Evaluation of pre-analytical variables in the quantification of dengue virus by real-time polymerase chain reaction.
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.
Anwar
Azlinda
A
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597.
Wan
Guoqiang
G
Chua
Kaw-Bing
KB
August
Joseph Thomas
JT
Too
Heng-Phon
HP
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003716:Dengue Virus / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D020871:RNA Stability; D012367:RNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012367:RNA, Viral