疫苗原辅料中猪圆环病毒1型、2型和猪细小病毒PCR检测方法的建立及验证  

Establishment and validation of PCR detection method for porcine circovirus type 1,type 2 and porcine parvovirus in vaccine raw materials

作　　者：施金荣 程小玲 申瑷琳 张雪婷 程尧 何婧雯 段凯 SHI Jin-rong;CHENG Xiao-ling;SHEN Ai-lin;ZHANG Xue-ting;CHENG Yao;HE Jing-wen;DUAN Kai(Quality control department,Wuhan Institute of Biological Products Co.,Ltd.,National Engineering Technology Research Center for Combined Vaccines,Wuhan 430207,Hubei Province,China;不详)

机构地区：[1]武汉生物制品研究所有限责任公司质量控制室,湖北武汉430207 [2]国家联合疫苗工程技术研究中心,湖北武汉430207

出　　处：《微生物学免疫学进展》2022年第5期11-15,共5页Progress In Microbiology and Immunology

基　　金：国家重点研发计划项目(2020YFC0841800)。

摘　　要：目的建立疫苗原辅料中猪圆环病毒1型(porcine circovirus type 1,PCV1)、2型(porcine circovirus type 2,PCV2)和猪细小病毒(porcine parvovirus,PPV)的聚合酶链式反应(polymerase chain reaction,PCR)检测方法并进行验证。方法根据NCBI公布的PCV1、PCV2和PPV基因序列设计了3对特异性引物,通过优化反应条件,建立起检测疫苗原辅料中PCV1、PCV2和PPV的PCR检测方法,并对方法的重复性、中间精密度、专属性、耐用性和检测限进行了验证。结果PCR反应的退火温度为55℃,特异性引物浓度为10μmol/L。该方法重复性、中间精密度、专属性和耐用性均良好,对PCV1、PCV2、PPV的最低检测限分别为1 pg/μL、100 fg/μL和10 fg/μL。结论PCR检测方法可以有效检测出疫苗原辅料中外源病毒污染情况,能为生产合格的疫苗提供质量保证。Objective To establish and validate a PCR method which was used for detection of porcine circovirus type 1(PCV1),type 2(PCV2)and porcine parvovirus(PPV)in vaccine raw materials.Method Three pairs of specific primers were designed according to the gene sequences of PCV1,PCV2,PPV registered in NCBI.By optimizing the reaction conditions,the PCR method was set up to detect PCV1,PCV2 and PPV in vaccine raw materials.The repeatability,intermediate precision,specificity,robustness and limit of detection were validated.Result The annealing temperature of PCR reaction was 55℃,and the specific primer concentration was 10μmol/L.This method was proved to have good repeatability,intermediate precision,specificity and robustness,and the limit of detection was 1 pg/μL for PCV1 and 100 fg/μL for PCV2,10 fg/μL for PPV,respectively.Conclusion The PCR detection method can effectively detect the contamination of exogenous virus in vaccine raw materials,which can provide quality assurance for the production of qualified vaccines.

关 键 词：疫苗原辅料 猪圆环病毒1型 猪圆环病毒2型 细小病毒 聚合酶链式反应 外源病毒 方法验证 质量保证 

分 类 号：R373.9[医药卫生—病原生物学]