Development and verification of droplet digital PCR for detection of baculovirus
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 char-acteristics 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 work-shop 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 reac-tion with BV nucleic acid as template,which showed that the method had good specificity.The CVs of intra-batch reproduci-bility 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.
NETL
NSTL
万方数据
