Abstract
BackgroundSimian immunodeficiency virus (SIV)-infected rhesus macaques constitute an excellent model of human HIV infection. Sensitive detection of SIV RNA in cell and tissue samples from infected animals subjected to treatment regimens becomes especially critical in determining which therapeutic attempts are successful, and consequently, which interventions should be prioritized in HIV cure research.ResultsIn this report, we describe the design and testing of a Raindance ddPCR platform-based, sensitive SIV reverse transcription droplet digital PCR (RT-ddPCR) assay by exploring the combinations of various priming conditions and reverse transcriptases, and testing one-step vs. two-step procedures, to eliminate background signal(s) and enable detection and quantification of low level target signals.ConclusionsSimilar reaction conditions and assay validation procedures can be explored for potential development of additional assays for other applications that require sensitive detection of low-level targets in RNA samples.
Highlights
Simian immunodeficiency virus (SIV)-infected rhesus macaques constitute an excellent model of human Human immunodeficiency virus (HIV) infection
Similar reaction conditions and assay validation procedures can be explored for potential development of additional assays for applications that require sensitive detection of low quantity target(s) from RNA samples derived from cell or tissue sources
One‐step Reverse transcriptase (RT)‐Droplet digital PCR (ddPCR) In the process of developing a ddPCR assay for quantifying SIV RNA, we first tested the option of one-step reverse transcription ddPCR (RT-ddPCR)
Summary
Simian immunodeficiency virus (SIV)-infected rhesus macaques constitute an excellent model of human HIV infection. Quantitative reverse transcription PCR (qRT-PCR) has established itself to be the benchmark for RNA target detection and quantification. Various qRT-PCR assays have been developed to detect and quantify negative-strand RNA viruses (including the measles and mumps viruses, and various viruses that target the respiratory tract) [6, 7], positive-strand RNA viruses (including rhino-, entero- and coronaviruses such as SARS-CoV-2) [8,9,10], double-stranded RNA viruses (such as human rotaviruses) [11], and retroviruses (HIV, HTLV, and related viruses in animal models, such as simian immunodeficiency virus (SIV)) [12,13,14,15,16]. Several assays have been critical for identifying, isolating and treating patients and defining the viral epidemiology during some of the recent and ongoing pandemics [18, 19]
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