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

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.