Title of Invention

"A PCR BASED METHOD FOR DIFFERENTIATING COW AND BUFFALO MILK"

Abstract

"A PCR based method for differentiating cow and buffalo milk" This invention relates to a PCR based process for the differentiation of cattle and buffalo milk samples by detecting segments of the mitochondrial D-loop gene comprising. a) Isolation of genomic DNA from predominantly leukocytes with small portion < 2% of epithelial cells of milk samples. b) Followed by PCR using two sets of self designed species specific PCR primers interms of the optimized reaction condition wherein touch down PCR is used with an initial annealing at 65°C and during following cycles the annealing temperature is gradually reduced by 1°C unitl it reached 55°C, using 25 cycles.

Full Text

Field of Invention; This invention relates to a PCR process for the differentiation of Cattle and buffalo milk samples by detecting segments of the mitochondrial D-loopgene. Background of the Invention; The production of buffalo milk in the Asia-Pacific region exceeds 45m tons annually. Over 30m tons are produced in India alone. This includes both cow and buffalo milk. Although there is no difference in the nutritive value and digestibility of milks from cow and buffalo, they differ substantially in composition and in the nature of the dairy product(s) made out of them. Buffalo milk contains 7-8% fat. The cholesterol content of buffalo milk is 0.65 mg/g as compared to 3.14 mg/g for cow milk. Buffalo milk has 11.42% higher protein than cow milk. Buffalo milk is superior to cow milk in calcium (+9%), iron (+37.7%) and phosphorus (+118%). Buffaloes metabolise all dietary carotenes into vitamin A, which is passed on to the milk. The presence of higher levels of immunoglobulins, lactoferrin, lysozyme and lactoperoxidase make buffalo milk suitable for special dietary and health foods. Buffalo milk is also very thick Difference exists in the quality of the dairy product made out of cattle and buffalo milk. Cheese made from buffalo milk displays typical body and textural characteristics. Authentic mozzarella is made from buffalo milk and more specifically, where chewing and stringing properties are specially desired. Rasogolla made out of cow milk has the highest organoleptic score. In India the mixing of cow and buffalo milk is common. It is difficult to determine the content of cow and buffalo milk from a pool of mixed milk. However, no scientific procedure/kit to differentiate cow milk from buffalo milk and vice versa, is currently available. Many methods based on DNA analysis have been employed in the food industry to monitor the adulteration of food products of animal origin. Among them, the most frequently used are: polymerase chain reaction (PCR) amplification of marker gene fragment(s) with universal primers, or amplification of DNA with species-specific primers. PCR-products of different origin can be discriminated by size, restriction fragment length polymorphism (RELP), or single stranded conformational polymorphism (SSCP) analysis. These methods have been used for the identification, and differentiation between, the animal origin raw or heat-treated meat and meat products (Lipkin et al, 1993, Chikuni et al. 1994; Lahiff et al., 2002, Lopez et al., 2004). These approaches are also applicable to the analysis of dairy products with a variable success rate. So far, no DNA-based techniques have been designed to detect the presence of bovine DNA in buffalo milk or buffalo milk in cattle milk. The method, employing buffalo and bovine-specific primers for amplification of specific mt-DNA, seems to be simple, fast, specific and sensitive. Chikuni, K. et al. (1994): Direct sequencing of the water buffalo (Bubalus bubalis)-casein gene. Anim. Sci. Technol. (Jpn.), 65, 652-655 Lahiff, S., M. Glennon, J. Lyng, T. Smith, N. Shilton and M. Maher, 2002. Real-time polymerase chain reaction detection of bovine DNA in meat and bone meal samples. J. Food Prot., 65: 1158-65. Lipkin, E., A. Shalom, H. Khatib, M. Seller and A. Friedmann, 1993. Milk as a source of deoxyribonucleic acid and as a substrate for thepolymerase chain reaction. J.Dairy Sci., 76:2025-2032. Lopez-Calleja, I., I. Gonzalez, V. Fajardo, M.A. Rodriguez, P.E. Hernandez, T. Garcia and R. Martin, 2004. Rapid Detection of Cows' Milk in Sheeps' and Goats' Milk by a Species-Specific Polymerase Chain Reaction Technique. J. Dairy Sci., 87:2839-2845. Existing methods for differentiating cow and buffalo milk; The presence of buffalo milk in cow milk is tested by the Hansa test. It is based on an immunological assay. One ml of milk is diluted with 4 ml of water and then treated with 1 ml of antiserum. The characteristic precipitation reaction indicates the presence of buffalo milk in the sample taken. (The antiserum is developed by injecting buffalo milk proteins into rabbits). Use of milk in a PCR reaction for the detection of microorganism; PCR was used in milk for the identification of various kinds of microorganisms present in it for a wholly different purpose. Brucella abortus (pathogenic abortive bacteria) can be detected by amplifying a segment (419 bp) of the OMP 25 kD gene by PCR from milk (Patent-CN1814785). A PCR-based method was used to detect the presence of nucleic acids from Mycobacterium aviwn ssp. Paratuberculosis (Mycobacterium paratuberculosis), in biological samples such as milk (EP1223225). The presences of traces of destructive bacteriophages of lactic acid bacteria (LAB) (Lactococcus, Streptococcus and Lactobacillus), which are used in industrial dairy fermentations, were reportedly detected by PCR (Patent-WO2006136640) No DNA based method is available to distinguish cow and buffalo milk on the basis of milk neutrophils and somatic cell content in milk. Drawback of the Hansa Test in distinguishing between cow and buffalo milk: The existing approaches require the raising of antibodies in the rabbit against buffalo milk proteins. We need a constant supply of the antisera for the detection of buffalo milk. The Hansa test is a qualitative test for buffalo milk. Therefore, the proportion of the cow and buffalo milk can not be determined by the Hansa test. The Hansa test requires invasive methods for injecting buffalo milk into rabbits and collection of serum for immunological test with milk. Other than the Hansa test, it is not possible to distinguish buffalo milk from cow milk. Only NDRI is makes antibodies against the buffalo milk proteins in rabbit. No commercial version of this test is readily available in the dairy industry. This in vitro selective PCR based method could be an alternate to the existing Hansa test. Problems encountered in using milk for PCR; Milk is a complex mixture of various biomolecules. It has a variable amount of proteinase activity. The fat content of the milk inhibits proper mixing with other biomolecules in small volumes and also inhibits the PCR. The Taq DNA polymerase is specifically reported to be inhibited by protease activity of the milk (Powell H A, Gooding C M, Garrett S D, Lund B M, McKee R A. Proteinase inhibition of the detection of Listeria monocytogenes in milk using the polymerase chain reaction. Lett Appl Microbiol. 1994; 18:59-61). The use of somatic cells and leukocytes in milk for a differentiation purpose has not been reported or used earlier. A short miniprep-DNA preparation using a low as 3 ml of milk was described to get DNA sufficient to use in various PCR reactions. The specificity and sensitivity of the designed oligonucleotide primers used in this process of selectively amplifying the cow or the buffalo sequence is the key component of this invention. Several common and cow/buffalo specific oligonucleotide primers are tested before they could to be used for this purpose. Objects of the Invention; The object of this invention is to develop an invitro DNA based test to differentiate cow and buffalo milk. Other object is to develop a completely non invasive laboratory test that can be implemented before the supplied milk is processed. Another object is to use milk somatic cells and leukocytes for the isolation of genomic DNA. Yet another object is to isolate DNA from milk in small quantity. Other object is to remove fat content so that any probable inhibitor of PCR is avoided. Yet another object is to detect cow and buffalo milk contaminants simultaneously. Another object is to amplify a target gene segment so as to determine the presence of cow or buffalo DNA by Judicious use of Primer Sets. Brief Description of the accompanying drawings; Figure 1: Determination of Buffalo DNA at 0%, 25%, 50%, 75% and 100% level. A total of 12 ng DNA from the milk mixture was used in each PCR reaction. M=100 bp DNA ladder. Figure 2: Determination of Buffalo DNA at 0%, 5%, 10% and 20% level. A total of 5 ng DNA from the milk mixture was used in each PCR reaction. M= 100 bp DNA ladder. Figure 3: Determination of Cattle DNA at 100%, 75%, 50%, 25% and 0% level A total of 10 ng DNA from the milk mixture was used in each PCR reaction. M=100 bp DNA ladder. Lane 1,2,3,4,5 and 6 contain 100%, 75%, 50%, 25% cattle DNA Figure 4: Determination of cattle DNA at 20%, 10%, 5% and 0% level. A total of 5 ng DNA from the milk mixture was used in each PCR reaction. M=PCR marker (80, 150, 300, 500 and 766bp). Lane 1,2,3,4,5 and 6 contain 20%, 10%, 5%, 0% and without template DNA. A total of S.OOng of genomic DNA was used in 30ul of PCR reaction mixture. Detailed description of the Invention; Milk contains a large but highly variable number of somatic cells, ranging from 104 to 107/ml, depending on the status of the cow/buffalo (parity, season, stage of lactation, and health). These cells are predominantly leukocytes but also include a small proportion ( 1. The ides of using milk somatic cells and leukocytes for the isolation of genomic DNA: PCR was used in milk for the identification of various kinds of microorganisms present in it for a wholly differently purpose. Brucella abortus /pathogenic abortive bacteria) can be detected by amplifying a segment (419 bp) of the OMP 25 kD gene by PCR from milk. A PCR-based method was used to detect the presence of nucleic acids from Mycobacterium aviwn ssp. Paratuberculosis (Mycobacterium paratuberculosis), in biological samples such as milk. The presence of traces of destructive bacteriophages of lactic acid bacteria (LAB) (Lactococcus, Streptococcus and Lactobacillus), which are used in industry dairy fermentations, were reportedly detected by PCR. However, no DNA based method is available to distinguish cow and buffalo milk on the basis of milk neutrophil and somatic cell content in milk. Extracting genomic DNA from a milk sample as template and carrying out a general PCR test with two sets of self designed primers in terms of the optimized reaction condition, which can be enlarged to amplify a target gene segment so as to determine the presence of cow or buffalo DNA. Procedure for the extraction of nucleic acids from milk: The milk samples (3ml) were centrifuged at 10,000g for 1 min and the supernatant was discarded. The pellet was washed once in normal saline solution. Finally, the pellet was dissolved in 50ml TENS (lOmM Tris, pH 8.0, 1mM EDTA; lOOmM Nacl and 0.5% SDS) and incubated at 50°C in a waterbath for 3 hr with proteinase K (100 ug/ml) for protein digestion. The samples were extracted once with phenol: chloroform (1:1) and DNA was precipated with 0.8 volumes of isopropanol in the presence of 0.1 volume of 3 M Sodium Acetate (pH 5.3). After washing with a 70% ethanol solution, the DNA pellet was dried and dissolved in 10 |al of sterile distilled water (De et al, 2000). Ouantitation of DNA by a UV-VIS spectrophotometer; The concentration of purified DNA was determined by measuring the absorbance at 260 nm and 280 nm in a UV-VIS spectrophotometer (Nanodrop R). The reading at 260 nm allows for the calculation of the concentration of nucleic acids in the sample. An OD reading of 1 corresponds to approximately 50 ng/mL for double-stranded DNA. Primer Details used for Cattle and Buffalo milk differentiation: (Table Removed) Example; 10 µL of purified DNA Measure absorbance of the 2 µL sample at 260 nm in Nanodrop. OD260 = 0.8 DNA concentration (µg/mL) = 50 |ag/mL x OD260 = 50 x 0.8 = 40 µg/mL Total Yield in 10 µL sample = DNA concentration x volume of sample in milliliters = 40 µg/mL x 0.01 mL =0.4 µg The DNA extracted from the mixed milk was diluted accordingly to get 10 ng or 12 ng/µL of diluted DNA stock before starting the PCR reaction. The 5ng DNA mix was prepared by mixing an appropriate quantity of cattle and buffalo DNA from their respective stocks to estimate the sensitivity of the reaction. This DNA was taken in 30 nL of the PCR master mix. PCR parameters; The touch-down PCR was used with an initial annealing at 65°C. During the following cycles, the annealing temperature was then gradually reduced by 1°C until it reached 55°C. At 55°C, 25 cycles were used. This method was used to avoid nonspecific PCR products in the presence of a complex genomic DNA mixture, where nonspecific annealing is more probable. Standard Tag DNA polymerase (0.5 unit/reaction, Bangalore Genei, Bangalore, India), MgCI2 (1.5 mM) and dNTP mix (200u) was used for the PCR amplification reaction. Results: A species specific polymerase chain reaction amplifying a fragment of the mitochondrial D-loop region gene was developed for the detection of bovine milk and buffalo milk in a milk mixture. Primer specificity and functionality was tested by amplifying reference DNA from the blood of both cow and water buffaloes. A schematic amplification plot was described diagrammatically in each figure. PCR products were subsequently analyzed by agarose gel electrophoresis and by the comparison of their migration patterns with a standard DNA marker. Buffalo milk Identification from a milk mix/pooled milk: The test process was divided into two components. In first part, the buffalo milk was identified. Subsequently the cow milk was identified. The buffalo milk was identified from a serial mixture of milk containing 0%, 25%, 50%, 75% and 100% buffalo milk. In Figure I, the buffalo component was detected specifically from a milk mixture containing 0%, 25%, 50%, 75% and 100% of buffalo milk. The primers 1 and 4 were used to selectively amplify a 226 bp mitochondrial D-loop reigion of buffalo. Although the bovine-to-buffalo milk ration was known, the DNA concentration in samples was also assessed prior to PCR reaction. The total genomic DNA was estimated to be 12 ng per 30 nl of the PCR mixture. Detection of Buffalo Milk (DNA) in a mixed Milk(Table Removed) Figure 1: Determination ofBuffalo DNA at 0%, 25%, 50%, 75% and 100% level. A total of 12 ng DNA from the milk mixture was used in each PCR reaction. M=1OO bp DNA ladder. To fine tune/validate this PCR based test to detect a very low lever of buffalo DNA, buffalo DNA was mixed with cattle DNA at 0%, 5%, 10% and 20% level (Figure 2). A second set of primers (2 and 5) was used to amplify another buffalo specific 283 bp region from the buffalo mitochondrial D-loop region. The total DNA in the PCR reaction was reduced to 5 nanograms per PCR reaction mix. The amplification was 13 found in a PCR reaction tube containing 0.25 nanograms (Figure 2 lane 2) of buffalo genomic DNA. Detection of Buffalo DNA in a mixed DNA(Table Removed) Figure 2: Determination ofBuffalo DNA at 0%, 5%, 1O% and 20% level. A total of 5 ng DNA from the milk mixture was used in each PCR reaction. M=1OO bp DNA ladder. Cow milk identification from the milk mix/pooled milk; The cow milk was identified from a serial mixture of milk containing 0%, 25%, 50%, 75% and 100% of cow milk respectively. In Figure 3, the cow component was detected specifically from a milk mixture containing 100%, 75%, 50%, 25% and 0% of cow milk. The primers 1 and 7 were used to selectively amplify a 126 bp mitochondiral D-loop region of cow. The total genomic DNA was estimated to be 10ng per 30µl of each PCR reaction mixture. Detection of Cattle Milk (DNA) in a mixed Milk(Table Removed) Figure 3: Determination of Cattle DNA at 10O%, 75%, 50%, 25% and 0% level. A total of 10 ng DNA from the milk mixture was used in each PCR reaction. M=100 bp DNA ladder. Lane 1,2,3,4,5 and 6 contain 100%, 75%, 50%, 25% cattle DNA. To validate this PCR based test to detect a very low level of cow DNA, the cow DNA was mixed with buffalo DNA at 0%, 5%, 10% and 20% levels respectively (Figure 4). A second set of primers (2 and 3) was used to amplify another cow specific 180 bp region from the bovine mitochondrial D-loop region. The total DNA in the PCR reaction was reduced to 5 nanograms per PCR reaction mixture. Amplification was obtained from a PCR reaction containing 0.25 nonograms (Figure 4 lane 3) of cow genomic DNA. Detection of Cattle DNA in a mixed DNA(Table Removed) Figure 4: Determination of cattle DNA at 20%, 10%, 5% and 0% level. A total of 5 ng DNA from the milk mixture was used in each PCR reaction. M=PCR marker (80, ISO, 300, 5OO and 766bp). Lane 1,2,3,4,5 and 6 contain 20%, 10%, 5%, 0% and without template DNA. A total of S.OOng ofgenomic DNA was used in 30ul of PCR reaction mixture. The possibility to detect small quantities is important, in addition to recognizing frauds, and also in protecting consumers allergic to cow milk or buffalo milk proteins. Due to its low detection limit, the assay here described is suitable to routine analysis and can be applied to detect fraudulent adulterations. 16 Summary of the detection/comments on specificity and sensitivity: 1. A total of two primer sets were tested in each case for the detection of either cattle or buffalo DNA in this study. All four sets were found specific for their respective species DNA. No cross-species amplification was encountered in this study. 2. Cow and Buffalo milk (without mixing) could be detected without any ambiguity (photograph not provided in this report). 3. In case of milk mixture, either cow or buffalo DNA could be detected at 0.25 ng (nanogram) of DNA in the 30 µl PCR reaction. This is evident from figure 2 and figure 4. 4. The PCR is very specific and no cross-species amplification was encountered in this study. It is to be noted that the formulation of the present invention is susceptible to modifications, adaptations and changes by those skilled in the art. Such variant formulations are intended to be within the scope of the present invention which is further set forth under the following claims:- We claim; 1. A PCR based process for the differentiation of cattle and buffalo milk samples by detecting segments of the mitochondrial D-loop gene comprising. a) Isolation of genomic DNA from predominantly leukocytes with small portion b) Followed by PCR using two sets of self designed species specific PCR primers interms of the optimized reaction condition wherein touch down PCR is used with an initial annealing at 65°C and during following cycles the annealing temperature is gradually reduced by 1°C unitl it reached 55°C, using 25 cycles. 2. The process for the differentiation of cattle and buffalo milk samples as claimed in claim 1, wherein total genomic DNA is estimated to be 12 ng per 30 µl of the PCR mixture. 3. The process for .the differentiation of cattle and buffalo milk samples as claimed in claim 1, wherein the primers 1 and 4 are used to selectively amplify a 226 bp mitochondrial D loop region of buffalo. 4. The process for the differentiation of cattle and buffalo milk samples as claimed in claim 1, wherein primers 2 and 5 are used to amplify another buffalo specific 283 bp region from the buffalo mitochondrial D-loop gene. 5. The process for the differentiation of cattle and buffalo milk samples as claimed in claim 1, wherein primers 1 and 7 are used to selectively amplify a 126 bp mitochondrial D-loop region of cow. 6. The process for the differentiation of cattle and buffalo milk samples as claimed in claim 1, wherein primers 2 and 3 are used to selectively amplify 180bp region from bovine mitochondrial D loop region.

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1385-del-2007-Abstract-(05-12-2012).pdf

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1385-delnp-2007-form-1.pdf

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