免疫磁珠分离-实时荧光PCR快速检测虾中沙门氏菌

Rapid Detection of Salmonella in Shrimp by Immunomagnetic Separation Combined with Real-time PCR

摘要:

建立了免疫磁珠分离（Immunomagnetic separation，IMS）联合实时荧光PCR（real time PCR）快速、灵敏地检测虾中沙门氏菌的方法。用纳米磁珠与抗沙门氏菌多克隆抗体制备免疫磁珠，优化反应条件，建立IMS方法。同时针对沙门氏菌ttr基因合成探针和引物，构建real time PCR体系，选4株代表性的沙门氏菌检测其特异性和灵敏度。结果显示，免疫磁珠的最适添加量为100 μL，免疫磁珠与样品菌液的最佳反应时间为30 min。建立的免疫磁珠分离-实时荧光PCR（IMS-real time PCR）方法特异性高，对于虾中沙门氏菌的检测限为鼠伤寒沙门氏菌5×101 CFU/25 g，猪霍乱沙门氏菌5×102 CFU/25 g，肠炎沙门氏菌和甲型副伤寒沙门氏菌1×102 CFU/25 g，检测全程可在6 h内完成。对40份实际样品，IMS-real time PCR方法与国标法检测结果完全一致。建立的IMS-Real time PCR方法用时短、灵敏度高，为水产品中沙门氏菌的快速检测提供了技术支撑，对沙门氏菌引起的食源性疾病的预防和控制具有现实意义。

Abstract:

A rapid and sensitive detection method of immunomagnetic separation (IMS) combined with real-time PCR for Salmonella in shrimp was established. The immunomagnetic beads were prepared by coupling anti-Salmonella polyclonal antibody to magnetic beads and the reaction conditions were optimized to establish the IMS method, and a probe and primers were synthesized for ttr gene of Salmonella to construct the real-time PCR system. The specificity and sensitivity of four representative Salmonella strains were examined. The results showed that the optimal dosage of immunomagnetic beads was 100 μL, and the optimal reaction time of immunomagnetic beads with sample bacteria was 30 min. The proposed IMS-real time PCR had a high specificity and the limits of detection were 5×101 CFU/25 g for Salmonella typhimurium, 5×102 CFU/25 g for Salmonella choleraesuis and 1×102 CFU/25 g for both Salmonella enteritidis and Salmonella paratyphi A, respectively, and the detection process could be completed within 6 h. The test results of IMS-real time PCR were same with those of the national standard for 40 collected shrimp samples. The established IMS-real time PCR need less time and had a high sensitivity, which provided technical support for the rapid detection of Salmonella in aquatic products as well as the practical significance for the prevention and control of foodborne diseases caused by Salmonella.