埃里希氏体病和莱姆病双重荧光定量PCR诊断方法的建立  

Establishment of a dual fluorescence quantitative PCR diagnostic method for Ehrlichiosis and Lyme disease

作　　者：常瑾 高超 于喜冰 李俊娇 穆佳琦 柴浩然 成姗育 尹仁福 CHANG Jin;GAO Chao;YU Xibing;LI Junjiao;MU Jiaqi;CHAI Haoran;CHENG Shanyu;YIN Renfu(Key Laboratory of Zoonosis Research,Ministry of Education,College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学动物医学学院人兽共患病研究教育部重点实验室,吉林长春130062

出　　处：《中国兽医学报》2021年第4期697-703,共7页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2016YFD0501005)。

摘　　要：埃里希氏体病及莱姆病均为由蜱传播的虫媒性传染病,为建立同时诊断埃里希氏体病和莱姆病的诊断方法,本研究选择埃里希氏体的groEL基因及伯氏疏螺旋体的ospA基因为检测对象,建立同时诊断埃里希氏体病和莱姆病的双重荧光定量PCR方法,并对其特异性、灵敏性、稳定性、与以往的普通PCR方法的一致性进行了研究。结果显示,本方法仅可特异性扩增埃里希氏体及伯氏疏螺旋体的基因,对钩端螺旋体、立克次体、大肠杆菌、绿脓杆菌基因组扩增均为阴性,最低核酸检测限为1×10^(1)拷贝/μL,批内与批间重复试验Tm值的变异系数均小于0.1%。利用此方法对25份采自黑龙江省佳木斯市的全沟硬蜱样品进行检测,其中埃里希氏体和伯氏疏螺旋体的检出率分别为28%和60%。结果表明,本方法特异性好、灵敏度高、稳定性强,能同时检测埃里希氏体和伯氏疏螺旋体,可用于蜱传疾病的分子流行病学调查、鉴别诊断以及疫病防控。Both Ehrlichiosis and Lyme disease are tick borne infectious diseases.In order to establish a diagnostic method for Ehrlichiosis and Lyme disease,we selected the groEL gene of Ehrlichia and the ospA gene of Borrelia burgdorferi as the detection objects to establish a double fluorescent quantitative PCR method for the diagnosis of Ehrlichiosis and Lyme disease.The specificity,sensitivity and stability,consistency with previous general PCR methods of this method were also studied.The results showed that,this method could only amplify the genes of Ehrlichia and Borrelia burgdorferi specifically,and it was negative for Leptospira,Rickettsia,Escherichia coli and Pseudomonas aeruginosa.The detection limit of nucleic acid was 1×10^(1) copies/μL,and the coefficient of variation of Tm value was less than 0.1%.The detection rate of Ehrlichia and Borrelia burgdorferi was 28%and 60%respectively.These results indicated that the method is specific,sensitive and stable,and can be used to detect both Ehrlichia and Borrelia burgdorferi simultaneously.It can be used for molecular epidemiological investigation,differential diagnosis and epidemic prevention and control of tick-borne diseases.

关 键 词：埃里希氏体 伯氏疏螺旋体 GROEL OSPA 双重荧光定量PCR 

分 类 号：R535[医药卫生—内科学] S852.61[医药卫生—临床医学]