| Avain leukosis(AL)is an immunosuppressive disease caused by avian leukosis virus(ALV),especially for chickens.The virus can spread horizontally or vertically.According to antibody neutralization test,the virus can be divided into 11 subgroups,of which 7subgroups can infect chickens,namely A～E,J and K types.At present,there are no effective vaccines and drugs to prevent the disease,so purifying chickens is the most important way of prevention and control.Establishing purification methods for different needs is the primary task of purifying the disease.Rapid and convenient detection methods are of great significance for the prevention and purification of ALV.In this study,ALV and J subgroup ALV isolated from Anhui province were studied in two aspects.First,the triple PCR method for simultaneous detection of A,J and general ALV was established,and the clinical test results were good.Firstly,the gp85 gene sequences of A and J subgroups and pol gene sequences of all subgroups were obtained from NCBI gene database.Three pairs of primers for A and J subgroups and two pairs of universal primers were designed by Oligo 7.0 software.The primers of subgroup A and subgroup J ALV isolated from Anhui province were verified and screened.Secondly,the three target gene fragments were recovered and ligated into p MD18-T to construct the recombinant plasmid,and the PCR method was optimized to establish the triple PCR detection method,and then the sensitivity and repeatability of the established method were tested.Finally,43 clinical samples were detected by the established method and compared with the results of the standardized kit.The results showed that the designed primers had strong specificity and conservation,and specific PCR bands could be amplified.The target fragments of A,J and universal primers were 274 bp,338 bp and 145 bp,respectively.The established triple PCR method could detect the minimum concentration of the template at 1×10~3copies/μL,without nonspecific amplification,and had good repeatability.The clinical sample detection results showed that the positive rate of the established method for sample detection was 83.7%,and the coincidence rate with the detection rate of the standardized kit was 92%.This shows that the established method has strong specificity,high sensitivity and low cost,which is suitable for qualitative detection of chicken farms of different sizes.Second,a dual fluorescent probe method for simultaneous detection of ALV and ALV of subgroup J was established,with high detection sensitivity and accuracy.Firstly,primers and probes for dual-fluorescence quantitative PCR were designed using Oligo 6.0 and Oligo 7.0 software according to the gp85 gene sequences of A and J subpopulation viruses.Secondly,the recombinant plasmid in the amplification region was designed and synthesized.The standard curve was established using the designed primers,and the reaction conditions were optimized.The sensitivity and stability were tested using probes.Finally,the designed primers and probes were used to detect 20 clinical samples.The results showed that the primers and probes of subtype A and J designed by the software were TQ-ALV-A-F,TQ-ALV-A-R and TQ-ALV-A-probe,TQ-ALV-J-F,TQ-ALV-J-R and TQ-ALV-A-probe,respectively,which could amplify 177 bp and 102 bp bands,respectively.The sensitivity of the established dual fluorescent probe method was 13copies/μL for type A,and 71 copies/μL for type J.The repeatability test showed that the coefficient of variation in each group was less than 1%.This shows that the established double fluorescent probe method has high specificity,sensitivity and repeatability,and can be used for egg purification.In conclusion,this study established a triple PCR method for simultaneous detection of ALV and a dual fluorescent probe method for simultaneous detection of A and J subtypes of ALV strains isolated from clinical practice.The specificity and sensitivity of clinical detection were high,which provided different methods for clinical purification of ALV. |
