SFTSV实时荧光定量RT-PCR检测方法的建立及应用  

Establishment and application of real-time fluorescent quantitative RT-PCR for the detection of SFTSV

作　　者：于志亚 杨鸣发 马云云 康洪涛 姜骞[2] 刘家森[2] 李广兴[1] 曲连东[1,2] YU Zhi-ya;YANG Ming-fa;MA Yun-yun;KANG Hong-tao;JIANG Qian;LIU Jia-sen;LI Guang-xing;QU Lian-dong(College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;State Key Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute,China Academy of Agricultural Sciences,Harbin 150001,China)

机构地区：[1]东北农业大学动物医学院,黑龙江哈尔滨150030 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《中国兽医科学》2022年第3期292-297,共6页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2017YFD0501801)。

摘　　要：为建立快速、灵敏的发热伴血小板减少综合征病毒(SFTSV)检测方法,根据GenBank中发热伴血小板减少综合征病毒RNA依赖性RNA聚合酶(RdRp)基因的保守序列设计引物和探针。通过对反应体系和反应条件的优化,建立了TaqMan实时荧光定量RT-PCR检测方法。当质粒标准品浓度为4.22×10^(2)~4.22×10^(9)copies/μL时,荧光定量标准曲线C;值与模板浓度呈良好的线性关系,相关系数可达0.9998。该方法对发热伴血小板减少综合征病毒最低检测限为4.22×10^(1)copies/μL,灵敏度是常规RT-PCR方法的100倍;该方法可特异性地检测出SFTSV,而对同科其他病毒检测结果均为阴性;批内、批间变异系数均小于2%。对采集的200只蜱进行检测,阳性检出率为3.5%,而常规RT-PCR方法的阳性检出率为2%。研究结果表明,本研究建立的TaqMan实时荧光定量RT-PCR检测方法特异性强、灵敏性高、重复性好,为发热伴血小板减少综合征的检测提供了技术手段。In order to establish a rapid and sensitive detection method for severe fever with thrombocytopenia syndrome virus(SFTSV),primers and probe were designed based on the highly conserved region of SFTSV RNA dependent RNA polymerase.Through optimization of reaction components and conditions,a real-time RT-PCR assay was successfully developed for detection of SFTSV.The results showed that the standard curve have a good linear relationship between C;value and the Rd Rp gene concentrations of the standard template at a range of 4.22×10^(2)copies/μL to 4.22×10^(9) copies/μL,and the correlation coefficient(r^(2))was 0.9998.The detection limit of the assay was 4.22×10^(1) copies/μL,which was 100 times more sensitive than the conventional RT-PCR.The results showed that the detection of SFTSV was specific,while other viruses in the same family were detected negative by this method.And the variable coefficient of intraand inter-reproducibility assay was below 2%.The 200 ticks collected were tested using the established real-time RT-PCR and the positive rate was 3.5%,while the positive rate was only 2%when these samples were detected by the conventional RT-PCR.The Taq Man real-time RT-PCR detection method has high specificity and sensitivity,good repeatability.The result provided a useful tool for clinical detection of SFTSV.

关 键 词：发热伴血小板减少综合征病毒 TAQMAN探针 实时荧光定量RT-PCR 

分 类 号：S852.659.5[农业科学—基础兽医学]