贝类中沙门氏菌、阪崎肠杆菌双重RT-PCR检测方法研究

Study on dual RT-PCR detection method for Salmonella and Enterobacter sakazakii in shellfish

旨在建立一种快速、高效、经济、实用的双重实时荧光PCR方法,能同时快速、灵敏、特异地检测贝类中沙门氏菌、阪崎肠杆菌的双重实时荧光PCR体系。本研究以水产品中贝类为目标,根据特定基因序列,设计特异性扩增引物和TaqMan荧光探针,通过优化反应体系和条件,每种引物探针体系均可以特异性检测目标致病菌类型。2套引物探针体系组合可同时检测沙门氏菌、阪崎肠杆菌。双重RT-PCR方法的最低检测限可低至0.002 ng或0.02 ng基因组/反应,变异系数均小于2%。利用建立的双重RT-PCR方法检测14份贝类产品样本,样品实测结果与国家标准方法的检测结果一致,符合率为100%。结果表明,本研究建立的双重RT-PCR方法具有特异性、敏感性高、重复性好等优点,为贝类产品中沙门氏菌、阪崎肠杆菌的快速检测提供了新的技术方法。

The aim is to establish a fast,efficient,economical,and practical dual real-time fluorescent PCR method that can simultaneously,sensitively,and specifically detect Salmonella and Enterobacter sakazakii in shellfish.This study targets shellfish in aquatic products and designs specific amplification primers and TaqMan fluorescent probes based on specific gene sequences.By optimizing the reaction system and conditions,each primer probe system can specifically detect the target pathogen type.The combination of two primer probe systems can simultaneously detect Salmonella and Enterobacter sakazakii.The minimum detection limit of the dual RT-PCR method can be as low as 0.002 ng or 0.02 ng genome/reaction,with coefficients of variation less than 2%.The established dual RT-PCR method was used to detect 14 samples of shellfish,and the measured results of the samples were consistent with the detection results of the national standard method,with a compliance rate of 100%.The results indicate that the dual RT-PCR method established in this study has the advantages of specificity,high sensitivity,and good repeatability,providing a new technical method for the rapid detection of Salmonella and Enterobacter sakazakii in shellfish.