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活性污泥高质量RNA快速提取方法研究 总被引:2,自引:0,他引:2
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. 相似文献
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Tineke H. Jones Alain Houde Elyse Poitras Pierre Ward Michael W. Johns 《Food and environmental virology》2009,1(2):57-65
There are increasing concerns of zoonotic transmission of some animal enteric viruses, such as calicivirus, hepatitis E virus,
and rotavirus, which are closely related to human pathogenic strains. Most enteric viruses are detected by molecular techniques
because they cannot be cultured. Surrogates such as F-RNA coliphages are cultivable but few molecular methods exist. Individual
real-time TaqMan RT-PCR assays for the replicase gene of F-RNA coliphage genogroups I and IV were developed and multiplexed
with a real-time TaqMan RT-PCR assay for feline calicivirus as a sample process control for the simultaneous detection and
enumeration of genogroup I and IV F-RNA coliphages. Genogroup IV were successfully detected with the multiplexed assay in
80% of fecal samples that contained F-RNA coliphage levels ≥3.2 log plaque forming units (pfu). F-RNA coliphage were at or
below the limit of detection in most fecal samples when levels were ≤4 log pfu/g. 相似文献