Development and application of a multiplex PCR method to differentiate Salmonella enterica serovar Typhimurium from its monophasic variants in pig farms

Salmonella enterica serovar Typhimurium monophasic variants (Salmonella 4,[5],12:i:-) has increased dramatically, causing human salmonellosis and colonization in pigs. With a difference to S. Typhimurium, the monophasic variants of S. Typhimurium lose the gene cassettes encoding the second phase flagellin. To establish a rapid method to detect and differentiate the two serotypes, we analyzed the published 679 genomes of S. Typhimurium and its monophasic variants and found that no Salmonella 4,[5],12:i:- strains carry both fljB and hin genes. Therefore, we established a novel multiplex PCR method using the fljB-hin region and mdh gene as target sequences to detect and differentiate both serotypes. This method can be used to specifically detect both serotypes with a detection limit for DNA concentration at 10 pg/μL. In addition, the PCR assay successfully differentiated 36 S. Typhimurium isolates from 62 isolates of monophasic variants preserved in our laboratory from 2009 to 2017, which corresponds to the whole-genome-based serotyping results. Application of the multiplex PCR method to 60 fecal samples from a pig farm identified 11.7% (7/60) of S. Typhimurium monophasic variants, which is consistent with the whole-genome-based serotyping results. The multiplex PCR assay is a rapid and precise method for the detection of S. Typhimurium monophasic variants from samples across food production chains.