长链醇类和油类物质洗涤液在磁珠法DNA和RNA共提取中的应用

Application of long-chain alcohols and oils as washing solution in DNA and RNA coextraction based on magnetic bead method

目的探讨长链醇类和油类物质作为洗涤液在磁珠法核酸提取中的应用价值。方法分别以5种长链醇类物质(十一醇、正癸醇、正辛醇、1-壬醇、2-十二烷醇)和3种油类物质(二甲基硅油、FC-40、石蜡油)作为洗涤液,采用磁珠法对102~105拷贝/μL的DNA和RNA混合物进行共提取,以传统的70%乙醇洗涤液作为对照。采用聚合酶链反应(PCR)或逆转录聚合酶链反应(RT-PCR)对提取的核酸进行扩增,采用循环阈值(Ct)评价不同洗涤液的核酸提取效率。以油类物质为洗涤液,比较不同试剂状态(干式试剂、湿式试剂)、不同混匀方式(涡旋混匀、移液枪混匀、颠倒混匀)的核酸提取性能,并分析其用于提取临床样本病原体基因组核酸的性能。结果8种洗涤液中,二甲基硅油和FC-40的核酸提取性能与传统的70%乙醇基本一致,可有效提取出102~105拷贝/μL DNA或RNA,对PCR和RT-PCR均无抑制作用。以70%乙醇洗涤液为对照,将二甲基硅油或FC-40作为核酸提取洗涤液时,无论是配合干式试剂还是湿式试剂,无论采用哪种混匀方式,提取103拷贝/μL DNA和RNA模板的Ct值差异均无统计学意义(P>0.05);提取肺炎支原体模拟临床样本DNA的Ct值差异无统计学意义(P>0.05),且与商品化核酸提取试剂盒基本一致。结论二甲基硅油和FC-40可作为磁珠法核酸共提取的洗涤液,并可用于微流控产品的开发。

Objective To investigate the application value of long-chain alcohols and oils as washing solution in nucleic acid extraction based on magnetic bead method.Methods Totally,102-105 copies/μL DNA and RNA mixtures were extracted by magnetic bead method,and 5 kinds of long-chain alcohols,including undecanol,decanol,octanol,1-nonanol and 2-dodecanol,and 3 kinds of oils,including dimethicone,FC-40 and paraffin oil,were determined as washing solution,with the traditional 70%ethanol as control.The extracted nucleic acids were amplified using polymerase chain reaction(PCR)or reverse transcription-polymerase chain reaction(RTPCR),and the nucleic acid extraction efficiency of different washing solutions was evaluated by cycle threshold(Ct).The nucleic acid extraction performance under different reagent status(dry reagents and wet reagents)and different mixing modes(vortex mixing,pipette mixing and inversion mixing)was compared using oils as washing solution,and the performance for extracting genomic nucleic acid from clinical pathogen samples was evaluated as well.Results The nucleic acid extraction performance of dimethicone and FC-40 among the 8 kinds of washing solutions was basically the same as that of traditional 70%ethanol.The 102-105 copies/μL DNA or RNA could be effectively extracted without inhibiting PCR and RT-PCR.When using 70%ethanol as control and dimethicone or FC-40 as washing solution,no statistical significance was found in Ct values of 103 copies/μL DNA and RNA amplification,regardless of the combination of reagent status or mixing mode(P>0.05).There was no statistical significance in Ct value of DNA extracted from Mycoplasma pneumoniae simulated clinical samples(P>0.05),and it was basically consistent with commercial nucleic acid extraction kits.Conclusions Dimethicone and FC-40 can be used as washing solutions for nucleic acid extraction by magnetic bead method and can be used for the development of microfluidic products.