Multiplexed and Extraction-Free Amplification for Simplified SARS-CoV-2 RT-PCR Tests

Byrnes, Samantha A.; Gallagher, Ryan; Steadman, Amy; Bennett, Crissa; Rivera, Rafael; Ortega, Corrie; Motley, S. Timothy; Jain, Paras; Weigl, Bernhard H.; Connelly, John T.

[Image: see text] The rapid onset of the global COVID-19 pandemic has led to challenges for accurately diagnosing the disease, including supply shortages for sample collection, preservation, and purification. Currently, most diagnostic tests require RNA extraction and detection by RT-PCR; however, extraction is expensive and time-consuming and requires technical expertise. With these challenges in mind, we report extraction-free, multiplexed amplification of SARS-CoV-2 RNA from 246 clinical samples, resulting in 86% sensitivity and 100% specificity. The multiplex RT-PCR uses the CDC singleplex targets and has an LoD of 2 c/μL. We also report on amplification using a range of master mixes in different transport media. This work can help guide which combinations of reagents will enable accurate results when availability of supplies changes throughout the pandemic. Implementing these methods can reduce complexity and cost, minimize reagent usage, expedite time to results, and increase testing capacity.