中国兽医杂志2026，Vol.62Issue(4)：37-45,9.DOI:10.20157/j.cnki.zgsyzz.2026.04.005
重组杆状病毒滴度qPCR快速检测方法的建立
Establishment of a Rapid qPCR Method for Determining Recombinant Baculovirus Titers
摘要
Abstract
To enable rapid and efficient determination of recombinant baculovirus(rBV)titers,thereby shortening its development cycle,enabling precise control of its production process,and supporting its preclinical safety evaluation,this study developed a real-time fluorescent quantitative polymerase chain reaction(qPCR)assay using the pFastBac Dual plasmid from the baculovirus expression system as the standard template and rBV genomic DNA as the detection template.Primers were designed against the transposon sequence shared by both the plasmid and rBV.After optimization of reaction conditions,the qPCR method for rBV detection was established and evaluated for specificity,repeatability,accuracy,and precision.Using immunostaining as a reference method and in combination with viral growth kinetics,the correlation,agreement,and equivalence between the two methods were assessed for practical application.The results showed that the optimal annealing temperature for the qPCR assay was 60℃,with optimal forward and reverse primer concentrations of 4 and 2 µmol/L,respectively.The assay specifically detected rBV.Both inter-assay and intra-assay coefficients of variation were less than 10%,indicating good repeatability.The relative error and relative standard deviation between theoretical and measured values were both below 10%,demonstrating good accuracy and precision.The standard curve exhibited good linearity over the range of 3.324×10³ to 3.324×10⁸ copies/µL,with a limit of detection of 33.24 copies/µL.Analysis of 34 rBV samples prepared in this laboratory showed that titers measured by qPCR were higher than those obtained by immunostaining;however,Pearson correlation analysis revealed a significant positive correlation between the two methods(r=0.891 9,P<0.000 1).Bland-Altman analysis further demonstrated good agreement.Comparison of rBV growth kinetics indicated no significant difference between the two methods(P>0.05),confirming their equivalence.These results demonstrate that a rapid and effective qPCR method was successfully established,enabling accurate determination of rBV titers within approximately 2 h.The method is broadly applicable to rBVs generated using the Bac-to-Bac shuttle expression system and provides a foundation for quantitative analysis,process optimization,and quality control of rBV.关键词
重组杆状病毒/细菌-杆状病毒穿梭表达系统(Bac-to-Bac)/实时荧光定量聚合酶链式反应(qPCR)/滴度Key words
recombinant baculovirus/Bac-to-Bac baculovirus expression system(Bac-to-Bac)/real-time fluorescent quanti-tative polymerase chain reaction(qPCR)/titer分类
农业科技引用本文复制引用
马文阁,戴海越,吴浩,刘宇昕,于越洋,王爽,孙玮玮,吴文学..重组杆状病毒滴度qPCR快速检测方法的建立[J].中国兽医杂志,2026,62(4):37-45,9.基金项目
国家重点研发计划(2022YFD1800700) （2022YFD1800700）
国家现代农业产业技术体系项目(CARS36) （CARS36）
中国博士后科学基金面上资助(2024M753562) （2024M753562）
