Establishment of one-step SYBR green-based real time-PCR assay for rapid detection and quantification of chikungunya virus infection

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus and one of the prevalent re-emerging arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America. It produces a spectrum of illness ranging from inapparent infection to moderate febrile illness as well as severe arthralgia or arthritis affecting multiple joints. In this study, a quantitative, one-step real-time SYBR Green-based RT-PCR system for the non-structural protein 2 (nsP2) of CHIKV that can quantify a wide range of viral RNA concentrations was developed. Comparisons between the conventional semi-quantitative RT-PCR assay, immunofluorescence detection method and the one-step SYBR Green-based RT-PCR assay in the detection of CHIKV infection revealed much rapid and increase sensitivity of the latter method. Furthermore, this newly developed assay was validated by in vitro experiments in which ribavirin, a well-known RNA virus inhibitor, showed a dose-dependent inhibition of virus replication on cells that was assessed by viral infectivity and viral RNA production. Our results demonstrate the potential of this newly developed one-step SYBR Green I-based RT-PCR assay may be a useful tool in rapid detection of CHIKV and monitoring the extent of viral replication possibly in patients' samples.

Figure 1

One-step SYBR green-based RT-PCR for detection of CHIKV infection. (A) Amplification profile and (B) the standard curve generated from the amplification profile of the one-step SYBR green-based quantitative RT-PCR of serially diluted CHIKV with known infective concentrations (10⁰ to 10⁵ PFU/ml) using the nsP2 primer set. A linear range of 6 logs of CHIKV dilution with a R² of 0.9899 is shown in B. (C) Melting curve analysis of the amplified product from two isolated strain of CHIKV using nsP2 primer set with a distinct melting peak (T_m value) at 83°C. Primer-dimer can also be observed from the non-template control (NTC) with T_m value below 76°C. (D) Semi-quantitative RT-PCR detection of serially diluted CHIKV with known infective concentrations (10⁰ to 10⁵ PFU/ml) using the nsP2 primer set.

Figure 2

Immunofluorescence assay (IFA) detection of viral antigen on CHIKV infected cells. Vero cells are infected with serially diluted CHIKV (expressed as PFU/ml) for 2 days and subjected to immunofluorescence staining with anti-alphavirus antibody. The CHIKV-infected cells are stained green and the cell nuclei are stained blue with the nuclear stain, DAPI. Mock-infected control and isotype control (cells are stained with secondary anti-mouse antibody conjugated with FITC) are included to ensure the specificity of the assay.

Figure 3

Inhibitory assay of CHIKV infection using ribavirin. (A) Dosage dependent inhibition of CHIKV in Vero cells treated with different concentrations of ribavirin (62.5 μg/ml to 750 μg/ml). The cellular supernatants containing the infectious virus titers are determined by plaque assays (expressed as Log PFU/ml) as well as the one-step SYBR green-based quantitative RT-PCR (expressed as Ct value). (B) Correlation between the viral titers expressed as Log PFU/ml obtained from plaque assays and the one-step SYBR green-based quantitative RT-PCR are shown. Results from three independent experiments are included and statistical analysis is performed with a non-parametric (Spearman's correlation) test.