Development of real-time fluorescent enzymatic recombinase amplification method for chikungunya virus detection
Objective To establish a rapid,accurate and highly sensitive detection method for chikungunya virus based on reverse transcription-enzyme recombinase amplification(RT-ERA)method.Methods Using the conserved sequence of chikungunya virus non-structural protein 1(NSP1)gene as the target,the fluorescent probes and primers were designed and synthesized according to the RT-ERA principle.The target plasmid was constructed using pUC18 as the carrier and transcribed in vitro.The product was used as the positive control.The combination of primers and fluorescent probes with the highest amplification efficiency was screened using serially diluted positive control.The combination of selected probe and primers was used in the fluorescent RT-ERA method to amplify chikungunya virus of different copy numbers to assess the lower detection limit of the method.The RNA templates of forest encephalitis virus,dengue virus,West Nile virus,Japanese encephalitis virus and yellopw fever virus were amplified by fluorescent RT-ERA to assess the specificity of the method.Results The fluorescent RT-ERA method could effectively amplify chikungunya virus RNA within 10 min at 40℃ and the lower detection limit was 10 copies/μL.The detection results of other viruses and only chikungunya virus was positive.Conclusions A fluorescence RT-ERA method for detection of Chikungunya virus NSP1 gene has been successfully established.The method is simple with low detection limit and good specificity.
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