KR20220112403A - Pork origin identification kit and pork origin identification method using the same - Google Patents

Abstract

The present invention relates to a simple kit for identifying the origin of pork. More specifically, the present invention relates to a method which analyzes the presence or absence of a swine fever antibody in pork by using a swine fever antigen and identifies whether the pork is domestic or imported. Accordingly, the present invention allows the origin of pork to be easily identified without expensive equipment and thus can be usefully applied to increase the efficiency of an origin labeling crackdown. In addition, during a crackdown on pork origin labeling, the crackdown takes place by using and providing the origin as scientific proof. Accordingly, the present invention can be utilized to increase the efficiency of law enforcement, protect consumers' right to know, and protect domestic pig farms.

Description

Pork origin identification kit and pork origin identification method using the same

The present invention relates to a kit for determining the origin of pork using immunochromatography and a method for determining the origin of pork using the same. More specifically, it relates to a kit for analyzing the presence or absence of antibodies to swine fever using a swine fever antigen and thereby discriminating between domestic pork and foreign pork, and a method for determining the origin of pork using the same.

Pork sold at butchers, restaurants, and supermarkets must indicate the country of origin in accordance with the 「Act on the Labeling of Origin of Agricultural and Fisheries Products」 and provide consumers with information on the country of origin. However, if the country of origin label is falsely displayed during the distribution process, it is impossible to visually distinguish the origin of pork. In addition, since the price of foreign pork is about 30% lower than that of domestic pork, there is a very high probability that it will be sold as domestically produced pork. The National Agricultural Products Quality Management Service cracks down on false indications of the origin of agricultural products, and this technology is needed to provide the scientific basis for determining the origin of agricultural products and to increase the effectiveness of the enforcement.

Recent pork production and imports (Unit: 1,000 tons) year 2014 2015 2016 2017 2018 2019 output 830 848 891 904 944 968 import volume 273 357 318 389 463 421

The causative agent of swine fever is an RNA virus (Classical Swine Fever Virus, CSFV) and is transmitted by direct contact or oral infection with a virus discharged in feces, urine, tears, or runny nose. Clinical symptoms include lack of appetite, coma, body temperature rise, and lymph node bleeding. Swine fever is classified as a Class A disease by the World Organization for Animal Health (OIE), and is also classified as a first-class livestock infectious disease in Korea.

The most effective way to control swine fever is to vaccinate. Piglets in Korea must be vaccinated once between 55 and 70 days of age and once a year for breeding pigs and breeding pigs in accordance with 「Swine Fever Prevention Implementation Guidelines (Ministry of Agriculture, Food and Rural Affairs Notification No. 2016-48)」. According to the above guidelines, the swine fever antibody positivity rate is monitored annually using pig blood, and the national antibody positivity rate from 2016 to 19 is very high, exceeding 97%.

However, for foreign pork, “sanitary conditions for importing pork and pig products by country (Ministry of Agriculture, Food and Rural Affairs Notice No. 2016-6, etc.)” Exports to Korea are possible only when the national disease-free condition (exporting country has no history of swine fever for one year prior to export, or the Korean government must be recognized as a blue-green country and not vaccinated) is satisfied. Therefore, foreign pork does not have swine fever antibodies.

Although an analytical method for discriminating domestic and foreign pork (Patent Publication No. 10-2017-0077261, Patent Publication No. 10-2019-0048343) has been developed, it is expensive to analyze the ratio of isotopes and inorganic elements. equipment is needed In addition, there are disadvantages in that the pretreatment cost and time are high, and it is difficult to process a large amount of samples at a time. Therefore, there is a need for a identification kit that can accurately determine the origin of pork without expensive equipment in the field.

Laid-Open Patent Publication No. 10-2017-0077261 Laid-Open Patent Publication No. 10-2019-0048343

In order to solve the above problems, the present inventors have developed a kit for discriminating domestic/foreign pork by using the antigen (E2) of swine fever (CSFV) in immunochromatography.

Accordingly, it is an object of the present invention to provide a kit for discriminating domestic/foreign pork containing gold particles bound to swine fever antigen.

Another object of the present invention is to provide a method for determining the origin (domestic or foreign) of pork using the kit.

In order to achieve the object of the present invention as described above, the present invention provides a discrimination kit for distinguishing domestic / foreign pork.

The kit may use a porcine antigen.

The porcine antigen is not limited, but may preferably be a swine fever antigen, and preferably may be an E2 antigen among swine fever antigens.

The swine fever antigen is a swine fever E2 antigen produced by a recombinant vector of SEQ ID NO: 5 disclosed in Bioapp Co., Ltd., trade name (cat. no: CSFV-E2H, product number: HERVANTI016) or Patent Publication No. 10-1732624. (Refer to identification number [0074] to [0091] of Patent Registration No. 10-1732624) may be.

The recombinant vector is described as SEQ ID NO: 1 in the present invention.

The kit may further include gold particles. The size of the gold particles may be 10 to 100 nm, and a specific example may be black nano gold particles, but is not limited thereto.

The present invention provides a method for determining the origin of domestic/foreign pork using the kit.

The method for determining the origin of pork according to the present invention may use immunochromatography.

The method for determining the origin of pork may include the following steps;

(a) attaching the swine fever antigen to the membrane;

(b) aggregating the pork juice to be analyzed with gold particles on the antigen-attached membrane.

The antigen in step (a) is not limited, but may preferably be a swine fever antigen, preferably an E2 antigen among swine fever antigens.

In addition, the membrane is preferably, but not limited to, a nitrocellulose membrane.

The broth of pork to be analyzed in step (b) may be a broth produced by crushing pork or a broth produced by pressing pork in a diluent.

In addition, the size of the gold particles may be 10 to 100 nm, but is not limited thereto. Examples of the gold particles may be black nano gold particles, but is not limited thereto. Gold particles have excellent reactivity and easy binding to proteins, so they are widely used in diagnostic kits.

In an embodiment of the present invention, the determination method is

(a) diluting Protein A and spraying it on a part of the nitrocellulose membrane, followed by drying to attach to the membrane;

(b) attaching the swine fever E2 antigen, which is an antigen of the antibody to be analyzed, to gold particles; and

(c) judging the subsequent agglutination reaction by comparing the results of standard positive pork and standard negative pork.

As described above, domestic/foreign pork origin discrimination through the present invention does not require expensive equipment because it is determined by visually observing whether the pork reacts with the swine fever antibody after binding the E2 antigen to gold particles. It can be carried out simply and economically.

In order to crack down on the case of selling foreign pork by falsely labeling it as domestic pork, it was complicated and expensive to analyze the origin of pork using equipment in a laboratory.

As described above, the present invention can be usefully used to increase the efficiency of control on country of origin indication by simply performing the identification of the origin of pork without expensive equipment.

1 shows the discrimination results of domestic and foreign pork using a kit according to the present invention.
2 is a cross-sectional view of a diagnostic kit according to the present invention.
3 shows the antibody reaction results (positive T-part antibody) of domestic pork belly and neck meat using the kit according to the present invention.
Figure 4 shows the antibody reaction results (antibody negative in the T part) of foreign pork belly and pork belly using the kit according to the present invention.
Figure 5 shows the antibody reaction results (positive) of domestically produced angiosperm, tenderloin, sirloin, ribs, and forelimbs using the kit according to the present invention.
Figure 6 shows the antibody reaction results (negative) of foreign-made antiseptic, tenderloin, sirloin, ribs, and forelimbs using the kit according to the present invention.
7 shows a process for determining the origin of pork according to the present invention.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

<Example> Manufacture of a simple kit for determining the origin of domestic/foreign pork

1. Test line: Protein A was diluted to 1.0 mg/ml in 1X PBS and then sprayed on a part of the nitrocellulose membrane at 1 ul/cm using a sprayer. The Protein A is a protein that binds to the IgG Fc region, and serves to capture pork IgG using this characteristic. Protein A used in this experiment was a product of Custom Monoclonals International.

2. Control line: After diluting anti-BSA (Anti-Bovine Serum Albumin) in 1X PBS, it was sprayed on a part of the nitrocellulose membrane at 1 ul/cm using a sprayer and dried.

3. Red nanogold-BSA binding: BSA was bound to a 40 nm gold colloid. This conjugate was diluted with a buffer containing 50 mM Tris-Cl, dispensed on a red nano-gold-junction pad, and dried.

4. Black nanogold-antigen binding: Swine fever antigen (E2) was bound to a colloid of 100 nm gold particles. The swine fever antigen was purchased from BioApp Co., Ltd. (cat. no: CSFV-E2H, product number: HERVANTI016), and black nano gold was used for gold particles. This binder was diluted with a buffer, dispensed on a black nano-gold-junction pad, and dried.

5. Assembly: A rod was prepared by attaching the prepared black nano-gold-E2 bonding pad, an absorbent pad, and a sample pad, and assembled in a plastic cassette to prepare a prototype.

<Experimental Example 1> Evaluation of the relative sensitivity and specificity of the kit according to the present invention

Using the kit of the present invention prepared in the above example, domestic and foreign pork were analyzed, and relative sensitivity and specificity were calculated. Sensitivity is the rate at which domestic pork is discriminated as domestic, and specificity is the rate at which foreign pork is discriminated as foreign.

Sensitivity and specificity determination result Test instrument result Check result Total positivity voice Positive (domestic) Jin Yang-seong (A) false positive (B) A+B Voice (foreign) false negative (C) True Voice (D) C+D Total A+C B+D

<Method for Calculating Sensitivity and Specificity>

1) Sensitivity (%) = true positive/(true positive + false negative) = A/(A+C) x 100

Pork Antibody Sensitivity: (191/191+2)x100 = 98.96 (99% rounded up)

2) Specificity (%) = true negative/(false positive + true negative) = D/(B+D) x 100

Specificity: (189/189+1)x100 = 99.47 (99.5% rounded off)

As a result of the experiment, as shown in Table 3 below, the relative sensitivity was 99.0% and the specificity was 99.5%.

Relative sensitivity and specificity of domestic and foreign pork origin origin domestic foreign pork origin
Identification kit domestic 191 One foreign 2 189 relative sensitivity 99.0% relatively special 99.5%

<Experimental Example 2> Determination of origin of pork using the kit according to the present invention

An experiment was conducted according to the procedure shown in FIG. 7 to determine the origin of pork using the identification kit of the present invention. First, about 0.4 g of the sample pork was put into 500 ul of the dilution solution, and the reaction solution was dropped into the sample injection part of the kit by 100 ul, and the result was checked after 5-8 minutes. If a band appeared on the control line and test line, it was domestic as an antibody-positive product, and if a band appeared only on the control line, it was determined as antibody-negative and foreign pork.

As a result of the experiment, it was possible to confirm the band of the control line in all domestic and foreign pork. Antigen*?* antibody reaction of the test line could be confirmed only in domestic pork, and it could be confirmed that there was no antigen*?* antibody reaction in the test line in foreign pork. It shows the antibody level of domestic pork (Figs. 1, 3, 4, 5, 6).

According to the present invention, since the origin of pork can be determined easily and inexpensively without analysis equipment, it can be usefully applied not only in the technical field to which the present invention belongs, but also in the process of cracking down on the indication of origin of agricultural and fishery products in the enforcement of the "Act on the Indication of Origin of Agricultural and Fisheries Products" can

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Claims (14)

Swine fever antigen for identification of pork produced by a recombinant vector having the sequence of SEQ ID NO: 1. A kit for determining the origin of pork containing the antigen of claim 1. According to claim 2, wherein the kit is a kit for determining the origin of pork, characterized in that the antigen of claim 1 is attached to the nitrocellulose membrane on the membrane. [Claim 4] The kit for determining the origin of discriminated pork according to claim 3, wherein an anti-BSA is additionally attached to the nitrocellulose membrane. The kit for determining the origin of pork according to claim 2, wherein the kit further comprises 10 to 100 nm gold particles. The kit for determining the origin of pork according to claim 2, wherein domestic pigs are inoculated with a swine fever vaccine to form antibodies, and foreign pigs are not vaccinated and thus antibodies are not formed. [Claim 3] The kit for determining the origin of pork according to claim 2, wherein the pork uses any one or more of samgyeopsal, neck meat, regular meat, tenderloin, sirloin, ribs, and front leg meat. The kit for determining the origin of pork according to claim 2, wherein the origin is determined as domestic or foreign. A method for determining the origin of pork using the identification kit according to any one of claims 2 to 8. (a) attaching the swine fever antigen of claim 1 to a nitrocellulose membrane;
(b) subjecting the pork juice to be analyzed to an aggregation reaction with gold particles on the antigen-attached membrane;
A method for determining the origin of pork, including The method of claim 10, wherein an anti-BSA is additionally attached to the nitrocellulose membrane. 11. The method of claim 10, wherein the size of the gold particles is 10 to 100 nm. 11. The method of claim 10, wherein the pork is pork belly, neck, whole meat, tenderloin, sirloin, ribs, and front leg meat using a nitrocellulose membrane swine fever antigen/antibody reaction to determine the origin. The method of claim 10, wherein the origin is determined as domestic or foreign.

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