夏青娟;徐艳玲;李树林;禇东琳;候丽娟;翁滨;肖想;邱璐;徐晓霞;刘令九;
,XIA Qing-juan;XU Yan-ling;LI Shu-lin;CHU Dong-lin;HOU Li-juan;WENG-Bin;XIAO Xiang;QIU Lu;XU Xiao-xia;LIU Ling-jiu;Changchun Institute of Biological Products Co. Ltd;

• 1:长春生物制品研究所有限责任公司
• 2:中国生物技术股份有限公司

摘要(Abstract)：

目的建立检测甲型肝炎减毒活疫苗病毒滴度的细胞培养/链特异性逆转录聚合酶链式反应(strand-specific reverse transcriptase-polymerase chain reaction,strand-specific RT-PCR)方法。方法根据甲型肝炎病毒(HAV)L-A-1株基因组序列,设计5条特异性引物。将甲型肝炎减毒活疫苗原液感染2BS细胞,收获增殖高峰期的HAV,提取RNA,进行2轮链特异性PCR扩增,检测HAV复制过程中的负链RNA,通过Reed-Muench公式计算HAV病毒滴度,并对建立的细胞培养/链特异性RT-PCR法进行特异性及敏感性验证,同时用该方法检测4批冻干甲型肝炎减毒活疫苗病毒滴度,并与《中国药典》三部(2010版)中甲型肝炎减毒活疫苗病毒滴度检测方法(细胞培养时间为28 d/ELISA法)进行比较。结果 HAV负链RNA经2轮扩增获得阳性扩增条带,大小与预期相符。该方法检测HAV复制过程中的负链RNA,特异性、敏感度均较好。两种方法检测的病毒滴度接近,差异无统计学意义(P>0.05)。结论建立的细胞培养/链特异性RT-PCR方法检测周期短,灵敏度、特异性好,可用于疫苗的滴度检测。
Objective To develop an integrated cell culture / strand-specific reverse transcriptase-polymerase chain reaction(ICC / strand-specific RT-PCR) method for determination of virus titer of live attenuated hepatitis A vaccine.Methods Five strand-specific primer sequences were designed according to the genome sequence of hepatitis A virus(HAV)L-A-1 strain. The 2BS cells were infected with bulk of HAV, and the HAV at peak period of propagation was harvested, from which RNA was extracted for two cycles of amplification by PCR. Negative stranded RNA during replication of HAV was determined. The titer of HAV was calculated by Reed-Muench method, and the developed method was verified for specificity and sensitivity. Four batches of freeze-dried live attenuated hepatitis A vaccine were determined for virus titer by the developed method, and the result was compared with that by the method(cell culture for 28 d / ELISA)in Chinese Pharmacopoeia(Volume Ⅲ, 2010 edition). Results Positive bands were obtained by two cycles of amplification of negative stranded RNA of HAV, of which the length was consistent with that expected. The developed method showed high specificity and sensitivity in test for negative stranded RNA during replication of HAV. The virus titers deter-mined by two methods showed no significant difference(P > 0. 05). Conclusion The ICC / strand-specific RT-PCR was time-saving, sensitive and specific, which might be used for determination of virus titer of vaccine.

关键词(KeyWords)： 甲肝减毒活疫苗;细胞培养/链特异性逆转录聚合酶链式反应;负链RNA;病毒滴度
Live attenuated hepatitis A vaccine;Integrated cell culture / stand-specific reverse transcriptase-polymerase chain reaction(ICC / strand-specific RT-PCR);Negative stranded RNA;Virus titer

Abstract:

Keywords:

基金项目(Foundation):

作者(Authors): 夏青娟;徐艳玲;李树林;禇东琳;候丽娟;翁滨;肖想;邱璐;徐晓霞;刘令九;
,XIA Qing-juan;XU Yan-ling;LI Shu-lin;CHU Dong-lin;HOU Li-juan;WENG-Bin;XIAO Xiang;QIU Lu;XU Xiao-xia;LIU Ling-jiu;Changchun Institute of Biological Products Co. Ltd;

DOI: 10.13200/j.cnki.cjb.000856

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