杆状病毒微滴式数字PCR检测方法的建立及验证

Development and verification of droplet digital PCR for detection of baculovirus

目的建立杆状病毒(baculovirus,BV)微滴式数字PCR检测方法,并对其退火温度、引物及探针浓度优化后,进行方法验证。方法根据GP64基因保守序列特征,设计特异性引物及TaqMan探针,建立靶向BV的微滴式数字PCR检测方法,优化体系的退火温度、引物及探针浓度,并进行方法的特异性、重复性、灵敏度、线性验证;采用建立的微滴数字式PCR法检测GMP中试生产车间24份样本,并与蚀斑法检测结果继续比较。结果建立了靶向BV的微滴式数字PCR检测方法,优化后的退火温度、引物及探针浓度分别为58.0℃、400 nmol/L、200 nmol/L。仅以BV核酸为模板的反应中检测到阳性液滴,方法特异性良好;批内重复性CV在2.78%~7.45%之间,批间重复性CV在2.28%~7.05%之间,均<10%,方法重复性良好;最低检测限为3.06 copies/μL,约为常规PCR的100倍,方法灵敏度高;24份样本检测结果与蚀斑法接近,相关系数(r)为0.913。结论成功建立了针对BV的优化后的微滴式数字PCR检测方法,该方法具有良好的特异性、重复性、灵敏度、快捷性,可用于生产中BV的快速检测。

Objective To develop a droplet digital PCR method for detection of baculovirus(BV),optimize the annealing temperature,primer and probe concentrations,and verify the method.Methods According to the conserved sequence characteristics of GP64 gene,specific primers and TaqMan probes were designed to develop a droplet digital PCR detection method for BV.The annealing temperature,primer concentration,and probe concentration of the system were optimized,and the specificity,reproducibility,sensitivity and linearity of the method were verified.The 24 samples from GMP pilot workshop were detected by the droplet digital PCR,and the results were compared with those detected by plaque method.Results A droplet digital PCR method targeting BV was developed.The optimized annealing temperature,primer and probe concentrations were 58.0℃,400 nmol/L,and 200 nmol/L,respectively.Positive droplets were detected only in the reaction with BV nucleic acid as template,which showed that the method had good specificity.The CVs of intra-batch reproducibility ranged from 2.78%to 7.45%,and those of inter-batch reproducibility ranged from 2.28%to 7.05%,both less than 10%,indicating that the method had good reproducibility.The minimum detection limit was 3.06 copies/μL,which was about 100 times higher than that of the ordinary PCR,showing the high sensitivity of the method.The results of 24 samples were close to those of plaque method,and the correlation coefficient(r)was 0.913.Conclusion An optimized droplet digital PCR assay for the detection of BV has been successfully developed,which has good specificity,reproduci-bility,sensitivity,and rapidness,and can be used for the rapid detection of BV in production.