| 摘要: |
| 【目的】建立一种基于E0基因高保守性的猪瘟病毒（CSFV）RT-nPCR检测方法，为临床诊断提供一种可靠方法，同时对获得的陕西猪瘟病毒E0基因进行序列分析，揭示猪瘟病毒基因的分子衍化特征，为防控猪瘟提供参考。【方法】根据GenBank中的猪瘟病毒参考序列，设计并合成了2对引物，建立猪瘟RT-nPCR检测方法，并对该方法的灵敏度和特异性进行检测。用该方法对采自陕西部分猪场的32份疑似猪瘟病料进行检测，并对获得的8株流行毒株E0基因进行测序及同源性分析。【结果】建立了猪瘟病毒RT-nPCR检测方法，该方法检测CSFV cDNA含量的最低极限为6.7×10-5 ng/L，从BVDV、PRRSV、PCV-2、PRV和PPV等参考毒株中提取或反转录获得的DNA/cDNA中不能扩增出目的条带，提示方法灵敏度高、特异性强。对32份疑似猪瘟组织病料的检测发现，有12份呈现阳性，阳性率为37.5％。序列分析表明，8株流行毒株间E0核苷酸、氨基酸同源性分别在97.0%～99.3%和94.8%～98.5%。与参考毒株的核苷酸同源性为83.4%～95.4%，氨基酸同源性为85.8%～98.1%。流行毒株与我国疫苗毒株HCLV、C HVRI的核苷酸同源性仅为83.4%～85.1%，氨基酸同源性仅为86.1%～89.1%，呈现出较明显的远离疫苗株的变异趋势。进化树分析发现，8个流行毒株均属于基因Ⅱ群。首次发现了1株流行毒株(SXWN02株)E0 Rnase活性基序位点第94位由E变异为K，其他位点未发生变异。【结论】建立的RT-nPCR检测方法灵敏度高、特异性强，可作为猪瘟病毒的临床诊断方法。陕西部分地区猪场猪瘟感染仍较严重，需要做好防控工作。流行毒株E0基因发生较大变异，尤其是在关键位点上出现变异毒株，需要密切关注。 |
| 关键词: 猪瘟 RT-nPCR方法 E0基因 分子特征 |
| DOI： |
| 分类号: |
| 基金项目:陕西省科学技术研究发展计划项目（2014K02-06-03）；咸阳职业技术学院重点科研项目(2013KYA02) |
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| Establishment of RT-nPCR method for detection of CSFV and analysis of CSFV E0 gene in some areas of Shaanxi province |
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WU Xu-jin,ZHU Xiao-fu
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| Abstract: |
| 【Objective】This study established an E0 gene based RT-nPCR method for detection of CSFV for reliable clinical diagnosis,and analyzed CSFV E0 gene sequences in some areas of Shaanxi province to reveal molecular characteristics of CSFV E0 gene and provide basis for CSFV prevention and control.【Method】According to CSFV reference sequences in GenBank,we designed and synthesized two pairs of primers,and established a classical CSFV RT-nPCR detection method.Its detection sensitivity and specificity were tested before being used to test suspected swine fever tissues collected from farms in Shaanxi province.The E0 gene sequences of obtained 8 strains were measured and homology analysis was conducted.【Result】RT-nPCR detection method for CSFV was established with the detection limit of 6.7×10-5 ng/L.Target genes could not be amplified using DNA/cDNA obtained from BVDV,PRRSV,PCV-2,PRV and PPV,indicating that the established method had high sensitivity and specificity.12 out of 32 suspected swine fever tissues were found positive,with the positive rate of 37.5%. Sequence analysis shows that nucleotide homology and amino acid homology of 8 prevalent strains E0 were 97.0%－99.3% and 94.8%－98.5%,respectively.Compared to the reference strains,the nucleotide homology was 83.4%－95.4%,and amino acid homology was 85.8%－98.1%.The nucleotide homology with vaccine strains HCLV and C HVRI was only 83.4%-85.1% and the amino acid homology was only 86.1%－89.1%,showing a significant variation trend away from the vaccine strain.Phylogenetic analysis reveals that the 8 pandemic strains belonged to genotype Ⅱ group.A pandemic strain (SXWN02) with mutated E→K at the 94th site of E0 Rnase active motifs was found for the first time.Other sites did not mutate.【Conclusion】The established RT-nPCR method had high sensitivity and specificity and can be used for clinical diagnosis.CSFV infection was serious in some areas of Shaanxi province and prevention and control should be well conducted.We found a greater mutation of pandemic strain E0 gene,especially variant strains appeared at key sites,which needs close attention. |
| Key words: classical swine fever RT-nPCR method E0 gene molecular characteristics |
