鼠疫耶尔森氏菌三色荧光定量PCR检测方法的建立

    潘光合1,谭勇1,吴文旺1,甘洁1,梁亮2,覃杨光1,王鸣柳3,万道正2,蓝翊文2,梁中平2

　　1.广西出入境检验检疫局，广西 南宁 530021；2.凭祥出入境检验检疫局；

　　3.广西壮族自治区疾病预防控制中心

　　摘要：目的  建立一种三色荧光定量PCR检测鼠疫耶尔森氏菌的方法。方法 根据鼠疫耶尔森氏菌pPCP1质粒上的pla基因、编码F1抗原在pMT1质粒上的caf1基因以及菌体染色体上的3a基因序列，设计特异性的引物和不同荧光标记的Taqman荧光探针，优化反应体系和扩增条件，考察该方法检测的敏感度、特异性、重复性和稳定性，最终建立三色荧光定量PCR检测方法。结果 本研究所建立的方法对鼠疫耶尔森氏菌DNA的扩增效率高，最低检出限为1×102copies/ml，特异性高，重复性的变异系数在合理范围内，稳定性好。结论  建立了一种同时检测鼠疫耶尔森氏菌pla、caf1和3a基因的三色荧光定量PCR方法，该方法敏感度和特异性高，能够实现快速检测的目的。

　　关键词：三色荧光；定量PCR方法；鼠疫耶尔森氏菌；检测

　　中图分类号：R446  文献标识码：B

　　Establishment of a three-color fluorescence quantitative PCRmethod

　　for detecting Yersinia pestis

　　PAN Guang-he﹡， TAN Yong， WU Wen-wang， GAN Jie， LIANG Liang， QINYang-guang， WANG Ming-liu，

　　WAN Dao-zheng， LAN Yi-wen， LIANG Zhong-ping

　　﹡Guangxi Entry-exit Inspection and Quarantine Bureau， Nanning，Guangxi 530021， China

　　Abstract: Objective   To establish a three-colorfluorescence quantitative PCR method to detect Yersinia pestis.  Methods   The specific primers and Taqman fluorescentprobes were designed according to pla gene on pPCP1 Plasmid， ca f1gene on pMT1 Plasmid and 3a gene on chromosome of Yersinia pestis.The reaction system and amplification conditions were optimized.The sensitivity， specificity，repeatability and stability of themethod were studied. The three-color fluorescent quantitative PCRmethod for detecting Yersinia pestis was eventually establishred.Results  The detecting ratios and amplification efficiency ofthe method for samples of Yersinia pestis DNA was high， the linedetecting limit of the method was 1×102 copies/ml， with betterspecificity， the repeatability was in the reasonable range， and thestability was good. Conclusion  The three-color fluorescentquantitative PCR method to detect the pla， ca f1 and 3a gene ofYersinia pestis is highly sensitive and specific， and can be usedto detect Yersinia pestis rapidly.

　　Key words: Three-color fluorescence； Quantitative PCR； Yersiniapestis； Detection

　　《中国国境卫生检疫杂志》2014年3期