Development of a sensitive, quantitative assay with broad subtype specificity for detection of total HIV-1 nucleic acids in plasma and PBMC

Development of a sensitive, quantitative assay with broad subtype specificity for detection of total HIV-1 nucleic acids in plasma and PBMC

Blog Article

Abstract An LTR-based quantitative PCR (qPCR) assay was modified and optimized for the quantification of total HIV-1 nucleic acids in plasma and PBMC.TaqMan qPCR primers and probes were designed against the NCBI/LANL HIV-1 compendium database by analyzing sequences used in Twin Platform Bed assays for sensitive cross-clade detection of HIV-1 as reported in the literature and elucidating regions of improved cross-subtype specificity.Inosine and mixed nucleotide bases were included at polymorphic sites.Real-time RT-qPCR and qPCR were performed on plasma viral RNA and cellular lysates.

A step-up amplification approach to allow binding of primers across polymorphic regions showed improved sensitivity compared to universal cycling.Unlike a lead competing laboratory-developed assay, all major HIV-1 subtypes, and a wide range of recombinants from a 127-member diversity panel were detected and accurately quantified in spiked plasmas.Semi-nested PCR increased detection sensitivity even further.The assay was able to detect down to 88 copies/mL of HIV-1 in plasma with 95% efficiency or the equivalent of a single infected cell.

The PCR assay will be valuable in studies that monitor very low viral levels including residual or break through HIV-1 in patients Lanyard receiving antiretroviral therapy, in HIV-1 cure, and in other research studies.