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目的建立多重实时荧光定量PCR(multiplex quantitative real-time PCR,multiplex qPCR)快速检测奶粉中金黄色葡萄球菌、沙门氏菌和克罗诺杆菌3种常见致病菌的方法。方法筛选目标菌株的特异性引物与探针,优化反应体系,建立稳定的多重q PCR反应体系。通过阳性菌株加标的方式验证体系的特异性,并确定人工污染奶粉的检出限。结果各对引物探针对目标菌株均能扩增,多重实时荧光PCR未发现交叉反应,对17株非目标菌进行检测均未检出,人工污染奶粉中克罗诺杆菌和沙门氏菌的检出限均为10~3 CFU/mL,金黄色葡萄球菌的检出限为10~4 CFU/mL。结论本研究方法可实现婴幼儿奶粉样品中3种致病菌qPCR高效率检测。
Objective To establish a multiplex quantitative real-time PCR (multiplex qPCR) method for the rapid detection of three common pathogens of Staphylococcus aureus, Salmonella and Cronobacter in milk powder. Methods The specific primers and probes of target strains were screened, and the reaction system was optimized to establish a stable multiplex q PCR reaction system. The specificity of the system was verified by spiked positive strains and the detection limit of artificial contaminated milk powder was determined. Results The primer pairs were able to amplify the target strains. No cross-reaction was detected by multiplex real-time PCR. The detection of 17 non-target bacteria were not detected. The detection limits of both Cronobacter and Salmonella in artificial contaminated milk powder 10 ~ 3 CFU / mL, the detection limit of Staphylococcus aureus is 10 ~ 4 CFU / mL. Conclusion The method can be used to detect high-efficiency qPCR of three pathogenic bacteria in infant milk powder samples.