title: Cross-subtype detection of HIV-1 using reverse transcription and recombinase polymerase amplification.
abstract: A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10-30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7%) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV.
authors.affiliation: PATH, 2201 Westlake Ave Suite 200, Seattle, WA 98121, USA.
authors.forename: Lorraine
authors.initials: L
authors.lastname: Lillis
authors.affiliation: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.
authors.forename: Dara A
authors.initials: DA
authors.lastname: Lehman
authors.affiliation: PATH, 2201 Westlake Ave Suite 200, Seattle, WA 98121, USA.
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Siverson
authors.affiliation: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.
authors.forename: Julie
authors.initials: J
authors.lastname: Weis
authors.affiliation: PATH, 2201 Westlake Ave Suite 200, Seattle, WA 98121, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Cantera
authors.affiliation: TwistDx Limited, Minerva Building, Babraham Research Campus, Babraham, Cambridge CB22, UK.
authors.forename: Mathew
authors.initials: M
authors.lastname: Parker
authors.affiliation: TwistDx Limited, Minerva Building, Babraham Research Campus, Babraham, Cambridge CB22, UK.
authors.forename: Olaf
authors.initials: O
authors.lastname: Piepenburg
authors.affiliation: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.
authors.forename: Julie
authors.initials: J
authors.lastname: Overbaugh
authors.affiliation: PATH, 2201 Westlake Ave Suite 200, Seattle, WA 98121, USA. Electronic address: dboyle@path.org.
authors.forename: David S
authors.initials: DS
authors.lastname: Boyle
mesh_terms: D015658:HIV Infections / Q000175:diagnosis* / Q000821:virology*; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D025202:Molecular Diagnostic Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D045522:Recombinases / Q000032:analysis / Q000235:genetics; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics
chemical_list: D045522:Recombinases; D014764:Viral Proteins
keywords: Diagnostic; Human immunodeficiency virus; Infant HIV; Point of care; Reverse transcription recombinase polymerase amplification
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