Multiplex PCR Assay Based on the Cite2 Gene and Intergenic Sequence for the Rapid Detection of Salmonella Pullorum in Chickens

Salmonella is one of the most common Gram-negative pathogens and seriously threatens chicken farms and food safety. This study aimed to establish a multiplex polymerase chain reaction (PCR) approach for the identification of different Salmonella enterica subsp. enterica. The citE2 gene and interval sequence of SPS4_00301-SPS4_00311 existed in all S. enterica subsp. enterica serovars by genomic comparison. By contrast, a 76 bp deletion in citE2 was found only in Salmonella Pullorum. Two pairs of special primers designed from citE2 and interval sequence were used to establish the multiplex PCR system. The optimized multiplex PCR system could distinguish Salmonella Pullorum and non-Salmonella Pullorum. The sensitivity of the optimized multiplex PCR system could be as low as 6.25 pg/μL and 104 colony-forming units (CFU)/mL for genomic DNA and Salmonella Pullorum cells, respectively. The developed multiplex PCR assay distinguished Salmonella Pullorum from 33 different Salmonella enterica subsp. enterica serotypes and 13 non-target species. The detection of egg samples artificially contaminated with Salmonella Pullorum, Salmonella Enteritidis, and naturally contaminated 69 anal swab samples showed that results were consistent with the culture method. These features indicated that the developed multiplex PCR system had high sensitivity and specificity and could be used for the accurate detection of Salmonella Pullorum in clinical samples.