基于免疫磁分离-双重荧光定量PCR的鲜猪肉中沙门氏菌和志贺氏菌的检测

Detection of Salmonella spp. and Shigella spp. in fresh pork using duplex real-time PCR based on immunemagnetic separation

中文关键词:  沙门氏菌  志贺氏菌  免疫磁分离  双重荧光定量PCR

英文关键词:Salmonella spp.  Shigella spp.  immunomagnetic separation  duplex real-time PCR

中文摘要:

      目的 使用免疫磁分离-双重荧光定量PCR方法实现对鲜猪肉中金黄色葡萄球菌和志贺氏菌的同时、快速检测。方法 在37 ℃的条件下, 利用特异性免疫磁球从250 mL循环体系中快速、有效地捕获目标菌。再通过特异性的引物与探针, 对沙门氏菌和志贺氏菌进行双重荧光定量PCR检测。结果 本研究方法针对鲜猪肉中沙门氏菌和志贺氏菌的检测限分别达到3.4 cfu/g和9.4 cfu/g。方法总体灵敏度、特异性和准确度均达到100%。此外, 通过对30组实际样品的检测, 该方法与传统标准方法的结果保持一致。结论 本研究建立的免疫磁分离-双重荧光定量PCR方法比国标方法更快速和高效, 适用于鲜猪肉中沙门氏菌和志贺氏菌的快速检测。

英文摘要:

      Objective To develop a rapid and simultaneous detection for Salmonella spp. and Shigella spp. from fresh pork samples by combining immunomagnetic separation (IMS) with duplex real-time PCR (RT-PCR). Methods Magnetic beads coated with speci?c antibodies were used to capture the pathogens from a 250 mL cycling system at 37 ℃ condition. Then, duplex RT-PCR was applied to detect Salmonella spp. and Shigella spp. with 2 sets of specific primers and probes. Results The limit of detections were 3.4 cfu/g for Salmonella spp., and 9.4 cfu/g for Shigella spp.. The sensitivity, specificity, and accuracy of IMS-multiplex RT-PCR method were all 100%. Moreover, the results of new method were consistent to those of traditional method by detecting 30 samples. Conclusion IMS-duplex RT-PCR method is more rapid and effective than GB method, and which is suitable for the rapid detection of Salmonella spp. and Shigella spp. in fresh pork.