猪细小病毒6型SYBR Green I real-time PCR检测方法的建立  

Development of a SYBR GreenⅠreal-time PCR method for detection of PPV6

作　　者：孙文超[1,2] 刘立明[1] 赵翠青[1] 汪伟 赵冠宇[2,4] 曹亮 张赫[2] 庄忻雨[2] 韩继成 鲁会军[1,2] 金宁一 SUN Wen-chao1,2 , LIU Li-ming1 , ZHAO Cui-qing1 , WANG Wei2 , ZHAO Guang-yu2,4 , CAO Liang2,3 , ZHANG He2,ZHUANG Xin-yu2, HAN Ji-cheng2,LU Hui-jun1,2 ,JIN Ning-yi1,2(1. Institute of Virology ,Wenzhou University ,Wenzhou , Zhejiang 325035, China ; 2. Institute of Military Veterinary, The Academy of Military Medical Sciences, Changchun 130122, China ;3. College of Anitaal Science and Technology, Jilin Agriculture University, Changchun 130118, China ; 4. College of Veterinary Medicine , Jilin University ,Changchun 130062 ,Chin)

机构地区：[1]温州大学病毒学研究所,浙江温州325035 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]吉林农业大学动物科技学院,吉林长春130118 [4]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2018年第3期442-445,452,共5页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划资助项目(2017YFD0500101)

摘　　要：采用PCR方法扩增PPV6保守区域基因252bp,将其克隆到pClon007载体,构建重组质粒作为标准阳性质粒。以质粒模板建立PPV6的SYBR GreenⅠreal-time PCR检测方法,并进行敏感性、特异性和重复性等验证。结果表明,重组质粒特异性好;建立的real-time PCR方法Ct值与标准品模板在8.80×10^9-8.80×10^1 copies/μL范围内呈良好的线性关系,相关系数为0.993,斜率为-4.320。该方法检测灵敏度可达8.80×10^1 copies/μL。该方法对JEV、PRV、PRRSV、PCV2、FMDV等猪常见病原检测均无特异性扩增;对临床样品的检测结果表明,所建立的荧光定量PCR阳性检测率为40.63%。本试验成功建立了PPV6SYBR Green I real-time PCR检测方法,可实现对PPV6快速灵敏的诊断。This study was to establish a real-time fluorescence quantitative PCR method which can quickly and accurately detect PPV6. The conserved region of PPV6 gene was amplified by PCR and cloned into pClon007 vector. The resulted pClon007-PPV6 plasmid DNA was used as template to optimize assay condition of developing a SYBR Green Ⅰ real-time PCR for detection of PPV6. The standard curve produced a good linear relationship between Ct value and initial amounts of total DNA at a range of 8.80 × 10^9 -8.80 × 10^1 copies per microlitre,and the correlation coefficient and slope were 0. 993 and -4. 320, respectively. The sensitivity of this method was 8.80 × 10^1 copies/gL. The specificity assay showed no amplification of PRRSV, PCV2 ,JEV, PRV, FMDV. All standards are specific narrow melting peak at 84.51℃. The positive rates were 40.63% in detected samples. The results indicated that SYBR Green I method was a rapidly specific and sensitive method for the detection of PPV6.

关 键 词：猪细小病毒6型 SYBR Green I 荧光定量PCR 

分 类 号：S858.28[农业科学—临床兽医学]