小反刍兽疫RT-PCR的鉴定及N基因序列分析

Identification of RT-PCR and Sequence Analysis of N Gene in Peste des Petits Ruminants Virus

摘要: 根据小反刍兽疫病毒(PPRV) P、N基因保守序列设计了两对引物，分别扩增部分P基因及N基因的全长阅读编码框(ORF)，对疑似小反刍兽疫病毒的样品抽取RNA并以其为模板，在RT-PCR的体系中能扩增出预期大小分别为766 bp、1578 bp的目的片段，同时对退火温度，敏感性及特异性等RT-PCR反应条件进行了优化。在最佳反应条件下，该RT-PCR能检出最低850拷贝的PPRV基因组。对N全基因进行RT-PCR扩增并对PCR产物克隆于pMD-19T载体，转化感受态细胞DH5α后，对重组质粒测序，结果表明N基因全长大小与预期完全一致。并且这两个基因与新疆伊犁、北京所发生的小反刍兽疫病毒同源性高达100%，说明该病毒在中国南北方都有存在，其亚型为基因4型。

Abstract: Two pairs of primers were designed to amplify the partial P gene and full-length N gene based on their own conserved sequences, respectively. RNA of suspected samples with PPRV was extracted and amplified by RT-PCR, from which target fragments of 766 bp and 1578 bp were obtained. Meanwhile, the RT-PCR reaction conditions, including annealing temperature and sensitivity, were optimized and 850 copies of PPRV can be tested under these conditions. PCR product of N gene was cloned into pMD-19T vector and the recombinant plasmid was transformed into DH5α competent cells and sequenced, which showed that N gene was consistent with that expected. Compared with other strains from Yili, Xinjiang province and Beijing, the homologies of N gene was almost 100%, which indicated that PPRV, as lineage 4, was endemic in North and South of China.Two pairs of primers were designed to amplify the partial P gene and full-length N gene based on their own conserved sequences, respectively. RNA of suspected samples with PPRV was extracted and amplified by RT-PCR, from which target fragments of 766 bp and 1578 bp were obtained. Meanwhile, the RT-PCR reaction conditions, including annealing temperature and sensitivity, were optimized and 850 copies of PPRV can be tested under these conditions. PCR product of N gene was cloned into pMD-19T vector and the recombinant plasmid was transformed into DH5α competent cells and sequenced, which showed that N gene was consistent with that expected. Compared with other strains from Yili, Xinjiang province and Beijing, the homologies of N gene were almost 100%, which indicated that PPRV, as lineage 4, was endemic in North and South of China.