Evaluation of Pre-Analytical Variables in the Quantification of Dengue Virus by Real-Time Polymerase Chain Reaction

Abstract

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°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. Dengue virus (DENV) has emerged as an important vector-borne viral disease, 1 usually afflicting rural areas of endemic countries and posing tremendous health problems in these regions.2 Hence, to prevent and control the progression of dengue disease, the World Health Organization has recommended the augmentation of active and accurate laboratory-based surveillance for early reporting of dengue virus infections to the public health authorities. Pre-analytical variables, including the storage and transport of patient samples, the stability of viral RNA in the samples and methods of isolating viral RNA of high yield and quality, have major impacts on the development and performance of any successful molecular diagnostics. 4,5 Hence, the technical challenges associated with these pre-analytical issues must first be identified and optimized. Various commercially available viral RNA extraction methods are currently in use for clinical diagnostics of viral diseases. These extraction kits are generally based on two methods-liquid phase partition (eg, TRIzol LS) and silica-based nucleic acid adsorption chromatography (eg, High Pure Viral RNA and QIAamp Viral RNA kits). The recoveries of viral RNA by these two methods differ substantially with different viruses, 6 -12 thus making the choice of any particular method for the isolation of viral RNA uncertain. While there were several studies on the isolation and stability of other viruses, few reports have focused on the systematic evaluation of the stability and recovery of DENV RNA from sera. [13][14][15] This study examined the contributions of various pre-analytical variables to the sensitivity of DENV RNA d...

Effects of Different Lysis Buffers of Nucleic Acid Purification Kit on the Stability of Influenza Virus RNA

“…The use of lysis buffers of NA isolation kits for stabilizing viral RNA has been investigated by Anwar et al [6], who found that viral RNA integrity assessed by rRT-PCR was maintained for at least 5 days at 25°C when samples were stored in lysis buffer, and by Blow et al [7], who found that viral RNA recovered from lysis buffer-stabilized samples was suitable for detection by rRT-PCR for up to 35 days at 4°C and -20°C, but degraded rapidly at room temperature and 32°C. Similar results were obtained in our study that, when analyzed by rRT-PCR, viral RNA from lysis buffer-stored samples did not show a significant reduction in relative yield within at least 30 days at 4°C and within 90 days or longer at -20°C, but decreased notably on day 7 at room temperature.…”

“…Earlier studies have shown that lysis buffers of NA extraction kits have the potential to be used as RNA stabilization agents at adverse temperatures and over an extended period of time [6,7], and have potential to inactivate viruses completely [8], which would reduce the biohazard risk from sample leakage during transit and simplify transportation requirements while preserving the integrity of viral RNA before testing. Nevertheless, these studies did not give a comprehensive picture of the abilities of lysis buffers in protecting viral RNA from degradation and there are still some questions that remain unanswered.…”

“…However, suboptimal storage and transport conditions of influenza virus (flu-v) samples will most likely result in viral RNA degradation [3][4][5], and in some settings it is difficult to ensure certain sample conditions required by WHO, such as ultra-low-temperature freezing, especially for resource-limited areas; consequently, the efficacy of the NA-based assays will be compromised. Thus, inexpensive, simple and efficient sample stabilization methods need to be developed for facilitating flu surveillance and diagnosis.Earlier studies have shown that lysis buffers of NA extraction kits have the potential to be used as RNA stabilization agents at adverse temperatures and over an extended period of time [6,7], and have potential to inactivate viruses completely [8], which would reduce the biohazard risk from sample leakage during transit and simplify transportation requirements while preserving the integrity of viral RNA before testing. Nevertheless, these studies did not give a comprehensive picture of the abilities of lysis buffers in protecting viral RNA from degradation and there are still some questions that remain unanswered.…”

“…Nevertheless, these studies did not give a comprehensive picture of the abilities of lysis buffers in protecting viral RNA from degradation and there are still some questions that remain unanswered. First, could intact viral RNA be recovered from the samples preserved in lysis buffer at adverse temperatures and over an extended period of time, considering that a real-time RT-PCR (rRT-PCR) targeting a shorter fragment was only employed to evaluate the presence, quantity and/or quality of viral RNA in these studies [6,7]? Second, what is the consequence of virus samples preserved in lysis buffer and subjected to repeated freeze-thaw cycles, For reprint orders, please contact: reprints@futuremedicine.com future science group reseArch Article Liu, Gan, Wu, Weng, Lei & Shen a frequently encountered problem during subsampling and sample transport?…”

Coleta, transporte e armazenamento de amostras para diagnóstico molecular

“…A natureza lábil do RNA dificulta grandemente a padronização desses testes. Além disso, um resultado negativo em uma amostra manuseada sem o devido cuidado pode ser decorrente da degradação do RNA-alvo e não pela ausência de doença (3) . Assim, torna-se relevante entender as possíveis causas de erro, na fase pré-analítica, mais frequentes no diagnóstico molecular.…”