林颖峥，林士佳，张冠楠，李树清，熊 炜，郭雨雁，严亚贤

（1.上海交通大学，上海 200240；2.上海出入境检验检疫局，上海 200135）

野兔热快速检测方法的建立

林颖峥1，林士佳2，张冠楠2，李树清2，熊 炜2，郭雨雁2，严亚贤1

（1.上海交通大学，上海 200240；2.上海出入境检验检疫局，上海 200135）

野兔热是由土拉弗朗西斯菌引起的兔的一种高度接触性、致死性传染病。为加强口岸对进境野生及实验用兔中野兔热的筛查和流行病学调查，基于土拉弗朗西斯菌保守基因序列，建立了PCR和实时PCR检测野兔热的方法。将建立的方法应用于临床样品检测发现经PCR检测，仅有土拉弗朗西斯菌DNA出现条带单一的特异性基因扩增，经实时PCR检测，土拉弗朗西斯菌DNA在10～12个循环处出现显著扩增，对照病毒样品未见扩增；PCR检测土拉弗朗西斯菌DNA模板的下限量为100～10 pg，而实时PCR检测的下限量为100～10 fg。检测结果证实两种检测方法具有良好的特异性、灵敏性和稳定性，适合应用于野兔热的快速检测。

野兔热；PCR；Real-time PCR；Taqman探针

Abstract：Tularemia is a highly contagious and lethal disease caused by Francisella tularensis. In order to investigate the epidemic character of Tularemia transmission among wild and experimental rabbits，methods of PCR and Real-time PCR to detect Tularemia were established，based on the conserved sequence of Francis tularensis strains. Both methods were applied to test clinical samples. By PCR detection，only Francis tularensis single band DNA showed bacteria specific gene amplification. By real-time PCR detection，tularemia bacteria DNA in 10-12 cycles Francis appeared significant amplification，and no amplification was found in the control virus samples. The lower limit of the amount of CR detected DNA template for tularemia bacteria Francis was 100-10 pg，and the lower limit of real-time PCR detection was 100-10 fg. The results showed that PCR and Real-time PCR methods were specific，sensitive and reproducible，thus both methods were suitable for Tularemia rapid detection.

Keywords：Tularemia；PCR；real-time PCR；taqMan probe

野兔热（Tularaemia）又称兔热病、土拉热、土拉杆菌病、土拉弗氏菌病，是由土拉弗朗西斯菌（Francisella tularensis）感染引起的一种急性人兽共患传染病。1912年，McCoy从美国土拉县的黄鼠中分离出该菌，并根据该地名将其命名为土拉菌。1919年，美国华盛顿公共卫生官员Francis被派到犹他州调查鹿蝇热病期间，对该菌做了大量的研究工作。鉴于其贡献，将该菌重命名为土拉弗朗西斯菌。根据菌株对家兔的毒力和能否分解甘油，将全世界已知菌株分为3个地理变种：美洲变种，能分解甘油，毒力最强；……