猪圆环病毒2型灭活疫苗中病毒抗原含量实时荧光定量PCR检测方法的建立

doi:10.12160/j.issn.1672-5190.2021.01.002

畜牧与饲料科学 ›› 2021, Vol. 42 ›› Issue (1): 8-13.doi: 10.12160/j.issn.1672-5190.2021.01.002

猪圆环病毒2型灭活疫苗中病毒抗原含量实时荧光定量PCR检测方法的建立

李雪峰¹, 康斌¹, 赵炳武¹, 武玉梅¹, 戴伶俐², 董鹏¹, 张建春³, 张春阳³, 斯琴高娃³

1.金河佑本生物制品有限公司,内蒙古呼和浩特 011517;
2.内蒙古自治区农牧业科学院,内蒙古呼和浩特 010031;
3.杭州佑本动物疫苗有限公司,浙江杭州 310018

收稿日期:2020-10-22 发布日期:2021-02-18
通讯作者: 康斌（1974-）,男,主要从事兽用生物制品开发和质量管理工作。
作者简介:李雪峰（1982-）,男,硕士,主要从事动物传染病分子生物学与免疫学研究工作。

Establishment of a Real-time Fluorescence Quantitative PCR Assay for Antigen Quantification of Inactivated Porcine Circovirus Type 2 Vaccines

LI Xue-feng¹, KANG Bin¹, ZHAO Bing-wu¹, WU Yu-mei¹, DAI Ling-li², DONG Peng¹, ZHANG Jian-chun³, ZHANG Chun-yang³, Siqingaowa³

1.Jinhe Youben Biological Products Co.,Ltd.,Hohhot 011517,China;
2.Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences,Hohhot 010031,China;
3.Hangzhou Youben Animal Vaccine Co., Ltd.,Hangzhou 310018,China

Received:2020-10-22 Published:2021-02-18

摘要/Abstract

摘要： 依据GenBank公布的猪圆环病毒2型Cap基因序列,在保守区域设计特异性引物和TaqMan探针,优化反应体系,建立评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,对方法的特异性、敏感性和重复性进行试验,并验证灭活剂用量和灭活时间对检测结果的影响。结果显示：该方法只对猪圆环病毒2型基因有特异性扩增,其他3种对照病毒基因的扩增结果均为阴性;检测灵敏度达到10^2.0 TCID₅₀/mL,比普通PCR方法高100倍;方法的重复性好,对同一样品进行10次检测,变异系数为2.28%;不同灭活剂用量和灭活时间对结果的影响较小,不会因各厂家使用的灭活剂用量和灭活时间不同,影响疫苗对比实验的公平性。该研究成功建立了一种评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,用于猪圆环病毒2型灭活疫苗样品中病毒抗原含量的定量,其结果可以反映不同猪圆环病毒2型灭活疫苗样品中抗原含量差异,为研究猪圆环病毒2型灭活疫苗病毒抗原含量评估方法提供了新的思路。

关键词: 实时荧光定量PCR, 猪圆环病毒2型, 灭活疫苗, Cap基因

Abstract: A real-time fluorescence quantitative PCR assay to quantify the antigen concentration of inactivated porcine circovirus type 2 vaccines was developed based on a set of primers and TaqMan probe designed targeting the Cap gene sequence of porcine circovirus type 2 from GenBank. After optimizing the reaction system, the specificity, sensitivity and repeatability of the established assay were assessed. Subsequently, the impacts of adding amount of inactivator and inactivation time on detection performance of the established assay were investigated. The results showed that positive amplification product was exclusively obtained for target gene of porcine circovirus type 2 by using the established real-time fluorescence quantitative PCR assay, while the other three kinds of porcine origin viruses had no expected amplification products; the sensitivity of the established assay reached up to 10^2.0 TCID₅₀/mL, which was 100 times higher than that of the ordinary PCR assay; the established assay had good repeatability with the variable coefficient of 2.28% in 10 repeated tests for the same one sample; the different adding amount of inactivator and inactivation time had limited impacts on detection performance of the established assay, in other words, the creditability of the inactivated vaccine comparison test was not affected by the adding amount of inactivator recommended by varied vaccine producers and different inactivation time. In this study, a real-time fluorescence quantitative PCR assay to quantify the antigen concentration of inactivated porcine circovirus type 2 vaccines is successfully developed. This established assay can quantitatively discriminate the antigen concentration of different inactivated porcine circovirus type 2 vaccine samples, providing novel insight into the development of quantitative evaluation method for antigen concentration of inactivated porcine circovirus type 2 vaccines.

Key words: real-time fluorescence quantitative PCR assay, porcine circovirus type 2, inactivated vaccines, Cap gene

中图分类号:

李雪峰, 康斌, 赵炳武, 武玉梅, 戴伶俐, 董鹏, 张建春, 张春阳, 斯琴高娃. 猪圆环病毒2型灭活疫苗中病毒抗原含量实时荧光定量PCR检测方法的建立[J]. 畜牧与饲料科学, 2021, 42(1): 8-13.

LI Xue-feng, KANG Bin, ZHAO Bing-wu, WU Yu-mei, DAI Ling-li, DONG Peng, ZHANG Jian-chun, ZHANG Chun-yang, Siqingaowa. Establishment of a Real-time Fluorescence Quantitative PCR Assay for Antigen Quantification of Inactivated Porcine Circovirus Type 2 Vaccines[J]. Animal Husbandry and Feed Science, 2021, 42(1): 8-13.

参考文献

[1] CHAE C.Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology[J].Veterinary Journal,2004,168(1):41-49.
[2] OPRIESSNIG T,XIAO C,HALBUR P G, et al.A commercial porcine circovirus (PCV) type 2a-based vaccine reduces PCV2d viremia and shedding and prevents PCV2d transmission to naïve pigs under experimental conditions[J].Vaccine,2017,35(2):248-254.
[3] CHAE C.Porcine circovirus type 2 and its associated diseases in Korea[J].Virus Research,2012,164(1/2):107-113.
[4] HANSEN M S, PORS S E, JENSEN H E, et al.An investigation of the pathology and pathogens associated with porcine respiratory disease complex in Denmark[J].Journal of Comparative Pathology,2010,143(2/3):120-131.
[5] MANKERTZ A.Molecular interactions of porcine circoviruses type 1 and type 2 with its host[J].Virus Research,2012,164(1/2):54-60.
[6] FENAUX M,OPRIESSNIG T,HALBUR P G, et al.A chimeric porcine circovirus (PCV) with the immunogenic capsid gene of the pathogenic PCV type 2 (PCV2) cloned into the genomic backbone of the nonpathogenic PCV1 induces protective immunity against PCV2 infection in pigs[J].Journal of Virology, 2004,78(12):6297-6303.
[7] GUO L J, FU Y J, HUANG L P, et al.A commercial PCV2a-based vaccine is effective in protection from experimental challenge of PCV2 mutant with two amino acids elongation in capsid protein[J].Vaccine, 2015, 33(31):3752-3757.
[8] XIA D, HUANG L, XIE Y, et al.The prevalence and genetic diversity of porcine circovirus types 2 and 3 in Northeast China from 2015 to 2018[J].Archives of Virology,2019,164(2):2435-2449.
[9] PARAGUISON R C, FLORES E B, CRUZ L C.Possible use of RNA isolate from inactivated vaccine for external positive control in reverse transcription-based detection of foot-and-mouth disease virus in bull semen[J].Biochemical and Biophysical Research Communications, 2010,392(4):557-560.
[10] LOURENÇ C M B, APARECIDA P L, LAPPAS G A P, et al.Development and validation of a real-time RT-PCR assay for the quantification of rabies virus as quality control of inactivated rabies vaccines[J].Journal of Virological Methods,2019,270(8):46-51.
[11] 姜立民,林晓波,高磊,等.β-丙内酯对狂犬病病毒的灭活效果[J].国际流行病学传染病学杂志,2014, (2):137-139.
[12] 李雪峰,赵炳武,康斌,等.一种检测油乳剂全病毒灭活疫苗中有效抗原含量的方法:CN201910280795[P].2019-06-04.

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猪圆环病毒2型灭活疫苗中病毒抗原含量实时荧光定量PCR检测方法的建立

Establishment of a Real-time Fluorescence Quantitative PCR Assay for Antigen Quantification of Inactivated Porcine Circovirus Type 2 Vaccines

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