title: Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment.
abstract: A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (C(T)) values (R(2) = 0.993), with a quantification range of 1 x 10(2) to 1 x 10(7) CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R(2) = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment.
authors.affiliation: National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, 2860 Søborg, Denmark. mhjo@food.dtu.dk
authors.forename: M H
authors.initials: MH
authors.lastname: Josefsen
authors.forename: C
authors.initials: C
authors.lastname: Löfström
authors.forename: T B
authors.initials: TB
authors.lastname: Hansen
authors.forename: L S
authors.initials: LS
authors.lastname: Christensen
authors.forename: J E
authors.initials: JE
authors.lastname: Olsen
authors.forename: J
authors.initials: J
authors.lastname: Hoorfar
mesh_terms: D000818:Animals; D001386:Azides / Q000378:metabolism*; D002167:Campylobacter / Q000302:isolation & purification*; D002645:Chickens / Q000382:microbiology*; D015169:Colony Count, Microbial / Q000379:methods; D050296:Microbial Viability; D016133:Polymerase Chain Reaction / Q000379:methods*; D011419:Propidium / Q000031:analogs & derivatives* / Q000378:metabolism; D018570:Risk Assessment / Q000379:methods; D013997:Time Factors
chemical_list: D001386:Azides; C533957:propidium monoazide; D011419:Propidium