猪繁殖与呼吸道综合征RT-PCR诊断方法的建立  

Establishment of RT PCR Method for Diagnosing Porcine Reproductive and Respiratory Syndrome

作　　者：娄高明 杜伟贤 谢明权 李雪梅[2] 林绍荣[3] 黄毓茂[3] 

机构地区：[1]广东省兽医研究所,广东广州510640 [2]沈阳农业大学畜牧兽医学院,辽宁沈阳110161 [3]华南农业大学动物医学系,广东广州510642

出　　处：《中国兽医学报》2000年第2期141-144,共4页Chinese Journal of Veterinary Science

摘　　要：根据猪繁殖与呼吸道综合征病毒 ( PRRSV)核衣壳蛋白 ORF7的序列 ,设计了 1套引物 ,建立了检测 PRRSV核酸的 RT-PCR方法。通过对猪繁殖与呼吸道综合征 ( PRRS)标准毒株的检测 ,证明该方法不仅能够扩增出特异性的核酸片段 ,同时可从基因水平上区分 PRRSV美洲型和欧洲型。使用该方法对国内临床上疑似为 PRRS的送检样品进行检测 ,结果为阳性 ,并确定其基因型均为美洲型。该研究建立的从组织中直接提取细胞总 RNA进行 RT-PCR的方法 ,为 PRRS的快速。One set of oligonucleotide primer was designed according to the sequence of the nucleocapsid protein gene of Quebec reference strain IAF exp91 of porcine reproductive and respiratory syndrome virus (PRRSV). The primers were used in reverse transcription and polymerase chain reaction (RT PCR) experiments for detection of viral genomic RNA from either infected HS 2H cells or tissues of domestic naturally infected pigs. The primers could amplify sequences from the American and European strains of PRRSV. When analyzed by agarose gel electrophoresis, the resulting bands displayed differences in electrophoretic mobilities due to the absence of 36 nucleotides in the European strain, thus allowing its differentiation from the American strain. No amplification was observed when RNA was extracted from either uninfected HS 2H cells or other porcine viruses. The sensitivity of the RT PCR for detection of PRRSV was measured to be a 50% tissue culture infective dose of 10. All of the detected domestic clinical samples were positive by the RT PCR and their strains belonged to the American type in gene level. It was first established that the direct extraction of the RNA from tissues and organs of naturally infected pigs and cell cultures was used in detection of RNA of PRRSV by RT PCR. The RT PCR is proved to be a specific, sensitive, rapid and simple method and can be used in research of epidemiology and early clinical diagnosis of PRRS in molecular level.

关 键 词：核酸检测 RT-PCR PRRS 诊断 

分 类 号：S858.285.3[农业科学—临床兽医学] S858.284.4[农业科学—兽医学]