非洲猪瘟病毒实时荧光定量PCR检测方法的建立及应用

Construction and Application of Real-time Quantitative PCR for Detection of African Swine Fever Virus

本研究为了建立一套检测非洲猪瘟病毒(African swine fever virus,ASFV)的实时荧光定量PCR检测方法,根据GenBank公布的23株编码ASFV结构蛋白p72的基因序列,设计引物和探针,优化退火温度、Mg2+浓度和引物、探针浓度,生成标准曲线,进行重复性、敏感性、特异性试验,并检测样品。结果显示,优化的退火温度为60℃,Mg2+终浓度为4mmol/L,引物、探针终浓度分别为0.8、0.3μmol/L。重复性试验变异系数均小于1.3%,敏感性试验最低能够检测到10拷贝/μL的质粒,以其他5种猪病病毒和ASFV质粒为模板进行特异性试验,只有ASFV质粒出现扩增曲线。结果表明,建立的实时荧光定量PCR方法是快速、灵敏、特异的检测ASFV的方法。

In order to construct real-time quantitative PCR assay for detection of African swine fever virus, this study was based on 23 isolates of gene sequence which encodes ASFV structural protein p72 in GenBank, then designing primers and probe. The reaction conditions were optimized by using different annealing temperature, different Mg^2＋ concentrations, differ- ent primers and probe concentrations. The real-time PCR system could automatically generate standard curve, testing repeat- ability, sensitivity and specificity. Wild boar samples were detected by this assay. The results showed optimal annealing tem- perature was 60℃, optimal Mg^2＋ concentration was 4 mmol/L, optimal primers and probe concentration were 0.8 and 0.3 μmol/L. The coefficients of variation of repeatability test were less than 1.3 M, sensitivity tests could detect 10 copies/μL plas mids, the specificity was tested by detecting five others swine viruses and ASFV plasmid, only detection of ASFV plasmid ap- pears amplification curve. In conclusion, constructed real-time quantitative PCR assay was rapid,sensitive and specific assay for detection of ASFV.