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

摘要

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. 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. 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. 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.