatitis assay conditions. Nucleic acid extracts of HIV-1-infected samples (44 plasma, 41 whole blood, 20 HIV-1 viral stocks) were tested on the TAC cards; 98 were reactive (92%) with 70 in multiple gene regions. Twenty-four of the 27 (89%) HIV-2 specimens (10 plasma, 1 PBMC lysate, 6 whole blood and 10 plasmids containing HIV-2 polymerase) were detected on TAC. No HIV or hepatitis virus sequences were detected in 30 HIV-negative samples (specificity 100%). Three HBV and 18 HCV co-infections were identified in the HIV-1-infected specimens. Multi-pathogen detection using TAC could provide a rapid, sensitive and more efficient method of surveying for a variety of infectious disease nucleic acids.