鼠伤寒沙门菌与沙门菌血清型4,[5],12:i:-双重RT-RPA快速检测方法的建立  

Development of a duplex real-time recombinase polymerase amplification assay for the rapid identification of Salmonella Typhimurium and its monophasic variant 4,[5],12:i:-

作　　者：蒋增海[1] 徐耀辉[1] 张晓战 彭志锋 陈益[1] 唐光武[1] 吕玉金[1] 乐敏[2] JIANG Zeng-hai;XU Yao-hui;ZHANG Xiao-zhan;PENG Zhi-feng;CHEN Yi;TANG Guang-wu;LüYu-jin;YUE Min(College of Veterinary Medicine,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;Molecular Microbiology and Food Safety Laboratory,Institute of Preventive Veterinary Medicine,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]河南牧业经济学院动物医药学院,河南郑州450046 [2]浙江大学动物预防医学研究所分子微生物学与食品安全研究室,浙江杭州310058

出　　处：《中国兽医科学》2023年第10期1254-1261,共8页Chinese Veterinary Science

基　　金：河南省科技攻关项目(212102110096);浙江省重点研发计划项目(2021C02008);河南牧业经济学院博士启动基金项目(2022HNUAHEDF027)。

摘　　要：为了保障食品公共卫生安全,基于实时荧光重组酶聚合酶扩增技术(RT-RPA),针对鼠伤寒沙门菌、沙门菌血清型4,[5],12:i:-共同携带的STM4495基因以及鼠伤寒沙门菌携带的flj B1,2基因,设计2条探针和一系列候选引物,并进行引物筛选、特异性试验、敏感性试验、各种方法比较检测和人工污染试验,建立一种快速检测鼠伤寒沙门菌与沙门菌血清型4,[5],12:i:-的双重RT-RPA方法。结果显示,该方法在39℃条件下20 min内完成检测,鼠伤寒沙门菌与沙门菌血清型4,[5],12:i:-的检测下限分别为25.6和251CFU/0.1 m L,不能检出其他肠道致病菌,针对猪产业链及临床样品来源的99株沙门菌与PCR、RT-PCR方法的检测结果一致,针对人工污染样品的检测结果与传统培养法一致。本研究成功建立了一种快速检测鼠伤寒沙门菌与沙门菌血清型4,[5],12:i:-的双重RT-RPA方法,具有特异性高、敏感性强的特点,快速简便,20min获取检测结果,在新鲜猪肉、临床病猪肝样品中得到初步应用。In order to ensure food public health safety,two probes and a series of candidate primers were designed for STM4495 gene carried by Salmonella Typhimurium and Salmonella serotype 4,[5],12:i:-,and flj B1,2 gene carried only by Salmonella Typhimurium in accordance with real-time fluorescent recombinase polymerase amplification(RT-RPA).A duplex RT-RPA was developed for the rapid identification of Salmonella Typhimurium and Salmonella serotype 4,[5],12:i:-by screening of primers,specificity,sensitivity,comparative detection of various methods and artificial contamination test.The results showed that the detection was completed within 20 min at 39℃,the detection limit of Salmonella Typhimurium and Salmonella serotype 4,[5],12:i:-was 25.6 CFU/0.1 m L and 251 CFU/0.1 m L,respectively,and other intestinal pathogenic bacteria could not be detected.The detection results of 99 Salmonella isolates from pig industry chain and clinical samples by RT-RPA were consistent with those of PCR and RT-PCR methods,and the detection results of artificially contaminated samples by RT-RPA were consistent with those of traditional culture methods.In this study,a dual RT-RPA assay for rapid detection of Salmonella Typhimurium and Salmonella serotype 4,[5],12:i:-was successfully developed,which had the characteristics of high specificity and sensitivity,was fast and simple,and of which the detection results were obtained in 20 min.This detection method was preliminarily applied in fresh pork and clinical sick pig liver samples.

关 键 词：鼠伤寒沙门菌 鼠伤寒单相变种 重组酶聚合酶扩增法 快速检测方法 

分 类 号：S852.612[农业科学—基础兽医学]