Development of a reverse-transcription loop-mediated isothermal amplification assay to detect avian influenza viruses in clinical specimens

In recent years, the avian influenza has brought not only serious economic loss to the poultry industry in China but also a serious threat to human health because of the avian influenza virus (AIV) gene recombination and reassortment. Until now, traditional RT-PCR, fluorescence RT-PCR and virus isolation identification have been developed and utilized to detect AIV, but these methods require high-level instruments and experimental conditions, not suitable for the rapid detection in field and farms. In order to develop a rapid, sensitive and practical method to detect and identify AIV subtypes, 4 specific primers to the conserved region of AIV M gene were designed and a loop-mediated isothermal amplification (RT-LAMP) method was established. Using this method, the M gene of H1-H16 subtypes of AIV were amplified in 30 min with a water bath and all 16 H subtypes of AIV were able to be visually identified in presence of fluorescein, without cross reaction with other susceptible avian viruses. In addition, the detection limit of the common H1, H5, H7, and H9 AIV subtypes with the RT-LAMP method was 0.1 PFU (plaque-forming unit), which was 10 times more sensitive than that using the routine RT-PCR. Further comparative tests found that the positivity rate of RT-LAMP on detecting clinical samples was 4.18% (14/335) comparing with 3.58% (12/335) from real-time RT-PCR. All these results suggested that the RT-LAMP method can specifically detect and identify AIV with high sensitivity and can be considered as a fast, convenient and practical method for the clinic test and epidemiological investigation of AIV.