摘要
目的 建立转染RNA拯救CVB3的方法,为优化CVB3的分离鉴定和体外培养提供技术参考.方法 比较Qiagen 57704、Qiagen 52904和Trizol三种核酸提取试剂提取CVB3基因组RNA的效率及Lipofectamine 2000和Lipofectamine 3000两种转染试剂转染病毒RNA的效率;探究提取RNA时增加洗脱次数对病毒拯救效率的影响;比较使用HeLa细胞、Vero细胞和HEK293T细胞拯救CVB3的效率.由此确定转染病毒RNA拯救CVB3的最适条件.结果 Qiagen 57704、Qiagen 52904、Tizol试剂盒提取RNA拯救病毒的噬斑数分别为13.33±1.53、150.00±15.00、1.67±0.58,三种方法提取CVB3 RNA拯救病毒RNA的效率差异具有统计学意义(F=268.920,P＜0.001);Lipofectamine3000、Lipofectamine2000 转染病毒 RNA 形成的噬斑数分别为 74.50±3.00、22.00±5.00,差异具有统计学意义(P＜0.01);HEK293T细胞培养病毒拯救CVB3的效率高于HeLa细胞和Vero细胞,三种细胞拯救病毒的拷贝数分别为6.09×107±8.00×105、5.18×103±6.17×102、0,差异具有统计学意义(F=17 383.644,P＜0.001),同时发现提取RNA时通过多次洗脱可以提高病毒拯救的效率.结论 本研究成功建立了一种优化的转染RNA拯救CVB3的方法,选用Qiagen 52904核酸提取试剂盒、增加洗脱次数、使用Lipofectamine 3000转染试剂、HEK293T细胞转染等方法,可以有效提升病毒的拯救效率.
Abstract
Objective To establish a method via transfection of RNA to rescue coxsackievirus B3 B3(CVB3).Methods The efficiency of CVB3 genomic RNA extraction from three nucleic acid extraction reagents,Qiagen 57704,Qiagen 52904,and Trizol,and the transfection efficiency of viral RNA with two transfection reagents(Lipofectamine 2000 and Lipofectamine 3000)were compared.The efficiency of CVB3 rescue in Vero cells and HEK293T cells to determine the optimal conditions for rescuing CVB3.Results The number of phagolysosomes for virus rescue by Qiagen 57704,Qiagen 52904,and Tizol kit extracted RNA was 13.33±1.53,150±15.00,and 1.67±0.58,respectively,and there was a statistically significant difference in the efficiency of the three method of extracting CVB3 RNA to rescue the viral RNA(F=268.920,P＜0.001);The number of phage spots formed by Lipofectamine3000 and Lipofectamine2000 transfected RNA was 74.50±3.00 and 22.00±5.00,respectively,and the difference was statistically significant(P＜0.01);Qiagen 52904 reagent extracted CVB3 nucleic acid more efficiently than Qiagen 57704 and Trizol reagents;the transfection efficiency of transfection reagent Lipofectamine 3000 was 3 times more than than that of Lipofectamine 2000,and the efficiency of virus rescue of CVB3 in HEK293T cell culture was higher than that of HeLa and Vero cells,and the copy numbers of the three kinds of viruses rescuing the virus were 6.09×107±8.00×105,5.18×103±6.17×102 and 0,the difference was statistically significant(F=17 383.644,P＜0.001),and it was also found that the efficiency of virus rescue could be improved by multiple elution when extracting RNA.Conclusions In this study,we successfully established the method of transfecting RNA to rescue CVB3,which can effectively improve the efficiency of virus rescue by choosing Qiagen 52904 nucleic acid extraction kit,increasing the number of elution,using Lipofectamine 3000 transfection reagent,and transfection of HEK293T cells.
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