PCR detection of cows’ milk in water buffalo milk and mozzarella cheese

López-Calleja, Inés; Alonso, Iago Justo; Fajardo, Violeta; Rodrı́guez, Miguel Á.; Hernández, Pablo E.; Garcı́a, Teresa; Martı́n, Rosario

doi:10.1016/j.idairyj.2004.12.003

Cited by 81 publications

(70 citation statements)

References 20 publications

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“…In this study, Capmp1a/Capmp1b and Capmp2a/ Capmp2b primers specific for C. abortus pmp genes were combined with bovine and ruminant host-spe- cific primers for mitochondrial 12S rDNA reported previously (Lopez-Calleja et al 2005). There are different strategies for using internal controls in PCR.…”

Section: Discussionmentioning

confidence: 99%

“…CPU 65942) and were designed in this study using Primer3 (Yuryev 2007). Bovine-(12SM-FW and 12SBT-REV2) and ruminant-specific primers (12S-FW and 12S-REV) were used as internal controls (Lopez-Calleja et al 2005). Primer properties are shown in Table 1. PCR procedures and protocols were optimised by adjusting the concentrations of PCR components such as MgCl 2, primers, Taq DNA polymerase and 10× PCR buffer concentrations in the mix and by testing different annealing temperatures and times according to the recommendations of Henegariu et al (1997).…”

Section: Methodsmentioning

confidence: 99%

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Development of Polymerase Chain Reaction assays with host-specific internal controls for Chlamydophila abortus

Cantekin¹,

Solmaz²,

Ergün³

et al. 2015

Vet. Med. - Czech

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ABSTRACT:Chlamydophila abortus (C. abortus) is one of the most important infectious agents causing abortion in ruminants. The bacterium is obligately intracellular, cannot grow on agar, but it needs cell culture or embryonated eggs for growth. Therefore, culture-independent detection methods such as the polymerase chain reaction are increasingly important and needed. The aim of this study was to develop a polymerase chain reaction assay with an internal control for detection of C. abortus in clinical samples. Using newly-designed two primer sets specific for C. abortus, the polymerase chain reaction assay was first tested with positive and negative control DNA and its sensitivity and specificity were determined. A new polymerase chain reaction protocol was developed by combining the new primer pair sets with bovine (12SM-FW and 12SBT-REV2) and ruminant host-specific primer sets (12S-FW and 12S-REV). In conclusion, the developed polymerase chain reaction assays can potentially be used for direct detection of Chlamydophila abortus in bovine and ruminant samples.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Development of Polymerase Chain Reaction assays with host-specific internal controls for Chlamydophila abortus

Cantekin¹,

Solmaz²,

Ergün³

et al. 2015

Vet. Med. - Czech

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“…To make up for this shortage and the high cost of buffalo milk, some breeders and dairy industries mix buffalo milk with milk from other species, especially cow milk (LÓPEZ-CALLEJA et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

Buffalo mozzarella chemical composition and authenticity assessment by electrophoretic profiling

Gonçalves

Silva

Conceição

et al. 2017

SCA

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Buffalo milk mozzarella is often adulterated by the addition of cow's milk. The aim of this study was to assess the quality of buffalo milk mozzarella by using electrophoresis (SDS-PAGE) to detect the addition of cow's milk. Reference cheeses were produced exclusively from combinations of buffalo and cow milk, and labeled reference treatment buffalo or cow (RTB and RTC, respectively). Standardized cheeses were made by combining buffalo milk and 2.5%, 5.0%, 10%, 20%, 30%, 40% or 50% cow's milk. A total of 9 cheese formulations were produced and either frozen immediately (time 0) or after 20 days (time 20). Eighteen commercial samples of buffalo mozzarella were sampled between 0-20 days of production. The chemical composition (moisture, ash, fat in dry matter, protein, total solids and defatted dry extract) and physicochemical characteristics (acidity) of the cheeses were evaluated. Proteins and water-soluble peptides (WSP) extracted from RTB, RTC and from the commercial samples, and were analyzed by SDS-PAGE electrophoresis under denaturing conditions. It was not possible to detect the inclusion of cow's milk by the cheeses' chemical and physicochemical properties. However, the separation and detection of peptide and protein fractions of the cheese was possible by electrophoresis. The results of the electrophoretic analysis suggest that 28% of the commercial samples considered here had evidence of the addition of cow's milk. The methodology described here is important to identify occurrences of fraud in buffalo mozzarella production. Key words: Adulteration. Peptides. Proteins. Cheese. SDS-PAGE. ResumoObjetivou-se com o presente estudo detectar fraudes por adição de leite de vaca em muçarela de búfala. Foram produzidos queijos exclusivamente com leite de búfala (TRB) e vaca (TRV) e com inclusões crescentes de leite bovino ao bubalino (2,5%, 5,0%, 10%, 20%, 30%, 40% e 50%), totalizando 9 formulações que foram congeladas no dia de fabricação (Tempo 0) e com 20 dias após a data de fabricação (Tempo 20). Foram coletadas 18 amostras comerciais de muçarela de búfala com até 20 dias de fabricação sendo congeladas no Tempo 20. Os queijos foram avaliados em relação à composição química (umidade, cinzas, gordura no extrato seco, proteínas, extrato seco total e extrato seco desengordurado) e característica físico-química (acidez titulável). Proteínas dos queijos muçarela produzidos e amostras comerciais e peptídeos solúveis em água (PSA) extraídos de TRB, TRV e amostras comerciais foram analisados por eletroforese sob condições desnaturantes por meio de dodecil sulfato de sódio (SDS-

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“…Article 6 of EC No 273/2008 described isoelectrofocusing of γ-casein as reference method for the detection of cows' milk in cheeses from other dairy species (i.e. ewes', goats', buffalos' milk or mixtures of them), but chemical methods may fail in species identification after excessive proteolysis or heat-induced denaturation of proteins indicator (López-Calleja et al, 2005a).…”

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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PCR detection of cows’ milk in water buffalo milk and mozzarella cheese

Cited by 81 publications

References 20 publications

Development of Polymerase Chain Reaction assays with host-specific internal controls for Chlamydophila abortus

Development of Polymerase Chain Reaction assays with host-specific internal controls for Chlamydophila abortus

Buffalo mozzarella chemical composition and authenticity assessment by electrophoretic profiling

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

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