A reverse transcription loop-mediated isothermal amplification for rapid visual detection of Crimean-Congo hemorrhagic fever virus

Abstract

Objective To develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid and visual detection of Crimean-Congo hemorrhagic fever virus. Methods The recombinant plasmid containing the S gene of Crimean-Congo hemorrhagic fever virus was constructed using in vitro gene synthesis. The most effective primers of the three sets of RT-LAMP primers, designed by Primer Explorer software 4.0, was selected using real-time turbidimetry. Hydroxynaphthol blue (HNB) as the indicator was used to judge the result. Results The result of real-time turbidimetry showed that the first set of primers had the highest amplification efficiency with a peak time of 20 min. The amplification efficiency was accelerated after adding loop primers, of which the peak time was 13min. The detection limit concentration of this method was 10copies/μl with a detection time of 30 min, which was 10-fold and 1000-fold higher than that of nests RT-PCR and quantitative real-time RT-PCR, respectively. This method has great stability. No cross-reactions were observed between Hantavirus, Marburg virus, severe fever with thrombocytopenia syndrome bunya virus, or Ebola virus. Conclusions The visual RT-LAMP assay could detect Crimean-Congo hemorrhagic fever virus with less labor and time consuming sensitively and specifically. This lower-cost method could effectively avoid aerosol pollution. It might be applicable for the routine surveillance of Crimean-Congo hemorrhagic fever in unequipped organizations or remote area. Clinical samples are warranted to evaluate the value of this method. Key words: Crimean-Congo hemorrhagic fever; RT-LAMP; Visual detection