腺病毒滴度不同测定方法比较

A comparative analysis of the methods for tittering adenoviruses

目的将目前测定腺病毒滴度常用的OD260法、荧光定量PCR法、绿色荧光蛋白标记法、壳蛋白免疫法、细胞病变(CPE)法进行比较研究,分析和讨论各种方法的优缺点。方法 4种重组腺病毒(Ad-G-AT2R-EGFP、Ad–CMV-EGFP、Ad-mif-shRNA-EGFP和Ad-CBA-GFP)扩增、纯化后,同时分别用OD260值法、荧光定量PCR法、绿色荧光蛋白标记法、壳蛋白免疫法及CPE法测定病毒滴度,并以壳蛋白免疫法测定结果作为标准,对各种方法所得结果进行比较研究。结果绿色荧光蛋白标记法、CPE法与壳蛋白免疫法同时测定4种腺病毒滴度,其结果无统计学差异(P>0.1)。荧光定量PCR法测定值明显高于壳蛋白免疫法(P<0.05),但与该法测定结果间存在良好的相关性(r=0.965),约为壳蛋白免疫法所测得结果的10倍。OD260法测得结果与壳蛋白免疫法测得结果间则无相关性。结论绿色荧光蛋白标记法与壳蛋白免疫法是测定腺病毒感染性滴度最为快速有效的方法,其结果客观可靠,能真实地反映病毒的实际感染力。荧光定量PCR法测定结果反映的是完整的病毒颗粒数,能够间接反映病毒的感染力,且该方法操作简单,耗时短,同样有较高的实用性。

Objective To compare different methods commonly used for tittering adenovirus and analyze the advantages and limitations of each method.Methods Four recombined adenoviruses（Ad-G-AT2R-EGFP,Ad-CMV-EGFP,Ad-mif-shRNA-EGFP and Ad-CBA-GFP） were amplified and purified,and each was tittered by optical absorbance,real-time PCR,green fluorescent protein（GFP）-labeled method,immunoassay,and cytopathic effect（CPE）.The results were then comparatively analyzed.Results No significant difference was found in the titer amounts derived from GFP-labeled method,immunoassay,and cytopathic effect method（P0.1）.A positive correlation was noted in the titer amounts determined by real-time PCR and immunoassay（r=0.965）,even though the value（vg/ml） obtained by real-time PCR was 10 times higher than that by immunoassay（ifu/ml）.Conclusion GFP-labeled method and immunoassay allow rapid determination of the adenoviral titer.Real-time PCR can not directly determine the real infectious titer of the adenovirus,but the result is well correlated to that of immunoassay and reflects,though indirectly,the actual infectious titer of adenovirus.Considering the procedural convenience and shorter time consumption,real-time PCR is still a practical method for adenoviral titration.