重组H5N1流感血凝素杆状病毒滴度荧光定量PCR检测方法的建立、验证及应用  

Developoment,validation and application of fluorescence quantitative PCR for detection of titer of recombinant baculovirus with H5N1 influenza hemagglutinin

作　　者：吴清胜 吴相英 姚春萍 李媛媛 WU Qing-sheng;WU Xiang-ying;YAO Chun-ping;LI Yuan-yuan(National Vaccine and Serum Institute,Beijing 101111,China;不详)

机构地区：[1]国药中生生物技术研究院有限公司,北京101111 [2]北京生物制品研究所有限责任公司,北京100176

出　　处：《中国生物制品学杂志》2021年第10期1227-1231,共5页Chinese Journal of Biologicals

基　　金：“重大新药创制”科技重大专项(2013ZX09402302)。

摘　　要：目的建立用于重组H5N1流感血凝素杆状病毒滴度检测的荧光定量PCR(qPCR)法,并进行验证及初步应用。方法利用Bac-to-Bac技术构建重组穿梭质粒(rBacmid-H5),以其为模板,梯度PCR扩增H5基因,优化退火温度和引物浓度,建立qPCR检测方法,对该方法的专属性、重复性进行验证,确定线性范围及检测限。应用建立的qPCR方法对H5杆状病毒连续传代4次以及连续培养0-6 d的样品进行检测,并与免疫荧光法进行比较。结果在退火温度55℃,1μL 10 mmol/L引物条件下,建立了qPCR法,该方法检测H5基因,专属性良好;模板浓度在1.15×10^(3)-4.25×10^(9)copies/μL之间,线性关系良好,R^(2)=0.999;6次重复检测重组H5杆状病毒培养3 d样品,RSD为1.01%,重复性较好。H5病毒连续传代4次,病毒滴度保持相对稳定,培养0-6 d病毒滴度在第3天达到较高值,且检测值均高于免疫荧光法。结论建立了qPCR法进行H5N1流感血凝素重组杆状病毒滴度的特异性检测。Objective To develop,validate and preliminarily apply a fluorescence quantitative PCR method for detection of recombinant baculovirus with H5N1 influenza hemagglutinin.Methods Recombinant shuttle plasmid rBacmid-H5 was constructed by Bac-to-Bac technique and used as a template for amplification of H5 gene by grident PCR.The annealing temperature and primer concentration were optimized,based on which a qPCR method was developed,validated for specificity and reproducibility,and determined for linear range and detection limit.The H5 baculovirus subcultured for four passages and cultured for 0 - 6 d were determined by the developed qPCR method,and the result was compared with that by fluorescence immunoassay.Results The qPCR method was developed at an annealing temperature of 55 ℃and a primer concentration of 1 μL × 10 mmol/L.The method showed good specificity in detection of H5 gene and good linearity at a template concentration of 1.15 × 10^(3)-4.25 × 10^(9) copies/μL(R^(2)=0.999).The RSD of six repeat test results of recombinant H5 baculovirus cultured for 3 d was 1.01%,indicating a good reproducibility.The titer of H5 Conclusion A fluorescence quantitative PCR method was established for the specific baculovirus was relatively stable after subculture for four passages,which reached the peak value on day 3 after culture,and all the measured values were higher than those by fluorescence immunoassay.

关 键 词：荧光定量PCR法 甲型流感H5N1病毒 血凝素 杆状病毒 滴度 

分 类 号：R392-33[医药卫生—免疫学]