【摘要】：本研究參考GenBank中山羊痘病毒基因組序列(AY077836)和綿羊痘病毒基因組序列(AY077832),針對A29L基因的核苷酸序列,設計1對特異性引物和兩條TaqMan探針,同時制備重組質粒標準品。通過退火溫度、引物濃度和探針濃度的篩選及優(yōu)化,將標準陽性質粒10倍倍比稀釋后,作實時熒光定量PCR標準曲線,建立了檢測羊痘病毒的實時熒光定量PCR方法,.對所建立的實時熒光定量PCR方法的特異性、靈敏性和重復性進行了評價,并用此方法對臨床樣品進行了檢測。結果顯示：建立的實時熒光定量PCR的反應條件為退火溫度58.4℃,引物濃度400 nmol/L,探針濃度200 nmol/L。該診斷方法與其它四種病毒不發(fā)生交叉反應,并且山羊痘病毒和綿羊痘病毒之間也不發(fā)生交叉反應。該方法的重復性試驗變異系數均低于2%,并且雙重實時熒光定量PCR最低濃度檢測限分別為470fg和440fg。對標準陽性模板進行定量檢測,生成的標準曲線相關系數分別為0.995和0.997。結果表明所建立的方法具有良好的特異性、靈敏性、穩(wěn)定性,可以對GTPV和SPPV進行準確的定量檢測。對19份臨床樣品進行檢測,GTPV陽性有14份,SPPV陽性有4份,GTPV和SPPV混合感染有1份。
[Abstract]:According to the genomic sequence of GenBank Zhongshan sheep pox virus (AY077836) and sheep pox virus (AY077832), a pair of specific primers and two TaqMan probes were designed for the nucleotide sequence of A29L gene. Through the screening and optimization of annealing temperature, primer concentration and probe concentration, a real-time fluorescent quantitative PCR method for the detection of sheep pox virus was established by diluting the standard positive plasmid by 10 times specific dilution and using real-time fluorescence quantitative PCR curve. The specificity, sensitivity and reproducibility of the established real-time fluorescent quantitative PCR method were evaluated, and the clinical samples were detected by this method. The results showed that the reaction conditions of real-time fluorescent quantitative PCR were: annealing temperature 58.4 鈩，

本文編號：2218842