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

作者: 李小鹏 :华南农业大学兽医学院,广东 广州;广东省农业科学院动物卫生研究所动物疫病诊断中心,广东 广州; 陈华良 , 翟俊琼 , 周 霞 , 程 松 , 张文娟 , 罗满林 :华南农业大学兽医学院,广东 广州; 翟少伦 :广东省农业科学院动物卫生研究所动物疫病诊断中心,广东 广州;

关键词: 小反刍兽疫病毒P、N基因逆转录–聚合酶联反应同源性序列分析Peste des Petits Ruminants Virus P/N Gene Reverse Transcription Polymerase Chain Reaction Homology Sequence Analysis

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.

文章引用: 李小鹏 , 陈华良 , 翟俊琼 , 周 霞 , 程 松 , 张文娟 , 罗满林 , 翟少伦 (2015) 小反刍兽疫RT-PCR的鉴定及N基因序列分析。 亚洲兽医病例研究, 4, 31-38. doi: 10.12677/ACRPVM.2015.44006

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