活性污泥高质量RNA快速提取方法研究

Rapid Method to Extract High-Quality RNA from Activated Sludge

通过比较RNA产量、纯度、降解程度、特定基因的扩增能力、微生物多样性等评价指标,考察和探讨了5种不同RNA提取方法对活性污泥总RNA提取效果的影响并最终建立了一种快速、有效的活性污泥RNA提取方法,即在TENP和PBS洗涤沉淀污泥的基础上,分别采用溶菌酶和TRIzol裂解活性污泥细菌、氯仿去除细菌裂解液中的蛋白和大部分DNA、异丙醇沉淀核酸和DNase I水解残留DNA后,最后进一步用离心柱纯化RNA.结果表明,这种方法可以有效提取高质量的菌群RNA,不仅提取的RNA总量多(每g污泥可提取169.6μg RNA)、纯度高、降解程度低、完整性好、具有丰富的生物多样性,而且可同时进行16SrRNA和amoA基因的RT-PCR扩增反应;与其它方法相比,性价比高,具有明显的优越性,适用于活性污泥RNA的大量提取,同时,T-RFLP结果证明RNA提取方法对分析样品的微生物种类和丰度分析结果影响较大,不同的RNA提取方法所获得的微生物群落基因多样性及其种类、丰度均不同.本研究建立了一种快速、有效的高质量RNA提取方法,将在监测活性污泥菌群动态变化、菌群代谢功能学、微生物群落芯片等研究上具有重要意义.

An effective and fast RNA isolation method of activated sludge was established and five different methods were compared based on RNA yield,purity,integrity,RT-PCR amplification of 16S rRNA genes and subsequent terminal restriction fragment length polymorphism（T-RFLP） analysis.That is,the precipitated activated sludge was washed with TENP and PBS buffer,followed by using lysozyme and TRIzol to direct lysis of microbial cells,chloroform to remove protein and most of the DNA from bacterial lysate,isopropanol to precipitate nucleic acid and DNase I to hydrolyze residual DNA.To further purify RNA,RNA purifying column was utilized.The results demonstrated that the extraction method,with the aid of TRIzol and RNA purification kit,can effectively extract high-quality RNA.It not only means low degradability and high quantity,purity and diversity,but also the genes of 16S rRNA and amoA can be amplified by RT-PCR.Compared with other methods,it showed great advantage of low cost and high efficienc.y and can be applied to RNA extraction of activated sludge in a large number.Furthermore,T-RFLP results indicated that the community composition as well as the abundance of individual members was affected by the kind of RNA extraction methods.This work established a rapid and effective method to extract high-quality RNA from activated sludge and would show great potential for monitoring microbial changes and studying metabolism and community array of activated sludge.