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.     

Simplex and duplex PCR assays for species specific identification of cattle and buffalo milk and cheese

A polymerase chain reaction, amplifying a fragment of the mitochondrial DNA D loop region was developed for species specific detection of cattle and buffalo milk. The method was simultaneously extended for detection of HTST pasteurized milk samples and cheeses of bovine and buffalo origin. A common forward primer was used with two different species specific reverse primers that resulted amplification of a 126 bp and 226 bp products for cattle and buffalo, respectively, in simplex as well as in multiplex polymerase chain reaction. The primers successfully amplified DNA extracted by conventional protocol from minimal amount of raw milk, heat treated milk and cheese of either bovine or buffalo origin. The primers showed a high degree of specificity. The sensitivity of the assay was excellent with detection level of 0.1 percent adulteration of cow and buffalo milk or cheese (0.15 ng buffalo and 0.04 ng cattle DNA). The assay represents a sensitive and simple method for identification of adulteration in milk and cheese.     

Biogenic amines content,histamine-forming bacteria,and adulteration of pork and poultry in tuna dumpling products

Nine tuna dumpling products were purchased at nine retail markets in southern Taiwan. Occurrence of biogenic amines, histamine-forming bacteria, and adulteration of pork and poultry were determined. This study showed the high contents of aerobic plate count, total coliform and Escherichia coli in tested tuna dumpling products. Average content of various biogenic amines in all tested samples was less than 2.0 mg/100 g. Fifteen histamine-producing bacterial strains isolated from tested samples produced 8.7–466 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH). Assay of multiplex polymerase chain reaction (PCR) revealed adulteration rates were 88.9% (8/9) and 33.3% (3/9) for pork and poultry, respectively, in tuna dumpling. In addition, six samples of tuna dumpling meat were identified as Thunnus obesus, while other three samples were identified as Thunnus thynnus.     

A multiplex-conventional PCR assay for bovine,ovine, caprine and fish species identification in feedstuffs: Highly sensitive and specific

A multiplex PCR assay was developed for rapid and reliable identification of bovine, ovine, caprine and fish species in feedstuffs simultaneously. The method merges the use of bovine, ovine, caprine and fish primers that amplify fragments (ovine; 119 bp, caprine; 142 bp, fish; 224 bp and bovine; 271 bp) of the mitochondrial t.glu gene forward and cyt b reverse, 12S rRNA, 12S rRNA, and ATPase subunit 8 genes respectively, and a universal 18S rRNA primers that amplifies a 99 bp from eukaryotic DNA. To evaluate the effect of heat treatment, a severe sterilization condition (133 °C at 300 kPa for 20 min) was applied. Multiplex analysis of the reference feedstuff samples showed that the detection limit of the assay was 0.01% for each species. Taken together, all data indicated that this multiplex PCR assay was a simple, rapid, sensitive, specific, and cost-effective detection method for bovine, ovine, caprine and fish species in feedstuffs.     

Development and application of molecular markers for poultry meat identification in food chain

Every year, large quantities of poultry and game meat are consumed. Thus, efficient techniques to identify the meat species origin are required which interest traders, consumers and organizations. In this study, two mitochondrial DNA (mtDNA) genes, Cytochrome b (Cyt b) and 12S ribosomal RNA (12S rRNA) were tested as putative discrimination markers in samples of raw and processed poultry meat (chicken, turkey, duck, goose, pheasant, partridge, woodcock, ostrich, quail and song thrush), applying the PCR–RFLP technique with universal primers and ten different restriction enzymes. Digestion of 12S rRNA by AciI successfully distinguished all avian species, producing species-specific patterns. We conclude that the 12S rRNA gene is more informative than Cyt b gene for avian species identification purposes. Moderate process treatment did not prevent the species identification, presenting similar patterns with the raw meat. Finally, this method was considered sufficient to detect mixtures of meat, making it a valuable tool for checking possible adulterations.     

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.     

Rapid and simple determination of chloropropanols (3-MCPD and 1,3-DCP) in food products using isotope dilution GC–MS

The paper describes simultaneous determination of 3-monochloropropanediol (3-MCPD) and 1,3-dichloropropanol (1,3-DCP) in small specimens of food products. 3-MCPD and 1,3-DCP were isolated from soy sauce and other food products using alumina column extraction. Sample portions (1–2 g) spiked with deuterated 3-MCPD-d5 and 1,3-DCP-d5 as internal standards were mixed with 2 g of aluminum oxide and packed into small disposable column. The compounds were eluted with 25 ml dichloromethane, concentrated and derivatized with heptafluorobutyryl anhydride. GC–MS analysis was done in SIM mode. Four ion fragments were used for identification of 3-MCPD and 3-MCPD-d5, three ions were used for 1,3-DCP. The limit of detection was 1 ng/g, the limit of quantitation was 3 ng/g, the recovery was around 80%. Observed matrix effects were negligible for 3-MCPD and amounted to around 20% signal decrease for 3-DCP.     

Biogenic amines content,histamine-forming bacteria and adulteration of bonito in tuna candy products

Thirty-four tuna candy products sold in retail markets and supermarkets in Taiwan were purchased and tested to determine the biogenic amine, histamine-forming bacteria, and adulteration of bonito meat. The levels of pH value, water activity (Aw), water content, total volatile basic nitrogen (TVBN), aerobic plate count (APC) and total coliform (TC) in all samples ranged from 5.3 to 6.1, 0.47 to 0.65, 7.37% to 17.32%, 12.1 to 54.6 mg/100 g, <1 to 6.2 log CFU/g and <3 to 1700 MPN/g, respectively. None of these sample contained Escherichia coli. The average content of various biogenic amines in all tested samples was less than 1.0 mg/100 g. Four histamine-producing bacterial strains isolated from tested samples produced 1.02–1.74 ppm of histamine in trypticase soy broth supplemented with 1.0% l-histidine (TSBH) after incubation at 35 °C for 24 h. Assay of multiplex polymerase chain reaction (PCR) revealed that adulteration rate of bonito meat was 26.5% (9/34) in tuna candy samples. Tuna species in tuna candy samples was identified as Thunnus albacares for 29 samples (85.3%), Thunnus alalunga for four samples (11.8%) and Thunnus obesus for one sample (2.9%) by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP).     

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.     

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).     

Pressurized liquid extraction coupled to liquid chromatography for the analysis of ochratoxin A in breakfast and infants cereals from Morocco

A sensitive and reliable method using pressurized liquid extraction (PLE) and liquid chromatography (LC) has been developed for the analysis of ochratoxin A (OTA) in breakfast and infants cereals. Influence of several extraction solvents that affect PLE efficiency was studied. The selected PLE operating method was: 10 g of sample was packed into 22 ml stainless-steel cell and OTA was extracted with acetonitrile/water (80:20) at 40 °C, 34 atm in one cycle of 5 min at 60% flush. The mean recovery of OTA was 82 ± 4 at fortification level of 3 ng/g OTA. The limit of quantification (LOQ) of OTA was 0.25 ng/g. The proposed method was successfully applied to the analysis of 68 samples of breakfast and infants cereals products collected from different supermarkets and pharmacies in Rabat. Results showed that all analyzed infant cereals were free of OTA contamination. However, four samples of breakfast cereals were contaminated with OTA. Levels of OTA in positive samples ranged between 5.1 and 224.6 ng/g. All positive samples (5.8% of total samples) were above the maximum level set by EU regulations for OTA in cereal derivatives products.     

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.     

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.     

Occurrence of diacetoxyscirpenol (anguidine) in processed cereals and pulses in Turkey by HPLC

The purpose of this study was to investigate the diacetoxyscirpenol (DAS, anguidine) level of contaminated cereal and pulse products in Turkey. DAS was detected using high performance liquid chromatography (HPLC) with diode array detector (DAD) at 205 nm and suspicious the results for two specimens suspected to contain DAS were confirmed by gas chromatography–mass spectrometry (GC–MS). An acetonitrile–water (21 + 4, v/v) extract of the sample was cleaned up on a column packed with alumina/charcoal (0.35 g + 0.40 g). The minimum detectable amount of DAS was 16 ng/injection, limit of detection was 0.8 μg/g for HPLC. A total of 85 commercially available cereal and pulse product samples, collected from markets and street bazaars, were analyzed. The recoveries for corn flour with the known amounts of DAS (2, 3, 4 μg/g) were 85.3% (SD 4.81, n = 5), 98.1% (SD 12.6, n = 5) and 96.4% (SD 3.2, n = 5), respectively. DAS was detected in none of the cereal and pulse products.     

Antimicrobial activity of clove (Syzgium aromaticum) oil in inhibiting Listeria monocytogenes on chicken frankfurters

The ability of Listeria monocytogenes to survive and grow at refrigeration temperature in some ready to eat (RTE) poultry products is a public health concern. The inhibitory effect of clove oil (1% and 2%, v/w) applied to the surface of RTE chicken frankfurters was determined on seven strains of L. monocytogenes inoculated at low (10²–10³ cfu/g) or high cell numbers (10⁴–10⁶ cfu/g), and stored at 5 °C for 2 weeks or at 15 °C for 1 week. All strains of L. monocytogenes survived and grew on control frankfurters at 5 °C and 15 °C but growth was inhibited under both storage conditions in the presence of either 1% or 2% clove oil. Depending on the sensory considerations, the addition of clove oil to frankfurters may be an effective strategy to control L. monocytogenes in chicken frankfurters.     

Inactivation of a non-pathogenic strain of E. coli by ionising radiation

Ionising radiation is an effective method to reduce pathogenic E. coli O157:H7 in meat and poultry products. Radiation sensitivity of bacteria, however, depends on several factors. After applying an irradiation dose of 1 kGy to cultures of the non-pathogenic strain of E. coli, DSM 498, grown and irradiated in nutrient broth, reductions of 3–4 decimal units were achieved (D₁₀ = 0.27 kGy). If grown on minced turkey meat, however, reduction rates were lower (D₁₀ = 0.47 kGy). Even lower reduction rates were obtained during irradiation of frozen meat (D₁₀ = 0.72 kGy) compared to treatments at cooling temperatures (D₁₀ = 0.48 kGy). For data evaluation, both, first order reduction kinetics and the Weibull model were compared. The results emphasise the necessity to determine inactivation kinetics in food matrices of target extrinsic factors (e.g. temperature).     

Estimate of aflatoxin M1 exposure in milk and occurrence in Brazil

The presence of aflatoxin M₁ (AFM₁) was investigated in 125 samples of powdered milk, pasteurized milk and ultra high treated (UHT) milk in the city of São Paulo, and estimates of AFM₁ intake were assessed. The samples were analysed using an immunoaffinity column for cleanup and a HPLC-FLD for determining AFM₁. The quantification limit was 10 ng/kg. AFM₁ was found in 119 (95.2%) at levels ranging from 10 to 200 ng/kg with mean concentration of 31 ng/kg. The average daily intake estimated for AFM₁ was 1 ng/kg bw per day for children and 0.188 ng/kg bw per day for adults.     

Histamine contents and histamine-forming bacteria in sufu products in Taiwan

Twelve white and ten brown sufu products sold in the supermarkets in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH, salt content, and aerobic plate count (APC) in all samples ranged from 4.6 to 6.6, 6.2% to 12.0%, and 3.0 to 7.9 log CFU/g, respectively. None of these samples contained total coliform and Escherichia coli. Although the average content for each of the nine biogenic amines in all samples was less than 5 mg/100 g, only one brown sufu sample had histamine content (15.8 mg/100 g) greater than the 5.0 mg/100 g allowable limit suggested by the US Food and Drug Administration. Two histamine-producing bacterial strains capable of producing 1.33 mg/100 ml and 1.34 mg/100 ml of histamine in trypticase soy broth (TSB) supplemented with 1.0% l-histidine (TSBH) were identified as Bacillus subtilis by 16S rDNA sequencing with PCR amplification.     

Histamine contents and histamine-forming bacteria in natto products in Taiwan

Thirty-six natto products imported from Japan and three domestic natto products sold in the supermarkets in southern Taiwan were purchased and tested to determine the occurrence of histamine and histamine-forming bacteria. The levels of pH and aerobic plate count (APC) in all samples ranged from 4.9 to 7.3 and 7.8 to 11.2 log CFU/g, respectively. None of these samples contained total coliform and E. coli. Although the average content for each of the nine biogenic amines in all samples was less than 5 mg/100 g, seven of them (17.9%) had histamine content greater than 5 mg/100 g, allowable limit suggested by the US Food and Drug Administration for scombroid fish and/or product. Four histamine-producing bacterial strains capable of producing 13.4 ppm to 17.5 ppm of histamine in trypticase soy broth (TSB) supplemented with 1.0% l-histidine (TSBH) were identified as Bacillus subtilis (two strains) and Staphylococcus pasteuri (two strains) by 16S rDNA sequencing with PCR amplification. To our knowledge, this is the first report to demonstrate the occurrence of histamine-forming bacteria in natto products.