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.     

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.     

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.     

Levels of aflatoxin M1 in milk,cheese consumed in Kuwait and occurrence of total aflatoxin in local and imported animal feed

A total of 321 milk samples (177 fresh, 105 long-life and 27 powdered milk, and 12 human milk), 40 cheese samples and 84 feed samples were analyzed for aflatoxin M1 (AFM1) and total aflatoxin. The samples were collected randomly during January 2005–March 2007, from Kuwaiti markets. The method used was ELISA technique. Results showed that all fresh milk samples except one were contaminated with AFM1 ranging from 4.9 to 68.7 ng/kg. Eight samples exceeded the EC’s regulatory limit. For the long-life milk samples, the ranges of AFM1 were from below the detection limit to 88.8 ng/kg, with four samples above the action limit of the EC. In the powdered milk samples, AFM1 ranged from 2.04 to 4.14 ng/kg. Of the human milk samples, only five were contaminated, with AFM1 levels ranging from 8.83 to 15.2 ng/kg with a mean 9.7 ng/kg. The cheese samples recorded 80% contamination with AFM1 with a range 23.8–452 and mean of 87.6 ng/kg, with one sample being above the regulatory limit (250 ng/kg) while the feed samples, showed 79.8% contamination with total aflatoxin.     

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.     

PCR-based methodology for the authentication of grouper (Epinephelus marginatus) in commercial fish fillets

In an attempt to authenticate commercial frauds in fish fillets of grouper (Epinephelus marginatus) by being substituted with those of Nile perch (Lates niloticus) and wreck fish (Polyprion americanus), a polymerase chain reaction (PCR) method, based on the design of specific primers of these species was explored here. The oligonucleotides, which were designed from the 12S ribosomal RNA gene, generated PCR fragments of 100, 138 and 169 bp length for grouper, wreck fish and Nile perch respectively. The specificity of the primers was tested against more than 50 different fish species. Moreover, in this work 70 commercial fish fillets samples were analysed by this methodology; 58 out of them confirmed to be incorrectly labelled. The results suggest that this method may be a reliable tool for the detection of grouper adulteration. Also, this technique may help implementation of traceability systems in the seafood industry.     

Aflatoxin M1 in raw,pasteurized and powdered milk available in the Syrian market

The incidence of contamination of aflatoxin M1 (AFM1) in milk samples collected from the Syrian market was investigated by using the competitive enzyme linked immunosorbent assay (ELISA) technique. A total of 126 samples composed of raw cow milk (74 samples), raw sheep milk (23), raw goat milk (11), pasteurized cow milk (10) and powdered milk (8) showed that 80% of tested samples were contaminated with various levels of AFM1 ranging from >20 to 765 ng/l. Percentages of AFM1-contaminated samples exceeding the American, Syrian and European tolerance limits were 22%, 38% and 52%, respectively.The range of contamination was relatively higher in pasteurized milk than in raw cow and sheep milk. 80% of AFM1-contaminated pasteurized cow milk samples exceeded the European tolerance limit with a range of contamination between 89 and 765 ng/l. Percentages of contaminated raw cow, sheep and goat milk exceeding the European tolerance limit were 59%, 24% and 14%, respectively.Milk powder was almost free of AFM1 contamination with only one sample containing a concentration lower than the European tolerance limit (12 ng/l).Extrapolation of aflatoxin B1 (AFB1) from AFM1 levels of contamination in milk samples indicates that contamination in dairy cattle feeds may range from 0.5 to 47.8 μg/kg.     

Targeting double genes in multiplex PCR for discriminating bovine,buffalo and porcine materials in food chain

Beef, buffalo and pork are the major meat of economic, religious and health concern. Current methods to authenticate these materials in food chain are based on mainly single gene targets which are susceptible to break down by food processing treatments. We, for the first time, described here a double gene targeting short-amplicon length multiplex polymerase chain reaction assay for discriminating bovine, buffalo and porcine materials in a single assay platform. The advantage of the assay is evidenced in terms of fidelity, cost and time since it is highly unlikely that two different targets would be missing even in a decomposed specimen. Detection of multiple targets in a single assay definitely saves analytical cost and time. Mitochondrial cytochrome b (cytb) and ND5 genes were targeted and six different targets (length: 90–146 bp), two for each of cow (120 and 106bp), buffalo (90 and 138bp) and pig (73 and 146bp), were amplified from raw, boiled, autoclaved and microwaved cooked meat under pure and mixed matrices. The detection limit was 0.02 ng DNA under pure states and 0.1% meat in binary mixtures and meatball products. Screening of Malaysian meatball products revealed all beef products were buffalo positive in which 35% were totally replaced. In contrast, all pork products were found uncontaminated from beef and buffalo.     

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.     

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).     

Characteristics of Batzos cheese made from raw,pasteurized and/or pasteurized standardized goat milk and a native culture

Experimental cheeses were made from raw (R), raw with starter (RS), pasteurized with starter (PS) and standardized, pasteurized with starter (PSS) goat milk to study the influence of the starter and pasteurization on the quality of Batzos cheese. Lactococcus lactis subsp lactis strains from raw milk cheese used as starter proliferated significantly (P < 0.05) only in PSS cheese. Heat treatment lowered (P < 0.05) the levels of most microbial groups. However, TC and LAB counts were higher (P < 0.05) in PSS cheese and this was accompanied by lower (P < 0.05) pH; thus, a higher rate of decrease of undesirable microorganisms in PSS cheese was recorded. Degradation of αs-casein was in R > RS and in PS > PSS, while a small reduction of β-casein during ripening and storage was recorded. The proportions of aminoacids and lipolysis products increased throughout ripening and storage.     

Aflatoxin M1 and ochratoxin A in human milk in Ribeirão Preto-SP,Brazil

The objective of this study was to determine the extent of aflatoxin M₁ (AFM₁) and ochratoxin A (OTA) contamination in human breast milk in the city of Ribeirão Preto, São Paulo State, Brazil. During 2012, 100 samples of human milk were collected at the local Human Milk Bank. The method comprised, immunoaffinity column purification and isolation, liquid chromatography separation and fluorescence detection. The average percentage recoveries of AFM₁ and OTA spiked at 20 and 50 ng/L in control human milk were 78.1 ± 11.7% and 73.7 ± 9.6%, respectively. The average relative standard deviations of AFM₁ and OTA spiked at the same levels were 11.7 and 9.6% respectively. The limits of detection was 0.3 ng/L for AFM₁ and OTA. The limit of determination was 0.8 ng/L for both mycotoxins. This method was used to analyze 100 human milk samples, of which, two samples were found to contain AFM₁ at level greater than 0.3 ng/L. OTA was detected in 66 samples (66%), wherein 32 were above the limit of detection and 34 were in the range of from 0.8 to 21 ng/L. Results of our study indicate that breast-fed Brazilian infants had only an insignificant exposure to AFM₁ and OTA.     

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.     

Changes in the concentration of aflatoxin M1 during manufacture and storage of White Pickled cheese

In this study White Pickled cheese was produced from cow’s milk contaminated artificially with aflatoxin M₁ (AFM1) at two different levels, 1.5 and 3.5 μg/kg (ppb), and the effects of process stages on the AFM1 contents were investigated. Pasteurization at 72 °C for 2 min caused losses of AFM1 about 12% and 9%, respectively, in milk contaminated with 1.5 μg/kg AFM1, and 3.5 μg/l AFM1. These losses were found to be statistically significant (P < 0.01). After the cheese production, about 56% and 59% of total AFM1 remained in cheese–curd while about 32% of total AFM1 transferred to the whey for both 1.5 μg/kg and 3.5 μg/kg AFM1 contaminated milk. After 3-month storage in brine, AFM1 content of cheeses produced from 1.5 and 3.5 μg/kg AFM1 contaminated milks decreased by 2.9% and 2.8%, respectively. Changes in AFM1 content of cheese samples were found statistically insignificant (P > 0.05 and P > 0.01) for 3-month storage periods.     

A Turkey survey of hygiene indicator bacteria and Yersinia enterocolitica in raw milk and cheese samples

In this research, a total of 200 dairy (raw milk, cheese) samples obtained from Ankara, were examined for the presence of Yersinia spp., total coliform and Escherichia coli. As expected, raw milk 55% (55/100) were significantly contaminated with Yersinia spp., than cheese samples 14% (14/100). Y. enterocolitica was the most commonly isolated species, and was recovered from 47.3% in raw milk 35.7% in cheese samples. The other Yersinia spp. were identified as Y. frederiksenii (31.0%, 21.4%), Y. kristensenii (12.7%), Y. intermedia (7.2%, 7.1%) and atypical Yersinia spp. (1.8%, 35.7%) in raw milk and cheese samples, respectively. All the samples of cheese examined were negative for Y. kristensenii. All Y. enterocolitica strains tested gave negative results in the autoagglutination tests and crystal violet binding test.Furthermore total coliform bacteria and E. coli were investigated in these samples. According to the analysis results, most of the samples containing Yersinia spp.were found high number of viable count cell total coliform than E. coli. Total coliform bacteria and E. coli, were detected (3.0 × 10⁴ cfu/ml and 1.5 × 10⁴ cfu/ml) in four milk samples containing Y. enterocolitica while, they were detected (2.5 × 10⁴ cfu/ml and 1.0 × 10⁴ cfu/ml) in eight milk samples containing Y. enterocolitica. In the present study, total coliform bacteria and E. coli were detected >1.1 × 10⁵ cfu/g in six cheese samples containing Y. enterocolitica and atypical Yersinia spp. The results indicate that Yersinia spp. is more likely to be isolated from foods with a high level of coliforms than from foods with low coliform counts.     

Determination of aflatoxin M1 in dairy cattle milk in Khartoum State,Sudan

Aflatoxins are a group of mycotoxins that contaminate various types of food and feedstuff leading to health risk in both humans and animals. Aflatoxin M1 (AfM1), the major metabolite of AfB1, was determined in dairy cattle milk samples of Khartoum State of Sudan using high-performance liquid chromatography (HPLC) with fluorescence detection. A total of 44 bulk dairy cattle milk samples were collected and analyzed. The percentage of AfM1 contamination has been found in 42/44 (95.45%) samples with contamination level ranging between 0.22 and 6.90 μg L⁻¹ and average concentration of 2.07 μg L⁻¹. AfM1 contamination in the samples of dairy cattle milk of Khartoum State of Sudan appears to be prevalent and may pose a public health problem at the moment. Awareness must be conveyed to producers, handlers and specialists.     

Aflatoxin M1 contamination in dairy products marketed in Iran during winter and summer

In the present study, 298 dairy product samples consisting of pasteurized milk (91 samples), yoghurt (68 samples), white cheese (72 samples), butter (31 samples) and ice cream (36 samples) collected from popular markets in four large Iranian cities were examined for aflatoxin M₁ (AFM₁) by thin layer chromatography (TLC) technique. The toxin was detected in 66 (72.5%) pasteurized milk samples (mean: 0.052 μg/l; range: 0.013–0.250 μg/l), 45 (66.1%) yoghurt samples (mean: 0.032 μg/kg; range: 0.015–0.119 μg/kg), 59 (81.9%) white cheese samples (mean: 0.297 μg/kg; range: 0.030–1.200 μg/kg), 8 (25.8%) butter samples (mean: 0.005 μg/kg; range: 0.013–0.026 μg/kg) and 25 (69.4%) ice cream samples (mean: 0.041 μg/kg; range: 0.015–0.132 μg/kg). The concentration of AFM₁ in 36.2%, 20.6%, 30.5%, 9.6% and 27.7% of pasteurized milk, yoghurt, white cheese, butter and ice cream samples, respectively, were higher than Iranian national standard limits. Levels of AFM₁ in samples of pasteurized milk, yoghurt, butter and ice cream collected in winter were significantly higher (P < 0.05) than those collected in summer. In the case of white cheese, level of AFM₁ was higher in winter than in summer, but the difference was not statistically significant (P > 0.05). The results indicated that the contamination of the dairy products in such a level could be a serious public health problem at the moment.     

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.     

Occurrence of aflatoxin M1 in raw milk in South Korea using an immunoaffinity column and liquid chromatography

The level of aflatoxin M₁ (AFM₁) contamination in raw milk produced in South Korea was investigated using immunoaffinity column chromatography and high performance liquid chromatography with fluorescence detector. A total of 100 raw milk samples were collected from 100 cattle ranches located in three different provinces of South Korea. Forty eight out of 100 raw milk samples contained AFM₁ at low level (0.002–0.08 μg/L) with mean value of 0.026 μg/L. Among the AFM₁ contaminated samples, 29 raw milk samples contained only traceable amount of AFM₁ below the limit of LOQ, 0.02 μg/L. None of samples exceeded the maximum level (0.5 μg/L) of Korean regulation for AFM₁ in milk. The limit of detection was 0.002 μg/L. The result of recovery test with 0.5 μg/L AFM₁ in raw milk sample was 96.3% (SD 3.6, n = 5). This is the first pioneering study to investigate the level of AFM₁ in raw milk used in dairy industries in South Korea.