A duplex fluorescence LAMP assay for identification of avian leukosis virus and chicken infectious anemia virus

【Objective】 To establish a duplex fluorescence loop-mediated isothermal amplification(LAMP) method for simultaneous identification of avian leukosis virus(ALV) and chicken infectious anemia virus(CIAV), and provide technical support for clinical rapid diagnosis and effective prevention and control of single or mixed infections of ALV and CIAV.【Method】 According to the conserved sequences of the ALV(A, B, C, D and J subgroups) pol gene and the CIAV VP2 gene, two sets of specific primers were designed for LAMP, and a double labeled probe was designed in the areas covered by F1c and B1c of ALV and CIAV sequences, respectivelyALV-probe(5' end was labeled with a FAM fluorescent dye, 3' end was labeled with a BHQ3 quencher dye) and CIAV-probe(5' end was labeled with a CY5 fluorescent dye, 3' end was labeled with BHQ3 quencher dye) . After the reaction in the Loopamp LA-320 C turbidimeter, the results could be observed in the polychromatic fluorescence system. The applicability and reliability of the duplex fluorescence LAMP test method were validated by specificity testing, sensitivity testing, and clinical sample testing.【Result】 The duplex fluorescence LAMP was optimized in 20 μL volume containing DNA/cDNA template 2.0 μL, 2×Reaction Mix 10.0μL, Bst DNA polymerase 0.8 μL, internal primers ALV-FIP, ALV-BIP, CIAV-FIP and CIAV-BIP(working concentration 40.0 μmol/L) 0.8 μL each. External primers were ALV-F3, ALV-B3, CIAV-F3 and CIAV-B3(working concentration 5.0μmol/L) 0.4 μL each. ALV-Probe(working concentration 0.5 μmol/L) 0.4 μL, CIAV-Probe(working concentration 0.5) μmol/L) 0.8 μL, making up to 20.0 μL volume with ddH₂ O. Amplification procedure: the reaction mixture was incubated at 62 ℃ for 60 min and inactivation at 80 ℃ for 5 min. The results showed that duplex LAMP detection method could simultaneously detect ALV and CIAV, and had no specific amplification for other avian pathogens;the minimum detection limit of single template of CIAV and ALV in sensitivity test were 10² copies/µL. The minimum detection limit of mixed template in sensitivity test was 10² copies/µL for ALV and 10³ copies/µL for CIAV;13 throat and cloacal swabs were detected by the duplex fluorescence LAMP. The results were consistent with those of ALV and CIAV by single PCR methods, and the coincidence rate was 100%.【Conclusion】 The duplex fluorescence LAMP method for simultaneous detection of ALV and CIAV in this study has the advantages of good specificity, high sensitivity and low pollution. The detection results can be observed by naked eyes in the polychromatic fluorescence system, which is applicable for rapid clinical screening of ALV and CIAV.