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 ؋ 10 2 CFU/ml could be detected without culture enrichment and amounts as low as 2.6 ؋ 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°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°C; 11% ؎ 4% and 10% ؎ 2%, respectively, at 20°C). This indicated that viable and VNC Campylobacter cells could be positively selected and quantified using the flotation method.

Estimation of Viable Salmonella Cell Numbers in Meat and Meat Product Using Real-Time PCR

“…The correlation coe$cients were all over 0.999. This coincidence among the standard curves is considered to be a result of the introduction of the clean-up procedure for the food samples 8) and the selectivity of EEM 7) . Here the ground beef and pork and the hamburger patty were initially contaminated at the aerobic bacterial count levels of 10 6.9 , 10 7.9 , and 10 4.7 CFU/g, respectively.…”

“…To suppress inhibition of PCR due to food constituents and microbial contaminants, we introduced a buoyant density centrifugation procedure 8) and then incubated the samples in a selective medium for Salmonella 7) . Further, we studied Salmonella growth in raw, ground chicken stored at a constant temperature.…”

Rapid Separation and Concentration of Food-Borne Pathogens in Food Samples Prior to Quantification by Viable-Cell Counting and Real-Time PCR

“…The benefits of BDC as a sample pretreatment method are well established and include (i) the possibility of separating biological matrix particles and microorganisms with different buoyant densities (12); (ii) elimination of parts of the PCR-inhibiting food substances (10); (iii) prevention of false-positive results due to DNA originating from dead cells, which has limited the use of quantitative PCR (qPCR) (22); (iv) the possibility of direct quantification of target organisms in the presence of a large background flora (22); (v) maintenance of cell viability, which allows isolation and analysis of the microorganisms (18); and (vi) speed and easy handling. However, even PCR methods after BDC detect at best 10 3 to 10 4 CFU/g of target pathogens (11,12,22,23). In this study we examined the use of a combination of several sample preparation methods, including filtration, lowspeed centrifugation, high-speed centrifugation, and finally two BDC methods, called (i) flotation, in which the top layer consists of a low-density solution and the bottom layer consists of a high-density solution of density gradient medium mixed with the sample, and (ii) sedimentation, in which density gradient solutions are rapidly and easily prepared without contamination from other food matrix in tubes.…”

“…However, there is still a lack of suitable separation and concentration methods that allow rapid quantification of the nucleic acids and removal of PCR inhibitors. Buoyant density centrifugation (BDC) has been used for rapid detection of food pathogens, such as Shigella flexneri (13), Escherichia coli (12), and Yersinia enterocolitica (10,11,13), by a sedimentation method and for rapid detection of Y. enterocolitica (22) and Campylobacter jejuni (23) by the flotation method. The benefits of BDC as a sample pretreatment method are well established and include (i) the possibility of separating biological matrix particles and microorganisms with different buoyant densities (12); (ii) elimination of parts of the PCR-inhibiting food substances (10); (iii) prevention of false-positive results due to DNA originating from dead cells, which has limited the use of quantitative PCR (qPCR) (22); (iv) the possibility of direct quantification of target organisms in the presence of a large background flora (22); (v) maintenance of cell viability, which allows isolation and analysis of the microorganisms (18); and (vi) speed and easy handling.…”

Desenvolvimento de métodos para a quantificação direta de Salmonella sp. por PCR-tempo real e por transcriptase reversa-PCR-tempo real

“…Para evitar este tipo de erro, técnicas diferentes têm sido sugeridas, como o emprego de flotation -uma variante da centrifugação por gradiente de densidade (Wolffs et al, , 2005a, ou o emprego de corante etídeo-monoazida (EMA). EMA pode entrar seletivamente nas células cuja membrana esteja danificada e ligar-se covalentemente ao DNA, inibindo a PCR (Nocker et al, 2006;Nogva et al, 2003).…”