Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597.
J Mol Diagn. 2009 Nov;11(6):537-42. doi: 10.2353/jmoldx.2009.080164. Epub 2009 Oct 8.
An accurate molecular diagnosis for viral pathogens is highly dependent on pre-analytical procedures. The efficiencies of two viral RNA extraction methods (liquid phase partition and silica-based adsorption chromatography) and the effects of handling and storage on the stability of RNA isolated from dengue virus (DENV) were studied. Viral RNA extracted from spiked sera or clinical samples characterized with DENV infection were quantified by TaqMan real-time PCR. The presence of high serum proteins severely affected the recovery of DENV RNA by the liquid phase partition, but not the silica-based method. The recovery with Trizol liquid phase partition method was significantly improved by a concomitant addition of a co-precipitant and the reduction of sera proteins, resulting in recoveries similar to that of the silica-based methods. Repeated freeze-thaw cycles did not affect the recovery of viral RNA. While intact DENV was found to be stable in serum for up to 2 hour at 25 degrees C, recovery of viral RNA from sera stored in the lysis/binding buffer was stable for up to 5 days. These data indicate that the choice of viral RNA extraction methods, the conditions for handling, and storing of clinical sera critically affect the quantification of viral nucleic acid from clinical samples. This will impact the accuracy and reproducibility of DENV diagnosis by PCR-based assays.
准确的病毒病原体分子诊断高度依赖于分析前程序。本研究考察了两种病毒 RNA 提取方法（液相分配和基于硅基的吸附色谱法）的效率，以及处理和储存对从登革热病毒（DENV）中分离的 RNA 稳定性的影响。通过 TaqMan 实时 PCR 对用 DENV 感染特征化的加标血清或临床样本中提取的病毒 RNA 进行定量。高血清蛋白的存在严重影响了液相分配法对 DENV RNA 的回收，但不影响基于硅基的方法。通过同时添加共沉淀剂和减少血清蛋白，Trizol 液相分配法的回收率显著提高，回收率与基于硅基的方法相似。反复冻融循环不影响病毒 RNA 的回收。虽然完整的 DENV 在 25°C 下的血清中稳定长达 2 小时，但储存在裂解/结合缓冲液中的血清中病毒 RNA 的回收在长达 5 天内保持稳定。这些数据表明，病毒 RNA 提取方法的选择、临床血清的处理和储存条件会严重影响从临床样本中定量病毒核酸。这将影响基于 PCR 的检测对 DENV 诊断的准确性和重现性。
