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Quantitative detection of chikungunya, Zika, and dengue viruses by one-step real-time PCR in different cell substrates

  • Bacterial, Fungal and Virus Molecular Biology - Research Paper
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Brazilian Journal of Microbiology Aims and scope Submit manuscript

Abstract

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 (R2) 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.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

All authors acknowledge the editorial fund and the Vicerrectoría de investigación (VCTI) from the UAN. The authors would like to thank Dr. Diana Diaz Arévalo and her student Ana María Concha, from the immunology research group of the Fundación Instituto de Inmunología de Colombia (FIDIC) for providing the P388D1 cell line; to Dr. Yulieth Upegui for DENV serotypes donation; to MSc. Luz D Nieves Barreto from the Diabetes, Lipids, and Metabolism research group and Dr. Karina Vargas from the Cellular Neurophysiology Laboratory of the Universidad de Los Andes for their technical assistance.

Funding

The research leading to these results received funding from Minciencias under Grant Agreement No. 124380864546—contract CT. FP 80740- 152–2019.

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Authors and Affiliations

  1. Faculty of Science, Universidad Antonio Nariño (UAN), Sede Circunvalar. Cra. 3 Este # 47A - 15, 110231, Bogotá, Colombia

    Andrés F. Cuellar-Quimbaya, Ana Luisa Muñoz, Anny K. Rodríguez & Mónica Losada-Barragán

  2. Fundación Banco Nacional de Sangre Hemolife, Bogotá, Colombia

    Ana Luisa Muñoz

  3. PhD Program in Biomedical and Biological Sciences, Universidad del Rosario, 111321, Bogotá, Colombia

    Yoelis Yepez-Perez

  4. Faculty of Science, Universidad Pedagógica y Tecnológica de Colombia (UPTC), 150003, Tunja, Colombia

    Ingrid DJiménez C & Nidya Alexandra Segura

  5. Faculty of Agricultural and Livestock Sciences, Program of Veterinary Medicine, Universidad de La Salle, 110141, Bogotá, Colombia

    Felio Bello

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  1. Andrés F. Cuellar-Quimbaya
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  2. Ana Luisa Muñoz
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  8. Mónica Losada-Barragán
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Contributions

All authors contributed to the study conception and design. Data collection: A.F.C.Q; Y.Y-P and I.D.J. Formal analysis: A.F.C.Q and M.L.B. Funding acquisition: A.L.M; A.K.R; N.A.S; F.B and M.L.B. Writing original draft: A.F.C.Q. Writing—review and editing: all authors. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Mónica Losada-Barragán.

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Cuellar-Quimbaya, A.F., Muñoz, A.L., Yepez-Perez, Y. et al. Quantitative detection of chikungunya, Zika, and dengue viruses by one-step real-time PCR in different cell substrates. Braz J Microbiol (2024). https://doi.org/10.1007/s42770-023-01226-5

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  • DOI: https://doi.org/10.1007/s42770-023-01226-5

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