Kit and detection method for detecting animal meat-derived components based on RPA-CRISPR-Cas12a

Abstract

The invention belongs to the technical field of meat product detection, particularly relates to a kit for detecting animal meat-derived components based on RPA-CRISPR-Cas12a and a detection method, and provides a combination of a primer pair suitable for isothermal amplification and a CRISPR / Cas12a nucleic acid detection system and a gene conserved sequence capable of specifically recognizing common meat-derived components. According to the method, RPA and CRISPR technologies are combined, the RPA primer and the crRNA sequence for detection are screened and optimized, the nucleic acid detection test strip and naked eye fluorescence color development are combined, meat-derived components in a sample can be rapidly identified without special professional equipment in the field detection process, and the method is suitable for rapid field detection of a base layer.

Description

Technical Field

[0001] The present invention belongs to the technical field of meat product detection, and specifically relates to a kit and a detection method for detecting animal meat-derived components based on RPA-CRISPR-Cas12a. Background Art

[0002] Meat is widely consumed around the world because it is rich in 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, that is, replacing, adding or changing high-quality meat with inferior products, is one of the most common forms of food fraud, but it is difficult to detect and has become a widespread problem around the world. For example, low-priced horse meat, duck meat, etc. are used to pass off as beef; the illegal inter-provincial transportation of cat meat and dog meat is widespread; more worryingly, undeclared wildlife ingredients (such as bat meat, fox meat, monkey meat, cat meat, rat meat, dog meat, etc.) are introduced into meat products. These practices not only violate ethical standards, but also pose serious health risks.

[0003] Although polymerase chain reaction (PCR), real-time fluorescence PCR, Sanger sequencing, etc. provide a basis for the identification of animal-derived ingredients. However, these methods are not suitable for on-site testing due to their technical complexity, because they require complex equipment, are not only time-consuming, but also require extremely stringent professional capabilities, which leads to the inability to detect multiple species at the same time. In addition, the current isothermal amplification technology that is more widely used includes loop-mediated isothermal amplification detection technology (LAMP). There have been some reports on the use of LAMP technology for the detection of multiple animal meat sources, such as the use of a constant temperature amplification primer set and its kit that can simultaneously detect pork and duck meat-derived ingredients (patent application number: 201610880177.4), etc. However, the LAMP primer pair is complex in design and contains six gene primers, which is not suitable 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 to ensure the safety and integrity of food supply. Summary of the invention

[0004] The object of the present invention is to provide a kit and a detection method for detecting animal meat-derived components based on RPA-CRISPR-Cas12a. The kit of the present invention includes a universal amplification primer pair suitable for isothermal amplification, a CRISPR-cas12a nine-meat-derived component-specific crRNA primer set, combined with nucleic acid detection test strips and naked eye fluorescence detection, which can not only quickly identify the nine meat-derived components, but also has high sensitivity, and the reaction results are readable by the naked eye, achieving multiple, rapid, sensitive, specific and other effects, and is very suitable for on-site detection, thereby achieving the purpose of the present invention.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] First, the present invention provides a composition for detecting animal meat-derived components by RPA-CRISPR / Cas12a, which is characterized by comprising an RPA primer pair and crRNA;

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

[0008] The crRNA is a primer of the CRISPR-Cas12a system constructed according to the sequences specific to the middle of animal mitochondria.

[0009] Preferably, the animal is one or more of chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog, and the crRNA primer sequences of chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog are shown as 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, which is characterized by comprising the above-mentioned composition.

[0011] Preferably, it further comprises the enzymes and buffers required for the RPA-CRISPR / Cas12a reaction.

[0012] Application of the above-mentioned RPA-CRISPR / Cas12a detection kit in detecting meat-derived genes.

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

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

[0015] S2: Using the DNA extracted in step S1 as a template, perform isothermal amplification of the genomic sequence of the test sample with the primer pair shown as SEQ ID NO.1 and SEQ ID NO.2;

[0016] S3: Add the amplified sequence to the CRISPR-cas12a reaction system and perform detection of meat-derived components in combination with the crRNA species-specific primer;

[0017] S4: Analyze the detection results.

[0018] Preferably, the detection system of RPA in step S2 is based on 20 μL, and the components are: 10 μL of 2X Reaction Buffer, 1 μL each of 20 μM forward primer and reverse primer, 2.5 μL of 10X Starter, 2 μL of DNA template, and made up to 20 μL with ultrapure water; the RPA amplification reaction conditions are: constant temperature of 39 °C for 18 - 25 min, and then the reaction is terminated.

[0019] Preferably, the detection system of the CRISPR-cas12a test strip in step S3 is based on 10 μL, and the components are: 1 μL of 10X Cleavage Buffer, 3.5 μL of 2 μM crRNA, 0.35 μL of 10 μM Cas12a Protein, 0.2 μL of 4 μM Reporter, 4 μL of RPA amplification product, and made up to 10 μL with ultrapure water. The reaction conditions of the CRISPR-Cas12a system are constant temperature of 45 °C for 10 - 30 min, and then the reaction is terminated;

[0020] The visual fluorescence detection system of CRISPR-Cas12a is based on 10 μL as the detection standard, and the components are: 1.1 μL of 10X NEBuffer r2.1, 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 made up to 10 μL with ultrapure water;

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

[0022] Preferably, the method for analyzing and detecting the results in step S4 is as follows: a: Use a colloidal gold nucleic acid test strip for result interpretation. If there is a band in the quality control C line but no band in the detection T line, it contains the target gene; if there are red bands in both the detection T line and the quality control C line, it does not contain the target gene; b: Use visual ultraviolet fluorescence for result reading. Positive samples show color under ultraviolet light, and negative samples are colorless.

[0023] Preferably, the colloidal gold nucleic acid test strip includes: a sample pad, a binding pad containing a complex of colloidal gold nanoparticles Au-NP-labeled with the fluorescent dye FAM and an 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 absorption pad for sucking liquid through the test strip.

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

[0025] (1) The detection primers for simultaneously detecting multiple animal meat-derived components provided by the present invention are beneficial for constructing universal primers for the conserved sequences at both ends and specific sequences in the middle of the mitochondria of common poultry and livestock, facilitating rapid isothermal amplification of multiple species.

[0026] (2) The selected sequence fragment is short, about 230 bp, suitable for fresh, processed, and slightly degraded meat-derived components, and is more applicable to the actual situation.

[0027] (3) The RPA isothermal amplification has a fast reaction speed, simple primer design, and low reaction temperature, making it more suitable for on-site use.

[0028] (4) According to the specific sequence in the middle of the mitochondria, the primer crRNA of the CRISPR-Cas12a system is constructed to specifically identify chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog-derived components.

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

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0031] Figure 1 It is the schematic diagram of the RPA reaction and the combination of CRISPR-Cas12a with a nucleic acid test strip detection and visual fluorescence reaction (drawn by biorender.com).

[0032] Figure 2 It is the gel electrophoresis diagram of the RPA universal primer amplifying 11 animals. Lane 1: Marker (StarMarker D750), 2: water, 3: chicken, 4: duck, 5: goose, 6: cattle, 7: pig, 8: sheep, 9: horse, 10: rabbit, 11: donkey, 12: cat, 13: dog.

[0033] Figure 3From top to bottom are the visual fluorescence and test strip reading results of the detection of specific components of chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog. From left to right in each figure are: water, chicken, duck, goose, cattle, pig, sheep, horse, rabbit, donkey, cat, and dog. If there are red bands in both the test line (T) and the control line (C), it proves to be a negative sample. If there is only a red band in the control line (C), it is a positive sample; under ultraviolet light, a colorless sample is a negative sample, and a highly bright sample is a positive sample.

[0034] Figure 4 From top to bottom are the sensitivity detections of chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog-derived components: from left to right are water, 10 0 -10 6 plasmid standards with concentration gradients of copies / μL. Detailed implementation mode

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

[0036] First, the present invention provides a composition for RPA-CRISPR / Cas12a detection of animal meat-derived components, which is characterized by comprising an RPA primer pair and a crRNA;

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

[0038] The crRNA is a primer for the CRISPR-Cas12a system constructed according to the sequences specific to the middle of the animal mitochondrion.

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

[0040] Preferably, the animals are chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog. Among them, chicken (Gallus Gallus), duck (Anas platyrhynchos), goose (Anser cygnoides), cattle (Bos taurus), pig (Sus scrofa), sheep (Ovis aries), horse (Equus caballus), rabbit (Oryctolagus cuniculus), and donkey (Equus asinus) were purchased from a Guangzhou farmers' market. Cat (Felis catus) and dog (Canis lupus familiaris) were provided by a Guangzhou pet hospital.

[0041] Secondly, the present invention also provides an RPA-CRISPR / Cas12a detection kit for detecting meat-derived components, which is characterized by comprising the above-mentioned composition.

[0042] Preferably, it further comprises enzymes and buffers required for RPA-CRISPR / Cas12a reaction.

[0043] Use of the above-mentioned RPA-CRISPR / Cas12a detection kit in detecting meat-derived genes.

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

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

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

[0047] S3: Add the amplified sequence to the CRISPR-cas12a reaction system, and perform detection of meat-derived components in combination with crRNA species-specific primers;

[0048] S4: Analyze the detection results.

[0049] Preferably, the detection system of RPA in step S2 is based on 20 μL as the standard, and the components are: 10 μL of 2X ReactionBuffer, 1 μL each of 20 μM upstream primer and downstream primer, 2.5 μL of 10X Starter, 2 μL of DNA template, and made up to 20 μL with ultrapure water; the RPA amplification reaction conditions are: constant temperature of 39 °C, 18 - 25 min, and then terminate the reaction, and preferably the RPA reaction time is 20 min.

[0050] Preferably, the test strip CRISPR-cas12a detection system in step S3 is based on 10 μL as the standard, and the components are: 1 μL of 10X Cleavage Buffer, 3.5 μL of 2 μM crRNA, 0.35 μL of 10 μM Cas12aProtein, 0.2 μL of 4 μM Reporter, 4 μL of RPA amplification product, and made up to 10 μL with ultrapure water. The reaction conditions of the CRISPR-Cas12a system are constant temperature of 45 °C, 10 - 30 min, and then terminate the reaction;

[0051] The CRISPR-Cas12a visual fluorescence detection system is based on a standard of 10 μL, and its components are: 1.1 μL of 10X NEBuffer r2.1, 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 made up to 10 μL with ultrapure water;

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

[0053] Preferably, the method for analyzing and detecting the results in step S4 is as follows: a: Use a colloidal gold nucleic acid test strip for result interpretation. If there is a band in the control C line but no band in the test T line, it contains the target gene; if there are red bands in both the test T line and the control C line, it does not contain the target gene (the line disappearance method); b: Use visual ultraviolet fluorescence for result reading. Positive samples show color under ultraviolet light, and negative samples are colorless.

[0054] Preferably, the colloidal gold nucleic acid test strip includes: a sample pad, a binding pad containing a complex of colloidal gold nanoparticles Au-NP labeled with the fluorescent dye FAM and an 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 absorption 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 in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0056] In the embodiments of the present invention, the production processes, experimental methods, or detection methods involved, unless otherwise specified, are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the art and are very clear and definite in the relevant application fields. Those skilled in the art can understand the conventional process steps according to the name and apply the corresponding equipment, and implement them under conventional conditions or conditions recommended by the manufacturer.

[0057] There are no special restrictions on the sources of various instruments, equipment, raw materials, or reagents used in the embodiments of the present invention. They are all conventional products that can be obtained through regular commercial channels, and can also be prepared according to the conventional methods well-known to those skilled in the art.

[0058] Example 1

[0059] Design of universal primers

[0060] Screen the mitochondria to perform sequence screening with interspecies conservation at both ends and species specificity in the middle among the above common animals. Search for mitochondrial gene information of chickens, ducks, geese, cows, pigs, horses, rabbits, cats, and dogs in NCBI, download the target genomes and save them in the ".FASTA" format. Analyze the mitochondrial gene information, perform sequence alignment through MegAlign, and finally select the 12s rDNA gene as the target gene sequence for RPA / CRISPR-Cas12a detection of these nine animals. Import the 12s rDNA genes of 11 species, namely chicken (Gallus Gallus), duck (Anas platyrhynchos), goose (Anser cygnoides), cow (Bos taurus), pig (Sus scrofa), sheep (Ovis aries), horse (Equus caballus), rabbit (Oryctolagus cuniculus), donkey (Equus asinus), cat (Felis catus), and dog (Canis lupus familiaris) into MegAlign for multiple sequence alignment, and select the sequences with interspecies conservation at both ends and species specificity in the middle.

[0061] RPA species universal primer sequence pair:

[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] Use Primer Premier5 software to design universal amplification primers for the conserved regions at both ends of the 12s rDNA gene screening sequence on the mitochondria of the above-mentioned meats, as shown in Figure 2 shown.

[0065] Use Oligo7 software and NCBI-Blast to verify the amplification primers for the conserved regions at both ends of the 12s rDNA on the mitochondria. Include

[0066] 1. Primer structure analysis: Ensure that the primers form dimers with each other to ensure that △G does not exceed 4.8 kcal / mol, 3-dimer < 2; the number of binding base pairs does not exceed 3;

[0067] 2. Prevent the formation of hairpin structures. The ΔG should not exceed 4.5 kcal / mol (preferably <2), and the number of paired base pairs 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 vary significantly.

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

[0070] Example 2

[0071] Design of the crRNA sequence

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

[0073] Using the benchling platform constructed by the Zhang Feng team, specifically designed crRNAs for the 12s rDNA screening gene sequences on the mitochondria of these nine animals. There should be a PAM sequence (TTTV) or sub-optimal PAM (TTTT, ATTA, CTTC) sequence upstream.

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

[0075] Verify the sequence specificity: Input the PAM + spacer of the designed chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog into NCBI - Blast. First match the target species and then the non-target species. mainly look at the matched identity. When the match is less than 18 nt, non-target species can be excluded (it is better to have at least three base mismatches with the non-target region).

[0076] Verify the sequence stability: Input the crRNA and its upstream stem-loop structure into the RNAWebSuite website to ensure that the stem-loop structure cannot be damaged and the Spacer region is easily opened; detect the GC content of the crRNA sequence at the sg.idtdna website. It is preferably between 40 - 60% to ensure stability.

[0077] The crRNA primer sets for detecting nine meat-derived components are shown in Table 1:

[0078] Table 1 crRNA primer sets for nine meat-derived components

[0079]

[0080]

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

[0082] Example 3

[0083] DNA Extraction and RPA Reaction

[0084] Using the lysis solution of the Magen kit, add 3 mg of animal tissue to 100 μL of the lysis solution, heat in a water bath at 95 °C for 15 mins and then centrifuge for standby, as shown in Figure 1 shown.

[0085] The OD260 / OD280 value measured by an ultraviolet spectrophotometer is 1.8 - 2.1.

[0086] For the RPA reaction, operate the reagents on ice, mix well and centrifuge before use. According to the following table, prepare the dosage of the experimental reagents for the RPA system:

[0087] Table 2 RPA Reaction System

[0088]

[0089] Among them, 10X Starter is added to the tube cap. Configure the above liquid into the recombinase of single-stranded nucleic acid (oligonucleotide primer), single-stranded DNA binding protein (SSB) and strand displacement DNA polymerase freeze-dried products required for the RPA reaction. After rapid centrifugation, place it in a 39 °C PCR instrument and react for 20 min. Take out the 8-tube strip at 5 min, invert it completely 8 - 10 times for mixing and briefly centrifuge, and then put it back into the PCR instrument until the reaction ends.

[0090] Example 4

[0091] CRISPR-Cas12a Reaction

[0092] Add the RPA product (target) obtained in Example 1 to the CRISPR-Cas12a system and perform specific detection according to the dosage of the experimental reagents for the CRISPR-Cas12a reaction system in Table 3:

[0093] Table 3 CRISPR-Cas12a Reaction System

[0094]

[0095] Perform RPA amplification on the gradient plasmids of the same species respectively, and then add the products (targets) to the CRISPR-Cas12a system for sensitivity detection (the system is the same as the specific detection, n = 8).

[0096] Fully centrifuge and mix the CRISPR-Cas12a system. Place it on a PCR instrument at 45 °C and react for 15 min.

[0097] After the reaction, centrifuge thoroughly and detect using a colloidal gold nucleic acid test strip: Mix the detection diluent and the reaction product of the CRISPR-Cas12a system at a ratio of 100:1 and centrifuge. Drop more than 50 μL of the diluted reaction solution onto the sample pad and read the result within five minutes. For the CRISPR-Cas12a system (visible fluorescence), directly read the result under an ultraviolet lamp.

[0098] Example 5

[0099] Interpretation of the results of the colloidal gold nucleic acid test strip

[0100] Positive reaction: When Cas12a detects the target nucleotide amplified by RPA, it will initiate single-strand cleavage activity and cleave the Reporter labeled with FAM and biotin. As a CRISPR / Cas12a detection system, the sequence of the substrate cleaved by Cas12a is TTATTT, with the 5' end labeled with the fluorophore FAM and the 3' end labeled with biotin Biotin.

[0101] When the Reporter is cleaved, FAM is separated from biotin. Therefore, in the reaction system, there are Au-NP-anti-FAM-FAM complexes, Au-NP-anti-FAM, and a biotin mixture. At the T line, streptavidin cannot capture the Au-NP-anti-FAM-FAM complexes and Au-NP-anti-FAM, so 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, since there is no cleavage of the reporter DNA, the reaction system contains Au-NP-anti-FAM-FAM-biotin complexes and Au-NP-anti-FAM. When the reaction system flows to the T line, the Au-NP-anti-FAM-FAM-biotin complexes are captured by streptavidin to form a band, and at the C line, the Au-NP-anti-FAM is captured by goat anti-mouse IgG to form a band.

[0103] 1. Specificity results

[0104] 1) The interpretation of the test strip results for the target species origin is as Figure 3 shown:

[0105] Negative control (water): The C and T lines show color;

[0106] Negative control: For other samples, the C and T lines show color;

[0107] Positive result: The target species origin component shows color only at the C line.

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

[0109] The negative control (water) is colorless;

[0110] Negative control: Other samples are colorless;

[0111] Positive result: Fluorescent color development of the target species origin component.

[0112] 2. Sensitivity results

[0113] 1) Interpretation of the test strip results of the target species origin is as follows Figure 4 and Table 4 shows:

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

[0115] 2) Interpretation of the visual fluorescence results of the target species origin is as follows Figure 4 and Table 4 shows:

[0116] For the target species plasmid with the lowest positive result, fluorescent color development occurs.

[0117] Table 4 Test strip and visual fluorescence results of the target species origin

[0118] Sensitivity (naked-eye fluorescence) Sensitivity (test strip) Chicken <![CDATA[10 0 > <![CDATA[10 0 > Duck <![CDATA[10 3 > <![CDATA[10 4 > Goose <![CDATA[10 3 > <![CDATA[10 4 > Cattle <![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] Determination and reporting

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

[0121] Components derived from chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog have good sensitivity.

[0122] As can be seen from Examples 1 - 5: Through the universal primers of the intermediate specific sequences designed based on the conservation at both ends of the mitochondrial DNA of poultry and livestock, the one - time detection of meat - derived components of multiple animals has been achieved. The present invention also adopts the RPA isothermal amplification technology, which is very suitable for on - site use due to its fast speed, simple primer design, and low reaction temperature. In combination with the CRISPR - Cas12a system, the specific crRNA is used to identify meat - derived components of multiple animals such as chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog, further improving the specificity and accuracy of the detection.

[0123] Although the above - mentioned embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments without creative efforts based on this embodiment, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A composition for RPA-CRISPR / Cas12a detection of animal meat-derived ingredients, characterized in that: Including RPA primer pairs and crRNA; The RPA primer pair sequences are shown in SEQ ID NO.1 and SEQ ID NO.2; The crRNA is a CRISPR-Cas12a system primer constructed based on the animal mitochondrial intermediate-specific sequence.

2. The composition for RPA-CRISPR / Cas12a detection of common animal meat-derived ingredients according to claim 1, characterized in that The animal is one or more of chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog, and the crRNA primer sequences of the chicken, duck, goose, cattle, pig, horse, rabbit, cat, and dog are shown in SEQ ID NO.3-11 respectively.

3. An RPA-CRISPR / Cas12a detection kit for detecting animal meat-derived ingredients, characterized in that: The invention comprises the composition described in claims 1-2.

4. The RPA-CRISPR / Cas12a detection kit according to claim 3, characterized in that Also included are the enzymes and buffers required for the RPA-CRISPR / Cas12a reaction.

5. Use of the RPA-CRISPR / Cas12a detection kit according to claim 3 or 4 in detecting meat-derived genes.

6. A method for detecting multiple meat-derived components, characterized in that: Comprising the RPA-CRISPR / Cas12a detection kit according to claim 3 or 4, the method comprises the following steps: S1: Extract DNA from the sample to be tested; S2: Using the DNA extracted in step S1 as a template, isothermally amplifying the genome of the test sample using the primers shown in SEQ ID NO.1 and SEQ ID NO.2; S3: Add the amplified sequence to the CRISPR-cas12a reaction system and combine it with crRNA species-specific primers to detect meat-derived ingredients; S4: Analyze the test results.

7. The detection method according to claim 6, characterized in that: The RPA detection system in step S2 is based on 20 μL as the standard, and the components are: 2X Reaction Buffer 10 μL, 20 μM upper primer and lower primer 1 μL each, 10X Starter 2.5 μL, DNA template 2 μL, and ultrapure water to 20 μL; the RPA amplification reaction conditions are: constant temperature 39°C, 18-25 minutes, and then terminate the reaction.

8. The detection method according to claim 6, characterized in that: The CRISPR-cas12a test strip detection system described in step S3 is based on 10 μL as the standard, and the components are: 10X Cleavage Buffer 1 μL, 2 μM crRNA 3.5 μL, 10 μM Cas12aProtein 0.35 μL, 4 μM Reporter 0.2 μL, RPA amplification product 4 μL, ultrapure water is added to 10 μL, and the reaction conditions of the CRISPR-Cas12a system are constant temperature 45 ° C, 10-30 min, and then the reaction is terminated; the CRISPR-Cas12a naked eye fluorescence detection system is based on 10 μL as the standard, and the components are: 10X NEBuffer r2.11 μL, 1 μM crRNA 3.5 μL, 10 μM Cas12aProtein 0.35 μL, 10 μM Reporter 1 μL, RPA amplification product 3 μL, ultrapure water is added to 10 μL; The CRISPR-Cas12a detection system also includes the RPA reaction product of step S1 and the CRISPR / Cas12a protein, the reporter sequence is TTATTT, the 5' end of the sequence is labeled with modified fluorescein FAM, and the 3' end is labeled with biotin Biotin.

9. The method according to claim 6, characterized in that The method of analyzing the test results in step S4 is as follows: a: using colloidal gold nucleic acid test strips to interpret the results, if the quality control C line has a band, but the detection T line has no band, the target gene is contained; if both the detection T line and the quality control C line have red bands, the target gene is not contained; b: The results are read using ultraviolet fluorescence from the naked eye. Positive samples show color under ultraviolet light, while negative samples are colorless.

10. The colloidal gold nucleic acid test strip according to claim 9, characterized in that: 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 absorption pad for attracting liquid through the test strip.

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