摘要
目的建立H5亚型禽流感病毒灭活疫苗中病毒核酸的特异、敏感、快速的定量检测方法,分析其与HI抗体效价之间的相关性。探索禽流感灭活疫苗效力体外检验的方法。方法选取H5亚型禽流感病毒(avian influenza virus,AIV)血凝素(hemagglutinin,HA)基因保守序列,使用Primer Express 2.0软件设计了特异性引物和TaqMan MGB(minor groove binder)探针,利用实时荧光PCR技术来定量检测禽流感灭活疫苗,通过体外转录,制备含有选定检测序列的RNA标准品,绘制标准曲线。对50批H5亚型禽流感灭活疫苗进行了荧光定量PCR检测,测定了疫苗的HA基因拷贝数,同时测定了50批疫苗相应的HI抗体效价。进行了疫苗的HA基因拷贝数与HI抗体效价之间相关性的分析。结果该方法的灵敏度为10拷贝/反应,标准曲线的相关系数为0.998003,对H9亚型禽流感灭活疫苗和其他禽病疫苗无交叉反应,特异性好、重复性佳。疫苗的HA基因拷贝数与HI抗体效价不相关。结论荧光定量PCR方法为H5亚型禽流感病毒灭活疫苗检测提供了一种特异、敏感、快速的定量检测方法。
Objective To establish a specific, sensitive, rapid method to detect virus gene of I-IS subtype inactivated vaccine and to analyze the correlation between the gene copy number and the HI antibody titer. Methods Specific primers and TaqMan MGB（minor groove binder） probes were designed with Primer Express 2.0 software according to the conserved region of the H5 subtype AIV hemagglutinin （HA） gene to make research of real-time fluorescent PCR in the quantitative detection of HS subtype avian influenza inactivated vaccine. A series of gradient diluted H5 RNA standard was prepared by in vitro transcription to draw a standard curve. Fifty batches of H5 subtype avian influenza inactivated vaccine were detected by fluorescence PCR and the copy number of HA gene of each vaccine was obtained. The correlation was analyzed between the copy number of HA gene and the HI antibody titers of 50 batches of vaccines. Results Tne standard curve showed the sensitivity was 10 copies/ reaction. Tne correlation coefficient of standard curve was 0.998 003. Tne result showed that the method was specifie and reproducible. No cross-reaction was discovered with other avian disease vaccines. There was no correlation between the copy number of HA gene and the HI antibody titers of the vaccine. Conclusion Real-time fluorescent PCR provided a specific, sensitive and rapid method for the quantitative detection of virus gene of H5 subtype avian influenza inactivated vaccine.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2007年第12期1139-1144,共6页
Chinese Journal of Microbiology and Immunology
基金
科技部高致病性禽流感防治专项(2004BA519A24)
