猪繁殖与呼吸综合征病毒欧洲株国家核酸标准物质的研制
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摘要
为了研制猪繁殖与呼吸综合征病毒(PRRSV)欧洲株国家核酸标准物质,将PRRSV欧洲株代表毒株LV接种Marc145细胞,待出现细胞病变后,反复冻融收集病毒液,并灭活分装。制备后进行均匀性评估、稳定性评估、数字PCR法定值、不确定度分析和临床试用。结果显示,制备的标准物质的定值为(1.83±0.22)×10~4 copies/μL;样品均匀;-20℃稳定保存12个月以上,4℃稳定2个月以上。经全国标准物质管理委员会的专家评审,该制备物达到了国家二级标准物质的要求,可以作为核酸扩增检测的PRRSV核酸标准物质。
To prepare the national reference material of EU-PRRSV for nucleic acid amplification technology,Marc145 cell lines were infected by EU-PRRSV(LV).After the cytopathic effect was observed,the viral fluid was harvested by freezing and thawing repeatedly,inactivated and sub-packed.The homogeneity and stability of the prepared reference materials were tested.Then,the prepared reference materials were valued using digital PCR and the clinical applicability was also evaluated.The results indicated that the value of the reference material was(1.83±0.22)×10~4 copies/μL.The prepared materials could be stable at-20℃ for at least 12 months,at 4℃ for more than 2 months.The prepared material of PRRSV passed the review of national administrative committee for CRM's,and reached the requirements of the national secondary reference.This reference material can be used as the standard for nucleic acid amplification test of EU-PRRSV.
To prepare the national reference material of EU-PRRSV for nucleic acid amplification technology,Marc145 cell lines were infected by EU-PRRSV(LV).After the cytopathic effect was observed,the viral fluid was harvested by freezing and thawing repeatedly,inactivated and sub-packed.The homogeneity and stability of the prepared reference materials were tested.Then,the prepared reference materials were valued using digital PCR and the clinical applicability was also evaluated.The results indicated that the value of the reference material was(1.83±0.22)×10~4 copies/μL.The prepared materials could be stable at-20℃ for at least 12 months,at 4℃ for more than 2 months.The prepared material of PRRSV passed the review of national administrative committee for CRM's,and reached the requirements of the national secondary reference.This reference material can be used as the standard for nucleic acid amplification test of EU-PRRSV.
引文
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[2] 原霖,韩焘,翟新验.核酸标准物质研究现状[J].中国畜牧兽医,2014,41(11):89-92.
[3] 詹成,燕丽,王琳,等.数字PCR技术的发展和应用[J].复旦学报(医学版),2015,42(6):786-789.
[4] 史艳宇,王莹,石虹,等.微滴数字PCR方法检测畜肉食品中鸭源性成分[J].食品安全质量检测学报,2018,9(3):583-588.
[5] 刘津,李婷,冼钰茵,等.转基因大豆MON89788双重数字PCR通用定量检测方法的建立[J].食品科学,2018,39(4):312-319.
[6] 于纪棉,李如松,倪健波,等.嗜肺军团菌微滴式数字PCR检测方法的建立[J].中国卫生检验杂志,2017,27(9):1233-1236.
[7] 叶子弘,赵煦泓,邹克琴,等.生物标准物质研究简析[J].中国计量,2009(4):67-69.
[8] 王露楠,吴健民,李金明,等.丙型肝炎病毒核酸检测的国家标准物质的研制[J].中华检验医学杂志,2006(4):354-357.
[9] 吴亚琼,高志强,吴延功,等.亚洲Ⅰ型口蹄疫病毒核酸检测标准物质的制备研究[J].中国兽医杂志,2012,48(8):8-12.
[10] 谷强,张伟,高志强,等.新城疫病毒核酸检测标准物质研究[J].中国动物检疫,2012,29(11):40-43.
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