基于重组酶聚合酶扩增-CRISPR/Cas12a的沙门氏菌可视化检测方法的建立

Establishment of visual detection method for Salmonella based on recombinase polymerase amplification-CRISPR/Cas12a

中文关键词:  沙门氏菌  成簇规律间隔短回文重复序列及其相关蛋白12a  试纸条  重组酶聚合酶扩增  可视化检测

英文关键词:Salmonella  clustered regularly interspaced short palindromic repeats/associated protein 12a  test strip  recombinase polymerase amplification  visual detection

中文摘要:

      目的 建立基于重组酶聚合酶扩增-CRISPR/Cas12a的沙门氏菌可视化检测方法。方法 本研究将重组酶聚合酶扩增(recombinase polymerase amplification, RPA)技术与成簇规律间隔短回文重复序列及其相关蛋白12a (clustered regularly interspaced short palindromic repeats/associated protein 12a, CRISPR/Cas12a)系统相结合, 以沙门氏菌invA作为目标基因, 设计并合成RPA引物和CRISPR引导RNA (CRISPR-derived RNA, crRNA), 通过荧光法和试纸条两种方法读取结果。对优化的RPA-CRISPR/Cas12检测体系进行特异性和灵敏度实验, 并应用于食品样本检测。结果 本研究成功研制纳米金核酸试纸条, 建立的RPA-CRISPR/Cas12a体系可在1.5 h内特异性完成沙门氏菌的检测, 灵敏度为80 CFU/mL。利用此方法对21个食品样本进行检测, 荧光法、试纸条法与国家标准法检测结果完全一致, 沙门氏菌检出率为4.8%。结论 本研究建立的沙门氏菌RPA-CRISPR/Cas12a可视化检测方法具有高特异性和灵敏度, 在沙门氏菌的现场快速检测方面具有应用价值。

英文摘要:

      Objective To establish a visual detection method of Salmonella based on recombinant enzyme polymerase amplification-CRISPR/Cas12a. Methods In this study, recombinase polymerase amplification (PRA) was combined with clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) system. RPA primers and CRISPR-derived RNA (crRNA) were designed and synthesized based on the target invA gene of Salmonella. Fluorescence and test strip methods were employed to read the results. The optimized RPA-CRISPR/Cas12 detection system were evaluated for specificity and sensitivity, and was applied to the detection of food samples. Results Nano gold test strip base on nucleic acid were developed. The established RPA-CRISPR/Cas12 detection method could specifically detecting Salmonella with a sensitivity of 80 CFU/mL within 1.5 h. Used this method to detect 21 kinds of food samples, the results from fluorescence and test strip read were consistent, with a Salmonella detection rate of 4.8%, which was completely consistent with the results of the national standard method. Conclusion The Salmonella RPA-CRISPR/Cas12a visual detection method established in this study has high specificity and sensitivity and is valuable for on-site rapid detection of Salmonella.