沙门氏菌重组酶聚合酶检测方法的建立及应用  

Development and Application of a Recombinase Polymerase Amplification Assay for Detection of Salmonella

作　　者：刘立兵[1,2] 耿云云 姜彦芬 刘思颖 孙晓霞[1,2] 南汇珠[1] 王建昌 LIU Libing;GENG Yunyun;JIANG Yanfen;LIU Siying;SUN Xiaoxia;NAN Huizhu;WANG Jianchang(Center of Inspection and Quarantine,Hebei Entry-Exit Inspection and Quarantine Bureau,Shijiazhuang 050051,China;Hebei Academy of Inspection and Quarantine,Shijiazhuang 050051,China;College of Life Science,Hebei Normal University Hebei,Shijiazhuang 050024,China)

机构地区：[1]河北出入境检验检疫局技术中心,河北石家庄050051 [2]河北省检验检疫科学技术研究院,河北石家庄050051 [3]河北师范大学生命科学学院,河北石家庄050024

出　　处：《食品科学》2019年第2期298-303,共6页Food Science

基　　金：国家质量监督检验检疫总局科研项目(2016IK107);河北师范大学博士基金项目(130401)

摘　　要：建立一种重组酶聚合酶扩增技术(recombinasepolymeraseamplification,RPA)检测沙门氏菌(Salmonella)的方法。本研究依据沙门氏菌侵袭蛋白A基因(invA)的保守序列设计特异性引物,通过对反应时间的优化,建立的RPA方法在38℃水浴锅中恒温反应20 min,即可实现对目的片段的有效扩增;除沙门氏菌外,其他26 种食源性致病菌均无扩增,具有良好的特异性;以沙门氏菌基因组DNA作为模板,该方法的检测灵敏度为1.1×10^(-3) ng/μL,与本研究应用的实时荧光聚合酶链式反应(real-time polymerase chain reaction,PCR)方法一致;人工污染实验表明,当羊肉、鸡肉和西兰花样品污染量为4 CFU/25 g,增菌8 h,即可通过RPA方法检出沙门氏菌。在人工污染实验中,RPA和PCR检测结果一致。本研究建立的沙门氏菌的RPA检测方法特异性强、操作简单、为食源性致病菌的鉴定提供了一种新的方向。The aim of the study was to develop a method for the determination of Salmonella by recombinase polymerase amplification (RPA). A specific primer pair was designed based on the conserved sequence of the invasion protein A gene (invA) of Salmonella. The reaction time was optimized and the RPA method was performed successfully at 38 ℃ for 20 min in a water bath: the target fragment was effectively amplified. The RPA reaction could specifically detect Salmonella rather than 26 other foodborne pathogens. The detection limit (LOD) of RPA was 1.1 × 10-3 ng/μL with the genomic DNA of Salmonella as a template, which was consistent with that of real-time PCR. For artificially contaminated lamp, chicken and broccoli samples with a bacterial concentration of 4 CFU/25 g, Salmonella could be detected by RPA after 8 hours of culture. Consistent results were obtained using real-time PCR. The RPA assay was specific, simple and rapid, and could represent a new direction for the detection of foodborne pathogens.

关 键 词：沙门氏菌 invA基因 重组酶聚合酶扩增 检测 

分 类 号：R155.5[医药卫生—营养与食品卫生学] Q93[医药卫生—公共卫生与预防医学]