基于TaqMan荧光PCR对副溶血性弧菌能力验证样品快速鉴定

Rapid Detection and Identification of Listeria Monocytogenes Proficiency Testing Samples by TaqMan Real-Time Fluorescence PCR

基于TaqMan荧光PCR对副溶血性弧菌能力验证样品进行快速筛查检测与鉴定。为验证方法的准确性,依据GB 4789.7—2013《食品安全国家标准食品微生物学检验副溶血性弧菌检验》进行传统培养法检测,通过增菌、分离、纯化得到疑似目标菌的纯菌落,通过初步鉴定和确定鉴进行研判,确定鉴定使用国产干制生化鉴定试剂盒、API20E试剂条及VITEK2全自动微生物鉴定系统。在传统培养法的同时,对3%氯化钠蛋白胨水培养物中的基因组DNA以及在分离平板上的菌落基因组DNA进行提取,设计特异性的Taqman引物和荧光探针,建立实时荧光PCR技术快速筛查与鉴定技术。结果表明:经干制生化试剂盒中6个生化试验鉴定,各项生化指标与标准一致,但鉴定试验项目少,需主观判定结果;经API20E试剂盒中21个生化试验鉴定后,通过菌库软件查得样本071为副溶血性弧菌,鉴定百分率为98.6%,T值为1;经VITEK2全自动微生物鉴定系统检测与分析,样本071检出副溶血性弧菌,样本276检出肠炎沙门菌和大肠埃希菌;经3%氯化钠蛋白胨水培养物和TCBS上的可疑菌落实时荧光PCR鉴定,样品071副溶血性弧菌检出,样品276副溶血性弧菌未检出。实时荧光PCR在增菌液快速筛查和可疑菌落的快速鉴定方面均与标准检验结果一致,可作为各种复杂样品的辅助检测与鉴定手段。

Rapid screening detection and identification of Vibrio parahaemolyticus proficiency validation samples based on TaqMan fluorescence PCR.In order to verify the accuracy of the method,according to the GB 4789.7—2013 National Food Safety Standard Food Microbiology Test-Vibrio parahaemolyticus Test for traditional culture detection,through enrichment,isolation,purification to obtain the pure colonies of suspected target bacteria,through preliminary identification and identification for research and judgment,to determine the identification of the use of domestic dried biochemical identification kit,API20E reagent strips and VITEK2 automatic microbial identification system.At the same time,as the traditional culture method,the genomic DNA in the aqueous culture of 3%sodium chloride peptone and the colony genomic DNA on the isolation plate were extracted,specific Taqman primers and fluorescent probes were designed,and the rapid screening and identification technology of real-time fluorescence PCR technology was established.The results showed that the six biochemical tests in the dried biochemical kit were consistent with the standards,but there were few identification test items,and the results needed to be subjectively determined.After the identification of 21 biochemical tests in the API20E kit,sample 071 was found to be Vibrio parahaemolyticus by the bacterial library software,with an identification percentage of 98.6%and a T value of 1.Detected and analyzed by VITEK2 automatic microbial identification system,Vibrio parahaemolyticus was detected in sample 071,and Salmonella enteritidis and Escherichia coli were detected in sample 276.Samples 071 were detected by fluorescence PCR identification of suspicious bacteria on 3%sodium chloride peptone aqueous culture and TCBS,and sample 276 samples of Vibrio haemolyticus were not detected.Real-time fluorescence PCR is consistent with the standard test results in the rapid screening of bacterial enrichment solution and the rapid identification of suspicious colonies,and can be used as an auxiliary detection and identification method for various complex samples.