Real-time PCR detection of Salmonella species using a novel target: the outer membrane porin F gene (ompF)

Abstract

Aims: To evaluate the outer membrane porin F gene (ompF) for the specific detection of Salmonella species by real‐time PCR assay. Methods and Results: Two hundred and eighteen isolates belonging to Salmonella enterica (subspecies I‐VI) and Salmonella bongori were examined using primers designed to detect the ompF gene. The DNA of the bacteria was extracted from pure culture. The target was present in all the 218 Salmonella isolates including all the subspecies of Salm. enterica and Salm. bongori. The ompF gene was absent in 180 non‐Salmonella strains tested. The limit of detection was determined to be three colony forming units per reaction in pure culture. In artificially contaminated food experiments with ten or less colony forming units per 25 g, the assay was successful in identifying the target in 100% of the samples after 22‐ to 24‐h incubation in enrichment broth. Conclusions: Based on this study, the ompF gene is 100% inclusive for Salmonella species and 100% exclusive for non‐Salmonella species for the strains tested. Significance and Impact of the Study: ompF gene was present in all the Salmonella strains tested and has the potential to be a good target for the rapid molecular identification of Salmonella.

Longitudinal prevalence, faecal shedding and molecular characterisation of Campylobacter spp. and Salmonella enterica in sheep

“…16S rRNA gene (287 bp 179 amplicon) using primers and PCR conditions described by Lubeck et al (2003). A subset of five 180 samples that were positive for S. enterica from each sampling on each farm (n = 120) were 181 subjected to PCR for the S. enterica ompF gene (578 bp amplicon) using primers and PCR 182 conditions described by Tatavarthy and Cannons (2010). PCR products were separated by gel 183 electrophoresis and purified using an in-house filter tip method (Yang et al, 2013 The overall prevalences of Campylobacter spp.…”

Development of a novel multiplex PCR assay for the identification of Salmonella enterica Typhimurium and Enteritidis

“…Milk samples were confirmed to be Salmonella free by standard culture method (Anonymous, 2008) prior to testing. Ten milliliter milk samples, and 1 mL of culture with approximately N, N Â 10, N Â 10 2 Salmonella cells (1 < N < 10), were added to 89 mL LB broth, and then incubated at 37 C for 12 h. DNA was extracted by the boiling method (Tatavarthy & Cannons, 2010) from 1 mL of the enrichment as a template for multiplex PCR. Results of the multiplex PCR assay were verified by standard culture method (Anonymous, 2008).…”

“…To determine the sensitivity, genomic DNA extracted by the phenol/chloroform method (Kalia et al, 1999) from S. Enteritidis (ATCC 13076) and S. Typhimurium (ATCC 14028) were serially diluted ten-fold with sterile ddH 2 O; overnight cultures of both strains were also serially diluted ten-fold to 10 À10 , respectively, and 1 mL of the dilution was used to extract DNA by the boiling method (Tatavarthy & Cannons, 2010). Finally, the templates were tested by the multiplex PCR in the presence of IAC.…”

“…Salmonella enterica, a common foodborne pathogen, causes gastroenteritis and septicemia in humans and even may lead to death (Alvarez et al, 2004;Tatavarthy & Cannons, 2010). In China, it is estimated that approximately 70%e80% of foodborne bacterial outbreaks are caused by Salmonella (Yang et al, 2010).…”

Antibiotic resistant Shigella is a major cause of diarrhoea in the Highlands of Papua New Guinea

“…Suspected isolates of Shigella and Salmonella were confirmed by real-time PCR [14][15][16] and polyvalent antisera when available (Remel, Thermo Scientific, Scoresby, Australia).…”