鼠伤寒沙门氏菌CRISPR/Cas12a检测方法的建立与应用

Establishment and application of CRISPR/Cas12a assay for the detection of Salmonella typhimurium

中文关键词:  CRISPR/Cas12a  重组酶聚合酶扩增  鼠伤寒沙门氏菌  快速检测

英文关键词:CRISPR/Cas12a  recombinase polymerase amplification  Salmonella typhimurium  rapid detection

中文摘要:

      目的 建立快速、超灵敏检测食品中鼠伤寒沙门氏菌的方法。方法 根据鼠伤寒沙门氏菌fliC基因保守片段设计并合成特异性CRISPR RNA (crRNA), 并根据crRNA所在的基因序列区域设计重组酶聚合酶扩增(recombinase polymerase amplification, RPA)特异性引物, 利用RPA技术扩增样本核酸, 并对扩增产物进行CRISPR/Cas12a荧光检测。结果 该检测方法克服了传统鼠伤寒沙门氏菌检测方法的缺陷, 耗时短且具有较高的灵敏度, 对鼠伤寒沙门氏菌的检出限低至1 copy/μL, 与乙型副伤寒沙门氏菌、金黄色葡萄球菌、单增李斯特菌、阪崎肠杆菌、大肠埃希氏菌等常见食源性致病菌核酸检测无交叉反应, 且建立的方法在检测人工污染的食品样品时具有较高的灵敏度和准确性。结论 本研究建立的检测方法耗时短、灵敏度高, 可用于鼠伤寒沙门氏菌的快速检测。

英文摘要:

      Objective To establish a rapid and ultrasensitive method for the detection of Salmonella typhimurium in food. Methods Specific CRISPR RNA (crRNA) was designed and synthesized based on the conserved fragment of fliC gene of Salmonella typhimurium, and recombinase polymerase amplification (RPA) specific primers was designed according to the gene sequence region where the crRNA was located. RPA specific primers were used to amplify the sample nucleic acid, and CRISPR/Cas12a fluorescence detection was performed on the amplified products. Results The method overcame the defects of traditional Salmonella typhimurium detection methods, with short time consumption and high sensitivity, the limit of detection ofSalmonella typhimurium was as low as 1 copy/μL, and there was no cross-reactivity with the nucleic acid detection of Salmonella paratyphi B, Staphylococcus aureus, Listeria monocytogenes, Enterobacter sakazakii, and Escherichia coli, and other common food-borne pathogens, moreover, the established method had high sensitivity and accuracy in detecting the artificially contaminated food samples. Conclusion The established method is quick and has high sensitivity, and can be used for rapid detection of Salmonella typhimurium.