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

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.

Keywords: CHIKV; DENV; RT-qPCR; ZIKV.

Fig. 1

Multiple sequence alignment of designed primers with CHIKV, ZIKV, and DENV genomic sequences from different isolates. (A) CHIKV, (B) ZIKV, (C) DENV-1 and (D) DENV-2. Dots indicate identity with the consensus sequence at the top of the alignment. Eighteen isolates of CHIKV and ZIKV and eleven isolates from DENV serotypes from various world regions were used. DENV-3 and -4 exhibited partial complementarity with the stated primers

Fig. 2

Standard curves of CHIKV, ZIKV, and DENV-2 at different hybridization temperatures. (A) nsP1 CHIKV and (B) NS5 ZIKV genes were amplified at 60ºC, 62ºC and 64º. (C) NS5 DENV-2 gene was amplified at 62ºC, 64ºC and 66ºC. Each point represents the mean Ct values of six replicates from the RNA of each virus in tenfold serial dilutions. The logarithm of the amount of sample is plotted against Ct data. The efficiency, coefficient of relation (R²), slope, and intercept are shown. Ct: the cycle number at which fluorescence increases significantly above background fluorescence