Lewandowski Kuiama, Bell Andrew, Miles Rory, Carne Simon, Wooldridge David, Manso Carmen, Hennessy Nicola, Bailey Daniel, Pullan Steven T, Gharbia Saheer, Vipond Richard
National Infection Service, Public Health England, Salisbury, United Kingdom.
National Infection Service, Public Health England, Salisbury, United Kingdom.
J Mol Diagn. 2017 Mar;19(2):303-312. doi: 10.1016/j.jmoldx.2016.10.005. Epub 2016 Dec 30.
Extraction of viral RNA and the storage of sample material are extremely important factors in the detection and whole genome sequencing (WGS) of viral pathogens. Although PCR-based detection methods focus on small amplicons, viral WGS applications require RNA of high quality and integrity for adequate sequence coverage and depth. This study examined the fitness of one manual and four automated RNA extraction platforms commonly used in diagnostic laboratories for use in metagenomic sequencing, how the practice of storing sample material in Qiagen buffer AVL before extraction affected the integrity of viral RNA and its suitability for use in amplicon-based WGS methods, and how the addition of Triton X-100 to buffer AVL affected the capability of the extraction platforms and the integrity of viral RNA in stored samples. This study found that the EZ1 platform gave the best performance of the automated platforms and gave comparable results to the frequently used manual Qiagen extraction protocol when extracted viral RNA was used in metagenomics sequencing. To maintain high levels of viral RNA integrity suitable for amplicon-based WGS, nucleic acid should be extracted from samples immediately, because even short storage periods in buffer AVL have a severe effect on integrity, and the addition of Triton X-100 had little effect on the quality of viral material for WGS.
病毒RNA的提取以及样本材料的储存是病毒病原体检测和全基因组测序（WGS）中的极其重要的因素。尽管基于PCR的检测方法侧重于小扩增子，但病毒WGS应用需要高质量和完整的RNA以实现足够的序列覆盖度和深度。本研究考察了诊断实验室常用的一个手动和四个自动化RNA提取平台用于宏基因组测序的适用性，在提取前将样本材料储存在Qiagen缓冲液AVL中对病毒RNA完整性及其在基于扩增子的WGS方法中使用的适用性有何影响，以及向缓冲液AVL中添加Triton X-100对提取平台的能力和储存样本中病毒RNA完整性有何影响。本研究发现，在宏基因组测序中使用提取的病毒RNA时，EZ1平台在自动化平台中表现最佳，并且与常用的手动Qiagen提取方案得到的结果相当。为了保持适合基于扩增子的WGS的高水平病毒RNA完整性，应立即从样本中提取核酸，因为即使在缓冲液AVL中短暂储存也会对完整性产生严重影响，并且添加Triton X-100对用于WGS的病毒材料质量影响很小。
