食品科学 ›› 2019, Vol. 40 ›› Issue (2): 298-303.doi: 10.7506/spkx1002-6630-20170906-094

• 安全检测 • 上一篇    下一篇

沙门氏菌重组酶聚合酶检测方法的建立及应用

刘立兵1,2,耿云云3,姜彦芬1,刘思颖3,孙晓霞1,2,南汇珠1,王建昌1,2,*   

  1. (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)
  • 出版日期:2019-01-25 发布日期:2019-01-22
  • 基金资助:
    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)

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

LIU Libing1,2, GENG Yunyun3, JIANG Yanfen1, LIU Siying3, SUN Xiaoxia1,2, NAN Huizhu1, WANG Jianchang1,2,*   

  1. (1. Center of Inspection and Quarantine, Hebei Entry-Exit Inspection and Quarantine Bureau, Shijiazhuang 050051, China; 2. Hebei Academy of Inspection and Quarantine, Shijiazhuang 050051, China; 3. College of Life Science, Hebei Normal University Hebei, Shijiazhuang 050024, China)
  • Online:2019-01-25 Published:2019-01-22

摘要: 摘 要:建立一种重组酶聚合酶扩增技术(recombinase polymerase amplification,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。

关键词: 沙门氏菌, invA基因, 重组酶聚合酶扩增, 检测

Abstract: 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.

Key words: Salmonella, invA, recombinase polymerase amplification (RPA), detection

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