25Nearly 400,000 people worldwide are known to have been infected with SARS-CoV-2 beginning 26 in December 2019. The virus has now spread to over 168 countries including the United States, where 27 the first cluster of cases was observed in the Seattle metropolitan area in Washington. Given the rapid 28 increase in the number of cases in many localities, the availability of accurate, high-throughput SARS-29CoV-2 testing is vital to efforts to manage the current public health crisis. In the course of optimizing 30 SARS-CoV-2 testing performed by the University of Washington Clinical Virology Lab (UW Virology Lab), 31 we evaluated assays using seven different primer/probe sets and one assay kit. We found that the most 32 sensitive assays were those that used the E-gene primer/probe set described by Corman et al. 33 (Eurosurveillance 25(3), 2020, https://doi.

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. MAINThe ongoing COVID-19 pandemic has put exceptional strain on public health laboratories, hospital laboratories, and commercial laboratories as they attempt to keep up with demands for SARS-CoV-2 testing. The current diagnostic testing methods recommended by the Centers for Disease Control and Prevention (CDC) in the United States and the World Health Organization (WHO) are traditional RT-qPCR assays that require two steps: first, an RNA extraction from patient nasopharyngeal (NP) swab material, followed by RT-qPCR amplification of the extracted RNA to detect viral RNA 1-3 . The major bottleneck to widespread SARS-CoV-2 testing lies at the RNA extraction step. The simplest manual kit (the Qiagen Viral RNA Mini) is no longer available, and reagents and supplies for the larger automated instruments are extremely limited with uncertain supply chains. While substitution of other RNA extraction kits 4,5 is possible, they too are in limited supply. The current bottleneck is not simply the availability of RNA extraction kits, but also the cost of the extraction assay, the labor and time required to perform it, and the fact that it is rate limiting compared to the downstream RT-qPCR analysis. To address this issue, we tested the unconventional approach of skipping the RNA extraction step altogether and directly loading patient swab material into the RT-qPCR mix. Herein, we report that this approach (which we refer to hereafter as "direct RT-qPCR") correctly identified 92% of samples (n =155) previously shown to be positive for SARS-CoV-2 RNA by conventional RT-qPCR featuring an RNA extraction. Thus, our results suggest that this streamlined assay could greatly alleviate constraints to COVID-19 testing in many regions of the world.

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends 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. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples ( n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.

Multiple rapid antigen (Ag) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have recently received emergency-use authorization (EUA) from the U.S. Food and Drug Administration (FDA). Although less sensitive than molecular detection methods, rapid antigen testing offers the potential for inexpensive, quick, decentralized testing. Robust analytical sensitivity data in comparison to reverse transcription-quantitative PCR (qRT-PCR) are currently lacking for many rapid antigen tests. Here, we evaluated the analytical sensitivity of the Abbott BinaxNOW COVID-19 Ag card using SARS-CoV-2-positive clinical specimens quantified by reverse transcription-droplet digital PCR (RT-ddPCR) and multiple FDA EUA qRT-PCR platforms using RNA standards. Initial and confirmatory limits of detection for the BinaxNOW COVID-19 Ag card were determined to be equivalent to 4.04 × 104 to 8.06 × 104 copies/swab. We further confirmed this limit of detection with 72 additional clinical samples positive for SARS-CoV-2 in either phosphate-buffered saline or viral transport medium. One hundred percent of samples with viral loads of >40,000 copies/swab were detected by rapid antigen testing. These data indicate that the BinaxNOW COVID-19 Ag card has an analytical sensitivity approximately equivalent to a generic qRT-PCR cycle threshold (CT) value of 29 to 30.

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