Simultaneous quantification of pathogenic Campylobacter and Salmonella in chicken rinse fluid by a flotation and real-time multiplex PCR procedure.
A procedure for simultaneous quantification of Campylobacter and Salmonella spp. in poultry skin rinse fluids by a flotation and real-time multiplex PCR method is described. Flotation of the target organisms in a discontinuous density gradient separated them from background microflora, particles from poultry skin, dead target cells and PCR inhibitors. Variation of the buoyant density between 1.052 to 1.106 g/ml was measured at different times for various Salmonella strains grown over a period of 4 weeks. This, and the results from earlier studies on the buoyant densities of Campylobacter spp., which were between 1.065 and 1.109 g/ml, led to design of an optimal discontinuous flotation method with three density layers, of 1.048, 1.109 and approximately 1.200 g/ml. This method preceded a real-time multiplex PCR assay using hybridization probes. The specificity of the PCR assay was confirmed on 73 target and non-target strains, and target organisms were detected at the level of one genome per PCR. Results obtained with the combined flotation and real-time multiplex PCR method showed that quantification in rinse fluids was possible down to 3.0+/-0.3 x 10(3) CFU/ml in the presence of other microorganisms at numbers up to 10(9) CFU/ml.
Wolffs
Petra F G
PF
Canadian Research Institute for Food Safety, Guelph University, Guelph, Canada. pwolf@lmib.azm.nl
Glencross
Kari
K
Norling
Börje
B
Griffiths
Mansel W
MW
D000818:Animals; D002167:Campylobacter / Q000302:isolation & purification*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D002645:Chickens / Q000382:microbiology*; D015169:Colony Count, Microbial / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis; D005506:Food Contamination / Q000032:analysis; D005511:Food Handling / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D012867:Skin / Q000382:microbiology
D004269:DNA, Bacterial