title: A reverse transcription loop-mediated isothermal amplification assay optimized to detect multiple HIV subtypes.
abstract: Diagnostic methods for detecting and quantifying HIV RNA have been improving, but efficient methods for point-of-care analysis are still needed, particularly for applications in resource-limited settings. Detection based on reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is particularly useful for this, because when combined with fluorescence-based DNA detection, RT-LAMP can be implemented with minimal equipment and expense. Assays have been developed to detect HIV RNA with RT-LAMP, but existing methods detect only a limited subset of HIV subtypes. Here we report a bioinformatic study to develop optimized primers, followed by empirical testing of 44 new primer designs. One primer set (ACeIN-26), targeting the HIV integrase coding region, consistently detected subtypes A, B, C, D, and G. The assay was sensitive to at least 5000 copies per reaction for subtypes A, B, C, D, and G, with Z-factors of above 0.69 (detection of the minor subtype F was found to be unreliable). There are already rapid and efficient assays available for detecting HIV infection in a binary yes/no format, but the rapid RT-LAMP assay described here has additional uses, including 1) tracking response to medication by comparing longitudinal values for a subject, 2) detecting of infection in neonates unimpeded by the presence of maternal antibody, and 3) detecting infection prior to seroconversion.
authors: Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors: Karen E
authors: KE
authors: Ocwieja
authors: Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors: Scott
authors: S
authors: Sherrill-Mix
authors: Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors: Changchun
authors: C
authors: Liu
authors: Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors: Jinzhao
authors: J
authors: Song
authors: Department of Mechanical Engineering and Applied Mechanics, School of Engineering and Applied Science, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors: Haim
authors: H
authors: Bau
authors: Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors: Frederic D
authors: FD
authors: Bushman
mesh_terms: D019295:Computational Biology / Q000379:methods; D005814:Genes, Viral; D005838:Genotype; D006678:HIV / Q000145:classification* / Q000235:genetics*; D015658:HIV Infections / Q000821:virology*; D015497:HIV-1 / Q000145:classification / Q000235:genetics; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results