Evaluation of real-time PCR assays for detection and quantification of fraudulent addition of bovine milk to caprine and ovine milk for cheese manufacture

Many typical Italian cheeses made from ovine milk are certified as Protected Designation of Origin (PDO). Because caprine and ovine milk production is limited, the fraudulent addition of cows' milk is widespread. In addition, some compounds in bovine milk have high allergenic potential; therefore, such fraud also has implications for consumer health. In this study, a real-time polymerase chain reaction (real-time PCR) test was developed to detect and quantify cow's milk in caprine and ovine cheeses, based on two target genes. The mitochondrial Cytochrome b gene (Cytb) of Bos taurus was used to detect and quantify bovine DNA. The nuclear gene myostatin (Myo), nuclear ribosomal gene 18S, or mitochondrial gene 16S were used alternatively as universal reference markers. Caprine (n = 30) and ovine (n = 51) cheese samples were purchased and analyzed and most were shown to be contaminated by bovine milk. Pairwise analysis of quantification data using a Spearmann Rank Correlation test demonstrated a highly significant correlation between data obtained with the different reference assays.     

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%.     

Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique

Koumiss is a popular dairy product in many lands, traditionally prepared from mare milk with spontaneous fermentation. Mare milk and its fermented derivates are more expensive than cow milk and its fermented derivates, and the possibility exists for producers and dealers to adulterate equine products with bovine items. In this work, we described the development of a triplex real-time PCR based on species-specific TaqMan probes for identification of bovine and equine DNA in milks and dairy products. In addition, a novel designed endogenous control was simultaneously amplified to eliminate possible false negatives. With this methodology, bovine and equine DNA were specifically identified by employing developed primers and probes. The limits of detection of this method were 0.001 ng for cow milk, yogurt, and mare milk, and 0.005 ng for sour soup and koumiss, respectively. In addition, the triplex real-time PCR assay for authentication of animal-derived products was effectively validated using binary DNA and milk mixtures, exhibiting well in terms of specificity, sensitivity, and reproducibility. In short, the triplex PCR assay was verified to be a time-saving and money-saving technique for the identification of bovine and equine DNA in milks and dairy products.     

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%.     

A competitive enzyme-linked immunosorbent assay for detection of bovine milk in ovine and caprine milk and cheese using a monoclonal antibody against bovine beta-casein.

A competitive ELISA (enzyme-linked immunosorbent assay) was performed to detect and quantify bovine milk in ovine and caprine milk and cheese using a monoclonal antibody (AH4 MAb) against bovine beta-casein. Ovine or caprine milk and cheese containing bovine milk were added simultaneously with the AH4 MAb to the wells of a microtiter plate that had been previously sensitized with commercial bovine beta-casein. The bovine caseins in milk or cheese samples compete with the bovine beta-casein bound to the plate for the AH4 MAb binding sites. Further immunorecognition of AH4 MAb bound to the bovine beta-casein immobilized onto the plate was attained with rabbit anti-mouse immunoglobulin conjugated to peroxidase. Subsequent enzymic conversion of the substrate showed clear differences in absorbance values during assay of mixtures of ovine and caprine milk and cheese containing various amounts of bovine milk. The competitive ELISA developed in this work allows the quantitative detection of bovine milk in ovine and caprine milk and cheese samples in the range of 0.5 to 25% of substitution.     

Quantitative detection of soybean in meat products by a TaqMan real-time PCR assay

In the present work, we propose a normalised real-time quantitative PCR assay to determine the addition of soybean to meat products. The method proved to be a powerful tool for the quantification of soybean protein (dry basis) in the range of 0.01% to 6%, being successfully in-house validated. Its application was effective in the analysis of several meat products, indicating 2% of non-compliance with the food allergen labelling legislation, and some inconsistencies when comparing the declared with estimated amounts of soybean. This work highlights the importance of efficient tools to assess labelling statements of meat products, avoiding fraudulent practices.     

肉制品中鸭源性成分TaqMan探针实时荧光PCR检测方法的建立与应用

目的 建立TaqMan探针实时荧光PCR法快速检测肉制品中鸭源性成分的分析方法.方法 以鸭生长激素(growth hormone,GH)基因为靶基因,基于特异性保守序列设计引物和TaqMan探针,优化反应体系和反应条件,建立了鸭源性成分实时荧光PCR(real-time PCR)检测方法.结果 所建立的real-tim...     

Development of triplex PCR for simultaneous detection of soybean and wheat

Food Science and Biotechnology - There is a constant demand for an effective detection method of major allergens, such as soybean (Glycine max) and wheat (Triticum aestivum). In this study, a...     

Duplex real-time PCR for authentication of anglerfish species

A duplex real-time PCR assay based on TaqMan probe technology was developed for the authentication of two commercially important anglerfish species (Lophius budegassa and L. piscatorius). This technique uses the cytochrome oxidase subunit I and allows the unambiguous identification of this species. It is notable for its simplicity, rapidity, highest potential for automation and minor risk of contamination. The TaqMan real-time PCR is currently the most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of these species. The method can be applied to all kind of products as fresh, frozen, and precooked products. The developed methodology, which uses two specific primer–probe sets, has been validated and subsequently applied to 40 commercial samples labeled as any anglerfish species in order to determinate whether the species used for their manufacturing correspond to these species. The methodology herein developed is appropriate to clarify questions related with the correct labeling of commercial products, traceability in commercial trade, and fisheries control.     

Isolation and characterisation of caseinmacropeptide from bovine,ovine, and caprine cheese whey

Based on the thermostability of caseinmacropeptide (CMP) and on the differences in molecular weight of its polymeric and monomeric forms, we have developed a method of isolating CMP from whey protein concentrate (WPC) and from liquid sweet cheese whey, particularly suited to large-scale industrial production. This procedure includes acidification and heating and ultrafiltration of cheese whey to give a CMP powder with a protein content from 75 to 79%. CMP obtained from WPC and from pure bovine, ovine, and caprine cheese whey were characterized. The CMP recovery was close to 71-76% and the purity determined by RP-HPLC ranged from 75 to 90%.     

A study of the growth of Lactobacillus acidophilus in bovine, ovine and caprine milk

The fermentation of reconstituted bovine, ovine and caprine milks with Lactobacillus acidophilus was investigated. Better growth of Lb. acidophilus was observed in ovine milk, but higher acidities developed in caprine milk. The high acidity produced after 12 h of fermentation (pH 3.9) created a hostile environment for the survival of the microorganism in caprine milk.     

实时荧光定量PCR技术在肉及肉制品中的应用

实时荧光定量PCR技术作为一种DNA定量检测的工具,可应用于肉制品种类鉴别和定量分析中。相比于传统感官和理化的鉴别方法,该技术具有灵敏性高、特异性强、操作简便、省时省力等特点。使用Taq Man荧光探针和荧光染料可以直接测定PCR循环后产物的总量,确定扩增DNA片段的种类及数量,从而确定肉品种类及添加量。因此,可以将该技术应用到肉制品成分及安全的检测中。本文介绍了实时荧光定量PCR技术的基本原理,主要综述了该技术在肉品种类鉴定方面的应用,简述其在肉制品细菌污染检测的应用,并对该技术在肉类研究中的应用进行展望。      

Feta cheese texture: the effect of caprine and ovine milk concentration

Feta cheese was produced commercially with different caprine to ovine milk ratios. Milk fat concentrations, moisture and salt contents were similar for all the batches. However, the hardness and adhesive characteristics of the cheeses differed in relation to the milk ratio. The hardness of the cheese appeared to be correlated to increased goat milk content. Cryo-scanning electron microscopy (cryo-SEM) of the cheese samples showed that feta cheese with a higher proportion of caprine milk had a more compact and less porous appearance than feta produced from purely ovine milk. This difference in cheese structure helps to explain the difference in hardness between the samples.     

Oxidative stability and antioxidant activity of bovine,caprine, ovine and asinine milk

This study compares the oxidative stability of fat isolated from bovine, caprine, ovine and asinine milk as well as the antioxidant activity of casein and whey from these milks. Fat from bovine and asinine milk showed the highest oxidative stability. The antioxidant activity of casein and whey was examined before and after an in vitro digestion process. Whey from asinine milk showed the highest antioxidant activity. The antioxidant activity of whey and casein increased after simulated intestinal digestion. In addition, the results of this study showed that asinine whey exhibited radical scavenging activity comparable with the strong synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).     

Evaluation of TaqMan PCR assay for detecting Salmonella in raw meat and shrimp.

We evaluated the TaqMan Salmonella amplification/detection kit from PE Applied Biosystems, which uses a polymerase chain reaction (PCR) assay for rapid detection of Salmonella in food samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The system's sensitivity and specificity were evaluated using 42 serotypes of 68 Salmonella strains isolated from fecal samples from patients in Tokyo, Japan, and 39 non-Salmonella strains in 22 genera. There were no false-negative or false-positive results. This PCR assay can detect 3 CFU per PCR tube of Salmonella in pure culture (120 CFU/ml of TSB culture). PCR signals were attenuated with artificially contaminated shrimp, but a similar detection limit was obtained. TaqMan's performance was tested with 100 meat and chicken samples purchased from stores in Tokyo. Overall, two of the DNA extraction protocols (the Chelex and EnviroAmp methods) worked equally well, with some exceptions. Of the 100 samples analyzed, 10 were positive for Salmonella with both conventional culture methods and the kit and 89 were negative with both. One sample was negative by the culture method but positive by the kit assay. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry. With this system, food samples can be analyzed for Salmonella in less than 20 h.     

Changes during ripening in chemical composition,proteolysis, volatile composition and texture in Kashar cheese made using raw bovine,ovine or caprine milk

Kashar cheeses were manufactured from pure ovine (OV), bovine (BV) and caprine (CP) milk, and the chemical composition, cheese yield, proteolysis, hardness, meltability and volatile composition were studied during 90 days. Gross chemical composition, cheese yield and level of proteolysis were higher in OV cheeses than those of BV or CP cheeses. Glu, Val, Leu, Phe and Lys were the most abundant free amino acids (FAA) in the samples, and the concentrations of individual FAA were at the highest levels in OV cheeses with following BV and CP cheeses. Urea‐PAGE patterns and RP‐HPLC peptide profiles of the BV cheeses were completely different from the small ruminants’ milk cheeses (OV or CP). Higher and lower hardness and meltability values were observed in CP cheeses, respectively. OV cheeses resulted in higher levels of the major volatile compounds. In conclusion, the Kashar cheese made using OV milk can be recommended due to high meltability, proteolysis and volatiles.     

An investigation of the plasmin–plasminogen system in caprine milk and cheese

The variability in activities of plasmin (PL) and plasminogen (PG) in bulk milk samples from different breeds of goats was investigated. The mean PL activity was higher (19.56±4.72 U g⁻¹) than in milk from other ruminant species, while PG-derived activity was, surprisingly, lower (12.84±5.31 U g⁻¹). No significant differences in PL and PG levels were observed among goat breeds, but PL activity and PL/PG ratio increased in late lactation compared with early lactation. PL activities in pilot-scale curds (acid or rennet) and in some Italian cheeses made from caprine milk were also measured. Generally, acid curds and cheeses had lower residual PL and PG-derived activities than rennet curds and semi-hard cheeses. In addition, the PL/PG ratio was greatly reduced in rennet curds, due to a higher extent of PG-derived activity. Measurement of proteolysis of β-casein in curds confirmed that more extensive proteolysis occurred in rennet curds, which had higher residual PL activities.     

Development of rapid and highly specific TaqMan probe-based real-time PCR assay for the identification and enumeration of Lactobacillus kefiri in kefir milk

Lactobacillus kefiri is one of the key functional lactic acid bacteria in kefir milk. We designed a novel real-time PCR primer/probe set, LK_508 targeting the recA gene, for the rapid identification and enumeration of L. kefiri. In inclusivity and exclusivity test using standard strains and kefir isolates, only the 9 tested L. kefiri strains were positive, with the remaining 38 closely related microorganisms testing negative, thus indicating 100% sensitivity and specificity of the assay. The population of L. kefiri was 3.77, 4.30, 4.79, and 5.63 log cfu mL⁻¹ of kefir milk fermented at 25 °C with 5% grain-milk ratio for 12, 24, 36, and 48 h, respectively. The newly developed qPCR assay could be applied to investigate the quantitative relationship of kefir microbiota in fermentation process.     

Comparison of the characteristics of halloumi cheese made from ovine milk, caprine milk or mixtures of these milks

The aim of this work was to study the rheological, physicochemical and organoleptic characteristics of four types of halloumi cheese made from pure ovine milk, pure caprine milk or mixtures containing 15 or 30% caprine milk. All the milks were standardized for casein to fat ratio. Pure ovine milk gave the highest yield of halloumi cheese and caprine the lowest. No significant differences in acidity, pH, moisture, total protein and ash contents of the four types of cheese were found. In contrast, significant differences in soluble proteins, non-protein nitrogen, acid degree value and calcium contents were noted. Rheological examinations showed the hardness and the force at the point of fracture of caprine halloumi cheese to be lower than those of the other types of cheese. The same cheese presented the highest compression at the point of fracture and cohesiveness, while there were no differences in gumminess or chewiness. Members of the assessment panel showed a preference for halloumi cheese made from 30% caprine milk and 70% ovine milk.     

Real-time TaqMan PCR for quantitative detection of cows’ milk in ewes’ milk mixtures

A real-time PCR based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene was developed and evaluated for the detection and quantification of cows’ milk in raw and heat-treated cow/sheep milk mixtures. The method combines the use of cow-specific primers that amplify a 252 bp fragment from cow DNA, and mammalian-specific primers amplifying a 428 bp fragment from mammalian species DNA, which is used as an endogenous control. The method measures PCR product accumulation through a 6-carboxyfluorescein-labeled fluorogenic probe (TaqMan). A comparison of the cycle number at which mammalian and cow-specific PCR products were first detected, in combination with the use of reference standards of known bovine content, allowed the determination of the percentage of cows’ milk in mixtures. Experimental raw and heat-treated binary mixtures were analyzed, demonstrating the specificity and sensitivity of the assay for detection and quantification of cows’ milk in the range 0.5–10%.