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首页> 外文期刊>Journal of Clinical Microbiology >Detection of porcine reproductive and respiratory syndrome virus and efficient differentiation between Canadian and European strains by reverse transcription and PCR amplification.
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Detection of porcine reproductive and respiratory syndrome virus and efficient differentiation between Canadian and European strains by reverse transcription and PCR amplification.

机译：通过逆转录和PCR扩增检测猪繁殖与呼吸综合征病毒并有效区分加拿大和欧洲菌株。

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Two sets of oligonucleotide primers (1008PS-1009PR and 1010PLS-1011PLR) were designed according to the sequence of the nucleocapsid protein (N) gene of Quebec reference strain IAF-exp91 of porcine reproductive and respiratory syndrome virus (PRRSV). The primers were used in reverse transcription and PCR (RT-PCR) experiments for detection of viral genomic RNA either from infected porcine alveolar macrophages (PAM) or tissues from experimentally infected specific-pathogen-free pigs. Considering the high degree of variation detected between the nucleotide sequences of the N genes of IAF-exp91 and Lelystad virus (LV) strains of PRRSV, the primers 1008PS-1009PR were referred to as the specific primers, since they were chosen in such a manner that they could amplify only sequences from IAF-exp91 RNA and not from LV. On the other hand, the primer pair 1010PLS-1011PLR was common to both strains of PRRSV. When analyzed by agarose gel electrophoresis, the products of RT-PCR from each set of primers were resolved as single band of the predicted size, the specificity of amplified products being confirmed by Southern blotting with a specific IAF-exp91 N gene probe. No amplification was observed when RNA was extracted from uninfected PAM or from other porcine viruses. As expected, only the common primer pair was able to amplify RNA from the Quebec reference strain and two European strains (LV and Weybridge). The resulting bands displayed differences in electrophoretic mobilities due to the absence of 37 nucleotides in both European strains, thus allowing their differentiation from the IAF-exp91 strain. Most of the tissue culture-adapted Quebec isolates were detected with both primer pairs. The sensitivity of the enzymatic amplification method for detection of PRRSV from lung tissues was a 50% tissue culture infective dose of 5. RT-PCR was found to be more sensitive than indirect immunofluorescence assay for detection of PRRSV in tissues from experimentally infected pigs and as sensitive as virus isolation in PAM, especially when combined with Southern blotting with the digoxigenin-labeled N probe and chemiluminescence detection.
机译：根据猪繁殖与呼吸综合征病毒（PRRSV）的魁北克参考菌株IAF-exp91的核衣壳蛋白（N）基因序列设计了两组寡核苷酸引物（1008PS-1009PR和1010PLS-1011PLR）。该引物用于逆转录和PCR（RT-PCR）实验中，用于检测感染的猪肺泡巨噬细胞（PAM）或实验感染的无特定病原体猪组织的病毒基因组RNA。考虑到IAF-exp91的N个基因的核苷酸序列和PRRSV的Lelystad病毒（LV）菌株之间检测到的高度变异，引物1008PS-1009PR被称为特异性引物，因为它们是按这种方式选择的他们只能从IAF-exp91 RNA扩增序列，而不能从LV扩增序列。另一方面，引物对1010PLS-1011PLR是PRRSV的两个菌株共有的。当通过琼脂糖凝胶电泳分析时，来自每组引物的RT-PCR产物被解析为预测大小的单条带，扩增产物的特异性通过特异性IAF-exp91 N基因探针的Southern印迹证实。从未感染的PAM或其他猪病毒中提取RNA时，未观察到扩增。正如预期的那样，只有通用引物对能够从魁北克参考菌株和两种欧洲菌株（LV和Weybridge）扩增RNA。由于在两个欧洲菌株中都不存在37个核苷酸，因此得到的条带在电泳迁移率上显示出差异，因此可以将它们与IAF-exp91菌株区分开。两种引物对均检测到大多数适应组织培养的魁北克分离物。酶促扩增法检测肺组织PRRSV的敏感性为50％组织培养感染剂量5。发现RT-PCR比间接免疫荧光检测法更灵敏地检测实验感染猪组织中的PRRSV。对PAM中的病毒分离敏感，尤其是与洋地黄毒苷标记的N探针进行Southern杂交和化学发光检测结合使用时。

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