BRPI1106427B1 - method and kit for quantification of material of bovine and buffalo origin in products of animal origin - Google Patents

Abstract

method and kit for quantifying bovine and buffalo material in animal products the present invention describes an ekit method for quantifying bovine and ebubaline material in dairy or meat food products and other animal products through a set of primers / probe specifics. for dna aquantification, the taqman pcr system in real time is used as a standard, but the method is also suitable for the sybr green system. the present invention also describes a new method for normalizing the total amount of dna present in a given sample and the construction of calibration curves according to the type of material to be analyzed.

Description

METHOD AND KIT FOR QUANTIFICATION OF MATERIAL OF BOVINE AND BUBBLE ORIGIN IN PRODUCTS OF ANIMAL ORIGIN

The present invention describes a method and kit for the quantification of material of bovine and buffalo origin in dairy or meat food products and other products of animal origin through a set of specific primers / probes. For DNA quantification, the TaqMan Real Time PCR system is used as a standard, but the methodology is also suitable for the SYBR Green system. The present invention also describes a new method for normalizing the total amount of DNA present in a given sample and the construction of calibration curves according to the type of material to be analyzed.

The authenticity of food products has been an ever-increasing concern for modern consumers, which puts increasing pressure on public policies to combat substitution or fraud and be effective at different levels of the food production chain (Herman, L. (2001). Determination of the animal origin of raw food by species-specific PCR. Journal of Dairy Research, 68 (03), 429-436. Retrieved May 3Q, 2011, from http://journals.cambridge.org .ez27.periodicos.capes.goy.br / abstract_SQ022029901004940). For example, the seasonal fluctuations in the availability of buffalo, goat and sheep milk, in addition to the higher price in relation to that of cows, are an incentive for these products to be adulterated with milk of greater availability and lower price, a fact that has been found in Brazil and other countries (Branciarí, R., Nijman, IJ, Pias, Μ. E., Di Antonio, E., & Lenstra, JA. (2000). Species Origin of Milk in Italian Mozzarella and Greek Feta Cheese, Journal of Food Protection®, 63 (3),

4. International Association for Food Protection. Retrieved May 25, 2011, from http: //www.ingentaconnect.eom/content/iafp/jfp/2000/00000063/00000003/art00 020; Egypt, A. $., Rosinha, GMS, Laguna, L. E „Miclo, L., Girardet, JM _f & Gaillard, JL (2006). Rapid electrophoretic method for detecting the adulteration of goat milk with bovine milk. Brazilian Archives of Veterinary Medicine and Animal Science, 58 (5), 932-939. doi: 10.1590 / S0102-

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09352006000500032 .; Lopparelli, R. M., Cardazzo, B., Balzan, S., Giaccone, V., & Novelli, E. (2007). Real-time TaqMan polymerase chain reaction detection and quantification of cow DNA in pure water buffalo mozzarella cheese: method validation and its application on commercial samples. Journal of agricultural and food chemistry, 55 (9), 3429-34. doi: 10.1021 / jf0637271.). Other products of animal origin, such as sausages, fresh meat and hides, are also susceptible to substitutions (Gonzalez-Cordova, F., Calderon de Ia Barca, M., Cota, M., & Vallejo-Cordoba, B. (1998). Immunochemical detection of 1st adulteration of chorizo of cerdo with soy proteins: Immunochemical detection of fraudulent adulteration of pork chorizo (sausage) with soy protein. Food Science and Technology International, 4 (4), 257-262. 10.1177 / 108201329800400404; Ballin, NZ, Vogensen, FK, & Karlsson, AH (2009). Species determination - Can we detect and quantify meat adulteration? Meat science, 83 (2), 165-174. Elsevier Ltd. doi:

10.1016 / j.meatsci.2009.06.003; Soares, S., Amaral, JS, Mafra, I., & Oliveira, Μ. BPP (2010). Quantitative detection of poultry meat adulteration with pork by a duplex PCR assay. Meat science, 85 (3), 531-6. Elsevier Ltd. doi: 10.1016 / j.meatsci.2010.03.001). Such adulterations have implications for human health, as, for example, in the case of consumers who are allergic to bovine milk, in addition to economic, ethical or even religious and cultural implications that can restrict the access of products to different markets (Branciari et al 2000; Woolfe, M., & Primrose, S. (2004). Food forensics: using DNA technology to combat misdescription and fraud. Trends in biotechnology, 22 (5), 222-6. Doi: 10.1016 / j.tibtech. 2004.03.010). Thus, adequate tools are needed for the detection of animal ingredients in foods and the identification of their origin (species) (Verkaar, E., Nijman, I., Boutaga, K., & Lenstra, J. (2002). cattle species in beef by PCR-RFLP of mitochondrial and satellite DNA.Meat Science, 60 (4), 365-369.doi: 10.1016 / S0309-1740 (01) 00144-9; López-Andreo, M., Lugo, L ., GarridoPertierra, A. _f Prieto, Μ. I., & Puyet, A. (2005). Identification and quantitation of species in complex DNA mixtures by real-time polymerase chain reaction. Analytical biochemistry, 339 (1), 73- 82. doi: 10.1016 / j.ab.2004.11.045).

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In recent years, several methods based on the analysis of lipids, proteins and DNA have been developed for the identification of mixtures of meat or milk of different species (Saeçd, T., Ali, SG, Rahman, HA, & Sawaya, WN (1989) Detection of pork and lard as adulterants in processed meat: 5 liquid chromatographic analysis of derivatized triglycerides. Journal Association of Official Analytical Chemists, 72 (6), 921-5. Retrieved May 26, 2011, from http: //www.ncbi .nlm.nih.goy / pubmed / 2592314; Gonzalez-Cordova et al 1998; Cçzzolino, R., Passalacqua, S., Salemi, S „& Garozzo, D. (2002). Identification of adulteration in water buffalo mozzarella and in ewe cheese by io using whey proteins as biomarkers and matrix-assisted laser desorption / ionization mass spectrometry. Journal of mass spectrometry: JMS, 37 (9), 985-91. doi; 10,1002 / jms.358; Egito et al 2006 ; Addeo, F., Pizzano, R., Nicolai, MA, Caira, $., & Chianese, L. (2009). Fast isoelectric focusing and anti pe ptide antibodies for detecting bovine casein in adulterated water buffalo 15 milk and derived mozzarella cheese. Journal of agricultural and food chemistry, 57 (21), 10063-6. doi: 10.1021 / jf9020009; Cuollo, M., Caira, S., Fierro, 0., Pinto, G., Picariello, G., & Addeo, F. (2010). Toward milk speciation through the monitoring of casein proteotypic peptides. Rapid communications in mass spectrometry: ROM, 24 (11), 1687-96. doi: 10.1002 / rcm.4564; Guarino, C., 20 Fuselli, F., La Mantia, A., Longo, L, Faberi, A., & Marianella, R. M. (2010).

Peptidomic approach, based on liquid chromatography / electrospray ionization tandem mass spectrometry, for detecting sheep’s milk in goat's and cow's cheeses. Rapid communications in mass spectrometry: RÇM, 24 (6), 705-13. doi: 10.1002 / rcm.4426; Branciari et al. 2000; Calvo, J. H., Zaragoza, P., Osta, 25 R. (2001). Technical note: A quick and more sensitive method to identify pork in processed and unprocessed food by PCR amplification of a new specific DNA fragment Journal of Animal Science, 79 (8), 2108-2112. Retrieved September 09, 2011, from http://www.ncbi.nlm.nih.gov/pubmed/11518219; Lanzilao, I., Burgalassi, F., Fancelli S., Settimelli M., Fani R. (2005). Polymerase chain 30 reaction-restriction fragment length polymorphism analysis of mitochondrial cytb gene from species of dairy interest. The Journal of AOAÇ International, 88 (1): 128-35. Retrieved September 09, 2011, from http: //www.ncbi

4/26 .nlm.nih.gov / pubmed / 15759735; Maccabiani, G., Pavoni, E., Tilola, M., Agnelli,

E., Simoni, M., D’Abrosca, F., Boni, P. (2005). Setting-Up a PCR-Based Method for Species Identification in Milk Products. Veterinary Research Communications, 29 (2), 327-329. doi: 10.1007 / s11259-005-0073-6; Zhang, C.,

Fowler, M., Scott, N., Lawson, G., & Slater, a. (2007). A TaqMan real-time PCR system for the identification and quantification of bovine DNA in meats, milks and cheeses. Food Control, 13 (9), 1149-1158. It hurts:

10.1016 / j.foodcont.2006.07.018; Reale, S., Campanella, A., Merigioli, A., Pilla,

F. (2008). A novel method for species identification in milk and milk-based products. Journal of Dairy Research, 75, 107-112.

doi: 10.1017 / S0022029907003020; Ramadan, H. A. I. (2011) Sequence of specific mitochondrial 16S rRNA gene fragment from Egyptian buffalo is used as a pattern for discrimination between river buffaloes, cattle, sheep and goats. Molecular Biology Reports (2011) 38: 3929-3934. DOI 10.1007 / s11033-01015 0509-0; Soares et al 2010). The detection of lipids and proteins is carried out by means of chromatographic techniques, immunoassays or, more recently, by mass spectrometry (Ashoor, SH, Monte, WC, & Stiles, PG (1988). Liquid chromatographic identification of meats. Journal - Association of Official Analytical Chemists, 71 (2), 397-403. Retrieved May 26, 2011, from 20 http://www.ncbi.nlm.nih.gov/pubmed/3384790; Saeed et al 1989; Chou, C -Ç., Lin, S.-P., Lee, K.-M, Hsu, C.-T., Vickroy, TW, & Zen, J.-M. (2007). Fast differentiation of meats from fifteen animal species by liquid chromatography with electrochemical detection using copper nanoparticle plated electrodes.Journal of chromatography.B, Analytical technologies in the biomedical and life 25 sciences, 846 (1-2), 230-9.doi: 10.1016 / j.jchromb.2006.09. 006; GonzalezCordova et al 1998; Addeo et al 2009; Cuollo et al., 2010; Guarino et al., 2010). However, these methods are laborious, generate results with ambiguous interpretation and / or cannot be applied to foods that have undergone heat treatments, such as cooking, or chemicals, such as fermentation (Mayer, H. (2005). Milk species identification in cheese varieties using electrophoretic, chromatographic and PCR techniques. International Dairy Journal, 15 (6-9), 595-604.doi: 10.1016 / j.idairyj.2004.10.012; López-Calleja, I.,

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Gonzalez, I., Fajardo, V., Martin, I., Hernandez, P. „Garcia, T., et al. (2007). Real-time TaqMan PCR for quantitative detection of cows 'milk in ewes' milk mixtures. International Dairy Journal, 17 (7), 729-736. It hurts:

10.1016 / j.idairyj.2006.09.005). In addition, chromatographic and immunological techniques may have little resolution in identifying phylogenetically close species, such as buffalo and cattle (Branciari et al. 2000; Bellis, C. (2003). A molecular genetic approach for forensic animal species identification. Forensic Science International, 134 (2-3), 99-108.doi: 10.1016 / 80379-0738 (03) 00128-2; Zhang et al 2007).

Comparatively, methods based on DNA detection are more practical, in addition to being more sensitive and robust, as they allow the analysis of foods processed by high temperatures, pressure and even chemical treatments (Branciari and ols. 2000; Lockley, K. , & Bardsley, RG (2000). DNA-based methods for food authentication. Trends in Food Science & Technology, 11 (2), 67-77. Doi: 10.1016 / 30924-2244 (00) 00049-2 .; Mayer 2005 ; Mafra, I., Ferreira, I., & Oliveira, B. (2007). Food authentication by PCR-based methods. European Food Research and Technology, 227 (3), 649-665. Doi: 10.1007 / s00217-OQ7 -0782-x .; Zhang et al 2007). Among these, the polymerase chain reaction (PCR) technique stands out, which uses pairs of primers with specific sequences for each species, allowing the amplification of minimal amounts of target DNA (Herman 20Q1; Mafra et al 2007) . PÇR can also be used as a quantitative tool, through the use of the Real Time PCR technique, which uses an agent or fluorescent probe that allows the detection of DNA as it is amplified (Lockley & Bardsley, 2000 ). A certain amount of DNA can, in this way, be quantified by analyzing the number of amplification cycles necessary to reach a certain level of fluorescence (called threshold). This number is called Cycle Threshold or Çt (Kubista, M., Andrade, JM, Bengtsson, M., Forootan, A., Jonák, J., Lind, K., et al. (2006). The real-time polymerase chain reaction.Molecular aspects of medicine, 27 (2-3), 95-125.

10.1016 / j.mam.2005.12.007). The use of a quantitative analytical method

6/26 not only increases the resolution of the technique, but can be useful in cases where the legislation allows a certain level of occasional contamination of the product (Sawyer, J. (2003). Real-time PCR for quantitative meat species testing. Food Control , 14 (8), 579-583.doi: 10.1016 / 30956-7135 (02) 00148-2; López-Çalleja et al 2007).

The TaqMan Real-Time PCR system uses an oligonucleotide © as a complementary probe to the internal region of the generated amplicon, that is, it nests in the region between the two primers used. This probe, which is linked to a fluorophore, is hydrolyzed every cycle by the 5 'nuclease activity of Taq DNA polymerase, which causes fluorescence emission proportional to the amount of amplified DNA and allows quantitative analysis of the material (Holland, PM, Abramson, RD, Watson, R., & Gelfand, DH (1991). Detection of specific polymerase chain reaction product by using the 5 '-— 3' exonuclease activity of Thermus aquaticus DNA polymerase. Proceedings of the National Academy of Sciences of the United States of America, 88 (16), 7276-80 Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi? Artid = 52277 & tool = pmcentrez & rendertype = abstract). This system is considered more specific and sensitive than others used for Real Time PCR (like the SYBR Green system) (Gasparic, Μ. B., Tengs, T., La Paz, J. L, Holst-Jensen, A., Pia, M., Esteve, T., et al. (2010). Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection. Analytical and bioanalytical chemistry, 396 (6), 2Q23-9. : 10.1Q07 / s002í6-009-3418-0), and has been used in food analysis for the detection of genetically modified organisms and contaminating microorganisms, as well as in the identification of species in meat and milk (Bahrdt, C., Krech, a B., Wurz, A., & Wulff, D. (2010). Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed. Analytical and bioanalytical chemistry, 396 (6 ), 2103-12.doi: 10.1007 / s00216-009-3380-x; Fujikawa, H „Shimojima, Y„ & Yano, K. (2006). Novel method for estimating viable Salmonella cell c ounces using real-time PCR. Shokuhin eiseigaku zasshi. Journal of the Food Hygienic Society of Japan, 47 (4), 151-6. Retrieved May 26, 2011, from

7/26 http://www.ncbi.nlm.nih.gov/pubmed/16984034; Kesmen, Z., Gulluce, a, Sahin, F., & Yetim, H. (2009). Identification of meat species by TaqMan-based realtime PCR assay. Meat science, 82 (4), 444-449. Elsevier Ltd. doi: 10.1016 / j.meatsci.2009.02.019; López-Calleja et al, 2007).

The quantification of DNA by means of Real Time PCR can be performed in several ways, among them the relative quantification through comparative Ct or ΔΔΟΤ. In this method the relative amount (RQ) of a specific DNA sequence present in each sample is calculated using the formula 2 ' ^ΔΔ0Τ . Another alternative is the relative standard curve methodology, which is more laborious, costly and less reproducible, and is normally used only when the amplification efficiency of the assay is low and / or variable. In both cases, the total amount of DNA in the sample, measured by amplifying a universal sequence, the normalizer, must be taken into account. In the case of species identification works in mixtures by means of Real Time PCR, the normalizer would be a DNA sequence shared by all species involved in the mixture. However, the amplification efficiency of universal primers can be affected by the presence of more than one type of DNA in the sample, or it can vary depending on the species present (López-Andreo et al, 2005). This can be caused by small sequence differences (mutations) present in certain species that can escape the control of the researcher, since a large number of species and lines must be evaluated. Taris et al. (Taris, N., Lang, R. P „& Camara, MD (2008). Sequence polymorphism can produce serious artefacts in real-time PCR assays: hard lessons from Pacific oysters. BMC genomics, 9, 234. doi : 10.1186 / 1471-2164-9234.) Showed how high levels of polymorphisms in individuals belonging to a single species can lead to errors in the analysis of real-time PCR experiments. The presence of genetic polymorphisms can be much greater when considering different species. In fact, when we tried to analyze the samples used in this study with universal initiators, considerable variations were registered in the amplification curves obtained with pure or mixed materials (data not

8/26 shown). Thus, in the present invention, we also innovated by developing a new method for normalizing the total amount of DNA present in a given sample.

The methodology of the present invention for normalizing the total amount of DNA is derived from the comparative Ct method, but does not use an invariable universal normalizer. Alternatively, specific primers capable of amplifying buffalo and bovine DNA molecules are used, and the total amount of DNA present in a given sample is calculated by adding the specific quantifications obtained with the two primers. In this way, only the DNA from the species in question is considered for the calculation of the relative quantifications.

The invention also includes the development of calibration curves built according to the type of material to be analyzed (for example, dairy products, meat or leather) and obtained from known amounts of material of bovine and buffalo origin. A software usable in Microsoft Excel® was also included in the technology and allows the quick calculation of the amount of bovine or buffalo material present in the tested samples, taking into account the deviations computed in the reference calibration curve. This makes the technology more precise and versatile than those currently available.

There are some studies already published that demonstrate the application of the Real Time PCR technique for the identification of species in meat and dairy products. However, this invention is unique in that it uses the calibration curves (controlled fraud curve). Such curves are capable of correcting errors arising from different biological characteristics that vary according to the species under study, such as, for example, the number of somatic cells available in milk and the number of mitochondria per cell. In addition, LópezAndreo and collaborators, in 2005, observed deviations in the amplification of the same gene between different species, and attributed such deviations to differences in the number of copies of the chosen target gene. The use of a calibration curve also allows you to correct them. The works published by Sawyer and

9/26 employees (2003) and Loparelli and employees (Lopparelli, RM, Cardazzo, B., Balzan, S., Giaccone, V., & Novell !, E. (2007). Real-time TaqMan polymerase chain reaction detection and quantification of cow DNA in pure water buffalo mozzarella cheese: method validation and its application on 5 commercial samples. Journal of agricultural and food chemistry, 55 (9), 3429-34.

doi: 10.1021 / jf0637271) use a controlled fraud curve to perform a relative quantification. However, the methodology described by them requires the construction of a curve for each experiment. In addition, the authors report the inefficiency of the methods proposed to deal with the species10 specific differences mentioned above. López-Andreo and collaborators, in 2005, used a controlled fraud curve, but only to test their quantification method. However, they also found errors much larger than those reported here, especially when the contamination was below 5%. In the case of López-Calleja and 15 collaborators (2007), the presented calibration curve is not complete and covers only samples that show contamination from 0.5 to 10%.

Patent document CN 101215603 - Bubalus products molecule diagnosis kit - describes KIT for differentiation between food products from buffaloes (Bubalus genus) and products from other 2Q genera, through the use of specific primers, PCR amplification and electrophoresis detection. This document differs from the present invention in that it does not use the quantitative Real-Time PCR technique and in that it uses different primers than those used in the present invention.

Patent document WO 02064822 - Method and kit for animal 25 species-specific dna identification of a sample - describes a method for detecting the presence or absence of species-specific DNA in a sample using PCR using specific primers for various animal species. The primers for amplification of bovine material (Bos genus) are similar to those used in the present invention (Bos primers). This document differs from the present invention in that it does not use specific initiators for Bubalus, does not quantify the material and does not present a calibration curve for normalizing the data.

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Patent document WO 03057913 - Method for the detection and / or identification of the original animal species in animal matter contained in a sample - describes a method for detecting or identifying the origin of animal samples. One of the initiators of this method resembles the 5 BosF primer of the present invention. This document differs from the present invention in that it does not use specific initiators for Bubalus, does not use the PCR technique in real time, does not quantify the material and does not present a calibration curve for data normalization.

Patent document US 2010025244/7582452/7927841 describes the method for qualitative and quantitative determination of material of animal origin. The experiments are based on specific characteristics of SINEs of pig, ox, bird and ruminant species. The SINEs are amplified and the result is evaluated qualitatively by electrophoresis or quantitatively by SYPR Green or TaqMan. This document differs from the present ₁₅ invention by using different primers and identify different species.

Patent document CN 101712996 - Method for quickly identifying categories of meat and dried meat products of five domestic animals - describes a method for identifying categories of products of animal origin 20 (pig, goat, ox, buffalo and yak), based on PCR - RFLP technique. This document differs from the present invention in that it uses differentiated detection technique and does not quantify the material.

The present invention allows the quantification of material of bovine and buffalo origin in dairy or meat products and other products of animal origin through a set of specific primers / probes, using the Real Time PCR TaqMan system, a reliable and efficient system for DNA quantification. Although the TaqMan system was chosen as a standard, the methodology also fits the SYBR Green system. The present invention also allows the normalization of the total amount of DNA present in a given sample, minimizing errors in the analysis of Real-Time PCR experiments.

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The technology present in this invention promotes a leap in quality with regard to the authentication of products of animal origin, causing important impacts on the production chain. It is possible to overcome the limitations of traditional identification methodologies through protein analysis. The test presented here, based on DNA, is quantitative, in addition to being more robust and sensitive, which allows the authentication of both In natura products and those already processed by thermal or chemical treatment.

The Quantification test of material of bovine and buffalo origin in products of animal origin by the Real Time PCR technique is innovative, mainly, in the following points:

1) It allows the quantitative detection of bovine and buffalo material present in food products and / or other products of animal origin;

2) It allows the analysis of both the raw material and the processed product that reaches the consumer (Ex: cheeses, hamburgers, sausages);

3) It allows the analysis of products processed both thermally and chemically (Ex: leather, cooked, smoked, fermented or treated with chemical preservatives).

4) Automated technology with greater scalability potential than traditional processes.

Adulterations in the composition of food with material from other species of greater availability or less cost, as well as mislabeling can cause health risks and economic losses. In the current globalized context, and due to the demands of the contemporary consumer market, this invention has wide application in the Identification of Origin and Purity Certification of products derived from buffalo.

In this context, the invention allows:

1) Detection of fraud or substitutions by material of bovine origin in products of buffalo origin (Ex: buffalo mozzarella). This application is of great value for certifying entities and inspection bodies.

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2) Certification of purity of dairy products (eg cheeses) and meat products (eg hamburgers) that are made from material of buffalo origin.

3) Specific identification of bovine and buffalo genetic material in different materials, whether pure or mixed.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 - Process of quantification of material of bovine and buffalo origin in food products by the Real Time PCR technique. Samples based on buffalo material that contain, or may contain, bovine material are submitted to DNA extraction protocols and Real-time PCR amplification. The result is submitted to a specific calibration curve for each product, which provides the actual percentage of bovine material present.

Figure 2 - Efficiency curves for Bub and Bos mixes with pure samples of meat tissue. Efficiency curves were constructed from triplicates of serial dilutions (: 2) of DNA extracted from pure samples of beef and buffalo. The samples were amplified by Real Time PCR, TaqMan system, with the set of primers / probe Bos (A) and Bub (B) and had their values of efficiency, inclination and R ² calculated by the 7500 Software (Applied Biosystems) according with manufacturer's instructions. The quantity values indicated refer to the spectrophotometric quantification of the samples.

Figure 3 - Efficiency curves for Bub and Bos mixes with pure mozzarella samples. Efficiency curves were constructed from triplicates of serial dilutions (: 2) of DNA extracted from pure samples of bovine and buffalo mozzarella. The samples were amplified by Real Time PCR, TaqMan system, with the set of primers / probe Bos (A) and Bub (B) and had their values of efficiency, inclination and R ² calculated by the 7500 Software (Applied Biosystems) according with manufacturer's instructions. The quantity values indicated refer to the spectrophotometric quantification of the samples.

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Figure 4 - Efficiency curves for Bub and Bos mixes using pure mozzarella samples and the SYBR Green system. Efficiency curves were constructed from triplicates of serial dilutions (: 2) of DNA extracted from pure samples of bovine and buffalo mozzarella. The samples were amplified by PCR in Real Time, SYBR Green system, with the set of primers Bos (A) and Bub (B) and had their values of efficiency, inclination and R ² calculated by the 7500 Software (Applied Biosystems) according with manufacturer's instructions. The quantity values indicated refer to the spectrophotometric quantification of the samples.

Figure 5 - Valid Mixed Food Detector Software. The Valid Mixed Food Detector Software was developed on Microsoft's Excel platform and has three screens: (A) on the first, called ΊηρυΓ, the user can upload the output file “Results of the 7500 Software (Applied Biosystems) (B) . The second screen (C), called “Experiment”, shows the results related to the 15 experiment in question and how the values obtained for each sample were interpellated on the calibration curve. In the last screen (D), called “Reference”, it is possible to observe the data of the controlled fraud curves used to construct the calibration curve.

Figure 6 - Observed and expected amounts of bovine DNA in the curves of controlled mozzarella fraud. Samples of two independent controlled fraud curves were obtained by weighing known amounts of pure bovine and buffalo mozzarella for DNA extraction. Each sample was dosed in a Nanovue spectrophotometer and 220 ng (in triplicates) were used in the Real Time PCR experiments. The calculation of the amount of bovine material was carried out according to Equation 1 described in the present invention.

Figure 7 - Calibration curve used for the actual quantification of bovine material present in a given sample. The calibration curve was obtained from two controlled fraud curves, constructed by weighing 30 known quantities of 100% bovine and 100% buffalo mozzarella. The average values obtained for each point of the calibration curve were plotted

14/26 against the real expected value for such points and a trend curve was plotted using Microsoft Excel 2010® software. The equation of the obtained line can now be used for the real quantification of bovine material present in the sample.

DETAILED TECHNOLOGY DESCRIPTION

The invention consists of a process of quantitative detection by Real Time PCR of bovine and buffalo material in food products. For this purpose, the invention uses specific primers for amplification of bovine and buffalo mitochondrial DNA. The comparison between the amounts of bovine DNA in relation to the amount of total DNA in the sample (given by the sum of the specific quantities of each of the analyzed species) allows the detection of the proportion of contaminating material that is present in the product. The invention also uses a versatile process of construction of calibration curves for each type of sample analyzed (ex: cheeses, hamburgers and smoked sausages), which makes the analysis more accurate because it takes into account deviations caused, for example, by the distinct number mitochondria / cell in different tissues or species, varying amounts of DNA present in the milk of different species or even chemical and thermal treatments. Figure 1 briefly illustrates the process that makes up this invention.

The present invention can be better understood, in a non-limiting way, through the following examples:

EXAMPLE 1: DESIGN OF INITIATORS AND PROBES FOR REAL-TIME PCR

The set of specific primers and probe for the amplification of bovine DNA (here called Bos set) (Table 1) was redesigned from those described by Zhang et al. (2007). The sequences of the oligonucleotides were optimized for the use of TaqMan probes connected to non-fluorescent quenchers of the type Minor Groove Biding (MGB) produced by the company Applied Biosystems (USA). Such adjustments were made with the aid of the Primer Express program (Applied Biosystems) and based on sequences from the mitochondrial CytB gene region of cattle,

15/26 buffalo, goats, sheep and pigs deposited in NCBI's GenBank sequence bank (Benson, DA, Karsch-Mizrachi, I., Lipman, DJ, Ostell, J., & Sayers, EW (2010). GenBank. Nucleic acids research, 39 (Database issue), D32-7.doi: 10.1093 / nar / gkq1079) and aligned using the ClustalW algorithm (Chenna, R., Sugawara, H., Koike, T., Lopez, R., Gibson, T. J „Higgins, DG, and ols. (2003). Multiple sequence alignment with the Clustal series of programs. Nucleic acids research, 31 (13), 3497-500. Retrieved June 9, 2011, from http: // www .pubmedcentral.nih.gov / articlerender.fcgi? artid = 168907 & tool = pmcent rez & rendertype = abstract) in the MEGA version 5 program (Tamura, K., Peterson, D., Peterson, N „Stecher, G., Nei, M„ & Kumar, S. (2011). MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution). The primers were designed in specific regions of the bovine gene, and the TaqMan probe linked to MGB (Applied Biosystems), in a region conserved between the different species.

The set of primers for the amplification of buffalo DNA (here called Bub set) (Table 1) was designed from the sequence of the 16S mitochondrial gene. As in the case of the set for the amplification of bovine DNA, sequences of this gene obtained from specimens of B, bubalis available in the GenBank sequence bank of NCBI (Benson et al 2010), were aligned and compared to the sequences of the same gene from Bps taurus taurus, Bos taurus indicus, Capra hircus, Ovis aríes and Sus scrofa species, obtained from the same bank. The design of the set of primers and probe was carried out with the aid of the Primer Express program (Applied Biosystems). This set was designed so that each of the primers has at least two nucleotides specific for the species B. bubalis among the last 5 nucleotides at the 3 'end. The probe, on the other hand, was designed over a conserved region among mammal species.

It is important to note that the two probes were built based on conserved regions between different species so that the same probe can be used in specific tests for different species. This strategy follows the precept that the specificity of a set

16/26 initiators / probe is given exclusively by the initiators (Kubista et al 2006). This type of primer / probe design allows them to be used for both the TaqMan and SYBR Green systems, which, although less accurate, are less expensive.

Table 1: Sets of primers and probes used in the present invention

Name SEQ ID Type ¹ Sequence (5'-3 ') ² Target BosF . SEQ ID N ° 1 Foward Initiator CGGAGTAATCCTT CTGCTCACAGT CytB (bovine) BosR SEQ ID N ° 2 Reverse Launcher GGATTGCTGATAAG AGGTTGGTG CytB (bovine) MamCytB SEQ ID N ° 3 TaqMan MGB probe FAMCATGAGGACAAAT ATC-MGB CytB (mammals) Bub2F SEQ ID N ° 4 Foward Initiator TCAGCCCAAAGAAA AATAAACCA 16S (buffalo) Bub2R SEQ ID N ° 5 Reverse Launcher GTCACCCCAACC GAAACTGT 16S (buffalo) Bub16S SEQlDN ° 6 TaqMan MGB probe FAMTAAGGARTAACAAC AMTCT-MGB ³ 16S (mammals)

- In the SYBR Green system, the probes must not be used, only the initiators. 2 - To standardize the present technology, the probes were marked with the fluorophore FAM, as indicated. Other fluorophores can also be adapted to replace them as needed. 3 - The probe was designed containing some degenerations: R indicates A or G, M indicates A or C.

EXAMPLE 2: SAMPLING, DNA EXTRACTION AND AMPLIFICATION

17/26

For the standardization, development and demonstration of the effectiveness of the quantification test of material of bovine and buffalo origin in food products by the Real Time PCR technique, samples of meat tissues (muscle) from 6 animals were used: 3 cattle (B. taurus taurus and / or B. taurus indicus) and 3 buffalo (B. bubalis). In addition, 2 samples of pure bovine mozzarella and 2 samples of pure buffalo mozzarella were used, produced from a mixture of milk from various animals.

DNA extraction from meat tissues was performed by adapting the technique described by Sambrook and collaborators (Sambrook, J „Fritsch, EF, & Maniatis, T. (1989). Molecular cloning: a laboratory manual (2nd ed.). Cold Spring Harbor, NY; Cold Spring Harbor Laboratories.). In this technique, the DNA of the meat sample is extracted, after digestion with proteinase K, using solutions containing phenol / chloroform / isoamyl alcohol, being subsequently precipitated and purified with ethanol.

DNA extraction from mozzarella samples was carried out by adapting the method described by Boom and collaborators (Boom, R., Sol, Ç. J., Salimans, Μ. M., Jansen, CL, Wertheim-van Dillen , PM, & Noordaa, J. van der. (1990). Rapid and simple method for purification of nucleic acids. Journal of clinical microbiology, 28 (3), 495-503. Retrieved from http: //www.pubmedcentral.nih .gov / articlerender.fcgi? artid = 269651 & tool = pmcent rez & rendertype = abstract). With this method the DNA of the samples is extracted and purified using a solution based on guanidine thiocyanate and silica prepared with an adequate electric charge to bind to the DNA fragments.

For the construction of calibration curves for mixtures of dairy products, in addition to extracting DNA from pure samples of each species, DNA was extracted from controlled frauds containing known values from the same pure samples.

All DNA samples were quantified in a Nanovue spectrophotometer (GE, USA) to determine their concentration (1 OD. At 260 nm is equal to 50 pg of DNA). After quantification, the DNA was diluted in sterile water for optimal concentration of analysis.

18/26

The standardization of this invention was performed on the 7500 RealTime PCR System (Applied Biosystems). The reactions were carried out in triplicates in 96-well plates. In the protocols described here, each sample is amplified in parallel by the sets of primers and probes specific for the amplification of bovine (BosF, BosR and MamCytB) and buffalo (Bub2F, Bub2R, Bub16S) DNA. In this way, it is possible to detect the proportion of bovine or buffalo material present in the sample in relation to the amount of total DNA from these two species.

Amplification reactions were performed in a total volume of 25 μ | _ containing 12.5 μΙ_ of TaqMan® Universal PCR Master Mix (Applied Biosystems) (which includes the heat-activated enzyme AmpliTaq Gold), 4Q0 nM of each oligonucleotide © initiator , 200 nM of the relevant probe and 2 μΙ_ of the target DNA (the concentrations used varied according to the experiment in question). For reactions using DNA from meat tissues, 5 pg of BSA (Bovine Serum Albumin) was added. The conditions for amplification were: 5 minutes at 50 ° C for AmpErase system action, 10 minutes at 95 ° C for enzyme activation and 40 cycles of 15 seconds at 95 ° C, 15 seconds at 58 ° C and 1 minute at 60 ° C. In the case of the analyzes performed using the SYBR Green system, 12.5 μΙ_ of the SYBR® Green PCR Master Mix (Applied Biosystems) and the same amounts of primers and DNA mentioned above were used. The reaction conditions were also the same as those described for the TaqMan system, with the addition of a dissociation curve stage (15 seconds at 95 ° C, 1 minute at 60 ° C, 30 seconds at 95 ° C and 15 seconds at 60 ° C) at the end of the amplification cycles. The dissociation curves of the final products were evaluated to confirm the presence of only one amplicon.

The reactions were controlled by the 7500 Software (Applied Biosystems). Readings were taken at each cycle and the log of the fluorescence increment was plotted against the cycle number. The correction of basal fluorescence was performed automatically by the program. The thresholds used were fixed for each pair of primers or primers / probe (0.127651 for the Bos set and 0.025158 for the Bub set). The number of cycles

19/26 necessary for a given sample to reach the established fluorescence limit (threshold), called Ct, was then computed and used in the calculation of relative quantification, as described below.

The quantifications of bovine and buffalo material present in samples of meat or dairy products were performed using the following equations:

2 “Ct Bos

_Qde bovine DNA = ₂ - _ΚΒΜ ; ₂ - _{ηΒΜ |>} (Equation 1)

OR

-, - CtBub

Buffalo DNA = ₂ _ _ctBo . _{+ 2} _ _ctB ^ (Equation 2) where Ct ^Bos and Ct ^Bub indicate the cycle in which the sample reached the pre-determined threshold for that specific set of primers or primers / probe. In this calculation, the amount of material of one species or another, present in a given sample, is relative. That is, the amount is expressed by the proportion of that species in relation to the amount of total bovine and buffalo DNA present in the sample.

The deduction of Equations 1 and 2 for the quantification of bovine material considers the method described by Schmittgen & Livak (Schmittgen, TD, & Livak, KJ (2008). Analyzing real-time PCR data by the comparative CT method. Online, 3 ( 6), 1101-1108.doi: 10.1038 / nprot.2008.73). According to these authors, when the normalizer used is variable, it is possible to calculate the amount of DNA by measuring individual non-normalized samples. Thus, for a reaction of efficiency equal to 100 ± 10%, the non-normalized N amount of initial specific DNA contained in each sample is given by:

N _b os = 2 ^_ctbos (Equation 3) or

N _bub = 2 ^-ctbub (Equation 4)

20/26

In our analyzes, for a given sample, the total amount of bovine and buffalo DNA is given by:

Ntotai = N _bos + N _bub (Equation 5)

Therefore, the percentage of bovine material present in the sample can be calculated as:

2 "Ctbos

Bovine DNA Q = (Equation 6) (Equation 1)

The same can be demonstrated for the amount of buffalo material present in the sample:

Nfe b ^ -Ctbub

DNAdebubaline = —— ^ - (Equation 7) = ₍₂ _ _ctb „ _{b ψ 2} _ _ct b57y (Equation 2)

In order to verify the amplification efficiency and identify the optimal amplification range for both sets of primers / probes, serial dilution curves (: 2) were performed for DNA extracted from each type of pure sample (from meat or dairy products). Efficiency curves for dairy products were also performed using the SYBR Green system and the Bos or Bub starter sets. Using the 7500 Software, the Cts obtained at each dilution (after adjusting the thresholds to 0.127651 and 0.025158 for Bos and Bub, respectively) were plotted on a graph according to the amount of DNA and the slope of the line was measured to evaluate the amplification efficiency (inclination of -3.32 = 100% efficiency). In addition, the R ² value was used as a parameter to assess the adequacy of the measured points to the equation of the obtained line, showing the reliability of the observed efficiency. The specificity limit of each set of primers / probe was measured using pure DNA from the other species (non-target).

Figures 2 and 3 show the efficiency curves for DNA samples extracted from meat tissues and mozzarella, respectively, for the sets of primers Bos (Figures 2A and 3A) and Bub (Figures 2B and 3B),

21/26 using the TaqMan system. Figure 2A shows that the Bos primer / probe set has an amplification efficiency of 94.8% (slope: -3.452; R ² = 0.984) when used to amplify DNA samples extracted from pure bovine meat tissues. The optimal efficiency range is from 2.5 ng to approximately 160 pg of DNA, according to the spectrophotometric dosage (260 nm). The limits for optimal detection expressed in Cts range from 27.8 to 32.6. To verify the specificity of the reaction, 2.5 ng of buffalo DNA derived from pure meat tissue were amplified under the same conditions and presented CT> 37, corresponding to less than 7 pg of bovine DNA. Figure 2B demonstrates the amplification efficiency with the Bub primer / probe set for DNA samples obtained from 100% buffalo meat (efficiency: 90.1%; slope: -3.583; R2 = 0.991). The optimum amount, according to spectrophotometric quantification, for amplification of DNA obtained from buffalo meat with the Bub mix, varies from 80 pg to approximately 5 pg, corresponding to a Ct of 28.5 to 32.9 (Fig. 1B). Twenty nanograms of bovine DNA were used to test the specificity of the reaction and showed a Ct> 35 when amplified with the Bub mix, equivalent to 1 pg of bovine DNA.

Figure 3A demonstrates that the Bos primer / probe set shows optimal amplification efficiency, with DNA samples extracted from bovine mozzarella, 91.6% (slope: -3.541; R2 = 0.993), in DNA amounts of 10 pg at approximately 78 ng. Expressed in Cts, these limits range from 16.8 to 24J. Ten micrograms of pure buffalo DNA samples amplified under the same conditions showed CT> 31, corresponding to less than 630 pg of bovine DNA, which is, therefore, the specificity limit of the reaction. Figure 3B demonstrates the amplification efficiency with the Bub primer / probe set for DNA samples obtained from 100% buffalo mozzarella (efficiency: 92.7%; slope: -3.51; R2 = 0.972). The optimum amount for amplification of DNA obtained from buffalo mozzarella is 10 pg to approximately 78 ng, with a detection limit in Cts ranging from 17.8 to 25.3. For the detection of the specificity of the reaction, 10 pg of DNA

22/26 bovine extracted from pure mozzarella were amplified with the Bub mix, and demonstrated a Ct> 35, corresponding to 80 pg of buffalo DNA.

To illustrate the versatility of the technique presented here, amplification efficiency curves of pure mozzarella samples were also made using the SYBR Green system and the Bos and Bub primer sets. It can be seen, through the analysis of Figure 4, that both sets have good amplification efficiency (93.1% for Bos and 92.9% for Bub) when we use this alternative system. For the Bos mix, the slope of the straight line was -3.498, with an R ² of 0.995; for Bub, we obtained a slope of -3.504 and an R ² of 0.976. The detection limits were 1.25 pg to 40 ng for Bos (Ct 16.7 to 22) and 1.25 pg to 80 ng for the Bub mix (with a Cts limit of 15.7 to 20).

EXAMPLE 3: CALIBRATION CURVES

One of the innovations proposed in this invention is the development of a calibration curve that correlates the amount obtained in a given experiment with the actual amount of material of a given species present in a sample. This process is necessary because, as already explained, it takes into account deviations caused, for example, by the number of mitochondria / cells present in different tissues or species and / or varying amounts of DNA present in the milk of different species (Lopparelli et al. 2007; Zhang et al 2007). In this example, the results obtained with the construction of a calibration curve from DNA extracted from mozzarella samples are described. Such samples were chosen because they potentially have a larger deviation between the observed and actual quantifications (when compared to meat tissue samples, for example), since the number of cells present in milk can vary from species to species (Boutinaud, Μ. B ., & Ammes, HJ (2002). Potential uses of milk epithelial cells: a review. Reproduction, Nutrition, Development, 42, 133-147. Doi: 10.1051 / rnd). In addition, cheeses are extremely complex matrices and undergo pasteurization processes that can lead to greater DNA damage when compared to those suffered in the production of hamburgers and

23/26 sausages, for example. It is important to note that the mozzarella cheese was chosen to carry out the analyzes described in this invention only because it represents a cheese traditionally made from buffalo milk and, therefore, subject to adulteration with cow's milk. However, the methodology 5 described and the calibration curve created are perfectly usable for the analysis of any other type of cheese. In fact, the technology has been tested and proved to be efficient for provolone, ricotta, curd and fresh cheese (data not shown). The use of it for other types of products of buffalo origin with production processes different from the cheeses mentioned here requires only the construction of new calibration curves, according to the material to be analyzed.

For the treatment of the data of the experiments and adjustment of the same in the calibration curve constructed from the controlled fraud curves, the Microsoft Excel 2010® software was used. A standard spreadsheet, consisting of 15 with three tabs, was developed and named Valid Mixed Food Detector

Software. The entire process was performed by a macro developed in Visual Basic for Applications (VBA), requiring the user only to indicate the data file of the experiment, generated automatically by the measuring equipment. The calibration curve can be updated at any time by the user, without the need to change the macro source code.

Valid Mixed Food Detector Software was added to this invention so that a given result is easily questioned on the calibration curve suggested here, generating a real quantification of the specific DNA 25 contained in that sample. Extremely easy to use, the only action required by the software user is to search and upload the reference file generated by the 7500 Software. The program was developed on Microsoft's Excel 2010® platform and has three screens, presented as different Excel tabs (Figure 5). It is interesting to note that the program was developed to use, as an input, the “Results” file generated by Applied Biosystems 7500 Software. The use of the same with files coming from systems

24/26 Real-Time PCR from other brands requires only simple modifications.

For data analysis using the Valid Mixed Food Detector Software, the data generated by the 7500 Software is imported into the “Input sheet sheet, in its original form. Automatically, in a second tab, the data are processed by separating the results corresponding to each sample and calculating, according to Equations 1 and 2, the measured quantities of each material evaluated. From the deviations obtained in the Ct measurements, the standard deviations in the material quantity values are calculated using error propagation in Equations 1 and 2. Finally, the calculated material quantity values are adjusted according to the stored reference curves. in the third tab of the spreadsheet and a results tab is created indicating the final values obtained and the reference curve used, in format for printing.

For the construction of calibration curves for mozzarellas, pure samples of each species (100% bovine or 100% buffalo mozzarellas) were weighed on an analytical balance and mixed in the proportions of 0%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% and 100% Bos / Bub, with a final weight of 6 g for each sample, to obtain the points in controlled fraud. The extraction of DNA from such samples, the amplification, as well as the calculations for quantification, were performed as previously described. It is important to note that the Cts obtained were always observed so that they did not fall outside the limits of the respective efficiency curves. For this, the amount of 220 ng / sample was fixed (2 pL of the sample at 110 ng / pL, according to the dosage in a spectrophotometer), which were amplified in two independent curves in triplicates by Real Time PCR. Figure 6 shows graphically the observed and expected bovine DNA values for each sample in both controlled fraud curves. The average of the relative amount of bovine DNA obtained for each of the points in the two curves was plotted on a graph against the actual weighted value for each sample. It was then set an equation of first degree (straight), via tool "Add trend line" available in Microsoft Excel 2010®. O

The result obtained is shown in Figure 7. An excellent correlation of the data was observed with the drawn line (trend curve), with a parameter R ² of curve adjustment greater than 0.99. From the constructed calibration curve, it is possible to infer the actual percentage of bovine material contained in the analyzed product.

To validate the efficiency of the invention proposed here, commercial samples of buffalo mozzarella from 9 different Brazilian brands were analyzed. For internal amplification control, a sample containing 50% Bos / Bub was used on each plate. In addition, 100% bovine and 100% buffalo samples were also used for amplification with both mixes, in order to control the cross-reaction limits for each. The values obtained after quantification by using the Valid Mixed Food Detector Software were then compared with the results obtained for these samples using a standard qualitative test used in routine analyzes in the laboratory. This qualitative test is based on the PCR-RFLP technique and was developed based on 15 modifications to the methodology described by Verkaar et al. (2002).

Table 2 shows the values obtained for each of the tested samples. It is possible to observe the high reproducibility of the control samples. Still, the results obtained for commercial samples are in agreement with those achieved with the standard technique used in the laboratory, which, despite being a qualitative technique, allows the detection of percentage ranges of contamination with bovine material (data not shown) . We emphasize that, in order to use the tool described here, it is necessary to observe the Cts presented by the sample in question. Samples with Cts above-or below the limits proposed by the efficiency curves of the Bos and Bub mix 25 should not be considered, with the exception of negative samples for a given species, which will demonstrate a high Ct value. In addition, it is important to include a negative control, without the presence of DNA (NTC), and the controls of 50% Bos / Bub, 100% Bos and 100% Bub, in each experiment, as performed for this analysis. The Ct observed for the 30 cross-amplification of a mix with the sample of the opposite species (ex: 100% bovine sample amplified with the Bub mix) must be included in the software as the detection limit of that species. In this way, samples

26/26 that reveal a Ct greater than that specified are considered negative for that species.

Table 2: Quantification of bovine material present in commercial samples of buffalo mozzarella

Sample % Bos% Bos Real Gross

AM2 04-04-11 0.00 0.00

AM 6 04-04-11 ..................... 0.93 .....

^: ”· .'Ρ.ΊΪ-ΐίϊ3 <Τ'Γ · ^ ®ϊ; ΐΐ ··· Β .. \ - - ::: - ... ¹ .

'·; · ..................-' .............. * ........... .

0.94

0.00 ·

AM8 04-04-11

0 _T 00

0.01

0% Bos 0.00 0.0Q

Claims (6)

1- METHOD FOR QUANTIFICATION OF MATERIAL OF BOVINE AND BUBBLE ORIGIN IN PRODUCTS OF ANIMAL ORIGIN BY THE REAL TIME PCR TECHNIQUE, characterized by using specific primers capable of amplifying buffalo and bovine DNA molecules, represented by SEQ ID N ° ^s 1,2 , 4 and 5. 2- METHOD FOR QUANTIFICATION OF MATERIAL OF BOVINE AND BUBBLE ORIGIN IN PRODUCTS OF ANIMAL ORIGIN BY THE REAL-TIME PCR TECHNIQUE, according to claim 1, characterized by using, preferably, PCR probes in real time represented by the sequences SEQ ID n ° 3 and 6. 3- METHOD FOR QUANTIFICATION OF MATERIAL OF BOVINE AND BUBBLE ORIGIN IN PRODUCTS OF ANIMAL ORIGIN BY THE REAL TIME PCR TECHNIQUE, according to claims 1 and 2, characterized by the probes being preferably marked with the fluorophore FAM. 4- METHOD FOR QUANTIFICATION OF MATERIAL OF BOVINE AND BUBBLE ORIGIN IN PRODUCTS OF ANIMAL ORIGIN BY THE REAL TIME PCR TECHNIQUE, according to claim 1, characterized by using, preferably, DNA interleaving reagent. 5- METHOD FOR QUANTIFICATION OF MATERIAL OF BOVINE AND BUBBLE ORIGIN IN PRODUCTS OF ANIMAL ORIGIN BY THE REAL TIME PCR TECHNIQUE, according to claims 1 to 4, characterized by the total amount of DNA present in a given sample to be calculated by adding the sum the measurements specific obtained with specific primers for amplifying bovine and buffalo DNA represented by SEQ ID ^NOS: 1, 2, 4 and 5, or primer / probe represented by SEQ ID NO : 1, 2, 3, 4, 5 and 6, using a reference calibration curve. 6- KIT FOR QUANTIFICATION OF MATERIAL OF BOVINE AND BUBBLE ORIGIN IN PRODUCTS OF ANIMAL ORIGIN BY TECHNIQUE 2/2 PCR IN REAL TIME, comprising primers specific for DNA amplification cattle and buffalo, represented by SEQ ID NO : 1, 2, 4 and 5, intercalating reagent DNA probes for real time PCR represented by the sequences SEQ ID NO : ° 3 and 6 and a 5 software that allows the calculation of the amount of bovine or buffalo material present in the tested samples, taking into account the deviations computed in the reference calibration curve.