A kit and method for detecting animal meat-derived components based on RPA-CRISPR-Cas12a

Abstract

The application belongs to the technical field of meat detection, and particularly relates to a kit and a detection method for detecting animal meat-derived components based on RPA-CRISPR-Cas12a, and provides a primer pair suitable for isothermal amplification and a CRISPR / Cas12a nucleic acid detection system which can specifically recognize a gene conservative sequence of a common meat-derived component. The application combines RPA and CRISPR technologies, screens and optimizes RPA primers and crRNA sequences for detection, and combines a nucleic acid detection test strip and naked-eye fluorescence color development, so that the meat-derived components in the sample can be rapidly identified in a field test process without special professional equipment, and the application is suitable for field rapid detection at the grassroots level.

Description

Technical Field

[0001] This invention belongs to the field of meat product testing technology, specifically relating to a kit and detection method for detecting animal meat-derived components based on RPA-CRISPR-Cas12a. Background Technology

[0002] Meat is widely consumed worldwide due to its rich content of essential nutrients such as protein, fat, carbohydrates, vitamins, and minerals. However, the complexity of the meat processing chain makes meat products vulnerable to various forms of fraud. Meat adulteration—the substitution, addition, or alteration of high-quality meat with inferior products—is one of the most common forms of food fraud, yet it is difficult to detect and has become a widespread problem globally. For example, cheap horse meat and duck meat are passed off as beef; the illegal inter-provincial transportation of cat and dog meat is prevalent; and more worryingly, undeclared wild animal ingredients (such as bat meat, fox meat, monkey meat, cat meat, rat meat, and dog meat) are introduced into meat products. These practices not only violate ethical standards but also pose serious health risks.

[0003] While polymerase chain reaction (PCR), real-time fluorescence PCR, and Sanger sequencing provide methods for identifying animal-derived components, these methods are technically complex and unsuitable for on-site testing. They require sophisticated equipment, are time-consuming, and demand highly skilled professionals, thus limiting the simultaneous detection of multiple species. Furthermore, loop-mediated isothermal amplification (LAMP) is widely used, and its application in detecting multiple animal meat sources has been reported. For example, a primer set and kit for simultaneously detecting pork and duck meat components (patent application number: 201610880177.4) have been developed. However, LAMP primer pairs are complex, containing six gene primers, making them unsuitable for efficient multi-species identification. Therefore, there is an urgent need to develop accurate and rapid analytical techniques to identify animal species in meat products, ensuring the safety and integrity of the food supply. Summary of the Invention

[0004] The purpose of this invention is to provide a kit and method for detecting animal meat-derived components based on RPA-CRISPR-Cas12a. The kit includes universal amplification primer pairs suitable for isothermal amplification, and CRISPR-Cas12a-specific crRNA primer sets for nine meat-derived components. Combined with nucleic acid detection strips and visual fluorescence detection, it can rapidly identify these nine meat-derived components with high sensitivity and visually readable results, achieving multiplex, rapid, sensitive, and specific effects. It is highly suitable for on-site detection, thus achieving the objective of this invention.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] First, the present invention provides a composition for the detection of RPA-CRISPR / Cas12a of animal meat-derived components, characterized in that it comprises an RPA primer pair and crRNA;

[0007] The RPA primer pair sequences are shown in SEQ ID NO.1 and SEQ ID NO.2;

[0008] The crRNA is a CRISPR-Cas12a primer system constructed based on a specific sequence in the middle of animal mitochondria.

[0009] Preferably, the animal is one or more of chicken, duck, goose, cow, pig, horse, rabbit, cat, and dog, and the crRNA primer sequences of the chicken, duck, goose, cow, pig, horse, rabbit, cat, and dog are shown in SEQ ID NO.3-11 in sequence.

[0010] Secondly, the present invention also provides an RPA-CRISPR / Cas12a detection kit for detecting meat-derived components, characterized in that it comprises the composition described above.

[0011] Preferably, it also includes the enzymes and buffer required for the RPA-CRISPR / Cas12a reaction.

[0012] The application of the RPA-CRISPR / Cas12a detection kit described above in the detection of meat-derived genes.

[0013] Finally, this invention provides a method for detecting multiple meat-derived components, including the RPA-CRISPR / Cas12a detection kit described above, the method comprising the following steps:

[0014] S1: Extract DNA from the sample to be tested;

[0015] S2: Using the DNA extracted in step S1 as a template, the genome of the test sample is amplified isothermally using the primers shown in SEQ ID NO.1 and SEQ ID NO.2;

[0016] S3: Add the amplified sequence to the CRISPR-cas12a reaction system and use crRNA species-specific primers to detect meat-derived components;

[0017] S4: Analyze the test results.

[0018] Preferably, the RPA detection system in step S2 is based on a 20 μL volume and consists of: 10 μL of 2X Reaction Buffer, 1 μL each of 20 μM upper and lower primers, 2.5 μL of 10X Starter, 2 μL of DNA template, and ultrapure water to a final volume of 20 μL. The RPA amplification reaction conditions are: constant temperature 39℃ for 18-25 min, followed by termination of the reaction.

[0019] Preferably, the CRISPR-Cas12a test strip detection system described in step S3 is based on a 10 μL standard and consists of: 10X Cleavage Buffer 1 μL, 2 μM crRNA 3.5 μL, 10 μM Cas12a Protein 0.35 μL, 4 μM Reporter 0.2 μL, RPA amplification product 4 μL, and ultrapure water to a final volume of 10 μL. The reaction conditions for the CRISPR-Cas12a system are a constant temperature of 45℃ for 10-30 min, followed by termination of the reaction.

[0020] The CRISPR-Cas12a visual fluorescence detection system uses 10 μL as the detection standard. The components are: 2.11 μL of 10X NEBuffer, 3.5 μL of 1 μM crRNA, 0.35 μL of 10 μM Cas12a Protein, 1 μL of 10 μM Reporter, 3 μL of RPA amplification product, and ultrapure water to a final volume of 10 μL.

[0021] The CRISPR-Cas12a detection system also includes the RPA reaction product from step S1 and the CRISPR / Cas12a protein. The reporter sequence is TTATTT, with the 5' end of the sequence labeled with fluorescein FAM and the 3' end labeled with biotin.

[0022] Preferably, the method of analysis and detection described in step S4 is as follows: a: interpret the results using colloidal gold nucleic acid test strips. If the control C line has a band but the test T line has no band, then the target gene is present; if both the test T line and the control C line have red bands, then the target gene is not present; b: read the results using ultraviolet fluorescence. Positive samples show color under ultraviolet light, while negative samples are colorless.

[0023] Preferably, the colloidal gold nucleic acid test strip comprises: a sample pad, a binding pad containing a complex of colloidal gold nanoparticles, Au-NP-fluorescein, FAM-labeled antibody, a T-line coated with streptavidin for capturing biotin, a C-line coated with goat anti-mouse IgG for capturing colloidal gold nanoparticles, and an absorbent pad for attracting liquid through the test strip.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) The detection primers provided by this invention for the one-time detection of multiple animal meat-derived components are beneficial for constructing universal primers with the conserved middle specific sequences at both ends of the mitochondria of common poultry and livestock, which facilitates rapid isothermal amplification of multiple species.

[0026] (2) The selected sequence fragments are short, around 230bp, which are suitable for fresh, processed, and slightly degraded meat-derived components and are more applicable to real-world situations.

[0027] (3) RPA isothermal amplification reaction is fast, primer design is simple, reaction temperature is low, and it is more suitable for field use.

[0028] (4) Construct CRISPR-Cas12a primers crRNA based on the mitochondrial intermediate specific sequence to specifically recognize chicken, duck, goose, cow, pig, horse, rabbit, cat and dog-derived components.

[0029] (5) This invention combines the advantages of RPA isothermal amplification and CRISPR-Cas12a system to construct a method for rapid on-site detection of animal-derived components, exploring new avenues for the detection of other animal-derived components. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 Schematic diagram of RPA reaction and CRISPR-Cas12a binding nucleic acid test strip detection and visual fluorescence reaction (drawn using biorender.com).

[0032] Figure 2 Gel electrophoresis images of 11 animal species amplified using universal RPA primers. Lanes 1: Marker (StarMarker D750), 2: Water, 3: Chicken, 4: Duck, 5: Goose, 6: Cow, 7: Pig, 8: Sheep, 9: Horse, 10: Rabbit, 11: Donkey, 12: Cat, 13: Dog.

[0033] Figure 3From top to bottom, the images show the results of visual fluorescence and test strip readings for specific components from chicken, duck, goose, cow, pig, horse, rabbit, cat, and dog. Each image, from left to right, represents: water, chicken, duck, goose, cow, pig, sheep, horse, rabbit, donkey, cat, and dog. A negative sample has red bands on both the test line (T) and the control line (C), while a positive sample has red bands only on the control line (C). A colorless result under UV light indicates a negative sample, while a bright result indicates a positive sample.

[0034] Figure 4 From top to bottom: sensitivity testing of chicken, duck, goose, cow, pig, horse, rabbit, cat, and dog-derived components; from left to right: water, 10... 0 -10 6 Plasmid standards with concentration gradients of copies / μL. Detailed Implementation

[0035] This invention provides a kit and detection method for detecting common meat-derived components based on RPA-CRISPR-Cas12a.

[0036] First, the present invention provides a composition for the detection of RPA-CRISPR / Cas12a of animal meat-derived components, characterized in that it comprises an RPA primer pair and crRNA;

[0037] The RPA primer pair sequences are shown in SEQ ID NO.1 and SEQ ID NO.2;

[0038] The crRNA is a CRISPR-Cas12a primer system constructed based on a specific sequence in the middle of animal mitochondria.

[0039] Preferably, the animal is one or more of chicken, duck, goose, cow, pig, horse, rabbit, cat, and dog, and the crRNA primer sequences of the chicken, duck, goose, cow, pig, horse, rabbit, cat, and dog are shown in SEQ ID NO.3-11 in sequence. Preferably, there are two or more animal species.

[0040] Preferably, the animals are chickens, ducks, geese, cattle, pigs, horses, rabbits, cats, and dogs. Among them, the chickens (Gallus Gallus), ducks (Anas platyrhynchos), geese (Anser cygnoides), cattle (Bos taurus), pigs (Sus scrofa), sheep (Ovis aries), horses (Equus caballus), rabbits (Oryctolagus cuniculus), and donkeys (Equus asinus) were purchased from Guangzhou farmers' markets. The cats (Felis catus) and dogs (Canis lupus familiaris) were provided by Guangzhou pet hospitals.

[0041] Secondly, the present invention also provides an RPA-CRISPR / Cas12a detection kit for detecting meat-derived components, characterized in that it comprises the composition described above.

[0042] Preferably, it also includes the enzymes and buffer required for the RPA-CRISPR / Cas12a reaction.

[0043] The application of the RPA-CRISPR / Cas12a detection kit described above in the detection of meat-derived genes.

[0044] Finally, this invention provides a method for detecting multiple meat-derived components, including the RPA-CRISPR / Cas12a detection kit described above, the method comprising the following steps:

[0045] S1: Extract DNA from the sample to be tested;

[0046] S2: Using the DNA extracted in step S1 as a template, the genome of the test sample is amplified isothermally using SEQ ID NO.1 and SEQ ID NO.2;

[0047] S3: Add the amplified sequence to the CRISPR-cas12a reaction system and use crRNA species-specific primers to detect meat-derived components;

[0048] S4: Analyze the test results.

[0049] Preferably, the RPA detection system in step S2 is based on a 20 μL volume and consists of: 10 μL of 2X Reaction Buffer, 1 μL each of 20 μM upper and lower primers, 2.5 μL of 10X Starter, 2 μL of DNA template, and ultrapure water to a final volume of 20 μL. The RPA amplification reaction conditions are: constant temperature 39℃ for 18-25 min, followed by termination of the reaction. Preferably, the RPA reaction time is 20 min.

[0050] Preferably, the CRISPR-Cas12a detection system of the test strip in step S3 is based on a 10 μL standard, and its components are: 10X Cleavage Buffer 1 μL, 2 μM crRNA 3.5 μL, 10 μM Cas12a Protein 0.35 μL, 4 μM Reporter 0.2 μL, RPA amplification product 4 μL, and ultrapure water to 10 μL. The reaction conditions of the CRISPR-Cas12a system are constant temperature 45℃ for 10-30 min, and then the reaction is terminated.

[0051] The CRISPR-Cas12a visual fluorescence detection system is based on a 10 μL volume and consists of: 2.11 μL of 10X NEBuffer, 3.5 μL of 1 μM crRNA, 0.35 μL of 10 μM Cas12a Protein, 1 μL of 10 μM Reporter, 3 μL of RPA amplification product, and ultrapure water to a final volume of 10 μL.

[0052] The CRISPR-Cas12a detection system also includes the RPA reaction product from step S1 and the CRISPR / Cas12a protein. The reporter sequence is TTATTT, with the 5' end of the sequence labeled with fluorescein FAM and the 3' end labeled with biotin.

[0053] Preferably, the analytical detection method described in step S4 is as follows: a: interpreting the results using colloidal gold nucleic acid test strips. If the control C line has a band but the test T line has no band, then the target gene is present; if both the test T line and the control C line have red bands, then the target gene is not present (escape method); b: reading the results using ultraviolet fluorescence. Positive samples show color under ultraviolet light, while negative samples are colorless.

[0054] Preferably, the colloidal gold nucleic acid test strip comprises: a sample pad, a binding pad containing a complex of colloidal gold nanoparticles, Au-NP-fluorescein, FAM-labeled antibody, a T-line coated with streptavidin for capturing biotin, a C-line coated with goat anti-mouse IgG for capturing colloidal gold nanoparticles, and an absorbent pad for attracting liquid through the test strip.

[0055] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0056] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0057] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art.

[0058] Example 1

[0059] Design of universal primers

[0060] Mitochondrial sequencing employed a sequence selection process in the aforementioned common animals, employing sequences that were conserved interspeciesly at both ends and species-specific in the middle. Mitochondrial gene information for chickens, ducks, geese, cattle, pigs, horses, rabbits, cats, and dogs was searched in NCBI, and the target genomes were downloaded and saved in ".FASTA" format. Analysis of the mitochondrial gene information was performed, including sequence alignment using MegAlign. Finally, the 12S rDNA gene was selected as the target gene sequence for RPA / CRISPR-Cas12a detection in these nine animals. The 12S rDNA genes of 11 species of chickens (Gallus Gallus), ducks (Anas platyrhynchos), geese (Anser cygnoides), cattle (Bos taurus), pigs (Sus scrofa), sheep (Ovis aries), horses (Equus caballus), rabbits (Oryctolagus cuniculus), donkeys (Equus asinus), cats (Felis catus), and dogs (Canis lupus familiaris) were introduced into MegAlign for multiple sequence alignment, and sequences with interspecific conservation at both ends and species-specific characteristics in the middle were selected.

[0061] RPA species universal primer sequence pairs:

[0062] The upstream primer sequence is: 5'-AGGGTTGGTAAATCTCGTGCCAGCCACCG-3' (SEQ ID NO.1);

[0063] The downstream primer sequence is: 5'-TTTAGGGCCAGGCATAGTGGGGTATCTAATC-3' (SEQ ID NO.2);

[0064] Universal primers for amplification of the conserved regions at both ends of the 12S rDNA gene selection sequence from meat mitochondria were designed using Primer Premier5 software, see [link to Primer software]. Figure 2 As shown.

[0065] Primers for amplifying the conserved regions at both ends of the 12S rDNA on mitochondria were validated using Oligo7 software and NCBI-Blast. (Including...)

[0066] 1. Primer structure analysis: Primers should form dimers to ensure that ΔG does not exceed 4.8 kcal / mol, and 3-dimers should be <2; the number of base pairs bound should not exceed 3.

[0067] 2. To prevent the formation of hairpin structures, the ΔG should not exceed 4.5 kcal / mol (preferably <2), and the number of base pairs bound should not exceed 3;

[0068] 3. The GC content is generally between 40% and 60%, and the GC content of the upstream and downstream primers should not differ too much;

[0069] 4. Tm is generally between 59 and 68℃, and the difference is similar between upstream and downstream. The Tm of the product is <94.

[0070] Example 2

[0071] crRNA sequence design

[0072] The Cas12a crRNA sequence consists of two parts: a repeat sequence (stem-loop structure) at the 5' end and a spacer sequence complementary to the target gene sequence at the 3' end.

[0073] Using the benchling platform constructed by Zhang Feng's team, specific crRNAs on the 12S rDNA screening gene sequences of the mitochondria of these nine animals were designed, with PAM sequences (TTTV) or suboptimal PAM sequences (TTTT, ATTA, CTTC) upstream.

[0074] A fixed stem-loop structure sequence was added upstream of the designed crRNA: 5'-UAAUUUCUACUAAGUGUAG AU-3'.

[0075] Verify sequence specificity: Input the designed PAM+spacer for chicken, duck, goose, cow, pig, horse, rabbit, cat, and dog into NCBI-Blast. First match the target species, then match the non-target species. The main focus is on the identity of the matched species. When the match is less than 18 nt, the non-target species can be excluded (it is better if there is at least a three-base mismatch with the non-target region).

[0076] Verify sequence stability: Input crRNA and its upstream stem-loop structure into RNAWebSuite, ensuring the stem-loop structure is not damaged and the spacer region is easily opened; check the GC content of the crRNA sequence on sg.idtdna, ideally between 40-60%, to ensure stability.

[0077] The crRNA primer set used to detect nine meat-derived components is shown in Table 1:

[0078] Table 1. Primer sets for crRNA of nine meat-derived components

[0079]

[0080]

[0081] Note: In the sequence listing XML format file, T is used instead of U in the RNA base sequence.

[0082] Example 3

[0083] DNA extraction and RPA reaction

[0084] Using the lysis buffer from the Magen kit, add 3 mg of animal tissue to 100 μL of lysis buffer, heat in a water bath at 95°C for 15 min, then centrifuge for later use. Figure 1 As shown.

[0085] Its OD260 / OD280 value was measured to be 1.8–2.1 using a UV spectrophotometer.

[0086] For RPA reactions, reagents should be handled on ice; mix thoroughly and centrifuge before use. Prepare the RPA system reagent quantities according to the table below:

[0087] Table 2 RPA reaction system

[0088]

[0089] Add 10X Starter to the tube cap. Add the above liquid to the lyophilized recombinase of single-stranded nucleic acid (oligonucleotide primers), single-stranded DNA-binding protein (SSB), and strand displacement DNA polymerase required for the RPA reaction. After rapid centrifugation, place the tube in a 39℃ PCR instrument and react for 20 min. After 5 min, remove the 8-tube strip, invert it completely 8-10 times to mix, and briefly centrifuge. Then return it to the PCR instrument until the reaction is complete.

[0090] Example 4

[0091] CRISPR-Cas12a reaction

[0092] The RPA product (target) obtained in Example 1 was added to the CRISPR-Cas12a system. Specificity was then determined according to the reagent amounts used in the CRISPR-Cas12a reaction system as specified in Table 3.

[0093] Table 3 CRISPR-Cas12a reaction system

[0094]

[0095] Gradient plasmids of the same species were amplified by RPA, and then the products (targets) were added to the CRISPR-Cas12a system for sensitivity detection (the system was the same as the specific detection, n=8).

[0096] Centrifuge the CRISPR-Cas12a system thoroughly to mix. Place it on a PCR instrument and incubate at 45°C for 15 min.

[0097] After the reaction, centrifuge thoroughly and then detect using a colloidal gold nucleic acid test strip: Mix the detection diluent and the CRISPR-Cas12a system reaction product at a ratio of 100:1 and centrifuge. Add at least 50 μL of the diluted reaction solution to the sample pad and read the results within five minutes. The CRISPR-Cas12a system (visible fluorescence) results can be read directly under a UV lamp.

[0098] Example 5

[0099] Interpretation of Colloidal Gold Nucleic Acid Test Strip Results

[0100] Positive reaction: Cas12a initiates single-strand cleavage activity upon detecting the target nucleotide amplified by RPA, cleaving the Reporter labeled with FAM and biotin. As a CRISPR / Cas12a detection system, the Cas12a cleavage substrate sequence is TTATTT, with the 5' end labeled with modified fluorescein FAM and the 3' end labeled with biotin.

[0101] When the Reporter is cleaved, FAM separates from biotin, resulting in a mixture of Au-NP-anti-FAM-FAM complex, Au-NP-anti-FAM, and biotin in the reaction system. At the T-line, streptavidin cannot capture the Au-NP-anti-FAM-FAM complex and Au-NP-anti-FAM, therefore no band appears at the T-line, while the C-line can be captured by goat anti-mouse IgG to form a band.

[0102] In negative samples, due to the absence of reporter DNA cleavage, the reaction system contained the Au-NP-anti-FAM-FAM-Biotin complex and the Au-NP-anti-FAM complex. When the reaction system flowed to the T line, the Au-NP-anti-FAM-FAM-Biotin complex was captured by streptavidin to form a band, while at the C line, the Au-NP-anti-FAM complex was captured by goat anti-mouse IgG to form a band.

[0103] 1. Specific results

[0104] 1) Interpretation of results from target species-derived test strips as follows: Figure 3 As shown:

[0105] The negative control (water) shows C and T lines.

[0106] Negative control: Other samples showed C and T lines;

[0107] Positive result: The target species-derived component only showed color on line C.

[0108] 2) Interpretation of the target species' visual fluorescence results as follows: Figure 3 As shown:

[0109] Negative control (water) is colorless;

[0110] Negative control: Other samples were colorless;

[0111] Positive result: fluorescence of the target species-derived component.

[0112] 2. Sensitivity Results

[0113] 1) Interpretation of results from target species-derived test strips as follows: Figure 4 As shown in Table 4:

[0114] The target species plasmid with the lowest positive result only shows color on the C line.

[0115] 2) Interpretation of the target species' visual fluorescence results as follows: Figure 4 As shown in Table 4:

[0116] The target species plasmid with the lowest positive result was fluorescently detected.

[0117] Table 4. Results of Target Species-Derived Test Strips and Macroscopic Fluorescence

[0118] chicken <![CDATA[10 0 ]]> <![CDATA[10 0 ]]> duck <![CDATA[10 3 ]]> <![CDATA[10 4 ]]> Goose <![CDATA[10 3 ]]> <![CDATA[10 4 ]]> ox <![CDATA[10 1 ]]> <![CDATA[10 2 ]]> pig <![CDATA[10 2 ]]> <![CDATA[10 2 ]]> horse <![CDATA[10 0 ]]> <![CDATA[10 1 ]]> rabbit <![CDATA[10 2 ]]> <![CDATA[10 3 ]]> cat <![CDATA[10 0 ]]> <![CDATA[10 0 ]]> dog <![CDATA[10 5 ]]> <![CDATA[10 5 ]]>

[0119] Judgment and Reporting

[0120] Components derived from chicken, duck, goose, cow, pig, horse, rabbit, cat, and dog can be specifically detected;

[0121] Chicken, duck, goose, cow, pig, horse, rabbit, cat, and dog-derived components have good sensitivity.

[0122] As demonstrated in Examples 1-5, universal primers designed to target the conserved middle sequences at both ends of mitochondrial DNA in poultry and livestock enable the one-time detection of various animal meat-derived components. This invention also employs RPA isothermal amplification technology, which, with its rapid speed, simple primer design, and low reaction temperature, is highly suitable for field use. Combined with the CRISPR-Cas12a system, specific crRNAs are used to recognize various animal-derived components from chickens, ducks, geese, cattle, pigs, horses, rabbits, cats, and dogs, further enhancing the specificity and accuracy of the detection.

[0123] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An RPA-CRISPR / Cas12a composition for the simultaneous detection of nine animal meat-derived components, characterized in that, Including universal RPA primer pairs and nine animal-specific crRNAs; The RPA primer pair sequences are shown in SEQ ID NO.1 and SEQ ID NO.2; The nine animals are chicken, duck, goose, cow, pig, horse, rabbit, cat and dog; The sequences of the nine animal-specific crRNAs are shown in SEQ ID NO.3-11, respectively.

2. An RPA-CRISPR / Cas12a detection kit for detecting nine animal meat-derived components, characterized in that, The composition includes that described in claim 1, wherein the nine animals are chicken, duck, goose, cow, pig, horse, rabbit, cat and dog.

3. The RPA-CRISPR / Cas12a detection kit according to claim 2, characterized in that, It also includes the enzymes and buffers required for the RPA-CRISPR / Cas12a reaction.

4. The application of the RPA-CRISPR / Cas12a detection kit according to claim 2 or 3 in the simultaneous detection of nine animal meat-derived components, characterized in that, The nine animals are chicken, duck, goose, cow, pig, horse, rabbit, cat, and dog.

5. A method for simultaneously detecting nine animal meat-derived components, characterized in that, Including the use of the RPA-CRISPR / Cas12a detection kit according to claim 2 or 3, the method includes the following steps: S1: Extract DNA from the sample to be tested; S2: Using the DNA extracted in step S1 as a template, RPA amplification was performed using the primer pairs shown in SEQ ID NO.1 and SEQ ID NO.2; S3: Add the amplification product to the CRISPR / cas12a reaction system and combine it with the nine animal-specific crRNAs described in claim 1 to detect meat-derived components; S4: Analyze the test results; The nine animals are chicken, duck, goose, cow, pig, horse, rabbit, cat and dog.

6. The detection method according to claim 5, characterized in that, The RPA detection system in step S2 is based on a 20 μL volume and consists of: 10 μL of 2× Reaction Buffer, 1 μL each of 20 μM RPA primers, 2.5 μL of 10× Starter, 2 μL of DNA template, and ultrapure water to a final volume of 20 μL. The RPA amplification reaction conditions are: constant temperature 39℃ for 18-25 min, followed by termination of the reaction.

7. The detection method according to claim 5, characterized in that, The reporter sequence used in the CRISPR / Cas12a reaction system described in step S3 is TTATTT, with the 5' end of the sequence labeled with fluorescein FAM and the 3' end labeled with biotin.

8. The method according to claim 5, characterized in that, The method for analyzing the test results in step S4 is as follows: a: The results are interpreted using colloidal gold nucleic acid test strips. If there is a band on the control C line but no band on the test T line, the target animal is present; if both the test T line and the control C line have red bands, the target animal is not present. b: Results are read using ultraviolet fluorescence with the naked eye. Positive samples show color under ultraviolet light, while negative samples are colorless. The colloidal gold nucleic acid test strip includes: a sample pad, a binding pad containing a complex of colloidal gold nanoparticles, Au-NP-fluorescein, FAM-labeled antibody, a T-line coated with streptavidin for capturing biotin, a C-line coated with goat anti-mouse IgG for capturing colloidal gold nanoparticles, and an absorbent pad for attracting liquid through the test strip.

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