为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法，本研究从GenBank数据库中获取PEDV N基因序列，扩增出PEDVN基因标准质粒，并在N基因的保守区域内设计了一对特异性荧光定量引物，成功建立了 SYBR Green Ⅰ实时荧光定量PCR检测方法。经过一系列试验表明，该检测方法线性关系良好，R2值为0。99;特异性强，敏感性高，最低可检测至2。23 copies/μL，比普通PCR灵敏约100倍;重复性好，组内变异系数为0。25％～0。43％，组间变异系数为0。67％～0。97％;对于各地区96份临床样品检测出PEDV阳性率为25％。本研究建立的实时荧光定量PCR检测方法为PEDV的临床诊断、流行病学调查以及定量研究提供了有效的检测工具。
Development of SYBR Green Ⅰ Real-Time PCR for Detection of Porcine Epidemic Diarrhea Virus
In order to develop an efficient and sensitive detection method for Porcine epidemic diarrhea virus(PEDV),the PEDV N gene sequence was obtained from GenBank database and a pair of specific primers were designed from its conservative region for development of a SYBR Green Ⅰ real-time PCR method.A series of experiments were performed for optimization and the optimal factors included the linear correlation coefficient at 0.99 and detection limit at 2.23 copies/μL,which was about 100 times more sensitive than conventional PCR.The coefficient of variation within groups was between 0.25％-0.43％and between groups was between 0.67％-0.97％,indicating its good repeatability.Total 96 clinical samples from various regions were tested using this method and the PEDV positive rate was 25％(24/96).The real-time PCR method developed in this study provided an effective tool for clinical diagnosis,epidemiological investigation and quantitative research of PEDV.
Porcine epidemic diarrhea virusN geneSYBR Green Ⅰreal-time PCR
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