Abstract: To shorten the detection cycle of subgroup J avian leukosis virus (ALV-J) and further accelerate the process of avian leukosis decontamination, this study combined SYBR Green Ⅰ qPCR and pooling sample assay to establish a blood nucleic acid screening technique (ALV-J-B-qPCR) for rapid screening of ALV-J in low prevalence. Based on the sequences of ALV-J pol and env genes in GenBank, the specific primers for ALV-J were designed and the reaction conditions were optimized to establish the ALV-J SYBR Green Ⅰ qPCR assay. Anticoagulant DNA, blood cell DNA, peripheral blood lymphocyte DNA, peripheral blood lymphocyte cDNA and plasma cDNA were respectively prepared to perform PCR detection of ALV-J, and the best assay templates were screened. Further, the accuracy of the three mixing methods of extracting DNA from mixed blood by red blood cell cracking method, the mixed method with 200 μL total volume of blood mixed sample and the mixed method with 10 μL total volume of blood mixed sample were compared, and the best mixing method was selected. The ALV-J-B-qPCR was established by combining the above qPCR method, assay template and mixing method. Simulated screening tests with expected prevalence scenarios of 1%-2% and 4%-5% were performed using ALV-J-B-qPCR respectively, and compared with the virus isolation method. Specificity, sensitivity and reproducibility tests of the qPCR method showed that the method specifically amplifies only ALV-J and has a minimum detection limit of 1×10² copies·μL^-1 for standard plasmids, with intra-and inter-batch coefficients of variation <1%. Results on 90 clinical samples showed that the detection rate of ALV-J by this qPCR method (15.6%) was higher than that of p27 antigen ELISA and PCR (12.2%). In the assay template screening test, anticoagulated blood DNA best met the requirements of high assay accuracy, operational complexity and low cost, and was the best assay template. In the mixing method screening test, the mixing method with a total mixing volume of 10 μL (mixing size <12) had the highest detection accuracy and was the best mixing method. In a simulated screening with a sample size of 400 and an expected prevalence of 4%-5%, the detection rate of ALV-J-B-qPCR was 6.25%, which was 2.00% higher than that of the virus isolation method; in a simulated screening with a sample size of 400 and an expected prevalence of 1%-2%, the detection rate of ALV-J-B-qPCR was 2.25%, which was 0.75% higher than that of the virus isolation method. The ALV-J-B-qPCR technique established in this study has the advantages of high sensitivity, rapid detection and cost saving, which provides a new idea and method to accelerate the ALV purification process in breeding poultry farms.

Key words: ALV-J, qPCR, pooling sample assay, screening technique