应用荧光定量逆转录PCR检测HAV在2BS细胞中的增殖动态  

Application of fluorescent quantitative reverse transcriptase PCR for determinating proliferation dynamics of HAV in 2BS cells

作　　者：夏青娟 周延彬 徐艳玲 张晶 李树生 徐国亮 李海燕 邵燕 于海龙 侯丽娟 

机构地区：[1]长春生物制品研究所有限责任公司疫苗二室,130062

出　　处：《国际生物制品学杂志》2017年第6期265-268,共4页International Journal of Biologicals

摘　　要：目的 利用荧光定量逆转录PCR（fluorescent quantitative reverse transcriptase PCR，qPCR）技术，从病毒基因组RNA复制水平研究HAV在2BS细胞中的增殖动态。方法 根据HAV基因组保守区序列设计引物和探针，构建重组HAV质粒作为工作标准品，建立检测HAV的qPCR法，并验证该法的灵敏度、特异性和重复性。同时用HAV病毒液感染2BS细胞，于感染后不同时间收病毒液，应用该法检测HAV cDNA。结果 该法在重组HAV质粒标准品为2.6×（10^1～10^7）拷贝/μl时具有良好的扩增曲线，检测灵敏度为2.6×10^1 拷贝/μl DNA质粒。该法可特异性检测HAV cDNA，且重复性良好，实验内和实验间对不同浓度的重组HAV质粒各检测3次获得的域值循环的变异系数分别为0.23%-1.41%和0.34%-4.82%。应用该法检测HAV在2BS细胞中的增殖显示，感染后6 d HAV开始增殖，感染后14-21 d HAV含量达到最高峰，并一直持续到第28天。结论 建立的方法具有良好的灵敏度、特异性和重复性，可用于实时监测甲型肝炎疫苗生产过程中的HAV增殖动态。Objective To detect HAV proliferation dynamics in 2BS cell by fluorescent quantitative reverse transcriptase PCR （qPCR） assay. Methods The specific primers and probes were designed according to the conservative region of HAV. The recombinant HAV plasmid was constructed as working standard to develop qPCR, and sensitivity, specificity and repeatability of the developed method were validated. Meanwhile, the 2BS cells were infected with HAV, and HAV bulks were harvested at different time points after infection. The HAV cDNA was detected using the developed method. Results The developed method had good amplification curve when the recombinant HAV plasmid standard concentrations were 2.6×（10^1-10^7） copies/μl. The sensitivity of this method was 2.6×10^1 copies/μl. The method could specifically detect HAV cDNA and had good repeatability.The coefficient variations of cycle threshold obtained by detecting the different concentrations of recombinant HAV plasmid for 3 times each in intra- and inter-assay were 0.23%-1.41% and 0.34%-4.82%, respectively. The results of HAV proliferation in 2BS cells detected by this method showed that HAV began to proliferate at 6 d after infection, and the HAV content in 2BS cells peaked at 14-21 d after infection and maintained until 28 d. Conclusion The developed method has good sensitivity and repeatability, and can be applied for monitoring the proliferation dynamics of HAV in preparation of hepatitis A vaccine.

关 键 词：逆转录聚合酶链反应 肝炎病毒 甲型 病毒培养 

分 类 号：R440[医药卫生—诊断学]