摘要
根据GenBank发表的猪流行性腹泻病毒(PEDV)CV777株和猪传染性胃肠炎病毒(TGEV)Purdue P115株的基因序列,针对其基因保守区,设计1条共用的下游引物和2条各自病毒的上游引物,可特异扩增出大小分别为213 bp和546 bp目的条带。经过对TaqDNA聚合酶和Mg2+浓度、退火温度(Tm)等PCR反应条件的优化,最终确定该双重RT-PCR的最佳条件为TaqDNA聚合酶浓度为0.03 U/μL,Mg2+浓度为0.05 mmol/L,dNTP浓度为200μmol/L,Tm为51℃。特异性和敏感性检测结果显示,所建立的检测方法具有很好的特异性以及较高的敏感性。
Based on the gene sequences of Porcine epidemic diarrhea virus （PEDV） CV777 strain and Transmissible gastroenteritis virus （TGEV） Purdue Pl15 strain reported in GenBank, a specific upper primer, and two down primers for PEDV and TGEV were designed,respectively. The PCR products of PEDV and TGEV had molecular sizes of 213 bp and 546 bp, respectively. The optimal reaction conditions of multiplex RT-PCR for TGEV and PEDV were determined through the optimizing concentrations of primers and Mg^2＋ , and annealing temperature. The optimal reaction system was carried out as followings： the concent＇rations of Taq DNA, Mg^2＋ and dNTPs were 0.03 U/μL, 0.05 mmol/L and 200μmol/L,respectively. The optimal annealing temperature was 51 ℃. The sensitivity test and specificity test results showed that 102 eopies/μL cDNA can be detected by the multiplex RT-PCR and no reations could be found. It indicated that the sensitivity and specificity of the multi RT-PCR were good through the primary application.
出处
《动物医学进展》
CSCD
北大核心
2009年第10期1-5,共5页
Progress In Veterinary Medicine
基金
浙江省科技计划重点项目(2007C22057)
关键词
猪流行性腹泻病毒
猪传染性胃肠炎病毒
双重RT—PCR
Porcine epidemic diarrhea virus
Transmissible gastroenteritis virus（TGEV）
double RT-PCR （PEDV）
