摘要
为建立快速检测HoBi瘟病毒的方法,根据GenBank提交的HoBi基因序列,使用软件设计2对引物和1条探针,通过体外转录获得定量标准品RNA。通过对荧光定量PCR将各反应条件进行优化,建立了HoBi瘟病毒一步法荧光定量PCR检测方法。该方法具有良好的特异性、敏感性和重复性,对牛病毒性腹泻病毒-1（BVDV1）-NADL、BVDV2-JN、猪瘟病毒（CSFV）、羊边界病毒（BDV）、小反刍兽疫病毒（PPRV）、牛轮状病毒（RV）和伪狂犬病病毒（PRV）核酸检测为阴性;最低核酸检测量为103拷贝/μL;组内变异系数和组间变异系数均小于2%。结果表明,所建立的HoBi瘟病毒一步荧光定量PCR检测方法具有特异性强、敏感性高、重复性好和快速等特点,可用于HoBi瘟病毒感染的流行病学调查和早期诊断。
To develop real-time fluorescent quantitative PCR for detection of HoBi-like pestivirus. Two pairs of primers and a TaqMan probe for conservative sequence of HoBi-like pestivirus were selected to develop TaqMan real-time PCR assay. The standard RNA used in real-time PCR was prepared by vitro transcript. TaqMan real-time fluorescent quantitation PCR for detection of HoBi- like pestivirus was established through optimizing the conditions. The real-time PCR had better specificity without cross reaction to BVDV1-NADL, BVDV2-JN, CSFV, BDV, PPRV, RV and PRV. The detection limite of HoBi-like real-time PCR was 103copies/μL. The coefficient of varia- tion of within group and between groups was all less than 2%. The result indicated that estab- lished HoBi-like pestivirus TaqMan real-time PCR is a specific, sensitive and repetitive method for epidemiological investigation and detection of HoBi-like pestivirus.
作者
时坤
李男
刁乃超
李大帅
姚泽慧
田甜
王春凤
杜锐
SHI Kun1 , LI Nan2 , DIAO Nai-chao1 , LI Da-shuai1 , YAO Ze-hui1, TIAN Tian1 , WANG Chun-feng3 , DU Rui1(1. College of Chinese Medicinal Materials, J ilin Agricultural University, Changchun 130118, China ; 2. Changchun Academ y of Agricultural Sciences, Changchun 130111, China ; 3. Col- lege of Animal Science and Technology ,Jilin Agricultural University ,Changchun 130118 ,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2018年第11期2051-2057,共7页
Chinese Journal of Veterinary Science
基金
国家自然科学基金面上资助项目(31372436,31672577)
