Lv Xinrui, Cao Weiwei, Zhang Huang, Zhang Yilin, Shi Lei, Ye Lei
Institute of Food Safety and Nutrition, Jinan University, Guangzhou 510632, China.
College of Food and Bioengineering, Guangdong Polytechnic of Science and Trade, Guangzhou 510640, China.
Foods. 2022 Jun 8;11(12):1681. doi: 10.3390/foods11121681.
is one of the major pathogenic species that contaminate seafood. Rapid and accurate detection is crucial for avoiding foodborne diseases caused by pathogens and is important for food safety management and mariculture. In this study, we established a system that combines chemically enhanced clustered regularly interspaced short palindromic repeats (CRISPR) and recombinase-aided amplification (RAA) (CE-RAA-CRISPR) for detecting in seafood. The method combines RAA with CRISPR-associated protein 12a (Cas12a) for rapid detection in a one-pot reaction, effectively reducing the risk of aerosol contamination during DNA amplifier transfer. We optimized the primers for , determined the optimal crRNA/Cas12a ratio, and demonstrated that chemical additives (bovine serum albumin and L-proline) could enhance the detection capacity of Cas12a. The limit of detection (at optimal conditions) was as low as 6.7 × 10 CFU/mL in pure cultures and 7.3 × 10 CFU/g in shrimp. Moreover, this method exhibited no cross-reactivity with other microbial pathogens. The CE-RAA-CRISPR assay was compared with the quantitative polymerase chain reaction assay using actual food samples, and it showed 100% diagnostic agreement.
是污染海产品的主要致病物种之一。快速准确的检测对于避免由病原体引起的食源性疾病至关重要，对食品安全管理和海水养殖也很重要。在本研究中，我们建立了一种将化学增强型成簇规律间隔短回文重复序列（CRISPR）与重组酶辅助扩增（RAA）相结合的系统（CE-RAA-CRISPR）用于检测海产品中的 。该方法将RAA与CRISPR相关蛋白12a（Cas12a）结合，在一锅反应中进行快速检测，有效降低了DNA扩增仪转移过程中气溶胶污染的风险。我们优化了针对 的引物，确定了最佳的crRNA/Cas12a比例，并证明化学添加剂（牛血清白蛋白和L-脯氨酸）可增强Cas12a的检测能力。在纯培养物中，（最佳条件下的）检测限低至6.7×10 CFU/mL，在虾中为7.3×10 CFU/g。此外，该方法与其他微生物病原体无交叉反应。使用实际食品样本将CE-RAA-CRISPR检测法与定量聚合酶链反应检测法进行比较，结果显示诊断一致性为100%。
