火鸡组织滴虫荧光定量PCR检测方法的建立及初步应用  

Establishment and application of real-time fluorescence quantitative PCR for the detection of Histomonas meleagridis

作　　者：陈乔光 孔令明 戎杰 候照峰 刘丹丹[1,2] 陶建平 许金俊[1,2] CHEN Qiao-guang;KONG Ling-ming;RONG Jie;HOU Zhao-feng;LIU Dan-dan;TAO Jian-ping;XU Jin-jun(College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,l^iigzhou 225009,China)

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]江苏动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国预防兽医学报》2020年第10期1014-1018,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金面上项目(31772727);扬州市重点研发计划(现代农业)项目(YZ2017051);江苏高校优势学科建设二期工程资助项目;扬州大学“青蓝工程”资助项目(2018年度);扬州大学专业学位研究生实践创新计划项目(XSJCX18_075)。

摘　　要：为建立火鸡组织滴虫的检测方法,本研究采用PCR扩增火鸡组织滴虫的18S rRNA基因片段,构建质粒标准品作为模板,建立火鸡组织滴虫的SYBR Green I荧光定量PCR检测方法,并进行了初步临床应用。结果显示:所建立的SYBR Green I荧光定量PCR检测方法Cq值与质粒标准品在4.6×106拷贝/μL^4.6×10^10拷贝/μL范围内呈良好的线性关系,相关系数为0.9939,斜率为-3.32;该方法对弓形虫、柔嫩艾美耳球虫、毒害艾美耳球虫、犬巴贝斯虫等常见原虫基因组DNA无扩增,对质粒标准品的检测下限可达4.6×10^2拷贝/μL,组内和组间变异系数分别为0.09%~0.33%和0.08%~0.21%,敏感性、特异性和重复性均较好。该方法对147份临床疑似火鸡组织滴虫感染样品的虫体总检出率高于普通PCR方法,其中对靶器官肝脏和盲肠的检出率(71.4%、66.7%)明显高于普通PCR方法的检出率(57.1%、52.4%)。本研究建立的火鸡组织滴虫SYBR Green I荧光定量PCR检测方法为临床开展组织滴虫病的诊断和流行病学调查提供了检测手段。To establish a sensitive and specific method for detecting the Histomonas meleagridis,the 18S rRNA gene fragment of H.meleagridis was amplified and cloned in a plasmid to establish a SYBR Green I fluorescence quantitative PCR method(qPCR)for detection of H.meleagridis.A pilot study of clinical samples by using the method was also conducted in the study.The results showed that the qPCR’s Cq value was linearly related with the concentration of the template in the range of 4.6×106-4.6×10^10 copies/μL,with a correlation coefficient of 0.9939 and a slope of-3.32.The specificity assay showed no amplification could be observed for the genomic DNA of common protozoa such as Toxoplasma gondii,Eimeria necatrix,Eimeria tenella,and Babesia canis.The detection limit could reach 4.6×10^2 copies/μL.The intra-group and inter-group coefficients of variation were 0.09%-0.33%and 0.08%-0.21%respectively.The total detection rate of H.meleagridis in 147 clinical suspected samples was higher than that of the conventional PCR method.Specifically,the detection rate in the target organ of liver and cecum(71.4%,66.7%)was significantly higher than that of the ordinary PCR method(57.1%,52.4%).The above results provide a useful detection tool for clinical diagnosis and epidemiological investigation of H.meleagridis infection in chicken.

关 键 词：火鸡组织滴虫 18S rRNA基因 PCR 荧光定量PCR 检测 

分 类 号：S855.9[农业科学—临床兽医学]