目的:建立并验证rAAV2-ND4中可复制型AAV2(rcAAV2)的qPCR检测方法。方法:rAAV2-ND4样品(3.0×1010 vg·mL-1,每孔接种100 μL)和wtAd5辅毒(6.0 ×109 TCID50·mL-1,每孔接种10 μL)同时感染HEK293细胞(6.0×105 cells·mL-1,每孔1 mL接种至12孔板中,37 ℃和5%二氧化碳条件培养24 h),经过2 h感染后,更换为细胞培养基(含10%FBS的 DMEM,每孔加入1 mL),继续在37 ℃和5%二氧化碳条件培养48 h。收集细胞并用细胞裂解液处理,细胞裂解物再经上述步骤进行1次感染和培养。收集第二次培养的细胞并加入裂解液56 ℃孵育30 min,之后90 ℃孵育10 min。处理后的细胞裂解物用qPCR方法进行rcAAV2检测,qPCR反应体系为2×T5 Fast qPCR Mix(Probe) 10 μL、引物混合液0.1 μL、探针1 μL、50×ROX Reference Dye I 0.4 μL、EASY Dilution 3.5 μL、处理后的细胞裂解物5 μL,qPCR扩增程序:95 ℃,10 min;[95 ℃,15 s;60 ℃,1 min]40个循环。对方法进行灵敏度、准确度、精密度和专属性验证。结果:所建立的检测rcAAV2的方法灵敏度可达到每108 vg<1 TCID50 rcAAV;qPCR方法回收率在77.6%~91.2%;每人3次(2人)实验均得到相同结果,方法专属性良好。结论:所建立的2轮感染扩增后qPCR检测的方法可应用于rAAV2-ND4样品中rcAAV2的检测。
Objective: To develop and validate a qPCR method for the determination of replication-competent AAV2(rcAAV2) in rAAV2-ND4. Methods: HEK293 cells (6.0×105 cells·mL-1, 1 mL per well, inoculated into a 12 well plate for 24 h at 37 ℃ and 5% carbon dioxide) were infected with rAAV2 ND4 sample (3.0×1010 vg·mL-1, with 100 μL per well) and wtAd5 (6.0×109 TCID50·mL-1, with 10 μL per well) simultaneously. After 2 h of infection, the infected medium was replaced with cell culture medium (DMEM containing 10% FBS, 1 mL per well), and then the infected cells were cultured at 37 ℃ and 5% carbon dioxide for another 48 h. The cells were collected and treated with cell lysate solution. The cell lysate was then subjected to the above steps for infection and culture. The cells from the second culture were collected and incubated with lysate at 56 ℃ for 30 min, followed by incubation at 90 ℃ for 10 min. The processed cell lysates were detected for rcAAV2 using qPCR method, the qPCR reaction system included 2×T5 Fast qPCR Mix (Probe) 10 μL, primer mixture 0.1 μL, probe 1 μL, 50×ROX Reference Dye I 0.4 μL, EASY Dilution 3.5 μL, treated cell lysate 5 μL, and qPCR amplification program was set as bellow: 95 ℃, 10 min; 40 cycles of [95 ℃, 15 s; 60 ℃, 1 min]. Then the sensitivity, accuracy, precision, and specificity of the method were verified. Results: The sensitivity of the established method for detecting rcAAV2 reached less than 1 TCID50 rcAAV per 108 virus genomes; the recovery rate of qPCR method ranged from 77.6% to 91.2%. The same results in three experiments per person (two people) was obtained, and the method specificity was good. Conclusion: The established qPCR method after two rounds of infection amplification can be applied to the detection of rcAAV2 in rAAV2 ND4 samples.
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