Primer composition for identifying or assisting in identifying plant protein raw material source and use thereof

Abstract

The application discloses a primer composition for identifying or assisting in identifying a plant protein raw material source and application thereof. The primer composition for identifying or assisting in identifying the plant protein raw material source comprises seven primer pairs in independent packages, and the names of the seven primer pairs are E1, E2, E3, E4, E5, E6 and E7. Seven primers are designed for specific genes of rapeseed, peanut, sunflower seed, oil palm, soybean, sesame and cotton seed of the plant protein raw material source, detection is carried out through a polymerase chain reaction (PCR) method, and the primers have strong specificity after verification, and can be used for identifying the types of plant source feed ingredients in feed.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a primer composition for identifying or assisting in the identification of plant protein raw material sources and its application. Background Technology

[0002] Feed ingredients refer to substances derived from animals, plants, microorganisms, or minerals, used in the processing of feed but not as feed additives. Currently, the scarcity of resources for livestock production has led to a supply shortage of feed ingredients in the livestock and poultry farming industry, resulting in the emergence of many non-grain protein sources, such as rapeseed meal, cottonseed meal, and palm oil meal. Plant-based feed ingredients are classified into energy sources and plant protein sources according to their function. Oilseeds generally belong to plant protein sources, while grains and root vegetables generally belong to energy sources. Testing methods for oilseed meals include sensory methods and microscopic examination, but these methods are time-consuming and have low accuracy.

[0003] Genetic detection technology is based on the relatively stable genetic material DNA for species identification. It can distinguish different plant species and requires small sample sizes, accuracy, and sensitivity. Currently, there are reports on the application of genetic detection methods in identifying animal-derived components and microbial species, but reports on their application in identifying the sources of plant feed ingredients are very rare. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to identify or assist in the identification of plant protein raw material sources.

[0005] To address the above technical problems, this invention proposes reagents or kits for identifying or assisting in the identification of plant-derived feed components.

[0006] The present invention provides a reagent or kit for identifying or assisting in the identification of plant-derived feed components, wherein the reagent or kit contains a primer composition; the plant is rapeseed ( Brassica napus ),peanut( Arachis hypogaea ), sunflower seeds ( Helianthus annuus ), oil palm ( Elaeis guineensis ), soybeans Glycine max ),Sesame( Sesamum indicum ), cottonseed ( Gossypium hirsutum The primer composition consists of seven primer pairs, named E1, E2, E3, E4, E5, E6, and E7.

[0007] The E1 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 15; the E2 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 16; the E3 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 17; the E4 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 18; the E5 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 19; the E6 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 20; and the E7 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 21.

[0008] In the above reagents or kits, the E1 primer pair consists of two single-stranded DNAs as shown in sequences 1 and 2 of the sequence listing; the E2 primer pair consists of two single-stranded DNAs as shown in sequences 3 and 4 of the sequence listing; the E3 primer pair consists of two single-stranded DNAs as shown in sequences 5 and 6 of the sequence listing; the E4 primer pair consists of two single-stranded DNAs as shown in sequences 7 and 8 of the sequence listing; the E5 primer pair consists of two single-stranded DNAs as shown in sequences 9 and 10 of the sequence listing; the E6 primer pair consists of two single-stranded DNAs as shown in sequences 11 and 12 of the sequence listing; and the E7 primer pair consists of two single-stranded DNAs as shown in sequences 13 and 14 of the sequence listing.

[0009] In the above reagents or kits, the seven primer pairs can be packaged independently.

[0010] The present invention also provides compositions for identifying or assisting in the identification of plant-derived feed components.

[0011] The composition is the primer composition described above; the plant is rapeseed, peanut, sunflower seed, oil palm, soybean, sesame and / or cottonseed; the primer composition consists of seven primer pairs, named E1 primer pair, E2 primer pair, E3 primer pair, E4 primer pair, E5 primer pair, E6 primer pair and E7 primer pair respectively;

[0012] The E1 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 15; the E2 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 16; the E3 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 17; the E4 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 18; the E5 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 19; the E6 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 20; and the E7 primer pair consists of primers specifically amplifying DNA molecules containing the nucleotide sequence SEQ ID No. 21.

[0013] The present invention also provides a method for preparing the above-mentioned reagents, kits, or PCR primer compositions.

[0014] The preparation method of the above-mentioned reagents, kits, or PCR primer compositions provided by the present invention includes the step of separately packaging the two single-stranded DNAs of each primer pair in the above-mentioned PCR primer compositions.

[0015] The present invention also provides the application of the above-mentioned reagents or kits or PCR primer compositions in identifying or assisting in the identification of plant-derived feed ingredients.

[0016] In the above applications, the plants are rapeseed, peanut, sunflower seed, oil palm, soybean, sesame and / or cottonseed.

[0017] The application of the above-mentioned reagents, kits, or PCR primer compositions in the preparation of products for identifying or assisting in the identification of plant-derived feed ingredients is also within the scope of protection of this invention.

[0018] This invention also provides a method for identifying or assisting in the identification of plant-derived feed ingredients.

[0019] In one embodiment, the method includes the following steps:

[0020] S1) Extract genomic DNA from the plant-derived feed components to be tested;

[0021] S2) Using the genomic DNA obtained in S1) as a template, PCR amplification is performed using the above primer combination to obtain amplified PCR products. The sequence or size of the PCR products is used to identify whether the feed to be tested or the raw materials of the feed to be tested contain the above-mentioned plant components.

[0022] In the above method, identifying whether the test feed or its raw materials contain the plant components based on the sequence or size of the PCR product includes at least one of the following:

[0023] S2-1) The PCR product obtained by the E1 primer pair contains a DNA molecule with the nucleotide sequence shown in SEQ ID No. 15. The feed to be tested contains or is candidate to contain components derived from rapeseed. The PCR product does not contain the DNA molecule. The feed to be tested does not contain or is candidate not to contain components derived from rapeseed.

[0024] S2-2) The PCR product obtained by the E2 primer pair amplification contains a DNA molecule with the nucleotide sequence shown in SEQ ID No. 16. The feed to be tested contains or is candidate to contain components derived from peanuts. The PCR product does not contain the DNA molecule. The feed to be tested does not contain or is candidate not to contain components derived from peanuts.

[0025] S2-3) The PCR product obtained by the E3 primer pair contains a DNA molecule with the nucleotide sequence shown in SEQ ID No. 17. The feed to be tested contains or is candidate to contain components derived from sunflower seeds. The PCR product does not contain the DNA molecule. The feed to be tested does not contain or is candidate not to contain components derived from sunflower seeds.

[0026] S2-4) The PCR product obtained by the E4 primer pair contains a DNA molecule with the nucleotide sequence shown in SEQ ID No. 18. The feed to be tested contains or is candidate to contain components derived from oil palm. The PCR product does not contain the DNA molecule. The feed to be tested does not contain or is candidate not to contain components derived from oil palm.

[0027] S2-5) The PCR product obtained by the E5 primer pair contains a DNA molecule with the nucleotide sequence shown in SEQ ID No. 19. The feed to be tested contains or is candidate to contain components derived from soybeans. The PCR product does not contain the DNA molecule. The feed to be tested does not contain or is candidate not to contain components derived from soybeans.

[0028] S2-6) The PCR product obtained by the E6 primer pair contains a DNA molecule with the nucleotide sequence shown in SEQ ID No. 20. The feed to be tested contains or is candidate to contain components derived from sesame. The PCR product does not contain the DNA molecule. The feed to be tested does not contain or is candidate not to contain components derived from sesame.

[0029] The PCR product obtained by amplification of the E7 primer pair (S2-7) contains a DNA molecule with the nucleotide sequence shown in SEQ ID No. 21. The feed to be tested contains or is candidate to contain components derived from cottonseed. The PCR product does not contain the DNA molecule. The feed to be tested does not contain or is candidate not to contain components derived from cottonseed.

[0030] In the above method, identifying whether the test feed or its raw materials contain the plant components based on the sequence or size of the PCR product includes at least one of the following:

[0031] S3-1) The PCR product obtained by the E1 primer pair amplification contains a 180 bp DNA fragment, and the feed to be tested contains or is candidate to contain components derived from rapeseed. The PCR product does not contain a 180 bp DNA fragment, and the feed to be tested does not contain or is candidate to not contain components derived from rapeseed.

[0032] S3-2) The PCR product obtained by amplification of the E2 primer pair contains a 139 bp DNA fragment, and the feed to be tested contains or is candidate to contain components derived from peanuts; the PCR product does not contain a 139 bp DNA fragment, and the feed to be tested does not contain or is candidate not to contain components derived from peanuts.

[0033] S3-3) The PCR product obtained by amplification of the E3 primer pair contains a 173 bp DNA fragment, and the feed to be tested contains or is candidate to contain components derived from sunflower seeds. The PCR product does not contain a 173 bp DNA fragment, and the feed to be tested does not contain or is candidate to not contain components derived from sunflower seeds.

[0034] S3-4) The PCR product obtained by the E4 primer pair contains a 249 bp DNA fragment, and the feed to be tested contains or is candidate to contain components derived from oil palm. The PCR product does not contain a 249 bp DNA fragment, and the feed to be tested does not contain or is candidate to contain components derived from oil palm.

[0035] S3-5) The PCR product obtained by amplification of the E5 primer pair contains a 343 bp DNA fragment, and the feed to be tested contains or is candidate to contain components derived from soybeans; the PCR product does not contain a 343 bp DNA fragment, and the feed to be tested does not contain or is candidate to contain components derived from soybeans.

[0036] S3-6) The PCR product obtained by the E6 primer pair contains a 200 bp DNA fragment, and the feed to be tested contains or is candidate to contain components derived from sesame. The PCR product does not contain a 200 bp DNA fragment, and the feed to be tested does not contain or is candidate to contain components derived from sesame.

[0037] The PCR product obtained by amplification of the E7 primer pair (S3-7) contains a 358 bp DNA fragment, and the feed to be tested contains or is candidate to contain components derived from cottonseed. If the PCR product does not contain a 358 bp DNA fragment, and the feed to be tested does not contain or is candidate to not contain components derived from cottonseed.

[0038] This invention designs seven pairs of primers targeting the specific genes of seven plant-derived feed ingredients: rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed. Detection was performed using polymerase chain reaction (PCR), and the primers were verified to have strong specificity and can be used for the identification of plant-derived feed ingredients. Attached Figure Description

[0039] Figure 1 This is an agarose gel electrophoresis image of the PCR product used in this invention to verify the specificity of rapeseed primers. Using genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed as templates, PCR amplification was performed using rapeseed-specific primers. Only rapeseed samples showed a band; no bands were observed in non-rapeseed components.

[0040] Figure 2 This is an agarose gel electrophoresis image of the PCR product used in this invention to verify the specificity of peanut primers. Using genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed as templates, PCR amplification was performed using peanut-specific primers. Only peanut samples showed a band; no bands were observed in non-peanut components.

[0041] Figure 3 This is an agarose gel electrophoresis image of the PCR product used in this invention to verify the specificity of sunflower seed primers. Using genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed as templates, PCR amplification was performed using sunflower seed-specific primers. Bands were amplified only in sunflower seeds; no bands were observed in non-sunflower seed components.

[0042] Figure 4 This is an agarose gel electrophoresis image of the PCR product used in this invention to verify the specificity of oil palm primers. Using genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed as templates, PCR amplification was performed using oil palm-specific primers. Only oil palm samples showed a band; no bands were observed in non-oil palm components.

[0043] Figure 5This is an agarose gel electrophoresis image of the PCR product used in this invention to verify the specificity of soybean primers. Using genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed as templates, PCR amplification was performed using soybean-specific primers. Only soybean samples showed a band; no bands were observed in non-soybean components.

[0044] Figure 6 This is an agarose gel electrophoresis image of the PCR product used in this invention to verify the specificity of sesame primers. Using genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed as templates, PCR amplification was performed using sesame-specific primers. Only sesame samples showed a band; no bands were observed in the non-sesame components.

[0045] Figure 7 This is an agarose gel electrophoresis image of the PCR product used in this invention to verify the specificity of cottonseed primers. Using genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed as templates, PCR amplification was performed using cottonseed-specific primers. Only cottonseed samples showed a band; no bands were observed in non-cottonseed components. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0048] The experimental materials used in the following examples, including rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed, were all purchased seeds of the plant.

[0049] In the following examples, unless otherwise specified, all experimental results were repeated three times.

[0050] Example 1: Primer Specificity Detection

[0051] 1. Sample collection and preservation

[0052] 1.1 Sampling

[0053] Samples of rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed were dried, ground into powder, and stored at room temperature.

[0054] 1.2 DNA template preparation

[0055] Take an appropriate amount of the plant powder sample (30±2 mg) from section 1.1 and place it in a 1.5 mL centrifuge tube. Use the Polysaccharide-Polyphenol Plant Genomic DNA Extraction Kit (TIANGEN) ® Genomic DNA extraction was performed, and the specific steps are as follows:

[0056] (1) Quickly add 500 μL of buffer GPS and 10 µL of LRNase A (10 mg / ml) to the centrifuge tube, vortex to mix, and incubate in a water bath at 65°C for 15 min. During the water bath, invert the centrifuge tube several times to mix the sample.

[0057] (2) Add 125 µL of buffer GPA, vortex for 1 min, centrifuge at 12,000 rpm for 5 min, transfer the supernatant to the filter column CS, centrifuge at 12,000 rpm for 1 min, and transfer the filtrate to a new centrifuge tube;

[0058] (3) Add an equal volume of anhydrous ethanol and mix thoroughly;

[0059] (4) Transfer the solution and flocculent precipitate obtained in the previous step to the RNase-Free adsorption column CR2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the RNase-Free adsorption column CR2 into the collection tube.

[0060] (5) Add 550 μL of protein removal solution RD to the RNase-Free adsorption column CR2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and put the RNase-Free adsorption column CR2 into the collection tube;

[0061] (6) Add 700 μL of washing buffer PW to the RNase-Free adsorption column CR2, centrifuge at 12,000 rpm (13,400×g) for 1 min, discard the waste liquid, and put the RNase-Free adsorption column CR2 into the collection tube; repeat step (6).

[0062] (7) Place the RNase-Free adsorption column CR2 back into the collection tube, centrifuge at 12,000 rpm for 2 min, discard the collection tube, and then transfer the RNase-Free adsorption column CR2 to a new centrifuge tube and air dry at room temperature for 5-10 min.

[0063] (8) Add 100 μL ddH2O to the RNase-Free adsorption column CR2, let it stand at room temperature for 3-5 min, centrifuge at 12,000 rpm for 2 min, and collect the solution into a centrifuge tube.

[0064] 1.3 Concentration Detection

[0065] DNA concentration and purity detection: Take 2.5 μL of DNA sample for concentration detection.

[0066] Table 1. Detection results of DNA concentration in plant protein raw materials.

[0067]

[0068] 2. PCR detection

[0069] 2.1 Preparation of PCR system

[0070] Table 2 PCR reaction system

[0071]

[0072] According to the experimental design, in a system using genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed as templates, seven PCR amplifications were performed using primer sequences (shown in Table 3):

[0073] 1) In the first PCR amplification reaction system, rapeseed-specific primer E1 (shown as 1-F and 1-R in Table 3) was used as primers F and R in Table 2 to prepare six PCR reaction systems for rapeseed-specific primer E1. The templates were genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed; ddH2O was used as a negative control.

[0074] 2) In the second PCR amplification reaction system, six PCR reaction systems using peanut-specific primer E2 (shown as 2-F and 2-R in Table 3) were prepared, using primers F and R from Table 2. The templates were: rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed genomic DNA; ddH2O was used as a negative control.

[0075] 3) In the third PCR amplification reaction system, sunflower seed-specific primer E3 (shown as 3-F and 3-R in Table 3) was used as primers F and R in Table 2 to prepare six PCR reaction systems for sunflower seed-specific primer E3. The templates were genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed; ddH2O was used as a negative control.

[0076] 4) In the fourth PCR amplification reaction system, six PCR reaction systems were prepared using oil palm-specific primer E4 (shown as 4-F and 4-R in Table 3) as primers F and R in Table 2. The templates were genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed; ddH2O was used as a negative control.

[0077] 5) In the fifth PCR amplification reaction system, six PCR reaction systems using soybean-specific primer E5 (shown as 5-F and 5-R in Table 3) were prepared, using primers F and R in Table 2. The templates were: rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed genomic DNA; ddH2O was used as a negative control.

[0078] 6) In the reaction system for the sixth PCR amplification, six PCR reaction systems were prepared using sesame-specific primer E6 (shown as 6-F and 6-R in Table 3) as primers F and R in Table 2. The templates were genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed; ddH2O was used as a negative control.

[0079] 7) In the seventh PCR amplification reaction system, six PCR reaction systems were prepared using cottonseed-specific primer E7 (shown as 7-F and 7-R in Table 3) as primers F and R in Table 2. The templates were genomic DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed; ddH2O was used as a negative control.

[0080] Table 3. Primers for identifying the sources of plant-derived feed components

[0081]

[0082] 2.2 Perform PCR reaction

[0083] Reaction program: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 60℃ annealing for 30 sec, 72℃ extension for 1 min, 30 cycles; 72℃ final extension for 10 min, 4℃ cooling for 10 min.

[0084] 3. Detection of PCR amplification products and interpretation of results

[0085] 3.1 Electrophoresis detection of PCR products

[0086] Weigh 1 g of agarose, add 100 ml of 1×TAE buffer, heat to boiling for 30 s, mix well, let cool slightly, add 10 μL of 10000×Gene Green nucleic acid dye, invert the gel plate, let stand for 30 min, and after solidification, place it in an electrophoresis tank containing 1×TAE for sample loading. Add 5 μL of PCR amplification product from 2.2 to each well, set the voltage to 110 V, and perform electrophoresis for 30-40 min. After electrophoresis, remove the gel block and image it using a gel imaging system.

[0087] 3.2 Result Judgment

[0088] The results are as follows Figure 1-7 As shown, Figures 1 to 7 In the middle, M represents the marker, and from bottom to top, they are 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 700 bp, 1000 bp, 1600 bp, 2000 bp, 5000 bp, and 8000 bp.

[0089] Rapeseed-specific primer E1 was used to amplify genomic DNA samples from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed. Only the PCR system using rapeseed genomic DNA as a template could amplify the rapeseed-specific DNA, which is 100-200 bp in size. The theoretical sequence is shown in sequence 15 of the sequence listing. Rapeseed-specific primer E1 could not obtain this specific DNA from peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed genomic DNA. This primer has specificity (…). Figure 1 ).

[0090] Peanut-specific primer E2 was used to amplify genomic DNA samples from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed. Only the PCR system using peanut genomic DNA as a template could amplify the peanut-specific DNA, which is 100-200 bp in size. The theoretical sequence is shown in sequence 16 of the sequence listing. Peanut-specific primer E2 could not obtain this specific DNA from rapeseed, sunflower seed, oil palm, soybean, sesame, and cottonseed genomic DNA. This primer has specificity. Figure 2 ).

[0091] The sunflower seed-specific primer E3 was used to amplify genomic DNA samples from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed. Only the PCR system using sunflower seed genomic DNA as a template could amplify the sunflower seed-specific DNA, which was approximately 200 bp in size. The theoretical sequence is shown in sequence 17 of the sequence listing. The sunflower seed-specific primer E3 could not obtain this specific DNA from rapeseed, peanut, oil palm, soybean, sesame, and cottonseed genomic DNA, indicating that this primer has specificity. Figure 3 ).

[0092] The specific primer E4 for oil palm was used to amplify genomic DNA samples from rapeseed, peanut, sunflower seed, soybean, sesame, and cottonseed. Only the PCR system using oil palm genomic DNA as a template could amplify the specific oil palm DNA, which is 200-300 bp in size. The theoretical sequence is shown in sequence 18 of the sequence listing. The specific primer E4 for oil palm could not obtain this specific DNA from rapeseed, peanut, sunflower seed, soybean, sesame, and cottonseed genomic DNA, indicating that this primer has specificity. Figure 4 ).

[0093] Soybean-specific primer E5 was used to amplify genomic DNA samples from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed. Only the PCR system using soybean genomic DNA as a template could amplify soybean-specific DNA, with a size of 300-400 bp. The theoretical sequence is shown in sequence 19 of the sequence listing. Soybean-specific primer E5 could not obtain this specific DNA from rapeseed, peanut, sunflower seed, oil palm, sesame, and cottonseed genomic DNA, indicating that this primer has specificity. Figure 5 ).

[0094] The sesame-specific primer E6 was used to amplify genomic DNA samples from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed. Only the PCR system using sesame genomic DNA as a template could amplify the sesame-specific DNA, which is 100-200 bp in size. The theoretical sequence is shown in sequence 20 of the sequence listing. The sesame-specific primer E6 could not obtain this specific DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, and cottonseed genomic DNA, indicating that this primer has specificity. Figure 6 ).

[0095] The cottonseed-specific primer E7 was used to amplify genomic DNA samples from rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed. Only the PCR system using cottonseed genomic DNA as a template could amplify the cottonseed-specific DNA, which is 300-400 bp in size. The theoretical sequence is shown in sequence 21 of the sequence listing. The cottonseed-specific primer E7 could not obtain this specific DNA from rapeseed, peanut, sunflower seed, oil palm, soybean, and sesame genomic DNA. This primer has specificity. Figure 7 ).

[0096] The above results indicate that the seven primer pairs designed for the specific genes of seven plant-derived feed components—rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and cottonseed—have been verified to have strong specificity and can be used for the identification of plant-derived feed components.

[0097] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A reagent for identifying a plant-derived feed ingredient, characterized in that: The reagent contains a primer composition; the plant is rapeseed, peanut, sunflower seed, oil palm, soybean, sesame and / or cottonseed; the primer composition consists of seven primer pairs, the names of the seven primer pairs are E1 primer pair, E2 primer pair, E3 primer pair, E4 primer pair, E5 primer pair, E6 primer pair and E7 primer pair respectively; The E1 primer pair consists of primers that specifically amplify DNA molecules containing the nucleotide sequence of SEQ ID No. 15, the E2 primer pair consists of primers that specifically amplify DNA molecules containing the nucleotide sequence of SEQ ID No. 16, the E3 primer pair consists of primers that specifically amplify DNA molecules containing the nucleotide sequence of SEQ ID No. 17, the E4 primer pair consists of primers that specifically amplify DNA molecules containing the nucleotide sequence of SEQ ID No. 18, the E5 primer pair consists of primers that specifically amplify DNA molecules containing the nucleotide sequence of SEQ ID No. 19, the E6 primer pair consists of primers that specifically amplify DNA molecules containing the nucleotide sequence of SEQ ID No. 20, and the E7 primer pair consists of primers that specifically amplify DNA molecules containing the nucleotide sequence of SEQ ID No. 21; The E1 primer pair consists of two single-stranded DNAs represented by sequence 1 and sequence 2 in the sequence listing; the E2 primer pair consists of two single-stranded DNAs represented by sequence 3 and sequence 4 in the sequence listing; the E3 primer pair consists of two single-stranded DNAs represented by sequence 5 and sequence 6 in the sequence listing; the E4 primer pair consists of two single-stranded DNAs represented by sequence 7 and sequence 8 in the sequence listing; the E5 primer pair consists of two single-stranded DNAs represented by sequence 9 and sequence 10 in the sequence listing; the E6 primer pair consists of two single-stranded DNAs represented by sequence 11 and sequence 12 in the sequence listing; and the E7 primer pair consists of two single-stranded DNAs represented by sequence 13 and sequence 14 in the sequence listing.

2. Composition for the identification of a feed ingredient of plant origin, characterized in that: The composition is the primer composition of claim 1; the plant is rapeseed, peanut, sunflower seed, oil palm, soybean, sesame and / or cottonseed; the primer composition consists of seven primer pairs, the names of the seven primer pairs are E1 primer pair, E2 primer pair, E3 primer pair, E4 primer pair, E5 primer pair, E6 primer pair and E7 primer pair respectively; The E1 primer pair consists of primers that specifically amplify a DNA molecule containing the nucleotide sequence of SEQ ID No. 15, the E2 primer pair consists of primers that specifically amplify a DNA molecule containing the nucleotide sequence of SEQ ID No. 16, the E3 primer pair consists of primers that specifically amplify a DNA molecule containing the nucleotide sequence of SEQ ID No. 17, the E4 primer pair consists of primers that specifically amplify a DNA molecule containing the nucleotide sequence of SEQ ID No. 18, the E5 primer pair consists of primers that specifically amplify a DNA molecule containing the nucleotide sequence of SEQ ID No. 19, the E6 primer pair consists of primers that specifically amplify a DNA molecule containing the nucleotide sequence of SEQ ID No. 20, and the E7 primer pair consists of primers that specifically amplify a DNA molecule containing the nucleotide sequence of SEQ ID No.

21.

3. A method for preparing the reagent of claim 1 or the composition of claim 2, comprising the step of separately packaging the two single-stranded DNAs of each primer pair in the primer composition of claim 1.

4. Use of the reagent of claim 1 or the primer composition of claim 2 in identifying the source of a feed ingredient derived from a plant, the plant being rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and / or cottonseed.

5. Use of the reagent of claim 1 or the primer composition of claim 2 in the preparation of a product for identifying the source of a feed ingredient derived from a plant, the plant being rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and / or cottonseed.

6. A method for identifying the origin of a feed ingredient of plant origin, characterized in that: The method comprises the following steps: S1), extracting genomic DNA of a plant-derived feed ingredient to be tested; S2), using the genomic DNA obtained in S1) as a template, performing PCR amplification with the primer composition of claim 3 to obtain an amplified PCR product, and identifying whether the feed to be tested or the raw material of the feed to be tested contains the plant ingredient of claim 1 according to the sequence or size of the PCR product; The plant is rapeseed, peanut, sunflower seed, oil palm, soybean, sesame, and / or cottonseed.

7. The method of claim 6, wherein: Identifying whether the feed to be tested or the raw material of the feed to be tested contains the plant ingredient of claim 1 according to the sequence or size of the PCR product comprises at least one of the following: S2-1) the PCR product amplified by the E1 primer pair contains a DNA molecule with the nucleotide sequence of SEQ ID No. 15, the feed to be tested contains or is suspected to contain an ingredient derived from rapeseed, and the PCR product does not contain the DNA molecule, the feed to be tested does not contain or is suspected to not contain an ingredient derived from rapeseed; S2-2) the PCR product amplified by the E2 primer pair contains a DNA molecule with the nucleotide sequence of SEQ ID No. 16, the feed to be tested contains or is suspected to contain an ingredient derived from peanut, and the PCR product does not contain the DNA molecule, the feed to be tested does not contain or is suspected to not contain an ingredient derived from peanut; S2-3) the PCR product amplified by the E3 primer pair contains a DNA molecule having the nucleotide sequence shown in SEQ ID No. 17, the feed to be tested contains or is suspected of containing a component derived from sunflower seed, and the PCR product does not contain the DNA molecule, the feed to be tested does not contain or is suspected of not containing a component derived from sunflower seed; S2-4) the PCR product amplified by the E4 primer pair contains a DNA molecule having the nucleotide sequence shown in SEQ ID No. 18, the feed to be tested contains or is suspected of containing a component derived from oil palm, and the PCR product does not contain the DNA molecule, the feed to be tested does not contain or is suspected of not containing a component derived from oil palm; S2-5) the PCR product amplified by the E5 primer pair contains a DNA molecule having the nucleotide sequence shown in SEQ ID No. 19, the feed to be tested contains or is suspected of containing a component derived from soybean, and the PCR product does not contain the DNA molecule, the feed to be tested does not contain or is suspected of not containing a component derived from soybean; S2-6) the PCR product amplified by the E6 primer pair contains a DNA molecule having the nucleotide sequence shown in SEQ ID No. 20, the feed to be tested contains or is suspected of containing a component derived from sesame, and the PCR product does not contain the DNA molecule, the feed to be tested does not contain or is suspected of not containing a component derived from sesame; S2-7) the PCR product amplified by the E7 primer pair contains a DNA molecule having the nucleotide sequence shown in SEQ ID No. 21, the feed to be tested contains or is suspected of containing a component derived from cottonseed, and the PCR product does not contain the DNA molecule, the feed to be tested does not contain or is suspected of not containing a component derived from cottonseed.

8. The method of claim 7, wherein: identifying whether the feed to be tested or the raw material of the feed to be tested contains the plant component described in claim 1 according to the sequence or size of the PCR product includes at least one of the following: S3-1) the PCR product amplified by the E1 primer pair contains a 180 bp DNA fragment, the feed to be tested contains or is suspected of containing a component derived from rapeseed, and the PCR product does not contain the 180 bp DNA fragment, the feed to be tested does not contain or is suspected of not containing a component derived from rapeseed; S3-2) the PCR product amplified by the E2 primer pair contains a 139 bp DNA fragment, the feed to be tested contains or is suspected of containing a component derived from peanut, and the PCR product does not contain the 139 bp DNA fragment, the feed to be tested does not contain or is suspected of not containing a component derived from peanut; S3-3) the PCR product amplified by the E3 primer pair contains a 173 bp DNA fragment, the feed to be tested contains or is suspected of containing a component derived from sunflower seed, and the PCR product does not contain the 173 bp DNA fragment, the feed to be tested does not contain or is suspected of not containing a component derived from sunflower seed; S3-4) the PCR product amplified by the E4 primer pair contains a 249 bp DNA fragment, the feed to be tested contains or is suspected to contain ingredients derived from oil palm, and the PCR product does not contain a 249 bp DNA fragment, the feed to be tested does not contain or is suspected not to contain ingredients derived from oil palm; S3-5) the PCR product amplified by the E5 primer pair contains a 343 bp DNA fragment, the feed to be tested contains or is suspected to contain ingredients derived from soybean, and the PCR product does not contain a 343 bp DNA fragment, the feed to be tested does not contain or is suspected not to contain ingredients derived from soybean; S3-6) the PCR product amplified by the E6 primer pair contains a 200 bp DNA fragment, the feed to be tested contains or is suspected to contain ingredients derived from sesame, and the PCR product does not contain a 200 bp DNA fragment, the feed to be tested does not contain or is suspected not to contain ingredients derived from sesame; S3-7) the PCR product amplified by the E7 primer pair contains a 358 bp DNA fragment, the feed to be tested contains or is suspected to contain ingredients derived from cottonseed, and the PCR product does not contain a 358 bp DNA fragment, the feed to be tested does not contain or is suspected not to contain ingredients derived from cottonseed.

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