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

Abstract

Salmonella enterica serovar Gallinarum biovars Pullorum (S. Pullorum) and Gallinarum (S. 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 Salmonella 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, stn, I137_08605 and ratA. Based on bioinformatics analysis, we found that gene I137_08605 was present only in S. Pullorum and S. Gallinarum, and a region of difference in ratA was deleted only in S. Pullorum after comparison with that of S. Gallinarum and other Salmonella 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 S. Pullorum and S. Gallinarum could be identified and discriminated accurately from all tested strains including 124 strains of various Salmonella serovars and 42 strains of different non-Salmonella 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 Salmonella isolates from a chicken farm. The results demonstrated that the established multiplex PCR was able to identify S. Gallinarum and S. 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 Salmonella biovars Gallinarum and Pullorum, which will be useful, particularly under massive screening situations.

Keywords: Salmonella Gallinarum; Salmonella Pullorum; accurate discrimination; multiplex PCR; one-step diagnostic PCR.

FIGURE 1

Primer design for the multiplex PCR method to distinguish Salmonella enterica serovar Gallinarum biovars Gallinarum (S. Gallinarum) and Pullorum (S. Pullorum) from other serovars. Gene I137_08605 exists only in the two Salmonella biovars, and gene ratA of S. Pullorum has a region of difference (ROD) compared with that of other serovars, which were exploited to design the primers. The red arrows indicate the positions of the designed primers.

FIGURE 2

Selectivity of the multiplex PCR method for the identification of S. Pullorum and S. Gallinarum. The multiplex PCR assays, using genomic DNA from various Salmonella and non-Salmonella strains, were conducted using the designed primers targeting stn (131 bp), I137_08605 (290 bp), and ratA ROD (571 bp). The three specific PCR products could be amplified in S. Gallinarum, while only stn and I137_08605 genes could be amplified in S. Pullorum.