Development of a SYBR GreenⅠReal-time PCR method for detection of goat Th1/Th2 cytokines
SUN Wen-chao1,2,5, ZHANG Shi-heng1,5, YI Chi-zhe2,4, HUANG Hai-xin1, ZHANG Hong-yun3, WANG Wei5, CAO Liang5, BI Jing-shan2, ZHENG Min2, LU Hui-jun1,5, WEI Xian-kai2, SU Jiao-xiu2, ZHAO Cui-qing1, CHEN Zheng1, WANG Mao-peng1
1. Institute of Virology,Wenzhou University, Wenzhou 325035, China; 2. Guangxi Center for Animal Disease Control and Prevention, Nanning 530001, China; 3. Yulin Center for Animal Disease Control and Prevention,Yulin 537000, China; 4. College of Animal Science and Technology, Guangxi University, Nanning 530004, China; 5. Institute of Military Veterinary,The Academy of Military Medical Sciences, Changchun 130122, China
Abstract：This study was to establish a real-time fluorescence quantitative PCR method for detection of goat's Th1/Th2 cytokines.The conserved region of goat's Th1/Th2 cytokines gene were amplified and cloned into pMD18-T vector.The plasmid DNA was used as template to optimize assay condition of developing a SYBR GreenⅠReal-time PCR for Th1/Th2 cytokines detection.The results showed that the Ct value of Th1/Th2 cytokines produced a good linear relationship, which R2 were all greater than 0.985.All standards were specific narrow melting peak and reproducetire and specific.The established assays were successfully used to detect IL-4 and IFN-γmRNA expression levels in peripheral blood mononuclear cells (PBMCs) in sheep experimentally infected with recombinant goat poxstrain (△N1L strain) and AV41 strain, both could stimulate Th1/Th2 cytokines with no difference in vivo. In this study, real-time fluorescence quantitative detection of goat Th1/Th2 cytokines was established, which laid the foundation for the research of goat pox genetic engineering vaccine.
孙文超, 张世亨, 易驰喆, 黄海鑫, 张红云, 汪伟, 曹亮, 闭璟珊, 郑敏, 鲁会军, 韦显凯, 苏姣秀, 赵翠青, 陈征, 王茂鹏. 山羊Th1/Th2细胞因子实时荧光定量检测方法的建立[J]. 中国人兽共患病学报, 2019, 35(2): 138-144.
SUN Wen-chao, ZHANG Shi-heng, YI Chi-zhe, HUANG Hai-xin, ZHANG Hong-yun, WANG Wei, CAO Liang, BI Jing-shan, ZHENG Min, LU Hui-jun, WEI Xian-kai, SU Jiao-xiu, ZHAO Cui-qing, CHEN Zheng, WANG Mao-peng. Development of a SYBR GreenⅠReal-time PCR method for detection of goat Th1/Th2 cytokines. Chinese Journal of Zoonoses, 2019, 35(2): 138-144.
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2019, Vol. 35 
