Development of a multiplex PCR assay for detection of Riemerella anatipestifer serotype 1 and serotype 2 strains

Abstract

Riemerella anatipestifer infection is one of the main infectious diseases that threaten the poultry industry at present. Serotypes 1 and 2 are the main serotypes causing global outbreaks of R. anatipestifer. In this study, we designed the primers specific for R. anatipestifer serotypes 1 and serotype 2 strains, and for R. anatipestifer 16S rRNA (for the species identification) to establish a multiplex PCR assay for rapid detection of the serotype 1 and serotype 2 strains. The specificity test showed that a 505 bp fragment was amplified from serotype 1 strains, and a 1125 bp fragment was amplified from serotype 2 strains. A 843 bp fragment of 16S rRNA was amplified from R. anatipestifer strains with different serotypes. No amplification band was shown for other species of bacterial pathogens, including Escherichia coli, Salmonella, Pasteurella multocida, Mycoplasma gallisepticum and Mycoplasma synoviae. The sensitivity test showed a detection limit of 10² CFU of the multiplex PCR assay. Furthermore, a total of 60 R. anatipestifer clinical isolates were tested for identification of the serotypes, and the results showed that a 843 bp 16S rRNA fragment was amplified from all 60 isolates, confirming they are R. anatipestifer strains. Moreover, a 505 bp serotype 1 fragment was amplified from 9 isolates, and a 1125 bp serotype 2 fragment was amplified from 28 isolates. R. anatipestifer serotype 1 and 2 strains accounted for 61.67 % of the isolates. The results were further validated by slide agglutination test, which showed 100 % consistency with the multiplex PCR.