利用mPCR方法检测巴氏奶中致病菌的鲁棒性研究

Robustness Study of mPCR for Pathogenic Bacteria Detection in Pasteurized Milk

为解决巴氏奶货架期短与致病菌传统检测方法耗时长相矛盾问题,建立一种检测巴氏奶中的阪崎克罗诺杆菌、大肠杆菌和沙门氏菌的鲁棒性多重聚合酶链式反应(multiple polymerase chain reaction,mPCR)方法。旨在常规mPCR的基础上深入研究,提高方法的准确性和稳定性。首先,针对每个目标菌株选择两种基因,并设计两组特异性引物,建立两套mPCR扩增体系,进行双重检测;然后,在不改变方法灵敏度的前提下,对优化了的退火温度进行范围稳定性选择;最后,将提出方法与国标方法 GB 4789.40-2016、GB 4789.38-2012、GB 4789.4-2016进行比较,同时检测人工污染样品并评价应用效果。结果表明,第一套mPCR检测巴氏奶中三种致病菌可在退火温度59~59.5℃(温差0.5℃)的范围内,具有较好的检测准确性和稳定性,灵敏度为10 CFU/mL。第二套mPCR检测巴氏奶中三种致病菌可在退火温度57.5~58.5℃(温差1℃)的范围内,不影响方法的准确性和稳定性,灵敏度为10 CFU/mL。采用建立的鲁棒性mPCR方法对人工污染的50份巴氏奶样品进行检测,检测结果与国标方法一致。在检测时效上需要4 h,较国标方法(检测时间为62~148 h)显著(P<0.05)缩短检测时间。该方法对微生物流行病学调查和研究,尤其是对巴氏奶中致病菌的快速检测和安全控制具有一定的实际应用价值。

To address the contradiction between the short shelf life of pasteurized milk and the time-consuming traditional methods for detecting pathogenic bacteria,a robust multiple polymerase chain reaction(mPCR)method was developed to detect Cronobacter sakazakii,Escherichia coli,and Salmonella in pasteurized milk.The aim was to conduct an in-depth research on the basis of routine mPCR,and improve the accuracy and stability of the method.Firstly,two genes were selected for each target strain,and two sets of specific primers were designed to establish two mPCR amplification systems for dual detection.Then,the range stability of optimized annealing temperatures was selected without changing the sensitivity of the method.Finally,the proposed method was compared with the national standard methods GB 4789.40-2016,GB 4789.38-2012,and GB 4789.4-2016 to simultaneously detect artificially contaminated samples and evaluate the application performance. The results showed that the first set of mPCR could detect three pathogenic bacteria in pasteurized milk with good accuracy and stability in the range of annealing temperature of 59~59.5 ℃ (temperature difference was 0.5 ℃), and the sensitivity was 10 CFU/mL. The second set of mPCR could detect three pathogenic bacteria in pasteurized milk with good accuracy and stability in the range of annealing temperature of 57.5~58.5 ℃ (temperature difference was 1 ℃), and the sensitivity was 10 CFU/mL. The established robust mPCR method was used to detect 50 artificially contaminated samples of pasteurized milk, and the results were consistent with the national standard method. The detection time was significantly reduced to 4 hours compared to the national standard method (detection time was 62~148 h). This method had practical application value for microbial epidemiological investigation and research, especially for the rapid detection and safety control of pathogenic bacteria in pasteurized milk.