体外转录法制备新城疫病毒荧光定量RT-PCR标准阳性模板

Preparation of Standard Positive Template with Transcription in Vitro for Real-time Quantitative Reverse Transcription PCR (qRT-PCR) for the Detection of Newcastle Disease Virus

本研究针对新城疫病毒M基因设计了1对荧光定量RT-PCR引物和Taqman探针,在上游引物的5′端引入T7启动子,采用RT-PCR方法获得了体外转录模板,并对转录产物RNA的浓度及D值进行了测定。线性试验和特异性试验结果表明,该模板具有良好的线性范围和特异性,模板稀释度为2.95×104～2.95×108拷贝/L时,相关系数为0.998。该模板稳定性好,-80℃保存30 d后无显著变化。浓度为2.95×104～2.95×108拷贝/μL的标准品的变异系数分别为0.20%、0.28%、1.00%、0.54%、0.52%,表明以此模板进行荧光定量PCR具有较好的重复性。

Primers and probe for qRT-PCR were designed based on the matrix protein gene sequence of Newcastle disease virus（NDV）,and T7 promoter was added to the 5＇ end of the forward primer.RT-PCR was used to get the template for transcription in vitro,and the concentration and the D value of the RNA were determined.The results of linear and specific test indicated that the template showed good linearity and specificity.The correlation coefficient was 0.998 when the template was diluted to 2.95×10^4 to 2.95×10^8 copies/μL.Storing at-80 ℃,the template still worked well after 30 days.And the coefficient of variation value of 2.95×10^4 to 2.95×10^8 copies/μL standard positive template were 0.20%,0.28%,1.00%,0.54% and 0.52% respectively,showing an excellent repetitiveness of the template.