单增李斯特菌PCR-ELISA快速检测技术研究

目的建立快速检测单增李斯特菌的PCR-ELISA方法,将其应用于人工污染的食物样品检测。方法根据GenBank数据库资料,应用分子生物学软件DNAMAN6.0和Primer Premier5.0,以单增李斯特菌的致病基因hlyA为基础,选用PCR引物,应用地高辛标记试剂盒,获得单增李斯特菌特异的地高辛标记片段。根据PCR目的片段序列,设计特异性捕获探针,建立了单增李斯特菌PCR-ELISA快速检测方法。应用该方法对不同血清型的单增李斯特菌食品分离株进行了检测,并将PCR方法与PCR-ELISA方法对人工污染单增李斯特菌的牛奶样品的检测敏感性进行了比较。结果应用单增李斯特菌特异的PCR-ELISA方法完成检测约需6h。该方法对单增李斯特菌分离株检测的结果,与国家食源性疾病检测网的鉴定结果100%符合。经过12h的增菌培养,PCR-ELISA方法最低可从25ml样品中检出1CFU,检测敏感性为传统PCR方法的10～100倍。结论建立了单增李斯特菌PCR-ELISA快速检测方法。该方法敏感性高、特异性强、可靠性好,对于提高食源性疾病预警、预测能力,增强检测的时效性和准确性,具有推广应用价值。

Study on rapid detection techniques of PCR-ELISA for Listeria monocytogenes

Objective To develop rapid PCR-ELISA methods for detecting Listeria monocytogenes, and detect food samples artificially contaminated with Listeria monocytogenes . Methods 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. Results It took 6 hours to detect Listeria monocytogenes in food samples by PCR-ELISA. The accordance rate to the bacteriological method was 100% . After 12h pre-enrichment, the detection limit of PCR-ELISA method was 1CFU/25ml milk. The sensitivity of PCR-ELISA method was 10 - 100 times as PCR. Conclusion 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.