title: A novel DANP-coupled hairpin RT-PCR for rapid detection of Chikungunya virus.
abstract: Chikungunya has re-emerged as an important arboviral infection of global health significance. Because of lack of a vaccine and effective treatment, rapid diagnosis plays an important role in early clinical management of patients. In this study, we have developed a novel molecular diagnostic platform that ensures a rapid and cost-effective one-step RT-PCR assay, with high sensitivity and specificity, for the early detection of the Chikungunya virus (CHIKV). It uses 2,7-diamino-1,8-naphthyridine derivative (DANP)-labeled cytosine-bulge hairpin primers to amplify the nsP2 region of the CHIKV genome, followed by measurement of the fluorescence emitted from DANP-primer complexes after PCRs. The detection limit of our assay was 0.01 plaque-forming units per reaction of CHIKV. Furthermore, the HP-nsP2 primers were highly specific in detecting CHIKV, without any cross-reactivity with the panel of RNA viruses validated in this study. The feasibility of the DANP-coupled hairpin RT-PCR for clinical diagnosis was evaluated using clinical serum samples from CHIKV-infected patients, and the specificity and sensitivity were 100% (95% CI, 80.0% to 100%) and 95.5% (95% CI, 75.1% to 99.8%), respectively. These findings confirmed its potential as a point-of-care clinical molecular diagnostic assay for CHIKV in acute-phase patient serum samples.
authors: Laboratory of Molecular RNA Virology and Antiviral Strategies, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
authors: Huixin
authors: H
authors: Chen
authors: Fumie
authors: F
authors: Takei
authors: Evelyn Siew-Chuan
authors: ES
authors: Koay
authors: Kazuhiko
authors: K
authors: Nakatani
authors: Justin Jang Hann
authors: JJ
authors: Chu
mesh_terms: D018354:Alphavirus Infections / Q000175:diagnosis*; D002646:Chikungunya virus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D006801:Humans; D055029:Inverted Repeat Sequences; D009287:Naphthyridines; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
chemical_list: C534173:2,7-diamino-1,8-naphthyridine; D017931:DNA Primers; D009287:Naphthyridines