多重-巢式PCR检测食品中单增李斯特菌研究

Rapid Detection of Listeria Monocytogenes in Food with Multiplex-nested PCR

〔目的〕建立多重—巢式PCR联合检测体系快速检测食品中的单增李斯特菌。〔方法〕针对单增李斯特菌中多个稳定的特异性基因(hlyA、plcB、prfAi、ap),设计并筛选出6对引物用于多重PCR、2对引物用于巢式PCR,组成多重—巢式PCR联合检测单增李斯特菌,并对珠海地区6类冷冻冷藏食品中单增李斯特菌的带菌情况进行了初步调查。〔结果〕建立的多重—巢式PCR联合检测体系特异性良好,多重PCR的灵敏度达到1×102cfu/ml,巢式PCR的灵敏度达到1×101cfu/ml。在检测的3439份样品中,单增李斯特菌阳性率达22.39%。〔结论〕该检测体系具有快速可靠、灵敏准确及特异性好的特点,有效缩短了检验周期,从传统的14d缩短到2d。对珠海地区6类冷冻冷藏食品中单增李斯特菌的带菌情况有了一定了解。

Objective To develop a sensitive multiplex-nested PCR to detect Listeria monocytogenes in foods. Method 4 special genes （gene hlyA, plcB, prfA and iap） of L. monocytogenes were selected as the target genes. And then 6 pairs and 2 pairs of primers were individually designed sceened out as the primers for multiplex PCR and nested PCR.The multiplex PCR and the nested PCR were applied together as the detection method of L. monocytogenes. Result The method was proved to be rapid, sensitive and specialized, of which lxllYcfu/ml and lxl01cfu/ml of L. monocytogenes could be individually detected for multiplex PCR and nested PCR, and only 2 days were needed as a whole.3 439 food samples of 6 different kinds of chilled and frozen food were detected to investigate the contamination status of L.monocytogenes in foods in the area of Zhuhai Guangdong,China,and the ratio of positive samples was 22.39%. Conculusion The method is proved to be applied,sensitive and specialized.The cycle of test would be shortened efficiently.