The Reverse Transcription Loop-mediated Isothermal Amplification(RT-LAMP) Method For The Detection Of Avian Leukosis Virus

Reverse transcription loop-mediated isothermal amplification(RT-LAMP) assay is performed under isothermal conditions(63℃~65℃) with a set of four specially designed primers that recognize six distinct sequences of the target.By selecting specific ALV RT-LAMP primers optimizing the ALV RT-LAMP assay and evaluating the stability and the specificity, the ALV RT-LAMP is successfully built up.As a specific, sensitive and rapid ALV diagnostic technology, it might provide a powerful method for AL surveillance and control, a rapid assay was developed for detection of ALV using RT-LAMP.Four sets of specific ALV RT-LAMP primers targeting the ALV gag gene were designed. A set of ALV specific primers named P1 was then selected for RT-LAMP assay which doesn’t amplify nucleic acid of IBDV,REV,NDV, IBV, EDSV and AIV.The reaction system(e.g. Betaine, Mg2+, dNTP, Bst DNA polymerase, AMV reverse polymerase and primers), and the reaction conditions(e.g. time and temperature) were optimized using an RNA template from ALV RAV-1. The ALV RT-LAMP assay could not amplify other avian disease viruses, showing that the method has high specificity for ALV. RT-LAMP amplification products could be detected by fluorescence detection or visual inspection of the turbility, a ladder-like appearance when electrophoresed on an agarose gel and white precipitates of magnesium pyrophosphate in the tubes. The simple end-point detection of the LAMP reaction can be made possible by judging whether the precipitate is formed.The test has a higher specificity than ELISA and PCR for ALV detection, and the minimum limit detected was 1pg nucleic acid or 0.2TCID50/0.1mL virus.Restricion endonucleases analysis of the LAMP products agreed with the expected conclusion,and the sequences of the cloned DNAs perfectly agreed with the expected nucleotide sequences.Finally the ALV RT-LAMP kit is successfully built up.The two methods of ELISA and ALV RT-LAMP were used to detect ALVs in 60 batchs of avian live viral vaccines. Among them, 11 batchs of the vaccines were positive by ALV RT-LAMP, 9 batchs of the vaccines were positive by ELISA. The level of agreement for ALV RT-LAMP was 91%, compared with ELISA.The ALV RT-LAMP is faster, easier and more sensitive than other methods. The method will be useful for ALV surveillance and rapid detection, and has great clinical potential.