| Avian leukemia is caused by Avian leukosis virus(ALV)of the genus Avian retrovirus of the retroviridae family,which can cause birds to suffer from different benign and malignant tumors.The physical function of poultry infected with ALV is inhibited,such as slow increase in body weight,decrease in egg production,immunosuppression and other subclinical symptoms.The economy of large-scale poultry industry in China suffered huge losses because of avian leukemia.Based on the culture characteristic,differences in the mutual interference mode and host range,and the diversity of antigen structure located on envelope protein,ALV can be divided into ten subgroups of A-J and newly identified K subgroup.Subgroup A,B,C,D,E,and J which were first isolated can infect poultry.Subgroup A and B are exogenous avian leukemia viruses,which are commonly found in commercial laying hens.The subgroup A leukemia virus mainly infects laying hens and mainly causes lymphomas.It is the main pathogen of avian leukemia.The J subgroup discovered late has the strongest infectivity and is currently the main epidemic.At present,there is no effective vaccine and drug for the prevention and control of ALV in clinic.Serum test of the virus is the only effective method for regular monitoring to clean the flock and strictly eliminate the positive chickens.But most of the detection methods for avian leukemia are time-consuming duration,tedious operation,low sensitivity of genotyping and low accuracy with high false positive rates.Therefore,it is urgent to establish new sensitive,specific,rapid,efficient and practical detection methods.The rapid RPA detection method established in this study was targeted on the conservative and specific gp85 fragment of A and J subgroup,in which multiple primers and probes were designed,respectively.Optimum primers and probes of the fluorescent RPA rapid detection method for A subgroups were screened by optimizing reaction time and the final reaction concentration.A number of avian viral nucleic acids were detected and only A subpopulations of viral nucleic acids were amplified,indicating that the method was highly specific and the sensitivity can reach 100 copies /μL.The coefficient of variation for repeated tests within batches was ranging from 1.14 % to 3.73 %,and the coefficient of variation for repeated tests between batches is 0.53 % to 3.69 %.repeatability of this method is good.The result of clinical sample test was consistent with that of ordinary PCR method,100 %coincidence rate.At the same time,the fluorescent RPA rapid detection method for J subgroup was also established in this study.The J subgroup was isolated and identified in this study,and a new epidemic strain was successfully isolated.J subgroup standard plasmid was constructed and applied to the establishment of the method.Detection of several avian viral nucleic acids and amplification of only J subpopulations of viral nucleic acids indicate that the method is highly specific and the minimum detection limit of the sensitivity test can reach 10 copies /μL.The coefficient of variation for repeated tests within batches was 0.59 %～3.82 %,and the coefficient of variation for repeated tests between batches was 0.64 %～4.61 %.And the repeatability is good.The coincidence rate with common PCR methods was 92% in clinical detection applications.The nucleic acid products of real time RPA positive samples were all ALV-J subgroups by sequencing analysis,and the method was more sensitive.The establishment of A,J subgroup real time RPA isothermal rapid detection method provides technology and support for ALV typing and field detection,and has important significance for ALV control and purification. |
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