Detection of Campylobacter jejuni in naturally contaminated chicken skin by melting peak analysis of amplicons in real-time PCR

Contamination of poultry by Campylobacter spp. is a significant source of human diarrheal diseases. Traditional methods currently used to detect Campylobacter in foods are time-consuming and labor-intensive. In this study, primers designed for the Campylobacter jejuni cadF gene sequence were used in a SYBR Green I real-time PCR assay as an alternative to a conventional bacteriological method for the rapid detection of C. jejuni from poultry. Twelve portions of chicken purchased from two local grocery stores and 39 portions obtained from a commercial processing plant were examined. Samples of the skin were enriched in Bolton broth at 37 degrees C for 3 h and then at 42 degrees C for 9, 21, or 45 h under microaerobic conditions. DNA was extracted from 1-ml aliquots of the enrichment cultures using 1% Triton X-100. The DNA was used as the template in a real-time polymerase chain reaction (PCR) assay. After 24 h of enrichment, C. jejuni was isolated from 13 samples and all of the positive cultures were also detected by the real-time PCR procedure. C. jejuni was detected by both methods from samples artificially contaminated with 1 or 10 CFU of C. jejuni per 10 g, after 24 h of enrichment. The real-time PCR method was found to be sensitive and specific. It significantly reduced the time required for the detection of C. jejuni in poultry following enrichment of samples.     

Detection of thermophilic Campylobacter spp. in blood-free enriched samples of inoculated foods by the polymerase chain reaction

The detection of thermophilic Campylobacter spp., as represented by Campylobacter jejuni, by the polymerase chain reaction (PCR) was investigated and compared with the selective agar isolation (SAI) method. Stationary-phase cultures of C. jejuni were inoculated into either blood-free enrichment broth (BFEB) or BFEB that contained 10% broccoli, crabmeat, mushroom, raw milk, and raw oyster rinses. Following a 48-h enrichment period, aliquots of food test portions were removed for simultaneous analysis by PCR and SAI. It was determined that the presence of charcoal and iron in the enrichment broth interfered with the PCR assay. Therefore, three DNA extraction techniques were developed and evaluated using a 16S rRNA primer pair in the PCR assay. The 50% end point (DL50) values (determined upon six initial C. jejuni spiking levels) were used to assess the frequency of isolation utilizing PCR versus SAI for the detection of this organism in the enrichment matrices. There were virtually no differences in detection of C. jejuni among enriched samples analyzed by PCR and SAI. Mean DL50 values (n = 3) for plain BFEB, broccoli, crabmeat, mushroom, raw milk, and raw oyster were, respectively, 0.02 (PCR) versus 0.01 (SAI), 0.01 versus 0.06, 0.07 versus 0.04, 0.03 versus 0.08, 0.01 versus 0.01, and 0.01 versus 0.01 CFU/5 g food. Significant variability in the detection limit of C. jejuni by PCR in the food enrichments was observed among DNA extraction techniques. Using 48-h enrichment cultures followed by PCR analysis could save 1 day of the time required for the presumptive identification of C. jejuni in suspected foods.     

Detection of Campylobacter jejuni and Campylobacter coli in foods by enrichment culture and polymerase chain reaction enzyme-linked immunosorbent assay

A polymerase chain reaction (PCR) assay based on a solution hybridization format with colorimetric end-point detection (PCR ELISA) was investigated for the specific detection of Campylobacter jejuni and Campylobacter coli in food samples following enrichment culture. One hundred fifteen samples of raw meat and offal (poultry, porcine, ovine, and bovine), raw shellfish, and artificially contaminated milk were enriched in blood-free Campylobacter Enrichment Broth for 48 h. Enrichment cultures were subcultured to Campylobacter blood-free selective agar plates, and presumptive isolates were identified by phenotypic methods. DNA was extracted from 1-ml aliquots of the enrichment cultures using a rapid extraction method, and the DNA was used as the template in a PCR ELISA. A comparison of the PCR ELISA with the enrichment culture and subculture to selective agar method showed that the results of 112 of the 115 samples tested were in agreement by both methods. Seventy-one of the various food samples were positive in the PCR ELISA, and 70 samples were positive by culture. The PCR ELISA had a sensitivity of 99% and a specificity of 96%, with a positive predictive value of 97% and a negative predictive value of 98%. The PCR ELISA is a rapid, sensitive, and specific method for the detection of C. jejuni and C. coli in foods following enrichment culture and significantly reduces the time required for their detection.     

Evaluation of selective enrichment PCR procedures for Yersinia enterocolitica

Four enrichment PCR protocols for detecting unlysed cells of pathogenic Yersinia enterocolitica were studied. First, the probability of detecting Y. enterocolitica cells of known concentrations by a multiplex PCR assay was determined, and it was found to follow a logistic regression model. From this model, the probability of detecting Y enterocolitica at a specific concentration could be estimated; for example, the detection probability of 10(4) CFU/ml was estimated to be 85.4%. The protocols were evaluated on enrichment cultures inoculated with 10(2) CFU/ml Y. enterocolitica and 10(2)-10(6) CFU/ml of a defined background flora. For each protocol, the time for sample withdrawal and the presence of background flora were studied with respect to PCR detection. The optimal point in time of sample withdrawal was found to be different for each protocol employed. Early detection was favoured by concentrating the target cells, and the most rapid PCR detection of Y. enterocolitica was achieved with enrichment in Yersinia-PCR-compatible-enrichment (YPCE) medium for 3 h at 25 degrees C, followed by a centrifugation prior to PCR analysis. For detection of Y. enterocolitica in the presence of high concentrations (10(6) CFU/ml) of background flora, a long incubation time followed by density centrifugation and a dilution step was most successful. The protocol that gave the most reliable PCR detection in the presence of 10(6) CFU/ml background flora included 24 h incubation in Yersinia-selective-enrichment (YSE) broth at 25 degrees C, followed by Percoll density centrifugation, and a 100 times dilution prior to PCR analysis.     

Improved PCR detection of Campylobacter jejuni from chicken rinses by a simple sample preparation procedure

Many food samples and enrichment media are inhibitory to the PCR, thereby lowering its detection capacity. A simple sample preparation method based on buoyant density centrifugation was examined for its application in PCR detection of Campylobacter jejuni from chicken rinse samples. Bacterial cells were spiked at different levels in a mixture of Preston broth and chicken rinse (4:1 ratio) and 0.9 ml of these mixtures were layered over 0.6 ml of gradient medium made from Percoll. PCR sensitivity for bacterial samples treated with this procedure was approximately 10-100 times higher than for samples without treatment. This sample preparation method allowed for the detection of C. jejuni from 26 of 31 naturally contaminated chicken samples after a 20-24-h enrichment period in Preston broth, compared with only 14 positives for untreated samples. In addition, the effect of Oxyrase on the growth and PCR detection of C. jejuni was examined. While Oxyrase significantly enhanced the growth and the PCR signals of C. jejuni in pure culture, it appeared not to improve the PCR detection of C. jejuni in naturally contaminated chickens.     

A charcoal- and blood-free enrichment broth for isolation and PCR detection of Campylobacter jejuni and Campylobacter coli in chicken

The microaerophilic nature of Campylobacter and its requirement of ～5% O(2) for growth have complicated its recovery from foods. The addition to the enrichment media of oxygen quenchers such as charcoal or blood could interfere with PCR for its detection. In this study, a two-step simple aerobic method for Campylobacter detection is proposed. A modification of the Tran blood-free enrichment broth (BFEB), in which charcoal was excluded from the medium (M-BFEB), was compared with the original formulation and other enrichment broths. Campylobacter jejuni and Campylobacter coli were screened by PCR directly from the enrichment media. Various levels of pure cultures of C. jejuni and C. coli combined with Escherichia coli were inoculated into Preston, Bolton, BFEB, and the modified BFEB (M-BFEB). In addition, Campylobacter was inoculated onto retail purchased chicken skin and recovery was quantified. Rates of recovery after 24 to 48 h of enrichment at 42 °C under aerobic incubation for BFEB and M-BFEB and microaerobic incubations for Preston and Bolton broths were determined. Overall, our results indicated that the most sensitive medium was Bolton's, followed by either BFEB or M-BFEB; the least sensitive was Preston's. M-BFEB was directly coupled to a PCR assay to detect Campylobacter, avoiding intermediate plating. Campylobacter was detected in the presence of up to 10(8) E. coli cells per ml. M-BFEB facilitated detection of both C. jejuni and C. coli artificially inoculated onto chicken skin samples. M-BFEB coupled to PCR is a rapid and attractive alternative for isolation and identification of C. coli and C. jejuni from poultry.     

Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR

Contamination of lettuce by Salmonella has caused serious public health problems. Polymerase chain reaction (PCR) allows rapid detection of pathogenic bacteria in food, but it is inaccurate as it might amplify DNA from dead target cells as well. This study aimed to investigate the stability of DNA of dead Salmonella cells in lettuce and to develop an approach to detecting viable Salmonella in lettuce. Salmonella-free lettuce was inoculated with heat-killed Salmonella Typhimurium cells and stored at 4 °C. Bacterial DNA extracted from the sample was amplified by real-time PCR targeting the invA gene. Our results indicate that DNA from the dead cells remained stable in lettuce for at least 8 d. To overcome this limitation, propidium monoazide (PMA), a dye that can selectively penetrate dead bacterial cells and cross-link their DNA upon light exposure, was combined with real-time PCR. Lettuce samples inoculated with different levels of dead or viable S. Typhimurium cells were treated or untreated with PMA before DNA extraction. Real-time PCR suggests that PMA treatment effectively prevented PCR amplification from as high as 10(8) CFU/g dead S. Typhimurium cells in lettuce. The PMA real-time PCR assay could detect viable Salmonella at as low as 10(2) CFU/mL in pure culture and 10(3) CFU/g in lettuce. With 12-h enrichment, S. Typhimurium of 10(1) CFU/g in lettuce was detectable. In conclusion, the PMA real-time PCR assay provides an alternative to real-time PCR assay for accurate detection of Salmonella in food.     

Evaluation of universal preenrichment broth for growth of heat-injured pathogens.

Universal preenrichment broth (UPB) was developed to enable enrichment of injured foodborne pathogens of different genera simultaneously in lieu of having to undergo separate simultaneous enrichment cultures for subsequent detection or isolation of each pathogen. Enrichment conditions in UPB for growth of injured pathogens to populations that will enable pathogen detection by rapid immuno-based or polymerase chain reaction (PCR)-based assays have not been defined. Hence, studies were done to determine recovery and growth rates of heat-injured Escherichia coli O157:H7, Salmonella enterica ser. Typhimurium, Salmonella enterica ser. Enteritidis. and Listeria monocytogenes in UPB. Bacterial cells were heat injured in tryptic phosphate broth at 57.2 degrees C and inoculated at populations of ca. 0.17 to 63 injured cells per ml with raw ground beef, fresh chicken, lettuce, and environmental sponge samples. Enrichment cultures were sampled at 1, 2, 3, 4, 5, 6, and 24 h at 37 degrees C postinoculation, and pathogens were enumerated on appropriate selective media. Results revealed that recovery and growth of pathogens during the first 6 h of enrichment were not sufficient to ensure adequate numbers of bacteria (> 10(3) CFU/ ml) for detection by most immunoassays or PCR assays. Cells often required 3 to 4 h for recovery before growth was initiated. Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes cell populations in enrichment cultures with ground beef or lettuce at 6 h were 0.5 to 2.9 log10 CFU/ml. At 24 h of incubation, cell counts of enrichment samples for the three pathogens from all food and environmental sponge samples ranged from 4.0 to 8.3 log10 CFU/ml. Enrichment in UPB at 37 degrees C of foods or environmental sponge samples containing heat-injured cells of Salmonella Typhimurium, Salmonella Enteritidis, E. coli O157:H7, or L. monocytogenes reliably provides at 24 h of incubation-but not at 6 h-sufficient cell populations for detection by rapid immunoassay or PCR assay procedures that can detect at least 4 log10 CFU/ml. These results raise questions regarding the sensitivity of rapid detection methods that employ an abbreviated enrichment protocol of 6 h or less.     

Combination of immunomagnetic separation with real-time PCR for rapid detection of Salmonella in milk, ground beef, and alfalfa sprouts

A rapid, specific, and sensitive method for detecting Salmonella spp. in pasteurized milk, ground beef, and alfalfa sprouts was developed. The method combined immunomagnetic separation with a real-time PCR assay based on the double-stranded DNA binding dye SYBR Green I. The primers used produced a product with a melting temperature of 87+/-0.5 degrees C during the PCR assay by amplifying a 284-bp sequence from the invasive gene (invA) of Salmonella. The method was successful in detecting 20 Salmonella strains, but the expected PCR product was not formed by any of 11 other bacterial strains. To test this combined method for the monitoring of Salmonella, Salmonella enterica serotype Newport was inoculated into 52 samples each of pasteurized milk, ground beef, and alfalfa sprouts. Following a 10-h nonselective enrichment step in buffered peptone water, cells were removed by immunomagnetic separation and DNA extracted using the High Pure PCR template preparation kit. The DNA produced was used as a template in the real-time PCR assay. When spiked pasteurized milk, ground beef, and alfalfa sprout samples were analyzed by this protocol, an initial inoculum of 1 CFU/ml, 25 CFU/25 g, and 1.5 CFU/25 g, respectively, was detectable within 13 h. These results indicate that the combination of immunomagnetic separation and real-time PCR assay was a highly specific and sensitive method for the rapid detection of Salmonella.     

Application of a molecular beacon-real-time PCR technology to detect Salmonella species contaminating fruits and vegetables

An oligonucleotide probe that becomes fluorescent upon hybridization to the target DNA (molecular beacon; MB) was evaluated in a real-time polymerase chain reaction (PCR) assay to detect the presence of Salmonella species. As few as 1-4 colony-forming units (CFU) per PCR reaction could be detected. The capability of the assay to detect Salmonella species from artificially inoculated fresh-cut produce such as cantaloupe, mixed-salad, cilantro, and alfalfa sprouts was demonstrated. A comparison of two commercially available kits utilizing MB-PCR (iQ-Check, Bio-Rad Laboratories) and conventional Association of Official Analytical Chemists (AOAC)-approved PCR (BAX, Dupont Qualicon) was performed on artificially inoculated produce. As few as 4 CFU/25 g of produce were detected after 16 h of enrichment in buffered peptone broth. These assays could be carried out entirely in sealed PCR tubes, enabling a rapid and high-throughput detection of Salmonella species in a large number of food and environmental samples. This is the first report of the application of MB probe being used for real-time detection of Salmonella species in whole and fresh-cut fruits and vegetables.     

Validation of an Improved Method for Detection of Campylobacter jejuni in Foods

ABSTRACT:  Campylobacter jejuni ATCC 29428 and 33560 were inoculated separately into beef muscle, ground beef, and chicken skin to yield approximately 10 to 100 CFU/g of food sample. The samples were stored at 4 °C for 10 d. On days 0, 3, 7, and 10, enrichment cultures in Bolton broth supplemented with antibiotics, with and without blood supplementation were made for each sample, for 24 and 48 h following the Food and Agricultural Products Center (FAPC) and the Food and Drug Administration (FDA) protocols. Enumeration of the organisms in the enrichment cultures was done on Campylobacter Karmali selective agar after 24 and 48 h of enrichment to compare the extent of growth in both protocols. There were no significant differences between counts recovered using the FDA and the FAPC methods for detection of Campylobacter jejuni for either strain in any of the food products tested ( P > 0.05). No significant differences were observed in performance of enrichment broth supplemented with and without blood ( P > 0.05). After 48 h of enrichment, the counts recovered were similar for all products. The organisms were detectable on all days of storage in raw chicken skin, beef, and ground beef samples after both 24 and 48 h of enrichment. The results from the FAPC method for detection of C. jejuni from food were not different from the FDA method. While in the proposed method incubation at 37 °C was adequate for the strains tested it is recommended that both enrichment temperatures be used for naturally contaminated samples to ensure detection of all strains that might be present.     

Quantification and differentiation of Campylobacter jejuni and Campylobacter coli in raw chicken meats using a real-time PCR method

Campylobacter species are one of the most common causes of bacterial diarrhea in humans worldwide. The consumption of foods contaminated with two Campylobacter species, C. jejuni and C. coli, is usually associated with most of the infections in humans. In this study, a rapid, reliable, and sensitive multiplex real-time quantitative PCR was developed for the simultaneous detection, identification, and quantification of C. jejuni and C. coli. In addition, the developed method was applied to the 50 samples of raw chicken meat collected from retail stores in Korea. C. jejuni and C. coli were detected in 88 and 86% of the samples by real-time quantitative PCR and the conventional microbiological method, respectively. The specificity of the primer and probe sets was confirmed with 30 C. jejuni, 20 C. coli, and 35 strains of other microbial species. C. jejuni and C. coli could be detected with high specificity in less than 4 h, with a detection limit of 1 log CFU/ml by the developed real-time PCR. The average counts (log CFU per milliliter) of C. jejuni or C. coli obtained by the conventional methods and by the real-time PCR assay were statistically correlated with a correlation coefficient (R2) between 0.73 and 0.78. The real-time PCR assay developed in this study is useful for screening for the presence and simultaneous differential quantification of C. jejuni and C. coli.     

Comparison of methods for the isolation of thermotolerant Campylobacter from poultry

Human campylobacteriosis has become the major cause of foodborne gastroenteritis in industrialized countries. Although there have been numerous studies investigating the prevalence of Campylobacter in animals and raw meat, sensitive and low-cost detection methods are needed to implement effective control measures during primary production and to use as tools in risk assessment studies. Thermophilic Campylobacter spp. in naturally contaminated (n = 64) and inoculated (n = 16) broiler samples were detected using two International Organization for Standardization (ISO)-approved methods. Both enrichment broths (those of Preston and of Park and Sanders) were inoculated with (i) Campylobacter jejuni ATCC35921, (ii) boneless breast from broilers, (iii) boneless breast rinse solution, (iv) boneless breast rinse solution inoculated with C. jejuni ATCC35921 before centrifugation, and (v) boneless breast rinse solution inoculated with C. jejuni ATCC35921 after centrifugation. The results indicated that the Park and Sanders broth was superior to the Preston broth for recovery of Campylobacter spp., and no significant differences (P > 0.05) were found between ISO (meat pieces) and modified ISO (centrifuged chicken rinse solution) methods for the detection of Campylobacter spp.     

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.     

Loop-Mediated Isothermal Amplification (LAMP) for the Rapid and Sensitive Detection of Salmonella Typhimurium from Pork

ABSTRACT: Loop-mediated isothermal amplification (LAMP) is a novel molecular detection method that is more rapid and simpler than PCR. Products can be detected by turbidity using one temperature without the need for expensive PCR equipment. Our objective was to sensitively detect Salmonella Typhimurium from pork products within 1 d using the LAMP assay. Pork chop and pork sausage samples (25 g) were inoculated with high (10⁸ to 10⁶ CFU) and low (10⁵ to 10⁰ CFU) inocula of S. Typhimurium. Serial dilutions in phosphate buffered saline were plated on XLT4 agar either immediately or after selective preenrichment in tetrathionate broth (225 mL) for 10-h at 37 °C. Nucleic acid was extracted using the TRIzol^® method from 1-mL samples. The LAMP assay using 6 specific invA gene primers and Bst DNA polymerase reaction mix was carried out at 62 °C for 90 min in a waterbath. Turbid products were detected visually and by agarose gel electrophoresis. Improved Salmonella detection at 10² CFU/25 g for both pork chop and sausage was obtained after 10-h enrichment and 10⁶ CFU/25 g without enrichment for both products. This assay can detect Salmonella from pork within 1 d, significantly faster than traditional methods that take >5 d. This method shows tremendous potential for routine diagnostics and monitoring of Salmonella by the pork industry. Practical Application: The novel loop-mediated isothermal amplification (LAMP) assay is a rapid, specific, and sensitive method that has potential application for routine diagnostics of Salmonella from pork products. The isothermal method does not require expensive equipment such as a PCR thermocylcer but only a simple waterbath for amplification within 90 min. Detection is even simpler by visual eye or turbidimeters that are less expensive than fluorescent spectrophotometers or real-time PCR machines. All these advantages make it a practical approach for routine use by processing industries to rapidly detect Salmonella in their environment and to implement appropriate control strategies. To improve detection sensitivities, preenrichment followed by selective enrichment may be necessary. Even so, the entire assay can be completed at the most within two 8-h working shifts.     

Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples

The objective of this study was to develop and evaluate a SYBR Green 1 real-time PCR method for the specific detection of Salmonella spp. in dairy farm environmental samples. Previously reported 119-bp invA gene was selected for specificity, and 124 Salmonella spp. including type strains and 116 non-Salmonella strains were evaluated. All Salmonella strains tested were invA-positive and all non-salmonella strains yielded no amplification products. The melting temperature (Tm=79 degrees C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the threshold cycle (C(T)) versus copy numbers of Salmonella Enteritidis showed good linearity in broth (R2=0.994; slope=3.256) and sterilized milk (R2=0.988; slope=3.247), and the minimum levels of detection were >10(2) and >10(3) colony forming units (CFU)/ml, respectively. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated samples. Lagoon water, feed/silage, bedding soil, and bulk tank milk samples obtained from dairy farms were spiked with 10(0) to 10(5) CFU/ml of Salmonella Enteritidis. Sensitivities for detecting Salmonella in these sources were 10(3) to 10(4) CFU/ml of inoculums in broth without enrichment. Detection limits were reduced to <10 CFU/ml of inoculum in broth after 18 h enrichment. Ninety-three environmental samples including fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding soil were analyzed for the presence of Salmonella by real-time PCR, results were compared with those obtained by conventional culture methods. All samples analyzed were negative for Salmonella by both real-time PCR and standard culture method. No false positive or false negative results were detected.     

食品中空肠弯曲菌荧光重组酶聚合酶扩增检测方法的建立

本研究为了实现空肠弯曲菌的快速和便捷检测,建立了一种基于荧光重组酶聚合酶扩增技术(exo-RPA)快速检测空肠弯曲菌的方法。通过对空肠弯曲菌和对照菌株的exo-RPA检测来判断该方法的特异性。用梯度稀释的空肠弯曲菌作为模板进行检测来分析exo-RPA方法的灵敏度。通过对模拟污染样品检测来分析exo-RPA的应用效果。分别以exo-RPA和荧光PCR检测实际食品样品来分析二者的检测效果。空肠弯曲菌exo-RPA方法可特异检出空肠弯曲菌,检测灵敏度达到6.0×102CFU/m L。在模拟污染试验中,含2.5×101 CFU/m L空肠弯曲菌的增菌液在培养24 h后可以被exo-RPA检测出阳性信号。exo-RPA和荧光PCR对于40份样品的检测结果相同。本研究建立的空肠弯曲菌exo-RPA具有特异、灵敏和抗干扰性强的特点,方法操作简便快速,具有较好的应用前景。     

PCR detection of Bacillus and Staphylococcus in various foods

A broad-range PCR assay for the detection of bacteria belonging to Bacillus and Staphylococcus genera was developed. Primers targeting the bacterial 16S rRNA gene were newly designed and used in a PCR assay. To determine the specificity of the assay, 81 different bacterial strains (of 50 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for every tested Bacillus, Staphylococcus, or Aerococcus strain. In addition, the result for Listeria grayi was positive with lower PCR product. For all other bacterial strains and eukaryotes tested, results were negative. Bacterial DNA was prepared with the use of achromopeptidase and Chelex 100 resin from culture after growth in brain heart infusion medium. To test the sensitivity of this PCR assay for Bacillus or Staphylococcus genus, either Bacillus cereus or Staphylococcus aureus was inoculated into various foods with undetectable levels of endogenous microbial contamination as an indicator. Inoculation of bacteria at 10 to 30 CFU/g of food was followed by a 5-h enrichment culture step after which the PCR assay allowed the detection of bacterial cells. When the inoculation (B. cereus or S. aureus) of 10 to 90 CFU/g into noodle foods containing endogenous microflora (10(3) to 10(5) CFU/g) was followed by a 6-h enrichment culture step, the PCR assay detected the bacteria. Including the enrichment culture step, the entire PCR detection process can be completed within 8.5 h.     

Rapid detection of Campylobacter jejuni in chicken rinse water by melting-peak analysis of amplicons in real-time polymerase chain reaction

Five DNA extraction protocols for the detection of Campylobacter spp. by polymerase chain reaction (PCR) were compared. A method involving Triton X-100 produced template DNA of sufficient quality to allow the detection of Campylobacter jejuni at levels of 100 CFU/ml in pure culture. Primers were designed on the basis of the cadF gene sequence. With a SYBR Green I real-time PCR assay, these primers amplified only sequences present in C. jejuni to produce a product with a melting temperature of 81.5 degrees C. None of the strains of Campylobacter coli, Campylobacter lari, or Campylobacter fetus tested produced this product during the PCR assay. Other noncampylobacter species tested were shown not to possess the cadF sequence. The real-time PCR combined with a rapid, simple Triton X-100 DNA extraction protocol made it possible to detect < 10 CFU of C. jejuni per ml of chicken rinse within 14 h.     

Comparison of a new enrichment procedure for Shiga toxin-producing Escherichia coli with five standard methods

A new procedure for enrichment of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli was compared to five standard methods: the British Public Health Laboratory Service, International Standard Method, U.S. Department of Agriculture, Canadian Health Products and Food Branch, and U.S. Food and Drug Administration. The new procedure was comparable to the standard methods in its ability to detect target cells inoculated into foods at approximately 1 CFU g(-1). Comparisons were also made of the ability of the six enrichment procedures to detect E. coli O157:H7 against a large background of competitor microorganisms. In these experiments the new procedure yielded more target cells than the other five enrichments by two to three orders of magnitude as determined by enumeration on sorbitol MacConkey agar with tellurite and cefixime and Rainbow agar with tellurite and novobiocin and by verification of presumptive colonies by real-time PCR. For example, the population of enterohemorrhagic E. coli strain 6341 recovered on sorbitol MacConkey agar with tellurite and cefixime after enrichment with the experimental method was 2.42 x 10(8) CFU ml(-1) and 1.80 x 10(6) CFU ml(-1) after enrichment with the Canadian Health Products and Food Branch method, the second most effective in this experiment. In addition, broth cultures resulting from each of the six enrichment procedures were used to prepare templates for real-time PCR detection of stx1/stx2. Resulting threshold cycle (Ct) values after the experimental enrichment were similar to positive control values, whereas the five standard methods produced delayed Ct values or were not detected.