Quantification of Campylobacter spp. in chicken rinse samples by using flotation prior to real-time PCR

Abstract

Real-time PCR is fast, sensitive, specific, and can deliver quantitative data; however, two disadvantages are that this technology is sensitive to inhibition by food and that it does not distinguish between DNA originating from viable, viable nonculturable (VNC), and dead cells. For this reason, real-time PCR has been combined with a novel discontinuous buoyant density gradient method, called flotation, in order to allow detection of only viable and VNC cells of thermotolerant campylobacters in chicken rinse samples. Studying the buoyant densities of different Campylobacter spp. showed that densities changed at different time points during growth; however, all varied between 1.065 and 1.109 g/ml. These data were then used to develop a flotation assay. Results showed that after flotation and real-time PCR, cell concentrations as low as 8.6 x 10(2) CFU/ml could be detected without culture enrichment and amounts as low as 2.6 x 10(3) CFU/ml could be quantified. Furthermore, subjecting viable cells and dead cells to flotation showed that viable cells were recovered after flotation treatment but that dead cells and/or their DNA was not detected. Also, when samples containing VNC cells mixed with dead cells were treated with flotation after storage at 4 or 20 degrees C for 21 days, a similar percentage resembling the VNC cell fraction was detected using real-time PCR and 5-cyano-2,3-ditolyl tetrazolium chloride-4',6'-diamidino-2-phenylindole staining (20% +/- 9% and 23% +/- 4%, respectively, at 4 degrees C; 11% +/- 4% and 10% +/- 2%, respectively, at 20 degrees C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method.

FIG. 1.

Changes in buoyant densities of three C. jejuni strains during a period of 14 days. (A) C. jejuni CCUG 10937; (B) C. jejuni ATCC 33291; (C) C. jejuni ATCC 29428. ▪, cell counts observed over time (y axis). The vertical lines represent the buoyant density window (z axis). *, faint bands due to low cell densities. The horizontal dotted lines indicate the limits between the two upper flotation solutions. To concentrate the target at the Campylobacter recovery location, the density has to fall between these two marked densities.

FIG. 2.

Overview of the flotation setup. This setup is used to recover viable and VNC cells with buoyant densities of 1.065 and 1.109 g/ml. The Campylobacter recovery location is at the upper interface. The buoyant densities of the different colloidal solutions used in the two setups are shown. *, the density of the lowest layer may vary slightly because it consists of sample mixed with colloidal silica solution. Since the density of the sample may vary, this can impact the final buoyant density of this layer.

FIG. 3.

Standard curve for hybridization probe Campylobacter real-time PCR assay. Data are the results from duplicate analyses.