【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(duì)A29L基因的核苷酸序列,設(shè)計(jì)1對(duì)特異性引物和兩條TaqMan探針,同時(shí)制備重組質(zhì)粒標(biāo)準(zhǔn)品。通過(guò)退火溫度、引物濃度和探針濃度的篩選及優(yōu)化,將標(biāo)準(zhǔn)陽(yáng)性質(zhì)粒10倍倍比稀釋后,作實(shí)時(shí)熒光定量PCR標(biāo)準(zhǔn)曲線(xiàn),建立了檢測(cè)羊痘病毒的實(shí)時(shí)熒光定量PCR方法,.對(duì)所建立的實(shí)時(shí)熒光定量PCR方法的特異性、靈敏性和重復(fù)性進(jìn)行了評(píng)價(jià),并用此方法對(duì)臨床樣品進(jìn)行了檢測(cè)。結(jié)果顯示：建立的實(shí)時(shí)熒光定量PCR的反應(yīng)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(yīng),并且山羊痘病毒和綿羊痘病毒之間也不發(fā)生交叉反應(yīng)。該方法的重復(fù)性試驗(yàn)變異系數(shù)均低于2%,并且雙重實(shí)時(shí)熒光定量PCR最低濃度檢測(cè)限分別為470fg和440fg。對(duì)標(biāo)準(zhǔn)陽(yáng)性模板進(jìn)行定量檢測(cè),生成的標(biāo)準(zhǔn)曲線(xiàn)相關(guān)系數(shù)分別為0.995和0.997。結(jié)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(duì)GTPV和SPPV進(jìn)行準(zhǔn)確的定量檢測(cè)。對(duì)19份臨床樣品進(jìn)行檢測(cè),GTPV陽(yáng)性有14份,SPPV陽(yáng)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(hào)：2218842