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犬瘟热病毒RT-nested PCR检测方法的建立与应用
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王凤雪，闫喜军，柴秀丽，罗国良，张海玲，邵西群，吴威，程世鹏
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（中国农业科学院 特产研究所，吉林 吉林132109）
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摘要：
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根据犬瘟热病毒（CDV）弱毒株Onderstepoort的核衣壳蛋白(NP)基因序列设计了套式引物，建立了RT-nested PCR检测方法。特异性试验表明，该法可以特异扩增出CDV NP基因片段，但从RNA病毒狂犬病病毒(RV)、犬副流感病毒(CPIV)、犬冠状病毒(CCV)，DNA病毒犬细小病毒(CPV)、犬腺病毒(CAV)及正常Vero细胞中均不能扩增出条带。敏感性试验表明，RT-PCR可以扩增10-6稀释度的病毒RNA，而建立的RT-nested PCR可以扩增到10-9稀释度的病毒RNA，后者的敏感性明显高于前者。用RT-nested PCR法检测了2006年我国东北地区临床表现犬瘟热症状的病例19例，检出率达90%，明显高于RT-PCR的检出率(45%)。部分病例扩增片段的测序结果表明,CDV NP基因出现个别碱基变异。研究结果表明，建立的犬瘟热病毒RT-nested PCR方法敏感、特异，适用于动物犬瘟热的快速诊断。 |
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关键词：
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犬瘟热病毒；反转录巢式PCR；反转录PCR；NP基因 |
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中图分类号：S 852.659.5：Q 503
文献标识码：
A 文章编号：1673-4696(2007)11-0955-05 |
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Development of RT-nested PCR for detecting
canine distemper virus |
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WANG Feng-xue,YAN Xi-jun,CHAI Xiu-li,LUO Guo-liang,ZHANG Hai-ling,
SHAO Xi-qun,WU Wei,CHENG Shi-peng |
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(Institute of Special Wild Economic Animal and Plant Science,Chinese Academy of Agricultural Sciences，Jilin 132109,China) |
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Abstract: A RT-nested PCR assay for detecting canine distemper virus (CDV) was established using 2 pairs of primers. The primers were designed using nucleocapsid protein (NP) gene information of CDV Onderstepoort strain in GenBank. The assay amplified only a NP gene from CDV; but not from other virus such as rabies virus (RV), canine parainfluenza virus (CPIV), canine parvovirus (CPV), canine adenovirus (CAV), or Vero cell. Serial 10-fold dilutions of 10-6 TCID50 CDV3 strain were used for both PCR and RT-nested PCR. The lower detection limit for CDV RNA was 10-9 and 10-6 dilution for the RT-nested PCR and the traditional RT-PCR, respectively. Of the 19 CD samples collected in Northeast region of China in 2006, the positive ratio was up to 90% and 45% for RT-nested PCR and the traditional RT-PCR, respectively. Sequences of parts of the amplified products from viscera samples were consistent with what have been reported although minor variations may occur occasionally. The results showed that the RT-nested PCR was easy, specific and sensitive for detecting CDV. It could be used for a quick diagnosis of CD. |
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Key words: canine distemper virus(CDV)；RT-nested PCR；RT-PCR;NP gene |
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