鼻气管鸟疫杆菌SYBRGreenⅠ荧光定量PCR检测方法的建立及应用技术报告.docVIP

鼻气管鸟疫杆菌SYBR GreenⅠ荧光定量PCR检测方法的建立及应用 摘要:建立鼻气管鸟疫杆菌SYBR Green I荧光定量PCR检测方法并应用于临床样品检测鼻气管鸟杆菌（GenBank登录号：JF810495.1）基因序列设计1对特异性引物经PCR扩增回收目的片段与pMD18-T Vector连接后转化到基因工程菌DH5α中提取重组质粒经PCR、酶切及测序鉴定后筛选阳性质粒作为模板SYBR GreenⅠ荧光定量PCR标准曲线，并做敏感性试验、重复性试验和特异性试验。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Tm值在8-83.2℃之间，灵敏度为×102拷贝/μL，特异性重复性。本试验建立了检测鼻气管鸟疫杆菌的SYBR GreenⅠ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。 关键词: 鼻气管鸟疫杆菌SYBR Green I；实时荧光定量PCR Development and application of SYBR GreenⅠreal-time quantitative PCR technique for Ornithobacterium rhinotracheale , DIAO You-xiang*, TANG Yi, JU Xiao-jun (College of Animal Science and Technology, Shandong Agricultural University, Tai’an, Shandong, 271018, China) Abstract:【Objective】Ornithobacterium rhinotracheale by SYBR Green I fluorescence quantitative PCR detection method and to application in clinical samples detection. 【】A pair of specific primers was designed according to the Ornithobacterium rhinotracheale（GenBank login number: JF810495.1）gene sequence. The target 214bp fragment was amplified in PCR gene amplification, and inserted into pMD18-T vector after being purified. Then the restructured vectors were transformed to E.coli DH5α. Positive plasmid was extracted as template to establish SYBR-Green Ⅰ fluorescence quantitative PCR standard curve after PCR identifying, enzyme cutting and sequencing. 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 template concentrationhe standard curve linear relationship was more than 0.99. The Tm was 81.3-83.2 and the sensitive degree is 1.44×102 copies per μL, Specific results showed that only amplification curve of Ornithobacterium rhinotracheale can be detectioned, repeatability test indicated the variation coefficient is less than 0.7%; clinical sample detection rate was 100%. 【】A SYBR Green Ⅰ fluorescent quantitative PCR assay for detecting Ornithobacterium