Journal
EBIOMEDICINE
Volume 61, Issue -, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.ebiom.2020.103036
Keywords
COVID-19; SARS-CoV-2; rRT-PCR; CRISPR diagnosis; Cas12a; SENA
Funding
- National Natural Science Foundation of China [31922046]
- National Key RAMP
- D Program of China [2019YFA0906000]
- NSFC [81772737, 81972830, 31670757]
- Shenzhen Sanming Project of Medicine in Shenzhen [SZSM201412018, SZSM201512037]
- Strategic Priority Research Program of the Chinese Academy of Sciences [XDB19040200]
- Shenzhen High-level Hospital Construction Fund
- Shanghai Rising Stars of Medical Talent Youth Development Program-Clinical Laboratory Practitioners Program
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Background: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. Methods: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a transcleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. Findings: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2 <= 1.6 <= 2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. Interpretation: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. (C) 2020 The Author(s). Published by Elsevier B.V.
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