Cao Wei, Wang Ning, Wang Xiaoying, Liu Hongsheng, Guo Yunchang
Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing.
Wei Sheng Yan Jiu. 2009 Nov;38(6):662-6.
To develop rapid PCR-ELISA methods for detecting Listeria monocytogenes, and detect food samples artificially contaminated with Listeria monocytogenes.
Specific primers for Listeria monocytogenes pathogenic gene hlyA were selected based on the Genbank data by using molecular biological software DNAman6.0. Digoxigenin-labeled hlyA fragments were obtained by using commercial kit. Specific capture probes were obtained by comparing bacterial pathogenic gene sequences in the Genbank. PCR-ELISA methods were developed and the Listeria monocytogenes isolates with different serotypes were detected. The sensitivity of PCR and PCR-ELISA was determined by artificially inoculating Listeria monocytogenes strains in milk.
It took 6 hours to detect Listeria monocytogenes in food samples by PCR-ELISA. The accordance rate to the bacteriological method was 100%. After 12 h pre-enrichment, the detection limit of PCR-ELISA method was 1 CFU/25 ml milk. The sensitivity of PCR-ELISA method was 10-100 times as PCR.
The PCR-ELISA method for rapidly detecting Listeria monocytogenes was established. The sensitivity, specificity and reliability of the method proved to be good. It would be valuable to improve the precaution and prediction abilities of food-borne diseases and enhance the chronergy and accuracy of detection method.
建立快速检测单核细胞增生李斯特菌的PCR-ELISA方法，并检测人工污染单核细胞增生李斯特菌的食品样本。
利用分子生物学软件DNAman6.0，根据Genbank数据选择单核细胞增生李斯特菌致病基因hlyA的特异性引物。使用商业试剂盒获得地高辛标记的hlyA片段。通过比较Genbank中细菌致病基因序列获得特异性捕获探针。建立PCR-ELISA方法并检测不同血清型的单核细胞增生李斯特菌分离株。通过在牛奶中人工接种单核细胞增生李斯特菌菌株来确定PCR和PCR-ELISA的灵敏度。
PCR-ELISA检测食品样本中的单核细胞增生李斯特菌耗时6小时。与细菌学方法的符合率为100%。经过12小时预增菌后，PCR-ELISA方法的检测限为1 CFU/25 ml牛奶。PCR-ELISA方法的灵敏度是PCR的10-100倍。
建立了快速检测单核细胞增生李斯特菌的PCR-ELISA方法。该方法的灵敏度、特异性和可靠性良好。对于提高食源性疾病的预防和预测能力以及提高检测方法的时效性和准确性具有重要价值。
