KR101209786B1 - PCR or real-time PCR primers for identification of animal species, the kit comprising the primers, and the method for identifying animal species using the primers or kits - Google Patents

Abstract

The present invention is a livestock discrimination PCR or real-time PCR primer composition for rapid identification of 10 livestock of cattle, pigs, chickens, ducks, sheep, goats, horses, dogs, turkeys and geese, livestock discrimination comprising the same Provided are a PCR kit for animal breeding using the same. Livestock differentiation PCR or real-time PCR primer composition according to the present invention and PCR or real-time PCR kit comprising the same can be used to ensure the stability of food by introducing into the label verification of food by applying to a single PCR, multiple PCR and real-time PCR .

Description

Livestock discrimination PCR or real-time PCR primer composition, livestock discrimination PCR kit comprising the same and livestock discrimination method using the same {PCR or real-time PCR primers for identification of animal species, the kit comprising the primers, and the method for identifying animal species using the primers or kits}

The present invention is a livestock discrimination PCR or real-time PCR primer composition for rapid identification of 10 livestock of cattle, pigs, chickens, ducks, sheep, goats, horses, dogs, turkeys and geese, livestock discrimination comprising the same Provided are a PCR kit for animal breeding using the same. Livestock differentiation PCR or real-time PCR primer composition according to the present invention and PCR or real-time PCR kit comprising the same can be used to ensure the stability of food by introducing into the label verification of food by applying to a single PCR, multiple PCR and real-time PCR .

As the industry develops and incomes rise, many consumers choose better foods, including livestock.

Meat consumption is increasing day by day to consume protein, but there are also consumers who have to eat meat like other produce due to allergic reactions or religion. It is increasing. In particular, among many foreigners who have moved from Korea to foreign countries, people who follow religious regulations such as Islam and vegetarians value information on meats used in food (Calvo et al., 2001, J Anim Sci 79: pp. 2108-2112).

In addition, the safety and illegal distribution of livestock products that may be caused by deceitful or unintentionally misrepresented meat products, inexpensive meat mixtures, indistinct or inadequate products, and the influx of meat from any animal that may be detrimental to human and animal health. There is a social need for test methods to prevent. Therefore, scientific tests have been developed by various researchers to objectively verify livestock labeling for livestock products to ensure consumer reliability and product stability. [Calvo et al., 2001, J Anim Sci 79: pp. 2108-2112; Dalmasso et al., 2004, Mol Cell Probes 18 (2): pp. 81-87; Lanzilao et al., 2005, J AOAC Int 88 (1): pp. 128-135; Mart'n et al., 2007a, J Anim Sci 85 (2): pp. 452-458; Mart'n et al., 2007b, J Anim Sci 85 (10): pp. 2734-2739; Pfeiffer et al., 2004, BMC Genet 5: p. 30; Wolf et al., 1999. J Agric Food Chem 47 (4): pp. 1350-1355].

As a result, the National Veterinary Research and Quarantine Service has established a meat discrimination method for processing standards and composition standards of livestock products, but its problems are as follows.

method of inspection Criteria Differential breeding Remarks Glycogen Test Horse (0.675%) Small (0-0.164%)
Veal (0%), Pig (0%) Horse, Cattle, Calf,
pig Criteria vague District inspection Duplicate targets all duplicated Horse, cow, sheep, pig Indeterminate Mercy Vapor smell - Tester's experience and subjectivity Serological test Check the sedimentation line - Heat treatment meat judgment difficulty Histidine
Dipeptide Content Determination Horse 134, Cow 8, Pig 30
Chicken 0.3, Duck 1.6, Turkey 0.07 6 breeders including horses Mixed product not judged Gene hybridization Color wheel and color intensity 9 breeders including horses Inspection period 5 days
Test method Difficult

The meat discrimination method, glycogen test method, fat test method and mercy method adopted by the National Veterinary Research and Quarantine Service published in the processing standard and ingredient standard are ambiguous or very subjective. In addition, serological test methods such as agar gel immunoassay and enzyme immunoassay are specific and sensitive, but cross-reaction occurs in some species [Berger et al., 1998, J Assoc Off Anal Chem 71 (2): 406-. P. 409; Hsieh et al., 1988, J Food Prot 61 (4): 476-481]. In addition, electrophoresis using protein in meat [Kim et al., 1986, J Food Sci 51 (3): pp. 731-741; Skarpeid et al., 1998, Electrophoresis 19 (18): 3103-3109], liquid chromatography [Chou et al., 2007, J Chromatogr B Analyt] Different types of assays can be used for differentiation, including Technol Biomed Life Sci 846 (1-2): pp. 230-239, but biological activity disappears after proteins are slaughtered, and their characteristics change according to cell classification. Breeder discrimination is known to be very difficult in processed specimens because it is denatured during processing [Montiel-Sosa et al., 2000, J Agric Food Chem 48 (7): 2829-2832].

On the other hand, in response to bovine spongiform encephalopathy, various countries have banned the feeding of mammalian substances to ruminants, and in 2004, it was forbidden to use leftover foods as feedstock for ruminants such as cattle. Although the Feed Management Act was revised and announced in December, the EU-approved microscopy method for the prevention of bovine spongiform encephalopathy requires a lot of time and professional skills as a method of examining the presence of animal scraps in feed [European Commission. Commission directive 98/88 / EC of 13 November 1998 establishing guidelines for the microscopic identification and estimation of constituents of animal origin for the official control of feedingstuffs. 1998. Off J Eur Comm L 318: pp. 45-50; European Commission. Commission regulation 1774/2002 / EC of 3 October 2002 laying down health rules concerning animal by-products not intended for human consumption. 2002, Off J Eur Comm L 273: pp. 1-95].

In addition, meat discrimination using the histological and anatomical characteristics unique to the breeding is possible only by the animal classification (mammals, birds and fishes), and the breeding species of its origin cannot be determined.

In order to solve this problem, a meat discrimination method by genetic testing using DNA from tissues such as cattle, sheep, pigs, and poultry, blood, and the like has been developed. See, eg, Polymerase chain reaction (PCR) [Calvo et al., 2001, J Anim Sci 79: 2108-2112; Mart'n et al., 2007a, J Anim Sci 85 (2): pp. 452-458; Mart'n et al., 2007b, J Anim Sci 85 (10): pp. 2734-2739], DNA hybridization [Lee Myung-hun et al., Journal of Veterinary Medicine 1999, 39 (3): 513-522], random amplified polymorphic DNA fingerprints (RAPD). ) -PCR [Lee et al., 1994, Forensic Sci Int 67 (2): pp. 103-107] has been developed and identified mutations in mitochondrial DNA (mt DNA) of livestock by PCR-restriction fragment length polymorphism (RFLP). Differentiation of livestock species has also been developed [Lanzilao et al., 2005, J AOAC Int 88 (1): pp. 128-135; Pfeiffer et al., 2004, BMC Genet 5: 30; Wolf et al., 1999. J Agric Food Chem 47 (4): pp. 1350-1355].

Real-time PCR has also been developed to test genes by measuring the amount of various fluorescent pigments in real time, which can be classified into two types and complementarily bind to PCR products such as TaqMan or FRET chemicals. Probes as described by Laube et al., 2003, International Journal of Food Science and Technology 38: 111-118; Rensen et al., 2005, Foodborne Pathog Dis 2 (2): pp. 152-159] and SYBR Green [Fajardo et al., 2008, J AOAC Int 91 (1): pp. 103-111]. It is divided into DNA intercalating dye which binds with. SYBR Green may be inaccurate to emit fluorescence by reacting with double helix regardless of the nucleotide sequence of the nucleus. The 5'-3 'exonuclease method based on the fluorescence probe is known to improve the specificity and sensitivity significantly compared to the conventional PCR test because it does not fluoresce nonspecifically, and it also has the advantage of reducing the time required for the test [Laube et al. , 2003, International Journal of Food Science and Technology 38: 111-118; Lpez-Andreo et al ., 2005, Anal Biochem 339 (1): pp. 73-82; Tanabe et al ., 2007, Biosci Biotechnol Biochem 71 (12): pp. 3131-3135].

However, in the case of differentiation of breeders using the conventional PCR technique, it is mostly differentiation using primers for a single breeder, so it is only possible to confirm whether the breeder is amplifying the breeder, and it is not easy to discriminate exactly what breeding species. In order to distinguish the breeding species for unknown breeding specimens, it was very cumbersome, such as conducting several PCR experiments to confirm the corresponding species for each breeding species.

Therefore, there has been a need in the art for the development of a method for differentiating several livestock under the same PCR conditions.

In general, PCR amplifies the gene by repeating denaturation, annealing, and polymerization. The annealing temperature at which the DNA backbone and the primer bind is determined by the GC / AT ratio between the backbone and the primer. Will be. Therefore, the design of primers for several livestock species used for food should be carried out by (i) PCR amplification by specifically binding to the DNA backbone of the livestock, and (ii) the same hybridization that binds all the primers designed for each livestock to the backbone The temperature must be shared and (iii) the PCR amplification products must have a size that can be distinguished between animal species. However, the breeders used for food are very diverse, and it was very difficult to design primers satisfying all the above conditions for all breeders.

As used herein, the term “primer set” refers to a set of forward and backward primers that can be bound to the DNA backbone in the hybridization step for PCR amplification.

As used herein, the term "primer composition" means a composition comprising the "primer set", and may or may not further include a solvent for dissolving the primer set or a component for stabilization of the primer molecule. The primer set may further include a probe or intercalator capable of binding to a PCR amplification product when the primer set is used for a real-time PCR amplification reaction.

The present inventors compared the nucleotide sequences of the 12S rRNA and the 16S rRNA gene of mt DNA by aligning the nucleotide sequences provided in the Genbank databases of the 10 livestocks, and designed the primers for the 10 livestocks. When using primers of (i) PCR amplification by binding specifically to the DNA backbone of the species, (ii) primers designed for each species share the same hybridization temperature that binds to the backbone, (iii) PCR The amplification products can be distinguished between various livestock species, and thus, the primers were appropriately selected according to the livestock species, and the present inventors have found that the type of livestock can be identified quickly and accurately even after performing one PCR.

Therefore, an object of the present invention is a PCR that can quickly and accurately discriminate livestock by performing only one PCR test under the same PCR conditions for 10 livestock of cattle, pigs, chickens, ducks, sheep, goats, horses, dogs, turkeys, and geese. It is to provide a breeding species PCR composition comprising a primer for the breeding species and PCR kit comprising the same.

In addition, another object of the present invention is to provide primers and probes for real-time PCR that can quickly and accurately discriminate and quantify livestock breeding with only one real-time PCR under the same PCR conditions for cattle, pigs, chickens, ducks, turkeys and geese. It is to provide a real-time PCR primer composition for discriminating livestock, and a real-time PCR kit for breeding including the same.

In order to achieve the above object, the breeding primer composition according to the present invention is a primer capable of specifically amplifying DNA obtained from cattle, sheep, goats, horses, dogs, pigs, chickens, ducks, geese or turkey specimens. Including a set, in the PCR primer composition for differentiating stocks used to discriminate one or more stocks selected from the group consisting of cattle, sheep, goats, horses, dogs, pigs, chickens, ducks, geese and turkeys through PCR amplification ,

The primer set is characterized in that it comprises one or more primer sets selected from the group consisting of (a) to (o):

(a) a set of primers for amplifying bovine DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 1 and 2;

(b) a set of primers for amplifying bovine DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 3 and 4;

(c) a primer set for positive DNA amplification, comprising a primer having a nucleotide sequence of SEQ ID NOs: 5 and 6;

(d) a set of primers for amplifying goat DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 7 and 8;

(e) a set of primers for amplifying DNA, comprising a primer having the nucleotide sequences of SEQ ID NOs: 9 and 10;

(f) a set of primers for DNA amplification, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 11 and 12;

(g) a primer set for amplifying pig DNA, comprising a primer having a nucleotide sequence of SEQ ID NOs: 13 and 14;

(h) a primer set for amplifying pig DNA, comprising a primer having the nucleotide sequences of SEQ ID NOs: 15 and 16;

(i) a primer set for amplifying pig DNA, comprising a primer having the nucleotide sequences of SEQ ID NOs: 17 and 18;

(j) a set of primers for DNA amplification of chicken, comprising a primer having the nucleotide sequences of SEQ ID NOs: 19 and 20;

(k) a set of primers for DNA amplification of chicken, comprising a primer having the nucleotide sequences of SEQ ID NOs: 21 and 22;

(l) a set of primers for DNA amplification of a duck, comprising a primer having the nucleotide sequences of SEQ ID NOs: 23 and 24;

(m) a set of primers for DNA amplification of a goose, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 25 and 26;

(n) a set of primers for DNA amplification of a goose, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 27 and 28;

(o) A primer set for DNA amplification of a turkey, comprising a primer having a nucleotide sequence of SEQ ID NOs: 29 and 30.

The nucleotide sequence of the primer set that can be used for single PCR and multiplex PCR for breeding is as follows.

Vine species designation The sequence (5 '- > 3') SEQ ID NO: Gene region Size of amplification product
(bp) 1 Forward ATTCTCTACACCAAGAGAATCAAGC SEQ ID NO: 1 12S rRNA 190 Reverse CCTCTCATGTAGCTAGTGCGTTTA SEQ ID NO: 2 2 Forward GCCAGAGTACTACTAGCAACTGGTTA SEQ ID NO: 3 12S rRNA 280 Reverse TTTCATAATAACTTTCGTGCTTGG SEQ ID NO: 4 Sheep Forward CACCCTCCTCAAGTAAATATGATAT SEQ ID NO: 5 12S rRNA
-16S rRNA 219 Reverse TATTTAAACTGGAGAGTGGGAGA SEQ ID NO: 6 Goat Forward AATACGAAAGCCATTATGAAATTAATG SEQ ID NO: 7 12S rRNA
-16S rRNA 350 Reverse GGTAAATGTTTTGTTTTAGACTGTTTT SEQ ID NO: 8 Words Forward AAGAACAAGAACTTTAACCCGG SEQ ID NO: 9 12S rRNA
-16S rRNA 467 Reverse TTTGCTGTTTAGTACTTTTAATGCA SEQ ID NO: 10 dog Forward CCTGCCCGGTGACACTTG SEQ ID NO: 11 16S rRNA 241 Reverse TGCCTTGTAGGTATCTAGTATCCATAAG SEQ ID NO: 12 Pig 1 Forward GAACAATAGTAAGCACAATCATAGCA SEQ ID NO: 13 12S rRNA 119 Reverse ATAAAAACTTTCGTGTGGTGGATATT SEQ ID NO: 14 Pig 2 Forward AACAATAGTAAGCACAATCATAGCA SEQ ID NO: 15 12S rRNA 193 Reverse CTTTACGCCGTGGATCTATTAAT SEQ ID NO: 16 Pig 3 Forward TATCAAAATCTCATTTGTCAGGGAA SEQ ID NO: 17 Fibroblast growth factor 7 153 Reverse ATGATACACTGTTATGCAGTATACTTTCA SEQ ID NO: 18 Chicken 1 Forward TGAGCTCAATAGCCCCTCG SEQ ID NO: 19 12S rRNA 171 Reverse CGTCTTAAAGTGAGCTTAGGGGG SEQ ID NO: 20 Chicken 2 Forward CAACAGTGAGCTCAATAGCCACTC SEQ ID NO: 21 12S rRNA 175 Reverse CGTCTTAAAGTGAGCTTAGGG SEQ ID NO: 22 duck Forward GTCAAGGTATAGCCTATCGGAC SEQ ID NO: 23 12S rRNA 229 Reverse CGACTTACCTCATCTTTGGCA SEQ ID NO: 24 Goose 1 Forward GACTTAGTTATAGCAACAGCCTAACTTC SEQ ID NO: 25 12S rRNA 111 Reverse CACTCTTTACGCCGTGTCAATA SEQ ID NO: 26 Goose 2 Forward ACGGAAAGAAGCGTGTATCCAC SEQ ID NO: 27 12S rRNA 166 Reverse CTTACCTCATCTTCAGCGTAATAAT SEQ ID NO: 28 Turkey Forward AACGCATCAAAGTACTAATAGTAATTT SEQ ID NO: 29 16S rRNA 268 Reverse CTTTCGTACTAGGAGGAGTTCTTA SEQ ID NO: 30 IPC Forward CGGTCTGAACTCAGATCACGTA SEQ ID NO: 31 18S rRNA 152? 3 Reverse CTAGGGATAACAGCGCAATC SEQ ID NO: 32

According to the present invention, the method for obtaining DNA from cattle, sheep, goats, horses, dogs, pigs, chickens, ducks, geese or turkey specimens can be carried out according to methods conventional in the art. See, eg, Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. It may be prepared as disclosed in Cold Spring Harbor Press (2001), but the present invention is not limited thereto.

On the other hand, cattle, sheep, goats, horses, dogs, pigs, chickens, ducks, geese or turkey specimens may be any species in which the carcasses of the livestock are used as food, such as Bos taurus , sheep ( Ovis aries ) , Goat ( Capra hircus ), Horse ( Equus caballus ), Dog ( Canis familiaris ), Pig ( Sus scrofa domestica ), Chicken ( Gallus gallus ), Duck ( Anas platyrhynchos; Cairina muschata ), Goose ( Anser anser ), Turkey ( Meleagris) gallipavo ), and the livestock specimens for obtaining DNA from these breeders may be cells, tissues, organs or body parts of the breeder, for example, a site containing a small amount of the breeder's DNA such as carcass, blood, and hair. It may be a portion, and may be preferably carcass. DNA obtained therefrom can be used as template DNA in performing all PCR such as single PCR, multiple PCR or real time PCR.

The breeder's PCR primer composition according to the present invention can be used to perform a PCR reaction by including in a PCR reaction solution, the PCR reaction is a denaturation step at 95 ° C, an annealing step at 57 ° C and 72 The cycle of the polymerization step at ° C. may be repeated. When the PCR reaction is completed, it is possible to determine the PCR amplification by checking the molecular size of the PCR amplification product through electrophoresis.

When the PCR primer composition for discriminating livestock according to the present invention includes only one primer set selected from the group consisting of the above (a) to (o), the unknown breeder corresponds to the breeder specifically amplified by the selected primer set. Can be used to determine if

Breeding discrimination PCR primer composition according to the invention comprises two or more primer sets selected from the group consisting of (a) to (o), cattle, sheep, goats, horses, dogs, pigs, chickens, ducks, geese And amplified DNA obtained from two or more livestock species selected from the group consisting of turkeys, and may include a primer set selected such that sizes of PCR amplification products are different from each other by livestock type.

For example, DNA obtained from two or more breeding species selected from the group consisting of cattle, sheep, goats, horses, dogs, pigs, chickens, ducks, geese and turkeys may be amplified for unknown specimens of unknown breeding species. In order to prepare a PCR primer composition for differentiation comprising two or more sets of primers selected from the group consisting of (a) to (o) to be included in the PCR reaction solution to perform a PCR reaction using a DNA obtained from an unknown sample as a main chain. If performed, it may be confirmed by one PCR whether the unknown sample corresponds to a breeder that specifically amplifies the selected primer set or two or more breeders are mixed.

In this case, the breeding PCR primer composition according to the present invention preferably comprises a primer set selected so that the size of the PCR amplification products are different from each other by breeding species. For example, the Goose 1 primer set and the Pork 1 primer set are almost similar in size to PCR amplification products, so instead of the goose 1 primer, instead of the goose 2 primer set or the pig 1 primer set to the pig 2 or pig 3 primer set By differentiating the size of the PCR reaction product for the two livestock species it is possible to distinguish between livestock species through a single PCR.

In order to confirm whether the PCR primer composition for breeding according to the present invention is performed in a normal PCR amplification reaction, a primer set for detecting eukaryotic cells having the nucleotide sequences of SEQ ID NOs: 31 and 32 as a control may be used for internal genes (Internal positive control; IPC) may further comprise a primer.

The present invention also provides a breeder discrimination PCR kit comprising a breeder discrimination PCR primer composition according to the present invention.

The kit according to the present invention may package the primer set in one reaction vessel, strip or microplate, and may be packaged by methods known in the art. The kit according to the present invention may include the components required for the PCR reaction in the PCR primer composition for different breeding species or provide them in a separate container.

In addition, the kit of the present invention may further include one or more selected from the group consisting of Taq polymerase, Taq polymerase, MgCl _2, and the like, dNTP, and stabilizer, in addition to those known in the art. Other reagents may be additionally included.

The kit of the present invention can accurately and simply confirm whether or not the authenticity of the livestock species used as food and / or other livestock species other than the labeling in the food.

According to the present invention, the minimum detection concentration of bovine and chicken genes in multiplex PCR is 100 fg / uL, and the minimum detection concentration of pig and duck genes is 1.0 pg / uL.

In addition, the present invention comprises the steps of (i) obtaining DNA from the livestock specimen; (ii) using the obtained DNA as a main chain and obtaining a PCR amplification product using the PCR primer composition for differentiation according to the present invention; (iii) electrophoresis the obtained PCR amplification product to determine the size of the amplification product to provide a method for discriminating the breeder comprising the step of discriminating the breeder of the sample.

The livestock differentiation method according to the present invention may be used to check whether mixed livestock is mixed with other livestock in addition to the livestock indicated in the product, even if the livestock is heat-treated to a food made of the material, or mixed in the state of the livestock product.

Meanwhile, the present invention provides a primer set capable of specifically amplifying DNA obtained from a bovine, pig, chicken, duck, goose or turkey sample, and a probe capable of specifically binding to a PCR amplification product by the primer set. Or an intercalator, comprising a livestock PCR real-time PCR primer composition for discriminating livestock selected from the group consisting of cows, pigs, chickens, ducks, geese, and turkeys. It provides a real-time PCR primer composition for breeding discrimination comprising at least one primer set selected from the group consisting of (i) to (ix):

(i) a set of primers for amplifying bovine DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 33 and 35;

(ii) a set of primers for amplifying bovine DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 34 and 35;

(iii) a set of primers for amplifying bovine DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 37 and 39;

(iv) a set of primers for amplifying bovine DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 38 and 39;

(v) a primer set for amplifying pig DNA, comprising a primer having a nucleotide sequence of SEQ ID NOs: 41 and 14;

(vi) a set of primers for DNA amplification of chickens comprising a primer having the nucleotide sequences of SEQ ID NOs: 43 and 44;

(vii) a set of primers for DNA amplification of a duck, comprising a primer having the nucleotide sequences of SEQ ID NOs: 45 and 46;

(viii) a set of primers for DNA amplification of a goose, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 25 and 26;

(ix) A primer set for DNA amplification of a turkey, comprising a primer having a nucleotide sequence of SEQ ID NOs: 47 and 48.

When the real-time PCR primer composition for discriminating livestock according to the present invention comprises a primer set selected from the group consisting of (i) to (ix), whether the unknown livestock corresponds to the breeding species specifically amplified by the selected primer set. Can be used to determine.

A probe capable of specifically binding to a PCR amplification product by a primer set selected from the group consisting of (i) to (ix) has the ability to complementarily bind to a sequence of a PCR amplification product and hybridization during PCR polymerization. It may be designed in an appropriate sequence in consideration of the annealing temperature.

As an example of such a probe sequence, when the primer set is (i) or (ii), the probe has the nucleotide sequence of SEQ ID NO: 36; When the primer set is (iii) or (iv), the probe has the nucleotide sequence of SEQ ID NO: 40; When the primer set is (v), the probe has a nucleotide sequence of SEQ ID NO: 42; When the primer set is (vi), (vii), (viii) or (ix), the probe may have a nucleotide sequence of SEQ ID NO: 49, but the present invention is not limited thereto.

The base sequences of the primer sets and probes used for real time PCR are for example:

Vine species designation The sequence (5 '- > 3') SEQ ID NO: Amplified products
Size (bp) R-S1 Forward 1 CACGAAAGTTATTATGAAACCAATAA SEQ ID NO: 33 166 bp Reverse 1 CCTCTCATGTAGCTAGTGCGTTTA SEQ ID NO: 35 Probe 1 CACCCTCCTCAAATAGATTCAGTGCATCT SEQ ID NO: 36 R-small 2 Forward 2 CATTCTCTACACCAAGAGAATCAAGC SEQ ID NO: 34 191bp Reverse 1 CCTCTCATGTAGCTAGTGCGTTTA SEQ ID NO: 35 Probe 1 CACCCTCCTCAAATAGATTCAGTGCATCT SEQ ID NO: 36 R-Small 3 Forward 3 TCGCTCGGAGGATTTTTTATT SEQ ID NO: 37 116 bp Reverse 2 GCTTTAACTAACCAACCCAAAGAG SEQ ID NO: 39 Probe 2 CCCAACCGAAACTACCAACTCATGGAGATT SEQ ID NO: 40 R-S4 Forward 4 TTTGGATCAATGAGCGATAGAG SEQ ID NO: 38 168 bp Reverse 2 GCTTTAACTAACCAACCCAAAGAG SEQ ID NO: 39 Probe 2 CCCAACCGAAACTACCAACTCATGGAGATT SEQ ID NO: 40 R-pig Forward GGAACAATAGTAAGCACAATCATAGCA SEQ ID NO: 41 121 bp

Reverse ATAAAAACTTTCGTGTGGTGGATATT SEQ ID NO: 14 Probe AGGTCAAGGTGTAGCTTATGGGTTGGA SEQ ID NO: 42 R-chicken Forward GACTTAGCCATAGCAACCCAG SEQ ID NO: 43 95 bp Reverse CACTCTTTACGCCGGGTA SEQ ID NO: 44 Probe TGCCAGCCACCGCGGTCAT SEQ ID NO: 49 R-Goose 1 Forward GACTTAGTTATAGCAACAGCCTAACTTC SEQ ID NO: 25 111 bp Reverse CACTCTTTACGCCGTGTCAATA SEQ ID NO: 26 Probe TGCCAGCCACCGCGGTCAT SEQ ID NO: 49 R-duck Forward TAGCAAGCCTCCACCCAA SEQ ID NO: 45 97 bp Reverse CACTCTTTACGCCGCTTGT SEQ ID NO: 46 Probe TGCCAGCCACCGCGGTCAT SEQ ID NO: 49 R-Turkey Forward AACTTGACTTAGCCATAGCAACTTTA SEQ ID NO: 47 201 bp Reverse CGTTAAGTTAAGATCATTTTTTAGGCT SEQ ID NO: 48 Probe TGCCAGCCACCGCGGTCAT SEQ ID NO: 49 R-IPC Forward CGGTCTGAACTCAGATCACGTA SEQ ID NO: 31 Reverse CTAGGGATAACAGCGCAATC SEQ ID NO: 32 Probe TGATCCAACATCGAGGTCGTAAACC SEQ ID NO: 50

The probe included in the real-time PCR primer composition for differentiation of livestock according to the present invention is labeled in a conventional manner such as connecting, binding or attaching the probe to the probe to enable detection of the amplification product during the real-time PCR reaction. Density, concentration, amount, etc. can be confirmed.

For example, it may be labeled with a commonly used fluorescent labeling factor, a luminescent material, a bioluminescent material, an isotope, etc., but the present invention is not limited thereto. It is preferred to be labeled with a fluorescent labeling factor.

Fluorescent labeling factors are now commercially available on the market and are readily available. For example, FAM, VIC, TAMRA, JOE, ROX, NED, HEX, TET, SYBR Green, Cy3, Texas Red, Cy5, RED 610, RED670, etc. may be labeled at the 5'-end of the probe. However, the present invention is not limited thereto.

In addition, the 3'-end of the probe is a quencher that suppresses the emission of the fluorescent labeling factor labeled at the 5'-end so that the luminescence is suppressed before binding to the PCR amplification product in order to emit light only when the PCR amplification reaction occurs It is preferable to label by (quencher).

If the excitation and emission wavelengths are different depending on the type of fluorescent labeling factor, the method of use is also different. Therefore, if two or more fluorescent labeling factors are used in one PCR reaction solution, it is determined whether the real-time PCR device has a separate detectable means. You must judge and use it. Specific matters and selection of the method of using the fluorescent labeling factors will be apparent to those skilled in the art.

For example, when two or more probes labeled with fluorescent labeling factors having different excitation and emission wavelengths are selected to hybridize to PCR amplification products for each species, separate real-time PCR reaction solutions are prepared to distinguish unknown species. Livestock differentiation can be performed more quickly and accurately by performing real time PCR through one real time PCR reaction solution.

Various methods can be used to confirm the real-time amplification of amplification products in the real-time PCR according to the present invention. For example, the interchelating method, the TaqMan ^™ probe method, and the molecular beacon method can be used. .

The intercalating method is a method of detecting fluorescence that develops with amplification by adding an interchelator (SYBR Green I, EtBr, etc.) that binds to double-stranded DNA to a PCR reaction. ) Binds to double-stranded DNA synthesized by a PCR reaction to emit fluorescence, and the amount of amplified product can be measured by detecting the fluorescence intensity.

TaqMan ^™ probe method is a method in which TaqMan ^™ probe is added to a PCR reaction solution by adding oligonucleotide (TaqMan ^™ probe) labeled with 5'-end as fluorescent labeling factor and 3'-end as quencher (eg TAMRA, etc.). TaqMan ^™ hybridizes specifically to template DNA in the hybridization step, but fluorescence is suppressed by the quencher on the probe and hybridized to the template by the 5 '→ 3'exonuclease activity of Taq DNA polymerase in the polymerization step. Since only the probe is decomposed and the fluorescent dye is released from the probe, the inhibition by the quencher is released to emit fluorescence.

Molecular beacon method is to add a oligonucleotide probe (Molecular Beacon probe) to the PCR reaction solution to form a hairpin secondary structure in which both ends are modified with a fluorescent labeling factors (e.g. FAM, TAMRA, etc.) and quencher (e.g. DABCYL, etc.) It is a way. Molecular beacon probes have a hairpin structure in the free state and are in close proximity to the fluorescent labeling factor and quencher material to inhibit fluorescence, but when hybridized specifically at the region complementary to the template DNA in the hybridization step, As the distance from is increased, the suppression by the quencher material is eliminated, so that the fluorescent dye on the probe fluoresces. However, unhybridized molecular beacon probes retain their hairpin structure and thus do not fluoresce.

According to the present invention, in consideration of reaction efficiency, time, fluorescent labeling factor type, etc., it is possible to select an appropriate real-time PCR amplification confirmation method among the methods listed above. In the present invention, it may be desirable to use the TaqMan ^™ probe method.

Real-time PCR method according to the present invention can be carried out according to a conventional real-time PCR method, for example, treated for 2 minutes at 50 ℃ for Uracil-DNA glycosylase (UDG) activity, initial denaturation (initial denaturation) at 95 ℃ After performing for 10 minutes, the conditions of denaturation (15 seconds at 94 ° C.), hybridization (annealing, 60 seconds at 55 ° C. or 60 ° C.) may be taken.

The real-time PCR method according to the present invention is a method for detecting and quantifying fluorescence that is performed in real time every cycle of PCR based on the principle of DNA polymerase and Fluorescence Resonance Energy Transfer (FRET). It can be identified and distinguished from nonspecific amplification products, and the results of analysis can be easily obtained in an automated manner.

Real-time PCR devices that can be used in the present invention include, but are not limited to, ABI Real-time PCR devices 7900, 7500, 7300, Stratagene Mx3000p, and BioRad Chromo 4 devices. At the end of the PCR, the laser of the real-time PCR device detects the fluorescent labeling factors labeled on the probes of the amplified PCR product and automatically analyzes the results by executing a program embedded in the device without electrophoresis.

The real-time PCR method can be distinguished by laser sensitization of the presence of small concentrations of DNA fragments or less than 100 bp PCR products that were difficult to distinguish by electrophoresis, and there is no concern about contamination by PCR products. Since the entire test process is operated by the automatic system until the inspection is completed, it is unlikely that a mistake of the investigator or an error of the inspection will occur, and it is easy to collect and analyze data and minimize the inspection time and labor required. Very useful in

The present invention provides a real-time PCR kit for animal breeding discrimination comprising a real-time PCR primer composition for animal breeding discrimination according to the present invention described above.

The kit according to the present invention may include components necessary for the real-time PCR reaction in addition to the real-time PCR primer composition of the present invention in the breeder's discrimination PCR primer composition or provide them in a separate container, and these components are conventional in the art. It is known as, and may include, for example, dNTP, PCR buffer, KCl, DNA polymerase and the like.

According to the present invention, the real-time PCR kit for breeding livestock for each livestock breeding through the real-time PCR for two or more livestock breeding selected from the group consisting of cattle, pigs, chickens, ducks, geese and turkeys It may comprise a real-time PCR primer composition for breeder discrimination. Each real-time PCR reaction solution was prepared using each real-time PCR primer composition, and then each reaction solution was added to a real-time PCR device, and the PCR reaction was performed at the same time to use a probe labeled with the same fluorescent labeling factor.

In this case, as a control for real-time PCR amplification confirmation, a set of primers for detecting eukaryotic cells, including a primer having a nucleotide sequence of SEQ ID NOs: 31 and 32, and a probe capable of specifically binding to a PCR amplification product by the primer set. It may further comprise a real-time PCR primer composition comprising a probe having a nucleotide sequence of SEQ ID NO: 50.

In this case, when the probe of SEQ ID NO: 50 uses a probe for differentiation of axial species and a fluorescence labeling factor having different excitation and emission wavelengths, a control primer set and a probe may be put in the same real-time PCR reaction solution, and a real-time PCR reaction may be performed. If the probe is labeled with the same fluorescent labeling factor it can be prepared in a separate real-time PCR reaction solution to perform a real-time PCR at the same time.

Kits according to the invention can be packaged in one reaction vessel, strip or microplate, and the primer set and / or probes can be packaged by methods known in the art. The kit according to the present invention may include the components required for the PCR reaction in the PCR primer composition for different breeding species or provide them in a separate container.

In addition, the kit of the present invention may further include one or more selected from the group consisting of Taq polymerase (Taq polymerase), MgCl ₂ and the like, dNTP and stabilizer (stabilizer), in addition to that known in the art Other reagents may be additionally included.

The kit of the present invention can accurately and simply confirm whether or not the authenticity of the livestock species used as food and / or other livestock species other than the labeling in the food.

According to the present invention, the minimum detection concentration of bovine and porcine chicken genes in real-time PCR is 1.0 pg / uL, and the minimum detection concentration of duck gene is 0.1 pg / uL.

In addition, when two or more livestock species are mixed in an unknown sample, the mixing ratio of the livestock species can also be determined by setting a baseline of fluorescence intensity and measuring the number of PCR cycles reaching the baseline.

The method of distinguishing 10 livestock breeding using a single PCR and / or multiple PCR and / or real-time PCR according to the present invention can be introduced as a livestock breeding method that can identify the livestock breeding in foods used as meat. Religious reasons to ban meat, protect the health of meat allergic patients and ensure the quality of products such as sausages and hams, as well as prevent low-cost meat mixtures, provide consumers with the right information and ensure food reliability and safety can do.

In addition, the 10 livestock single or multiple detection sets of the present invention, and the 10 livestock single or multiple discrimination method using the same, can not only significantly reduce the accuracy, time and cost of the existing livestock discrimination method, and as a result, Can be quickly confirmed by electrophoresis or real time.

Figure 1 shows the process of designing a primer and TaqMan for identifying four species of birds (chicken, duck, goose, turkey) for real-time PCR.
Figure 2 shows the process of designing a single PCR, multiple PCR, IPC primer and TapMan probe for real-time PCR.
3a and 3b show single PCR results for 11 livestock using bovine primers and IPC primers.
Figure 4 shows a single PCR results for 11 livestock using cotton wool and IPC primers.
Figure 5 shows a single PCR results for 11 livestock using goat primers and IPC primers.
Figure 6 shows a single PCR results for 11 livestock using horse and IPC primers.
Figure 7 shows a single PCR results for 11 livestock using dog primers and IPC primers.
8A to 8B show single PCR results for 11 livestock using a pig primer and an IPC primer.
Figure 8c shows the results of a single PCR for 11 livestock using a pig primer.
Figure 9a shows a single PCR results for 11 livestock using chicken primers and IPC primers.
9B shows single PCR results for 11 livestock using chicken primers.
Figure 10 shows a single PCR results for 11 livestock using duck primers and IPC primers.
Figure 11a shows a single PCR result for 11 livestock using goose primer and IPC primers.
11B shows single PCR results for 11 livestock using goose primers.
Figure 12 shows a single PCR results for 11 livestock using the turkey primer and IPC primers.
Figure 13 shows multiple PCR results for 11 livestock using cattle, pigs, chickens, duck primers and IPC primers.
Figure 14 shows the multiple PCR results for 11 livestock using four bird (chicken, duck, goose, turkey) primer.
Figure 15a shows the results of real-time PCR reaction using the real-time PCR detection set using the R-So1 or R-So2 primer and probe of the present invention.
Figure 15b shows the results of real-time PCR reaction using the real-time PCR detection set using the R-So3 or R-So4 primer and probe of the present invention.
Figure 15c shows the results of real-time PCR reactions for all livestock DNA using the real-time PCR detection set comprising the R-So1 primer and probe of the present invention.
Figure 16 shows the results of real-time PCR reaction using the real-time PCR detection set containing the R-pig primer and probe of the present invention.
17-21 shows the results of real-time PCR reaction using a real-time PCR detection set comprising the R-chicken, R-duck, R-goose 1, R- turkey primer and probe of the present invention.
Figure 22 shows the results of the detection limit of the beef in the growth by containing the content of beef in 0.1% to 50.0% of pork using pork, so primer and IPC primer.
Figure 23 shows the results of the detection limit of the beef in the heat-treated meat so that the content of beef in the pork using 0.1% to 50.0% of pork using a pig, so primer and IPC primer.
Figure 24 shows the results of the detection limit of pork in the growth by containing the pork content of 0.1% to 50.0% in beef using a pig, so primer and IPC primer.
Figure 25 shows the results of the detection limit of the pork in the heat-treated meat so that the content of pork in the beef using 0.1% to 50.0% of pork using the pig, so primer and IPC primer.
Figure 26 shows the results of the detection limit of pork in growth by containing the content of pork in the chicken 0.1 to 50.0% using chicken and pig primer and IPC primer.
Figure 27 shows the experimental results of the detection limit of pork in the heat-treated meat by using the chicken and pork primer and IPC primer so that the content of pork in the chicken 0.1% to 50.0%.
Figure 28 shows the results of the detection limit of the chicken in the growth by containing the content of chicken from 0.1% to 50.0% of pork using chicken and pig primer and IPC primer.
Figure 29 shows the results of the detection limit of the chicken in the heat-treated meat by using the chicken and pork primer and IPC primer so that the content of chicken in the pork from 0.1% to 50.0%.
Figure 30 shows the results of the detection limit of beef in growth by containing the content of beef in 0.1% to 50.0% of pork using the R-So1 primer and probe of the present invention.
Figure 31 shows the results of the detection limit of the beef in the heat-treated meat by using the R-so1 primer and probe of the present invention so that the content of beef in the pork contained 0.1% to 50.0%.
Figure 32 shows the results of the detection limit of pork in growth by containing the content of pork in 0.1% to 50.0% of beef using the R-pig primer and probe of the present invention.
Figure 33 shows the results of the detection limit of pork in the heat-treated meat by using the R-pig primer and probe of the present invention so that the content of pork in 0.1% to 50.0% of beef.
Figure 34 shows the results of the detection limit of pork in the growth by containing the pork content of 0.1% to 50.0% in chicken meat using the R-pig primer and probe of the present invention.
Figure 35 shows the results of the detection limit of the pork in the heat-treated meat by using the R-pig primer and probe of the present invention so that the content of pork in the chicken 0.1 to 50.0%.
36 shows the experimental results of the detection limit of the chicken in the growth by containing the chicken content of 0.1% to 50.0% in pork using the R-chicken primer and probe of the present invention.
37 shows the results of the detection limit of the chicken in the heat-treated meat by using the R-chicken primer and probe of the present invention so that the content of chicken in the pork from 0.1% to 50.0%.

The technical spirit of the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Experimental material and genomic DNA extraction

In the following examples, genomic DNA of each species is described in Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. It was isolated according to the method disclosed in Cold Spring Harbor Press (2001), and the test materials used were beef, sheep blood, goat blood, horse stool, pig blood, dog blood, cat blood, chicken blood, duck blood, goose blood, turkey Genomic DNA corresponding to each livestock was isolated from blood.

Genomic DNA Concentration Measurement

In the following Examples, the concentration of genomic DNA isolated from each livestock was quantified at 260: 280 nm using DNA NanoDrop ™ ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, USA) extracted according to the pest die-based protocol. Will. The genomic DNA solution separated with sterile tertiary distilled water was quantified at a concentration of 10-100 ng / μl and stored at -20 ° C until use.

Example 1 PCR Primer Design

After comparing and analyzing the nucleotide sequences of 12S rRNA and 16S rRNA genes of mt DNA provided in the Genbank databases of the 10 breeders as shown in FIG. 1, a primer for discriminating the breeders for each breeder was designed and Primer sequences for generating PCR amplification products specifically for are as shown in Table 1 below.

On the other hand, the PCR primer for pig breeder discrimination was designed by analyzing the fibroblast growth factor 7 gene provided by the Genbank database, the primer base sequence in Table 1 is as shown in Table 1 below.

In addition, in order to confirm that the desired gene is amplified by the extracted DNA as a template, an internal positive control (IPC) which generates the same PCR amplification products common to the 10 livestocks by comparing the nucleotide sequences of the 18S rRNA genes. ) Primers were designed and used as control PCR primers when performing single PCR, multiple PCR and real-time PCR as axial discrimination tests. Primers having the sequence was prepared by requesting Bioneer.

Vine species designation The sequence (5 '- > 3') Gene region Size of amplification product
(bp) 1 Forward ATTCTCTACACCAAGAGAATCAAGC 12S rRNA 190 Reverse CCTCTCATGTAGCTAGTGCGTTTA 2 Forward GCCAGAGTACTACTAGCAACTGGTTA 12S rRNA 280 Reverse TTTCATAATAACTTTCGTGCTTGG Sheep Forward CACCCTCCTCAAGTAAATATGATAT 12S rRNA
-16S rRNA 219 Reverse TATTTAAACTGGAGAGTGGGAGA Goat Forward AATACGAAAGCCATTATGAAATTAATG 12S rRNA
-16S rRNA 350 Reverse GGTAAATGTTTTGTTTTAGACTGTTTT Words Forward AAGAACAAGAACTTTAACCCGG 12S rRNA
-16S rRNA 467 Reverse TTTGCTGTTTAGTACTTTTAATGCA dog Forward CCTGCCCGGTGACACTTG 16S rRNA 241 Reverse TGCCTTGTAGGTATCTAGTATCCATAAG Pig 1 Forward GAACAATAGTAAGCACAATCATAGCA 12S rRNA 119 Reverse ATAAAAACTTTCGTGTGGTGGATATT Pig 2 Forward AACAATAGTAAGCACAATCATAGCA 12S rRNA 193 Reverse CTTTACGCCGTGGATCTATTAAT Pig 3 Forward TATCAAAATCTCATTTGTCAGGGAA Fibroblast growth factor 7 153 Reverse ATGATACACTGTTATGCAGTATACTTTCA Chicken 1 Forward TGAGCTCAATAGCCCCTCG 12S rRNA 171 Reverse CGTCTTAAAGTGAGCTTAGGGGG Chicken 2 Forward CAACAGTGAGCTCAATAGCCACTC 12S rRNA 175 Reverse CGTCTTAAAGTGAGCTTAGGG duck Forward GTCAAGGTATAGCCTATCGGAC 12S rRNA 229 Reverse CGACTTACCTCATCTTTGGCA Goose 1 Forward GACTTAGTTATAGCAACAGCCTAACTTC 12S rRNA 111 Reverse CACTCTTTACGCCGTGTCAATA Goose 2 Forward ACGGAAAGAAGCGTGTATCCAC 12S rRNA 166 Reverse CTTACCTCATCTTCAGCGTAATAAT Turkey Forward AACGCATCAAAGTACTAATAGTAATTT 16S rRNA 268 Reverse CTTTCGTACTAGGAGGAGTTCTTA IPC Forward CGGTCTGAACTCAGATCACGTA 18S rRNA 152? 3 Reverse CTAGGGATAACAGCGCAATC

Example 2 Differentiation of Livestock Species by PCR

Genomic DNA was purified from the ten livestock specimens, respectively.

Using genomic DNA purified as template DNA, the PCR reaction solution of any one of Tables 2 to 5 was prepared, and PCR amplification was performed according to the reaction conditions of Table 6 below.

Vine species designation density content primer Forward 10 pmol / μl 1 μl Reverse 10 pmol / μl 1 μl IPC Primer Forward 10 pmol / μl 0.5 μl Reverse 10 pmol / μl 0.5 μl DMSO 0.4 μl Template DNA 2 μl Distilled water 14.6 μl Total amount 20 μl Maxime PCR PreMix Kit i -StarTaq, iNtRON biotechnology, Korea

In Table 2, the primers are small primers (SEQ ID NOs: 1 and 2), small primers (SEQ ID NOs: 3 and 4), cotton primers (SEQ ID NOs: 5 and 6), goat primers (SEQ ID NOs: 7 and 8), horse primers (SEQ ID NOs: 9 and 10), dog primers (SEQ ID NOs: 11 and 12), pig 1 primers (SEQ ID NOs: 13 and 14), pig 2 primers (SEQ ID NOs: 15 and 16), chicken 1 primers (SEQ ID NOs: 19 and 20) , Duck primers (SEQ ID NOs: 23 and 24) and goose 1 primers (SEQ ID NOs: 25 and 26).

Vine species designation density content primer Forward 10 pmol / μl 1 μl Reverse 10 pmol / μl 1 μl DMSO 0.4 μl Template DNA 2 μl Distilled water 15.6 μl Total amount 20 μl Maxime PCR PreMix Kit i -StarTaq, iNtRON biotechnology, Korea

In Table 3, the primer is one selected from Pork 3 primer (SEQ ID NOs: 17 and 18), Chicken 2 primer (SEQ ID NOs: 21 and 22), Goose 2 primer (SEQ ID NOs: 27 and 28), and turkey primer (SEQ ID NOs: 29 and 30). .

Vine species designation density content SO1 primer
Forward 10 pmol / μl 1 μl Reverse 10 pmol / μl 1 μl Pig 1 Primer
Forward 10 pmol / μl 1 μl Reverse 10 pmol / μl 1 μl Chicken 1 Primer
Forward 10 pmol / μl 1 μl Reverse 10 pmol / μl 1 μl Duck primer
Forward 10 pmol / μl 1 μl Reverse 10 pmol / μl 1 μl IPC Primer
Forward 10 pmol / μl 0.5 μl Reverse 10 pmol / μl 0.5 μl DMSO 0.4 μl Template DNA 2 μl Distilled water 8.6 μl Total amount 20 μl Maxime PCR PreMix Kit i -StarTaq, iNtRON biotechnology, Korea

Vine species designation density content Chicken 1 Forward 10 pmol / μl 1 μl Reverse 10 pmol / μl 1 μl duck Forward 10 pmol / μl 1 μl Reverse 10 pmol / μl 1 μl Goose 1 Forward 10 pmol / μl 1 μl Reverse 10 pmol / μl 1 μl Turkey Forward 10 pmol / μl 1 μl Reverse 10 pmol / μl 1 μl DMSO 0.4 μl Template DNA 2 μl Distilled water 9.6 μl Total amount 20 μl Maxime PCR PreMix Kit i -StarTaq, iNtRON biotechnology, Korea

Temperature time cycle 95 ℃ 5 minutes 1 time 95 ℃ 30 seconds 35 times 57 ℃ 35 seconds 72 ℃ 35 seconds 72 ℃ 5 minutes 1 time

After PCR, the electrophoresis was performed using a QIAxcel DNA High Resolution kit with a QIAxcel system (Qiagen, USA) to identify the amplified gene region, and analyzed using the Bio Cal-culator software supplied by the manufacturer. As molecular size markers were used 50-800bp or pUC18 / HaeIII DNA ladder (Qiagen, USA), into the alignment markers were used as C ₁ (15bp) and C ₂ (1,000bp).

The results are shown in FIGS. 3 to 14.

In Figures 3 to 14, lane M in each figure is 50-800bp DNA ladder or pUC18 / HaeIII, lane 1 is the result of PCR reaction using bovine genomic DNA as template DNA, and lane 2 is the genomic DNA of sheep as template DNA. Lane 3 is the PCR reaction using goat genomic DNA as template DNA, lane 4 is the PCR reaction using genomic DNA of horse as template DNA, and lane 5 is the template DNA. PCR result using lane 6 is the PCR reaction using genomic DNA of cat as template DNA, lane 7 is PCR result using genomic DNA of pig as template DNA, lane 8 is template DNA using genomic DNA of chicken PCR result using genomic DNA of duck as template DNA, lane 10 is PCR result using genomic DNA of goose as template DNA, and lane 11 template of genomic DNA of turkey With DNA Yonghan a PCR reaction results.

In Figures 3 to 12, lane 12 of each figure is distilled water.

In FIG. 13 to FIG. 14, lane 13 of each drawing is distilled water.

In Figure 13, lane 12 is a PCR reaction using genomic DNA mixture of cattle, pigs, chickens and ducks as template DNA.

In Figure 14, lane 12 is a PCR reaction using genomic DNA mixture of chicken, duck, goose and turkey as template DNA.

Figure 3a is a PCR result using the PCR anti-solution in Table 2 using the small primers and IPC primers.

Figure 3b is a PCR result using the PCR anti-solution in Table 2 using small 2 primer and IPC primer.

Figure 4 is a PCR result using the PCR anti-solution in Table 2 using a cotton primer and an IPC primer.

5 is a PCR result using a PCR anti-solution in Table 2 using a goat primer and an IPC primer.

6 shows PCR results using PCR anti-solutions in Table 2 using horse primers and IPC primers.

7 is a PCR result using the PCR anti-solution of Table 2 using the dog primer and the IPC primer.

Figure 8a is a PCR result using the PCR anti-solution of Table 2 using pig 1 primer and IPC primer.

Figure 8b is a PCR result using the PCR anti-solution of Table 2 using pig 2 primer and IPC primer.

Figure 8c is a PCR result using the PCR anti-solution of Table 3 using the pig 3 primer.

Figure 9a is a PCR result using the PCR anti-solution of Table 2 using chicken 1 primer and IPC primer.

Figure 9b is a PCR result using the PCR anti-solution in Table 3 using chicken 2 primer.

10 is a PCR result using the PCR anti-solution of Table 2 using a duck primer and an IPC primer.

Figure 11a is a PCR result using the PCR anti-solution of Table 2 using goose 1 primer and IPC primer.

Figure 11b is a PCR result using the PCR anti-solution in Table 3 using goose 2 primer.

12 is a PCR result using a PCR anti-solution in Table 3 using a turkey primer.

FIG. 13 shows PCR results using the PCR anti-solution shown in Table 4.

FIG. 14 shows PCR results using the PCR anti-solution shown in Table 5.

As a result of amplification by primer sets for each livestock species, as shown in FIGS. 3 to 14, the small primers were 190bp amplification products specific to the species, and the small primers were 280bp amplification products specific to the species, respectively. The primer is a 219 bp amplification product, goat goat is a 350 bp amplification product, a horse primer is a 467 bp amplification product, and a dog primer is a 241 bp amplification product. , Pig 1 primer is 119 bp amplification product, Pig 2 primer is 193 bp amplification product, Pig 3 primer is 153 bp amplification product, and Chicken 1 primer is phytospecific. 171bp amplification product, chicken 2 primer is breeder-specific 175bp amplification product, duck primer is breeder-specific 229bp amplification product, goose 1 primer is breeder-specific 111 bp amplification product, goose 2 primer for 166 bp amplification product, turkey primer for 268 bp amplification product, and IPC primer for 152-3 bp amplification product for all 11 livestock. Generated. In addition, as shown in Figure 13, the results of lane 12 shows that the DNA of the cattle, pigs, chickens and ducks were all amplified. As shown in Figure 14, lane 12 shows that the DNA of chicken, duck, goose, turkey all amplified. Therefore, it was confirmed that the primer for discriminating the breeder according to the present invention specifically amplifies DNA for the breeder.

Example 3 Primer and Probe Design for Real-Time PCR

The base sequences of primers for real-time PCR of cattle, pigs, chickens, ducks, geese, and turkeys were compared by analyzing the 12S rRNA gene sequences of bovine, swine, chicken, duck, goose, and turkey mt DNA. As designed. Probe sequences that specifically bind to them were designed.

The goose and internal positive control (IPC) primers, ie the primers for R-goose and R-IPC, are identical in sequence to the goose 1 primer and IPC primer of Table 1, respectively.

Vine species designation The sequence (5 '- > 3') Amplified products
Size (bp) R-S1 Forward 1 CACGAAAGTTATTATGAAACCAATAA 166 bp Reverse 1 CCTCTCATGTAGCTAGTGCGTTTA Probe 1 FAM-CACCCTCCTCAAATAGATTCAGTGCATCT-BHQ1 R-small 2 Forward 2 CATTCTCTACACCAAGAGAATCAAGC 191bp Reverse 1 CCTCTCATGTAGCTAGTGCGTTTA Probe 1 FAM-CACCCTCCTCAAATAGATTCAGTGCATCT-BHQ1 R-Small 3 Forward 3 TCGCTCGGAGGATTTTTTATT 116 bp Reverse 2 GCTTTAACTAACCAACCCAAAGAG Probe 2 FAM-CCCAACCGAAACTACCAACTCATGGAGATT-BHQ1 R-S4 Forward 4 TTTGGATCAATGAGCGATAGAG 168 bp Reverse 2 GCTTTAACTAACCAACCCAAAGAG Probe 2 FAM-CCCAACCGAAACTACCAACTCATGGAGATT-BHQ1 R-pig Forward GGAACAATAGTAAGCACAATCATAGCA 121 bp Reverse ATAAAAACTTTCGTGTGGTGGATATT Probe FAM-AGGTCAAGGTGTAGCTTATGGGTTGGA-BHQ1 R-chicken Forward GACTTAGCCATAGCAACCCAG 95 bp Reverse CACTCTTTACGCCGGGTA Probe FAM-TGCCAGCCACCGCGGTCAT-BHQ1 R-Goose 1 Forward GACTTAGTTATAGCAACAGCCTAACTTC 111 bp Reverse CACTCTTTACGCCGTGTCAATA Probe FAM-TGCCAGCCACCGCGGTCAT-BHQ1 R-duck Forward TAGCAAGCCTCCACCCAA 97 bp Reverse CACTCTTTACGCCGCTTGT Probe FAM-TGCCAGCCACCGCGGTCAT-BHQ1 R-Turkey Forward AACTTGACTTAGCCATAGCAACTTTA 201 bp Reverse CGTTAAGTTAAGATCATTTTTTAGGCT Probe FAM-TGCCAGCCACCGCGGTCAT-BHQ1 R-IPC Forward CGGTCTGAACTCAGATCACGTA Reverse CTAGGGATAACAGCGCAATC Probe JOE-TGATCCAACATCGAGGTCGTAAACC-BHQ1

Example 4 Classification of Livestock Species Using Real-Time PCR

Genomic DNA was purified from the six livestock specimens, respectively.

For each DNA livestock sample, R-cow 1, R-cow 2, R-cow 3, R-cow 4, R-pig, R-chicken, R-duck, R-goose 1, R- turkey primer and probe, And a real-time PCR reaction solution containing an R-IPC primer and a probe, and a control real-time PCR reaction solution containing only distilled water without template DNA. The real-time PCR reaction solution of R-So1, R-So2, R-So3, R-So4 and R-Pig primers and probes is shown in the Table 8 below, R-Chicken, R-Duck, R-Goose 1, for the R- turkey primer and probe was prepared in the composition of Table 9.

For each real-time PCR reaction solution, real-time PCR was performed under the conditions shown in Table 10 below.

Vine species designation density content Cow or pig
Primers and Probes Forward 10 pmol / μl 0.6 μl Reverse 10 pmol / μl 0.6 μl Probe 10 pmol / μl 0.4 μl IPC
Primers and Probes Forward 10 pmol / μl 0.4 μl Reverse 10 pmol / μl 0.4 μl Probe 10 pmol / μl 0.4 μl 2 × master mix 10 μl Template DNA 1 μl Distilled water 6.2 μl Total amount 20 μl Taqman Gene Expression master kit (2X, Applied biosystems)

Vine species designation density content Chicken or duck or goose or turkey primer and probe Forward 10 pmol / μl 0.6 μl Reverse 10 pmol / μl 0.6 μl Bird probe 10 pmol / μl 0.4 μl IPC
Primers and Probes Forward 10 pmol / μl 0.4 μl Reverse 10 pmol / μl 0.4 μl Probe 10 pmol / μl 0.4 μl 2 × master mix 10 μl Template DNA 1 μl Distilled water 6.2 μl Total amount 20 μl Taqman Gene Expression master kit (2X, Applied biosystems)

Temperature time cycle 50 ℃ 2 minutes 1 time 95 ℃ 10 minutes 1 time 95 ℃ 15 seconds 40 times 55 ° C 60 seconds

The results are as shown in FIGS. 15 to 21.

FIG. 15a shows that a template DNA was used as a bovine, and R-so1, R-so2 primers and probes were amplified by livestock-specific PCR.

FIG. 15b shows that R-so3, R-so4 primers, and probes were amplified by real-time PCR.

15c and 16 to 20 are the results of real-time PCR using the DNA of cattle, pigs, chickens, ducks, geese, turkeys as template DNA, respectively, primer and probe set according to the present invention is a species-specific template It can be seen that the amplification of DNA.

Figure 15c confirms the amplification curve of the cow using R-So1 primer and probe, Figure 16 confirms the amplification curve of the pig, Figure 17 confirms the amplification curve of the chicken, Figure 18 confirms the amplification curve of the duck, Figure 19 Is a goose amplification curve, Figure 20 is a turkey amplification curve.

FIG. 21 shows genomic DNA obtained from 11 livestock (cows, pigs, chickens, ducks, geese, turkeys, sheeps, goats, horses, dogs, and cats) as template DNA, using a primer and probe for IPC. Amplification curves were observed for pigs, chickens, ducks, geese, turkeys, sheep, goats, horses, dogs, and cats.

Example 5 Limits of Detection of Livestock Breeds in Mixed Meat Using Multiplex PCR

Samples were prepared by cutting the specimens with scissors for detection limit testing of mixed meats of beef and pork, and mixed meats of chicken and pork. In order to confirm the detection limit according to the meat content, 0.1 g of beef and chicken samples were prepared in 0.1, 1, 2, 10 and 50% of pork, respectively. Purified genomic DNA from growth mixture meat and purified genomic DNA from heat-treated meat samples heated for 30 minutes at 120 ° C. of each specimen for detection experiments by heat treatment were prepared.

Each DNA sample extracted from the mixed meat was prepared by multiple PCR reaction composition of Table 4 using multiple PCR to confirm DNA of cattle, pigs, and chickens, and then PCR was performed under the PCR reaction conditions of Table 6.

After multiple PCR types, electrophoresis was performed using a QIAxcel DNA High Resolution kit with a QIAxcel system (Qiagen, USA) to identify the amplified gene region. Obtained data were analyzed using the Bio Calculator software supplied by the electrophoretic manufacturer. 50-800bp DNA ladder (Qiagen, USA) was used as the molecular size marker, and C ₁ (15bp) and C ₂ (1,000bp) were used as internal alignment markers.

The results are as shown in FIGS. 22 to 29.

Figure 22 shows the results of the detection limit of the beef in the growth by containing the content of beef in 0.1% to 50.0% of pork using pork and cattle primer and IPC primer.

Figure 23 shows the results of the detection limit of the beef in the heat-treated meat by using the pork and cow primer and IPC primer so that the content of beef in the pork contained 0.1% to 50.0%.

Figure 24 shows the results of the detection limit of pork in the growth by containing the pork content of 0.1% to 50.0% in beef by using a pig and cow primer and IPC primer.

Figure 25 shows the results of the detection limit of the pork in the heat-treated meat by using the pork and bovine primer and IPC primer so that the content of pork in the beef contained 0.1% to 50.0%.

Figure 26 shows the results of the detection limit of pork in growth by containing the content of pork in the chicken 0.1 to 50.0% using chicken and pig primer and IPC primer.

Figure 27 shows the experimental results of the detection limit of pork in the heat-treated meat by using the chicken and pork primer and IPC primer so that the content of pork in the chicken 0.1% to 50.0%.

Figure 28 shows the results of the detection limit of the chicken in the growth by containing the content of chicken from 0.1% to 50.0% of pork using chicken and pig primer and IPC primer.

Figure 29 shows the results of the detection limit of the chicken in the heat-treated meat by using the chicken and pork primer and IPC primer so that the content of chicken in the pork from 0.1% to 50.0%.

In Figures 22-23, lane M of each figure is a 50-800bp DNA ladder, lane 1 is beef content 0.1%, lane 2 is beef content 1.0%, lane 3 is beef content 2.0%, lane 4 is beef content 10.0% and lane 5 is 50% beef.

In Figures 24 to 27, lane M in each figure is a 50-800bp DNA ladder, lane 1 is pork content 0.1%, lane 2 is pork content 1.0%, lane 3 is pork content 2.0%, lane 4 Is pork content 10.0%, lane 5 is pork content 50%.

In Figures 28-29, lane M in each figure is a 50-800bp DNA ladder, lane 1 is 0.1% chicken, lane 2 is 1.0% chicken, lane 3 is 2.0% chicken, lane 4 is chicken 10.0% and lane 5 is 50% chicken.

As a result of testing the detection limits of cattle, pigs and chickens in mixed meats by multiplex PCR, the raw meats and heat-treated meats of ground mixed meats containing 0.1, 1, 2, 10 and 50% of beef contained in pork in FIGS. 22 to 23. The detection limit of beef was 0.1%. In FIGS. 24 to 25, the detection limit of pork was 0.1% in the raw meat and the heat-treated meat of the ground mixed meat by the content of pork in beef. In FIGS. 26 to 27, the detection limit of pork was 1.0% in the non-heated and heat-treated meats of ground mixed meats containing 0.1, 1, 2, 10 and 50% of pork in chicken. In FIGS. 28 to 29, the detection limit of chicken meat in ground crushed mixed meat of pork was 0.1% in raw meat and heat-treated meat.

Example 6 Limits of Detection of Livestock Breeds in Mixed Meat Using Real-Time PCR

Samples were prepared by cutting the specimens with scissors for detection limit testing of mixed meats of beef and pork, and mixed meats of chicken and pork. In order to confirm the detection limit according to the meat content, 0.1 g of beef and chicken samples were prepared in 0.1, 1, 2, 10 and 50% of pork, respectively. Purified genomic DNA from growth mixture meat and purified genomic DNA from heat-treated meat samples heated for 30 minutes at 120 ° C. of each specimen for detection experiments by heat treatment were prepared.

Each DNA sample extracted from the mixed meat was prepared by using real-time PCR for real-time PCR to identify DNA of cattle, pigs, and chickens, and then PCR was performed under real-time PCR reaction conditions of Table 10.

Specific classification of cattle, pigs and chickens used in mixed meat by real-time PCR was performed using amplification curve.

The results are as shown in FIGS. 30 to 37.

Figure 30 shows the results of the detection limit of the beef in growth by containing the content of beef in 0.1% to 50.0% of pork using R-So1 primer and probe.

Figure 31 shows the results of the detection limit of the beef in the heat-treated meat by containing the content of beef in 0.1% to 50.0% of pork using R-So1 primer and probe.

Figure 32 shows the results of the detection limit of pork in growth by containing the content of pork in 0.1% to 50.0% of beef using the R-pig primer and probe.

Figure 33 shows the results of the detection limit of the pork in the heat-treated meat by using the R-pig primer and probe so that the content of pork in 0.1% to 50.0% of beef.

Figure 34 shows the results of the detection limit of the pork in the growth by containing the content of pork in the chicken 0.1% to 50.0% using R-pig primer and probe.

Figure 35 shows the results of the detection limit of the pork in the heat-treated meat by using the R-pig primer and probe so that the content of pork in the chicken 0.1 to 50.0%.

36 shows the experimental results of the detection limit of the chicken in the growth by containing the chicken content of 0.1% to 50.0% in pork using the R-chicken primer and probe of the present invention.

37 shows the results of the detection limit of the chicken in the heat-treated meat by using the R-chicken primer and probe of the present invention so that the content of chicken in the pork from 0.1% to 50.0%.

In Figures 30 to 31, lane 1 of each figure is beef content 50.0%, lane 2 is beef content 10.0%, lane 3 is beef content 2.0%, lane 4 is beef content 1.0%, lane 5 is beef content 0.1 %, Lane 6 is beef content 0.0%. In Figures 32 to 35, lane 1 of each figure is pork content 50.0%, lane 2 is pork content 10.0%, lane 3 is pork content 2.0%, lane 4 is pork content 1.0%, lane 5 Is pork content 0.1%, lane 6 is pork content 0.0%. In Figures 36 to 37, lane 1 of each diagram is chicken content 50.0%, lane 2 is chicken content 10.0%, lane 3 is chicken content 2.0%, lane 4 is chicken content 1.0%, lane 5 is chicken content 0.1 %, Lane 6 is the chicken content 0.0%.

As a result of experiments on detection limits of cattle, pigs and chickens in mixed meat by real-time PCR, the raw meat and heat-treated meat of ground mixed meat containing 0.1, 1, 2, 10 and 50% of beef in pork in FIGS. The detection limit of beef was 1.0%. 32 to 35, the limit of detection of pork in raw meat and heat-treated meat of ground mixed meat by the amount of pork in beef or chicken was 1.0%. 36 to 37, the limit of detection of chicken meat in ground mixed meat by the amount of chicken in pork was 0.1% in raw meat and heat-treated meat.

Attach an electronic file to a sequence list

Claims (11)

Use to discriminate one or more livestock selected from the group consisting of cows, pigs, chickens and ducks through PCR amplification, including primer sets capable of specifically amplifying DNA obtained from cattle, pigs, chickens or duck samples In the PCR primer composition for differentiation of livestock,
Said primer set comprises the following primer set (a), primer set (g), primer set (j) and primer set (l), characterized in that the PCR primer composition for breeding discrimination:
(a) a set of primers for amplifying bovine DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 1 and 2;
(g) a primer set for amplifying pig DNA, comprising a primer having a nucleotide sequence of SEQ ID NOs: 13 and 14;
(j) a set of primers for DNA amplification of chicken, comprising a primer having the nucleotide sequences of SEQ ID NOs: 19 and 20;
(l) a set of primers for DNA amplification of a duck, comprising a primer having the nucleotide sequences of SEQ ID NOs: 23 and 24; The method of claim 1,
Cattle, sheep, goats, horses, through PCR amplification, including primer sets capable of specifically amplifying DNA obtained from cattle, sheep, goats, horses, dogs, pigs, chickens, ducks, geese or turkey specimens In the PCR primer composition for breeding stock used to discriminate one or more breeding stocks selected from the group consisting of dogs, pigs, chickens, ducks, geese and turkeys,
Said primer set comprises a primer set of the following primer set (a) to (o) for breeding discrimination PCR primer composition:
(a) a set of primers for amplifying bovine DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 1 and 2;
(b) a set of primers for amplifying bovine DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 3 and 4;
(c) a primer set for positive DNA amplification, comprising a primer having a nucleotide sequence of SEQ ID NOs: 5 and 6;
(d) a set of primers for amplifying goat DNA, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 7 and 8;
(e) a set of primers for amplifying DNA, comprising a primer having the nucleotide sequences of SEQ ID NOs: 9 and 10;
(f) a set of primers for DNA amplification, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 11 and 12;
(g) a primer set for amplifying pig DNA, comprising a primer having a nucleotide sequence of SEQ ID NOs: 13 and 14;
(h) a primer set for amplifying pig DNA, comprising a primer having the nucleotide sequences of SEQ ID NOs: 15 and 16;
(i) a primer set for amplifying pig DNA, comprising a primer having the nucleotide sequences of SEQ ID NOs: 17 and 18;
(j) a set of primers for DNA amplification of chicken, comprising a primer having the nucleotide sequences of SEQ ID NOs: 19 and 20;
(k) a set of primers for DNA amplification of chicken, comprising a primer having the nucleotide sequences of SEQ ID NOs: 21 and 22;
(l) a set of primers for DNA amplification of a duck, comprising a primer having the nucleotide sequences of SEQ ID NOs: 23 and 24;
(m) a set of primers for DNA amplification of a goose, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 25 and 26;
(n) a set of primers for DNA amplification of a goose, the set comprising primers having the nucleotide sequences of SEQ ID NOs: 27 and 28;
(o) A primer set for DNA amplification of a turkey, comprising a primer having a nucleotide sequence of SEQ ID NOs: 29 and 30. The PCR primer composition of claim 1 or 2, further comprising a primer set including a primer for detecting eukaryotic cells having the nucleotide sequences of SEQ ID NOs: 31 and 32 as a control for PCR amplification confirmation. Breeding discrimination PCR kit comprising a PCR primer composition for discriminating the breeder according to claim 1. (i) obtaining DNA from the livestock specimen;
(ii) using the obtained DNA as a main chain and obtaining a PCR amplification product using the PCR primer composition for discriminating the stock species according to claim 1 or 2;
(iii) a livestock differentiation method comprising electrophoresis the obtained PCR amplified product to determine the size of the amplified product to discriminate the livestock of the sample. delete delete delete delete delete delete

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