Development of a real-time multiplex PCR assay for detection of Salmonellae in chicken samples

Abstract

Salmonellosis is one of the important bacterial diseases affecting poultry. Its importance is derived from the loss in productivity of affected birds. Salmonella Typhimurium is the most Salmonella serovars causing acute gastroenteritis and diarrhea. Serovar 1, 4, [5], 12: i: - is considered a monophasic variant of S. Typhimurium that is increasingly important as public health risk. A total of 58 Salmonella strains were serologically reidentified according to the White-Kauffmann- Le Minor scheme using slide agglutination test. Phase inversion method is performed to detect negative H phase for biphasic strains. A duplex real time PCR was applied for detection of genus Salmonella and S. Typhimurium using 16S rRNA and fliC genes respectively. All 58 strains were positive by SYBER green real time - PCR using 16S rRNA. Twenty one strains harbor fliC gene that include S. Typhimurium (12), S. Kentucky (6) , S.1,4,[5],12:i:- (1) , S. Lagos (1) and S. Kedougou (1), which possess the first flagellar antigen i. A duplex Tagman real-time PCR was performed for differentiation between biphasic S. Typhimurium and monophasic variant 1,4,[5],12:i:- using fljB1,2 and fliB / IS200 in the Fli A- B intergenic region. Fifteen strains were positive with fljB 1,2 gene .Ten strains of Salmonella Typhimurium harbor fljB1,2 gene encodes for phase 2 flagellar antigen .It was noticed that other serovars S. Paratyphi B (2) & S. Newport (1)and S. Virginia (2) also possesses fljB 1,2 . While two strains S. Typhimurium and S.1,4,[5],12:i:- don't have this gene. Concerning fli B/IS200, thirteen strains possessed fli B/IS200 (12 S. Typhimurium and the variant strain S.1,4,[5],12:i:-). The fli B / IS200 is a specific gene for S .Typhimurium (biphasic and monophasic). Two strains S. Typhimurium and strain S.1,4,[5],12:i:- don’t harbor fljB1,2,but possess fliB/IS200 so these three strains are monophasic variants of S. Typhimurium. Sequencing of fliC gene for two strains (S. Typhimurium and monophasic variant S.1,4,[5],12:i:-) reporting accession no (Mk103394) and (Mk103395) respectively in gen Bank. Inspections of the sequence alignments show a high degree of identity. There are greater than 98% amino acid sequence identity.