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

López-Calleja, Inés; González, Isabel; Fajardo, Violeta; Martín, Irene; Hernández, Pablo E.; Garcı́a, Teresa; Martı́n, Rosario

doi:10.1016/j.idairyj.2006.09.005

Cited by 55 publications

(36 citation statements)

References 25 publications

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“…4b the corresponding standard curve (considering 50 ng DNA in the PCR system as 100%). The lowest quantifiable level of bovine DNA was about 5 pg; linearity (R 2 00.999) and efficiency (93.5%) were acceptable and comparable to other reported results [19,20].…”

Section: Detection Of Cow's Milk In Cheese Using Quantitative Real-tisupporting

confidence: 86%

“…As all methods based on milk fat composition have the great disadvantage that any adulteration with skimmed cow's milk cannot be detected, most techniques currently used in adulteration control are based on the analysis of the protein fraction, the main ones are immunology, electrophoresis, and chromatography [2,11,12]. In recent years, DNAbased methods (e.g., species-specific PCR, allele-specific PCR, qPCR) are more and more used both for qualitative detection of cow's milk and also for quantitative determination of cow's milk percentage in mixed-milk dairy products [14,[19][20][21][22]26]. Moreover, due to the current trend towards soy milk (as alternative for people suffering from milk protein allergy), these dairy-like products have also to be controlled by food authorities.…”

Section: Discussionmentioning

confidence: 99%

“…With special regard to the critical range from 40% to 60% (important for the decision whether a commercial sample is adulterated or not), the respective log (DNA) of 1.602 to 1.778 correspond to C t values of 22.23 to 21.57 only. This means that these two "threshold" levels (40% and 60% cow's milk, respectively) are very close together (within 0.66 C t values only), which is still a challenge for food analysts using qPCR as molecular biological tool for quantitative determination of cow's milk percentage in mixed-milk cheese samples [19][20][21][22]30].…”

Section: Detection Of Cow's Milk In Cheese Using Quantitative Real-timentioning

confidence: 99%

“…A high number of other analytical techniques (e.g., electrophoretic, chromatographic, immunological, and molecular biological methods) have also been used for qualitative (and partly also quantitative) species authentication in dairy products [2,[11][12][13][14][15][16]. Moreover, peptidomic approaches based on liquid chromatography and mass spectrometry [17,18], and also quantitative real-time polymerase chain reaction (qPCR) methods for the quantitative determination of milk of different species were reported recently [19][20][21][22].…”

Section: Introductionmentioning

confidence: 99%

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Quantification of cow’s milk percentage in dairy products – a myth?

2012

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The substitution of ewe's and goat's milk for cheaper cow's milk is still a fraudulent practice in the dairy industry. Moreover, soy-based products (e.g., soy milk, yoghurt) have to be checked for cow's milk as they are an alternative for people suffering from an allergy against bovine milk proteins. This work reports the evaluation of different protein-based electrophoretic methods and DNA-based techniques for the qualitative detection as well as the quantitative determination of cow's milk percentage in dairy and soy milk products. Isoelectric focusing (IEF) of γ-caseins using an optimized pH gradient was appropriate not only for the detection of cow's milk, but also for an estimation of cow's milk percentage in mixed-milk cheese varieties. Urea-polyacrylamide gel electrophoresis (PAGE) proved the method of choice to detect cow's milk in soy milk products, whereas IEF and SDS-PAGE of proteins were not applicable due to false-positive results. Polymerase chain reaction (PCR) analysis was used to confirm the results of protein-based electrophoretic methods. Problems inherent in quantitative analysis of cow's milk percentage using protein-based techniques and even more using DNA-based methods were emphasized. Applicability of quantitative real-time PCR for the determination of cow's milk percentage in mixed-milk cheese was shown to be hampered by several factors (e.g., somatic cell count of milk; technological parameters influencing the final DNA concentration in ripened commercial cheese samples). The implementation of certified reference standards (of major relevant cheese groups) containing 50% cow's milk was urgently recommended to enable at least a yes/no decision in commercial mixed-milk cheese samples.

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Section: Detection Of Cow's Milk In Cheese Using Quantitative Real-tisupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Section: Detection Of Cow's Milk In Cheese Using Quantitative Real-timentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Quantification of cow’s milk percentage in dairy products – a myth?

2012

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“…Among the genetic markers used for species traceability of dairy products, the mitochondrial DNA (mtDNA) was mainly studied due to its unique characteristics among species (Bottero et al, 2003;European Commission, 2008;Plath et al, 1997). In fact, using species-specific primers, several authors have used different types of PCR to differentiate cows', goats', and ewes' milk or cheeses (Bottero et al, 2003;Dalmasso et al, 2011;Feligini et al, 2005;Mafra et al, 2004;Mafra et al, 2007;Maudet and Caberlet,2001;Mayer, 2005;López-Calleja et al, 2004;López-Calleja et al, 2005b;López-Calleja et al, 2007b;López-Calleja et al, 2007c).…”

Section: Introductionmentioning

confidence: 99%

12S rRNA mitochondrial gene as marker to trace Sicilian mono-species dairy products

et al. 2016

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A B S T R A C TFor a rapid, specific and sensitive identification of cows', ewes' and goats' milk in mono-species Sicilian dairy products, species-specific duplex-PCR protocol was applied. DNA samples from blood and experimental cheeses of Sicilian autochthonous breeds were extracted to amplify the 12S rRNA (and part of 16S rRNA in case of Ovis aries) mitochondrial species-specific gene fragment. The use of species-specific primers for Bos taurus, Capra hircus and Ovis aries species, after electrophoresis on agarose gel, yielded fragments of 256 bp, 326 bp and 172 bp, respectively. Amplification by duplex-PCR of DNA pools from two species showed detection thresholds of 0.1% of "contaminant" DNA in each mixture. Finally, duplex-PCR assay was applied to experimental cheeses in order to detect the minimum threshold of DNA belonging to one species in cheese made with milk of two species. The results showed a sensitive threshold of 0.1% of ewes' milk in cows' and goats' cheeses, 0.1% of cows' milk in ewes' and goats' cheeses, and finally 0.1% of goats' milk in cows' and ewes' cheeses. The proposed assay represents a rapid and straightforward method of species traceability for the detections of adulteration in Sicilian mono-species dairy products.

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Detection of animal allergens in foods

Rizou¹

2013

Food Allergen Testing

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

Cited by 55 publications

References 25 publications

Quantification of cow’s milk percentage in dairy products – a myth?

Quantification of cow’s milk percentage in dairy products – a myth?

12S rRNA mitochondrial gene as marker to trace Sicilian mono-species dairy products

Detection of animal allergens in foods

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