A molecular biological method for identifying pigeon meat and chicken meat and its application

Abstract

The present invention discloses a molecular biology method and application for identifying pigeon meat and chicken. The method uses primers shown in SEQ ID NO.1 and SEQ ID NO.2 to perform PCR amplification on the genomic DNA of the sample to be detected, and after capillary electrophoresis, sequence alignment is performed. When the peak value is 259bp, it is pigeon meat; when the peak value is 235bp, it is chicken. The present invention has the advantages of being able to quickly and accurately identify pigeon meat and chicken, and combating unscrupulous merchants who sell inferior products as good ones. In addition, the method is simple to operate, easy to operate in the laboratory, and convenient for grassroots promotion and use, and has broad market application prospects. In addition, the detection kit developed based on the method of the present invention can generate considerable economic benefits and good social value.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a molecular biology method for identifying pigeon meat and chicken meat. Background Art

[0002] Pigeon meat is known as the "ginseng of animals". It contains a lot of high-quality protein, a small amount of sugar, phospholipids, iron, calcium, vitamin A, vitamin B1, vitamin D and other nutrients. It is easy to digest and absorb, and is a high-quality nutritional product for pregnant women, children, patients, etc. At the same time, pigeons are mainly fed with raw grains, have strong disease resistance, and use less vaccines and drugs. It is a natural green product.

[0003] Since pigeons have a low reproductive efficiency, it usually takes about half a year for them to reach sexual maturity. A pair of pigeons can only lay about 30 eggs per year, which can produce 18 to 20 offspring. Chickens, on the other hand, have a very high reproductive efficiency, laying about 300 eggs per year, which can produce more than 280 offspring. Summary of the invention

[0004] One of the purposes of the present invention is to provide a molecular biology method for identifying pigeon meat and chicken in view of the shortcomings of the prior art.

[0005] A second object of the present invention is to provide primers for detecting the above-mentioned molecular markers.

[0006] The third object of the present invention is to provide the use of the above-mentioned molecular markers.

[0007] A fourth object of the present invention is to provide a detection method using the above-mentioned molecular markers.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A molecular marker for identifying pigeon meat and chicken meat, wherein the molecular marker uses the peak value of capillary electrophoresis to distinguish whether the meat contains pigeon meat and chicken meat. When the peak value is 259 bp, it is pigeon meat; when the peak value is 235 bp, it is chicken meat. A method for identifying pigeon meat and chicken meat, comprising extracting genomic DNA of meat to be tested, amplifying it by PCR and performing capillary electrophoresis, and then performing peak value analysis. When the peak value is 259 bp, it is pigeon meat; when the peak value is 235 bp, it is chicken meat.

[0010] The present invention also provides a primer pair for identifying molecular markers of pigeon meat and chicken, wherein:

[0011] Forward primer (SEQ ID NO.1): 5'-CTAGCCTGTCAAAAATACG-3'

[0012] Reverse primer (SEQ ID NO. 2): 5'-CCAGAAAGAAATTCAAGAA -3'.

[0013] The invention also provides a kit for identifying pigeon meat and chicken meat, which contains the primer pair.

[0014] The invention also provides application of the molecular marker or primer or kit pair in identifying pigeon meat and chicken meat.

[0015] The invention also provides application of the molecular marker or primer or kit pair in screening pigeon meat.

[0016] The present invention also provides the use of the molecular marker or primer or kit pair in screening chicken.

[0017] The present invention also provides a method for identifying pigeon meat and chicken meat. The duck genome DNA is amplified by primer pair SEQ ID NO.1 / SEQ ID NO.2, and peak analysis is performed after capillary electrophoresis. When the peak value is 259 bp, it is pigeon meat; when the peak value is 235 bp, it is chicken meat.

[0018] In a specific embodiment, the present invention also provides a rapid identification method for detecting pigeon meat and chicken, comprising the following steps:

[0019] 1) Extracting genomic DNA from the meat to be tested;

[0020] 2) using the DNA extracted in step 1) as a template and the primer pair of the present invention to perform a PCR amplification reaction;

[0021] 3) The PCR reaction system in step 2) is: 25 μL of 2×PCR Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 1 μL of each 10 μmol / L forward primer, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water;

[0022] 4) The PCR reaction procedure in step 2) is:

[0023] 5) After the reaction is completed, the PCR product is subjected to capillary electrophoresis and peak analysis is performed.

[0024] 6) When the peak is 259bp, it is pigeon meat; when the peak is 235bp, it is chicken.

[0025] Beneficial effects:

[0026] The invention utilizes the peak value of capillary electrophoresis to identify pigeon meat and chicken, and has the advantage of being able to quickly and accurately identify pigeon meat and chicken. The method is simple to operate, easy to operate in the laboratory, and convenient for grassroots promotion and use, and has broad market application prospects. In addition, the detection kit developed based on the method of the invention can generate considerable economic benefits and good social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Chicken peak diagram.

[0028] Figure 2 Pigeon Peak Diagram. DETAILED DESCRIPTION

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0030] Example 1

[0031] 1. Detection of different breeds of pigeons and chickens

[0032] 1.1 Sample collection

[0033] Pigeon meat from four breeds, namely, White King Pigeon, Silver King Pigeon, White Kanu Pigeon, and Taishen Pigeon; and small broiler chickens from three breeds, namely, WOD168, Yisheng 909, and 817 broilers were collected; 30 pigeons from each breed were used to extract genomic DNA using a kit (Tiangen Biotechnology Co., Ltd.).

[0034] 1.2 PCR amplification

[0035] The primers designed by the present invention are used for PCR amplification.

[0036] Forward primer: 5'-CTAGCCTGTCAAAAATACG-3'; FAM fluorescent label was added to the 5' end.

[0037] Reverse primer: 5′-CCAGAAAGAAATTCAAGAA-3′.

[0038] The PCR reaction system is:

[0039] 2×PCR Mix (Nanjing Novogene Biotech Co., Ltd.) 25 μL, 10 μmol / L forward and reverse primers 1 μL each, 50-100 μg / ml template DNA 2 μL, ultrapure water 21 μL.

[0040] The PCR reaction program was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 58°C for 30 s, 72°C for 30 s), and 72°C for 3 min.

[0041] 1.3 Capillary electrophoresis

[0042] After the reaction, the PCR products were subjected to capillary electrophoresis on an ABI 3730 sequencer.

[0043] 1.4 Peak Statistics

[0044] The peak distribution ratios of different groups are shown in Table 1. Figure 1 and Figure 2 ,

[0045] Table 1 Detection of different varieties of pigeon meat and chicken

[0046]

[0047] Note: Capillary electrophoresis has "slip", and 1b is within the normal error range. The result may show a peak of 234bp or 236bp, which is counted as 235bp; the result may show a peak of 258bp or 260bp, which is counted as 259bp.

[0048] Example 2

[0049] 2. Identification of authentic "pigeon meat"

[0050] 2.1 Sample collection

[0051] Ten cheap frozen "meat pigeons" were purchased from the wholesale market, and genomic DNA was extracted from the pigeon meat using a kit (Tiangen Biotechnology Co., Ltd.).

[0052] 2.2 PCR amplification

[0053] The primers designed by the present invention are used for PCR amplification.

[0054] Forward primer: 5'-CTAGCCTGTCAAAAATACG-3'; FAM fluorescent label was added to the 5' end.

[0055] Reverse primer: 5′-CCAGAAAGAAATTCAAGAA-3′.

[0056] The PCR reaction system is:

[0057] 2×PCR Mix (Nanjing Novozyme Biotechnology Co., Ltd.) 25 μL, 10 μmol / L forward and reverse primers 1 μL each, 50-100 μg / ml template DNA 2 μL, ultrapure water 21 μL.

[0058] The PCR reaction program was: 95°C for 5 min, 35 cycles of (95°C for 30 s, 58°C for 30 s, 72°C for 30 s), and 72°C for 3 min.

[0059] 2.3 Capillary electrophoresis

[0060] After the reaction, the PCR products were subjected to capillary electrophoresis on an ABI 3730 sequencer.

[0061] 2.4 Peak Statistics

[0062] The peak value of the 10 "meat pigeons" was 235bp. After tracing the merchant's animal quarantine certificate, it was confirmed that they were counterfeit chicken products.

[0063] In summary, it can be seen that the molecular marker has no difference in pigeon breeds or chicken breeds, but has specificity between pigeons and chickens. At the same time, the efficiency of the present invention in distinguishing pigeon meat and chicken meat is proved.

Claims

1. A method for identifying pigeon meat and chicken, characterized in that: The genomic DNA of the sample to be tested was amplified by PCR using the primers shown in SEQ ID NO.1 and SEQ ID NO.

2. After capillary electrophoresis, sequence comparison was performed. When the peak value was 259 bp, it was pigeon meat; when the peak value was 235 bp, it was chicken.

2. The method for identifying pigeon meat and chicken according to claim 1, characterized in that: The pigeons are white feather king pigeons, silver king pigeons, white Kanu pigeons and Taishen pigeons.

3. The method for identifying pigeon meat and chicken according to claim 1, characterized in that: The chickens are WOD168, Yisheng 909 and 817 broilers.

4. The method for identifying pigeon meat and chicken according to claim 1, characterized in that: The PCR reaction system was: 2×PCRMix 25 μL, 1 μL of each 10 μmol / L forward primer, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

5. The method for identifying pigeon meat and chicken according to claim 1, characterized in that: The PCR reaction program is:

6. A method for screening pigeon meat and chicken, characterized in that: The genomic DNA of the sample to be tested was amplified by PCR using the primers shown in SEQ ID NO.1 and SEQ ID NO.

2. After capillary electrophoresis, sequence comparison was performed. When the peak value was 259 bp, it was pigeon meat; when the peak value was 235 bp, it was chicken.

7. The method for screening pigeon meat and chicken according to claim 6, characterized in that: The PCR reaction system was: 25 μL of 2×PCRMix, 1 μL of each 10 μmol / L forward primer, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

8. The method for screening pigeon meat and chicken according to claim 6, characterized in that: The PCR reaction program is:

9. The method for screening pigeon meat and chicken according to claim 6, characterized in that: The pigeons are white feather king pigeons, silver king pigeons, white Kanu pigeons and Taishen pigeons.

10. The method for screening pigeon meat and chicken according to claim 6, characterized in that: The chickens are WOD168, Yisheng 909 and 817 broilers.

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