Molecular biological identification method for different types of meat ducks and application

Abstract

The invention discloses a molecular biological identification method for different types of meat ducks and application of the molecular biological identification method. PCR amplification and capillary electrophoresis are carried out on genome DNA of meat ducks to be detected through primers shown in SEQ ID NO.1 and SEQ ID NO.2, when the allele is 1150bp, the meat ducks are domestic ducks, when the allele is 1156bp, the meat ducks are muscovy ducks, and when the allele is 1150bp and 1156bp, the meat ducks are half muscovy ducks. The detection kit developed on the basis of the method 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 primer pair for identifying different types of meat ducks.

[0014] The invention also provides application of the molecular marker or primer or kit pair in distinguishing domestic ducks from Muscovy ducks.

[0015] The present invention also provides application of the molecular marker or primer or kit pair in distinguishing domestic ducks and Mule ducks.

[0016] The present invention also provides application of the molecular marker or primer or kit pair in distinguishing Muscovy ducks and semi-Muscovy ducks.

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

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

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

[0020] A method for identifying different types of meat ducks, wherein the different types of meat ducks refer to domestic ducks, Muscovy ducks or semi-Muscovy 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 products, if the PCR products contain the 1150bp allele, they are domestic ducks; if the PCR products contain the 1156bp allele, they are Muscovy ducks; and if the PCR products contain both the 1150bp and 1156bp alleles, they are semi-Muscovy ducks.

[0021] A method for screening different types of meat ducks, wherein the different types of meat ducks refer to domestic ducks, Muscovy ducks or semi-Muscovy ducks; PCR amplification is performed on the genomic DNA of the meat ducks to be detected by using the primers shown in SEQ ID NO.1 and SEQ ID NO.2; after capillary electrophoresis of the PCR products, if the PCR products contain the 1150bp allele, they are domestic ducks; if the PCR products contain the 1156bp allele, they are Muscovy ducks; and if the PCR products contain both the 1150bp and 1156bp alleles, they are semi-Muscovy ducks.

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

[0023] 1) extracting genomic DNA from the duck meat to be tested, wherein the duck meat to be tested refers to domestic duck, Muscovy duck or semi-Muscovy duck;

[0024] 2) Using the DNA extracted in step 1) as a template, a PCR amplification reaction was performed using the primer pair shown in SEQ ID NO.1 and SEQ ID NO.2. After capillary electrophoresis of the PCR product, if the 1150bp allele is contained, it is a domestic duck; if the 1156bp allele is contained, it is a Muscovy duck; if the 1150bp and 1156bp alleles are contained at the same time, it is a semi-Muscovy duck.

[0025] In some embodiments, the PCR reaction system in step 2) of the present invention is: 2×PCR Mix (Nanjing Novozyme Biotechnology Co., Ltd.) 25 μL, 1 μL 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) is completed, the PCR product is subjected to capillary electrophoresis and then allele analysis is performed.

[0029] The domestic duck breeds described in the present invention include Beijing duck, Cherry Valley duck, Jinding duck, and Gaoyou duck; the Muscovy duck breeds described in the present invention include Wen's White Muscovy Duck No. 1 and Yongqiang Muscovy Duck; the semi-Muscovy duck breed described in the present invention is the Zhongchu Landrace semi-Muscovy duck.

[0030] Beneficial effects:

[0031] The present invention utilizes alleles to identify domestic ducks, Muscovy ducks and semi-Muscovy ducks, and has the advantage of being able to quickly and accurately identify domestic ducks, Muscovy ducks and semi-Muscovy ducks, and can be used to identify ducklings and duck meat in the market, combating bad merchants who sell inferior goods 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Allele map of domestic duck.

[0033] Figure 2 Muscovy duck allele map.

[0034] Figure 3 Allele map of the Mule duck. DETAILED DESCRIPTION

[0035] 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.

[0036] Example 1

[0037] 1Identification of different breeds of domestic ducks, Muscovy ducks and semi-Muscovy ducks

[0038] 1.1 Sample collection

[0039] The main domestic duck breeds raised in my country were collected: Beijing duck, Cherry Valley duck, Jinding duck, Gaoyou duck, Muscovy duck breeds: Wenshi White Muscovy Duck No. 1, Yongqiang Muscovy Duck, and semi-Muscovy duck breeds: Zhongchu Landrace Semi-Muscovy Duck; blood was collected from the wing vein of 30 ducks of each breed, and genomic DNA was extracted using a kit (Tiangen Biotechnology Co., Ltd.).

[0040] 1.2 PCR amplification

[0041] The primers designed by the present invention are used for PCR amplification.

[0042] Forward primer: 5'-AGAGTTGGTAAGTGTTGGTTT-3'; FAM fluorescent label added to the 5' end.

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

[0044] The PCR reaction system is:

[0045] 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.

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

[0047] 1.3 Capillary electrophoresis

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

[0049] 1.4 Allele statistics

[0050] The allele distribution ratios of different populations are shown in Table 1 and Figure 1-3 shown.

[0051] Table 1 Allele distribution of different types of meat ducks

[0052]

[0053] Note: Capillary electrophoresis has "slip", and ±1bp is within the normal error range. The result may show a peak chart of 830 or 832bp, which is counted as 831bp; a peak chart of 839 or 841bp, which is counted as 840bp; a peak chart of 1149 or 1151bp, which is counted as 1150bp; a peak chart of 1155bp or 1157bp, which is counted as 1156bp.

[0054] Example 2 Identification of Muscovy Ducklings

[0055] 2.1 Sample collection

[0056] Thirty Muscovy ducklings were purchased from an e-commerce platform, and genomic DNA was extracted using a kit (Tiangen Biotechnology Co., Ltd.).

[0057] 2.2 PCR amplification

[0058] The primers designed by the present invention are used for PCR amplification.

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

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

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

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

[0063] 2.3 Capillary electrophoresis

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

[0065] 2.4 Allele statistics

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

[0067] Table 2 Allele distribution of Muscovy ducklings

[0068]

[0069] 2.5 Feeding observation

[0070] After being raised to 3 months of age, all 30 domestic ducks were observed by body shape and appearance, and the results were completely consistent with those of the present invention.

[0071] Example 3 Identification of ducklings mixed with mule ducks

[0072] 3.1 Sample collection

[0073] During the duckling sorting process, due to a worker's mistake, 50 Muscovy ducklings were accidentally put into 150 semi-Muscovy ducklings. Feathers of 200 ducklings were collected and genomic DNA was extracted using a kit (Tiangen Biotechnology Co., Ltd.).

[0074] 3.2 PCR amplification

[0075] The primers designed by the present invention are used for PCR amplification.

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

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

[0078] The PCR reaction system was: 25 μL of 2×PCR Mix (Nanjing Novozyme Biotechnology Co., Ltd.), 1 μL 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, 35 cycles of (95°C for 30 s, 60°C for 30 s, 72°C for 30 s), 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] According to statistics, there are 50 ducklings containing the 1156bp allele, and 150 ducklings containing both the 1150bp and 1156bp alleles.

[0084] 3.5 Feeding observation

[0085] The ducklings with two different alleles were grouped and raised until they were 3 months old. Among the ducks with the 1156bp allele group, one died. Through observation of body shape and appearance, all 49 were Muscovy ducks. Among the ducks with the 1150bp and 1156bp allele groups, two died. Through observation of body shape and appearance, all 148 were semi-Muscovy 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 semi-Muscovy ducks. The molecular markers have no difference between ducks, Muscovy ducks and semi-Muscovy ducks. At the same time, the invention proves the high efficiency of 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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