16S rRNA gene probe quantitates residual host cell DNA in pharmaceutical-grade plasmid DNA.
The development and widespread use of DNA-based vaccination against infectious pathogens have been a great triumph of medical science. Quality control of DNA vaccines as biopharmaceutical productions is a problem to solve. Residual genomic DNA of engineering bacteria has been identified as a potential risk factor, so whose level must be controlled under the regulatory standards. We report a dot-blot hybridization method to detect residual host cell DNA in purified DNA vaccines. The assay utilizes PCR amplified and digoxigenin-labeled Escherichia coli 16S rRNA gene as probe. The sensitivity of the dot-blot hybridization assay with E. coli 16S rRNA gene probe was evaluated in comparison with single copy UidR gene probe. The optimized dot-blot hybridization assay had both low background and a suitable sensitivity, detecting 10 pg of residual E. coli DNA. The method is suitable in the routine use of measuring the levels of residual E. coli DNA in the pharmaceutical-grade DNA vaccine.
Department of Medical Genetics, Second Military Medical University, 800 Xiang'Yin Road, Shanghai 200433, China.
Kai-Yu
KY
Wang
Ying-Jun
YJ
Guo
Shu-Han
SH
Sun
Ke
K
Shi
Shu
S
Zhang
Kai-Hui
KH
Wang
Yi-Zhang
Zu-Huan
ZH
Chen
D004247:DNA / Q000032:analysis*; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis; D004076:Digoxigenin / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D010957:Plasmids / Q000235:genetics; D012336:RNA, Ribosomal, 16S
D015342:DNA Probes; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D004247:DNA; D004076:Digoxigenin