Establishment of one-step SYBR green-based real time-PCR assay for rapid detection and quantification of chikungunya virus infection.
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.
Ho
Phui San
PS
School of Applied Science, Republic Polytechnic, 9 Woodlands Avenue 9, 738964, Singapore . ho_phui_san@rp.sg.
Ng
Mary Mah Lee
MM
Chu
Justin Jang Hann
JJ
D018354:Alphavirus Infections / Q000175:diagnosis* / Q000821:virology*; D052160:Benzothiazoles; D002646:Chikungunya virus / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D006801:Humans; D009930:Organic Chemicals / Q000378:metabolism; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000235:genetics
D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C098022:SYBR Green I