摘要
根据GenBank公布的副鸡禽杆菌hagA基因序列设计特异性引物,经PCR扩增出了123bp的片段。产物经回收与pMD18-T Vector连接后转化到基因工程菌DH5α中,提取重组质粒,经PCR及测序鉴定后,作为阳性模板建立SYBR GreenⅠ荧光定量PCR标准曲线,并做敏感性试验、特异性试验、重复性试验和临床检测应用。结果显示,标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数R2=0.999 8;溶解曲线特异,产物Tm在81.3~81.5℃之间;最低检测限为0.18拷贝/μL,其敏感性比常规PCR至少高100倍;该方法不与其细菌发生交叉反应,呈现很好的特异性;重复性试验中,批内和批间变异系数均小于1%,说明该方法重复性很好;临床副鸡禽杆菌样本的检测结果表明所建立的荧光定量PCR方法的检测率明显高于常规PCR方法,为该病的早期快速诊断,并定量分析副鸡禽杆菌感染程度奠定了基础。
A pair of primers were designed specificly for hagA gene of Apg according to the published nucleotide sequence(GenBank).The about 123 bp expected fragment was amplified using PCR from extracted DNA.The PCR product of target gene was cloned into pMD18-T vector and then transfered into DH5 a.Positive recombinant plasmid was screened through gel electrophoresis and sequencing,then it was used as a positive quantitative template to establish a standard curve,along with sensitivity test,specificity test,repeatability test and clinical applications.The results showed that the threshold of standard curve and template concentration showed a good linear correlation,correlation coefficient was 0.999 8;the sensibility of this assay attained 0.18copies/μL of plasmid DNA,which was 100 times higher than that of routine PCR assay,Tm value was between 81.3℃ and 81.5℃.The CVof the intra and inter assay was no more than 1%,indicating that the developped fluorescence quantitive PCR assay is stable,reproducible and specific.Compared to rountine PCR,the developed method could be used for the rapid diagnosis and quantitative detection of Avibacterium paragallinaruminfection degree.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2015年第1期57-62,共6页
Chinese Journal of Veterinary Science
