一种甲型肝炎灭活疫苗灭活验证试验方法的建立  

Effective inactivation test of inactivated hepatitis A vaccine

作　　者：刘令九[1] 徐晓霞[1] 王桂荣[1] 李娜[1] 孔雪[1] 段雪峰[1] 邵燕[1] 尚瑞琴[1] 于海龙[1] 夏青娟[1] 

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130062

出　　处：《微生物学免疫学进展》2015年第4期26-30,共5页Progress In Microbiology and Immunology

摘　　要：目的探讨细胞培养/链特异性RT-PCR方法可否作为甲型肝炎(甲肝)灭活疫苗(L-A-1减毒株)的病毒灭活验证试验方法。方法根据甲肝病毒(HAV)L-A-1株基因组序列,设计5条基因特异性引物,提取HAV基因组RNA,应用设计的正向引物进行反转录,再进行两轮PCR扩增,通过检测HAV复制过程中的负链中间体,对甲肝灭活疫苗灭活验证试验方法进行探讨,并与《中华人民共和国药典》甲肝灭活疫苗HAV灭活验证试验方法进行比较。结果细胞培养/链特异性RT-PCR方法对HAV负链RNA特异、敏感。通过方法学验证试验表明,该方法的特异性、敏感性和重复性均良好。利用此方法对5批甲肝灭活疫苗(L-A-1减毒株)进行检测,结果全为阴性,与《中华人民共和国药典》甲肝灭活疫苗HAV灭活验证试验测定结果相同。结论细胞培养/链特异性RT-PCR方法快速、灵敏可靠,可作为检测甲肝灭活疫苗(L-A-1减毒株)HAV灭活验证试验的方法。Objective To investigate the integrated cell culture / strand specific reverse transcriptase-polymerase chain reaction（ ICC / strand-specific RT-PCR） method can be used as hepatitis A（ HAV） inactivated vaccine（ L-A-1 attenuated strain） virus inactivation test method validation. Methods Five primers were designed according to genomic sequence of HAV L-A-1 attenuated strain,and the genomic RNA was extracted. Using designed positive primer to reverse transcription and two rounds of PCR amplification to detect the negative-strand intermediate. The effective inactivation tests of inactivated hepatitis A vaccine were carried out using ICC / strand-specific RT-PCR assay and compared with the method described in Pharmacopoeia of the People＇s Republic of China on inactivated hepatitis A vaccine inactivated HAV verification test. Results The ICC / strand-specific RT-PCR had high specificity and sensitivity for HAV negative-strand RNA. The results showed that the specificity,sensitivity and repeatability of the assay were good. Five batch samples were all negative results detected by both ICC / strand-specific RT-PCR and the method described in Pharmacopoeia of the People＇s Republic of China. Conclusions The ICC / strand-specific RT-PCR is a novel,rapid and reliable method to test the effective inactivation test of inactivated hepatitis A vaccine. It could be used to evaluate the availability of routine inactivated hepatitis A test.

关 键 词：甲型肝炎灭活疫苗 细胞培养/链特异性RT-PCR 灭活验证试验 

分 类 号：R392[医药卫生—免疫学]