食品科学 ›› 2020, Vol. 41 ›› Issue (24): 304-309.doi: 10.7506/spkx1002-6630-20191018-185

• 安全检测 • 上一篇    下一篇

实时PCR检测食品中荧光假单胞菌方法的建立

闵可,张政,周雨蕾,侯温甫,王宏勋,周敏   

  1. (武汉轻工大学食品科学与工程学院,湖北 武汉 430023)
  • 出版日期:2020-12-25 发布日期:2020-12-28
  • 基金资助:
    “十三五”国家重点研发计划重点专项(2016YFD0401202)

Establishment of a Real-time PCR Method for Detecting Pseudomonas fluorescens in Food Samples

MIN Ke, ZHANG Zheng, ZHOU Yulei, HOU Wenfu, WANG Hongxun, ZHOU Min   

  1. (School of Food Science and Engineering, Wuhan Polytechnic University, Wuhan 430023, China)
  • Online:2020-12-25 Published:2020-12-28

摘要: 针对荧光假单胞菌分子检测靶点gyrB基因设计引物和探针,制备标准质粒,绘制标准曲线,建立荧光假单胞菌实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)检测体系。特异性评价表明该体系能够特异性检测荧光假单胞菌,其他细菌均无检出。灵敏性检测结果表明纯DNA水平灵敏度可达14.3 fg/μL,纯培养物水平上检测灵敏度为3.0×102 CFU/mL。抗干扰性实验结果表明当加入低浓度背景干扰菌时,对上述体系检测灵敏度没有影响。人工污染样品检测表明适当增菌可检测到荧光假单胞菌,说明TaqMan探针real-time PCR法特异性强、灵敏度高、抗干扰性能力强

关键词: 荧光假单胞菌;实时聚合酶链式反应;gyrB基因

Abstract: In this study, we designed primers and probes targeting the gyrB gene of Pseudomonas fluorescens, prepared standard plasmids, drew standard curves, and established a real-time polymerase chain reaction (PCR) system for the detection of P. fluorescens. Our results confirmed that the PCR system was specific to P. fluorescens without detection of other tested bacteria. The sensitivity was 14.3 fg/μL and 3.0 × 102 CFU/mL for pure DNA and pure culture, respectively, and was not affected by the background interference at low concentrations. In artificially contaminated samples, P. fluorescens could be detected by appropriate enrichment. The TaqMan-based real-time fluorescence quantitative PCR method proved to be highly specific, sensitive and resistant to interferences.

Key words: Pseudomonas fluorescens; real-time polymerase chain reaction; gyrB gene

中图分类号: