| 猪瘟病毒野毒株和兔化弱毒疫苗株RT-PCR-RFLP鉴别检测方法的建立 |
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| 引用本文： | 胡继明,杨培培,王颖,孙海芳,黄娟,陈俏俏,杨瑞梅,张传美,秦晓冰,单虎.猪瘟病毒野毒株和兔化弱毒疫苗株RT-PCR-RFLP鉴别检测方法的建立[J].畜牧兽医学报,2012,43(6):943-949. |
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| 作者姓名： | 胡继明 杨培培 王颖 孙海芳 黄娟 陈俏俏 杨瑞梅 张传美 秦晓冰 单虎 |
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| 作者单位： | 1. 青岛农业大学山东省预防兽医学重点实验室,青岛,266109
2. 莱阳市畜牧局,莱阳,265200 |
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| 基金项目： | 国家科技支撑计划，科技基础性工作专项，山东省生猪产业创新团队建设，青岛市公共领域科技支撑计划 |
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| 摘 要： | 本研究旨在建立猪瘟病毒野毒株和兔化弱毒疫苗株RT-PCR-RFLP鉴别检测方法。根据Shimen株设计1对特异性引物,建立猪瘟病毒RT-PCR-RFLP检测方法;对20份疑似猪瘟临床样品进行检测,并对检出的山东8株流行野毒株和2株疫苗株PCR产物进行克隆与序列分析,验证上述方法。结果RT-PCR扩增片段为825bp,产物经RFLP分析,野毒株的PCR产物能被ApaⅠ酶切为322bp和503bp 2个片段,兔化弱毒疫苗株则不能被酶切,检测出RNA的最低浓度为0.028 6μg.mL-1;8株流行野毒株都含GGGCCC序列(ApaⅠ酶切位点),2株疫苗株相应序列为GAGCCC,不能被ApaⅠ酶切;8株流行野毒株属于基因2群,2株疫苗株与HCLV遗传关系近,为基因1群。建立了可鉴别猪瘟病毒野毒株和兔化弱毒疫苗株RT-PCR-RFLP检测方法,为猪瘟的防控提供有效手段。
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| 关 键 词： | 猪瘟病毒 野毒株 兔化弱毒疫苗株 RT-PCR-RFLP |
Development of RT-PCR-RFLP for Detection and Differentiation of Wild-type and Vaccine Viruses of Classical Swine Fever Virus |
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| HU Ji-ming
,
YANG Pei-pei
,
WANG Ying
,
SUN Hai-fang
,
HUANG Juan
,
CHEN Qiao-qiao
,
YANG Rui-mei
,
ZHANG Chuan-mei
,
QIN Xiao-bing
,
SHAN Hu.Development of RT-PCR-RFLP for Detection and Differentiation of Wild-type and Vaccine Viruses of Classical Swine Fever Virus[J].Acta Veterinaria et Zootechnica Sinica,2012,43(6):943-949. |
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| Authors: | HU Ji-ming YANG Pei-pei WANG Ying SUN Hai-fang HUANG Juan CHEN Qiao-qiao YANG Rui-mei ZHANG Chuan-mei QIN Xiao-bing SHAN Hu |
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| Institution: | 1*(1.Shandong Key Laboratory of Preventive Veterinary Medicine,Qingdao Agricultural University, Qingdao 266109,China;2.Laiyang Farming Bureau,Laiyang 265200,China) |
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| Abstract: | A RT-PCR-RFLP assay was developed for the detection and differentiation between wild-type and rabbit attenuated vaccine viruses of classical swine fever virus(CSFV).A pair of specific primers was designed based on the Shimen strain,and used to verify the assay.Eight epidemic wild-type strains isolated from 20 clinical pathological samples of suspected swine fever and 2 vaccine strains were amplified by RT-PCR,cloned and sequenced.The results showed that a fragment of 825 bp was amplified from genomic RNA of CSFV wild-type which could be digested with ApaⅠ into two fragments of 322 bp and 503 bp when analyzed with RFLP,while the vaccine strain could not be digested with ApaⅠ.The lowest concentration of RNA could be detected in the present assay is 0.028 6 μg·mL-1.Eight epidemic wild-type strains contained the ApaⅠ recognition sequence GGGCCC while the two vaccine strains contained the sequence GAGCCC.Eight epidemic wild-type strains belonged to Genomic Group 2.On the contrary,two vaccine strains with close genetic relationship to HCLV belonged to Genomic Group 1.The developed RT-PCR-RFLP could be used to detect and differentiate wild-type CSFV from pig vaccinated with the vaccine viruses. |
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| Keywords: | classical swine fever virus wild-type vaccine viruses RT-PCR-RFLP |
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