Pork quality evaluation kit and pork quality determination method using same

Abstract

The present invention relates to a pork quality evaluation kit comprising a composition for pork quality evaluation comprising a first primer set and a second primer set, wherein the composition for pork quality evaluation is in a powder form.

Description

Pork meat quality diagnostic kit and method for determining pork meat quality using the same

[0001] Cross-reference to related applications

[0002] This application claims the benefit of priority to Korean Patent Application No. 2023-0166426, filed November 27, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] The present invention relates to a pork meat quality diagnostic kit and a pork meat quality determination method using the same.

[0004] The pork industry has primarily focused on increasing productivity (quantity) and fresh meat distribution, which has often resulted in a decline in pork quality (Cameron, 1990; Cliplef and McKay, 1993). In Korea, pork consumption patterns are dominated by grilled and boiled pork, leading to a high consumption of fresh meat.

[0005] Fundamentally, meat quality is a key criterion for meat purchase. Inconsistent meat quality reduces consumer confidence and their willingness to choose high-quality meat (Jung et al., 2011). Therefore, to overcome these issues, efforts have been made to develop meat quality trait improvement systems to meet consumer demands for pork quality (Lee et al., 2012).

[0006] Recently, to improve pork quality, pigs have been raised and scientifically managed to meet established standards, and the resulting pork has been branded. A variety of brands of pork are already commercially available. However, it is difficult for the average person to distinguish between branded and unbranded pork with the naked eye.

[0007] Meanwhile, most economic traits in pigs are known to be quantitative traits influenced by various genes. Active research is underway to identify the causal genes involved in these traits and explore their genetic locations. In particular, to identify trait-related genetic markers, single nucleotide polymorphism (SNP) identification and trait-related studies are actively underway targeting functional genes. To date, a variety of trait-related SNPs have been discovered.

[0008] However, the PCR (Polymerase Chain Reaction)-RFLP (Restinction Fragment Length Polymorphism)-based analysis method for predicting pork meat quality traits using SNP markers requires a process of diagnosing genotypes by treating with restriction enzymes after PCR amplification, which takes approximately two days for the final analysis, making it unapplicable for field analysis. In addition, the use of restriction enzymes increases analysis costs. In addition, there was a problem that additional analysis was necessary because there was a high possibility of errors in genotype interpretation results depending on the activity or treatment time of the restriction enzyme used.

[0009] Therefore, there is still a need in the art for a method for accurately determining pork quality based on PCR techniques without the use of restriction enzymes.

[0010] [Prior Art Literature]

[0011] [Patent Document]

[0012] (Patent Document 1) Republic of Korea Patent No. 10-1941893 (announced on January 25, 2019)

[0013] (Patent Document 2) Republic of Korea Patent No. 10-2019997 (announced on September 10, 2019)

[0014] The problem to be solved by the present invention is to provide a pig meat quality diagnostic kit including a pig meat quality diagnostic composition capable of quickly and accurately determining the meat quality of a pig by utilizing the difference in the length of an amplification product derived from a pig meat quality causative gene.

[0015] Another problem that the present invention seeks to solve is to provide a method for quickly and accurately determining the meat quality condition of a pig using a kit including a primer set composed of primers produced using the MYH3 gene, which is a gene causing meat quality in pigs.

[0016] One aspect of the present invention is a pig meat quality diagnostic kit comprising a pig meat quality diagnostic composition comprising a first primer set and a second primer set, wherein the pig meat quality diagnostic composition is in powder form.

[0017] In the present invention, the first primer set may be composed of base sequences represented by sequence numbers 1 and 2, and the second primer set may be composed of base sequences represented by sequence numbers 3 and 4.

[0018] In the present invention, the composition can amplify the MYH3 gene, which is a target gene, and produce an amplification product that exhibits a length difference during electrophoresis.

[0019] In the present invention, the composition can be determined as low-quality pork if the length of the amplification product is 311 bp, medium-quality pork if the length of the amplification product is 311 bp and 650 bp, and high-quality pork if the length of the amplification product is 650 bp.

[0020] In the present invention, meat quality may be related to one or more meat quality trait factors selected from intramuscular fat content, meat color, shear force, water-holding capacity, and fatty acid composition.

[0021] In the present invention, the composition for diagnosing pork meat quality may further include a reaction buffer, Taq DNA polymerase, dNTP, and loading dye.

[0022] In the present invention, the kit may further include instructions for use.

[0023] Another aspect of the present invention is a method for determining pork meat quality, comprising the steps of: a) extracting genomic DNA from a pig sample; b) performing an amplification reaction using the separated genomic DNA as a template and a pig meat quality diagnostic kit; and c) performing electrophoresis on the amplified product to determine the meat quality of the pork by confirming the size difference of the amplified product.

[0024] In the present invention, as a result of the electrophoresis in step c), if the amplification product appears as a 311 bp single band, it can be determined as low-quality pork, if it appears as a 650 bp single band, it can be determined as high-quality pork, and if both 311 bp and 650 bp bands appear, it can be determined as medium-quality pork.

[0025] According to the present invention, there is an advantage in that the pork quality can be accurately judged into grades of low-quality, medium-quality, and high-quality by only determining the difference in length of the amplified product by electrophoresis after PCR amplification without restriction enzyme treatment.

[0026] The present invention has the advantage of being able to obtain reading results only through electrophoresis after PCR amplification, so that the quality of pork can be accurately determined within a short period of time, approximately 3 hours, and the unit cost of analysis is very low because expensive restriction enzymes are not used.

[0027] In addition, the present invention is convenient because it is possible to determine pork meat quality without errors in reading the genotype causing the meat quality using only PCR amplification, and therefore there is no need to perform additional or supplementary analysis separately.

[0028] In addition, the present invention can prevent errors occurring in previous simple meat quality diagnosis methods and accurately determine pork meat quality by grade into low-quality, medium-quality, and high-quality types.

[0029] In addition, unlike conventional liquid samples, the composition of the diagnostic kit of the present invention is formed into a powder, making it easy to distribute and store.

[0030] Figure 1 is a drawing showing a pork meat quality diagnostic kit of the present invention.

[0031] Figure 2 is a drawing showing a method for diagnosing pork meat quality using the pork meat quality diagnosis kit of the present invention.

[0032] Figure 3 shows mutations within the genetic location for predicting pork meat quality through next-generation sequencing (NGS) analysis.

[0033] Figure 4 shows candidate genes related to pork meat quality within the locations identified in the Ensembl Data base.

[0034] Figure 5 shows the mutation region within the gene location.

[0035] Figure 6 shows the results of meat quality determination of pigs using a kit according to the present invention.

[0036] Figure 7 shows the results of comparing a conventional HpyCH4-RFLP method, a conventional simple identification composition, and a pork meat quality identification method using the kit of the present invention.

[0037] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in various different embodiments and is not limited to the embodiments described herein. Like reference numerals designate similar parts throughout the specification.

[0038] Additionally, when the terms “about,” “approximately,” or similar expressions such as “at least” are used in connection with a numerical value herein, it is intended that the numerical value be allowed a theoretical, experimental, statistical, or empirical error of ±10%, ±7%, ±5%, ±3%, ±2%, or ±1% based on that numerical value.

[0039]

[0040] The present inventors have conducted extensive research to develop a method for judging the meat quality of pigs through genetic traits, and as a result, have identified the MYH3 gene as a gene that determines the meat quality traits of pigs, and have confirmed that this can be used to predict the genetically determined meat quality traits of pigs.

[0041] In the present invention, the "MYH3 gene" means a gene encoding myosin heavy chain 3, which is one of the heavy chain proteins included in myosin that constitutes animal muscles, and its base sequence can be obtained from known databases such as NCBI's GenBank (GenBank Accession No. KX549311 (LAND), KX549312 (KNP)).

[0042] The present inventors have confirmed that the meat quality traits of pigs, such as intramuscular fat content and redness, are determined by the MYH3 gene, and have revealed that since there is a mutation in the gene between native breeds with excellent meat quality traits and foreign breeds with poor meat quality traits, it is possible to determine the meat quality traits of pigs by detecting a genetic marker containing the mutation.

[0043] In relation to this, QTL analysis and Linkage and linkage disequilibrium (LALD) mapping were performed on offspring obtained by crossing Jeju native pigs with Landrace or Duroc pigs, and the results revealed that the MYH3 gene located on chromosome 12 is the causal gene involved in the meat quality traits of pigs.

[0044] Meanwhile, the "PCR-RFLP" method is a technique that analyzes the polymorphism (Restriction Fragment length polymorphism) of gene fragments treated with restriction enzymes by combining PCR technology among various DNA analysis technologies for improving the genetic characteristics or abilities of livestock. When a restriction enzyme recognition site exists at a specific point mutation site, the length of the fragment produced by the restriction enzyme cuts differently depending on the difference in the genotype of each individual, making it a method that can determine the genotype of the analysis target more quickly and accurately.

[0045] The PCR-RFLP method is widely used for pork meat quality analysis. However, this method requires approximately two days from gene amplification to genotyping, limiting its applicability to on-site analysis. Furthermore, because this existing method relies on restriction enzymes, there's always a risk of error in interpretation depending on enzyme activity and other factors. Furthermore, the use of restriction enzymes increases analysis costs.

[0046] Accordingly, the inventors of the present invention designed primer(s) that can accurately diagnose the meat quality of pigs into grades of low, medium, and high meat quality simply by analyzing the difference in length of amplified products by PCR-electrophoresis based on the MYH3 gene identified as a meat quality causative gene, and confirmed that the primer set designed in this way can easily and quickly determine the meat quality of pigs.

[0047] In addition, the inventors of the present invention discovered that some errors occurred in the previous simple diagnosis method, and as a result of careful research efforts, they discovered that errors occurring in the previous simple diagnosis method could be resolved by designing a primer set using a new mutation region rather than an existing mutation region, leading to the present invention.

[0048] Figure 1 is a drawing showing a pork meat quality diagnostic kit of the present invention.

[0049] Referring to FIG. 1, one aspect of the present invention is a pig meat quality diagnostic kit comprising a pig meat quality diagnostic composition comprising a first primer set and a second primer set, wherein the pig meat quality diagnostic composition is in powder form.

[0050] In the present invention, the first primer set may be composed of base sequences represented by sequence numbers 1 and 2, and the second primer set may be composed of base sequences represented by sequence numbers 3 and 4.

[0051] In addition, in the present invention, the composition for diagnosing pork meat quality has a powder form, so it is easy to distribute and store compared to conventional liquid samples, and can be diagnosed without problems even if it is kept at room temperature for 5 days or more, preferably 7 days or more.

[0052] The term "primer" as used herein refers to a short sequence of bases having a short free 3' hydroxyl group at the end, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. In the present invention, a primer used for amplifying a genetic marker, an appropriate component such as an appropriate buffer (for example, four different nucleoside triphosphates and a polymerizing agent such as DNA, RNA polymerase or reverse transcriptase), and a single-stranded oligonucleotide that can serve as a starting point for template-directed DNA synthesis under an appropriate temperature may be used. The appropriate length of the primer may vary depending on the intended use. The primer sequence does not need to be completely complementary to a polynucleotide containing a genetic marker or its complementary polynucleotide, but may be used as long as it is sufficiently complementary to hybridize.

[0053] Additionally, primers can be modified, including, but not limited to, methylation, capping, substitution of nucleotides, or modification between nucleotides, such as modification with uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

[0054] In the present invention, the primers may be polynucleotides each consisting of a base sequence represented by sequence number 1, 2, 3 or 4.

[0055] A person skilled in the art can design an appropriate variant based on the sequence information (including the nucleotide connection order and sequence length) of a primer consisting of a base sequence represented by SEQ ID NO: 1, 2, 3, or 4 disclosed in the present invention, so the scope of the primer according to the present invention is not necessarily limited to primers having a base sequence of SEQ ID NO: 1, 2, 3, or 4.

[0056] The primers constituting the primer set according to the present invention can amplify the MYH3 gene, which is a target gene, and produce an amplification product that exhibits a difference in length during electrophoresis.

[0057] In one embodiment of the present invention, a kit including a composition including a first primer set consisting of base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2; and a second primer set consisting of base sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4 was used to amplify a target gene related to meat quality or meat quality traits of a pig, i.e., the MYH3 gene, and then electrophoresis was performed, and it was confirmed that the meat quality of a pig can be determined based on the difference in the length of the amplification product that appears (Fig. 6, Fig. 7).

[0058] Specifically, after amplifying the target gene MYH3 gene using the kit according to the present invention and then performing electrophoresis, if the length of the amplification product is 311 bp, it can be determined as low-quality pork (qq), if the length of the amplification product is 311 bp and 650 bp, it can be determined as medium-quality pork (qQ, Qq), and if the length of the amplification product is 650 bp, it can be determined as high-quality pork (QQ).

[0059] Therefore, the pork meat quality diagnostic kit according to the present invention can accurately diagnose, determine, or predict the meat quality grade of the target pig by further subdividing it into high-quality, medium-quality, and low-quality types through electrophoresis alone after PCR amplification, and can also shorten the analysis time and enable on-site analysis. Furthermore, since the present invention does not use restriction enzymes, it also has the effect of reducing analysis costs.

[0060] According to an embodiment of the present invention, the pork meat quality described above may be related to meat quality traits including, but not limited to, intramuscular fat content, meat color, shear force, water-holding capacity, and fatty acid composition.

[0061] The term "meat quality trait" used herein refers to various expressive traits indicating the condition of slaughtered meat excluding bones obtained from pigs, and these expressive traits may be, but are not particularly limited to, intramuscular fat content, meat color, water holding capacity, shear force, etc. Intramuscular fat content refers to the content of fat contained in the muscle, meat color refers to the color of meat as judged by the naked eye, water holding capacity refers to the ability of meat to retain moisture, and shear force refers to the degree of toughness of meat when torn. The higher the intramuscular fat content, the redder the meat color, and the lower the water holding capacity and shear force, the higher the quality of meat can be determined to be.

[0062] In the present invention, the composition for diagnosing pork meat quality may further include Taq DNA polymerase, dNTP, and loading dye. The Taq DNA polymerase, dNTP, and loading dye may be used without limitation as long as they are widely known in the art.

[0063] In the present invention, the meat quality diagnostic composition, Taq DNA polymerase, dNTP, and loading dye can be mixed in the following contents.

[0064] Specifically, 0.1 to 0.5 μl of Taq DNA polymerase, 1 to 3 μl of dNTP, 0.1 to 1 μl each of the forward primer and reverse primer of the two pairs of primer sets included in the meat quality diagnostic composition (total 0.4 to 4 μl), 1 to 3 μl of loading dye, and 10 to 15 μl of DW (distilled water) are placed in a tube for reaction, and then the mixture can be manufactured into a powder form in a freeze dryer.

[0065] Preferably, 0.3 ㎕ of Taq DNA polymerase, 2 ㎕ of dNTP, 0.75 ㎕ each of the forward primer and reverse primer of the two pairs of primer sets included in the meat quality diagnosis composition, 2 ㎕ of loading dye, and 12.7 ㎕ of DW (distilled water), for a total of 20 ㎕, are added to a tube for reaction, and then the mixture can be manufactured into a powder form in a freeze dryer, and within the above range, pork quality can be easily determined.

[0066] In the present invention, the kit may include instructions for use. The kit of the present invention can determine the level of meat quality traits in pigs by confirming the MYH3 marker gene, which is a pig meat quality causative gene, as well as other marker genes through PCR amplification, or by confirming the mRNA expression level of these marker genes.

[0067] Additionally, the kit of the present invention may further include a test tube or other suitable container.

[0068] In addition, in the present invention, the kit can determine pork quality through PCR and electrophoresis after adding sterile distilled water and a DNA sample.

[0069]

[0070] *Figure 2 is a drawing showing a method for diagnosing pork meat quality using the pork meat quality diagnosis kit of the present invention.

[0071] In another aspect of the present invention, the present invention provides a method for determining the meat quality of pork, comprising the steps of: a) extracting genomic DNA from a pig sample; b) performing an amplification reaction using the separated genomic DNA as a template and a pig meat quality diagnostic kit; and c) performing electrophoresis on the amplified product to determine the meat quality of the pork by confirming the size difference of the amplified product.

[0072] In step b), when using a pork meat quality diagnostic kit, an amplification reaction can be performed by adding genomic DNA and distilled water.

[0073] In the meat quality determination method of the present invention, the pig sample may be a sample such as hair, urine, blood, various body fluids, isolated tissue, isolated cells, or saliva, but is not particularly limited thereto.

[0074] In the present invention, nucleic acid means not only DNA but also cDNA and RNA molecules synthesized from mRNA.

[0075] In one embodiment of the present invention, the primer set included in the kit used in step b) may be produced based on the MYH3 gene.

[0076] In one embodiment of the present invention, electrophoresis in step c) may be performed on a commonly used agarose gel. Electrophoresis may be performed within 50 minutes, preferably within 40 minutes, and more preferably within 30 minutes.

[0077] In one embodiment of the present invention, as a result of the electrophoresis in step c), if the amplification product appears as a 311 bp single band, it can be determined as low-quality pork, if it appears as a 650 bp single band, it can be determined as high-quality pork, and if both 311 bp and 650 bp bands appear, it can be determined as medium-quality pork.

[0078] In one embodiment of the present invention, the entire process of judging pork quality according to the present invention may vary depending on the sample condition, the skill of the equipment operator, etc., but the pork quality can be completed within approximately 3 hours, and at the latest within 5 hours.

[0079] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0080]

[0081] Example 1. Preparation of pigs

[0082] The experimental animals used in the present invention were 32 pigs (11) of low-fat type, 46 pigs (12) of medium-fat type, and 30 pigs (12) of high-fat type, and a total of 108 pigs were prepared as shown in Tables 1 and 2 below.

[0083]

[0084] 1112221LD21-125F15-8549F15-752LD21-326F15-9650F15-803LD21-727F15-10351F15 -894LD21-1728F15-10852F15-915D23-129LD21-453F15-976D23-1830LD21-854F15-10 17D23-2331LD21-1455F15-1098D23-4532LD21-1856F15-1149L22-6033L22-8457LD21- 210L22-7234L22-8958LD21-2311L22-8035L22-9359LD21-3112L22-8736L23-4860LD21 -3713L23-7337L23-5261L23-11014L23-7838L23-7462L23-11615L23-8439L23-8263L2 3-12216L23-12840L23-8564D22-4017D22-3641D22-3565D22-5618D22-4342D22-5166D 23-619D22-4643D22-6367D23-1720D22-5044D23-468D23-2421D22-5945D23-569D23-3 922D22-6546D23-1670D23-5323D22-6847D23-4371D23-5524D22-7148D23-4672D23-65

[0085] 1112221D20-1959D20-8431DK21-2882D20-20010D20-9032DK21-2893D22-6511D20-10633DK21-2904 D22-6712D20-10734DK21-2915DK21-33213D20-10835DK21-2936DK21-33314D20-11236DK21-2967DK 21-33415D20-1148DK21-33516D22-6417D22-6918DK21-8119DK21-9220DK21-29221DK21-29422DK21 -29523DK21-33624DK21-33725DK21-33826DK21-33927DK21-34028DK21-34129DK21-34230DK21-343

[0086] Example 2. DNA isolation

[0087] In this study, pigs (test stocks) were used for genetic analysis, with DNA extracted from the jugular vein or the tails of piglets produced. DNA isolation was performed using a modified sucrose-proteinase K method (Sambrook et al., 1989). The isolated DNA was measured for absorbance using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA), and genomic DNA with an A260 / A280 ratio of 1.8 or higher was used as a template for polymerase chain reaction (PCR).

[0088]

[0089] Example 3. Extraction and analysis of base sequences including primers for judging pork meat quality.

[0090] Next-generation sequencing (NGS) revealed a region (s55372428) with a higher P-value than the existing high-meat-quality SNP (s55373709) (Figs. 3, 4, Table 3). Functional analysis revealed that the MYH3 gene is the causal gene that determines pork meat quality (intramuscular fat content and red meat). Functional analysis confirmed that the SNP at 55373709 was the causal mutation, and the distance between the two SNPs was approximately 1.3 Kb.

[0091]

[0092] SNP NAMEpvaluePPs549283912.50E-710.2033s549293366.00E-710.2179s549802296.55E-850.0536s5 50010643.31E-720.7553s550231571.32E-740.3297s550283051.56E-760.1188s550284391.13E-74 0.0602s550305064.03E-740.2072s550307883.13E-740.2091s550634633.05E-710.1978s5508649 84.55E-690.0814s552816876.08E-700.2432s553724283.08E-850.6782s553737094.52E-780.0247

[0093] Example 4. Production of primers for judging pork meat quality and production of a pork meat quality judgment kit Based on the sequence information (Table 4) of the MYH3 gene containing SNPs (mutations 1 (SSC12: 55,373,090; 55,373,067; 55,373,066; 55,373,052), 2 (SSC12: 55,372,428), 3) discovered in Example 3, primers were produced as follows (Fig. 5). Thereafter, a first primer set consisting of sequence numbers 1 and 2, a second primer set consisting of sequence numbers 3 and 4, Taq DNA polymerase (Genet Bio, Prime Taq DNA Polymerase), dNTPs (Genet Bio, Prime Taq DNA Polymerase), and loading dye (Genet Bio, Prime Taq DNA Polymerase) were mixed. Specifically, 0.3 μl of Taq DNA polymerase, 2 μl of dNTP, 3 μl of each of the forward and reverse primers of the first primer set consisting of SEQ ID NOs: 1 and 2 and the second primer set consisting of SEQ ID NOs: 3 and 4 (0.75 μl each), 2 μl of loading dye, and 12.7 μl of DW (distilled water) (20 μl in total) were added to a tube for reaction, and then prepared in powder form in a freeze dryer. Freeze-drying was performed at -20°C, and a kit was prepared after preparing the powder in a 50 μl PCR tube.

[0094]

[0095]

[0096] Primer Design:

[0097] MYH3_3del_KF: CTT-CCC-GGG-CAG-GGT-CCC-TTC (SEQ ID NO: 1)

[0098] MYH3_3del_KR: CCG-GGG-GGG-AAC-CAG-TAC-CC (SEQ ID NO: 2)

[0099] MYH3_int40_qF: TAC-GTG-GTC-CCC-CGG-CAC-CAG-GT (SEQ ID NO: 3)

[0100] MYH3_int40_qR: CTC-ACT-GTG-GGA-CCC-GCC-TTT-GCT (SEQ ID NO: 4)

[0101]

[0102] Comparative Example. Preparation of a Composition for Judging Pork Meat Quality

[0103] Based on the sequence information (Table 4) of the MYH3 gene containing the SNP (existing analysis region (SSC12:55,393,709)) discovered in Example 3, primers were prepared as follows (Fig. 7). Then, a third primer set consisting of SEQ ID NOs: 5 and 6, a fourth primer set consisting of SEQ ID NOs: 7 and 8, Taq DNA polymerase (GenetBio, Prime Taq DNA Polymerase), dNTP (GenetBio, Prime Taq DNA Polymerase), and loading dye (GenetBio, Prime Taq DNA Polymerase) were mixed. Specifically, 0.3 μl of Taq DNA polymerase, 2 μl of dNTP, 3 μl of each of the forward and reverse primers of the third primer set consisting of SEQ ID NOs: 5 and 6 and the fourth primer set consisting of SEQ ID NOs: 7 and 8 (0.75 μl each), 2 μl of loading dye, and 12.7 μl of DW (distilled water) (20 μl in total) were added to a tube for reaction, followed by preparation into powder form in a freeze dryer. Freeze-drying was performed at -20°C, and a kit was prepared after preparation into powder form in a 50 μl PCR tube.

[0104]

[0105] Primer Design:

[0106] MYH3_1Q_F: AGG-ACG-TGT-GTT-CCC-CAC-AC (SEQ ID NO: 5)

[0107] MYH3_1Q_R: GGA-ACC-AGT-ACC-CTG-GAG-ATG-G (SEQ ID NO: 6)

[0108] MYH3_1q_F: ACg-GCT-GAC-ACC-AGA-ATG-AAC (SEQ ID NO: 7)

[0109] MYH3_1q_R: GGG-GAA-CAA-CAG-CAG-TCC-TCC (SEQ ID NO: 8)

[0110]

[0111] Test Example 1. Meat quality determination using a kit according to the present invention

[0112] Fifteen pork samples (5 low-quality, 5 medium-quality, and 5 high-quality) were subjected to PCR amplification using the kit manufactured in Example 4 and then analyzed.

[0113] Specifically, 2.0 μl of DNA isolated from pig blood and 18 μl of sterilized deionized water were added to the kit, and PCR reaction was performed as shown in Table 5 below. PCR amplification was performed using a mastercycler X50 (Eppendorf, Germany). The amplified products were electrophoresed on a 0.8-1.2% agarose gel, and the genotype was identified using the presence or absence of gene amplification and length under UV.

[0114]

[0115] Temp(℃)TimeCycles955 min19510 sec306515 sec7230 sec725 min125forever1

[0116] As shown in Figure 6, it can be confirmed that a 311bp band was specifically observed in the 5 heads of the low-meat type (qq), a 650bp band was specifically observed in the 5 heads of the high-meat type (QQ), and both 311bp and 650bp bands were observed in the 5 heads of the medium-meat type (qQ).

[0117]

[0118] Test Example 2. Accuracy Analysis of the Pork Meat Quality Determination Method According to the Present Invention

[0119] 107 pork samples (31 low-quality pork, 46 medium-quality pork, and 30 high-quality pork) were subjected to PCR amplification using the kit according to Example 4 and Comparative Example, and then analyzed, and genotypes were diagnosed using the conventional PCR-RFLP method.

[0120] Specifically, 2.0 μl of DNA isolated from pig blood and 18 μl of sterilized deionized water were added to the kit, and the PCR reaction was performed as shown in Table 5 above. PCR amplification was performed using a mastercycler X50 (Eppendorf, Germany). The amplified products were electrophoresed on a 0.8-1.2% agarose gel, and the genotype was identified using the presence or absence of gene amplification and length under UV.

[0121] The PCR-RFLP method is as follows. Specifically, the part including the above base mutation was amplified using primers (forward: 5'-TGG TCT TTC CTA ATT GGT GAC AT-3' (SEQ ID NO: 10), reverse: 5'-AGT TTT GAG CAA GGC TTT TGT T-3' (SEQ ID NO: 11). 2.0 μl of DNA isolated from the blood of each pig, 10.0 μl of 2X HS Prime Taq Premix, 0.5 μl each of forward and reverse primers, 2.0 μl of DMSO (Dimethyl sulfoxide), and 5 μl of sterilized deionized water were added to perform the PCR reaction in a volume of 20 μl as shown in Table 5. PCR amplification was performed using a mastercycler X50 (Eppendorf, Germany).

[0122] The amplified PCR product was digested using the restriction enzyme HpyCH4Ⅳ (recognition site: A▼CGT), and the restriction enzyme reaction conditions were as follows: 3 ㎕ of the PCR amplified product, 1 ㎕ of 10X buffer, 0.3 ㎕ of the restriction enzyme, and 5.7 ㎕ of distilled water were mixed and reacted at 37℃ for 12 hours. The cleavage pattern of the MYH3 gene by the restriction enzyme was confirmed by electrophoresis on a 1.5% agarose gel containing EtBr (ethidium bromide). The results are shown in Table 6 and Fig. 5 below.

[0123]

[0124] Number of segments analyzed myh3_genotypeqqQqQQComparative example 107114056HpyCH4-RFLP 107314630Example 5107314630

[0125] qq: low-quality meat, qQ: medium-quality meat, QQ: high-quality meat

[0126]

[0127] Referring to Figure 7 and Table 6, it can be confirmed that when the kit was used in the comparative example, errors occurred in all of the low-quality, medium-quality, and high-quality types, but when the kit according to the present invention was used, it can be confirmed that the same results as the conventional HpyCH4-RFLP method were shown.

[0128] In addition, as a result of the analysis of the difference in the length of the amplified product based on PCR-electrophoresis according to the present invention, it can be confirmed that low-quality pork (qq) shows a 311 bp band, medium-quality pork shows 311 bp and 650 bp bands, and high-quality pork (QQ) shows a 650 bp band.

[0129]

[0130] The present invention can accurately determine the meat quality of pork into low-quality, medium-quality, and high-quality grades within a short period of time by only determining the difference in length of amplified products by electrophoresis after PCR amplification without restriction enzyme treatment, and can exhibit superior effects compared to existing primer sets.

Claims

1. A pig meat diagnostic kit comprising a pig meat diagnostic composition comprising a first primer set and a second primer set, A pork quality diagnostic kit, wherein the above pork quality diagnostic composition is in powder form.

2. In paragraph 1, The above first primer set consists of base sequences represented by sequence number 1 and sequence number 2, A pig meat quality diagnostic kit, wherein the second primer set comprises base sequences represented by sequence numbers 3 and 4.

3. In paragraph 1, The above composition is a pig meat quality diagnostic kit capable of amplifying the MYH3 gene, which is a target gene, and generating an amplification product showing a difference in length during electrophoresis.

4. In paragraph 3, The above composition determines that the pork is of low quality if the length of the amplified product is 311 bp. If the length of the amplified product is 311 bp and 650 bp, it is judged as intermediate meat type pork. A pork quality diagnostic kit that determines that pork is high-quality if the amplification product is 650 bp long.

5. In paragraph 1, A pork meat quality diagnostic kit, wherein meat quality is related to one or more meat quality trait factors selected from intramuscular fat content, meat color, shear force, water-holding capacity, and fatty acid composition.

6. In paragraph 1, A pig meat quality diagnostic kit, wherein the above pig meat quality diagnostic composition further comprises Taq DNA polymerase, dNTP, and loading dye.

7. In paragraph 1, A kit for judging the meat quality of pork, wherein the above kit further includes an instruction manual. 8.a) Step of extracting genomic DNA from a pig sample; b) a step of performing an amplification reaction using the kit according to paragraph 1 using the separated genomic DNA as a template; and c) A method for determining pork quality, comprising the step of determining pork quality by performing electrophoresis on the amplified product and confirming the difference in the size of the amplified product.

9. In paragraph 8, A method for judging pork quality, wherein if the electrophoresis result of step c) shows that the amplification product is a single band of 311 bp, it is judged as low-quality pork, if it shows that it is a single band of 650 bp, it is judged as high-quality pork, and if both bands of 311 bp and 650 bp show up, it is judged as medium-quality pork.

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