摘要
【目的】建立一种能够应用于临床样本猪瘟病毒(CSFV)检测的快速、简便且标准化程度高的检测技术,以满足畜牧业生产和兽药市场的需求。【方法】根据GenBank中猪瘟病毒及近源病毒的基因组全序列,选择特异保守区域设计了2对猪瘟病毒引物,借助3′端引物碱基错配对PCR扩增效率的影响,优化筛选了能够鉴别猪瘟兔化弱毒疫苗和强毒疫苗的PCR退火温度,经多重试验组合建立了一套鉴别猪瘟强毒和弱毒的多重RT-PCR鉴别诊断方法。【结果】扩增反应条件的确定试验表明,退火温度为55℃;特异性分析表明,从弱毒C株和石门强毒株的基因组中分别扩增出1条大小为492和178 bp的特异性片段,从强弱毒株混合物中1次扩增出2条大小为492和178 bp的特异性片段,检测不到伪狂犬病病毒的RNA;敏感性分析表明,该方法能检测出0.8 pg的猪瘟病毒RNA;应用试验表明,8份疑似猪瘟病料中5份为强毒感染,2份为弱毒感染,1份为弱毒和强毒的混合感染。【结论】研究建立的多重RT-PCR方法具有敏感性、特异性强、重复性好的特点,对养猪业的发展具有十分重要的意义。
[Objective] This study established a rapid,simple, high standardized technique for the clinical detection of classic swine fever virus （CSFV） to meet the development of animal husbandry and the demand of veterinary drugs market. [Method] Two pairs of primers were designed and synthesized according to the highly conserved regions of CSFV and near origin virus genome sequence in Genabank. The anneal temperature of polymerese chain reaction （PCR） was optimized with the effaction of the mismatch of 3＇ basic group of primers on the amplification efficiency of PCR. Then the multiplex RT-PCR method for the differentiation of wild-type and vaccine viruses of CSFV was established after tests. [Result] The confirmed test of reacting condition showed that the anneal temperature was 55℃. The specificity test showed that a fragment of 492 bp or 178 bp was respectively amplified from genomic RNA of C-strain or Shimen strain,two fragments of 492 bp and 178 bp were simultaneously amplified from the mixed RNA sample of C-strain and Shimen strain,and no amplification was achieved from Pseudorabies virus RNA. The sensitivity test showed that the RT-PCR could detect 0.8 pg of CSFV RNA. The amplication test showed that five of eight field samples suspected of CSF were found to be Shimen-like, 2 to be C-strain-like, and 1 to be Shimen-like and C-strain-like mixture by this method. [Conclusion] The result indicated RT-PCR method had specificity,sensitive, repetitive characteristics, and was significant for the development of pig industry.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2008年第11期47-51,56,共6页
Journal of Northwest A&F University(Natural Science Edition)
基金
河南省高等学校青年骨干教师资助计划项目(2005461)
关键词
猪瘟病毒
多重反转录聚合酶链式反应
鉴别诊断
classical swine fever virus
multiplex RT-PCR
detection and differentiation
