Development of a Sensitive, Quantitative Assay with Broad Subtype Specificity for Detection of Total HIV-1 Nucleic Acids in Plasma and PBMC

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Title: Development of a Sensitive, Quantitative Assay with Broad Subtype Specificity for Detection of Total HIV-1 Nucleic Acids in Plasma and PBMC
Authors: Kibirige, C
Manak, M
King, D
Abel, B
Hack, H
Wooding, D
Liu, Y
Fernandez, N
Dalel, J
Steve, K
Imami, N
Jagodzinski, L
Gilmour, J
Item Type: Journal 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 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.
Date of Acceptance: 28-Oct-2021
DOI: 10.1038/s41598-021-03016-1
ISSN: 2045-2322
Publisher: Nature Publishing Group
Journal / Book Title: Scientific Reports
Volume: 12
Copyright Statement: © The Author(s) 2022, corrected publication 2022. Tis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
Sponsor/Funder: St Stephen's Aids Trust
Funder's Grant Number: N/A
Keywords: Science & Technology
Multidisciplinary Sciences
Science & Technology - Other Topics
Publication Status: Published
Appears in Collections:Department of Infectious Diseases
Faculty of Medicine

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