Avian Leukosis (AL) is an infectious tumour disease affecting poultry caused by avian leukosis viruses (ALVs) belonging to the Alpharetrovirus genus of the family Retroviridae . Chickens infected with ALVs suffer from decreased performance and immunosuppression, leading to tumours and even death , and it is one of the most important diseases endangering the healthy development of the poultry industry in China . Based on the envelope glycoprotein (gp85) associated with subgroup specificity, ALVs from naturally infected chicken flocks can be divided into seven subgroups (A, B, C, D, E, J and K) , among which endogenous subgroup E viruses with little to no pathogenicity are present in nearly all chicken lines, while the others are exogenous and pathogenic [5, 6]. Subgroup A and B viruses mainly cause lymphocytic leukemia and myeloid leukosis in field flocks [7, 8], while flocks naturally infected with subgroup C and D are rarely observed in the field [9, 10].
Avian leukosis virus subgroup J (ALV-J) was first isolated and identified in broiler chickens in 1988 . Chickens infected with ALV-J can suffer from medulloblastoma and decreased production performance, causing huge economic losses to the poultry breeding industry across the world . ALV-J was originally thought only to infect meat-type chickens , but its host range is now known to include layer chickens and local broiler breeders in most parts of China [14, 15], and the disease has even spread to waterfowl and wild birds . Most large foreign breeder companies had eradicated classic exogenous ALVs by the end of the 1980s, but ALV-J remains widespread in China .
Effective vaccines and drugs have not yet been reported for AL. Obtaining a population of breeding poultry without exogenous ALV through continuous purification is therefore the main method for disease prevention and control, for which virus detection is crucial. A variety of methods for detecting and differentially diagnosing ALV have been established in various regions of the world, mainly involving virus isolation and identification , pathological diagnosis , serological detection methods such as enzyme-linked immunosorbent assay (ELISA) , and PCR-based molecular biological detection . However, existing detection methods have some limitations and shortcomings. For example, traditional virus isolation and identification is too time-consuming to meet the requirements of rapid diagnosis, and PCR and real-time PCR (RT-PCR) require expensive instruments and equipment which are not conducive to fieldwork. Additionally, while the loop-mediated isothermal amplification (LAMP) detection method does not require specific instruments or equipment , it is highly prone to false positives, weak positive results tend to be inaccurate, and contamination by aerosols can occur easily.
Cross-priming amplification (CPA) is a rapid detection method developed in recent years [23–25]. The CPA utilizes multiple primers and probes (4 s, 5a, 2a1s, 3a, and 2a) to amply nucleotide sequence isothermally. By labelling primer 2a with biotin, primer 3a with FITC, then the amplified products were recognized by anti-biotin and anti-FITC monoclonal antibodies on the test line, where gold nanoparticles (AuNPs) were fixed, CPA amplified products could be visualized in nucleic acid test strip . It benefits from high specificity and sensitivity, does not require expensive instruments, the operation process is relatively simple, amplification is rapid, and it can be used with disposable nucleic acid detection strip in closed tubes, thereby avoiding aerosol pollution, making results more intuitive and objective . This method has been applied to the diagnosis of infectious diseases and the detection of multiple pathogens [24, 28]. In the present study, we developed a CPA assay for ALV-J, which would be a promising and user-friendly rapid detection method.