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CRISPR/Cas12a-based SARS-CoV-2 Rapid Visual Detection and Clinical Application
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  • Jingsong Xu,
  • Xi Wang,
  • Xiaowei Ma,
  • Zhaoyang Sun,
  • Qian Liu,
  • Min Li,
  • Hua Wang
Jingsong Xu
Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Laboratory Medicine

Corresponding Author:[email protected]

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Xi Wang
Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Laboratory Medicine
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Xiaowei Ma
Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Laboratory Medicine
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Zhaoyang Sun
Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Laboratory Medicine
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Qian Liu
Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Laboratory Medicine
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Min Li
Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Laboratory Medicine
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Hua Wang
Shanghai Jiao Tong University School of Medicine Affiliated Renji Hospital Department of Laboratory Medicine
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Abstract

Background: Currently, rapid and widespread testing is essential to promote early intervention and curb the ongoing COVID-19 pandemic. The reverse transcription-polymerase chain reaction (RT-PCR) for detecting novel coronavirus (SARS-CoV-2) is restricted to professional laboratories and well-trained personnel, thus, limiting its widespread use in in resource-limited conditions. Methods: To overcome these challenges, we developed a rapid and convenient assay using recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology for the rapid detection SARS-CoV-2, which was named as Visual Detection of RPA-amplified Products (VDAP). The reaction conditions of the VDAP were optimized and selected using pure SARS-CoV-2 RNA standards and the sensitivity and specificity of the VDAP were further determined. Finally, the VDAP was verified on clinical specimens. Results: The VDAP was performed at 37 °C for 15 min, and the result was visible by the naked eye. The limits of detection (LODs) of the VDAP for the target ORF1ab and N genes are 70 and 500 copies, respectively. No cross-reactivity was observed with the RNA standard samples of four respiratory viruses or clinical samples of common respiratory viral infections. These results confirmed that the assay was highly specific. Thirty SARS-CoV-2 positive and 30 SARS-CoV-2 negative patient specimens were analyzed. We compared these results to RT-PCR, the overall sensitivity and specificity of the VDAP compared to RT-PCR for detection SARS-CoV-2 were 93.3% and 100.0%, respectively. Conclusions: The VDAP is a simple, highly specific, and convenient assay for the detection of SARS-CoV-2 in resource-limited conditions