Development and Application of Loop-mediated Isothermal Amplification Assay(LAMP)for Detection of Influenza Virus Subtype H_3

Objective To provide service of rapid diagnosis of influenza virus in the lab at grass root level by setting up the Loop-mediated isothermal amplication assey to detect the nucleic acids of infmenza virus sbutype H3. Methods Two pairs of specific primers and a loop primer were designed.Nucleic acids of influenza virus H3 were amplified using reverse-transcription and loop-mediated isothermal amplification technology(RT-LAMP).The reaction systems were optimized.Results Sensitivity of the RT-LAMP assay was 0.01TCID50/reaction which was as same as that of the real-time RT-PCR assay.There was no crossing-reaction with other aspiratory viruses including influenza virus subtype H1 and H5,influenza B,Respiratory Syncytial Virus(RSV),Measles virus,Rubella virus,Mumps virus,adenovirus and para-influenza virus which indicated the good specificity of the RT-LAMP.58 of clinical samples were analyzed by real-time PCR and RT-LAMP.The positive rate of virus isolation was 37.9%. The real-time PCR and RT-LAMP assays were 53.45% and 51.72% of respectively. The sensitivity of two assey was 100 times higher than that of the viral isolation. Conclusions Thit loop-mediated isothermal amplification assay for detection of influenza virus H3 was not only rapid,specific and sensitive,but also simple and cheap.It was very suitable for the labs at grass root level.