植物RNA作为载体RNA在病毒核酸提取过程中的保护效果研究

Protective effect of plant RNA as carrier RNA during viral nucleic acid extraction

目的探究植物RNA作为载体RNA在病毒核酸提取过程中对目标核酸的保护效果。方法分别提取绿萝、水稻、竹子3种常见植物中的RNA作为载体RNA,同时以商业试剂Takara载体RNA为对照,提取甲型流感病毒(Flu A)、乙型流感病毒(Flu B)、呼吸道合胞病毒A型(RSV A)核酸,并利用实时荧光定量反转录聚合酶链反应(RT-qPCR)定量分析提取得到的病毒核酸浓度,以评估植物RNA在核酸提取中对病毒核酸的保护效果。结果经琼脂糖凝胶电泳检测,所提取植物RNA的28S rRNA、18S rRNA条带清晰明亮,无弥散现象,所得RNA具有良好的完整性。且经验证,植物RNA对病毒核酸扩增无干扰。裂解液中分别加入0、1000、3000、5000、7000、9000 ng绿萝RNA进行Flu A RNA提取时,提取物经RT-qPCR扩增后的Ct值分别为35.06±0.14、33.01±0.42、32.45±0.33、31.95±0.34、31.74±0.28、31.92±0.24,使用Takara载体RNA提取核酸的Ct值为31.96±0.34。Ct值随着绿萝RNA添加量增加而降低,当添加量≥5000 ng时,Ct值接近使用Takara载体RNA的提取组,继续提高绿萝RNA用量,Ct值趋于稳定,且与使用TaKaRa载体RNA的Ct值比较,差异无统计学意义(P>0.05)。添加5000 ng绿萝RNA与添加0 ng绿萝RNA相比,Ct值相差3.11,根据浓度差等于2ΔCt计算可知添加5000 ng绿萝RNA提取得到的Flu A核酸浓度比不添加载体RNA高了8.63倍。进一步研究发现,添加绿萝RNA、水稻RNA、竹子RNA及Takara载体RNA,提取得到的Flu A、Flu B和RSV A核酸经RT-qPCR扩增后得到的Ct值与不添加载体RNA比较,差异均有统计学意义(P<0.05)。绿萝RNA作为载体RNA在提取Flu A、Flu B、RSV A混合病毒核酸时,对比无载体RNA的对照组,分别降低了2.61、2.90、1.45个Ct值,即添加了绿萝RNA后提取Flu A、FluB、RSV A所得的核酸浓度分别提高了6.11、7.46、2.73倍。且添加了水稻RNA和竹子RNA提取混合病毒核酸所得的核酸浓度也分别至少提高了2.63倍和2.60倍。结论在病毒核酸提取过程中植物RNA具有保护病毒核酸的作用,可以提高提取得到的目的病毒RNA浓度,可用作病毒核酸提取时的载体RNA,为核酸提取中的载体RNA选择提供更多来源。

Objective To explore the protective effect of plant RNA as carrier RNA on target nucleic acid in the process of viral nucleic acid extraction.Methods RNA from three commonly known plants,namely,epipremnum aureum,rice plants and bamboo,was extracted as carrier RNA.Meanwhile,nucleic acids of influenza A virus(Flu A),influenza B virus(Flu B),respiratory syncytial virus A(RSV A)were extracted employed commercial reagent Takara carrier RNA as control.Real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-qPCR)was employed to quantitatively analyze the concentration of viral nucleic acid to assess the protective effect of plant RNA on viral nucleic acid during nucleic acid extraction.Results By means of agarose gel electrophoresis,the bands of 28S rRNA and 18S rRNA of the extracted plant RNA were distinct and bright,without any dispersion phenomenon,and the obtained RNA exhibited excellent integrity.And it be demonstrated that plant RNA does not interfere with the amplification of viral nucleic acid.Flu A virus nucleic acid was extracted by adding 0,1000,3000,5000,7000 and 9000 ng of epipremnum aureum RNA into the lysate,respectively.The RT-qPCR Ct values of extracts were 35.06±0.14,33.01±0.42,32.45±0.33,31.95±0.34,31.74±0.28,31.92±0.24 respectively.The control Ct values of nucleic acid extracted by Takara carrier RNA was 31.96±0.34.The Ct value decreased with the increase of epipremnum aureum RNA.When the supplemental level was≥5000 ng,the Ct value was close to the Takara carrier RNA group,and continued to increase,it tended to be stable,and there was no statistical significance compared with the Ct value of Takara carrier RNA control group(P>0.05).The difference in Ct values between the 5000 ng group and the 0 ng group was 3.11.According to the calculation that the concentration difference was equal to 2ΔCt,the concentration of Flu A nucleic acid extracted by adding 5000 ng epipremnum aureum RNA was 8.63 times higher than that without adding carrier RNA.Further,the RT-qPCR amplification Ct values of the extracted Flu A,Flu B and RSV A nucleic acids with the addition of epipremnum aureum RNA,rice RNA,bamboo RNA and Takara carrier RNA were significantly different from the control group without carrier RNA(P<0.05).In the extraction of nucleic acids from Flu A,Flu B and RSV A mixed sample,the Ct values decreased by 2.61,2.90 and 1.45 respectively,compared with the control group without carrier RNA.Mean that the nucleic acid concentration of Flu A,FluB and RSV A increased by 6.11,7.46 and 2.73 times respectively.Moreover,the nucleic acid concentration was at least 2.63 times and 2.60 times higher when rice RNA and bamboo RNA acted as carrier RNA during extraction respectively.Conclusion Plant RNA plays a protective role in the process of viral nucleic acid extraction,which can improve the concentration of the target viral RNA obtained.Plant RNA can be used as a carrier RNA in viral nucleic acid extraction,and provide more sources for the selection of carrier RNA in nucleic acid extraction.