P594 Comparison of real-time PCR and direct culture for the detection of Campylobacter spp. from human faecal samples

Safe water is a global concern, and methods to accurately monitor quality of water are vital. To assess the risks related to bacterial pathogen load in Lake Vomb that provides drinking water to the southern part of Sweden, this study combined molecular analyses of enterobacteria and bacterial pathogens in water using quantitiative real-time PCR with hydrodynamic modeling and quantitative microbial risk assessment (QMRA). A real-time PCR assay to detect enterobacteria was set up by primers targeting ssrA. Between February 2015 and May 2016, presence of ssrA gene copies as well as Campylobacter spp., Salmonella spp., and EHEC O157 DNA was analyzed by real-time PCR at several locations in the catchment of Lake Vomb and its tributaries Björkaån, Borstbäcken, and Torpsbäcken. Björkaån had the highest detected concentrations of the ssrA gene and, according to the results of hydrodynamic modeling, contributed most to the contamination of the water intake in the lake. None of the water samples were positive for genes encoding EHEC O157 and Campylobacter spp., while invA (Salmonella spp.) was present in 11 samples. The QMRA showed that the suggested acceptable risk level (daily probability of infection <2.7 × 10−7) is achieved with a 95% probability, if the Salmonella concentrations in the water intake are below 101 bacteria/100 mL. If a UV-disinfection step is installed, the Salmonella concentration at the water intake should not exceed 106 bacteria/100 mL.

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Strategies for the inclusion of an internal amplification control in conventional and real time PCR detection of Campylobacter spp. in chicken fecal samples

Molecular and Cellular Probes ◽

10.1016/j.mcp.2005.10.002 ◽

2006 ◽

Vol 20 (2) ◽

pp. 92-99 ◽

Cited By ~ 27

Author(s):

M. Lund ◽

M. Madsen

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pcr Detection ◽

Internal Amplification Control ◽

Fecal Samples ◽

Campylobacter Spp

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Rapid diagnosis of toxinogenic Clostridium difficile in faecal samples with internally controlled real-time PCR

Clinical Microbiology and Infection ◽

10.1111/j.1469-0691.2005.01301.x ◽

2006 ◽

Vol 12 (2) ◽

pp. 184-186 ◽

Cited By ~ 41

Author(s):

R.J. van den Berg ◽

E.J. Kuijper ◽

L.E.S. Bruijnesteijn van Coppenraet ◽

E.C.J. Claas

Keyword(s):

Clostridium Difficile ◽

Real Time ◽

Real Time Pcr ◽

Rapid Diagnosis ◽

Faecal Samples

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Isolation and Identification of Campylobacter spp. from Poultry and Poultry By-Products in Tunisia by Conventional Culture Method and Multiplex Real-Time PCR

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-321 ◽

2017 ◽

Vol 80 (10) ◽

pp. 1623-1627 ◽

Cited By ~ 3

Author(s):

Hela Jribi ◽

Hanen Sellami ◽

Siala Mariam ◽

Salma Smaoui ◽

Asma Ghorbel ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Poultry Meat ◽

Culture Methods ◽

Isolation And Identification ◽

Conventional Culture ◽

Multiplex Pcr Assay ◽

Thermophilic Campylobacter ◽

By Products ◽

Campylobacter Spp

ABSTRACT Thermophilic Campylobacter spp. are one of the primary causes of bacterial human diarrhea. The consumption of poultry meats, by-products, or both is suspected to be a major cause of human campylobacteriosis. The aims of this study were to determine the prevalence of thermophilic Campylobacter spp. in fresh poultry meat and poultry by-products by conventional culture methods and to confirm Campylobacter jejuni and Campylobacter coli isolates by using the multiplex PCR assay. Two hundred fifty fresh poultry samples were collected from a variety of supermarkets and slaughterhouses located in Sfax, Tunisia, including chicken (n =149) and turkey (n =101). The samples were analyzed using conventional microbiological examinations according to the 2006 International Organization for Standardization method (ISO 10272-1) for Campylobacter spp. Concurrently, a real-time PCR was used for identification of C. jejuni and C. coli. Of the 250 samples of poultry meat and poultry by-products, 25.6% (n = 64) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (26.8%) followed by turkey meat (23.7%). Among the different products, poultry breasts showed the highest contamination (36.6%) followed by poultry by-products (30%), poultry wings (28%) and poultry legs (26%) showed the lowest contamination, and no contamination was found on neck skin. Of the 64 thermophilic Campylobacter isolates, C. jejuni (59.7%) was the most frequently isolated species and 10.9% of the isolates were identified as C. coli. All of the 64 Campylobacter isolates identified by the conventional culture methods were further confirmed by PCR. The seasonal peak of Campylobacter spp. contamination was in the warm seasons (spring and summer). The study concluded that high proportions of poultry meat and poultry by-products marketed in Tunisia are contaminated by Campylobacter spp. Furthermore, to ensure food safety, poultry meats must be properly cooked before consuming.

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Detection and quantification of Campylobacter in foods: New analytic approaches to detect and quantify Campylobacter spp. in food samples

Italian Journal of Food Safety ◽

10.4081/ijfs.2020.8591 ◽

2020 ◽

Vol 9 (2) ◽

Author(s):

Maria Francesca Peruzy ◽

Yolande Thérèse Rose Proroga ◽

Federico Capuano ◽

Federica Corrado ◽

Serena Santonicola ◽

...

Keyword(s):

Real Time ◽

Campylobacter Jejuni ◽

Internal Control ◽

Real Time Pcr ◽

Quantitative Methods ◽

Bacterial Load ◽

Digital Pcr ◽

Food Samples ◽

Meat Samples ◽

Campylobacter Spp

The aim of the present study was to develop rapid qualitative and quantitative methods based on the use of Real-Time PCR and Droplet Digital PCR (ddPCR), in order to have reliable techniques to detect and quantify Campylobacter spp. in food samples. The gene 16S-rRNA was used as specific target for Campylobacter spp. Real- Time PCR evaluation assay and a not competitive internal control was ushered in it. To investigate the selectivity of the method, 26 Campylobacter strains and 40 non-Campylobacter strains were tested and in order to verify the application of Real-Time PCR method, 5 pork meat samples were experimentally inoculated with a Campylobacter jejuni strain. Subsequently, dilutions with a bacterial load of Campylobacter jejuni within 10-106 CFU/mL were chosen for the optimization of the ddPCR assay. Lastly, a total of 54 naturally contaminated foods samples were analyzed through molecular (Real-Time PCR and ddPCR) and traditional methods. The Real-Time PCR protocol demonstrated to amplify only the Campylobacter spp. strains and when Campylobacter jejuni was experimentally inoculated in meat samples the pathogen was always detected. The ddPCRs assay allowed to quantify a level of contamination of 10 CFU/mL, but it was unable to quantify levels of 105 – 106 CFU/mL. Lastly, Campylobacter spp. was never detected in the 54 samples tested. In conclusion, the novel analytic approach proposed, based on an initial screening of the samples with Real-Time PCR and then on quantification of Campylobacter spp. with a ddPCR on those positive, represents a quick monitoring tool and, if used correctly, it would allow the implementation of food safety.

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Quantification of Campylobacter spp. in Chicken Rinse Samples by Using Flotation prior to Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.5759-5764.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 5759-5764 ◽

Cited By ~ 27

Author(s):

Petra Wolffs ◽

Börje Norling ◽

Jeffrey Hoorfar ◽

Mansel Griffiths ◽

Peter Rådström

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Quantitative Data ◽

Buoyant Density ◽

Tetrazolium Chloride ◽

Viable Cells ◽

Flotation Treatment ◽

Flotation Method ◽

Cell Concentrations ◽

Campylobacter Spp

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 × 102 CFU/ml could be detected without culture enrichment and amounts as low as 2.6 × 103 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.

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