免疫磁捕获-荧光定量PCR快速检测食品中沙门氏菌

Rapid detection of Salmonella in food by immunomagnetic separation-real-time PCR assay

建立了免疫磁珠分离(Immunomagnetic separation,IMS)联合荧光定量PCR(Real-time PCR)技术准确、快速检测食品中沙门氏菌(Salmonella spp)的方法。采用亲和纯化的羊抗沙门氏菌抗体与Dynabeads M-280磁珠制备沙门氏菌免疫磁珠,优化反应条件,建立免疫磁珠分离方法。同时根据沙门氏菌特异性ttr基因,建立Real-time PCR体系,并检测其特异性及敏感性。结果显示,沙门氏菌可产生荧光信号,而其他细菌均未见明显荧光信号。利用所建立的免疫磁捕获-荧光定量PCR(IMS-real-time PCR)方法可以检测初始含菌量最低为6.5CFU/25g的食品样品,全过程需时约8h。150份实际食品样品中,IMS-real-time PCR方法检出27份阳性样品,免疫磁珠联合XLT4平板培养法检出18份阳性样品,传统国标法检出14份阳性样品。

The aim of this study was to develop a rapid and specific method for detection of Salmonella in food using immunomagnetic separation combined with real-time PCR （IMS-real-time PCR）. The immunomagnetic beads used in the study were prepared by coating goat anti-Salmonella antibody to Dynabeads M-280 tosylactivated,and the separation conditions were further optimized. Specifically designed primers and a probe target the ttr locus were employed in real-time PCR. A preenrichment step,an IMS, DNA extraction,and hybridization probe-based real-time PCR analysis were performed in the order for the IMS-real-time PCR assay. The results showed that primers and probe could specifically detect Salmonella in our experiments. A result within 8h could be achieved with IMS-real-time PCR assay,and the detection limit was as low as 6.5CFU/25g. Compared with the reference method on 150 collected samples,27 positive samples were detected with the IMS-real-time PCR assay,whereas only 18 for IMS/culture and 14 for the conventional standard methods.