Avaliação de métodos para a extração de RNA do vírus da dengue

Abstract

Dengue virus (DENV) is one of the main arboviruses, being responsible for a disease that clinically resembles the infection caused by other viruses, such as Zika and Chikungunya, especially during the onset of symptoms, however it can progress to severe stages like the dengue hemorrhagic fever (DHF). Therefore, it is important to carry out laboratory tests to aid in the disease diagnosis. Among the available diagnostic tests, RT-qPCR is a fast, sensitive, and highly specific method, which identifies any DENV serotype through the amplification of its genetic material, though it is expensive and requires high-quality RNA to be performed. In this context, this study aimed to evaluate different RNA extraction methods and analyze which one have the best cost/benefit ratio. Fifteen serum samples, with 1 ml each, from patients with the infection caused by the dengue virus, were used. Three commercial kits (QIAamp, Biogene and Biopur) were compared. The extracted RNA obtained from the three methods under analysis had its quality and quantity assessed by a Nanodrop® One Espectrophotometer. The High-Capacity cDNA Reverse Transcription kit (Applied Biosystems) was used to convert the viral RNA into cDNA, and DENV detection was performed using the 7500 fast real-time PCR system (Applied Biosystems), using technical triplicates and the Power SYBR Green Master Mix (Thermo Fisher) kit. All methods generated an average RNA concentration greater than 100 ng/μl. The A260/A230 and A260/A280 purity ratios were higher than those recommended, what was probably caused by the addition of carrier RNA. The DENV (serotype 2) was detected in all samples, indicating that all tested kits allowed the isolation of viral RNA from the serum samples. The comparison of the cycle of quantification (Cq) values from each sample showed that the QIAamp kit displayed the best sensitivity among the tested kits. However, when comparing the cost from each methodology used, the QIAamp kit had the highest cost per sample, being 64.3% and 80.2% more expensive than the Biopur and BioGene kits, respectively. Thus, all tested methods can be used for the isolation DENV from serum samples. The QIAmp method allowed the generation of lower Cqs values on the RT-qPCR analysis, when compared to the other RNA isolation methods, suggesting that it may present a higher sensitivity on the target detection, however, the BioGene method showed a best cost/benefit ratio.