ABSTRACT
The ongoing COVID-19 pandemic has caused an unprecedented need for rapid diagnostic testing. The Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) recommend a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. We hypothesized that SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether, and tested this hypothesis on a series of blinded clinical samples. The direct RT-qPCR approach correctly identified 92% of NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Thus, direct RT-qPCR could be a front-line approach to identify the substantial majority of COVID-19 patients, reserving a repeat test with RNA extraction for those individuals with high suspicion of infection but an initial negative result. This strategy would drastically ease supply chokepoints of COVID-19 testing and should be applicable throughout the world.
Footnotes
The original version of this manuscript reported that addition of nasopharyngeal (NP) swab diluent from COVID-19 positive donors directly into a RT-qPCR reaction (ie. skipping the RNA extraction step) was a streamlined approach that could identify SARS-CoV-2 RNA in NP swab samples. Our initial finding was based on a single patient NP sample (which is shown in Figure 1a of this revision). The remainder of the data in this revision is new. Specifically, we have now optimized conditions for this direct RT-PCR test (new Table 1), and replicated our result using 154 additional clinical samples (new Figures 1b,c,d and 2, new Table 2, and new Supplementary Table 1). Furthermore, we have included COVID-19 negative samples, which enables control for the presence of RNA in the swab specimens to rule out missing a positive due to an absence of intact RNA in the specimen (Figure 1d). Importantly, new collaborators (The Jerome laboratory at UW Seattle) joined the UVM team to generate this new data and are included as coauthors. Note that this revision removes Figures 2 and 3 of the original version (dealing with the use of alternative RNA extraction kits).