| Avian infectious bronchitis（IB）is an acute and highly infectious respiratory disease caused by avian infectious bronchitis virus（IBV）,which mainly affects the respiratory and genitourinary systems of chickens and causes secondary infection,which brings serious economic losses to the poultry industry.Although there are many commercial vaccines,the IBV genome is prone to mutation,resulting in constant appearance new genotype or serotype IBVs,and cross-protection between different serotypes is limited,so immunization failure occurs frequently.In addition,the clinical manifestations and anatomical lesions of the disease are similar to other respiratory diseases,which make the diagnosis and prevention of the disease more difficult.Therefore,it is particularly important to establish rapid,sensitive and specific methods for pathogen and antibody detection.Many studies have shown that the prevailing dominant IBV genotype in China is LX4 type,and there is a lack of research on LX4 type specific LAMP method.In view of this,IBV LX4 specific LAMP method was established in this study,and an indirect ELISA method based on the the eukaryotic expression S1 protein of GX-YL5,a representative srtain of dominant serotype in Guangxi,was established.The details are as follows.Based on the S1 gene differential sequence of IBV LX4 type strains and other strains in Gen Bank,a set of specific LAMP primers were designed and screened.After the optimization of the reaction system and reaction conditions,the IBV LX4 type specific LAMP method was established and its specificity and repeatability were analyzed,and the method was applied to the diagnosis of clinical samples.The results showed that the established LAMP method was positive for the amplification of LX4 genotype IBV strains,and the amplification was completed at 62-64℃for 1 h,while the amplification results for other genotypes IBV,Newcastle disease virus（NDV）,and infectious bursal disease virus（IBDV）,avian leukemia virus（ALV）,Marek’s disease virus（MDV）were negative.The sensitivity test showed that the minimum detection concentration of this method was 8×10-5 ng/μL,which was 100times higher than that of ordinary PCR.The repeatability test showed that the LX4 genotype IBV nucleic acid could be detected by repeated tests for 3 times.The established IBV LX4 type-specific LAMP method was used to detect the 22 IBV tissue samples stored by the research group,and the results showed that 9 of them were LX4 type,which was consistent with the previous S1 gene analysis results of these strains.In this study,LX4 type specific LAMP method for avian infectious bronchitis virus was established for the first time.This method was specific,sensitive,stable,fast,cheap,practical and suitable for application in farms.A large number of S1 proteins of GX-YL5,a representative IBV srtain of dominant serotype in Guangxi,were obtained according to the eukaryotic expression conditions optimized by the research group,and the purified S1 protein was used as an antigen.An indirect ELISA method based on the eukaryotic expression S1 protein of GX-YL5 strain was successfully established through the optimization of antigen coating concentration,serum dilution of primary antibody,optimal working concentration of HRP goat anti-chicken,and the optimal time for blocking,color rendering,primary antibody and secondary antibody incubation.And its specificity,sensitivity and repeatability were analyzed.In addition,the developed type-specific LAMP was applied to the clinical samples and compared with the IDEXX commercial kit.The results showed that this method had no cross-react with the positive sera of NDV,IBDV,ALV and MDV;the lowest serum dilution that could be detected was 1:800;the coefficient of variation between batches and within batches was less than 10%.The method was applied to the detection of120 serum samples,the positive rate of S1-ELISA method was 87.5%（105/120）.The positive rate of the IDEXX commercial kit was 92.5%（111/120）,and the positive coincidence rate between the established method and the commercial kit was 94.6%（105/111）,with a high coincidence rate.Therefore,the established indirect ELISA based on IBV S1 protein has good specificity,sensitivity and stability,which could meet the needs of large-scale IBV antibody detection in farms.In summary,LX4 type specific LAMP method was established for the first time in the present study,and the established LAMP method was specific,fast,and simple,which provided a new detection method for rapid diagnosis and epidemiological investigation of IBV.Furthermore,an indirect ELISA method based on the eukaryotic expression S1 protein from the representative strain of dominant serotype in Guangxi was successfully established.This method has low cost,high specificity,high sensitivity and good reproducibility.It was suitable for the detection of antibodies in large-scale serum samples,and provided a new method for immune monitoring,diagnosis and serological investigation of IBV,which was worth popularizing and applying. |
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