Establishment Of LAMP Assay For Brucella Spp. Differential Diagnosis

Brucellosis is an important zoonotic infectious disease,which not only causes great economic losses to the development of animal husbandry,but also seriously threatens human health.At present,the commonly used serological diagnosis methods of brucellosis can not effectively distinguish the sick animals in the immune herd,and can not carry out the quarantine and purification of the herd.In this study,the specific gene GL?0002189 and TrbJ were screened from the genome sequencing data of Brucella S2 attenuated vaccine,the eryC gene deletion fragment was screened from the genome sequencing data of S19 attenuated vaccine.Based on Loop Mediated Isothermal Amplification(LAMP),the differential detection method was studied to solve the technical problem of lack of differential diagnosis method in production practice.The main research results are as follows.According to the specific gene of Brucella S2 vaccine strain GL?0002189 sequence designed three groups of LAMP primers to detect S2 vaccine strain,and finally determined the best group of primers to establish S2-LAMP detection system.The concentration of MgSO₄,d NTPs,F3/B3,FIP/BIP,LF/LB and reaction temperature were optimized.The optimal reaction temperature was 65?.The specificity,sensitivity and repeatability of the method were evaluated.The results showed that the primer could specifically detect S2 vaccine strain,and negative reaction was found in Brucella A19,S19,Rev.1,M28,Staphylococcus aureus,Streptococcus,Salmonella and other strains.The detection limit was 68.9 pg/?L compared with ordinary PCR,the S2 DNA template with the same sensitivity can be obtained.The repeatability of three different batches of LAMP reagents showed good repeatability.Three groups of LAMP primers were designed and screened according to the TrbJ sequence of S2 vaccine specific gene was used to establish LAMP detection method.The results showed that the best reaction temperature of LAMP method for TrbJ gene detection was 67?.It had good specificity for several other Brucella strains and several pathogenic strains.The lowest sensitivity was 6.89pg/?L DNA template,which was 100 times higher than that of ordinary PCR.Five groups of LAMP primers were designed and screened with eryC gene deletion of Brucella S19 vaccine as the target sequence.The DNA of A19 vaccine strain was used as template to optimize the reaction conditions and reaction components.The results showed that the optimal reaction temperature of A19-LAMP detection method was 65?,and the specificity of the tested strains was consistent with the theoretical value.The sensitivity of LAMP detection was the same as that of ordinary PCR,and the minimum detectable DNA template was 17pg/?L.There was good specificity between the reagents.