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

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 m L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。

ABSTRACT: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 specific 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.