Specific detection ofSalmonella entericaserotype Enteritidis using the polymerase chain reaction

Abstract

SUMMARYAn assay was developed for the specific detection of Salmonella enterica serotype Enteritidis, using a novel application of the polymerase chain reaction (PCR). This PCR assay is based on the mismatch amplification mutation assay, an allele-specific reaction, and can discriminate Enteritidis from all other salmonella. PCR primers were selected to amplify a 351-base pair (bp) DNA fragment from the salmonella plasmid virulence A (spvA) gene of Enteritidis. A single base difference at position 272 is present between the nucleotide sequence of the spvA gene of Enteritidis and other salmonellae. The downstream PCR primer, that encompasses position 272 of the Enteritidis spvA gene, was designed to contain a single base mismatch at the penultimate position, resulting in a 1-base mismatch with Enteritidis and a 2-base mismatch with other salmonellae that harbour the virulence plasmid. The upstream primer was completely homologous with the region immediately 5' to the spvA gene. When these primers were used and the annealing and extension reactions were performed at the same temperature, the PCR assay was specific for Enteritidis; no PCR product was detected for 40 other serotypes and 28 different genera examined. In pure culture, 120 colony forming units (c.f.u.) could be detected; a PCR product was observed from template derived from a 5 h enrichment broth culture of chicken seeded with 1 c.f.u. per gram of Enteritidis. This PCR assay is specific, reproducible, and less time consuming than the standard bacteriological methods used to detect Enteritidis.

Development of a PCR assay for the identification of Salmonella enterica serovar Brandenburg

“…This procedure is time-consuming, laborious and costly. Rapid and inexpensive PCR assays for the detection of Salmonella at the genus level have been developed (Rahn et al, 1992), but the number of assays to determine the Salmonella serotype is limited and includes those to detect serotypes Enteriditis (Lampel et al, 1996), Gallinarum (Shah et al, 2005), Typhi (Farrell et al, 2005;Kumar et al, 2006) and Typhimurium (Leon-Velarde et al, 2004). To date, there is no serotype-specific PCR assay for the detection of S. Brandenburg.…”

Use of Polymerase Chain Reaction and Restriction Enzyme Analysis to directly detect and identifySalmonella typhimuriumin food

“…The method potentially allows amplification of the target DNA from as few as one copy (Zhu et al 1996). Several PCR methods of detecting Salmonella have been published utilizing a specific gene sequence for targeting (Way et al 1993;Stone et al 1994;Lin and Tsen 1996;Lampel et al 1996;Zhu et al 1996;Kwang et al 1996).…”

Applied Microbiology

“…For pathogenic organisms virulence determinants are frequently used and often allow a very specific identification of a bacterial species (differentiation between C. jejuni and C. coli, Gonzalez et al, 1997) or a serotype (specific identification of S. enteritidis, Lampel et al, 1996 andof S. enteritidis andS. typhimurium, Soumet et al, 1999).…”

“…typhimurium, Soumet et al, 1999). Some of these PCR identification systems , Lampel et al, 1996, however, were not sufficiently validated on field strains and have to be seen more as an indication than as a real identification (Heyndrickx & Herman, personal communication) A lot of these virulence genes are located on plasmid DNA. When possible a chromosomal localisation is preferred because of the instability of plasmids during lab manipulations, This is seen for plasmid bearing Yersinia enterocolitica, where loss of the virulence plasmid during enrichment and isolation complicates the detection of the pathogen in food products (Bhaduri & Cottrell, 1997).…”