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.

Chikungunya fever: Epidemiology, clinical syndrome, pathogenesis and therapy

“…Ribavirin is known to inhibit in cellulo a large range of RNA viruses and notably viruses in the genus Alphavirus, such as Semliki forest virus (SFV) and CHIKV (Ho et al, 2010;Smee et al, 1988). In animal models, ribavirin is effective against a more limited set of viruses, mainly RNA viruses.…”

Detection of chikungunya virus‐specific IgM on laser‐cut paper‐based device using pseudo‐particles as capture antigen

“…Arboviruses are mainly prevalent in developing countries of tropical and subtropical areas and represent a serious public health concern for these countries . Diagnostic assays, such as reverse‐transcriptase polymerase chain reaction (RT‐PCR), plaque reduction neutralization test (PRNT) or enzyme‐linked immunosorbent assay (ELISA) can provide sensitive results and help to optimize patient care . However, these assays are time‐consuming, rarely affordable, and require infrastructure and qualified operators, making them accessible to relatively few low‐income countries …”

Neurological manifestations of Chikungunya and Zika infections

“…The molecular assays are a rapid and sensitive technique for the diagnosis of CHIKV infection during the early stages of illness. Conventional reverse transcription polymerase chain reaction (RT-PCR) 32 are available, along with other RT-PCR real-time assays 33 More recently, a one-step SYBR green-based real-time assay targeting the non-structural nsp2 gene has been described 34 . Virus isolation from the serum of infected patients can also be performed.…”

“…The detection of viral nucleic acid or the infectious virus in serum samples is useful during the initial viremic phase, at the onset of symptoms, and usually for the following five to 10 days, when CHIKV RNA reaches very high levels. After this, the diagnosis depends on the detection of specific immune responses by serological methods 32,33,34 . The molecular assays are a rapid and sensitive technique for the diagnosis of CHIKV infection during the early stages of illness.…”