Validation and application for determination of residual host cell DNA in enterovirus 71 inactivated vaccine by magnetic bead based extraction combined with quantitative PCR

Objective To verify the detection method for residual host cell DNA in enterovirus 71 inactivated vaccine (Vero cell) (EV71 vaccine) by magnetic bead based extraction combined with quantitative PCR (qPCR). Methods Residual host cell DNA in samples was extracted utilizing the specific binding of magnetic bead to DNA in protein solution. qPCR was performed on extracted DNA and serial diluted standards simultaneously. Residual DNA in samples was quantitatively analyzed according to the linear relationship between cycle threshold values and concentrations of standards. Results The standard detection range was 0.03-3 000.00 pg/reaction. The correlation coefficient of standard curve was >0.980, with amplification efficiency at 90.0%-110.0%. The recovery rates of spiked samples were 50%-150%, and the relative standard deviation was <30%. All parameters of results were within the required range. Conclusions The magnetic bead based extraction method can solve technical difficulties in sample pretreatment for residual DNA assay. qPCR is a simple, rapid and accurate method for quantitation of residual DNA in EV71 vaccine. This method is suitable for quality control of EV71 vaccine in production, and may provide indication for quality control of other same cell based viral vaccines. Key words: Quantitative polymerase chain reaction; Vero cells; Residual DNA; Magnetic bead based extraction; Host cell