Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species.
OBJECTIVE
Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples.


RESULTS
Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria.


CONCLUSIONS
Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples.


CONCLUSIONS
This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.
Ziemer
C J
CJ
Swine Odor and Manure Management Research Unit, National Swine Research and Information Center, ARS, USDA, Ames, IA 50011, USA. ziemer@nsric.ars.usda.gov
Steadham
S R
SR
D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D026923:Amino Acid Transport Systems, Basic / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D017931:DNA Primers; D004275:DNA, Ribosomal / Q000235:genetics; D004768:Enterotoxins / Q000235:genetics; D005798:Genes, Bacterial; D007422:Intestines / Q000382:microbiology*; D008565:Membrane Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity; D012680:Sensitivity and Specificity; D015534:Trans-Activators / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D017931:DNA Primers; D004275:DNA, Ribosomal; D004768:Enterotoxins; C102236:HilA protein, Salmonella; D008565:Membrane Proteins; C094578:Salmonella invasion protein C; D015534:Trans-Activators; D037521:Virulence Factors; C082864:enterotoxin, Salmonella; C094579:invasion protein B, Salmonella typhimurium; C075518:invA protein, Bacteria; C043828:histidine permease, Bacteria