發(fā)布時(shí)間：2018-08-11 13:23

【摘要】：本實(shí)驗(yàn)為了建立一種快速準(zhǔn)確、特異性強(qiáng)、敏感性高的鑒別CSFV和BVDV的雙重實(shí)時(shí)熒光PCR方法,根據(jù)GenBank上已發(fā)表的豬瘟病毒和牛病毒性腹瀉-黏膜病1型病毒的全基因序列,進(jìn)行對(duì)比分析,分別設(shè)計(jì)合成兩對(duì)能特異性擴(kuò)增CSFV和BVDV1的2對(duì)引物及2條探針。制備標(biāo)準(zhǔn)陽(yáng)性質(zhì)粒DNA模板,對(duì)雙重實(shí)時(shí)熒光定量PCR反應(yīng)參數(shù)和反應(yīng)條件進(jìn)行優(yōu)化,并制備實(shí)時(shí)熒光PCR標(biāo)準(zhǔn)曲線,評(píng)價(jià)所建立的雙重實(shí)時(shí)熒光定量PCR反應(yīng)體系的敏感性、特異性、重復(fù)性,并用本試驗(yàn)建立的鑒別CSFV和BVDV1的雙重實(shí)時(shí)熒光定量PCR方法對(duì)86份臨床樣品進(jìn)行檢測(cè)。結(jié)果顯示：引物濃度400 nmol/L,探針濃度為300 nmol/L,退火溫度58℃C時(shí)所建立的雙重實(shí)時(shí)熒光定量PCR方法達(dá)到最佳的反應(yīng)效果。并且此診斷方法可以特異的檢測(cè)CSFV和BVDV1,并能與綿羊痘病毒、山羊痘病毒、羊口瘡病毒、小反芻獸疫病毒、牛傳染性鼻氣管炎病毒區(qū)分。另外,此建立的方法Ct值的變異系數(shù)均小于0.01,CSFV和BVDV1的最小檢出量分別為1.04x102拷貝數(shù)／μL、1.15×102拷貝數(shù)/μL。表明所建立的雙重實(shí)時(shí)熒光PCR方法具有較強(qiáng)的穩(wěn)定性、較高的敏感性。運(yùn)用所建立的PCR方法檢測(cè)臨床樣品,可以快速、準(zhǔn)確的檢測(cè)CSFV和BVDV1。
[Abstract]:In order to establish a rapid, accurate, specific and sensitive dual real-time fluorescent PCR method for the identification of CSFV and BVDV, the whole gene sequence of CSFV and BVD-1 virus published on GenBank was established. Two pairs of primers and two probes for specific amplification of CSFV and BVDV1 were designed and synthesized. The standard positive plasmid DNA template was prepared to optimize the reaction parameters and reaction conditions of double real-time fluorescent quantitative PCR, and the standard curve of real-time fluorescent PCR was prepared to evaluate the sensitivity and specificity of the dual real-time fluorescent quantitative PCR reaction system. The reproducibility of 86 clinical samples was detected by double real time fluorescence quantitative PCR method which was established in this study to identify CSFV and BVDV1. The results showed that the double real-time PCR method with primer concentration of 400 nmol / L, probe concentration of 300 nmol / L and annealing temperature of 58 鈩，

本文編號(hào)：2177118