Establishment and application of a new one-step multiplex PCR for the accurate detection and differentiation of Salmonella Gallinarum biovars Pullorum and Gallinarum

Salmonella enterica serovar Gallinarum biovars Pullorum and Gallinarum lead to pullorum disease and fowl typhoid in avian species and have been of substantial economic significance to the poultry industry. Serotyping and biochemical assays are complicated for use in the differentiation of these two biovars owing to their antigenic similarity. The aims of this study were to establish a new multiplex polymerase chain reaction (PCR) method to detect and differentiate the two Salmonella biovars, Gallinarum and Pullorum. The novel multiplex PCR was established on the basis of three genes, SG1445, I137_14465, and stn. In silico analysis indicated the SG1445 gene exists in all Salmonella serovar, apart from S. Pullorum, which allowed the use of this gene for S. Pullorum identification. The I137_14465 gene is absent in S. Gallinarum and present in all other Salmonella serovars, allowing this gene to be utilized for S. Gallinarum detection. stn was employed as a control gene for the Salmonella genus. The multiplex PCR could identify and distinguish the two pathogens accurately from 74 strains of Salmonella, including 29 different serovars and without false positive reactions from 44 non-Salmonella bacteria. The specificity was verified with a broad collection of Salmonella isolates. The detection limit of the multiplex PCR was as low as 22.5 pg/μL genomic DNA of the two pathogens and 100 CFU of bacterial cells. Detection of clinical isolates from a chicken farm was carried out using this method with good agreement with detection based on traditional Salmonella serotyping and biochemical identification. The findings of this study showed that the developed multiplex PCR technique could sensitively detect and specifically differentiate the two biotypes of S. Gallinarum in laboratory and clinical samples.