Real-time PCR method for Salmonella spp. targeting the stn gene.
OBJECTIVE
To develop a real-time PCR assay for Salmonella spp. targeting the stn gene.


RESULTS
The presence of stn in the Salmonella bongori genome was found by a BLAST with Salmonella enterica stn sequence. Manual alignment of stn sequences showed that Salm. bongori had 88% sequence identity with Salm. enterica. Two primers (stnL-433 and stnR-561) and a probe (stnP-452) were designed to target conserved regions in stn and meet the requirements of a 5'-nuclease assay. The primers and probe were evaluated against 353 isolates, including 255 Salm. enterica representing 158 serotypes, 14 Salm. bongori representing 12 serotypes and 84 non-Salmonella representing 56 species from 31 genera. All isolates were correctly identified, with the exception of three isolates of Citrobacter amalonaticus, which gave false positives. The limit of detection with cultured Salmonella was 3 CFU per reaction.


CONCLUSIONS
The stn real-time PCR method had 100% inclusivity, 96.4% exclusivity and a level of detection of 3 CFU per reaction for cultured Salmonella spp.


CONCLUSIONS
The study showed that stn is present in Salm. bongori and is a valid target for both species of Salmonella. The Salmonella s tn real-time PCR is a useful method for identifying Salmonella spp.
Moore
M M
MM
Seafood Products Research Center, Pacific Regional Laboratory Northwest, US Food and Drug Administration, Bothell, WA 98021, USA. michelle.moore@fda.hhs.gov
Feist
M D
MD
D001483:Base Sequence; D015169:Colony Count, Microbial; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis*; D004768:Enterotoxins / Q000235:genetics*; D005516:Food Microbiology; D006801:Humans; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012475:Salmonella / Q000235:genetics*; D012480:Salmonella Infections / Q000382:microbiology*; D019779:Salmonella enterica / Q000235:genetics; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
D017931:DNA Primers; D004269:DNA, Bacterial; D004768:Enterotoxins; C082864:enterotoxin, Salmonella