摘要
该研究旨在建立一种快速、敏感和特异性检测新型鸭呼肠孤病毒（Novel duck reovirus,NDRV）的荧光定量PCR诊断方法。本实验以感染新型鸭呼肠孤病毒的鸭组织RNA提取物为模板,根据Gen Bank数据库中呼肠孤病毒S1基因全序列,设计合成了一对特异性引物,PCR扩增基因片段,将其克隆至p ET-30a载体,重组质粒测序并进行同源性分析;以阳性质粒为模板,建立SYBR Green II荧光定量PCR检测方法,并进行敏感性和特异性检测。经测序证实扩增片段与预期目的片段相符,所建立的SYBR Green II荧光定量PCR检测S1的反应在101～108 copies/u L之间具有良好的线性关系,反应的检出下限为10 copies/μL,而H5型禽流感病毒、H9型禽流感病毒、鸡传染性支气管炎病毒、C型鸭肝炎病毒、新城疫病毒、鹅细小病毒、鸭瘟病毒等病毒的检测为阴性,表明该方法敏感、特异。本研究成功建立了SYBR Green II荧光定量PCR检测新型鸭呼肠孤病毒的方法,为新型鸭呼肠孤病毒致病机制和机体免疫保护机制的研究提供了技术平台。
To develop a fast, sensitive, specifi c SYBR Green II fl uorescent quantitative PCR assay for detecting Novel duck reovirus（NDRV） infection, S1 gene was amplifi ed in RT-PCR from the NDRV infected-duck tissues, and cloned into p ET-30 a vector. The resulting recombinant plasmid was used as the template for making a standard curve. Subsequently, the sensitivity and specifi city of the SYBR Green II fl uorescent quantitative PCR assay that was developed were determined. The results showed that the NDRV real-time PCR assay had a dynamic range of detection between 101 and 108 copies/μL with a sensitivity of 10 copies/μL. There was no cross reaction with H5 subtype Avian infl uenza virus, H9 subtype Avian infl uenza virus, Infectious bronchitis virus, Duck hepatitis virus type C, New castle disease virus, Goose parvovirus and Duck viral enteritis. In conclusion, a SYBR Green II fl uorescent quantitative PCR assay has been developed for quantifi cation of NDRV, which can be sued for investigating the pathogenesis of NDRV.
出处
《中国动物传染病学报》
CAS
北大核心
2016年第1期7-14,共8页
Chinese Journal of Animal Infectious Diseases
基金
上海市科委创新项目(13391901600)
国家自然科学基金项目(31270194)
