重组杆状病毒滴度qPCR快速检测方法的建立OA

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.