Quantification of total viable bacteria on fish fillets by using ethidium bromide monoazide real-time polymerase chain reaction

Real-time PCR based on universal primers for amplification of a highly conserved bacterial 16S rDNA sequence was utilized in conjunction with the treatment of extracted bacterial cells with ethidium bromide monoazide (EMA) for the differential enumeration of viable and dead cells on cod fillets. Amplification of DNA from dead bacterial cells was successfully inhibited by EMA, whereas the DNA from viable cells was readily amplified. The detection range of the EMA real-time PCR assay was linear from 1 x 10(1) to 1 x 10(5) mixed bacterial genomic targets per PCR derived from broth cultures of fish tissue. The minimum detection limit of bacteria was found to be 1 x 10(1) genomic units/real-time PCR, equivalent to 1 x 10(5) CFU per gram of tissue. The EMA real-time PCR allowed construction of a standard curve obtained by plotting the log of genomic targets from strictly viable cells against resulting PCR cycles (Ct values) that facilitated quantification of total viable bacteria from fish fillets. The log of the total number of genomic DNA targets from EMA treated cells and plate counts from six randomly procured cod fillets were found not to be statistically different with the exception of one fillet. The process of freezing and thawing fillet tissue resulted in a drop in mean colony forming units (CFU) detected by plate counts from log 8.5+/-0.2 to log 8.1+/-0.1. A similar reduction in genomic targets from 8.5+/-0.1 to 8.0+/-0.16 was detected by EMA real-time PCR.     

Monitoring beef carcass surface microbial contamination with a luminescence-based bacterial phosphatase assay.

A commercially available microbial phosphatase test kit (Fast Contamination Indicator; FCI) was evaluated as a rapid method for estimating microbial contamination levels on beef carcass tissues. A set of actual beef carcass surface sample swabs (n = 70) was tested using the assay as a means to rapidly (10 min) monitor carcass swab sample microbial contamination. A regression equation was developed in experiment 1 and tested on an independent population. There was agreement between this assay and the conventional plating method for total aerobic mesophilic bacteria (r = 0.93). The predicted total mesophilic aerobic bacteria count generated from the fitted regression line (predicted log10 CFU/cm2 = 0.7505 x log10 FCI microbial phosphatase test values + 0.6726) showed a high correlation with actual aerobic mesophilic total counts (r = 0.88). The FCI test offers a simple and rapid method to estimate microbial contamination levels on beef carcasses.     

Practical Molecular Detection Method of Beef and Pork in Meat and Meat Products by Intercalating Dye Based Duplex Real-Time Polimerase Chain Reaction

In this study, a rapid, specific, and low-cost duplex-detection technique of pork and beef was developed by a real-time polimerase chain reaction assay based on fluorescence. Deoxyribonucleic acid was extracted from variable mixtures of pork and beef in sausage and industrial products to develop the duplex assay using the GIDAGEN^® Multi-Fast DNA Isolation Kit. Identification of genomes was accomplished in the same tube by their distinctive melting peak, which was 87.5°C for pork and 80.5°C for beef, respectively. The detection limit of the method was 0.01 ng/µL deoxyribonucleic acidor 0.001% target pork and beef in sausage. The results showed that the intercalating dye based duplex real-time polimerase chain reaction is a potentially sensitive, reliable, and practical assay for the detection of meat species adulterated with beef and pork.     

Real-Time TaqMan PCR for Rapid Detection and Quantification of Coliforms in Chilled Meat

Coliforms are a major group of bacteria associated with spoilage of refrigerated pork. It is necessary to develop an efficient and fast method to detect total coliforms in chilled pork because the traditional methods are laborious and time-consuming. In this study, a quantitative real-time polymerase chain reaction (qRT-PCR) targeting the lacZ gene was developed for rapid enumeration of coliforms from chilled pork. Of the 106 strains tested, 35 coliform strains and one Shigella sonnei strain were correctly identified compared with 70 control strains which failed to amplify. Detection sensitivity was 112 CFU/g. The assay was able to detect and accurately quantify the total coliforms in chilled meat within 2 h without preenrichment. The results indicate that the real-time PCR is an efficient and time-saving method that could be adopted by the meat industry to detect coliforms to replace the traditional most probable number and the rapid coliform count plate methods.     

TaqMan real-time PCR for the detection and quantitation of pork in meat mixtures

A rapid and highly specific real-time quantitative PCR, based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene (rRNA), has been developed for the quantitation of pork (Sus scrofa) in binary pork/beef muscle mixtures. The method combines the use of pork-specific primers, that amplify a 411bp fragment from pork DNA, and mammalian-specific primers amplifying a 425-428bp fragment from mammalian species DNA, which are used as endogenous control. An internal fluorogenic probe (TaqMan), that hybridizes in the "pork-specific" and also in the "mammalian" DNA fragments is used to monitor the amplification of the target gene. A comparison of the cycle number (C(t)) at which mammalian and pork-specific PCR products are first detected, in combination with the use of reference standards of known pork content, allows the determination of the percentage of pork in a mixed sample. Analysis of experimental pork/beef muscle binary mixtures demonstrated the specificity and sensitivity of the assay for detection and quantitation of pork in the range 0.5-5%.     

Microbiological sampling of carcasses by excision or swabbing

Groups of 25 carcasses were obtained by random selection of carcasses at the end of each of eight commercial processes for the dressing or cooling of carcasses. Samples were collected from six groups of pig or beef carcasses by excision or swabbing with sponge, gauze, or cotton wool, with one sample obtained by each of the four methods from a separate, randomly selected site on each carcass. Total aerobic counts, coliforms, and Escherichia coli from each sample were enumerated. Values for the mean log10, log10 mean, and log10 total numbers recovered were calculated for each set of total aerobic counts. Those statistics indicated that the numbers of bacteria recovered by excision or swabbing with sponge or gauze were similar, while the numbers recovered by swabbing with cotton wool were at the lower end of or below the range of the numbers recovered by the other methods. The numbers of coliforms or E. coli recovered from carcasses by sampling areas up to 100 cm2 were too few for the estimation of log mean numbers. Sampling of two groups of carcasses by swabbing with gauze indicated that each 10-fold increase in the area sampled, from 10 to 1,000 cm2, approximately doubled the number of samples from which coliforms or E. coli were recovered. Sampling of six groups of carcasses from one process indicated that the sizes of swabs and volumes of diluent used for processing swabs did not have to be increased proportionally to the area of carcass surface sampled to recover numbers of E. coli proportional to the sampled area. It therefore appears that carcass sampling techniques can be varied widely without compromising the recovery of bacteria, and that the relative efficiencies with which bacteria are recovered by different techniques can be assessed by sampling each carcass in a group of 25 by each of the methods to be compared.     

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.     

Analysis of pork adulteration in commercial meatballs targeting porcine-specific mitochondrial cytochrome b gene by TaqMan probe real-time polymerase chain reaction

A test for assessing pork adulteration in meatballs, using TaqMan probe real-time polymerase chain reaction, was developed. The assay combined porcine-specific primers and TaqMan probe for the detection of a 109 bp fragment of porcine cytochrome b gene. Specificity test with 10 ng DNA of eleven different species yielded a threshold cycle (Ct) of 15.5 ± 0.20 for the pork and negative results for the others. Analysis of beef meatballs with spiked pork showed the assay can determine 100-0.01% contaminated pork with 102% PCR efficiency, high linear regression (r(2) = 0.994) and ≤ 6% relative errors. Residuals analysis revealed a high precision in all determinations. Random analysis of commercial meatballs from pork, beef, chicken, mutton and goat, yielded a Ct between 15.89 ± 0.16 and 16.37 ± 0.22 from pork meatballs and negative results from the others, showing the suitability of the assay to determine pork in commercial meatballs with a high accuracy and precision.     

Direct quantification and distribution of tetracycline-resistant genes in meat samples by real-time polymerase chain reaction

The evolution of antimicrobial-resistant bacteria has become a threat to food safety and methods to control them are necessary. Counts of tetracycline-resistant (TR) bacteria by microbiological methods were compared with those obtained by quantitative PCR (qPCR) in 80 meat samples. TR Enterobacteriaceae counts were similar between the count plate method and qPCR (P= 0.24), whereas TR aerobic mesophilic bacteria counts were significantly higher by the microbiological method (P < 0.001). The distribution of tetA and tetB genes was investigated in different types of meat. tetA was detected in chicken meat (40%), turkey meat (100%), pork (20%), and beef (40%) samples, whereas tetB was detected in chicken meat (45%), turkey meat (70%), pork (30%), and beef (35%) samples. The presence of tetracycline residues was also investigated by a receptor assay. This study offers an alternative and rapid method for monitoring the presence of TR bacteria in meat and furthers the understanding of the distribution of tetA and tetB genes.     

Analysis of Pork Adulteration in Commercial Burgers Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by TaqMan Probe Real-Time Polymerase Chain Reaction

A TaqMan probe real-time polymerase chain reaction assay was developed for the determination of pork adulteration in commercial burgers. The assay combined porcine-specific primers and TaqMan probe for the selective amplification and detection of a 109-bp fragment of swine cytochrome b (cytb) gene. Specificity test with 10 ng DNA of 11 different meat-providing animal and fish species yielded a quantification cycle (Cq) of 15.5 ± 0.20 for the pork and negative results for the others in a 40-cycle reaction with a change of analysts and sources. Analysis of beef burger formulations with spiked pork showed the assay can determine 100–0.01% contaminated pork with a PCR efficiency (E) of 93.8% and a correlation coefficient (R ²) of 0.991. A plot of actual value against real-time PCR-predicted value also yielded a good linear regression, R ² 0.998, and small root mean square error of calibration, RMSEC 0.42. A strong correlation was found between the partial least square (PLS)-predicted values and real-time PCR-determined values. The accuracy of the method was ≥90% in all determinations of the standard set. Residual analysis also revealed a high precision in all determinations. Finally, a random analysis of 10 ng DNA of commercial burgers from pork, beef, chicken, mutton, and chevon yielded a Cq of 15.56 ± 0.22 to 16.24 ± 0.35 from pork burgers, and negative results from the others, showing the suitability of the assay to determine pork in commercial burgers with a high accuracy and precision.     

Decontamination of carcasses by vacuum-hot water cleaning and steam pasteurizing during routine operations at a beef packing plant

The microbiological effects of three operations for cleaning areas on dressed beef carcasses with vacuuming equipment which also applies hot water to the carcass, and of an operation for pasteurizing beef carcass sides with steam, were assessed. All four operations were routine in a commercial carcass dressing process. For each operation, swab samples were obtained from randomly selected carcasses, with a single sample being collected from each carcass, from a site selected at random from those affected by the operation. For the cleaning operations, 25 samples were obtained before and 25 after each operation. For the pasteurizing operation, 50 samples were obtained before and 50 after the operation. In addition, 50 samples were obtained from beef sides after the carcass cooling process which followed the pasteurizing operation. Total aerobic counts, coliforms and Escherichia coli from each sample were enumerated. The cleaning operations generally reduced the log mean numbers of bacteria on treated areas by ≤ 0.5 and had no discernible effect on the overall microbiological condition of the carcasses emerging from the process. The pasteurizing operation reduced the log mean numbers of total aerobic bacteria on carcasses by about 1, and the log mean numbers of coliforms and E. coli by > 2. The cooling process had no affect on the total counts, but further reduced the log mean numbers of coliforms and E. coli, apparently by about 1, to give beef sides from which E. coli were not recovered.     

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.     

Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction

Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the C _q (quantification cycle) from 10 ng/μl porcine DNA is (18.70 ± 0.12 to 19.08 ± 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r ² = 0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork–beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification.     

Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat

In this study, a combined enrichment/real-time PCR method for the rapid detection of Salmonella on fresh meat carcasses, was designed, developed and validated in-house following requirements outlined in ISO 16140:2003. The method included an 18 h non-selective enrichment in buffered peptone water (BPW) and a 6 h selective enrichment in Rappaport Vasilliadis Soya (RVS) broth, based on the traditional culture method, ISO 6579:2002. The real-time PCR assay included an internal amplification control (IAC), was 100% specific and was sensitive to one cell equivalent. The alternative method was validated against the traditional culture method and relative accuracy of 94.9%, sensitivity of 94.7% and specificity of 100% were determined using 150 fresh meat carcass swabs. This alternative method had a detection limit of 1–10 CFU/100 cm² for fresh meat carcass swabs and was performed in 26 h. Following further inter-laboratory studies, this alternative method could be suitable for implementation in testing laboratories for the analysis of carcass swabs.     

Decontamination of beef carcass surface tissue by steam vacuuming alone and combined with hot water and lactic acid sprays

Hot beef carcass surface regions (outside round, brisket, and clod) contaminated with feces spread over a 5-cm2 (1-in2) area were cleaned using a steam-vacuum spot-cleaning system alone or combined with subsequent sanitizing treatments of hot water (95 degrees C at the nozzle), or warm (55 degrees C) 2% lactic acid spray, or combinations of these two sanitizing methods. These treatments were compared for effectiveness in reducing aerobic plate counts (APC) and counts of Enterobacteriaceae, total coliforms, thermotolerant coliforms, and Escherichia coli. All treatments significantly reduced the numbers of each group of bacteria on beef carcass surfaces. However, reductions obtained by steam vacuuming were significantly smaller than those obtained by a combination of steam vacuuming with any sanitizing treatment. No differences in bacterial reductions were observed between different carcass surface regions. Steam vacuuming reduced the number of different indicator organisms tested by ca. 3.0 log cycles but also spread the bacterial contamination to areas of the carcass surface adjacent to the contaminated sites. This relocated contamination after steam vacuuming was most effectively reduced by spraying with hot water and then lactic acid. This combined treatment consistently reduced the numbers of Enterobacteriaceae, total and thermotolerant coliforms, and E. coli to undetectable levels (<1.0 log10 CFU/cm2) on areas outside the initial 5-cm2 inoculated areas.     

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%.     

基于DNA染料EMA的RT-PCR技术定量检测海产品中病原性副溶血弧菌活细胞

将一种DNA染料叠氮溴化乙锭(ethidium bromide monoazide,EMA)与实时定量聚合酶链式反应(RT-PCR)技术相结合,建立一种能选择性定量检测牡蛎中trh阳性副溶血弧菌活细胞的新方法。结果表明：使EMA成功插入死细胞DNA并且光解溶液中游离EMA的最佳曝光时间为20min;不抑制副溶血弧菌活细胞DNA扩增的最大EMA质量浓度为2.0μg/mL;完全抑制热致死细胞DNA扩增的最小EMA质量浓度为1.0μg/mL;人工污染牡蛎样品,不经过富集,在2.0×103～2.0×107CFU范围内细胞数的常用对数值与Ct值之间呈严格的负相关性,并且纯培养与人工污染牡蛎样品的RT-PCR检测限均为2×103CFU,即人工污染牡蛎样品的RT-PCR检测灵敏度为每克牡蛎样品400个活细胞;冻融实验表明,在温度低于55℃的水浴中对冷冻海产品进行解冻时,冻融过程对副溶血弧菌活细胞几乎没有影响。该方法是一种快速、灵敏且能有效鉴别并定量检测病原活细胞的新方法。     

Selective Detection of Mixed Bacterial Survivors from Fish Fillets after Freezing and Thawing by Ethidium Bromide Monoazide Real-Time PCR

The effect of freezing and thawing on the survival of mixed bacteria from cod fillets was studied using ethidium bromide monoazide (EMA) real-time PCR (EMA Rti-PCR). The permeability of cell membranes, media selection, EMA treatment, addition of glycerol, and the number of freeze-thaw cycles were investigated to discriminate and enumerate viable from dead bacteria following freezing and thawing. Universal primers were used to amplify the sequence of a conserved region of all eubacterial 16S rDNA for EMA Rti‐PCR. Amplification of DNA from frozen-thawed bacterial cells was significantly inhibited by EMA, whereas the DNA without EMA from frozen-thawed cells was readily amplified. Freezing and thawing resulted in damage to the cell membrane resulting in increased EMA uptake as revealed by confocal microscopy. The effect of glycerol on survival of the frozen-thawed cells was determined by plate counts and EMA Rti-PCR. Mixed bacteria from fish fillets suspended in saline containing glycerol (5–20%) resulted in greater survival following freezing and thawing than when bacteria were suspended in saline alone during freezing and thawing. Increasing the number of freeze-thaw cycles resulted in a decrease in the log CFU and an increase in Ct value.     

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.