多重RT-PCR检测猪传染性胃肠炎病毒和猪流行性腹泻病毒  

Differentiation Between Porcine Epidemic Diarrhea Virus and Transmissible Gastroenteritis Virus by Multiplex RT-PCR

作　　者：陈芳 刘惠莉[2] 孙红立 曹祥荣[3] 李震[2] 赵立平[4] 

机构地区：[1]上海市农业遗传育种重点实验室,上海201106 [2]上海市农业科学院畜牧兽医研究所,上海201106 [3]南京师范大学生命科学学院,江苏南京210097 [4]上海交通大学生命科技学院,上海200030

出　　处：《中国兽医学报》2005年第1期9-12,共4页Chinese Journal of Veterinary Science

基　　金：上海市农委科技攻关项目

摘　　要：为了检测、区分猪传染性胃肠炎 (TGE)和猪流行性腹泻 (PED) ,建立了同时检测这 2种疾病病原体的多重 PCR方法。参考 Gen Bank登录的 TGEV和 PEDV纤突蛋白 S基因序列 ,经 DNAsis 2 .0比较分析 ,分别筛选出 TGEV、PEDV S基因相对保守区。以 TGEV TH- 98株 (Gen Bank登陆号为 AF4 94 337)和 PEDV CV777株 (Gen Bank登陆号为 NC0 0 3436 )为参考模板 ,利用软件 Primer 3设计合成了 2对寡核苷酸引物 ,以 ST细胞培养的 TGEV和 PEDV毒株核酸为模板 ,利用所设计的 2对引物进行多重 RT- PCR扩增 ,结果同时得到与试验设计相符的 4 99bp(TGEV)和199bp(PEDV)的扩增条带 ,而对其他 3种猪病原的扩增结果均为阴性。敏感性试验结果表明 ,建立的多重 RT- PCR方法可检测出 10 pg TGEV RNA和 10 0 pg PEDV RNA。A multiplex RT-PCR method was developed to simultaneously detect two pathogen of Porcine-TGEV and PEDV.According to conservative sequence of the spike protein gene of TGEV and PEDV at the GenBank,two sets of specific primers were designed by the software Primer 3.TGEV and PEDV RNA was extracted and amplified by the multiplex RT-PCR using these two sets of primers.The result showed that the two specific fragments of TGEV 499 bp and PEDV 199 bp were amplified,but negative results were gotten with other three porcine pathogenic viruses.The sensitivity assay indicated that 10 pg of TGEV and 100 pg PEDV RNA could be detected using this method.

关 键 词：TGEV 猪流行性腹泻病毒 猪传染性胃肠炎病毒 RT-PCR检测 引物 纤突蛋白 猪病 扩增 RNA 阴性 

分 类 号：R446[医药卫生—诊断学] S858.28[医药卫生—临床医学]