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Updated: Nov 17, 2025
Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
Published on: March 31, 2021
Development of a reverse transcription droplet digital PCR (RT-ddPCR) assay for sensitive detection of simian
Samuel Long1, Brian Berkemeier2
1AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Frederick, MD, 21702, USA. Samuel.Long@nih.gov.
Background:
Simian 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.
Results:
In 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.
Conclusions:
Similar 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.
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