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

• Published in: Scientific reports 2022-01, Vol.12 (1), p.1550-16, Article 1550
• Main Authors: Kibirige, C. N., Manak, M., King, D., Abel, B., Hack, H., Wooding, D., Liu, Y., Fernandez, N., Dalel, J., Kaye, Steve, Imami, N., Jagodzinski, L., Gilmour, J.
• Format: Article
• Language: English
• Subjects: 631/1647; 631/337; 692/308; 692/4017; 692/420; 692/53; 692/699; 692/700; Antiretroviral agents; Antiretroviral therapy; DNA probes; HIV; HIV Infections - blood; HIV Infections - diagnosis; HIV Infections - virology; HIV Long Terminal Repeat - genetics; HIV-1 - genetics; HIV-1 - isolation & purification; Human immunodeficiency virus; Humanities and Social Sciences; Humans; Leukocytes, Mononuclear - virology; Lysates; multidisciplinary; Nucleic acids; Peripheral blood mononuclear cells; Plasma; Real-Time Polymerase Chain Reaction - methods; Recombinants; RNA, Viral - blood; RNA, Viral - genetics; Science; Science (multidisciplinary); Sensitivity and Specificity
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-021-03016-1

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 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 receiving antiretroviral therapy, in HIV-1 cure, and in other research studies.