华支睾吸虫囊蚴SYBR GreenⅠ实时荧光定量PCR方法的建立  

Establishment of SYBR Green I real-time fluorescent quantitative PCR method for Clonorchis sinensis metacercariae

作　　者：苑淑贤[1] 侯绪森 宫鹏涛 郭衍冰[1] 董航[1] 孙兴忠 王楠 曹利利[1] YUAN Shuxian;HOU Xusen;GONG Pengtao;GUO Yanbing;DONG Hang;SUN Xingzhong;WANG Nan;CAO Lili(Jilin Academy of Animal Husbandry and Veterinary Medicine,Changchun 130062,China;College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林省畜牧兽医科学研究院,长春130062 [2]吉林大学动物医学学院,长春130062

出　　处：《黑龙江畜牧兽医》2023年第17期7-12,139,140,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：吉林省科技发展计划技术攻关项目(20190303109SF)。

摘　　要：为了建立一种特异且敏感的华支睾吸虫囊蚴SYBR GreenⅠ实时荧光定量PCR方法,试验针对华支睾吸虫线粒体细胞色素c氧化酶亚基1(CSCOⅠ)基因设计特异性引物,筛选最佳引物浓度和退火温度,绘制标准曲线,优化扩增体系和程序建立SYBR GreenⅠ实时荧光定量PCR方法,并评价该方法的敏感性、特异性、重复性及符合性。结果表明:最佳引物浓度为750 nmol/L,最佳退火温度为57℃。标准曲线的斜率为-3.21,截距为35.23,相关系数(R^(2))为0.9823。该方法的最低检出限为1×10^(2)copies/μL;批间和批内变异系数分别为1.53%~2.59%和0.36%~0.76%,表现出良好的重复性;仅标准阳性质粒、华支睾吸虫囊蚴及肝片吸虫出现特异性扩增曲线,具有较好的特异性;与传统压片镜检法比较符合率为96%。说明试验建立的SYBR GreenⅠ实时荧光定量PCR方法敏感、特异、稳定,为后续开展淡水鱼感染华支睾吸虫囊蚴的流行病学调查提供了技术支持。To establish a specific and sensitive SYBR Green I real-time fluorescent quantitative PCR method for metacercariae of Clonorchis sinensis,the specific primers for the mitochondrial cytochrome c oxidase submit I of Clonorchis sinensis were designed;the standard curve was drawn;the amplification system and procedure were optimized,and the SYBR CGreen I real-time fluorescence quantitative PCR method was established,and its sensitivity,specificity,repeatability and compliance were evaluated.The results showed that the optimal concentration of primers was 750 nmol/L and the optimal annealing temperature was 57 C.The slope of the standard curve was-3.21;the intercept was 35.23,and the correlation coefficient R?was 0.9823.The minimun detection limit of this method was 1×10^(2)copies/μL.The inter-and intra-batch coefficients of variation were 1.53%-2.59%and 0.36%-0.76%,respectively,showing good repeatability.Only the standard positive plasmids C.sinensls metacercarae and Fasciola hepatica showed specific amplification curves,with good specificity.The coincidence rate was 96%compared with the traditional compression microscopy method.It indicated that the SYBR Green I real-time fluorescence quantitative PCR method was sensitive,specific and stable,which provided technical support for the subsequent epidemiological investigation of C.sinensis metacercariae infection in freshwater fish.

关 键 词：华支睾吸虫 囊蚴 CSCOⅠ SYBR GreenⅠ 实时荧光定量PCR 

分 类 号：S855.9[农业科学—临床兽医学]