Real_timePCR检测核酸疫苗中宿主基因组残留DNA_李亮助.pdfVIP

2007 6 20 6 Chin J Biologicals June 2007, Vo.l 20N o. 6 # 43 # R392 A 1004-5503(2007) 06-439-04 Rea-l timePCR DNA 1, 2 2 2 2 1 2 李亮助 刘勇 王贻杰 程海 孙茂盛 邵一鸣 Real-tmi e PCR, DNA L ightcycler , 23S RNA , SYBR Green Real-tmi e PCR , 30 m in, , 10 fg /L ,l - 01 DNA Rea-l tmi e PCR; DNA; Determination ofResidual ostGenomicDNA inDNA Vaccine byRea-l timePCR v v LI L iang-zhu , LIU Yong, WANG Y -i jie, et al ( Institute of M ed ical B iology, Chinese Academy of M ed ical Science Peking UnionM edical College, K unm ing 650118, China) Abstract Objective To develop a rea-l tmi e PCR for determ ination of residual host genom icDNA in DNA vaccine. Methods Amp lify residual host genom ic DNA from DNA vaccine by PCR using designed prmi ers target toE. coli 23S ribosom e RNA gene and de- term ine the real-tmi e fluorescence intensity of am plified product. Determ ine the intermed iate product during purification of DNA vaccine by the developed rea-l tmi e PCR. Results Thewhole determ inationw as com pletedw ith in 30 m in. The developed rea-l tmi e PCR show ed good specificity and a sensitivity of 10 fg/L l. The correlation coefficient of standard curve of them ethod was - 01 . Conclusion The developed real-tmi e PCR m ay be used for the determ ination of residual host genom ic DNA in DNA vaccine. Keywords Real-tmi e PCR; H ost genom ic DNA; DNA vaccine , DNA pGP140 ( 7 074 bp) , DNA ; , DH5A Invitrogen 21 [ 1, 2] m TM DNA , SYBR Prem ix Ex Taq ( DNA