One-tube real-time isothermal amplification assay to identify and distinguish human immunodeficiency virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG

To halt the human immunodeficiency virus type 1 (HIV-1) epidemic requires i nterventions that can prevent transmission of numerous HTV-1 subtypes. The most frequently transmitted viruses belong to the subtypes A, B, and C and the circulating recombinant forms (CRFs) AE and AG. A fast one-tube assay t hat identifies and distinguishes among subtypes A, B, and C and CRFs AE and AG of HIV-1 was developed. The assay amplifies a part of the gag gene sequ ence of the genome of all currently known HIV-1 subtypes and can identify a nd distinguish among the targeted subtypes as the reaction proceeds, becaus e of the addition of subtype-specific molecular beacons with multiple fluor ophores. The combination of isothermal nucleic acid sequence-based amplific ation and molecular beacons is a new approach in the design of real-time as says. To obtain a sufficiently specific assay, we developed a new strategy in the design of molecular beacons, purposely introducing mismatches in the molecular beacons. The subtype A and CRF AG isolates reacted with the same molecular beacon. We tested the specificity and sensitivity of the assay o n a panel of the culture supernatant of 34 viruses encompassing all HIV-1 s ubtypes: subtypes A through G, CRF AE and AG, a group O isolate, and a grou p N isolate, Assay sensitivity on this panel was 92%, with 89% correct subt ype identification relative to sequence analysis. A linear relationship was found between the amount of input RNA in the reaction mixture and the time that the reaction became positive. The lower detection level of the assay was approximately 10(3) copies of HIV-1. RNA per reaction. In 38% of 50 ser um samples from EW-l-infected individuals with a detectable amount of virus , we could identify subtype sequences with a specificity of 94% by using se quencing and phylogenetic analysis as the "gold standard." In conclusion, w e showed the feasibility of the approach of using multiple molecular beacon s labeled with different fluorophores in combination with isothermal amplif ication to identify and distinguish subtypes A, B, and C and CRFs AE and AG of HIV-1. Because of the low sensitivity, the assay in this format would n ot be suited far clinical use but can possibly be used for epidemiological monitoring as well as vaccine research studies.