Establishment and verification of DNA residue detection method of oral hexavalent reassortant live attenuated rotavirus vaccine(Vero cells)
Objective To establish a method for the detection of DNA residues in the stock solution and finished product of oral hexavalent reassortant live attenuated rotavirus(RV)vaccine(Vero cells)by magnetic bead separation method combined with fluorescent staining method,and to verify the method.Methods The residual Vero cell DNA in the sample was extracted by the host cell residual DNA sample preparation kit(magnetic bead method),and then the residual DNA in the sample was determined by fluorescence staining,and the standard curve was fitted by linear regression and the residual amount of DNA in the sample was analyzed.The established method was validated for the linear range,precision,accuracy,robustness,specificity,and limit of quantification(LOQ),and the residual host cell DNA in the monovalent stock solutions and finished products of six serotypes was determined.Three different methods(fluorescent staining,qPCR and molecular hybridization)were used for detection,and the consistency of detection results was compared.Results The LOQ for the determination of DNA residue content in oral hexavalent reassortant live attenuated RV vaccine(Vero cells)by this method was 1.25 ng/mL,and the linearity was good at the standard concentration in the range of 1.25-80 ng/mL,with the linear correlation coefficient(r)of 1.00.The spike recoveries were 93.8％-132.1％for six repeated tests of the monovalent stock solution and 66.0％-90.0％for the finished product.The relative standard deviations(RSDs)of the same operator for six repeated tests of the monovalent stock solution and finished product were between 5.5％and 16.1％,and the RSDs of different personnel for monovalent stock solution and finished product in six repeated tests were 7.7％and 15.6％,respectively.When the standard was diluted to 25 µg/mL with interference solution and RNase-free Water respectively,the recoveries of DNA residue detection results of Vero cells were between 98.0％and 138.8％,RSD＜12％.The RSDs of the test results of the monovalent stock solution and finished product with different incubation times were less than 16.3％and 8.6％,respec-tively.The RSDs of the detected DNA residues in 6 different serotypes of stock solution and 3 batches of finished products were less than 7.1％and 6.1％at different times and laboratories,respectively.The detection results of the same sample by the three different methods were consistent.Conclusion The combination of magnetic bead separation and fluorescent staining can be used for the detection of DNA residues in oral hexavalent reassortant live attenuated RV vaccine(Vero cells),and can also be used as an evaluation for key steps such as intermediates in the process.
DNA residuesFluorescent staining methodMagnetic bead separation methodVero cellsRotavirus(RV)
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