Unknown

Dataset Information

0

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


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 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.

SUBMITTER: Kibirige CN 

PROVIDER: S-EPMC8799642 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

altmetric image

Publications

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

Kibirige C N CN   Manak M M   King D D   Abel B B   Hack H H   Wooding D D   Liu Y Y   Fernandez N N   Dalel J J   Kaye Steve S   Imami N N   Jagodzinski L L   Gilmour J J  

Scientific reports 20220128 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  ...[more]

Similar Datasets

| S-EPMC3535555 | biostudies-literature
| S-EPMC3502729 | biostudies-literature
| S-EPMC9937448 | biostudies-literature
| S-EPMC7181152 | biostudies-literature
| S-EPMC2853127 | biostudies-literature
| S-EPMC3868369 | biostudies-literature
| S-EPMC5899152 | biostudies-literature
| S-EPMC3565980 | biostudies-literature
| S-EPMC5986701 | biostudies-literature
| S-EPMC3167487 | biostudies-literature