Detection of avian influenza virus-H5N1 by multiplex reverse transcription and multiple cross displacement amplification combined with lateral flow biosensor technology
Objective To establish of a new method for the rapid detection of H5N1-Avian Influenza virus by combining reverse transcription(RT),multiple cross displacement amplification(MCDA)and nanoparticle-based lateral flow biosensor(LFB).Methods MCDA primers were designed based on gene sequences specific to Hemagglutinin(HA)and Neuraminidase(NA)in H5N1 avian influenza virus.The target genes HA and NA were amplified through reverse transcription and MCDA in one reaction system.Results were displayed by LFB.The assay was named as H5N1-mRT-MCDA-LFB.The reaction conditions of the H5N1-RT-MCDA-LFB method were optimized,and the sensitivity and specificity were also assessed.Results The H5N1-RT-MCDA-LFB assay could achieve good amplification effect at a constant temperature of 65 ℃ for 40 minutes.The method had a lower limit of detection of 100 fg per reaction with 100-fold higher sensitivity than that of the RT-qPCR(lower limit of detection 10 pg per reaction).The assay was negative in detecting 28 common viruses,mycoplasmas,chlamydias,bacterias and funguses,except for H5N1.In addition,the H5N1-RT-MCDA-LFB method showed better validation in simulated clinical samples with a lower limit of detection at 1×102copies/ml.Conclusion The H5N1-RT-MCDA-LFB assay is a valuable molecular diagnostic technique for detecting H5N1 avian influenza virus due to its simplicity,rapidity,sensitivity and specificity.
Influenza A virus,H5N1 subtypeReverse transcriptionThermostatic amplificationLateral flow biosensorDiagnostic method
万方数据
维普
