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.

Beyond Fever and Pain: Diagnostic Methods for Chikungunya Virus

“…A variety of molecular assays for CHIKV have been published or are commercially available (see Table S1 in the supplemental material). Reported assays include conventional RT-PCR (69, 108-112), real-time RT-PCR (rRT-PCR) (102,105,(113)(114)(115)(116)(117)(118)(119)(120), isothermal methods (110,(121)(122)(123)(124), and multiplex assays (125)(126)(127)(128)(129)(130)(131)(132)(133)(134)(135)(136)(137)(138). No molecular gold standard by which to evaluate reported assays in practice exists, and the decision to implement a particular test depends on the relative advantages and disadvantages of the method along with the capabilities in a given laboratory.…”

“…Consistent with molecular diagnostics in general, real-time methods for CHIKV have proven more sensitive than conventional RT-PCR (112,115,116,121), though this has not been shown in all studies (111). Comparisons between real-time methods have not demonstrated clear differences in assay performance (105,118,124,132,136), and CHIKV detection in published multiplex assays does not appear to be decreased relative to that in monoplex tests (125,126,133,135,137,138). Multiplex assays facilitate testing for a set of pathogens in all patients, and the utility of this approach has been demonstrated in regions with transmission of multiple arboviruses and/or malaria (7,34).…”

RT‐Hpro‐PCR: A MicroRNA Detection System Using a Primer with a DNA Tag

“…The Hpro can either form a secondary hairpin structure containing a C‐bulge or hybridize with the tag sequence to create a fully matched Hpro‐tag duplex. When the DANP binds the C‐bulge, the DANP ⋅ C‐bulge complex emits fluorescence at approximately 430–450 nm, with a 30 nm bathochromic shift from the fluorescence of the free, unbound state . The open form of the probe with the primer tag and the hairpin structure of the probes with DANP are metastable at room temperature; in other words, this PCR system monitors the deflection of the equilibrium between the Hpro and the Hpro associated with the tag of the primer duplex.…”

“…We first used miRNA‐122a as a template and designed sequences for RT and a tagged forward primer, each containing a consensus sequence that allowed for the progression of the RT‐PCR (Table ). The tag‐probe set used was the same as that reported previously . The reaction system included 1) a forward primer (mir122F‐11‐Tag) consisting of 11 nt of the RT primer (bold), a T4 linker, and a DNA tag (underlined), and 2) a reverse primer (mir122R‐Tag) consisting of an 11‐mer primer site (bold), a T4 linker, and the same DNA tag.…”

“…The amplicons obtained after 35 PCR cycles were analyzed by native polyacrylamide gel electrophoresis (PAGE, Figure A). Approximately 50 bp of the complex was the forward or reverse primer with Hpro . A 70 bp product was obtained after 25 PCR cycles.…”