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目的:建立并确证实时定量PCR(RT-PCR)定量检测生物基质中质粒DNA的方法,并应用于治疗性核酸疫苗HIV-PV的体内生物分布研究。方法:建立SYBR Green荧光定量PCR绝对定量方法(Q-PCR),考察方法的特异性、灵敏度、精密度、准确度以及生物基质对PCR的抑制效应。小鼠尾静脉注射裸质粒DNA、小鼠尾静脉注射和灌胃2种途径免疫HIV-PV DNA疫苗,考察建立的Q-PCR方法对于不同形式的质粒DNA疫苗以及不同免疫方式的适用性。结果:Q-PCR方法可特异检测目标DNA,定量检测范围为10~107拷贝/100 ng基因组DNA(gDNA),定量下限(LLOQ)为10拷贝/100 ng gDNA。批内和批间精密度分别<11%和8%;由高至低4种浓度(107,104,100,10拷贝/100 ng gDNA)QC样品准确度分别为(1.57±0.02)%,(1.60±0.01)%,(2.20±0.02)%和(2.28±0.02)%。用Q-PCR方法成功得到不同形式DNA疫苗和不同免疫途径(尾静脉注射和灌胃HIV-PV疫苗)下该质粒DNA的生物分布模式。结论:利用RT-PCR技术,建立了符合DNA疫苗生物分布研究要求的定量检测生物基质中质粒DNA的方法学,并成功应用于不同质粒DNA疫苗形式和不同免疫途径下的DNA疫苗的生物分布研究。
Objective: To establish and confirm real-time quantitative PCR (RT-PCR) quantitative detection of plasmid DNA in biological matrix, and applied to the in vivo biodistribution study of therapeutic nucleic acid vaccine HIV-PV. Methods: Quantitative SYBR Green quantitative PCR (Q-PCR) was established to investigate the specificity, sensitivity, precision and accuracy of PCR and the inhibitory effect of biomatrix on PCR. Immunization of HIV-PV DNA vaccine with naked plasmid DNA, mouse tail vein injection and gavage by mouse tail vein was carried out to investigate the applicability of Q-PCR method to different forms of plasmid DNA vaccine and different immunization methods. Results: The Q-PCR method could detect the target DNA specifically. The quantitative detection range was 10 ~ 107 copies / 100 ng genomic DNA (gDNA), and the limit of quantification (LLOQ) was 10 copies / 100 ng gDNA. Intra-assay and inter-assay precision were <11% and 8%, respectively. The accuracy of QC samples from high to low (107,104,100,10 copies / 100 ng gDNA) were (1.57 ± 0.02)% and (1.60 ± 0.01) %, (2.20 ± 0.02)% and (2.28 ± 0.02)% respectively. The biodistribution pattern of this plasmid DNA under different forms of DNA vaccine and different immunization routes (tail vein injection and gavage of HIV-PV vaccine) was successfully obtained by Q-PCR. Conclusion: The method of quantitative detection of plasmid DNA in biological matrices that meets the requirements of DNA vaccine biodistribution research was established by RT-PCR and successfully applied in the study of biodistribution of DNA vaccines under different forms of DNA vaccines and different immunization routes .