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CRISPR/Cas12a-Based Versatile Method for Checking Quantitative Polymerase Chain Reaction Samples with Cycles of Threshold Values in the Gray Zone

文献类型： 外文期刊

作者： Chen, Yanju 1 ; Wu, Shuyue 2 ; Wu, Hui 1 ; Cheng, Peng 3 ; Wang, Xiaofu 3 ; Qian, Siwenjie 1 ; Zhang, Mengyao 1 ; Xu, Jun 1 ;

作者机构： 1.Zhejiang Univ, Coll Biosyst Engn & Food Sci, Hangzhou 310058, Peoples R China

2.Zhejiang Univ Co Ltd, Architectural Design & Res Inst, Hangzhou 310028, Peoples R China

3.Zhejiang Acad Agr Sci, State Key Lab Managing Biot & Chem Threats Qual &, Hangzhou 310021, Peoples R China

4.Zhejiang Univ, Affiliated Hosp 1, Sch Med, Hangzhou 310003, Zhejiang, Peoples R China

5.Minist Agr, Key Lab Site Proc Equipment Agr Prod, Hangzhou 310058, Peoples R China

关键词： qPCR; Ct values; CRISPR; gray zone; African swine fever virus

期刊名称：ACS SENSORS （ 影响因子：7.711； 五年影响因子：8.162 ）

ISSN： 2379-3694

年卷期： 2021 年 6 卷 5 期

页码：

收录情况： SCI

摘要： Quantitative polymerase chain reaction (qPCR) is widely applied in foodborne pathogen detection and diagnosis. According to the cycles of threshold (Ct) values of qPCR testing, samples are judged as positive or negative. However, samples with Ct values in the gray zone are classified as "possibly positive" and required to be tested again. Repetitive qPCR may not eliminate the uncertain results but increase the workload of detection. CRISPR/Cas12a can specifically recognize the nucleic acid of the nM level and then indiscriminately slash the single-strand DNA with multiple turnovers. In this way, the detection signals can be greatly amplified. Here, we propose a CRISPR-based checking method to solve gray zone problems. After qPCR testing, the screening gray zone samples can be successfully checked by the CRISPR/Cas12a method. Furthermore, to conduct CRISPR reaction assay more conveniently and prevent possible aerosol contamination in the operational process, a gray zone checking cassette is designed. African swine fever virus (ASFV) is selected as an example to demonstrate the feasibility of the CRISPR-based checking method. Of 28 real swine blood samples, 6 ASFV qPCR gray zone samples are successfully checked. The CRISPR-based checking method provides a novel solution to eliminate gray zone sample problems with no additional effects on the PCR, which is operable and applicable in practical detection. The entire process can be completed within 10-15 min. This method will be a good supplementary and assistance for qPCR-based detection, especially in the diagnosis of diseases such as COVID-19.

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