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Rapid Detection of 2019 Novel Coronavirus SARS-CoV-2 Using a CRISPR-based 1 DETECTR Lateral Flow Assay 2 3
Abstract
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virus (ZIKV), and most recently SARS-CoV-2 1,2 . All of these epidemics presumably 46 resulted from an initial zoonotic animal-to-human transmission event, with either 47 clinically apparent or occult spread into vulnerable human populations. Each time, a 48 lack of rapid, accessible, and accurate molecular diagnostic testing has hindered the 49 public health response to the emerging viral threat. 50
In early January 2020, a cluster of cases of pneumonia from a novel coronavirus, 51 SARS-CoV-2 (with the disease referred to as COVID-19), was reported in Wuhan, 52
China 1,2 . This outbreak has spread rapidly, with over 90,000 reported cases and 3,000 53 deaths as of March 4th, 2020 3 . Person-to-person transmission from infected individuals 54 with no or mild symptoms has been reported 4,5 . Assays using quantitative reverse 55 transcription-polymerase chain reaction (qRT-PCR) approaches for detection of the 56 virus in 4-6 hours have been developed by several laboratories, including an 57
Emergency Use Authorization (EUA)-approved assay developed by the US CDC 6 . 58
However, the typical turnaround time for screening and diagnosing patients with 59 suspected SARS-CoV-2 has been >24 hours given the need to ship samples overnight 60 to reference laboratories. To accelerate clinical diagnostic testing for COVID-19 in the 61 United States, the FDA on February 28th, 2020 permitted individual clinically licensed 62 laboratories to report the results of in-house developed SARS-CoV-2 diagnostic assays 63 while awaiting results of an EUA submission for approval 7 . 64
Here we report the development and initial validation of a CRISPR (clustered 65 regularly interspaced short palindromic repeats)-Cas12 based assay 8-11 for detection of 66 SARS-CoV-2 from extracted patient sample RNA in ~30 min, called SARS-CoV-2 67 DETECTR. This assay performs simultaneous reverse transcription and isothermal 68 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.06.20032334 doi: medRxiv preprint target region for the CDC assay is no longer being used due to concerns regarding 92 flaws in manufacturing reagents and potential decreased sensitivity) 14 . 93
We next compared the analytic limits of detection (LoD) of the RT-LAMP/Cas12 94 DETECTR assay relative to the US FDA Emergency Use Authorization (EUA)-approved 95 CDC assay for detection of SARS-CoV-2 (Table 1; Fig. 2d) . A standard curve for 96 quantitation was constructed using 7 dilutions of a control IVT viral nucleoprotein RNA 97 ("CDC VTC nCoV Transcript") 6 , with 3 replicates at each dilution (Fig. 2d, left; 98 Extended Data 1). Ten two-fold serial dilutions of the same control nucleoprotein RNA 99
were then used to run the DETECTR assay, with 6 replicates at each dilution (Fig. 2d, 100 right; Supplementary Fig. 3) . The estimated LoD for the CDC assay tested by 101
California Department of Public Health was 1 copy/µL reaction, consistent with the 102 analytic performance in the FDA package insert, versus 10 copies/µL reaction for the 103 DETECTR assay. 104
We then assessed the capability of the RT-LAMP assay to amplify SARS-CoV-2 105 nucleic acid directly from raw sample matrix consisting of nasopharyngeal swabs from 106 asymptomatic donors placed in universal transport medium (UTM) or phosphate 107 buffered saline (PBS) and spiked with SARS-CoV-2 IVT target RNA. Assay 108 performance was degraded at reaction concentration of ≥ 10% UTM and ≥ 20% PBS by 109 volume, with estimated limits of detection decreasing to 500 and 1,500 copies/µL, 110 respectively (Supplementary Fig. 4) . 111
Finally, we tested extracted RNA from 11 respiratory swab samples collected 112 from 6 PCR-positive COVID-19 patients (COVID19-1A/B to COVID19-5A/B, where 113
A=nasopharyngeal swab and B=oropharyngeal swab and COVID19-6, a single 114 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.06.20032334 doi: medRxiv preprint nasopharyngeal swab) and 12 nasopharyngeal swab samples from patients with 115 influenza (n=4), common human seasonal coronavirus infections (n=3, representing 116 OC43, HKU1, NL63), and healthy donors (n=5) (Fig. 2e, f; Supplementary Fig. 5) . 117
Relative to the CDC qRT-PCR, SARS-CoV-2 DETECTR was 90% sensitive and 100% 118 specific for detection of the coronavirus in respiratory swab samples, corresponding to 119 positive and negative predictive values of 100% and 91.7%, respectively (Fig. 2g) . 120
Here we combined isothermal amplification with CRISPR-Cas12 DETECTR 121 technology to develop a rapid (~30 min) and low-cost test for detection of SARS-CoV-2 122
in clinical samples. The use of existing qRT-PCR based assays is hindered by the need 123 for expensive lab instrumentation, and availability is currently restricted to public health 124 laboratories. Importantly, the DETECTR assays developed here have comparable 125 accuracy to qRT-PCR and are broadly accessible, as they use routine protocols and 126 commercially available, "off-the-shelf" reagents. Key advantages of our approach over 127 existing methods such as qRT-PCR include (1) isothermal signal amplification for rapid 128 target detection obviating the need for thermocycling, (2) single nucleotide target 129 specificity (guide RNAs at the N2 site can distinguish SARS-CoV-2 from SARS-CoV 130 and MERS-CoV), (3) integration with portable, low-cost reporting formats such as lateral 131 flow strips, and (4) quick development cycle to address emerging threats from novel 132 zoonotic viruses (<2 weeks for SARS-CoV-2, Supplementary Fig. 6) . 133
Although most of the cases of COVID-19 infection during the first month of the 134 epidemic were traced to the city of Wuhan and Hubei province in China, the ongoing 135 rise in cases now appears to be driven to local community transmission 15,16 . For a 136 number of reasons, there is an urgent public health need for rapid diagnostic tests for 137 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.06.20032334 doi: medRxiv preprint
162 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.06.20032334 doi: medRxiv preprint CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.06.20032334 doi: medRxiv preprint 181 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.06.20032334 doi: medRxiv preprint interest. First a PCR step was performed on the synthetic gene fragment with a forward 232 primer that contained a T7 promoter. Next, the PCR product was used as the template 233
for an in-vitro transcription (IVT) reaction at 37°C for 2 hours. The IVT reaction was then 234 treated with TURBO DNase (Thermo) for 30 min at 37°C, followed by a heat-235 denaturation step at 75°C for 15 min. RNA was purified using RNA Clean and 236
and diluted in nuclease-free water to working concentrations. 238 239 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity. CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.06.20032334 doi: medRxiv preprint Extended Data 1 ("Extended_Data_1.xlsx"). Standard curve generated by running 361 seven 5-or 10-fold dilutions of the CDC N2 qRT-PCR assay, with 3 replicates each 362 dilution. The R-squared measure corresponding to the regression line is 0.9981. 363 364 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.06.20032334 doi: medRxiv preprint Extended Data 2 ("Extended_Data_2.xlsx"). Primer, reporter molecules, target gene 365 fragments, and guide RNAs used in this study. 366 367 368 369 . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.06.20032334 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
is the (which was not peer-reviewed) The copyright holder for this preprint . https://doi.org/10.1101/2020.03.06.20032334 doi: medRxiv preprint
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