A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species

A novel common primer multiplex PCR (CP-M-PCR) was applied to detect four kinds of meats (chicken, cattle, pig and horse) as raw materials. A common adapter was designed in the 5′-end of species-specific reverse primers which matched with the species-specific DNA sequences for each species and also used as the common primer (CP). CP-M-PCR primers were designed to uncover different length fragments of 239, 292, 412, and 451 bp from chicken, cattle, pig and horse meats, respectively. The bands of specific DNA fragments amplified by CP-M-PCR method still appeared until the concentration of species-specific primers diluted to 0.015 pmol and primer sensitivity was increased by 100 times compared with conventional multiplex PCR without CP. CP-M-PCR detection limit of the DNA samples was 0.1 ng (36.4 copies) for single kind of meat as well as four kinds of meats. CP-M-PCR method simplified the PCR reaction system and conquered the disparate amplified efficiency from different primers. The CP-M-PCR method could be widely applied in practical detection for simultaneous identification of other meat species and their products.     

Rapid and sensitive identification of buffalo’s,cattle’s and sheep’s milk using species-specific PCR and PCR–RFLP techniques

For the rapid, specific and sensitive identification of buffalo’s, cattle’s and sheep’s milk, species-specific PCR and PCR–RFLP techniques were developed. DNA from small amount of fresh milk (100 μL) was extracted to amplify the gene encoding species-specific repeat (SSR) region and the mitochondrial DNA segment (cytochrome-b gene). PCR amplification size of the gene encoding SSR region was 603 bp in both buffalo’s and cattle’s milk, while in sheep’s milk was 374 bp. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique was used to discriminate between buffalo’s and cattle’s milk. Restriction analysis of PCR–RFLP of the mitochondrial cytochrome-b segment (359 bp) analysis showed difference between buffalo’s and cattle’s milk. Where, the fragment length (bp) generated by TaqI PCR–RFLP were 191 and 168, whereas no fragments were obtained in cattle’s milk for cytochrome-b gene (359 bp). The proposed PCR and PCR–RFLP assays rep resent a rapid and sensitive method applicable to the detection and authentication of milk species-specific.     

Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods

The aim of our work was to evaluate a new commercial test kit for the detection of Listeria monocytogenes by PCR, using different DNA extraction methods. Food samples (pork sausage and “mozzarella” cheese) were spiked with known concentrations of L. monocytogenes and culture-enriched for 24 h. DNA extracted using three commercial kits and two standard methods, was amplified in species-specific PCR employing a L. monocytogenes PCR Detection Kit (Diatheva). The PCR-based method proved to be a reliable means of detecting the pathogen in food samples independently from the extraction procedure used, even for a contamination cell number of 1 cfu/g before culture enrichment. The molecular assay, showing perfect agreement with standard microbiological tests and a considerably shortened analysis time, provides a sensitive and rapid alternative for applications in the testing of foods for microbiological contamination, and highlights the potential of PCR technology in routine food control.     

Meat species identification based on the loop mediated isothermal amplification and electrochemical DNA sensor

An easy, rapid and sensitive method of detection of the presence of meat species in raw or processed foods is important from cultural, religious, health and commercial perspectives. In this study we have tried to distinguish species-specificity in control and processed pork, chicken and bovine meats using loop mediated isothermal amplicons (LAMP) and disposable electrochemical printed (DEP) chips. LAMP is a nucleic acid amplification method that amplifies target DNA with high specificity, efficiency and rapidity under isothermal condition (63 °C). Electrochemical genosensor with the DEP chips detects the amplicons by Linear Sweep Voltammetry (LSV) observation of DNA–Hoechst33258 interaction on the chip surface. Hoechst33258 interacts with DNA in solution without immobilization of DNA onto the electrode surface eliminating the time consuming probe immobilization step. Our method is more specific and free of unwanted amplifications compared to Multiplexed PCR (M-PCR) method and gave limits of detection of ∼20.33 ng/μl (3 × 10⁴ copies/reaction), ∼78.68 pg/μL (3 × 10² copies/reaction) and ∼23.63 pg/μL (30 copies/reaction) for pork, chicken and bovine species, respectively. Our method of detection is quick, taking only an hour, and it may be useful for food administration laboratories to carry out meat species identification in raw and processed foods.     

Application of an optimized 18-h method involving one step culturing and single primer-based PCR assay for detection of Salmonella spp. in foods

A polymerase chain reaction (PCR) using 20-mer oligonucleotide single primer (named primer 3) randomly designed on the basis of Salmonella Typhimurium gatD gene encoding galactitol-1-phosphate dehydrogenase could produce the specific DNA product of approximate 770-bp in all 38 Salmonella strains used. No 770-bp DNA band was amplified from any DNA samples of 20 non-Salmonella bacteria. The DNA band was detected by 1% agarose gel electrophoresis and ethidium bromide staining. The sensitivity of the RAPD–PCR assay for detection of pure genomic DNA from S. Typhimurium ATCC 13311 and Salmonella Enteritidis DMST 15676 was as few as 0.01 ng. When simple boiling in TE buffer for 5 min was used for extraction both Salmonella spp. DNA, the detection limits were at least 230 and 320 cells, respectively. By using the RAPD–PCR assay following at least 14 h pre-enrichment in nutrient broth (NB), as few as 1 CFU of S. Typhimurium ATCC 13311 or S. Enteritidis DMST 15676 per 25 g of autoclaved chicken meat was detected. When the optimized 18-h method involving pre-enrichment in NB and DNA extraction by boiling protocol followed by RAPD–PCR using primer 3 was evaluated in comparison with the conventional method on 195 possibly naturally-contaminated food samples, 36 samples were found positive by both methods. In addition, the results of the developed RAPD–PCR-based assay proved to be identical to those by the conventional method. The optimized 18-h method was simple, rapid and sensitive, achieved the same detection limit as the conventional method and produced a zero level of false-negative results.     

Quantitative detection of goats’ milk in sheep’s milk by real-time PCR

The need to support food-labeling legislation has provided a driving force for development of analytical techniques for the analysis of food ingredients. In this study, the development of a method for quantification of goats’ milk in sheep’s milk mixtures is described. The technique involves the use of a real time PCR technique, based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene (rRNA). The method combines the use of goat-specific primers that amplify a 171 bp fragment from goat DNA, and mammalian-specific primers amplifying a 119 bp fragment from mammalian species DNA, which are used as endogenous control. An internal fluorogenic probe (TaqMan) that hybridizes in the “goat-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 goat-specific PCR products are first detected, in combination with the use of reference standards of known caprine content, allows the determination of the percentage of goats’ milk in a milk mixture. The assay was used to analyze raw and heat-treated milk binary mixtures (goat/sheep), enabling the quantification of goats’ milk in the range 0.6–10%. The reported PCR assay may represent a rapid and straightforward tool applicable to the authentication of milk and other dairy products.     

Inactivation of foodborne pathogens in raw milk using high hydrostatic pressure

Plate counting, viability test, pulse field gel electrophoresis (PFGE) and transmission electron microscopy (TEM) were used to investigate the effect of high hydrostatic pressure (HHP) on Salmonella, Escherichia coli, Shigella and Staphyloccocus aureus in raw milk in order to determine the optimal inactivation conditions, and further understand the mechanisms of HHP on pathogens inactivation in food. The results exhibited that 300 Mpa treatment with 30 min duration at 25 °C was the optimal condition for Salmonella, E. coli, Shigella and S. aureus inactivation. Damage of the cell wall, cell membrane and cytoplasmic components by high pressure treatment can be observed in TEM micrographs. The injured cells could not be recovered, the growth rate of survivors was much lower than that of the untreated cells. PFGE showed neither corresponding DNA bands with same molecular weight nor DNA bands with same brightness could be found in the lanes between HHP treated pathogens and untreated ones. The results indicated that HHP processing can be applied to inactivate pathogens in food, the inactivation is mainly due to cell membrane damage, cell wall rupture and chromosome DNA degradation.     

A HPLC with fluorescence detection method for the determination of tetracyclines residues and evaluation of their stability in honey

A high-performance liquid chromatography with fluorescence detection method for the simultaneous determination of oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) residues in honey was developed and validated. Sample preparation was done in a single solid phase extraction step. The chromatographic separation was performed on a C₈ column. Matrix matched calibration curves showed linearity higher than 0.99. The accuracy values lay between 86% and 111% and the precision was lower than 11%. Limits of detection and quantitation were 8 μg kg⁻¹ and 25 μg kg⁻¹, respectively. The method was employed to evaluate the stability of the tetracyclines in honey during 60 days of storage. The residue levels of OTC, TC and CTC were reduced 97%, 76% and 71%, respectively. Honey samples (n = 22) collected from the retail market were analyzed. No tetracyclines residues were detected.     

Development of a novel target-enriched multiplex PCR (Tem-PCR) assay for simultaneous detection of five foodborne pathogens

In the present study, a novel target-enriched multiplex PCR (Tem-PCR) assay was developed for simultaneous detection of Salmonella spp., Listeria (L.) monocytogenes, Staphylococcus (S.) aureus, Escherichia (E.) coli O157:H7 and Shigella spp.. DNA primers including universal primer and composite primer pairs were used in this Tem-PCR assay. Of which, the universal primer was linked to the 5'-end of each specific primer designed targeting Salmonella invA gene, Staphylococcus aureus femA gene, Shigella ipaH gene, Listeria monocytogenes hly gene and Escherichia coli O157:H7 rfbE gene, respectively, generating the composite primers. During PCR amplification, the composite primer pairs were employed to enrich target genes of different pathogens in initial cycles and the universal primer was employed to enrich the amplicons produced with the composite primers priming in later PCR cycles. Significantly, the Tem-PCR assay overcomes the amplification disparity resulting from primers competition observed in conventional multiplex PCR assay. With the evaluation of the Tem-PCR assay for the detection of these five foodborne pathogens, the assay showed high specificity to the target bacteria with an analytical detection limit of <2.0 × 10² CFU/mL for each, and practical detection capability, suggesting a novel multiplex PCR method for detecting foodborne pathogens.     

Detection of hazelnut (Corylus spp.) in processed foods using real-time PCR

Hazelnut seeds (Corylus spp.) are source of allergens and could cause severe adverse reactions in sensitized subjects, even if consumed in traces. This work presents two sensitive real-time PCR methods to quantify hazelnut in foods, one using the Sybr green dye and one based on hazelnut-specific Taqman probe designed on Cor a1.04 gene (specific amplicon: 82 bp). The sensibility and the robustness of the method were estimated analyzing spiked samples and some commercial hazelnut-containing creams. The lowest detection limit was 0.1 ng of genomic DNA. A qualitative specific PCR and a comparison of different DNA extraction protocols are also discussed.     

Extraction and detection of endogenous soybean DNA from fermented foods

We examined rapid and simple genomic DNA extraction method to detect material soybeans from natto (fermented soybeans) and soy sauce. Genomic DNA was extracted from natto and soy sauce by CTAB or alkaline lysis methods. Genomic DNA solutions from natto by both extraction methods were of adequate purity and yield for applying PCR, resulting that amplifications of the expected 100 bp fragment using the soybean specific primer pair were detected. When genomic DNA was extracted from soy sauce, the DNA solutions by alkaline lysis method were of a sufficient purity (A₂₆₀/A₂₈₀ = 1.5–2.0), but those by CTAB method were insufficient (A₂₆₀/A₂₈₀ = 1.2–1.7). In addition, the amplification products from the genomic DNA solutions by CTAB method were not detected. Thus, these results suggested that the desirable genomic DNA was not extracted from soy sauce by CTAB method, although it was possible to extract it by alkaline lysis method. Furthermore, when genomic DNA was extracted from natto and soy sauce using FTA^® card, which can preserve nucleic acids dried on the card just by applying a sample to it, the amplification was detected. It is found that an endogenous gene (lectin 1 gene) of material soybeans can be extracted by rapid and simple methods, and detected from fermented foods as natto and soy sauce.     

The incidence of significant foodborne pathogens in domestic refrigerators

The interior surfaces of household refrigerators are at risk of becoming contaminated with foodborne pathogens, increasing the risks of cross-contamination to other food items, including higher risk ready-to-eat foods. This study determined the incidence of a number of significant foodborne pathogens, and the general hygienic status (as estimated by total viable counts (TVCs), and total coliform counts (TCCs)) on the interior surfaces of domestic refrigerators (n = 342). Campylobacter spp., Salmonella spp. and Escherichia coli O157:H7 were not recovered from any refrigerators, but Staphylococcus aureus was recovered from 6.4%, Listeria monocytogenes and E. coli from 1.2% and Yersinia enterocolitica from 0.6% of examined refrigerators. As the recovered species can survive and grow under refrigeration or conditions of mild temperature abuse, such pathogens may transfer to (and develop to clinically significant numbers in) food in domestic fridges. Such risks are of particular concern in relation to “ready-to-eat” foods, which will not receive further bactericidal treatments (cooking) before consumption. The study estimated TVCs ranging from 2.91 log₁₀ cfu/cm² to 8.78 log₁₀ cfu/cm² and TTCs ranging from 0.045 log₁₀ cfu/cm² to 5.96 log₁₀ cfu/cm² indicating very poor standards of consumer refrigerator management and hygiene, and posing risks to consumer health. The study findings highlight the importance of adequate temperature control and thorough, regular cleaning of domestic refrigerators to ensure food safety, and of effective cooking as the last link in the domestic food service chain.     

Simple and rapid detection of Listeria monocytogenes in fruit juice by real-time PCR without enrichment culture

We developed a simple, rapid procedure for the detection of Listeria monocytogenes in unpasteurized fruit juice using real-time PCR without enrichment culture. PCR inhibitors were removed from fruit juices using Chelex resin followed by gel filtration with a Sephadex column. The purification step can be completed in 20 min, and purified juice samples can be used directly as PCR templates without further dilution. PCR conditions were optimized and maximum sensitivity (ca. 1 cell/reaction) was achieved. This convenient method should prove useful for high-throughput surveillance of L. monocytogenes as well as other food-borne pathogens that may contaminate fruit juices.     

Comparative study of different methods for the extraction of DNA from fish species commercially available in South Africa

This study describes the comparison of five DNA extraction methods (urea-SDS-proteinase K; phenol-chloroform; salt extraction; SureFood^® PREP Allergen kit and Wizard^® Genomic DNA Purification kit) in terms of their ability to extract high yields of pure, readily amplifiable DNA from the muscle tissue of 29 fish species available in South Africa. Although variations in the yield and purity of extracted DNA were observed between methods, all five methods produced DNA suitable for PCR amplification. Overall, the SureFood^® PREP method was of the simplest and least hazardous to perform, extracting significantly (P < 0.05) higher DNA yields than all other methods evaluated.     

Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus,Listeria monocytogenes and Salmonella spp. in raw shrimp

Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. are important pathogens contaminating seafood in China. In this study, we developed an efficient multiplex real-time PCR for the simultaneous detection of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in raw shrimp without a prior enrichment step. In a test using 28 target and non-target strains only the targets were detected and two calibration curves, for pure cultures and artificially contaminated samples, were used to evaluate the efficiencies of this method. Amplification efficiencies of this multiplex real-time PCR were excellent in pure cultures and artificially contaminated shrimps. The limits of detection in artificially contaminated shrimps were 112 CFU/g for V. parahaemolyticus, 158 CFU/g for L. monocytogenes and 103 CFU/g for Salmonella spp. We validated this multiplex real-time PCR method on 48 commercial samples and the results were comparable to standard culture methods. This efficient multiplex real-time PCR, where each test takes only 50 min after DNA extraction, is a useful tool for high-throughput surveillance of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in seafood products.     

A multiplex PCR assay for fraud identification of deer products

Attempts were made to established one-step multiplex PCR assay for the fraud identification of the mostly used species in deer products (bovine, ovine, porcine and poultry). Primers were selected from published papers or designed in the well-conserved region of tRNA-Val and 16S rRNA mitochondrial genes after alignment of the available sequences in the GenBank database. The primers generated specific fragments of 124, 183, 225 and 290 bp length for bovine, poultry, ovine and porcine, respectively. The detection limit was 1 ng for porcine and ovine primers, 5 ng for poultry primers and 0.5 ng for bovine primers. The results demonstrated that fraud phenomena are very epidemic in the deer products, especially heart, blood, penis and antler products.The multiplex PCR described in this study, proved to be very sensitive and reliable in species identification, could be considered as a further improvement of traditional methods based assay for the identification of deer products.     

Simultaneous determination of melamine and related compounds by capillary zone electrophoresis

A simple, rapid and accurate method for the simultaneous determination of melamine and related compounds (ammeline, ammelide and cyanuric acid) by capillary zone electrophoresis with diode array detection (CZE–DAD) was developed and successfully applied in egg, dairy products and pet feed. Real samples were extracted with acetonitrile/water/diethylamine (80/18/2, v/v/v) and analyzed by CZE method directly after centrifuging and filtering. Separation was performed by CZE using 40 mM disodium hydrogen phosphate buffer (pH 9.0) as running buffer, an applied voltage of +30 kV, and UV detection at 214 nm. Four targets were separated completely within 10 min. The optimized method demonstrated good performance concerning linearity (r ⩾ 0.9964), precision (⩽4%), accuracy (82.2–101.6%), and enough sensitivity. And the proposed method was proved to have the merits of good separation, easy operation, short analysis time and low cost.     

Presence of aflatoxin M1 in milk and infant milk products in Tehran,Iran

Aflatoxins are highly toxic, mutagenic, teratogenic and carcinogenic compounds. The purpose of this survey was to determine natural occurrence and level of AFM₁ in pasteurized liquid milk, infant formula and milk-based cereal weaning food consumed in Tehran, Iran.A total of 328 branded milk products and liquid milk samples were collected and investigated by Enzyme Linked Immuno Sorbent Assay (ELISA).The samples of pasteurized liquid milk (n = 128), infant formula (n = 120) and milk-based cereal weaning food (n = 80) showed that the incidence of contamination with AFM₁ is 96.3%, the presence of AFM₁ in each group was 72.2 ± 23.5, 7.3 ± 3.9 and 16.8 ± 12.5 ng/kg, ranging between 31–113, 1–14 and 3–35 ng/kg, respectively.In general, the amount of AFM₁ in 100 (78%) of liquid milk samples and 24 (33%) of milk-based weaning food was higher than the maximum tolerance limit accepted by European Union, but in all of the infant formula samples was lower (European Communities and Codex Alimentarius has prescribed a limit of 50 ng/kg for AFM₁ in milk and 25 ng/kg in infant milk products).     

Detection of gluten-containing cereals in flours and “gluten-free” bakery products by polymerase chain reaction

A polymerase chain reaction-based method for the detection of gluten-containing cereals in flours and “gluten-free” bakery products was optimized and its intralaboratory validation was carried out. The optimized method involved DNA isolation by chaotropic solid-phase extraction and PCR with primers of Dahinden et al. [Dahinden I., von Büren M., Lüthy J., 2001. A quantitative competitive PCR system to detect contamination of wheat, barley and rye in gluten-free food for coeliac patients. European Food Research and Technology 212, 228–233]. Using purified DNA, intrinsic detection limit of 42 ± 12 pg was determined, which corresponds to 10° genome copies. By the analysis of a panel of 26 European wheat cultivars and flours from six non-gluten-containing plants, which are commonly used for the production of gluten-free bakery products, inclusivity of 100% and exclusivity of 100% were determined. By the analysis of model samples of soya flour and cakes, detection limit of 0.1% (w/w) of fine wheat flour was determined, which is suitable for the analysis of “gluten-free” food products, as it is approximately equivalent to the limit of 10 mg per 100 g for gluten stated by Codex Alimentarius. The method was successfully applied to four samples of flours and 13 brands of biscuits designated “gluten-free”, out of which two flours and one brand of biscuits were found positive for gluten-containing cereals. The method proved to be suitable for routine use, it was relatively straightforward and could be completed in one working day.     

Non-thermal inactivation of Escherichia coli K12 in buffered peptone water using a pilot-plant scale supercritical carbon dioxide system with a gas–liquid porous metal contactor

This study evaluated the effectiveness of a supercritical carbon dioxide (SCCO₂) system, with a gas–liquid porous metal contactor, for reducing Escherichia coli K12 in diluted buffered peptone water. 0.1% (w/v) buffered peptone water inoculated with E. coli K12 was processed using the SCCO₂ system at CO₂ concentrations of 3.1–9.5 wt%, outlet temperatures of 34, 38, and 42 °C, a system pressure of 7.6 MPa, and a flow rate of 1 L/min. Increased CO₂ concentrations and temperatures significantly (P < 0.05) enhanced microbial reduction. A maximum reduction of 5.8-log was obtained at 8.2% CO₂ and 42 °C. To achieve a 5-log reduction of E. coli K12 in 0.1% buffered peptone water, minimum CO₂ concentrations of 9.5%, 5.5%, and 5.3% were needed at 34, 38, and 42 °C, respectively. Further reductions of cells were observed after storage for 7 days at 4 °C. But storage at 25 °C increased the number of viable cells to 8-log cfu/mL after 7 days. This study showed the potential of the pilot scale SCCO₂ system with a gas–liquid porous metal contactor for microbial inactivation in liquid food.