title: Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection.
abstract: A molecular diagnostic platform with DANP-anchored hairpin primer was developed and evaluated for the rapid and cost-effective detection of Chikungunya virus (CHIKV) with high sensitivity and specificity. The molecule 2, 7-diamino-1, 8-naphthyridine (DANP) binds to a cytosine-bulge and emits fluorescence at 450 nm when it is excited by 400 nm light. Thus, by measuring the decline in fluorescence emitted from DANP-primer complexes after PCR reaction, we could monitor the PCR progress. By adapting this property of DANP, we have previously developed the first generation DANP-coupled hairpin RT-PCR assay. In the current study, we improved the assay performance by conjugating the DANP molecule covalently onto the hairpin primer to fix the DANP/primer ratio at 1:1; and adjusting the excitation emission wavelength to 365/430 nm to minimize the background signal and a 'turn-on' system is achieved. After optimizing the PCR cycle number to 30, we not only shortened the total assay turnaround time to 60 minutes, but also further reduced the background fluorescence. The detection limit of our assay was 0.001 PFU per reaction. The DANP-anchored hairpin primer, targeting nsP2 gene of CHIKV genome, is highly specific to CHIKV, having no cross-reactivity to a panel of other RNA viruses tested. In conclusion, we report here a molecular diagnostic assay that is sensitive, specific, rapid and cost effective for CHIKV detection and can be performed where no real time PCR instrumentation is required. Our results from patient samples indicated 93.62% sensitivity and 100% specificity of this method, ensuring that it can be a useful tool for rapid detection of CHIKV for outbreaks in many parts of the world.
authors: Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
authors: Huixin
authors: H
authors: Chen
authors: School of Applied Science, Republic Polytechnic, Singapore.
authors: Mariya
authors: M
authors: Parimelalagan
authors: The National Defense Medical College, Tokorozawa, Saitama, Japan.
authors: Fumie
authors: F
authors: Takei
authors: Environmental Health Institute, National Environment Agency, Singapore.
authors: Hapuarachchige Chanditha
authors: HC
authors: Hapuarachchi
authors: Molecular Diagnosis Centre, Department of Laboratory Medicine and Pathology, Yong Loo Lin School of Medicine, National University Health System, Level 3, National University Hospital Main Building, Singapore.
authors: Evelyn Siew-Chuan
authors: ES
authors: Koay
authors: Environmental Health Institute, National Environment Agency, Singapore.
authors: Lee Ching
authors: LC
authors: Ng
authors: School of Applied Science, Republic Polytechnic, Singapore.
authors: Phui San
authors: PS
authors: Ho
authors: The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Japan.
authors: Kazuhiko
authors: K
authors: Nakatani
authors: Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore.
authors: Justin Jang Hann
authors: JJ
authors: Chu
mesh_terms: D065632:Chikungunya Fever / Q000175:diagnosis*; D002646:Chikungunya virus / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics*; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D009287:Naphthyridines / Q000737:chemistry*; D012367:RNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D009287:Naphthyridines; D012367:RNA, Viral