配方乳粉中6种致病菌双重酶促等温扩增快速检测方法的建立

A dual enzymatic recombinase amplification method for rapid detection of six foodborne pathogens in formula milk powder

摘要:

目的 建立一种双重快速检测方法，旨在快速筛查配方乳粉中克罗诺杆菌、大肠埃希氏菌O157:H7、蜡样芽孢杆菌、金黄色葡萄球菌、沙门氏菌和单核增生李斯特氏菌等6种常见食源性致病菌。方法 酶促等温扩增(enzymatic recombinase amplification, ERA)技术是一种新型等温扩增技术，能在25-42 ℃的条件下，仅需10-30 min完成对微量DNA或RNA的指数级扩增。基于该技术，本研究设计了针对克罗诺杆菌的ERA检测引物探针，同时筛选适用于ERA检测体系的大肠埃希氏菌O157:H7、蜡样芽孢杆菌、金黄色葡萄球菌、沙门氏菌和单核增生李斯特氏菌的引物探针，确定了每种单一菌种的ERA检测引物探针。通过引物探针的两两组合分析及方法优化，建立双重ERA检测方法。通过人工模拟污染试验和实际样品检测，确定双重ERA检测方法的检出限和准确性。结果 建立了3组双重ERA法，能够快速检测6种食源性致病菌，检测时间仅需16 min 10 s。灵敏度检测结果显示，克罗诺杆菌和大肠埃希氏菌O157:H7、蜡样芽孢杆菌和金黄色葡萄球菌2种组合的DNA检测灵敏度均为1 ng/μL，而沙门氏菌和单核增生李斯特氏菌的DNA检测灵敏度为10^-1 ng/μL。人工模拟污染试验显示，该方法能检出的最低菌浓度为1 CFU/mL。在37份临期市售配方乳粉中，蜡样芽孢杆菌和单核增生李斯特氏菌的检出率分别为37.84%和21.62%。与行业标准实时荧光PCR法的检测结果比对，本研究建立的双重ERA检测方法结果一致，证实了其准确性。结论 本研究建立的双重ERA方法具有较高的特异性和灵敏度，从DNA提取到最终获得检测结果该方法仅需约20 min，并能同时检测6种致病菌，显著提高了检测效率，对食源性致病菌的快速筛查具有重要意义。

Abstract:

Objective To establish a dual detection method for contaminations by six foodborne pathogens (Cronobacter, Escherichia coli O157:H7, Bacillus cereus, Staphylococcus aureus, Salmonella, and Listeria monocytogenes) in formula milk powder in a rapid manner.Methods Enzymatic recombinase amplification (ERA) is a novel isothermal amplification technology that exponentially amplifies trace amounts of DNA or RNA in 10-30 min at 25-42 ℃. The primers and probe of ERA for the detection of Cronobacter were designed. Meanwhile, the ERA primers and probes suitable for the detection of E. coli O157:H7, B. cereus, S. aureus, Salmonella, and L. monocytogenes were screened. Further, through pairwise combination and cross-reactivity analysis, as well as method optimization, the dual ERA detection system was established. The limit of detection and accuracy of the method were determined by application of this method in the detection of simulated contaminations and actual samples.Results Three groups of dual ERA systems were established, achieving the detection of six pathogens in 16 min 10 s. The established method showed the sensitivity of 1 ng/μL in the DNA detection of the combinations of Cronobacter with E. coli O157:H7, B. cereus, and S. aureus, while it showed the sensitivity of 10^-1 ng/μL in the DNA detection of Salmonella and L. monocytogenes. The results of the simulation contaminations showed that the limit of detection of the method was 1 CFU/mL. The dual ERA method established in this study was then adopted to detect 37 commercially available formula milk powder samples near the expiration date. The detection rates of B. cereus and L. monocytogenes were 37.84% and 21.62%, respectively. The results were consistent with those of the real-time PCR (industry standard method), confirming the accuracy of the dual ERA method established in this study.Conclusion The dual ERA method established in this study exhibits high specificity and high sensitivity. Moreover, it takes merely approximately 25 min from DNA extraction to obtaining the detection results, and it is capable of simultaneously detecting six pathogens, demonstrating high efficiency. This method is of importance for the rapid screening of foodborne pathogens.