Establishment Of Latex Agglutination And Colloidal Gold Immunochromatographic Assay Based On The Riemerella Anatipestifer MotB Protein

Riemerella anatipestifer(RA)disease is a poultry infectious disease in the world caused by the Riemerella anatipestifer,and it has a high incidence.Twenty-one serotypes have been reported and almost all serotypes only react with isotype antisera specifically.Clinical laboratory methods such as ELISA and PCR take a lot of time and require professional technicians.This study analyzed the prevalence of RA serotypes and establishes a latex agglutination test that can detect mainstream serotype RA strains.At the same time,the clinical,fast and simple latex agglutination test and the immunocolloid gold test strip were established based on RA MotB protein and rabbit anti-RA MotB polyclonal antibody,to detect the mainstream RA serotypes and RA strains.This study conducted serotype identification and analysis of 373 RA strains isolated from various provinces and cities in China from 2014 to 2019.The main epidemic serotypes have changed to serotype 2(37.8%),serotype 11(25.7%)and serotype 1(16.3%).Then the study used the main epidemic serotypes(1,2,and 11 RA serotype)to establish latex agglutination test to detect RA strains(1,2,and 11 serotype respectively).Optimizing the quality of latex after sensitization,the result showed that there is no self-curing phenomenon of sensitization latex and the latex could specifically detect RA strains.Latex also can be stored at 4℃for more than 6 months with a high specificity and stability.The minimum detection limit for RA of the latex is 10~7 colony forming units.The identity rate with serum agglutination is 100%,and the agglutination effect is more obvious than serum agglutination.PCR amplified the RA MotB gene by RA ATCC 11845 DNA,and expressing the vector p ET-32a-MotB.Then plasmid p ET-32a-MotB was converted into the protein-expressing strain,and the IPTG induced the recombinant protein.Then used the protein RA MotB to establish a rapid latex agglutination test assay to detecte the RA serum.Then this study tested the quality after sensitization optimization.The result showed that there is no self-aggregation appearance of latex.No agglutination was detected with sera from ducks inoculated with other pathogens,the lowest limit of positive serum detection was up to 1:128.LAT also can be keep in 4℃,25℃and 37℃go beyond 8 months with a high specificity and stability and has a good consistency with ELISA detection method.It can detect RA positive sera of type 1,2,6,7,8,9,10 and 11.The rabbit anti-RA MotB polyclonal antibody were generated by immunization of rabbit with purified RA MotB protein.The titer of antiserum was assessed over 1:1600 by ELISA.The study used the rabbit anti-RA MotB polyclonal antibody to establish a immunocolloid gold test strip to detecte the RA.Then test the quality after sensitization optimization.The result showed that there are no self-curing phenomenon of sensitization latex and it could specifically detect RA strains.It also has a good repeatability between batches and can be stored at 4℃,25℃and 37℃for go beyond 6 months with a high quality specificity and durability.The minimum detection limit for RA of the latex was 10~6colony forming units and has a good consistency with PCR detection method.It can detect almost RA strains(1,2,6,7,8,9,10 and 11 serotypes).