非洲猪瘟病毒核酸能力验证样品的制备及初步应用

Development and Application of Samples for Proficiency Testing of African Swine Fever Virus Nucleic Acid Detection

核酸检测技术在非洲猪瘟的诊断和监测中应用普遍,是国际兽医组织和我国指定的检测技术。非洲猪瘟病毒(African swine fever,ASFV)VP72基因是其核酸检测的通用靶基因,参照GenBank中AFSV Georgia 2007毒株VP72基因序列,体外合成VP72全基因片段,克隆于pMD20-T载体,经工艺研究,研制了ASFV核酸能力验证样品,均匀性和稳定性检验结果表明,样品均匀、稳定,满足中国合格评定国家认可委员会对能力验证样品的要求。为评价我国各级兽医实验室对非洲猪瘟的核酸检测能力,组织实施了能力验证计划《非洲猪瘟病毒核酸检测》(CNCA-17-A03)。将能力验证样品编号后下发给参试实验室,共有20个省、市的30家实验室参试,结果显示所有实验室均获得满意结果。同时,能力验证计划中也发现存在缺乏荧光PCR国家标准,核酸标准样品供应不足等问题。建议及时修订完善国家标准,并尽快研制非洲猪瘟(AFS)核酸有证标准样品。

Gene amplification-based methods including conventional and real-time PCR assay,recommended by OIE and the Chinese authorities,are widely used in African swine fever virus(ASFV)diagnosis and surveillance.The highly conserved VP72 gene is commonly exploited for detection of ASFV.In this study,the full-length ASFV VP72 gene was synthesized with reference to the corresponding sequence of the Georgia2007 isolate in GenBank and then cloned into vector pMD20-T,which was applied to the development of a proficiency test for ASFV detection.The sample homogeneousness and stability met the standard for proficiency testing samples of the China National Accreditation Service for Conformity Assessment(CNAS).The proficiency testing project"The detection of ASFV nucleic acids"(CNCA-17-A03)was launched among veterinary sectors of government to evaluate their ability in the identification of ASFV.Thirty laboratories from 20 Provinces participated in this project,and obtained the expected results.However,some problems were discovered during the project,including the absence of a national standard for real-time PCR assay for ASFV detection,and insufficient nucleic acid standard samples preparation,which must be resolved by developing an augmented National Standard and developing certified ASFV nucleic acid standard samples,respectively.