目的:探索采用多重数字PCR(dPCR)技术评价重组腺相关病毒(recombinant adeno-associated virus, rAAV)基因组完整性的可行性。方法:采用芯片式dPCR系统,针对rAAV基因组的末端反向重复序列(ITR)区和目的基因(或3’非翻译区)建立双重PCR法,通过适量稀释使模板DNA在PCR芯片中呈理论单拷贝分布(阳性孔低于20%),单阳性孔代表非完整片段,双阳性孔代表2组引物对之间的序列完整,通过测试单、双阳性孔的比例,评价rAAV基因组的完整性;尝试采用不同样品前处理方式(病毒基因组DNA、病毒颗粒、DNaseⅠ处理),并比较测试结果。结果:双重PCR测得拷贝数与单重PCR基本一致,无明显干扰;在一定范围内,样本的实测拷贝数与模板量呈线性相关,但单阳、双阳比例基本一致;提取的病毒基因组DNA实测拷贝数减少,但双阳率高于未处理病毒样本,DNase Ⅰ处理后的病毒样本实测拷贝数明显减少,单阳、双阳比例与未处理病毒样本基本一致。结论:多重dPCR技术可用于评价rAAV产品基因组完整性,具有良好的应用前景。
Objective: To explore the feasibility of multiplex PCR technique in recombinant adeno-associated virus(rAAV) genome integrity evaluation. Methods: A chip dPCR system was used to establish a dual PCR method targeting ITR region and gene of interest (or the 3’untranslated region) of rAAV genome. By proper dilution, a theoretical single copy distribution of template DNA was achieved in the PCR chip wells (the parentage of positive wells was less than 20%), the single positive wells represented the incomplete fragment, and the double positive wells represented the complete sequence between two sets of primer pairs. The integrity of rAAV genome was evaluated by testing the ratio of double positive wells. Different sample pretreatment methods (virus genomic DNA, virus particles, DNase Ⅰ treatment) were conducted to compare the test results. Results: The copy number measured by duplex PCR was basically consistent with that of single PCR, showing no obvious interference. Within a certain range, the measured copy number of samples was linearly correlated with the amount of templates, but the proportions of single positive and double positve was basically the same. Compared with untreated viral particles, the measured copy number of the extracted viral genome DNA was reduced, but the double positive rate was higher. The measured copy number of the virus samples treated with DNaseⅠ was significantly reduced, and the proportions of single positive and double positive were basically consistent with those of the untreated viral samples. Conclusion: Multiplex dPCR technology could be used to evaluate the genome integrity of rAAV products, showing a good application prospect.
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