Rapid detection of the common avian leukosis virus subgroups by real-time loop-mediated isothermal amplification

Abstract

BACKGROUND: Subgroups A, B, E and J are the major subgroups of avian leukosis virus (ALV) infecting chickens. ALV infection has become endemic in China and has a significant negative effect on the poultry industry. Consequently, there is an urgent need for a specific, sensitive and rapid method for diagnosis and eradication of ALV. Therefore, we developed a simple and rapid real-time loop-mediated isothermal amplification (LAMP) reaction for the timely detection of the common ALV subgroups, whereby the amplification can be obtained in 35 min under isothermal conditions at 63 °C, ability to specific, sensitive and rapid detect all the common ALV subgroups. METHODS: A set of four specific primers was designed to target the sequences of the pol gene of ALV, and the loop-mediated isothermal amplification (LAMP) assay were developed and compared with PCR and virus isolation methods. RESULTS: The results from specificity of the LAMP assay showed that only target ALVs DNA was amplified. The LAMP assay demonstrated a sensitivity of 20 copies/reaction of ALV DNA, which was 10 times higher than the conventional PCR measurement. To further evaluate the reliability of the method, the assay was evaluated with ALV DNA from a panel of 81 clinical samples suspected of ALV infection. The results verify that the LAMP method was more sensitive than the conventional PCR and virus isolation method. CONCLUSION: In conclusion, the developed LAMP assay was a simple, inexpensive, sensitive method for the rapid detection of the most common subgroups of ALV, and it provided a useful and practical tool in the eradication program for ALV in the poultry industry.