食源性肠球菌荧光定量PCR检测方法的建立与评价

Development and Evaluation of a Real-Time PCR System for Enterococcus spp. Detection in Food Samples

利用基因组序列比对分析等生物信息学方法发掘肠球菌新的属特异性靶点,根据42个候选靶点序列设计50对引物,结合普通PCR初筛和荧光定量PCR复筛,挑选特异性和灵敏度等检测性能最佳的引物,建立相应的荧光定量PCR检测方法,并对该方法应用于食品中肠球菌检测时的效果作出评价。分析结果显示,特异性最强的引物为EF1902,利用该引物建立的荧光定量体系检测肠球菌时均产生特异性扩增信号,而检测非肠球菌菌株时均无特异性扩增信号形成。经优化PCR体系后,该方法的基因组DNA检测灵敏度为13.78拷贝/PCR,纯培养物灵敏度为38.4 cfu/PCR。以肠球菌人工污染牛奶,当初始接菌量为2.63 cfu/mL时,只需增菌6 h即可用该方法检出肠球菌。对52份食品样品进行检测准确率为94.23%,证实了该方法可应用于食源性肠球菌的快速检测。综上所述,作者建立的肠球菌荧光定量PCR方法,特异性强且灵敏度高,可应用于食品中肠球菌的快速检测。

The aim of the study was to explore new detective targets for Enterococcus spp.,with better performance in specificity and sensitivity,and subsequently to develop a real-time PCR method which could be effectively applied to rapid quantitative detection of enterococci.Genomic comparative analysis among Enterococcus and non-Entercoccus strains was conducted to screen the specific targets for enterococci,and 42 target fragments were selected for the design of 50 primer pairs.Their specificity and sensitivity were then evaluated by regular PCR and real-time PCR,respectively.Finally,the method was effectively evaluated while it was used in the detection of food samples.Analysis results showed that primer EF1902 was confirmed as the best primer due to its high specificity and sensitivity in real-time PCR tests.The results showed a specific amplification from 44 Enterococcus strains,whereas no amplification from 23 non-Enterococcus strains.A detection limit of 13.78 copies/PCR for purified genomic DNA and 38.4 cfu/PCR for bacterial cultures could be achieved after PCR parameter optimization.Artificial contamination assays showed that Enterococcus could be detected by real-time PCR after 6 h enrichment,with an initial spike of 2.63 cfu/mL bacterium in milk samples.The real-time PCR method developed in this study was also applied to detect various food samples,and the positive rate of detection was 57.69%.The accuracy was 94.23% compared with the conventional standard method,which demonstrates its potential for practical application.In conclusion,the real-time PCR system developed in this study was suitable for the rapid detection of Enterococcus spp.in foods for its advantages in specificity,sensitivity,rapidness and accuracy.