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.

Analysis of antibody response (IgM, IgG, IgG3) to Chikungunya virus using panel of peptides derived from envelope protein for serodiagnosis

“…Diagnostic methods available for the detection of CHIKV includes viral isolation [18], RT-PCR [19], real-time loop-mediated isothermal PCR [20], and antigen capture ELISA [21]. In one of the studies, a single step RT-PCR has developed which uses 2, 7-diamino-1, 8-naphthyridine derivative (DANP)-labeled cytosinebulge hairpin primers to amplify the nsP2 (non-structural protein) region of the CHIKV genome that act as a potential clinical molecular diagnostic assay for CHIKV in acute phase patient serum samples [22]. These methods are highly sensitive but can be used only within the first week of disease onset.…”

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection

“…The detection limit of the assay was 0.001 PFU per reaction that is lower than that of the previous DANP coupled assay [13] and is comparable to real time RT-PCR assays developed by other groups [22, 23]. A side-by-side comparison of our assay with the ab TES DEN 5 qPCR I Kit (Cat: 300152) from AIT biotech, a Taqman probe-based multiplex real time RT-PCR for DENV/CHIKV detection.…”

“…The viral titers were determined by plaque forming assay. [13] Ross River virus (RRV), Sindbis virus (SINV), Kunjin virus (KUNV, MRM 61C strain), West Nile virus (WNV, Sarafend strain), Zika virus (ZIKV, MR 766 strain), DENV-1 (S144 strain), DENV-2 (New Guinea C strain), DENV-3 (Eden 130/05 strain), DENV-4 (S8976 strain), Influenza A virus subtype H1N1, H3N2, Poliovirus type 1 (PV1, Sabin strain), type 2 (PV2, Sabin strain), type 3 (PV3, Sabin strain), Human enterovirus 71 (HEV71, AF316321 strain), Coxsackie B2 virus (CB2), Coxsackie A16 virus (CA16, WHO strain) and Enteric cytopathic human orphan virus 7 (Echo7) were also used to examine the cross-reactivity of this assay. The ZIKV, DENV1-4, PV1-3, HEV71, CB2, CA16 and Echo7 viruses were maintained in the laboratory.…”

“…[13] In brief, DANP molecule contains a naphthyridine ring which enables it to bind specifically to a cytosine-bulge in a hairpin structure of the PCR primer by hydrogen bonds. [13] The binding of DANP molecule to DNA gives rise to a 400 nm excitation and 450 nm emission property to the bound DANP molecule. As PCR proceeds, the primer is incorporated into double stranded DNA and the hairpin is opened, causing the release of DNAP molecule and thereby decreasing the fluorescence intensity.…”

“…As PCR proceeds, the primer is incorporated into double stranded DNA and the hairpin is opened, causing the release of DNAP molecule and thereby decreasing the fluorescence intensity. [13] The utilization of DANP coupled hairpin PCR has also been demonstrated in a single-nucleotide polymorphism study of the cytochrome P450 gene 2C9*3 by Takei and colleagues. [14] However, the binding of DANP molecule to the hairpin-primer is in an equilibrium manner, so that excess DANP molecules must be added to ensure a detectable fluorescence intensity.…”