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Establishment of a Dual-Fluorescence RT-RAA Assay for the Detection of Five Mosquito-Borne Viruses_Zhou Donggen.pdf
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Abstract: Objective To establish a recombinase-aided isothermal amplification (RAA) method for the rapid screening of five mosquito-borne viruses: dengue virus (DENV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), yellow fever virus (YFV), and chikungunya virus (CHIKV). Methods The conserved genes of the five viruses were analyzed as target regions, and the ribonuclease P (RNaseP) gene was incorporated as an internal control. Six sets of specific primers and probes were designed, and based on bioinformatics analysis of primer dimers and mismatches, these six primer-probe combinations were grouped into three dual-fluorescent RT-RAA detection systems, which were then evaluated under identical reaction conditions. The sensitivity of the three fluorescent RT-RAA detection systems was validated using in vitro-transcribed RNAs of the five viruses, while their specificity was assessed using nucleic acids from Plasmodium, hantavirus, Seoul virus, and influenza virus. The repeatability of the three dual-fluorescent RT-RAA detection systems was further evaluated using in vitro-transcribed RNAs of the five viruses at medium concentration (10^4 copies/mL) and low concentration (10^2 copies/mL). Results Bioinformatics analysis enabled the combination of the five viruses and RNaseP into three dual-fluorescent RT-RAA detection systems: DENV-ZIKV, JEV-YFV, and CHIKV-RNaseP. Each system was incubated at 39°C for 15 minutes. The sensitivity of the three dual-fluorescent RT-RAA detection systems for detecting in vitro-transcribed RNAs of the five viruses ranged from 1 to 10^4 copies/mL; specifically, the detection limits for DENV, ZIKV, and YFV were 1 copy/mL, for JEV it was 10 copies/mL, and for CHIKV it was 10^2 copies/mL. None of the three fluorescent RT-RAA detection systems showed cross-reactivity with other viruses. The detection rates of the five viruses at medium concentration reached 100.0%, and even at low concentration, the detection rates remained at 83.3%. Conclusion The detection method established in this study exhibits excellent sensitivity, specificity, and repeatability, making it suitable for the detection of the five mosquito-borne viruses in both human and mosquito samples.
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