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

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.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 microg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 microg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 microg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 x 10(2) to 1 x 10(5) genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 x 10(5) CFU/ml at 4, 20, and 37 degrees C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 degrees C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 degrees C to a minority of the mixed population compared to membrane damage.     

Rapid separation and concentration of food-borne pathogens in food samples prior to quantification by viable-cell counting and real-time PCR

Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low- and high-speed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Vibrio cholerae O139, Vibrio parahaemolyticus O3K6, Vibrio vulnificus, Providencia alcalifaciens, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 10(1) to 10(3) CFU/g using the RTi-qPCR assay, and amounts as small as 10(0) to 10(1) CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 10(1) to 10(2) CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak.     

Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR

AIMS: Surface contamination by Listeria monocytogenes of gouda-like cheeses during processing represents a potential public health problem. The aim of this work was to develop novel real-time PCR diagnostics to detect the presence of viable, dead or viable but not culturable (VBNC) cells on gouda-like cheeses. METHODS AND RESULTS: We used ethidium monoazide bromide (EMA)-PCR for direct quantification of viable and dead cells, while semiquantitative detection of culturable cells below the PCR detection limit (c. 100 CFU g(-1)) was obtained by combining growth and real-time PCR. We were able to quantify the fraction of >0.5% viable cells in a background of dead cells by EMA-PCR, given that the viable cell concentration was above the PCR detection limit. The combined growth and real-time PCR complemented the EMA-PCR, and enabled semiquantitative detection of low levels of culturable cells (10 and 100 CFU g(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: The significance of this work is that we have developed a novel concept for detection of viable and potentially infectious L. monocytogenes.     

Discrimination of viable and dead Vibrio vulnificus after refrigerated and frozen storage using EMA, sodium deoxycholate and real-time PCR

The effect of refrigerated and frozen storage on the viability of Vibrio vulnificus was evaluated using cell suspensions (1 × 10⁸ CFU/ml). Ethidium bromide monoazide (EMA) was utilized to selectively allow real-time (Rti) PCR amplification of target DNA from viable but not dead cells. Bacterial survivors from the EMA Rti-PCR were evaluated by comparison with the plate count assay following different temperature exposures (− 20 and 4 °C) every 24 h for 72 h. The log CFU values from the EMA Rti-PCR assays were erroneously higher than that from plate counts. DNA amplification was not completely suppressed by EMA treatment of low temperature destroyed cells suggesting that membrane damage was not sufficient to allow effective EMA penetration into the cells. The optimal concentration of sodium deoxycholate (SD) was also determined to enhance discrimination of viable and dead cells following exposure of cells to low temperatures. The use of 0.01% or less of SD did not inhibit the Rti-PCR amplification derived from viable bacterial cells. A rapid decrease of the log CFU was observed with cell suspensions subjected to frozen storage and a slow decline in the log CFU occurred at 4 °C. The combination of SD and EMA treatments applied to cells of V. vulnificus held at − 20 °C and 4 °C resulted in a high level of correlation between the log of CFU (plate counts) and the log of the number of viable cells determined from SD+EMA Rti-PCR.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 μg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 μg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 μg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 × 10² to 1 × 10⁵ genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 × 10⁵ CFU/ml at 4, 20, and 37 °C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 °C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 °C to a minority of the mixed population compared to membrane damage.     

Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log(10) was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.     

Use of Ethidium Monoazide and PCR in Combination for Quantification of Viable and Dead Cells in Complex Samples

The distinction between viable and dead cells is a major issue in many aspects of biological research. The current technologies for determining viable versus dead cells cannot readily be used for quantitative differentiation of specific cells in mixed populations. This is a serious limitation. We have solved this problem by developing a new concept with the viable/dead stain ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR). A dynamic range of approximately 4 log₁₀ was obtained for the EMA-PCR viable/dead assay. Viable/dead differentiation is obtained by covalent binding of EMA to DNA in dead cells by photoactivation. EMA penetrates only dead cells with compromised membrane/cell wall systems. DNA covalently bound to EMA cannot be PCR amplified. Thus, only DNA from viable cells can be detected. We evaluated EMA-PCR with the major food-borne bacterium Campylobacter jejuni as an example. Traditional diagnosis of this bacterium is very difficult due to its specific growth requirements and because it may enter a state where it is viable but not cultivable. The conditions analyzed included detection in mixed and natural samples, survival in food, and survival after disinfection or antibiotic treatment. We obtained reliable viable/dead quantifications for all conditions tested. Comparison with standard fluorescence-based viable/dead techniques showed that the EMA-PCR has a broader dynamic range and enables quantification in mixed and complex samples. In conclusion, EMA-PCR offers a novel real-time PCR method for quantitative distinction between viable and dead cells with potentially very wide application.     

Detection of Pasteurella multocida in experimentally infected embryonated chicken eggs by PCR assay

Applicability of polymerase chain reaction (PCR) assay to detect Pasteurella multocida in experimentally infected embryonated chicken egg was assessed in the present study. PCR assay rapidly and specifically detected the genome of P. multocida in amniotic fluid, allantoic fluid and homogenates of infected embryo and its membranes. The sensitivity of detection was as low as 20 bacterial cells/ml of allantoic or amniotic fluids. Detection of P. multocida in dead embryos by PCR was possible up to 6 and 30 days or more following storage of dead embryos at 37 degrees C, and at 4 degrees C as well as at -20 degrees C, respectively. The study revealed that PCR assays could be employed directly for detection and confirmation of P. multocida infection in experimentally infected chicken embryos.     

Development of a rapid and sensitive method combining a cellulose ester microfilter and a real-time quantitative PCR assay to detect Campylobacter jejuni and Campylobacter coli in 20 liters of drinking water or low-turbidity waters

Investigations of Campylobacter jejuni and Campylobacter coli in samples of drinking water suspected of being at the origin of an outbreak very often lead to negative results. One of the reasons for this failure is the small volume of water typically used for detecting these pathogens (10 to 1,000 ml). The efficiencies of three microfilters and different elution procedures were determined using real-time quantitative PCR to propose a procedure allowing detection of Campylobacter in 20 liters of drinking water or low-turbidity water samples. The results showed that more than 80% of the bacteria inoculated in 1 liter of drinking water were retained on each microfilter. An elution with a solution containing 3% beef extract, 0.05 M glycine at pH 9, combined with direct extraction of the bacterial genomes retained on the cellulose ester microfilter, allowed recovery of 87.3% (±22% [standard deviation]) of Campylobacter per 1 liter of tap water. Recoveries obtained from 20-liter volumes of tap water spiked with a C. coli strain were 69.5% (±10.3%) and 78.5% (±15.1%) for 91 CFU and 36 CFU, respectively. Finally, tests performed on eight samples of 20 liters of groundwater collected from an alluvial well used for the production of drinking water revealed the presence of C. jejuni and C. coli genomes, whereas no bacteria were detected with the normative culture method in volumes ranging from 10 to 1,000 ml. In the absence of available epidemiological data and information on bacterial viability, these last results indicate only that the water resource is not protected from contamination by Campylobacter.     

Concurrent quantitation of total campylobacter and total ciprofloxacin-resistant campylobacter loads in rinses from retail raw chicken carcasses from 2001 to 2003 by direct plating at 42 degrees C

This is the first report on the use of a normally lethal dose of ciprofloxacin in a Campylobacter agar medium to kill all ciprofloxacin-sensitive Campylobacter spp. but allow the selective isolation and quantitation of naturally occurring presumptive ciprofloxacin-resistant Campylobacter CFU in rinses from retail raw chicken carcasses (RTCC). Thermophilic-group total Campylobacter CFU and total ciprofloxacin-resistant Campylobacter CFU (irrespective of species) were concurrently quantified in rinses from RTCC by direct plating of centrifuged pellets from 10 or 50 ml out of 400-ml rinse subsamples concurrently on Campylobacter agar and ciprofloxacin-containing Campylobacter agar at 42 degrees C (detection limit = 0.90 log(10) CFU/carcass). For 2001, 2002, and 2003, countable Campylobacter CFU were recovered from 85%, 96%, and 57% of RTCC, while countable ciprofloxacin-resistant Campylobacter CFU were recovered from 60%, 59%, and 17.5% of RTCC, respectively. Total Campylobacter CFU loads in RTCC rinses ranged from 0.90 to 4.52, 0.90 to 4.58, and 0.90 to 4.48 log(10) CFU/carcass in 2001, 2002, and 2003, respectively. Total ciprofloxacin-resistant Campylobacter CFU loads in RTCC rinses ranged from 0.90 to 4.06, 0.90 to 3.95, and 0.90 to 3.04 log(10) CFU/carcass in 2001, 2002, and 2003, respectively. Overall, total Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, 4 to 5 log(10) CFU/carcass, respectively, were recovered from 16%, 32%, 26%, and 5% of RTCC tested over the 2-year sampling period. For the same period, total ciprofloxacin-resistant Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, and 4 to 5 log(10) CFU/carcass, respectively, were recovered from 24%, 11%, 7%, and 0.2% of RTCC tested. There was a steady decline in total Campylobacter and total ciprofloxacin-resistant Campylobacter loads in RTCC rinses from 2001/2002 to 2003.     

An advanced PCR method for the specific detection of viable total coliform bacteria in pasteurized milk

Pasteurized milk is a complex food that contains various inhibitors of polymerase chain reaction (PCR) and may contain a large number of dead bacteria, depending on the milking conditions and environment. Ethidium monoazide bromide (EMA)-PCR is occasionally used to distinguish between viable and dead bacteria in foods other than pasteurized milk. EMA is a DNA-intercalating dye that selectively permeates the compromised cell membranes of dead bacteria and cleaves DNA. Usually, EMA-PCR techniques reduce the detection of dead bacteria by up to 3.5 logs compared with techniques that do not use EMA. However, this difference may still be insufficient to suppress the amplification of DNA from dead Gram-negative bacteria (e.g., total coliform bacteria) if they are present in pasteurized milk in large numbers. Thus, false positives may result. We developed a new method that uses real-time PCR targeting of a long DNA template (16S-23S rRNA gene, principally 2,451?bp) following EMA treatment to completely suppress the amplification of DNA of up to 7?logs (10(7)?cells) of dead total coliforms. Furthermore, we found that a low dose of proteinase K (25?U/ml) removed PCR inhibitors and simultaneously increased the signal from viable coliform bacteria. In conclusion, our simple protocol specifically detects viable total coliforms in pasteurized milk at an initial count of ≥1?colony forming unit (CFU)/2.22?ml within 7.5?h of total testing time. This detection limit for viable cells complies with the requirements for the analysis of total coliforms in pasteurized milk set by the Japanese Sanitation Act (which specifies <1?CFU/2.22?ml).     

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

Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low- and high-speed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Vibrio cholerae O139, Vibrio parahaemolyticus O3K6, Vibrio vulnificus, Providencia alcalifaciens, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 10¹ to 10³ CFU/g using the RTi-qPCR assay, and amounts as small as 10⁰ to 10¹ CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 10¹ to 10² CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak.     

基于EMA-qPCR的茄科青枯菌活体检测技术的建立

【目的】利用特异性核酸染料叠氮溴乙锭(Ethidium monoazide bromide, EMA)与实时荧光定量PCR技术相结合, 建立一种能有效区分青枯菌死活细胞的检测方法。【方法】样品DNA制备前经EMA渗透预处理, 再进行实时荧光定量PCR特异扩增菌体DNA。【结果】终浓度为2.0 mg/L的EMA能有效排除1.0×107 CFU/mL灭活青枯菌细胞DNA的扩增, 对活细胞和不可培养状态(Viable but non-culturable, VBNC)活菌的DNA扩增均没有影响。当每个定量PCR反应体系中的活细胞在5.0×100?5.0×104 CFU范围内时, 扩增Ct值与定量PCR反应体系中活细胞CFU对数值呈良好的负相关性(R2=0.992 5)。比较EMA-qPCR法和平板计数法对经过不同温度短期保存的青枯菌检测结果发现, 待检样品可在24 °C与4 °C冷藏条件下短期保存。【结论】本研究建立的EMA-qPCR方法能有效检测青枯菌VBNC细胞和有效区分死活菌, 避免或减少青枯菌PCR检测的假阳性和假阴性。     

Bactericidal effect of solar water disinfection under real sunlight conditions

Batch solar disinfection (SODIS) inactivation kinetics are reported for suspensions in water of Campylobacter jejuni, Yersinia enterocolitica, enteropathogenic Escherichia coli, Staphylococcus epidermidis, and endospores of Bacillus subtilis, exposed to strong natural sunlight in Spain and Bolivia. The exposure time required for complete inactivation (at least 4-log-unit reduction and below the limit of detection, 17 CFU/ml) under conditions of strong natural sunlight (maximum global irradiance, approximately 1,050 W m(-2) +/- 10 W m(-2)) was as follows: C. jejuni, 20 min; S. epidermidis, 45 min; enteropathogenic E. coli, 90 min; Y. enterocolitica, 150 min. Following incomplete inactivation of B. subtilis endospores after the first day, reexposure of these samples on the following day found that 4% (standard error, 3%) of the endospores remained viable after a cumulative exposure time of 16 h of strong natural sunlight. SODIS is shown to be effective against the vegetative cells of a number of emerging waterborne pathogens; however, bacterial species which are spore forming may survive this intervention process.     

Development of a quantitative competitive PCR assay for detection and quantification of Escherichia coli O157:H7 cells.

A quantitative competitive PCR (QC-PCR) assay was developed to detect and quantify Escherichia coli O157:H7 cells. From 10(3) to 10(8) CFU of E. coli O157:H7 cells/ml was quantified in broth or skim milk, and cell densities predicted by QC-PCR were highly related to viable cell counts (r(2) = 0.99 and 0.93, respectively). QC-PCR has potential for quantitative detection of pathogenic bacteria in foods.     

Specific Detection of Viable Legionella Cells by Combined Use of Photoactivated Ethidium Monoazide and PCR/Real-Time PCR

Legionella organisms are prevalent in manmade water systems and cause legionellosis in humans. A rapid detection method for viable Legionella cells combining ethidium monoazide (EMA) and PCR/real-time PCR was assessed. EMA could specifically intercalate and cleave the genomic DNA of heat- and chlorine-treated dead Legionella cells. The EMA-PCR assay clearly showed an amplified fragment specific for Legionella DNA from viable cells, but it could not do so for DNA from dead cells. The number of EMA-treated dead Legionella cells estimated by real-time PCR exhibited a 10⁴- to 10⁵-fold decrease compared to the number of dead Legionella cells without EMA treatment. Conversely, no significant difference in the numbers of EMA-treated and untreated viable Legionella cells was detected by the real-time PCR assay. The combined assay was also confirmed to be useful for specific detection of culturable Legionella cells from water samples obtained from spas. Therefore, the combined use of EMA and PCR/real-time PCR detects viable Legionella cells rapidly and specifically and may be useful in environmental surveillance for Legionella.     

Rapid and quantitative detection of blood Serratia marcescens by a real-time PCR assay: its clinical application and evaluation in a mouse infection model

Large-scale nosocomial outbreaks of Serratia marcescens septicaemia in Japan have had a fatality rate of 20-60% within 48 h. As a countermeasure, a real-time PCR assay was constructed for the rapid diagnosis of S. marcescens septicaemia. This assay indeed detected S. marcescens in clinical blood specimens (at ca. 10(2)CFU ml(-1)), at a frequency of 0.5% in suspected cases of septicaemia. In mice, the assay provided estimates of blood S. marcescens levels at various infectious stages: namely, 10(7) to 10(8)CFU ml(-1) at a fatal stage (resulting in 100% death), 10(4)-10(5)CFU ml(-1) at a moderately fatal stage (resulting in 50% or more death), and <10(3)CFU ml(-1) at a mild stage (resulting in 100% survival), consistent with actual CFU measurements. Blood bacterial levels could be an important clinical marker that reflects the severity of septicaemia. The simultaneous detection of S. marcescens and the carbapenem resistance gene was also demonstrated.     

Use of PCR for direct detection of Campylobacter species in bovine feces

This study reports on the use of PCR to directly detect and distinguish Campylobacter species in bovine feces without enrichment. Inhibitors present in feces are a major obstacle to using PCR to detect microorganisms. The QIAamp DNA stool minikit was found to be an efficacious extraction method, as determined by the positive amplification of internal control DNA added to bovine feces before extraction. With nested or seminested multiplex PCR, Campylobacter coli, C. fetus, C. hyointestinalis, and C. jejuni were detected in all fecal samples inoculated at approximately 10(4) CFU g(-1), and 50 to 83% of the samples inoculated at approximately 10(3) CFU g(-1) were positive. At approximately 10(2) CFU g(-1), C. fetus, C. hyointestinalis, and C. jejuni (17 to 50% of the samples) but not C. coli were detected by PCR. From uninoculated bovine feces, a total of 198 arbitrarily selected isolates of Campylobacter were recovered on four commonly used isolation media incubated at three temperatures. The most frequently isolated taxa were C. jejuni (152 isolates) and C. lanienae (42 isolates), but isolates of C. fetus subsp. fetus, Arcobacter butzleri, and A. skirrowii also were recovered (相似文献