AUTHOR=Xiong Dan , Song Li , Pan Zhiming , Jiao Xinan 

TITLE=Identification and Discrimination of Salmonella enterica Serovar Gallinarum Biovars Pullorum and Gallinarum Based on a One-Step Multiplex PCR Assay

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01718

DOI=10.3389/fmicb.2018.01718

ISSN=1664-302X

ABSTRACT=<p><italic>Salmonella enterica</italic> serovar Gallinarum biovars Pullorum (<italic>S.</italic> Pullorum) and Gallinarum (<italic>S.</italic> Gallinarum) can result in pullorum disease and fowl typhoid in avian species, respectively, and cause considerable economic losses in poultry in many developing countries. Conventional <italic>Salmonella</italic> serotyping is a time-consuming, labor-intensive and expensive process, and the two biovars cannot be distinguished using the traditional serological method. In this study, we developed a rapid and reliable one-step multiplex polymerase chain reaction (PCR) assay to simultaneously identify and discriminate the biovars Pullorum and Gallinarum. The multiplex PCR method focused on three specific genes, <italic>stn</italic>, <italic>I137_08605</italic> and <italic>ratA</italic>. Based on bioinformatics analysis, we found that gene <italic>I137_08605</italic> was present only in <italic>S.</italic> Pullorum and <italic>S.</italic> Gallinarum, and a region of difference in <italic>ratA</italic> was deleted only in <italic>S.</italic> Pullorum after comparison with that of <italic>S.</italic> Gallinarum and other <italic>Salmonella</italic> serovars. Three pairs of primers specific for the three genes were designed for the multiplex PCR system and their selectivity and sensitivity were determined. The multiplex PCR results showed that <italic>S.</italic> Pullorum and <italic>S.</italic> Gallinarum could be identified and discriminated accurately from all tested strains including 124 strains of various <italic>Salmonella</italic> serovars and 42 strains of different non-<italic>Salmonella</italic> pathogens. In addition, this multiplex PCR assay could detect a minimum genomic DNA concentration of 67.4 pg/μL, and 100 colony forming units. The efficiency of the multiplex PCR was evaluated by detecting natural-occurring <italic>Salmonella</italic> isolates from a chicken farm. The results demonstrated that the established multiplex PCR was able to identify <italic>S.</italic> Gallinarum and <italic>S.</italic> Pullorum individually, with results being consistent with traditional serotyping and biochemical testing. These results demonstrated that a highly accurate and simple biovar-specific multiplex PCR assay could be performed for the rapid identification and discrimination of <italic>Salmonella</italic> biovars Gallinarum and Pullorum, which will be useful, particularly under massive screening situations.</p>