Application of a multiplex PCR for the simultaneous detection of Escherichia coli O157:H7, Salmonella and Shigella in raw and ready-to-eat meat products

Escherichia coli O157:H7, Salmonella and Shigella might contaminate similar types of meat products and cause deadly diseases in humans. Traditional microbiological analyses to detect these pathogens are labor-intensive and time-consuming. The objective of this study was to apply a multiplex PCR for simultaneous detection of the pathogenic bacteria in certain raw and ready-to-eat meat matrices. The tested samples had aerobic plate counts ranging from non-detectable, in chicken nuggets and salami, to 8.36log(10)CFU/g in ground pork. The pH of homogenates spanned from 6.86, in ground beef, to 7.17 in salami. Following a 24-h enrichment, the multiplex PCR assay could concurrently detect the three pathogens at 0.2log(10)CFU/g in ground beef, roast beef, beef frankfurters, chicken nuggets, salami and turkey ham, and 1.2log(10)CFU/g in ground pork. This multiplex PCR offers an efficient microbiological tool for presumptive detection of E. coli O157:H7, Salmonella and Shigella in meat.     

Lead and cadmium in meat and meat products consumed by the population in Tenerife Island, Spain

The aim of this study was to determine the levels of lead and cadmium in chicken, pork, beef, lamb and turkey samples (both meat and meat products), collected in the island of Tenerife (Spain). Lead and cadmium were measured by graphite furnace atomic absorption spectrometry (GFAAS). Mean concentrations of lead and cadmium were 6.94 and 1.68 µg kg^-1 in chicken meat, 5.00 and 5.49 µg kg^-1 in pork meat, 1.91 and 1.90 µg kg^-1 in beef meat and 1.35 and 1.22 µg kg^-1 in lamb meat samples, respectively. Lead was below the detection limit in turkey samples and mean cadmium concentration was 5.49 µg kg^-1. Mean concentrations of lead and cadmium in chicken meat product samples were 3.16 and 4.15 µg kg^-1, 4.89 and 6.50 µg kg^-1 in pork meat product, 6.72 and 4.76 µg kg^-1 in beef meat product and 9.12 and 5.98 µg kg^-1 in turkey meat product samples, respectively. The percentage contribution of the two considered metals to provisional tolerable weekly intake (PTWI) was calculated for meat and meat products. Statistically significant differences were found for lead content in meats between the chicken and pork groups and the turkey and beef groups, whereas for cadmium concentrations in meats, significant differences were observed between the turkey and chicken, beef and lamb groups. In meat products, no clear differences were observed for lead and cadmium between the various groups.     

Meat species identification and Halal authentication using PCR analysis of raw and cooked traditional Turkish foods

The method performance characteristics of commercially available PCR kits for animal species identification were established. Comminuted meat products containing different levels of pork were prepared from authentic beef, chicken, and turkey. These meat products were analysed in the raw state and after cooking for 20 min at 200 °C. For both raw and cooked meats, the PCR kit could correctly identify the animal species and could reliably detect the addition of pork at a level below 0.1%. A survey of 42 Turkish processed meat products such as soudjouk, salami, sausage, meatball, cured spiced beef and doner kebap was conducted. Thirty-six samples were negative for the presence of pork (< 0.1%) and four were found to be correctly labelled as containing pork. However, one sausage sample was labelled as containing 5% beef, but beef DNA was not detected and a meatball sample labelled as 100% beef was found to contain chicken. Another turkey meatball sample was predominantly chicken.     

Quantification of beef,pork, chicken and turkey proportions in sausages: use of matrix-adapted standards and comparison of single versus multiplex PCR in an interlaboratory trial

Quantitative PCR methods for the determination of beef, pork, chicken and turkey proportions in sausage were tested in an interlaboratory trial. Twelve different laboratories analysed six meat products each made of different compositions of beef, pork, chicken and turkey. Two kinds of calibrators were used: sausages of known proportions of meat and DNA from muscle tissue. Results generated using calibration sausages were more accurate than those resulting from the use of muscle tissue DNA. Regardless of the method used (either multiplex or single PCR), when using calibration sausages, it was always possible to quantify the proportions of meats in the unknown samples (in the range of 0.5–80%) with high precision and accuracy.     

Species identification in meat products using real-time PCR

One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.     

Species identification in meat products using real-time PCR.

One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.     

Determination of soybean proteins in commercial heat-processed meat products prepared with chicken,beef or complex mixtures of meats from different species

The addition of foreign proteins (mainly soybean proteins and milk proteins) to heat-processed meat products is a common practice. This work approaches the determination of additions of soybean proteins in heat-processed meat products prepared with chicken meat, beef meat, and complex mixtures of meats from different species (chicken, pork, beef, and turkey) by perfusion reversed-phase high-performance liquid chromatography. The applied method was previously developed for the determination of soybean proteins in pork and turkey meat products but it has never been tested for the determination of soybean proteins in other heat-processed meat products containing other kinds of meats. This paper demonstrates the validity of this method for the detection of soybean proteins in heat-processed meat products containing different varieties of meats and even in the presence of other foreign proteins such as milk proteins. The specificity and existence of matrix interferences have been checked for these samples and accuracy has been evaluated by the comparison of the soybean protein contents determined by the proposed method and the official ELISA method.     

Species identification in meat products using real-time PCR

One of the most convenient methods for the identification of animal species in processed meat products is the examination of DNA sequences. Real-time polymerase chain reaction (qPCR) techniques are particularly suitable because even small fragments of DNA formed during heat processing of the meat can be amplified and identified. A real-time PCR method has been developed and evaluated for the identification of processed meat products. In test mixtures containing beef, pork, horse, mutton, chicken and turkey, it was possible to identify these species down to a level of 0.05%. By adjusting the number of cycles, it was possible to detect levels as low as 0.01% of these species. Cross-reactivity between these species was not found, except for pure horsemeat (250 ng DNA) in the assay for turkey meat. Cross-reactivity of deer, roe, ostrich, kangaroo, goat, domestic duck, mallard, goose, pigeon, guinea fowl, quail and pheasant was also investigated and it was found that amounts as high as 250 ng DNA of these species in the reaction vial did not result in (false) positive signals except for amounts higher than 125 ng deer DNA and higher than 50 ng pigeon DNA in the determination of chicken and beef, respectively. More than 150 meat samples were examined using DNA hybridization and real-time PCR. A comparison of the results showed a better performance of the real-time procedure compared to DNA hybridization.     

Antimicrobial resistance in Enterococcus spp. strains isolated from organic chicken, conventional chicken, and turkey meat: a comparative survey

The mean counts of Enterococcus spp. were determined for 30 samples each of organic chicken meat, conventional chicken meat, and turkey meat, and differences for Enterococcus contamination in meat were determined. Two enterococci strains from each sample were isolated to obtain a total of 180 strains, and resistance to ampicillin, chloramphenicol, doxycycline, ciprofloxacin, erythromycin, gentamicin, nitrofurantoin, and vancomycin was determined by a disk diffusion method. Average counts obtained showed that Enterococcus mean counts from organic chicken meat (3.18 log CFU/g) were significantly higher than those obtained from conventional chicken meat (2.06 log CFU/g) or conventional turkey meat (1.23 log CFU/g). However, the resistance data obtained showed that isolates from organic chicken meat were less resistant than enterococci isolates from conventional chicken meat to ampicillin (P = 0.0067), chloramphenicol (P = 0.0154), doxycycline (P = 0.0277), ciprofloxacin (P = 0.0024), erythromycin (P = 0.0028), and vancomycin (P = 0.0241). In addition, isolates from organic chicken were less resistant than conventional turkey meat isolates to ciprofloxacin (P = 0.001) and erythromycin (P = 0.0137). Multidrug-resistant isolates were found in every group tested, but rates of multidrug-resistant strains were significantly higher in conventional chicken and turkey than those obtained from organic chicken meat. Enterococcus faecalis was the most common species isolated from organic chicken (36.67%), whereas Enterococcus durans was the most common species isolated from conventional chicken (58.33%) and turkey (56.67%). The rates obtained for antimicrobial resistance suggest that although organic chicken meat may have higher numbers of Enterococcus, these bacteria present a lower level of antimicrobial resistance.     

Characterization of antimicrobial resistance and virulence genes in Enterococcus spp. isolated from retail meats in Alberta, Canada

The objective of this study was to characterize antimicrobial resistance (AMR) and virulence genotypes of Enterococcus spp. particularly Enterococcus faecalis isolated from retail meats purchased (2007-2008) in Alberta, Canada. Unconditional statistical associations between AMR pheno- and genotypes and virulence genotypes were determined. A total of 532 enterococci comprising one isolate from each positive sample were analyzed for antimicrobial susceptibility. A customized enterococcal microarray was used for species identification and the detection of AMR and virulence genes. E. faecalis was found in >94% of poultry samples and in about 73% of beef and 86% of pork samples. Enterococcus faecium was not found in turkey meat and its prevalence was 2% in beef and pork and 4% in chicken samples. None of the enterococci isolates were resistant to the clinically important drugs ciprofloxacin, daptomycin, linezolid and vancomycin. Multiresistance (≥3 antimicrobials) was more common in E. faecalis (91%) isolated from chicken and turkey (91%) than those isolated from beef (14%) or pork (45%). Resistance to aminoglycosides was also noted at varying degrees. The most common resistance genes found in E. faecalis were aminoglycosides (aac, aphA3, aadE, sat4, aadA), macrolides (ermB, ermA), tetracyclines (tetM, tetL, tetO), streptogramin (vatE), bacitracin (bcrR) and lincosamide (linB). Virulence genes expressing aggregation substances (agg) and cytolysin (cylA, cylB, cylL, cylM) were found more frequently in poultry E. faecalis and were unconditionally associated with tetM, linB and bcrR resistance genes. Other virulence genes coding for adhesion (ace, efaAfs), gelatinase (gelE) were also found in the majority of E. faecalis. Significant statistical associations were found between resistance and virulence genotypes, suggesting their possible physical link on a common genetic element. This study underscores the importance of E. faecalis as a reservoir of resistance and virulence genes and their potential transfer to humans through consumption of contaminated undercooked meat.     

市售鸡肉及内脏中磺胺耐药菌污染特征

为评价抗生素耐药基因通过市售鸡肉产品进行迁移的风险,从上海5个大型超市和综合市场采集的9个鸡肉样品和4个内脏样品中分离对磺胺甲噁唑/甲氧苄啶不敏感的细菌(Sul/Trir),研究其耐药特征,并用Southern杂交对耐药基因进行定位。结果表明,所有样品均分离到了Sul/Trir菌,菌落数为2.0×102~1.0×107 CFU/g,占总可培养菌的1.42%~82.00%。297株Sul/Trir菌的多重耐药现象普遍,耐药基因sul1、sul2、tetA、tetB、tetC、tetE、tetG、tetL、tetM和intI的检出率分别为11.33%、32.81%、6.64%、12.89%、2.73%、6.25%、9.38%、18.75%、39.84%和28.52%。耐药基因的寄主菌包括Escherichia sp.、Pedobacter sp.、Staphylococcus sp.、Enterococcus sp.、Sphingobacterium sp.和Acinetobacter sp.等,大多数的磺胺最小抑菌浓度大于512 μg/mL,其中一株葡萄球菌的质粒上有sul2基因。综上,耐药菌和耐药基因可能通过市售鸡肉及内脏产品进行迁移。     

Quantitative Detection of Poultry in Cooked Meat Products

ABSTRACT: There is a growing awareness of perceived harm from meat species adulteration, both intentional and accidental. The present study developed a monoclonal antibody (Mab)-based enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of chicken and turkey meat adulterated in cooked (100 °C, 15 min) mammalian meat. The specificity of Mab 5D2 to different species (pork, beef, lamb, deer, horse, duck, chicken, and turkey) and tissues (serum, gizzard, heart, and liver) was studied by noncompetitive ELISA. The detection of cooked chicken in beef, and turkey in pork was accomplished by competitive and noncompetitive ELISAs. Both ELISAs were optimized to quantify cooked poultry in red meats. The new Mab-based ELISAs enabled the detection of cooked poultry in red meats at levels as low as 1% (v/v) or better. The correlation ( r > 0.994) between chicken or turkey concentrations and ELISA signals permitted the quantification of poultry adulterants in cooked non-poultry meats.     

Phenotypic and genetic characterization of antimicrobial resistance in Salmonella serovars isolated from retail meats in Alberta, Canada

This study determined the prevalence of Salmonella serovars, antimicrobial resistance (AMR) and resistance genes in Salmonella isolated from retail meats purchased in Alberta, Canada. Samples were collected during one year period (May 2007–April 2008) on weekly basis from 19 census divisions in Alberta. A total of 564 samples including chicken (n = 206), turkey (n = 91), beef (n = 134) and pork (n = 133) were purchased. Salmonella were recovered from chicken (40%), turkey (27%) and pork (2%) samples and was not found in ground beef. A total of 21, 8, and 3 different serovars were recovered from chicken, turkey and pork meats, respectively. Salmonella Hadar was most common in chicken whereas S. Heidelberg was common in turkey meat. Overall 29% (32/110) of isolates were susceptible to tested antimicrobials and resistance to ciprofloxacin, amikacin and nalidixic acid was not found in any isolate. Multiresistance (≥2 antimicrobials) was found in 56% of isolates. Resistance to amoxicillin–clavulanic acid (AMC), ceftiofur (TIO), and ceftriaxone (CRO) was found in about 21% of chicken and 25% of turkey isolates. Resistance to either of tetracycline (TET), streptomycin (STR) or ampicillin (AMP) was unconditionally associated with S. Hadar but resistance to either of TET, AMP, AMC, TIO, CRO or cefoxitin was associated with S. Heidelberg. The strA/B (42% isolates), tet(A) (28% isolates), bla_CMY-2 (21% isolates) and bla_TEM (17% isolates) were the most common resistance genes found. The bla_CMY-2 and bla_TEM genes were unconditionally associated with S. Heidelberg; tet(A) and strA/B with S. Hadar and tet(B) gene with S. Kentucky. The strA/B genes were not associated with S. Heidelberg. Our data suggests that the prevalence of Salmonella serovars varied by the meat type and that AMR and resistance genes varied by the Salmonella serovars.     

Detection of meat species using TaqMan real-time PCR assays

Species-specific real-time PCR (TaqMan) assays were developed for detection of beef, pork, lamb, chicken and turkey. Assays were developed around small (amplicons <150 base pairs) regions of the mitochondrial cytochrome b (cytb) gene. Speciation was achieved using species-specific primers. For detection purposes, two TaqMan probes were developed; the first was specific to the mammalian species (beef, lamb and pork), the second to the poultry species (chicken and turkey). Normal end-point TaqMan PCR conditions were applied; however, PCR was limited to 30 cycles. Applying the assays to DNA extracts from raw meat admixtures, it was possible to detect each species when spiked in any other species at a 0.5% level. The absolute level of detection, for each species, was not determined; however, experimentally determined limits for beef, lamb and turkey were below 0.1%.     

Occurrence of pathogens in raw and ready-to-eat meat and poultry products collected from the retail marketplace in Edmonton, Alberta, Canada

A total of 800 meat and poultry products were purchased from the retail marketplace in Edmonton, Alberta, Canada. The products consisted of raw ground beef, chicken legs, pork chops, and ready-to-eat fermented sausage, roast beef, processed turkey breast, chicken wieners, and beef wieners. The samples were analyzed to determine the prevalence of Shiga toxin-producing Escherichia coli, Salmonella, Campylobacter spp., and Listeria monocytogenes. Shiga toxin-producing E. coli 022: H8 was found in one raw ground beef sample. Salmonella and Campylobacter were found in 30 and 62% of raw chicken legs, respectively. L. monocytogenes was found in 52% of raw ground beef, 34% of raw chicken legs, 24% of raw pork chops, 4% of fermented sausages, 3% of processed turkey breast, 5% of beef wieners, and 3% of chicken wieners. The occurrence of pathogens in this study is similar to that in retail products in many other international locales.     

Polybrominated diphenyl ethers (PBDEs) in US meat and poultry: 2012–13 levels,trends and estimated consumer exposures

ABSTRACT

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants whose use has contaminated foods and caused subsequent human exposures. To address the issue of possible human exposure, samples from a 2012–13 US meat and poultry (beef, pork, chicken, turkey) study were analysed for seven PBDEs. The mean summed concentrations of the seven BDE congeners (ΣPBDE) from beef, pork, chicken and turkey were 0.40, 0.36, 0.19, and 0.76 ng g^–1 lipid weight (lw). The range of ΣPBDEs for all meat classes was 0.01–15.78 ng g^–1 lw. A comparison of this study with a 2007–08 study revealed a decline in the median ΣPBDEs for all four meat classes, a reduction of 25.9% to 70.0%, with pork, chicken and turkey PBDE residues being statistically lower relative to the 2007–08 study. BDEs 47 and 99 contributed the most to the ΣPBDE concentrations, indicating likely animal exposures to the penta-BDE formulation. Based on the reported data an estimate of US consumer daily intake of PBDEs from meat and poultry was 6.42 ng day^–1.     

Evaluation of the petrifilm plate method for the enumeration of aerobic microorganisms and coliforms in retailed meat samples.

This study was designed to compare the effectiveness and applicability of the Petrifilm plate method with the Association of Official Analytical Chemists' (AOAC) standard aerobic count method and violet red bile agar method for meat products. The comparison was carried out using 303 meat samples collected from various retailers: 110 pork samples, 87 chicken samples, and 107 beef samples. In the comparison of the correlation coefficient (R) between the conventional method and the Petrifilm plate method by a linear regression analysis, the correlation coefficient in total microorganisms was 0.99, 0.95, and 0.94 in pork, beef, and chicken samples, respectively. The correlation coefficient in coliform count was 0.83, 0.96, and 0.81 in pork, beef, and chicken samples, respectively. Based on the high correlation in the total microorganism count, it might be possible to replace the conventional methods with the Petrifilm plate method. For coliform counts, the Petrifilm plate method also showed a generally high correlation coefficient, except for pork samples, which are more subject to contamination. The Petrifilm plate method was simpler and less time-consuming in sample preparation and, in procedures, faster than the conventional method. These results suggested that the 3M Petrifilm plate method could replace the conventional methods in the analysis of microorganism contamination measurement in meat products.     

Species identification in food products using the bioMerieux FoodExpert-ID<Superscript>®</Superscript> system

Animal feeds and meat mixtures were analysed using the bioMerieux FoodExpert-ID^® system. The system utilises a reverse dot technique on a DNA microarray to allow the identification of over 30 species of fish, birds and mammals. DNA is amplified by PCR (polymerase chain reaction) then hybridised to the microarray chip. Using this technique, turkey and chicken were correctly identified in 100% of feed samples that contained these species above a level of 0.1%. Pig, lamb and cow could not be reliably detected below a level of 1% in feed samples. For meat mixtures, a level of 0.2% pork or chicken could be correctly identified when mixed with 50% beef or pork, respectively. When a baked or canned meat mixture was investigated, a level of 5% pork, beef or chicken could be correctly identified, following either process. The bioMerieux FoodExpert-ID^® system can therefore be used as a general screen to identify likely species present in a sample, the level of which can be confirmed using other methods.     

Optimization of emulsion characteristics of beef, chicken and turkey meat mixtures in model system using mixture design

Emulsion pH (pHe), emulsion capacity (EC), emulsion stability (ES), emulsion density (ED) and apparent yield stress of emulsion (raw emulsion, AYSe) and emulsion gel (cooked emulsion, AYSg) of beef, chicken and turkey meats and their mixtures were studied using a model system.

Turkey meat homogenate was found to have higher protein concentration than chicken or beef homogenates. The highest pHe, EC and ES values and the lowest ED and AYSe values were found in chicken meat. However, the highest AYSg value was found in chicken–turkey meat mixture. Generally, the increasing amount of chicken meat in mixtures increased EC and ES, and decreased ED and AYSe values. Also, chicken–turkey meat mixtures had lower ES values than the mixtures containing only chicken or only turkey meat. With beef, the addition of chicken and turkey meats improved emulsion characteristics significantly. Optimum levels of beef, chicken and turkey meats were found to be 0–23%, 9–30% and 53–91% respectively.