College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China.
The Architectural Design & Research Institute of Zhejiang University Company Ltd., Hangzhou 310028, China.
ACS Sens. 2021 May 28;6(5):1963-1970. doi: 10.1021/acssensors.1c00515. Epub 2021 May 11.
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.
实时荧光定量聚合酶链反应（qPCR）广泛应用于食源性致病菌的检测和诊断。根据 qPCR 检测的阈值（Ct）值循环，将样本判断为阳性或阴性。然而，Ct 值处于灰色地带的样本被归类为“可能阳性”，需要再次进行检测。重复 qPCR 可能无法消除不确定的结果，反而会增加检测的工作量。CRISPR/Cas12a 可以特异性识别纳摩尔级别的核酸，然后进行多次循环的无差别切割单链 DNA。这样可以大大放大检测信号。在这里，我们提出了一种基于 CRISPR 的检测方法来解决灰色地带问题。在 qPCR 检测之后，可以通过 CRISPR/Cas12a 方法成功检查筛选出的灰色地带样本。此外，为了更方便地进行 CRISPR 反应检测，并防止操作过程中可能的气溶胶污染，设计了一个灰色地带检测盒。我们选择非洲猪瘟病毒（ASFV）作为示例来验证基于 CRISPR 的检测方法的可行性。在 28 份实际的猪血液样本中，有 6 份 ASFV qPCR 灰色地带样本被成功检查。基于 CRISPR 的检测方法为消除 PCR 无额外影响的灰色地带样本问题提供了一种新的解决方案，在实际检测中具有可操作性和适用性。整个过程可以在 10-15 分钟内完成。该方法将成为 qPCR 检测的良好补充和辅助手段，特别是在 COVID-19 等疾病的诊断中。
