摘要
目的建立1种特异、灵敏、高效的检测IBRV gE基因的荧光定量PCR方法,并为鉴别IBRV野毒株和gE基因缺失疫苗株的感染提供技术手段。方法根据IBRV gE基因保守序列,设计一对引物,建立荧光定量PCR反应体系和反应条件,利用10倍梯度稀释病毒DNA进行荧光定量PCR扩增,以检测本试验建立的荧光定量PCR的灵敏度。用IBRV、牛病毒性腹泻病毒、牛轮状病毒、MDBK细胞、牛血清等进行特异性检测。用模拟样品进行临床应用试验。结果本试验建立的荧光定量PCR方法,其灵敏度约为70 DNA拷贝/μL,除了IBRV从其他病毒和样品未扩增出曲线,临床模拟样品检测结果为0.03TCID50。结论本试验建立的荧光定量PCR方法具有特异性强、灵敏性高、快速,低污染等优点,因此临床上可以用于IBRV的检测和病原学调查。
Purpose To establish the Real-time PCR for detection of IBRV gE gene,and to distinguish between the IBRV wild strain and gE deletion IBRV vaccine strain.Methods A pair of primer were designed based on the IBRV gE gene conservative region.The real-time PCR method was developed by the optimization of the reaction conditions.The viral 10-fold serial dilution DNA samples and IBRV,BVDV,bovine rotavirus,MDBk cells and bovine serum were used to test the sensitivity and specificity of the method.Result The real-time PCR method had been developed,and it could detect 70 copies/μL.The result of the clinical simulation detection was 0.03TCID50.Conclusion： This method characterized by high specific,sensitive,short testing time and lower pollution.In conclusion,this method could be used for rapid quantitative detection of IBRV in clinical samples.
出处
《内蒙古农业大学学报（自然科学版）》
CAS
北大核心
2012年第3期5-8,共4页
Journal of Inner Mongolia Agricultural University(Natural Science Edition)
基金
国家自然科学基金项目(30860202)
内蒙古自然科学基金项目(2010BS0407)
