2种常用核酸提取方法的比较

Comparison on 2 common methods of nucleic acid extraction

目的通过对PCR监测中2种常用核酸提取方法的比较,为不同的需要选择病毒核酸检测实验中的最佳提取方法。方法利用国家流感网络实验室分离到的10株甲型H3N2流感病毒毒株,分别采用滤膜法和磁珠法进行核酸提取,对收集到的核酸分别进行扩增比对分析;用一株甲型H3N2流感病毒毒株以10倍为单位进行10~90倍稀释,获得10组不同的稀释标本,滤膜法和磁珠法分别进行同步提取,比对扩增结果。结果磁珠法在核酸提取中的效率明显高于滤膜法,结果差异均值为1.29个Ct值,有统计学意义(P <0.05)。滤膜法的核酸提取过程中,经反复冲洗后仍可获得较理想的Ct值,前3次的冲洗Ct值差值均值分别为1.39和1.49,差异有统计学意义(P <0.05);第三次和第四次的冲洗,Ct值差值均值为0.17,无统计学意义(P>0.05)。结论磁珠法的提取效率明显高于传统滤膜法,在敏感性较高的疫情处置中及处于灰区或者阴性的标本复核中,可利用磁珠法提高结果的准确性;滤膜法可在标本量较少的情况下经多批次的冲洗后,能为进一步的科研分析与多项目检测提供较多的核酸提取物。

Objective By comparing 2 common nucleic acid extraction methods in PCR monitoring,the best extraction method in virus nucleic acid detection experiment was selected for different needs. Methods 10 influenza A H3 N2 virus strains isolated by national influenza network laboratory was selected,the RNA were extracted by filtration membrane method and magnetic beads method respectively,analyze the RNA amplifie efficiency.A strain of influenza A H3 N2 virus was diluted 10-90 times with 10 times as the unit to obtain 10 groups of different diluted samples,the filter membrane method and magnetic bead method were used for simultaneous extraction,and the amplification results were compared.Results The efficiency of magnetic bead method in nucleic acid extraction was significantly higher than filter membrane method,the results showed that the mean difference was 1.29 Ct values(P <0.05)with statistically significant difference.In the nucleic acid extraction process of filter membrane method,the ideal Ct value could still be obtained after repeated washing.The mean difference of Ct values of the first 3 washing were 1.39 and 1.49 with statistically significant difference(P <0.05),there was no significant difference between the third flush and the fourth flush,the mean difference of CT value was 0.17(P >0.05). Conclusion The extraction efficiency of magnetic bead method is significantly higher than traditional filter membrane method.Magnetic bead method can be used to recheck in the epidemic situation with high sensitivity,And in the recheck of samples in gray area or negative area detected by filter membrane method to improve the accuracy of results.Filter membrane method can provide reference for further scientific research analysis and multi item detection after multiple batches of washing in the case of small amount of samples for more nucleic acid extract.More suitable nucleic acid extraction methods can be selected according to different needs.