A molecular biology method for simultaneous detection of duck and goose derived components and its application

Abstract

The present invention discloses a molecular biology method and application capable of simultaneously detecting duck and goose derived components. The genomic DNA of a sample to be detected is amplified by PCR using primers shown in SEQ ID NO.1 and SEQ ID NO.2. After capillary electrophoresis, sequence alignment is performed. When the peak value is 999 bp, it indicates that the sample contains duck derived components. When the peak value is 992 bp, it indicates that the sample contains goose derived components. When the peak values ​​are 999 bp and 992 bp, it indicates that the sample contains both duck and goose derived components. 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 capable of simultaneously detecting duck- and goose-derived components. Background Art

[0002] The duck in the present invention is a domestic duck, belonging to the family Anatidae, the genus Anas, and the main domestically raised species include Cherry Valley, Beijing duck, Jinding duck, Gaoyou duck, etc. The goose belongs to the family Anatidae, the genus Anser, and the main domestically raised species include Taihu goose, Yangzhou goose, Zhedong white goose, Sichuan white goose, etc.

[0003] Although ducks and geese are both waterfowl, ducks are much more efficient than geese in terms of production efficiency. For example, the annual egg production of breeding ducks can reach more than 250 eggs, while the annual egg production of breeding geese is only about 70 eggs at most; ducks can generally be marketed in about 40 days, while geese generally take about 70 days. Driven by profit, adulteration of duck and goose products occurs frequently. For example, famous down product brands such as Canada Goose, Columbia, and Converse have all used "duck down" to impersonate "goose down"; duck meat impersonating goose meat, beef, and mutton is even more common in the market.

[0004] At present, the methods for simultaneously detecting multiple poultry-derived ingredients are mostly to design a pair of primers or probes for each type of poultry, and then perform multiple amplification. Different primers and probes will interfere with each other, affecting the accuracy of the results. Therefore, a fast, accurate and easy-to-operate technology is urgently needed to detect duck and goose-derived ingredients to ensure the orderly, healthy and stable development of my country's poultry industry and meet the people's diversified poultry product needs. Summary of the invention

[0005] One of the purposes of the present invention is to provide a molecular biology method capable of simultaneously detecting duck- and goose-derived components in view of the deficiencies of the prior art.

[0006] A second object of the present invention is to provide primers for detecting the above-mentioned molecular markers.

[0007] The third object of the present invention is to provide the use of the above-mentioned molecular markers.

[0008] A fourth object of the present invention is to provide a detection method using the above-mentioned molecular markers.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A molecular marker capable of simultaneously detecting duck and / or goose derived components, the molecular marker utilizes capillary electrophoresis peaks to distinguish whether duck and goose derived components are contained; when the peak is 999 bp, duck derived components are contained; when the peak is 992 bp, goose derived components are contained; when the peaks are 999 bp and 992 bp, both duck and goose derived components are contained.

[0011] The present invention also provides a primer pair capable of simultaneously detecting molecular markers of duck- and goose-derived components, wherein:

[0012] Forward primer (SEQ ID NO. 1): 5'-TATTCAGGTTTGTGTTACTGGAA-3'

[0013] Reverse primer (SEQ ID NO. 2): 5'-GATGCATGCAAAGAACTCCAGC-3'.

[0014] The present invention also provides a kit containing the primer pair for simultaneously detecting duck- and goose-derived components.

[0015] The present invention also provides the use of the above molecular markers or primers or kit in detecting duck-derived components.

[0016] The present invention also provides the use of the above molecular markers or primers or kit in detecting goose-derived components.

[0017] The present invention also provides the use of the above molecular markers or primers or kit in the simultaneous detection of duck- and goose-derived components.

[0018] A method for simultaneously detecting duck and / or goose derived components, comprising: performing PCR amplification on genomic DNA of a sample to be detected using primers shown in SEQ ID NO.1 and SEQ ID NO.2, and performing sequence alignment after capillary electrophoresis. When a peak value is 999 bp, it indicates that a duck derived component is contained; when a peak value is 992 bp, it indicates that a goose derived component is contained; and when peak values ​​are 999 bp and 992 bp, it indicates that both duck and goose derived components are contained.

[0019] The present invention also provides a method for screening duck or goose derived components, wherein PCR amplification is performed on the genomic DNA of the sample to be detected using the primers shown in SEQ ID NO.1 and SEQ ID NO.2, and sequence comparison analysis is performed after capillary electrophoresis. When the peak value is 999 bp, it indicates that the sample contains duck derived components; when the peak value is 992 bp, it indicates that the sample contains goose derived components; and when the peak values ​​are 999 bp and 992 bp, it indicates that the sample contains both duck and goose derived components.

[0020] In some embodiments, the genomic DNA of the sample to be detected in the present invention can be selected from samples commonly used in the art, such as feathers, muscles, blood, or oral swabs.

[0021] In some embodiments, the PCR reaction system of the present invention is: 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; in some embodiments, the PCR reaction procedure of the present invention is:

[0022]

[0023] The duck sources described in the present invention include Beijing ducks, Cherry Valley ducks, Jinding ducks, and Gaoyou ducks; the goose sources described in the present invention include Taihu geese, Yangzhou geese, East Zhejiang white geese, and Sichuan white geese.

[0024] Beneficial effects:

[0025] The present invention uses peak value to detect whether duck and goose derived ingredients are contained, and has the advantage of being able to quickly and accurately detect duck and goose derived ingredients in down products and meat products, and combating unscrupulous 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

[0026] Figure 1 Peak diagram of duck-derived ingredients.

[0027] Figure 2 Peak diagram of goose-derived ingredients.

[0028] Figure 3 Contains both duck and goose derived components. 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 ducks and geese

[0032] 1.1 Sample collection

[0033] The main domestic duck breeds raised in my country were collected: Beijing duck, Cherry Valley duck, Jinding duck, Gaoyou duck, and goose breeds: Taihu goose, Yangzhou goose, Zhedong white goose, and Sichuan white goose. Thirty geese of each breed were collected and genomic DNA was extracted 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'-TATTCAGGTTTGTGTTACTGGAA-3'; FAM fluorescent label added to the 5' end.

[0037] Reverse primer: 5′-GATGCATGCAAAGAACTCCAGC-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, 60°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 Tables 1 and Figure 1-3

[0045] Table 1 Peak distribution of different breeds of ducks and geese

[0046]

[0047]

[0048] Note: Capillary electrophoresis has "slip", and 1b is within the normal error range. The result may show a peak of 1000bp or 998bp, which is counted as 999bp; the result may show a peak of 991bp or 993bp, which is counted as 992bp.

[0049] The peak values ​​of all duck species are all 999bp, and the peak values ​​of all goose species are all 992bp. The molecular marker has no difference between duck or goose species. At the same time, the high efficiency of the present invention in detecting duck and goose derived components is proved.

[0050] Example 2

[0051] 2. Detection of duck and goose down products

[0052] 2.1 Sample collection

[0053] Three down products with goose down filling in the labels were selected, and 10 down feathers were collected from each down product. Genomic DNA was extracted using a kit (Tiangen Biotechnology Co., Ltd.).

[0054] 2.2 PCR amplification

[0055] The primers designed by the present invention are used for PCR amplification.

[0056] Forward primer: 5'-TATTCAGGTTTGTGTTACTGGAA-3'; FAM fluorescent label added to the 5' end.

[0057] Reverse primer: 5′-GATGCATGCAAAGAACTCCAGC-3′.

[0058] The PCR reaction system is:

[0059] 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.

[0060] 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.

[0061] 2.3 Capillary electrophoresis

[0062] After the reaction, the PCR products were subjected to capillary electrophoresis on an ABI 3730 sequencer.

[0063] 2.4 Peak Statistics

[0064] The peak values ​​of different samples are shown in Table 2

[0065] Table 2 Peak distribution of duck and goose derived ingredients

[0066] variety Peak distribution Sample 1 999bp Sample 2 999 / 992bp Sample 3 992bp

[0067] Sample 1 only contains duck-derived ingredients and no goose-derived ingredients, which is a fake or inferior product; Sample 2 contains both duck-derived and goose-derived ingredients, which is a fake or inferior product; Sample 3 only contains goose-derived ingredients and is a qualified product. This detection method was later verified by the detection method of "Down and Feather Inspection Method (GB / T 10288-2016)", and the results were accurate, proving the high efficiency of the present invention in identifying and detecting duck and goose-derived ingredients.

Claims

1. A method for simultaneously detecting duck and / or goose derived components, 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 alignment was performed. When the peak value was 999 bp, it indicated that duck-derived components were contained; when the peak value was 992 bp, it indicated that goose-derived components were contained; when the peak values ​​were 999 bp and 992 bp, it indicated that both duck-derived and goose-derived components were contained; the duck-derived component was down, and the goose-derived component was down.

2. The method for simultaneously detecting duck and / or goose derived components according to claim 1, characterized in that: The PCR reaction system was: 25 μL of 2×PCR Mix, 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.

3. The method for simultaneously detecting duck and / or goose derived components according to claim 1, characterized in that: The PCR reaction program is: .

4. The method for simultaneously detecting duck and / or goose derived components according to claim 1, characterized in that: The duck sources include Beijing ducks, Cherry Valley ducks, Jinding ducks, and Gaoyou ducks; the goose sources include Taihu geese, Yangzhou geese, East Zhejiang white geese, and Sichuan white geese.

5. A method for screening duck-derived or goose-derived ingredients, 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 analysis was performed. When the peak value was 999 bp, it indicated that duck-derived components were contained; when the peak value was 992 bp, it indicated that goose-derived components were contained; when the peak values ​​were 999 bp and 992 bp, it indicated that both duck-derived and goose-derived components were contained; the duck-derived component was down, and the goose-derived component was down.

6. The method for screening duck-derived or goose-derived components according to claim 5, characterized in that: The PCR reaction system was: 25 μL of 2×PCR Mix, 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.

7. The method for screening duck-derived or goose-derived components according to claim 5, characterized in that: The PCR reaction program is: .

8. The method for screening duck-derived or goose-derived components according to claim 5, characterized in that: The duck sources include Beijing ducks, Cherry Valley ducks, Jinding ducks, and Gaoyou ducks; the goose sources include Taihu geese, Yangzhou geese, East Zhejiang white geese, and Sichuan white geese.

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