摘要
本试验旨在建立快速诊断牛瑟氏泰勒虫的荧光定量PCR检测方法。以常规PCR方法扩增牛瑟氏泰勒虫P33基因,将其克隆至载体pMD-18T,然后进行质粒标准品的构建,以此为模板建立牛瑟氏泰勒虫P33基因TaqMan荧光定量PCR和SYBR Green荧光定量PCR检测方法。结果显示:2种方法均能检测到2.26~2.26×10^(8)copies/μL范围,特异性试验中与其他病原体均无特异性扩增曲线,批内和批间变异系数都小于4%。鉴于2种方法均具有较好的敏感性、特异性和重复性,适用于牛瑟氏泰勒虫P33基因的临床实验室鉴别。
This study aimed to establish real-time PCR for rapid detection of Theileria sergenti. The P33 gene was amplified from T. sergenti by PCR, and then inserted into pMD-18 T vector for use as plasmid standard. TaqMan real-time PCR and SYBR Green real-time PCR methods were set up by using the plasmid standard containing T. sergenti P33 gene. The results showed that both TaqMan and SYBR Green real-time PCR methods detected the copy number in the range of 2.26-2.26×10^(8)copies/μL, produced no amplification curve for other pathogens in the specific test, and had intra-assay and the inter-assay coefficients of variation less than 4%. Given their excellent sensitivity, specificity and reproducibility, TaqMan and SYBR Green real-time PCR methods can be applied for clinical laboratory identification of T. sergenti.
作者
王轶男
胡诗悦
陈洪涛
金春梅
金一
于龙政
WANG Yi-nan;HU Shi-yue;CHEN Hong-tao;JIN Chun-mei;JIN Yi;YU Long-zheng(Agricultural College,Yanbian University,Yanji 133002,China;Changchun Animal Disease Prevention and Control Centre,Changchun 130000,China;Government of Quangou Town,Xintai City,Shandong Province,Xintai 271207,China)
出处
《中国兽医杂志》
CAS
北大核心
2021年第11期33-37,共5页
Chinese Journal of Veterinary Medicine
基金
国家自然科学基金项目(32060807)
吉林省教育厅项目(JJKH20200522KJ)
高等学校学科创新引智计划资助(D20034)。