M.M. Daude, E.N. Barros, G.C.A. Santos, H.G. Barreto
Published: February 01, 2023
Genet. Mol. Res. 22(1): GMR19075
DOI: https://doi.org/10.4238/gmr19075
Cite this Article:
M.M. Daude, E.N. Barros, G.C.A. Santos, H.G. Barreto (2023). Dengue virus RNA quantification through PCR: what is the best cost-effective approach?. Genet. Mol. Res. 22(1): GMR19075. https://doi.org/10.4238/gmr19075
About the Authors
M.M. Daude, E.N. Barros, G.C.A. Santos, H.G. Barreto
Corresponding Author
Email: horllys@uft.edu.br
ABSTRACT
Detection via PCR is a fast, sensitive, and highly specific method. However, the cost for testing through this technique is quite high, mainly because of the costs of the kits that are used. We looked for the best cost-effective alternative for Dengue virus (DENV) detection via PCR through the evaluation, optimization, and comparison of RT-PCR (Reverse Transcription – PCR) and RT-qPCR (Reverse Transcription–qPCR) detection kits. The biological material was samples of blood serum collected from 40 Brazilian patients suspected of DENV infection. Two reaction final volumes were tested for diagnosis via RT-PCR, 12.5 µL and 25 µL, and diagnosis via RT-qPCR was performed using the two-step approach with the Sybr Green detection system. An analysis of the associated cost for each approach was also made. Analysis via RT-PCR allowed viral RNA amplification from 27 samples, independent of the final reaction volume tested. Diagnosis via RT-qPCR enabled virus identification from 33 samples. The costs per reaction for the RT-PCR technique were US$ 2.91 and US$ 2.41 American dollars for the final reaction volumes of 25 µL and 12.5 µL, respectively. For the RT-qPCR technique, the reaction cost was found to be US$ 2.30. The comparison between the techniques showed that RT-qPCR was more sensitive, allowing virus detection in a larger number of samples. However, results indicated that RT-PCR (12.5 µL) can be used as a screening method, considering its lower reaction cost. The cost analysis showed that RT-qPCR had the best cost-benefit ratio, since it allowed virus detection from a larger number of samples with a cost similar to RT-PCR. We also found that optimization of the cDNA (complementary DNA) synthesis step can significantly affect the final diagnosis cost for both techniques.
Key words: Flavivirus, Molecular diagnosis, RT-PCR, RT-qPCR, Viral RNA.