B亚型禽偏肺病毒SYBRGreenI荧光定量PCR检测方法的建立及应用

根据GenBank登录的B亚型禽偏肺病毒（JN224985．1）F基因序列设计1对特异性引物，经RT—PCR扩增出214bp的目的片段。回收目的片段并与pMD18-T载体连接后转化到基因工程菌DH5a中，提取重组质粒，经PCR、酶切及测序鉴定后，筛选出阳性质粒作为模板，建立SYBRGreenI荧光定量PCR标准曲线，并做敏感性试验、特异性试验和重复性试验。结果表明，标准曲线循环阈值与模板浓度呈良好的线性关系，产物T值在83．0～83．7℃之间，灵敏度为7．9×10^2拷贝／μL，特异性和重复性较好。本试验建立了检测B亚型禽偏肺病毒的sYBRGreenI荧光定量PCR方法，为该病的早期诊断以及感染程度的定量分析奠定了基础。

Development and application of SYBR Green I real-time quantitative PCR technique for avian metapneumovirus subgroup B

A pair of specific primers was designed according to the avian metapneumovirus subgroup B strain （JN224985.1）F conserved gene sequence available in GenBank. The target 214 bp fragment was amplified in RT-PCR experiments, and inserted into pMD18-T vector after being purified. Then the restructured vectors were transformed to E. coli DH5a. After PCR identifying, enzyme cutting and sequencing, positive plasmid was extracted as template to establish SYBR Green I fluorescence quantitative PCR standard curve. Sensitivity,reproducibility and specificity of the method were determined. The results indicated that standard curve established by recombinant plasmid showed a good linear relationship between threshold cycle and templat concentration,the T was 83.0-83.7℃ and the sensitive degree is 7.9×10^2 copies per/μL,and the quantitative PCR was highly reproducibility and more specificity than traditional PCR. A SYBR Green I fluorescent quantitative RT-PCR assay for detecting F gene of aMPV subgroup B was developed. It lays for the basis of the early detection and quantitative analyzing of the aMPV infection degree.