Development of PCR Primers for the Detection of Salmonella enterica Serovar Choleraesuis Based on the fliC Gene

Abstract

Salmonella enterica serovar Choleraesuis may cause swine salmonellosis and human infection. Because the conventional method for detection of this Salmonella serovar may take 3 to 5 days, a PCR method for detection was evaluated. By comparing the sequence of the phase 1 flagellin (fliC) gene of Salmonella Choleraesuis with that of other Salmonella serovars and of other bacteria species available in GenBank, two PCR primers (flinC-F and flinC-R) were designed. Using these primers, all 97 Salmonella Choleraesuis strains assayed generated the expected PCR product, with a molecular mass of 963 bp. Except for S. enterica Paratyphi C, Salmonella isolates other than Salmonella Choleraesuis and non-Salmonella isolates, including strains of Enterobacteriaceae, all generated negative PCR results. Salmonella Paratyphi C could be differentiated from Salmonella Choleraesuis through the use of primers designed from the viaB gene. When Salmonella Choleraesuis isolates from swine stool, pork, liver, feed, and human whole blood samples were assayed with a preenrichment step, as low as 1 CFU/g or ml of the original sample could be detected.

Pathology of Salmonella choleraesuis Related Respiratory Infection in Piglets, Its Isolation, Identification and Antibiogram

“…This was in accordance with Chiu et al, (2005) and Bose (2015). Bose (2015) reported extra intestinal isolation of Salmonella from lungs, heart blood and bronchial lymph node of pigs based on conventional and PCR method.…”

“…Isolates positive in conventional methods were subjected to fliC based S. choleraesuis specific primer as per Chiu et al, (2005). Forward primer 5'-AAG GAA AAG ATC ATG GCA CAA-3'and reverse primer 5'-GAA CCC ACC ATC AAT AAC TTT G-3' were used to get an amplicon size of 963bp.…”

Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a Broad Spectrum of Samples

“…We verified that the PNA-FISH method was able to detect Salmonella at the end of the preenrichment step in BPW, avoiding the selective enrichment need. This means that this method is capable of detecting Salmonella in less than 20 h, resulting in the saving of at least 3 days compared to the ISO assay and matching the best times reported for PCR-based techniques (10,13,36,40). Moreover, we did not have problems with inhibitory substances affecting the hybridization procedure, as reported in some studies using molecular approaches for Salmonella detection (28,36).…”

“…The PNA-FISH protocol presented in this work is technically less demanding. Although an enrichment step is needed, the total time required for the PNA-FISH assay (less than 20 h, except for PIF, which takes 12 h) is similar to or even better than the times reported for PCR-based methods (10,13,36,40).…”

“…A rapid and reliable tool to assist disease control management should aim to reduce salmonellosis in both people and animals. For this purpose a number of assays, such as the enzyme-linked immunosorbent assay (ELISA), PCR, and fluorescence in situ hybridization (FISH), have been developed to decrease the time required to identify Salmonella in food, feces, water, and other clinical samples (8,10,14,15,25,26,31,41).…”