CAJ | 학술논문

根据GenBank登录的猪流行性腹泻病毒（PEDV）毒株（登录号KJ960180）的M基因保守序列，设计1对扩增片段大小为299 bp的特异性引物，经PCR扩增、克隆、测序鉴定后，提取质粒作为阳性标准品，建立了PEDV的SYBR GreenⅠ荧光定量RT-PCR检测方法。该方法在1.21×103～1.21×108拷贝/μL范围内呈现良好的线性，相关系数（R2）为0.999，扩增效率为99%，扩增产物的熔解曲线为单个特异峰，产物Tm值为85.5～86℃，最低检测限为1.21×101拷贝/μL。本研究建立的SYBR GreenⅠ实时荧光定量RT-PCR检测方法特异性强、重复性好、成本低且操作简单，适用于PEDV的早期诊断、定量研究和流行病学监测。
근거GenBank등록적저류행성복사병독（PEDV）독주（등록호KJ960180）적M기인보수서렬，설계1대확증편단대소위299 bp적특이성인물，경PCR확증、극륭、측서감정후，제취질립작위양성표준품，건립료PEDV적SYBR GreenⅠ형광정량RT-PCR검측방법。해방법재1.21×103～1.21×108고패/μL범위내정현량호적선성，상관계수（R2）위0.999，확증효솔위99%，확증산물적용해곡선위단개특이봉，산물Tm치위85.5～86℃，최저검측한위1.21×101고패/μL。본연구건립적SYBR GreenⅠ실시형광정량RT-PCR검측방법특이성강、중복성호、성본저차조작간단，괄용우PEDV적조기진단、정량연구화류행병학감측。
According to the porcine epidemic diarrhea virus (PEDV)strain (Accession No. KJ960180) M conserved gene sequence available in GenBank,a pair of specific primers was designed and amplified 299 bp fragment.After PCR Amplification,cloning and sequencing,as a positive standard template to establish SYBR GreenⅠfluorescence RT-PCR detection for the quantization of PEDV.The standard curve generated had a wide dynamic range from 1.21×103~1.21×108 DNA copies/μL with a linear correlation (R2) of 0.999 and efficiency of 0.99.The melting curve analysis showed one specific peak with melting temprature (Tm) of 85.5~86 ℃,and no primer-dimers peak represented,and the sensitive degree is 1.21×101 copies/μL,and showed excellent specificity and reproducibility.A SYBR GreenⅠfluorescent quantitative RT-PCR assay for detecting M gene of PEDV was developed for the early detection,quantitative analyzing and epidemiological surveillance.