三重微滴数字PCR定量检测即食米粉中致病菌方法研究

Research on the quantification method to detect pathogenic bacteria in instant rice noodles by 3-plex droplet digital polymerase chain reaction test

目的建立三重微滴数字PCR(ddPCR)方法同时定量检测即食食品中的沙门菌、蜡样芽胞杆菌和单核细胞增生李斯特菌。方法选择以沙门菌ttrA/ttrC、蜡样芽胞杆菌essC、单核细胞增生李斯特菌侵袭相关内肽酶基因等3个单拷贝基因对应的3对引物探针,采用实时荧光定量PCR验证引物/探针特异性后,建立三重ddPCR方法同时定量检测3种致病菌的拷贝数。结果该方法的线性范围分别为:沙门菌25~22687 copies/20μL;蜡样芽胞杆菌19~15620 copies/20μL;单核细胞增生李斯特菌18~23373 copies/20μL,线性相关因子r^(2)≥0.999,6个浓度3次重复测定3种菌的相对标准偏差(RSD)≤12%,重复性好,对于上述菌株的最低检出限分别为6、3和7 copies/20μL;采用已建立的ddPCR方法和平板计数方法对模拟染菌米粉样品进行检测,两种方法测定值结果RSD小于9%,结果一致性较好。结论本研究建立的三重ddPCR同时定量检测即食食品中沙门菌、蜡样芽胞杆菌和单核细胞增生李斯特菌的方法与平板计数法相比,更快速、灵敏,结果准确。

Objective This study aimed to establish a quantitative 3-plex droplet digital polymerase chain reaction(ddPCR)method for simultaneously detecting the copy numbers of Salmonella,Bacillus cereus,and Listeria monocytogenes in instant food.Methods Three pairs of primers and probes corresponding to three single-copy-genes were selected as target genes.The genes were the essC gene in Bacillus cereus,ttrA/ttrC gene in Salmonella,and invasionassociated endopeptidase gene in Listeria monocytogenes.The specificity of the primers and probes were verified by real-time fluorescence quantitative PCR separately.A 3-plex ddPCR method was constructed to detect the copy numbers of three pathogenic bacteria simultaneously.Results The linear ranges were:25-22687 copies/20μL for Salmonella,19-15620 copies/20μL for Bacillus cereus,and 18-23373 copies/20μL for Listeria monocytogenes.The three linear correlation coefficients were r≥0.999.relative standard deviation(RSD)≤12%at six concentrations and repeated thrice,indicating favorable repeatability.The minimum detection limits were six copies/20μL for Salmonella,three copies/20μL for Bacillus cereus,and seven copies/20μL for Listeria monocytogenes.When a simulated sample of contaminated rice noodles was detected by 3-plex ddPCR and the plate counting method,the deviation between these two methods was<9%,indicating a good consistency in the results.Conclusion The 3-plex ddPCR method for the simultaneous and quantitative detection of Salmonella,Bacillus cereus,and Listeria monocytogenes in instant food was quicker,more sensitive,and more accurate than the plate-counting method.