Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA, USA.
J Appl Microbiol. 2011 Jul;111(1):216-23. doi: 10.1111/j.1365-2672.2011.05029.x. Epub 2011 May 5.
Viral pathogens in environmental media are generally highly diffuse, yet small quantities of pathogens may pose a health risk. This study evaluates the ability of TransPlex™ whole transcriptome amplification (WTA) to amplify small quantities of RNA viruses from complex environmental matrices containing background nucleic acids.
DNA extracts from mock drinking water samples containing mixed microbial populations were spiked with small quantities of echovirus type 13 (EV) RNA. Samples were amplified using a Transplex™ WTA kit, and EV-specific quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify target pathogens before and after application of WTA. Samples amplified by WTA demonstrated a decreased limit of detection. The log-linear relationship between serial dilutions was maintained following amplification by WTA.
WTA is able to increase the quantity of target organism RNA in mixed populations, while maintaining log linearity of amplification across different target concentrations.
WTA may serve as an effective preamplification step to increase the levels of RNA prior to detection by other molecular methods such as PCR, microarrays and sequencing.
环境介质中的病毒病原体通常高度分散，但少量病原体可能构成健康风险。本研究评估 TransPlex™ 全转录组扩增 (WTA) 从含有背景核酸的复杂环境基质中扩增少量 RNA 病毒的能力。
从含有混合微生物种群的模拟饮用水样本的 DNA 提取物中掺入小量肠道病毒 13 型 (EV) RNA。使用 TransPlex™ WTA 试剂盒对样品进行扩增，并用 EV 特异性定量逆转录聚合酶链反应 (qRT-PCR) 在 WTA 应用前后定量检测目标病原体。WTA 扩增的样品显示出检测限降低。WTA 扩增后，连续稀释之间的对数线性关系得以维持。
WTA 能够增加混合群体中目标生物 RNA 的数量，同时保持不同靶浓度扩增的对数线性。
WTA 可以作为一种有效的预扩增步骤，在其他分子方法（如 PCR、微阵列和测序）检测之前提高 RNA 的水平。
