Primer composition for identifying or assisting in identifying plant energy raw material components and its application

Abstract

The present invention discloses a primer composition for identifying or assisting in identifying plant energy raw material components and its application. The primer composition for identifying or assisting in identifying plant energy raw material components provided by the present invention and its application are composed of six primer pairs in independent packaging, and the names of the six primer pairs are E1, E2, E3, E4, E5 and E6; the present invention designs 6 pairs of primers by specific genes of corn, rice, barley, cassava, potato and wheat in plant energy raw material components, and detects them by polymerase chain reaction (PCR) method. It has been verified that it has strong specificity and can be used for the identification of types of plant-derived energy raw materials in feed.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and in particular relates to a primer composition for identifying or assisting in identifying plant energy raw material components and an application thereof. Background Art

[0002] As the cheapest energy substance in aquatic feed, carbohydrates not only provide energy in the physiological activities of aquatic animals, but also serve as structural materials of organisms and information molecules for cell recognition; and the appropriate level of carbohydrate sources can also save protein and fat. Starch and other carbohydrate sources are not only one of the economical and efficient energy sources in aquatic feed, but also a good feed binder. Appropriate addition can improve the stability of aquatic feed in water, so they are widely used in fish feed. my country has a wide range of starch sources and rich sugar types, which have different prices due to different geographical divisions of major crops. At present, carbohydrate substances in aquatic feed in my country mainly come from crops with high starch content, such as rice, wheat, corn, cassava, potato, barley, sorghum and oats. Since different fish have different utilization rates of feed sugar sources, it is particularly important to add the correct type of sugar source feed. At present, the identification of plant-based feed is generally carried out by color, smell, microscopy and other methods, which requires the testers to have a certain knowledge reserve.

[0003] Genetic testing technology is based on relatively stable genetic material DNA for species identification. It can distinguish different plant species and requires a small sample size. It is accurate and sensitive. At present, there are reports on the use of genetic testing methods in the identification of animal-derived ingredients and microbial species, but there are very few reports on its application in the identification of plant feed ingredient sources. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to identify or assist in identifying the source of plant feed ingredients.

[0005] In order to solve the above problems, the present invention provides a primer composition for identifying or assisting in identifying the source of plant-based feed ingredients, which consists of six primer pairs, and the names of the six primer pairs are E1 primer pair, E2 primer pair, E3 primer pair, E4 primer pair, E5 primer pair and E6 primer pair.

[0006] The plants are corn (Zea mays), rice (Oryza sativa), barley (Hordeum vulgare), cassava (Manihot esculenta crantz), potato (Solanum tuberosum) and wheat (Triticum aestivum).

[0007] The six primer pairs can be packaged independently.

[0008] The primer pair named E1 is a primer pair for identifying or assisting in identifying corn, the primer pair named E2 is a PCR primer pair for identifying or assisting in identifying rice, the primer pair named E3 is a PCR primer pair for identifying or assisting in identifying barley, the primer pair named E4 is a PCR primer pair for identifying or assisting in identifying cassava, the primer pair named E5 is a PCR primer pair for identifying or assisting in identifying potato, and the primer pair named E6 is a PCR primer pair for identifying or assisting in identifying wheat;

[0009] The E1 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.13, the E2 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.14, the E3 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.15, the E4 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.16, the E5 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.17, and the E6 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.18.

[0010] In the above primer composition, the specific amplification is a polymerase chain reaction.

[0011] In the above primer composition, the E1 primer pair consists of two single-stranded DNAs shown in sequence 1 and sequence 2 in the sequence list; the E2 primer pair consists of two single-stranded DNAs shown in sequence 3 and sequence 4 in the sequence list; the E3 primer pair consists of two single-stranded DNAs shown in sequence 5 and sequence 6 in the sequence list; the E4 primer pair consists of two single-stranded DNAs shown in sequence 7 and sequence 8 in the sequence list; the E5 primer pair consists of two single-stranded DNAs shown in sequence 9 and sequence 10 in the sequence list; and the E6 primer pair consists of two single-stranded DNAs shown in sequence 11 and sequence 12 in the sequence list.

[0012] The invention also provides a reagent or a kit for identifying or assisting in identifying the source of plant feed ingredients.

[0013] The present invention provides a reagent or a kit for identifying or assisting in identifying the source of a plant feed ingredient, wherein the plant feed ingredient is corn, rice, barley, cassava, potato and / or wheat.

[0014] The use of the above primer combination, reagent or kit in identifying or assisting in identifying the source of plant feed ingredients also falls within the protection scope of the present invention.

[0015] The plant is corn, rice, barley, cassava, potato and / or wheat.

[0016] The present invention also provides the use of the primer combination, reagent or kit in the preparation of products for identifying or assisting in identifying the source of plant feed ingredients.

[0017] In the above application, the plant is corn, rice, barley, cassava, potato and / or wheat.

[0018] The present invention also provides a method for identifying or assisting in identifying the source of a plant feed ingredient.

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

[0020] S1), extracting genomic DNA from the feed to be tested or the raw material of the feed to be tested;

[0021] S2), using the genomic DNA obtained in S1) as a template, using the above primer combination to perform PCR amplification to obtain amplified PCR products, and identifying whether the feed to be tested or the raw material of the feed to be tested contains plant components based on the sequence or size of the PCR products, wherein the plant is corn, rice, barley, cassava, potato and / or wheat.

[0022] In the above method, identifying whether the feed to be tested or the raw material of the feed to be tested contains plant components according to the sequence or size of the PCR product includes at least one of the following:

[0023] S2-1) the PCR product obtained by amplification by the E1 primer pair contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 13, and the feed to be tested contains or may contain a component derived from corn, and the PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from corn;

[0024] S2-2) the PCR product obtained by amplification by the E2 primer pair contains a DNA molecule having a nucleotide sequence as shown in SEQ ID No. 14, and the feed to be tested contains or may contain a component derived from rice, and the PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from rice;

[0025] S2-3) the PCR product obtained by amplification by the E3 primer pair contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 15, and the feed to be tested contains or may contain a component derived from barley, and the PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from barley;

[0026] S2-4) the PCR product obtained by amplification by the E4 primer pair contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 16, and the feed to be tested contains or may contain a component derived from cassava, and the PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from cassava;

[0027] S2-5) the PCR product obtained by amplifying the E5 primer pair contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 17, and the feed to be tested contains or may contain a component derived from potato; the PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from potato;

[0028] S2-6) The PCR product obtained by amplifying the E6 primer pair contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No.18, and the feed to be tested contains or may contain a component derived from wheat. The PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from wheat.

[0029] In the above method, identifying whether the feed to be tested or the raw material of the feed to be tested contains plant components according to the sequence or size of the PCR product includes at least one of the following:

[0030] S3-1) The PCR product obtained by amplification by the E1 primer pair contains a 163 bp DNA fragment, and the feed to be tested contains or may contain a component derived from corn; the PCR product does not contain a 163 bp DNA fragment, and the feed to be tested does not contain or may not contain a component derived from corn;

[0031] S3-2) the PCR product obtained by amplification by the E2 primer pair contains a 239 bp DNA fragment, and the feed to be tested contains or may contain a component derived from rice; the PCR product does not contain a 239 bp DNA fragment, and the feed to be tested does not contain or may not contain a component derived from rice;

[0032] S3-3) the PCR product obtained by amplification by the E3 primer pair contains a DNA fragment of 178 bp, and the feed to be tested contains or is likely to contain a component derived from barley; the PCR product does not contain a DNA fragment of 178 bp, and the feed to be tested does not contain or is likely to not contain a component derived from barley;

[0033] S3-4) the PCR product obtained by amplification by the E4 primer pair contains a 287 bp DNA fragment, and the feed to be tested contains or is likely to contain a component derived from cassava; the PCR product does not contain a 287 bp DNA fragment, and the feed to be tested does not contain or is likely to not contain a component derived from cassava;

[0034] S3-5) the PCR product obtained by amplification with the E5 primer pair contains a 503 bp DNA fragment, and the feed to be tested contains or may contain a component derived from potato; the PCR product does not contain a 503 bp DNA fragment, and the feed to be tested does not contain or may not contain a component derived from potato;

[0035] S3-6) The PCR product obtained by amplifying the E6 primer pair contains a DNA fragment of 812 bp, and the feed to be tested contains or may contain a component derived from wheat. The PCR product does not contain a DNA fragment of 812 bp, and the feed to be tested does not contain or may not contain a component derived from wheat.

[0036] The present invention designs 6 pairs of primers for the specific genes of 6 plants, namely corn, rice, barley, cassava, potato and wheat, in plant-based feed raw materials. Through polymerase chain reaction (PCR) detection, all six primer pairs have strong specificity and can be used for type identification of plant-based feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the agarose gel electrophoresis diagram of the PCR product of the corn primer E1 specific verification in the present invention. Using corn, rice, barley, cassava, potato, and wheat genomic DNA as templates, PCR amplification was performed using corn-specific primers. Among all samples, only the corn was amplified with bands, and no bands were found in non-corn components.

[0038] Figure 2 This is the agarose gel electrophoresis diagram of the rice primer specific verification PCR product in the present invention. Using corn, rice, barley, cassava, potato, and wheat genomic DNA as templates, PCR amplification was performed using rice specific primers. Among all samples, only rice was amplified with bands, and no bands were found in non-rice components.

[0039] Figure 3 This is the agarose gel electrophoresis diagram of the PCR product of the barley primer specificity verification in the present invention. Using corn, rice, barley, cassava, potato, and wheat genomic DNA as templates, PCR amplification was performed using barley specific primers. Among all samples, only the barley was amplified with bands, and no bands were found in non-barley components.

[0040] Figure 4 This is the agarose gel electrophoresis diagram of the PCR product of the cassava primer specificity verification in the present invention. Using corn, rice, barley, cassava, potato, and wheat genomic DNA as templates, cassava-specific primers were used for PCR amplification. Among all samples, only cassava was amplified with bands, and no bands were found in non-cassava components.

[0041] Figure 5This is the agarose gel electrophoresis diagram of the potato primer specific verification PCR product in the present invention. Using corn, rice, barley, cassava, potato, and wheat genomic DNA as templates, PCR amplification was performed using potato specific primers. Among all samples, only the potato was amplified with bands, and no bands were found in non-potato components.

[0042] Figure 6 This is the agarose gel electrophoresis diagram of the wheat primer specific verification PCR product in the present invention. Using corn, rice, barley, cassava, potato, and wheat genomic DNA as templates, PCR amplification was performed using wheat specific primers. Among all samples, only wheat was amplified with bands, and no bands were found in non-wheat components. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0044] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0045] In the following examples, all experimental results were repeated three times unless otherwise specified.

[0046] Example 1: Primer specificity detection

[0047] 1. Sample collection and storage

[0048] 1.1 Sampling

[0049] Corn seeds, rice seeds, barley seeds, cassava rhizomes, potato rhizomes, and wheat seed samples were dried at 60°C, ground into powder, and stored at room temperature.

[0050] 1.2 DNA template preparation

[0051] Take an appropriate amount of 30±2 mg of the powdered sample in 1.1 and place it in a 1.5 mL centrifuge tube using the Polysaccharide and Polyphenol Plant Genomic DNA Extraction Kit. To extract genomic DNA, the specific steps are as follows:

[0052] (1) Quickly add 500 μL of GPS buffer and 10 μL of RNase A (10 mg / mL) to a centrifuge tube, vortex to mix, and incubate at 65°C in a water bath for 15 min. Invert the tube several times to mix the sample during the incubation period.

[0053] (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.

[0054] (3) Add an equal volume of anhydrous ethanol and mix thoroughly.

[0055] (4) Transfer the solution and flocculent precipitate obtained in the previous step to RNase-Free adsorption column CR2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the RNase-Free adsorption column CR2 in a collection tube.

[0056] (5) Add 550 μL of deproteinized solution RD to the RNase-Free adsorption column CR2, centrifuge at 12,000 rpm for 1 min, discard the waste liquid, and place the RNase-Free adsorption column CR2 in a collection tube.

[0057] (6) Add 700 μL of rinse solution PW to the RNase-Free adsorption column CR2, centrifuge at 12,000 rpm (13,400 × g) for 1 min, discard the waste liquid, and place the RNase-Free adsorption column CR2 in a collection tube. Repeat step (6).

[0058] (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 dry it at room temperature for 5-10 min.

[0059] (8) Add 100 μL ddH2O to the RNase-Free adsorption column CR2, place at room temperature for 3-5 min, centrifuge at 12,000 rpm for 2 min, collect the solution into a centrifuge tube, and obtain rice, corn, barley, cassava, potato, and wheat genomic DNA, respectively.

[0060] 1.3 Concentration detection

[0061] DNA concentration and purity detection: Take 2.5 μL of DNA sample for concentration detection.

[0062] Table 1 Sample DNA concentration detection

[0063]

[0064]

[0065] 2. PCR detection

[0066] 2.1 Preparation of PCR system

[0067] Table 2 PCR reaction system

[0068]

[0069] The maize-specific primer E1 shown in 1-F and 1-R in Table 3 was used as the primer F and primer R in Table 2 to prepare 6 PCR reaction systems of maize-specific primer E1. The 6 PCR systems of maize-specific primer E1 had the same components except for the different templates. The templates were the maize, rice, barley, cassava, potato, and wheat genomic DNA obtained in step 1. Maize-maize specific primer E1 system ( Figure 1 The template is corn genomic DNA), rice-corn specific primer E1 system ( Figure 1 The template is rice genomic DNA), barley-corn specific primer E1 system ( Figure 1 Barley is referred to as barley in Chinese, and the template is barley genomic DNA), cassava-corn specific primer E1 system ( Figure 1 Cassava is referred to as cassava in Chinese, and the template is cassava genomic DNA), potato-corn specific primer E1 system ( Figure 1 The template is potato genomic DNA), wheat-corn specific primer E1 system ( Figure 1 Wheat is referred to as wheat in Chinese, and the template is wheat genomic DNA).

[0070] The rice-specific primer E2 shown in 2-F and 2-R in Table 3 was used as the primer F and primer R in Table 2 to prepare 6 PCR reaction systems of rice-specific primer E2. The 6 PCR systems of rice-specific primer E2 had the same components except for the different templates. The templates were the genomic DNA of corn, rice, barley, cassava, potato, and wheat obtained in step 1. Corn-rice specific primer E2 system ( Figure 2 The template is corn genomic DNA), rice-rice specific primer E2 system ( Figure 2 The template is rice genomic DNA), barley-rice specific primer E2 system ( Figure 2 Barley is referred to as barley in Chinese, and the template is barley genomic DNA), cassava-rice specific primer E1 system ( Figure 2 Cassava is referred to as cassava in Chinese, and the template is cassava genomic DNA), potato-rice specific primer E2 system ( Figure 2 The template is potato genomic DNA), wheat-rice specific primer E2 system ( Figure 2 Wheat is referred to as wheat in Chinese, and the template is wheat genomic DNA).

[0071] The barley-specific primer E3 shown in 3-F and 3-R in Table 3 was used as the primer F and primer R in Table 2 to prepare 6 PCR reaction systems of the barley-specific primer E3. The 6 PCR systems of the barley-specific primer E3 had the same components except for the different templates. The templates were the genomic DNA of corn, rice, barley, cassava, potato, and wheat obtained in step 1. The corn-barley specific primer E3 system ( Figure 3 The template is corn genomic DNA), rice-barley specific primer E3 system ( Figure 3 The template is rice genomic DNA), barley-barley specific primer E3 system ( Figure 3 Barley is referred to as barley in Chinese, and the template is barley genomic DNA), cassava-barley specific primer E3 system ( Figure 3 Cassava is referred to as cassava in Chinese, and the template is cassava genomic DNA), potato-barley specific primer E3 system ( Figure 3 The template is potato genomic DNA), wheat-barley specific primer E3 system ( Figure 3 Wheat is referred to as wheat in Chinese, and the template is wheat genomic DNA).

[0072] The cassava-specific primer E4 shown in 4-F and 4-R in Table 3 was used as the primer F and primer R in Table 2 to prepare 6 PCR reaction systems of cassava-specific primer E4. The 6 PCR systems of cassava-specific primer E4 had the same components except for the different templates. The templates were the genomic DNA of corn, rice, barley, cassava, potato, and wheat obtained in step 1. The corn-cassava specific primer E4 system ( Figure 4 The template is corn genomic DNA), rice-cassava specific primer E4 system ( Figure 4 Rice is referred to as rice in Chinese, and the template is rice genomic DNA), barley-cassava specific primer E4 system ( Figure 4 Barley is referred to as barley in Chinese, and the template is barley genomic DNA), cassava-cassava specific primer E4 system ( Figure 4 Cassava is referred to as cassava in Chinese, and the template is cassava genomic DNA), potato-cassava specific primer E4 system ( Figure 4 The template is potato genomic DNA), wheat-cassava specific primer E4 system ( Figure 4 Wheat is referred to as wheat in Chinese, and the template is wheat genomic DNA).

[0073] The potato-specific primer E5 shown in 5-F and 5-R in Table 3 was used as the primer F and primer R in Table 2 to prepare 6 PCR reaction systems of potato-specific primer E5. The 6 PCR systems of potato-specific primer E5 had the same components except for the different templates. The templates were the genomic DNA of corn, rice, barley, cassava, potato, and wheat obtained in step 1. The corn-potato specific primer E5 system ( Figure 5 The template is corn genomic DNA), rice-potato specific primer E4 system ( Figure 5 The template is rice genomic DNA), barley-potato specific primer E5 system ( Figure 5 Barley is referred to as barley in Chinese, and the template is barley genomic DNA), cassava-potato specific primer E5 system ( Figure 5 Cassava is referred to as cassava in Chinese, and the template is cassava genomic DNA) Potato-potato specific primer E5 system ( Figure 5 The specific primer E5 system of wheat-potato (referred to as potato in Chinese, and the template is potato genomic DNA) Figure 5 Wheat is referred to as wheat in Chinese, and the template is wheat genomic DNA).

[0074] The wheat-specific primer E6 shown in 6-F and 6-R in Table 3 was used as the primer F and primer R in Table 2 to prepare 6 PCR reaction systems of wheat-specific primer E6. The 6 PCR systems of wheat-specific primer E6 had the same components except for the different templates. The templates were the genomic DNA of corn, rice, barley, cassava, potato, and wheat obtained in step 1. Corn-wheat specific primer E6 system ( Figure 6 The template is corn genomic DNA), rice-wheat specific primer E6 system ( Figure 6 The template is rice genomic DNA), barley-wheat specific primer E6 system ( Figure 6 Barley is referred to as barley in Chinese, and the template is barley genomic DNA), cassava-wheat specific primer E6 system ( Figure 6 The specific primer E6 system of potato-wheat ( Figure 6 The specific primer E6 system of wheat-wheat (referred to as potato in Chinese, and the template is potato genomic DNA) Figure 6 Wheat is referred to as wheat in Chinese, and the template is wheat genomic DNA).

[0075] The above PCR systems were all based on ddH 2 O is the negative control.

[0076] Table 3 Primers for identifying the sources of plant-derived energy feed ingredients

[0077]

[0078] 2.2 PCR reaction

[0079] Reaction procedure: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 sec, annealing at 60°C for 30 sec, extension at 72°C for 1 min, 30 cycles; final extension at 72°C for 10 min, cooling at 4°C for 10 min.

[0080] 3. PCR amplification product detection and result determination

[0081] 3.1 Electrophoresis detection of PCR products

[0082] 3.1 Electrophoresis detection of PCR products

[0083] Weigh 1g agarose, add 100mL 1×TAE buffer, heat and boil for 30s, mix well, wait until slightly cool, add 10μL 10000×Gene Green nucleic acid dye, invert the gel plate, let it stand for 30min, and after solidification, place it in an electrophoresis tank containing 1×TAE for spotting. Add 5μL of PCR amplification product in 2.2 to each gel well, set the voltage to 110V, and perform electrophoresis for 30-40min. After the electrophoresis is completed, take out the gel block and image it using a gel imaging system.

[0084] 3.2 Result determination

[0085] The results are as follows Figure 1-6 As shown, Figures 1 to 6 The M in the middle represents Marker, and from bottom to top they are 100bp, 200bp, 300bp, 400bp, 500bp, 700bp, 1000bp, 1600bp, 2000bp, 5000bp, and 8000bp respectively.

[0086] The PCR amplification of rice, corn, barley, cassava, potato and wheat genomic DNA samples was performed using corn specific primer E1. Only the PCR system with corn genomic DNA as the template (corn-corn specific primer E1 system) can amplify corn specific DNA with a size of 100-200 bp. The theoretical sequence is shown in sequence 13 in the sequence table. Corn specific primer E1 cannot obtain the specific DNA from rice, barley, cassava, potato and wheat genomic DNA. The primer has specificity ( Figure 1 ).

[0087] The rice specific primer E2 was used to perform PCR amplification on rice, corn, barley, cassava, potato and wheat genomic DNA samples respectively. The PCR system with only rice genomic DNA as a template (rice-rice specific primer E2 system) can amplify rice specific DNA with a size of 200-300bp. The theoretical sequence is shown in sequence 14 in the sequence table. The rice specific primer E2 cannot obtain the specific DNA from corn, barley, cassava, potato and wheat genomic DNA. The use of this primer has specificity ( Figure 2 ).

[0088] The barley specific primer E3 was used to perform PCR amplification on rice, corn, barley, cassava, potato and wheat genomic DNA samples respectively. Only the PCR system with rice genomic DNA as the template (barley-barley specific primer E3 system) can amplify the barley specific DNA with a size of about 100-200 bp. The theoretical sequence is shown in sequence 15 in the sequence table. The barley specific primer E3 cannot obtain the specific DNA from the rice, corn, cassava, potato and wheat genomic DNA. The primer has specificity ( Figure 3 ).

[0089] Cassava specific primer E4 was used to perform PCR amplification on rice, corn, barley, cassava, potato and wheat genomic DNA samples respectively. Only the PCR system with cassava genomic DNA as template (cassava-cassava specific primer E4 system) can amplify cassava specific DNA with a size of 200-300 bp. The theoretical sequence is shown in sequence 16 in the sequence table. Cassava specific primer E4 cannot obtain the specific DNA from rice, corn, barley, potato and wheat genomic DNA. The primer has specificity ( Figure 4 ).

[0090] The potato specific primer E5 was used to perform PCR amplification on rice, corn, barley, cassava, potato and wheat genomic DNA samples. Only the PCR system with potato genomic DNA as the template (potato-potato specific primer E5 system) could amplify the barley specific DNA with a size of about 500 bp. The theoretical sequence is shown in Sequence 17 in the sequence table. The potato specific primer E5 could not obtain the specific DNA from the rice, corn, barley, cassava and wheat genomic DNA. The primer has specificity ( Figure 5 ).

[0091] The wheat specific primer E6 was used to perform PCR amplification on rice, corn, barley, cassava, potato and wheat genomic DNA samples respectively. Only the PCR system with potato genomic DNA as the template (wheat-wheat specific primer E6 system) could amplify the barley specific DNA with a size of 700-1000 bp. The theoretical sequence is shown in sequence 18 in the sequence table. The wheat specific primer E5 could not obtain the specific DNA from the rice, corn, barley, cassava and potato genomic DNA. The primer has specificity ( Figure 6 ).

[0092] The above results show that the six pairs of primers designed for the specific genes of six plants, rice, corn, barley, cassava, potato and wheat, which are the ingredients of plant-based energy feed, amplified specific bands through PCR, indicating that the six pairs of primers developed have strong specificity and can be used to identify the types of plant-based feed.

[0093] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A primer composition for identifying the source of a plant-derived feed ingredient, comprising six primer pairs, the six primer pairs being named E1 primer pair, E2 primer pair, E3 primer pair, E4 primer pair, E5 primer pair and E6 primer pair; the plant being corn, rice, barley, cassava, potato and / or wheat; The primer pair named E1 is a primer pair for identifying corn, the primer pair named E2 is a PCR primer pair for identifying rice, the primer pair named E3 is a PCR primer pair for identifying barley, the primer pair named E4 is a PCR primer pair for identifying cassava, the primer pair named E5 is a PCR primer pair for identifying potato, and the primer pair named E6 is a PCR primer pair for identifying wheat; The E1 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.13, the E2 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.14, the E3 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.15, the E4 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.16, the E5 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.17, and the E6 primer pair consists of primers for specifically amplifying a DNA molecule containing a nucleotide sequence of SEQ ID No.

18.

2. The primer composition according to claim 1, characterized in that: The specific amplification is a polymerase chain reaction.

3. The primer composition according to claim 2, characterized in that: The E1 primer pair consists of two single-stranded DNAs shown in sequence 1 and sequence 2 in the sequence list; the E2 primer pair consists of two single-stranded DNAs shown in sequence 3 and sequence 4 in the sequence list; the E3 primer pair consists of two single-stranded DNAs shown in sequence 5 and sequence 6 in the sequence list; the E4 primer pair consists of two single-stranded DNAs shown in sequence 7 and sequence 8 in the sequence list; the E5 primer pair consists of two single-stranded DNAs shown in sequence 9 and sequence 10 in the sequence list; and the E6 primer pair consists of two single-stranded DNAs shown in sequence 11 and sequence 12 in the sequence list.

4. A reagent or kit for identifying the source of a plant feed ingredient, characterized in that: The reagent or kit contains the PCR primer pair composition according to any one of claims 1 to 3; the plant is corn, rice, barley, cassava, potato and / or wheat.

5. Use of the primer composition according to any one of claims 1 to 3 or the reagent or kit according to claim 4 in identifying the source of plant feed ingredients, wherein the plant is corn, rice, barley, cassava, potato and / or wheat.

6. Use of the primer composition according to any one of claims 1 to 3 or the reagent or kit according to claim 4 in the preparation of a product for identifying the source of a plant feed ingredient, wherein the plant is corn, rice, barley, cassava, potato and / or wheat.

7. A method for identifying the source of a plant feed ingredient, characterized in that: The method comprises the following steps: S1) extracting genomic DNA from the feed to be tested or the raw material of the feed to be tested; S2) using the genomic DNA obtained in S1) as a template, performing PCR amplification using the primer combination described in any one of claims 1 to 3 to obtain amplified PCR products, and identifying whether the feed to be tested or the raw material of the feed to be tested contains plant components based on the sequence or size of the PCR products, wherein the plant is corn, rice, barley, cassava, potato and / or wheat.

8. The method according to claim 7, characterized in that: Identifying whether the feed to be tested or the raw material of the feed to be tested contains plant components according to the sequence or size of the PCR product includes at least one of the following: S2-1) the PCR product obtained by amplification by the E1 primer pair contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 13, and the feed to be tested contains or may contain a component derived from corn, and the PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from corn; S2-2) the PCR product obtained by amplification by the E2 primer pair contains a DNA molecule having a nucleotide sequence as shown in SEQ ID No. 14, and the feed to be tested contains or may contain a component derived from rice, and the PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from rice; S2-3) the PCR product obtained by amplification by the E3 primer pair contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 15, and the feed to be tested contains or may contain a component derived from barley, and the PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from barley; S2-4) the PCR product obtained by amplification by the E4 primer pair contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 16, and the feed to be tested contains or may contain a component derived from cassava, and the PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from cassava; S2-5) the PCR product obtained by amplifying the E5 primer pair contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 17, and the feed to be tested contains or may contain a component derived from potato; the PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from potato; S2-6) The PCR product obtained by amplifying the E6 primer pair contains a DNA molecule with a nucleotide sequence as shown in SEQ ID No.18, and the feed to be tested contains or may contain a component derived from wheat. The PCR product does not contain the DNA molecule, and the feed to be tested does not contain or may not contain a component derived from wheat.

9. The method according to claim 7, characterized in that: Identifying whether the feed to be tested or the raw material of the feed to be tested contains plant components according to the sequence or size of the PCR product includes at least one of the following: S3-1) The PCR product obtained by amplification by the E1 primer pair contains a 163 bp DNA fragment, and the feed to be tested contains or may contain a component derived from corn; the PCR product does not contain a 163 bp DNA fragment, and the feed to be tested does not contain or may not contain a component derived from corn; S3-2) the PCR product obtained by amplification by the E2 primer pair contains a 239 bp DNA fragment, and the feed to be tested contains or may contain a component derived from rice; the PCR product does not contain a 239 bp DNA fragment, and the feed to be tested does not contain or may not contain a component derived from rice; S3-3) the PCR product obtained by amplification by the E3 primer pair contains a DNA fragment of 178 bp, and the feed to be tested contains or is likely to contain a component derived from barley; the PCR product does not contain a DNA fragment of 178 bp, and the feed to be tested does not contain or is likely to not contain a component derived from barley; S3-4) the PCR product obtained by amplification by the E4 primer pair contains a 287 bp DNA fragment, and the feed to be tested contains or is likely to contain a component derived from cassava; the PCR product does not contain a 287 bp DNA fragment, and the feed to be tested does not contain or is likely to not contain a component derived from cassava; S3-5) the PCR product obtained by amplification with the E5 primer pair contains a 503 bp DNA fragment, and the feed to be tested contains or may contain a component derived from potato; the PCR product does not contain a 503 bp DNA fragment, and the feed to be tested does not contain or may not contain a component derived from potato; S3-6) The PCR product obtained by amplifying the E6 primer pair contains a DNA fragment of 812 bp, and the feed to be tested contains or may contain a component derived from wheat. The PCR product does not contain a DNA fragment of 812 bp, and the feed to be tested does not contain or may not contain a component derived from wheat.

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