A molecular biological identification method for different types of meat ducks and its application

Abstract

The present invention discloses a method for molecular biological identification of different types of meat ducks and its application. PCR amplification and capillary electrophoresis are performed on the genomic DNA of the meat ducks to be detected using the primers shown in SEQ ID NO.1 and SEQ ID NO.2. When the allele is 1150bp, it is a domestic duck; when the allele is 1156bp, it is a muscovy duck; when the alleles are 1150bp and 1156bp, it is a mule duck. The detection kit developed based on the method of the present invention can generate considerable economic benefits and good social values.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a molecular biology identification method for different types of meat ducks and applications thereof. Background Art

[0002] In 2023, my country will produce 4.218 billion commercial meat ducks, and duck meat has become the third largest source of meat protein in my country after pork and chicken. my country's meat ducks are mainly composed of domestic ducks (mainly white-feathered meat ducks), Muscovy ducks and semi-Muscovy ducks. Domestic ducks belong to the Anatidae family and the Anas genus. The main varieties include Cherry Valley, Beijing duck, Jinding duck, Gaoyou duck, etc. Muscovy ducks, also called knob-headed ducks, belong to the Anatidae family and the Cairina genus. The main varieties include Wen's White Muscovy Duck No. 1 and Yongqiang Muscovy Duck. Semi-Muscovy ducks are the hybrid offspring of Muscovy ducks and white-feathered meat ducks. The main varieties include the Changbai semi-Muscovy duck, etc.

[0003] Since the breeding efficiency of domestic ducks is higher than that of other types of meat ducks, many unscrupulous merchants use domestic ducks to impersonate Muscovy ducks or semi-Muscovy ducks in the sale of ducklings and duck meat, causing market chaos. Therefore, a fast, accurate and easy-to-operate technology is urgently needed to identify domestic ducks, Muscovy ducks and semi-Muscovy ducks, so as to ensure the orderly, healthy and stable development of my country's meat duck industry and meet the people's diversified demand for poultry products. Summary of the invention

[0004] One of the purposes of the present invention is to provide a molecular biological identification method for different types of meat ducks 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 different types of meat ducks. The molecular marker uses the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 to amplify meat duck genomic DNA. After capillary electrophoresis of the PCR product, if it contains the 1150bp allele, it is a domestic duck; if it contains the 1156bp allele, it is a Muscovy duck; if it contains both the 1150bp and 1156bp alleles, it is a semi-Muscovy duck.

[0010] The present invention also provides a primer pair for identifying molecular markers of different types of meat ducks, wherein:

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

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

[0013] The present invention also provides a kit containing the above primer pair for differentiating different types of meat ducks.

[0014] The present invention also provides the application of the above molecular marker or primer or kit in differentiating domestic ducks and muscovy ducks.

[0015] The present invention also provides the application of the above molecular marker or primer or kit in differentiating domestic ducks and mule ducks.

[0016] The present invention also provides the application of the above molecular marker or primer or kit in differentiating muscovy ducks and mule ducks.

[0017] The present invention also provides the application of the above molecular marker or primer or kit in screening domestic ducks.

[0018] The present invention also provides the application of the above molecular marker or primer or kit in screening muscovy ducks.

[0019] The present invention also provides the application of the above molecular marker or primer or kit in screening mule ducks.

[0020] A method for differentiating different types of meat ducks, where the different types of meat ducks refer to domestic ducks, muscovy ducks or mule ducks; PCR amplification is performed on the genomic DNA of the meat duck to be detected using the primers shown in SEQ ID NO.1 and SEQ ID NO.2. After capillary electrophoresis of the PCR product, when the allele of 1150bp is present, it is a domestic duck; when the allele of 1156bp is present, it is a muscovy duck; when both the alleles of 1150bp and 1156bp are present, it is a mule duck.

[0021] A method for screening different types of meat ducks, where the different types of meat ducks refer to domestic ducks, muscovy ducks or mule ducks; PCR amplification is performed on the genomic DNA of the meat duck to be detected using the primers shown in SEQ ID NO.1 and SEQ ID NO.2. After capillary electrophoresis of the PCR product, when the allele of 1150bp is present, it is a domestic duck; when the allele of 1156bp is present, it is a muscovy duck; when both the alleles of 1150bp and 1156bp are present, it is a mule duck.

[0022] In a specific embodiment, the present invention also provides a rapid identification method for different types of meat ducks, including the following steps:

[0023] 1) Extract genomic DNA from the duck meat to be tested, where the duck meat to be tested refers to domestic ducks, muscovy ducks or mule ducks;

[0024] 2) Using the DNA extracted in step 1) as a template, perform a PCR amplification reaction with the primer pairs shown in SEQ ID NO.1 and SEQ ID NO.2. After capillary electrophoresis of the PCR product, when the 1150bp allele is present, it is a domestic duck; when the 1156bp allele is present, it is a muscovy duck; when both the 1150bp and 1156bp alleles are present, it is a mule duck.

[0025] In some embodiments, the PCR reaction system in step 2) of the present invention is: 2×PCR Mix (Nanjing Novoprotein Biological Co., Ltd.) 25 μL, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50 - 100 μg / ml template DNA, and 21 μL of ultrapure water.

[0026] In some embodiments, the PCR reaction procedure in step 2) is:

[0027]

[0028] In some embodiments, after the reaction in step 2) ends, after capillary electrophoresis of the PCR product, allele analysis is performed.

[0029] The domestic duck breeds described in the present invention include Beijing ducks, Cherry Valley ducks, Jinding ducks, and Gaoyou ducks; the muscovy duck breeds described in the present invention include Wen's White Muscovy Duck No. 1 and Yongqiang Muscovy Ducks; the mule duck breed described in the present invention is Zhongchu Changbai Mule Duck.

[0030] Beneficial effects:

[0031] The present invention uses alleles to identify domestic ducks, muscovy ducks and mule ducks. The advantage is that it can quickly and accurately identify domestic ducks, muscovy ducks and mule ducks, and can be used for the identification of ducklings and duck meat in the market to combat unscrupulous merchants who pass off inferior goods as good ones. And this method is simple to operate, easy to perform in the laboratory, convenient for grass-roots popularization 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 of the drawings

[0032] Figure 1 Allele map of domestic ducks.

[0033] Figure 2 Allele map of muscovy ducks.

[0034] Figure 3 Allele map of mule ducks. Detailed implementation manners

[0035] The following examples are used to illustrate the present invention, but not 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.

[0036] Example 1

[0037] 1 Identification of Different Varieties of Domestic Ducks, Muscovy Ducks and Mule Ducks

[0038] 1.1 Sample Collection

[0039] Collect the main domestic duck varieties raised in China: Beijing Duck, Cherry Valley Duck, Jinding Duck, Gaoyou Duck, Muscovy duck varieties: Wen's White Muscovy Duck No. 1, Yongqiang Muscovy Duck, mule duck variety: Zhongchu Changbai Mule Duck; collect blood from the wing vein, 30 individuals are collected for each variety, and genomic DNA is extracted using a kit (Tiangen Biotech Co., Ltd.).

[0040] 1.2 PCR Amplification

[0041] Use the primers designed by the present invention for PCR amplification.

[0042] Forward primer: 5'-AGAGTTGGTAAGTGTTGGTTT-3'; add FAM fluorescence label at the 5' end.

[0043] Reverse primer: 5'-AGACAGGATCTTTCTCATAG-3'.

[0044] The PCR reaction system is as follows:

[0045] 2×PCR Mix (Nanjing Novoprotein Biological Co., Ltd.) 25 μL, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50 - 100 μg / ml template DNA, 21 μL of ultrapure water.

[0046] The PCR reaction program is: 95°C for 5 min, (95°C for 30 s, 60°C for 30 s, 72°C for 30 s) for 35 cycles, 72°C for 3 min.

[0047] 1.3 Capillary Electrophoresis

[0048] After the reaction, the PCR products are subjected to capillary electrophoresis on an ABI 3730 sequencer.

[0049] 1.4 Allele Statistics

[0050] The distribution ratios of alleles in different populations are shown in Table 1 and Figures 1-3 as shown.

[0051] Table 1 Allele Distribution of Different Types of Meat Ducks

[0052]

[0053] Note: There is "slip" in capillary electrophoresis, and ±1bp belongs to the normal error range. The peak pattern may show 830 or 832bp, which is statistically counted as 831bp; 839 or 841bp peak pattern, statistically counted as 840bp; 1149 or 1151bp peak pattern, statistically counted as 1150bp; 1155bp or 1157bp peak pattern, statistically counted as 1156bp.

[0054] Example 2 Identification of Muscovy Duck Ducklings

[0055] 2.1 Sample Collection

[0056] Purchase 30 "Muscovy duck" ducklings from an e-commerce platform, and extract genomic DNA using a kit (Tiangen Biotech Co., Ltd.).

[0057] 2.2 PCR Amplification

[0058] Use the primers designed in the present invention for PCR amplification.

[0059] Forward primer: 5'-AGAGTTGGTAAGTGTTGGTTT-3'; FAM fluorescence label is added to the 5' end.

[0060] Reverse primer: 5'-AGACAGGATCTTTCTCATAG-3'.

[0061] The PCR reaction system is: 2×PCR Mix (Nanjing Novoprotein Biotech Co., Ltd.) 25μL, 1μL each of 10μmol / L forward and reverse primers, 2μL of 50 - 100μg / ml template DNA, and 21μL of ultrapure water.

[0062] The PCR reaction program is: 95℃ for 5min, (95℃ for 30s, 60℃ for 30s, 72℃ for 30s) for 35 cycles, and 72℃ for 3min.

[0063] 2.3 Capillary Electrophoresis

[0064] After the reaction, the PCR product is subjected to capillary electrophoresis on an ABI 3730 sequencer.

[0065] 2.4 Allele Statistics

[0066] As can be seen from Table 2, all 30 "Muscovy duck" ducklings are domestic ducks.

[0067] Table 2 Allele Distribution of Muscovy Duck Ducklings

[0068]

[0069] 2.5 Feeding Observation

[0070] Raised until 3 months old, through observation of body shape and appearance, 30 domestic ducks were determined, which was completely consistent with the results of the present invention.

[0071] Identification of domestic duck mixed with mule duckling in Example 3

[0072] 3.1 Sample collection

[0073] During the sorting process of ducklings, due to the mistake of the worker, 50 muscovy ducklings were accidentally put into 150 mule ducklings. The feathers of 200 ducklings were collected, and genomic DNA was extracted using a kit (Tiangen Biotech Co., Ltd.).

[0074] 3.2 PCR amplification

[0075] PCR amplification was carried out using the primers designed in the present invention.

[0076] Forward primer: 5'-AGAGTTGGTAAGTGTTGGTTT-3'; FAM fluorescence label was added to the 5' end.

[0077] Reverse primer: 5'-AGACAGGATCTTTCTCATAG-3'.

[0078] The PCR reaction system was: 2×PCR Mix (Novoprotein Scientific Inc.) 25 μL, 1 μL each of 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

[0079] The PCR reaction program was: 95°C for 5 min, (95°C for 30 s, 60°C for 30 s, 72°C for 30 s) for 35 cycles, and 72°C for 3 min.

[0080] 3.3 Capillary electrophoresis

[0081] After the reaction, the PCR products were subjected to capillary electrophoresis on an ABI 3730 sequencer.

[0082] 3.4 Allele statistics

[0083] After statistics, there were 50 ducklings with the 1156bp allele, and 150 ducklings with both the 1150bp and 1156bp alleles.

[0084] 3.5 Feeding observation

[0085] The ducklings with two different alleles were raised in groups until 3 months old. In the group with the 1156bp allele, 1 died. Through observation of body shape and appearance, all 49 were muscovy ducks; in the group with both the 1150bp and 1156bp alleles, 2 died. Through observation of body shape and appearance, all 148 were mule ducks, which was completely consistent with the results of the present invention.

[0086] In summary, the method described in this patent can effectively identify domestic ducks, muscovy ducks and mule ducks. This molecular marker has no difference among duck, muscovy duck and mule duck breeds. At the same time, it proves the high efficiency of the present invention in identifying different types of meat ducks.

Claims

1. A method for identifying different types of meat ducks, characterized in that: The different types of meat ducks refer to domestic ducks, Muscovy ducks and / or semi-Muscovy ducks; the genomic DNA of the meat duck to be tested is amplified by PCR and capillary electrophoresis using the primers shown in SEQ ID NO.1 and SEQ ID NO.

2. When the allele is 1150bp, the gene is a domestic duck; when the allele is 1156bp, the gene is a Muscovy duck; when the alleles are 1150bp and 1156bp, the gene is a semi-Muscovy duck.

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

3. The method for identifying different types of meat ducks according to claim 1, characterized in that: The PCR reaction program is:

4. The method for identifying different types of meat ducks according to claim 1, characterized in that: The domestic ducks include Beijing ducks, Cherry Valley ducks, Jinding ducks, and Gaoyou ducks; the Muscovy ducks include Wen's White Muscovy Duck No. 1 and Yongqiang Muscovy Duck; and the semi-Muscovy ducks are Landrace semi-Muscovy ducks.

5. A method for screening different types of meat ducks, characterized in that: The different types of meat ducks refer to domestic ducks, Muscovy ducks and / or semi-Muscovy ducks; the genomic DNA of the meat duck to be tested is amplified by PCR using the primers shown in SEQ ID NO.1 and SEQ ID NO.

2. When the allele is 1150bp, the gene is a domestic duck; when the allele is 1156bp, the gene is a Muscovy duck; when the alleles are 1150bp and 1156bp, the gene is a semi-Muscovy duck.

6. The method for screening different types of meat ducks according to claim 5, characterized in that: The PCR reaction system was: 25 μL of 2×PCRMix, 1 μL of each 10 μmol / L forward and reverse primers, 2 μL of 50-100 μg / ml template DNA, and 21 μL of ultrapure water.

7. The method for screening different types of meat ducks according to claim 5, characterized in that: The PCR reaction program is:

8. The method for screening different types of meat ducks according to claim 5, characterized in that: The domestic ducks include Beijing ducks, Cherry Valley ducks, Jinding ducks, and Gaoyou ducks; the Muscovy ducks include Wen's White Muscovy Duck No. 1 and Yongqiang Muscovy Duck; and the semi-Muscovy ducks are Landrace semi-Muscovy ducks.

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