EBioMedicine · 2020
A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection.
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Huang Weiren, Yu Lei, Wen Donghua, Wei Dong, Sun Yangyang, Zhao Huailong, Ye Yu, Chen Wei, Zhu Yongqiang, Wang Lijun, Wang Li, Wu Wenjuan, Zhao Qianqian, Xu Yong, Gu Dayong, Nie Guohui, Zhu Dongyi, Guo Zhongliang, Ma Xiaoling, Niu Liman, Huang Yikun, Liu Yuchen, Peng Bo, Zhang Renli, Zhang Xiuming, Li Dechang, Liu Yang, Yang Guoliang, Liu Lanzheng, Zhou Yunying, Wang Yunshan, Hou Tieying, Gao Qiuping, Li Wujiao, Chen Shuo, Hu Xuejiao, Han Mei, Zheng Huajun, Weng Jianping, Cai Zhiming, Zhang Xinxin, Song Fei, Zhao Guoping, Wang Jin
Аннотация
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 trans-cleavage 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.
FUNDING: Detailed funding information is available at the end of the manuscript.
METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage 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.
FUNDING: Detailed funding information is available at the end of the manuscript.
Предметные рубрики MeSH
Bacterial ProteinsBetacoronavirusCOVID-19CRISPR-Associated ProteinsClustered Regularly Interspaced Short Palindromic RepeatsCoronavirus InfectionsCoronavirus Nucleocapsid ProteinsEndodeoxyribonucleasesHumansLimit of DetectionNasal CavityNucleic Acid Amplification TechniquesNucleocapsid ProteinsPandemicsPhosphoproteinsPneumonia, ViralPolyproteinsRNA, ViralReal-Time Polymerase Chain ReactionReference StandardsSARS-CoV-2Viral Proteins