Quantitative detection of chikungunya, Zika, and dengue viruses by one-step real-time PCR in different cell substrates.
Andrés F Cuellar-QuimbayaAna Luisa MuñozYoelis Yepez-PerezIngrid DJiménez CAnny K RodríguezNidya Alexandra SeguraFelio BelloMónica Losada-BarragánPublished in: Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology] (2024)
Chikungunya (CHIKV), Zika (ZIKV), and dengue viruses (DENV) are vector-borne pathogens that cause emerging and re-emerging epidemics throughout tropical and subtropical countries. The symptomatology is similar among these viruses and frequently co-circulates in the same areas, making the diagnosis arduous. Although there are different methods for detecting and quantifying pathogens, real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for detecting viruses. However, the currently developed assays frequently involve probes and high-cost reagents, limiting access in low-income countries. Therefore, this study aims to design and evaluate a quantitative one-step RT-qPCR assay to detect CHIKV, ZIKV, and DENV with high specificity, reproducibility, and low cost in multiple cell substrates. We established a DNA intercalating green dye-based RT-qPCR test that targets nsP1 of CHIKV, and NS5 gene of ZIKV, and DENV for the amplification reaction. The assay exhibited a high specificity confirmed by the melting curve analysis. No cross-reactivity was observed between the three viruses or unspecific amplification of host RNA. The sensitivity of the reaction was evaluated for each virus assay, getting a limit of detection of one RNA copy per virus. Standard curves were constructed, obtaining a reaction efficiency of ~ 100%, a correlation coefficient (R 2 ) of ~ 0.97, and a slope of -3.3. The coefficient of variation (CV) ranged from 0.02 to 1.43. In addition, the method was optimized for viral quantification and tested in Vero, BHK-21, C6/36, LULO, and the Aedes cell lines. Thus, the DNA intercalating green dye-based RT-qPCR assay was a highly specific, sensitive, reproducible, and effective method for detecting and quantifying CHIKV, ZIKV, and DENV in different cell substrates that could also be applied in clinical samples.
Keyphrases
- dengue virus
- zika virus
- aedes aegypti
- real time pcr
- high throughput
- nucleic acid
- single cell
- low cost
- cell therapy
- high resolution
- label free
- small molecule
- gene expression
- single molecule
- gram negative
- magnetic resonance imaging
- loop mediated isothermal amplification
- climate change
- bone marrow
- computed tomography
- stem cells
- wastewater treatment
- antimicrobial resistance
- diffusion weighted imaging
- data analysis
- dna methylation
- multidrug resistant
- structural basis
- cell free