A composition for meat adulteration identification, a kit containing the same and applications thereof

Abstract

The application belongs to the technical field of gene detection, and particularly relates to a composition for meat adulteration identification, a kit containing the same and application. The application provides a composition for meat adulteration identification, which comprises a first nucleic acid composition, a second nucleic acid composition and a fluorescent probe group. The nucleotide sequence of the first nucleic acid composition is shown as SEQ ID NO:1-6, and the nucleotide sequence of the second nucleic acid composition is shown as SEQ ID NO:8-13. The composition has high sensitivity, high detection efficiency and high specificity, and can be used for meat adulteration identification.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, specifically relating to a composition for identifying adulterated meat, a kit containing the composition, and its application. Background Technology

[0002] The cytochrome b (cytb) gene is one of the most commonly used species-specific genes. It encodes a subunit of the gene that produces cytochrome b oxidase on the inner mitochondrial membrane, and is encoded by the H strand of mtDNA. The coding sequence region of this gene evolves slowly and is relatively conserved; however, the variable regions within the conserved regions evolve rapidly and exhibit significant differences between species. Therefore, sequence differences can be used to distinguish species. Numerous studies have demonstrated that cytb gene detection can be an important tool in species identification.

[0003] Methods for identifying adulterated meat include sensory, physical, spectroscopic, immunological, and DNA analysis methods. Among these, traditional morphological identification methods, relying primarily on sensory perception and experience, are insufficient for accurately identifying processed meat products. Spectroscopic methods, such as Raman spectroscopy and infrared spectroscopy, identify adulterated meat by analyzing the content and composition of metabolites like amino acids, phenols, and sugars; however, these methods involve expensive equipment and are difficult to popularize. Immunological methods, such as enzyme-linked immunosorbent assays (ELISA), utilize antigen-antibody interactions to detect species-specific proteins; however, these methods suffer from low sensitivity and specificity due to the susceptibility of proteins to high temperatures and their poor stability.

[0004] DNA molecules exist in all tissues and are thermostable at high temperatures. DNA analysis methods detect adulteration by analyzing differences in mitochondrial or nuclear DNA sequences between species, offering high sensitivity and specificity, making them a core method for identifying adulterated meat. In actual meat identification, PCR is the most widely used method due to its high sensitivity and good reproducibility, but its experimental procedures are complex, cumbersome, and time-consuming. Real-time quantitative PCR (RT-PCR) can be divided into dye-based and probe-based methods. The probe method achieves higher specificity through template-probe binding and hydrolysis, and it simplifies the process by eliminating the need for electrophoresis, allowing for the simultaneous identification of multiple meats using a single reaction. However, both PCR and RT-PCR are PCR-based detection methods, and their sensitivity and specificity are relatively limited, potentially leading to false positives for trace amounts of target genes in the sample.

[0005] LAMP technology is a highly specific and sensitive isothermal amplification method. Multiplex LAMP systems can detect multiple targets within a single system, but the DNA polymerase used in LAMP systems lacks 5'-3' exonuclease activity, preventing the hydrolysis of probes and the release of fluorescence. This presents a technical bottleneck for the quantitative and real-time detection of multiplex LAMP amplification. Therefore, existing research reports on fluorescent probe-based multiplex LAMP systems mainly rely on strand substitution activation, but the specificity remains insufficient. Furthermore, this technology cannot distinguish highly similar SNPs. For example, Chinese patent application 201180040896.0 discloses a gene-based diagnostic method that can rapidly distinguish selected strains of a chosen pathogen from other populations within the same species. This method uses oligonucleotide probes, called "assimilation probes," to perform real-time monitoring of the sequence specificity of DNA LAMP. These assimilation probes contain two oligonucleotide chains, one containing a quencher (called a quenching probe) and the other containing a fluorophore (called a fluorescent probe). During the LAMP reaction, when the two chains substitute for each other... A fluorescent signal is generated. Chinese patent application 202110824236.7 discloses a primer-probe combination, kit, and detection method for detecting the novel coronavirus based on LAMP technology. The primer-probe combination includes an N gene primer-probe combination and / or an E gene primer-probe combination. The probe contains two oligonucleotide chains, wherein the first oligonucleotide chain contains a quencher at the 3' end, and the second oligonucleotide chain contains a fluorescent group at the 5' end and is complementary to the first oligonucleotide chain at its 5' portion. During the loop-mediated isothermal amplification reaction, a fluorescent signal is generated when the two oligonucleotide chains replace each other.

[0006] Therefore, a simple, rapid, sensitive, and specific technical solution is needed to achieve rapid detection of the source of meat and meat products. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention provides a composition for identifying adulterated meat and a kit containing the composition.

[0008] This invention introduces loop-mediated isothermal amplification (LAMP) technology to amplify target genes, achieving more convenient and efficient nucleic acid amplification. However, traditional LAMP detection methods cannot simultaneously detect multiple targets. To further simplify the operation and enable simultaneous detection of multiple targets in a single sample, a single-system multiplex LAMP strategy based on DNA glycosylase-activated probe fluorescence is designed, enabling rapid simultaneous detection of beef and pork. This probe incorporates nucleotides containing modified bases, which can be specifically recognized by DNA glycosylases and activated to produce fluorescence, further improving the specificity of the reaction compared to existing fluorescent probes based solely on strand substitution reactions. Furthermore, since fluorescence generation is not affected by primer dimer formation, sensitivity can be improved by increasing primer concentration.

[0009] Furthermore, the species-specific gene cytb selected in this invention is located on circular mitochondrial DNA with a high copy number, which is more resistant to degradation than nuclear DNA. It can be used to detect target DNA with a higher abundance, thus making it more suitable for degradation and trace samples.

[0010] The technical solution provided by this invention is as follows:

[0011] In a first aspect, the present invention provides a composition for identifying adulteration in meat, the composition comprising a first nucleic acid composition, a second nucleic acid composition, and a fluorescent probe set;

[0012] The first nucleic acid composition includes:

[0013] cytb-F3-1, cytb-B3-1, cytb-FIP-1, cytb-BIP-1, cytb-LF-1, cytb-LB-1;

[0014] The second nucleic acid composition includes:

[0015] cytb-F3-2, cytb-B3-2, cytb-FIP-2, cytb-BIP-2, cytb-LF-2, cytb-LB-2;

[0016] The nucleotide sequence of the cytb-FIP-1 is shown in SEQ ID NO:1;

[0017] The nucleotide sequence of cytb-BIP-1 is shown in SEQ ID NO:2;

[0018] The nucleotide sequence of cytb-F3-1 is shown in SEQ ID NO:3;

[0019] The nucleotide sequence of cytb-B3-1 is shown in SEQ ID NO:4;

[0020] The nucleotide sequence of cytb-LF-1 is shown in SEQ ID NO:5;

[0021] The nucleotide sequence of cytb-LB-1 is shown in SEQ ID NO:6;

[0022] The nucleotide sequence of the cytb-FIP-2 is shown in SEQ ID NO:8;

[0023] The nucleotide sequence of the cytb-BIP-2 is shown in SEQ ID NO:9;

[0024] The nucleotide sequence of cytb-F3-2 is shown in SEQ ID NO:10;

[0025] The nucleotide sequence of cytb-B3-2 is shown in SEQ ID NO:11;

[0026] The nucleotide sequence of the cytb-LF-2 is shown in SEQ ID NO:12;

[0027] The nucleotide sequence of the cytb-LB-2 is shown in SEQ ID NO:13.

[0028] In one embodiment, the GC content of the first nucleic acid composition and the second nucleic acid composition is between 30% and 70%.

[0029] In one implementation, the Tm values ​​of cytb-FIP-1, cytb-BIP-1, cytb-FIP-2 and cytb-BIP-2 are between 55°C and 70°C.

[0030] In one implementation, the T of cytb-F3-1, cytb-B3-1, cytb-F3-2 and cytb-B3-2 m The value is between 50℃ and 65℃.

[0031] In one implementation, the T of cytb-LF-1, cytb-LB-1, cytb-LF-2 and cytb-LB-2 m The value is between 50℃ and 65℃.

[0032] In one embodiment, the fluorescent probe set includes at least two fluorescent probes:

[0033] At least one first fluorescent probe targeting the bovine cytb gene, and at least one second fluorescent probe targeting the porcine cytb gene;

[0034] The first fluorescent probe is used in combination with the first nucleic acid composition, and the second fluorescent probe is used in combination with the second nucleic acid composition.

[0035] In one embodiment, the fluorescent probe binds to the loop region of the LAMP amplicon.

[0036] In one embodiment, the T of the fluorescent probe m The value is between 55℃ and 70℃.

[0037] In one embodiment, the GC content of the fluorescent probe is between 30% and 70%.

[0038] In one embodiment, the length of the fluorescent probe is between 20 and 60 bases.

[0039] In one embodiment, the fluorescent probe contains a base that has been replaced with a modified deoxyribonucleotide.

[0040] In one embodiment, the deoxyribonucleotide is flanked by a fluorescent reporter group and a quencher group.

[0041] In one embodiment, the modified base is selected from one of 7,8-dihydroxy-8-oxoguanine, 8-hydroxyadenine, fapy-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine, 5-hydroxycytosine, and 5-hydroxyuracil.

[0042] In one embodiment, the fluorescent reporter group is selected from at least one of FAM, SIMA, HEX, ROX, TAMRA, Texas Red, and CalFluor610.

[0043] In one embodiment, the quenching group is selected from at least one of BHQ1, BHQ2, BHQ3, MGB, Dabcyl, and Eclipse.

[0044] In one embodiment, the fluorescent reporter group and the quencher group are spaced 1-5 bases apart.

[0045] In one embodiment, the 3' end of the fluorescent probe is modified with a blocking group selected from at least one of C3Spacer, C6 Spacer, phosphate group, amino group, biotin-TEG, polyhexaethylene glycol, reverse dT, reverse dG, or dideoxynucleotide.

[0046] In one embodiment, the nucleotide sequence of the first fluorescent probe is shown in SEQ ID NO:7, and the nucleotide sequence of the second fluorescent probe is shown in SEQ ID NO:14.

[0047] In a second aspect, the present invention provides a kit for identifying adulterated meat, the kit comprising the composition described in the first aspect.

[0048] In one embodiment, the kit further includes a sample processing solution, Mg 2+ At least one of dNTPs, DNA polymerase, DNA glycosylase and UDG enzyme.

[0049] In one embodiment, the dNTPs may include 0.1mM-10mM dATP, 0.1mM-10mM dTTP, 0.1mM-10mM dCTP, 0.1mM-10mM dGTP, and 0.1mM-10mM dUTP.

[0050] In one embodiment, the sample processing solution may be an inorganic salt solution of 1 mM to 50 M and / or an organic solution of 5% to 95%.

[0051] In one embodiment, the inorganic salt solution may be selected from at least one of NaCl solution, KCl solution, LiCl solution, NaOH solution, KOH solution and CaOH solution.

[0052] In one embodiment, the organic solution may be selected from at least one of SDS solution, EDTA solution, Triton X-100 solution, Tween-20 solution, Tween-80 solution, PEG-200 solution, and PEG-400 solution.

[0053] In a preferred embodiment, the DNA polymerase may be 1-50 U of Bst DNA Polymerase.

[0054] In a preferred embodiment, the DNA glycosylase may be an 8-oxoguanine DNA glycosylase of 0.1 U-80 U, which has two enzymatic activities: DNA N-glycosylase activity and AP lyase activity.

[0055] In a preferred embodiment, the UDG enzyme can be a 0.1U-50U uracil-DNA glycosylation enzyme.

[0056] In a third aspect, the present invention provides the application of the composition or the kit in the identification of adulterated meat.

[0057] In a fourth aspect, the present invention provides a method for identifying adulterated meat, the method comprising the following steps:

[0058] (1) Add sample processing solution to the sample to be tested to extract or release nucleic acid;

[0059] (2) Amplify the nucleic acid obtained in step (1) using the composition described in the first aspect or the kit described in the second aspect;

[0060] (3) Result interpretation.

[0061] In one implementation, the sample to be tested includes DNA samples, tissue samples, blood samples, or bone samples, etc.

[0062] In one implementation, the amplification can be loop-mediated isothermal amplification.

[0063] In a preferred embodiment, the amplification system comprises 0.1 mM-200 mM Tris-HCl, 1 mM-400 mM KCl, 1 mmol-80 mmol NaCl, 0.5 mM-50 mM MgSO4, 5 mM-300 mM MgCl2, 0.1%-10% Tween-20, 0.2 mM-200 mM (NH4)2SO4, 0.2 mM-20 mM dNTP mix, 0.1-50 U Bst DNA Polymerase, 0.1 U-120 U DNA glycosylase, and 0.2-60 U UDG enzyme.

[0064] In one embodiment, the amplification reaction conditions are constant temperature of 58°C-68°C.

[0065] Compared with the prior art, the compositions and / or kits provided by the present invention have at least the following beneficial effects:

[0066] (1) High sensitivity, capable of detecting target DNA down to 100 copies;

[0067] (2) High detection efficiency, using direct amplification technology for samples;

[0068] (3) High specificity: the amplification kinetic curve corresponding to each probe is monitored in real time by fluorescent probes, or the final fluorescence signal of the reaction is detected directly.

[0069] (4) Prevent aerosol pollution. Attached Figure Description

[0070] Figure 1 The amplification kinetics curves of bovine species-specific primer and probe set I are shown.

[0071] Figure 2 The amplification kinetics curves of bovine species-specific primer-probe set II are shown.

[0072] Figure 3 The amplification kinetics curves of pig species-specific primer and probe set I are shown.

[0073] Figure 4The amplification kinetics curves of the pig species-specific primer and probe set II are shown.

[0074] Figure 5 The results of specificity verification of the bovine species-specific primer-probe set of the present invention are shown.

[0075] Figure 6 The results of specificity verification of the pig species-specific primer and probe set of the present invention are shown.

[0076] Figure 7 The results of specificity verification of bovine cytb target genes by a dye method without fluorescent probes are shown.

[0077] Figure 8 The results of specificity verification of porcine cytb target genes by a dye method without fluorescent probes are shown.

[0078] Figure 9 The results of sensitivity verification of the bovine species-specific primer and probe set of the present invention are shown.

[0079] Figure 10 The results of sensitivity verification of the pig species-specific primer and probe set of the present invention are shown.

[0080] Figure 11 The results show the sensitivity verification of the dye method without fluorescent probes for bovine cytb target genes.

[0081] Figure 12 The results of sensitivity verification of the dye method without fluorescent probes for porcine cytb target genes are shown.

[0082] Figure 13 The amplification kinetics curves of the primer and probe set of the present invention on beef samples are shown.

[0083] Figure 14 The amplification kinetics curves of the primer and probe set of the present invention on pork samples are shown.

[0084] Figure 15 The amplification kinetics curves of the primer and probe set of the present invention using a simulated sample of adulterated beef as a template are shown.

[0085] Figure 16 The amplification kinetics curves of the primer and probe set of the present invention using chicken samples as templates are shown.

[0086] Figure 17 The amplification kinetics curves of the primer and probe set of the present invention with a negative control as a template are shown. Detailed Implementation

[0087] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0088] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0089] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0090] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in many publications.

[0091] This invention provides a DNA glycosylase-activated multiplex LAMP fluorescent probe and a rapid detection method for meat adulteration, comprising the following steps:

[0092] Step 1: Take a small amount of sample, add 10μL-100μL of sample processing solution, let stand for 1min-5min, and then use it directly as a template for the next reaction;

[0093] Step 2: For cytb used for species identification, design bovine species-specific primer and probe sets (three pairs of primers: outer primer F3 / B3, inner primer FIP / BIP, loop primer LF / LB; and a probe complementary to the amplicon loop region) and swine species-specific primer and probe sets (three pairs of primers: outer primer F3 / B3, inner primer FIP / BIP, loop primer LF / LB; and a probe complementary to the amplicon loop region) to identify and amplify the target genes in bovine and swine. The two probes are labeled with different fluorescent reporter groups and quencher groups, as well as a modified base site.

[0094] Step 3: For the identification of meat species for each sample, loop-mediated isothermal amplification (LAMP) reaction was performed in one tube, with bovine species-specific primer and probe sets and swine species-specific primer and probe sets added. The tube was incubated on a real-time quantitative PCR instrument or incubated in a water bath before being detected by an enzyme-linked immunosorbent assay (ELISA) reader. The amplification reaction system is shown in Table 1.

[0095] Step 4: Based on the real-time detection of amplification products by the real-time fluorescence quantitative PCR instrument or the endpoint fluorescence detection, interpret the meat species identification results. If there is fluorescence excitation from bovine species-specific probes but no fluorescence excitation from swine species-specific probes, it indicates that the sample contains bovine components; if there is fluorescence excitation from swine species-specific probes but no fluorescence excitation from bovine species-specific probes, it indicates that the sample contains swine components; if both probes are excited, it indicates that the sample contains both bovine and swine components; if neither probe is excited, it indicates that the sample does not contain either bovine or swine components.

[0096] Table 1. Amplification reaction system of the present invention

[0097]

[0098] In step one, the sample type can be a DNA sample, a tissue sample, a blood sample, or a bone sample.

[0099] The sample processing solution in step one includes 0.1mM-100mM inorganic salt solution and / or 1%-99% organic solution.

[0100] Example 1: Primer Design and Optimization

[0101] Primer and probe sets were designed for the species-specific gene cytb: The cytb gene sequences of 10 animal species (cattle, pigs, horses, donkeys, sheep, dogs, rabbits, chickens, ducks, and geese) were identified using the NCBI database. Based on these sequences, bovine and swine species-specific primer and probe sets were designed and synthesized. The bovine and swine species-specific primer and probe sets were used to identify and amplify the target gene cytb. Each primer and probe set was complementary to the corresponding template sequence. To facilitate subsequent multiplex LAMP amplification, fluorescent reporter and quencher groups were added to both sets of probes, and modified bases were substituted. To increase amplification specificity and detection resolution, multiple primer sets were designed and screened experimentally.

[0102] The optimal primer-probe set was selected based on the following objectives: (1) High amplification specificity: Both bovine and swine species-specific primer-probe sets can only amplify specific target bands for the corresponding templates. (2) High amplification sensitivity: It can effectively amplify the target fragment even when the target sequence is present in the sample in quantities as low as one ten-thousandth. (3) High adaptability to sample types: It is not only suitable for common genomic DNA samples, but also for meat, blood, and bone samples.

[0103] To ensure amplification accuracy and specificity, screening was conducted as follows: Genomic DNA from cattle, pigs, horses, donkeys, sheep, dogs, rabbits, chickens, ducks, and geese was used as templates. Multiple sets of designed primers and probes were employed to amplify these templates using a multiplex LAMP method, with negative controls implemented. The accuracy and specificity of the designed primers and probes were determined by real-time monitoring of the amplification kinetics curves triggered by probe fluorescence excitation.

[0104] Two sets of bovine species-specific primer and probe sets were designed as alternatives: primer and probe set I and primer and probe set II. Genomic DNA from the aforementioned 10 animal species was used as templates for amplification. The results showed that primer and probe set I exhibited non-specific amplification when using genomic DNA from horses, dogs, and rabbits as templates. Figure 1 Primer-probe set II showed specific amplification when bovine genomic DNA was used as a template, but no non-specific amplification was observed when genomic DNA from the other nine animal species was used as a template. Figure 2 Two sets of pig species-specific primer and probe sets were designed as alternatives: primer and probe set III and primer and probe set IV. Genomic DNA from the aforementioned 10 animal species was used as templates for amplification. The results showed that primer and probe set III exhibited non-specific amplification when using donkey and rabbit genomic DNA as templates. Figure 3 Primer-probe set IV showed specific amplification when using porcine genomic DNA as a template, but no non-specific amplification was observed when using genomic DNA from the other nine animal species as templates. Figure 4 ).

[0105] Experimental results showed that primer and probe sets II and IV, after amplification of the samples, could distinguish samples from different species that contained the bovine cytb targeting gene or the swine cytb targeting gene. Furthermore, no false positives or false negatives were observed, consistent with the expected results.

[0106] Therefore, primer and probe sets II and IV, obtained from screening, were ultimately selected as the bovine and swine species-specific primer and probe sets, respectively. The nucleotide sequences of the bovine species-specific primer set (first nucleic acid composition) are shown in SEQ ID NO:1-SEQ ID NO:7, respectively:

[0107] The nucleotide sequence of cytb-FIP-1 is shown in SEQ ID NO:1:

[0108] 5'-AGCTCCGTTTGCGTGTATGTATCGATACACTACACATCCGACACAACAA-3';

[0109] The nucleotide sequence of cytb-BIP-1 is shown in SEQ ID NO:2:

[0110] 5'-ATATGCACGTAGGACGAGGCTTAAGCAGAAGGATTACTCCAATATTTC-3';

[0111] The nucleotide sequence of cytb-F3-1 is shown in SEQ ID NO:3;

[0112] 5'-ATCCTCACAGGCCTATTCCCTAG-3';

[0113] The nucleotide sequence of cytb-B3-1 is shown in SEQ ID NO:4:

[0114] 5'-TCCTATAAATGCTGTGGCTATTAC-3';

[0115] The nucleotide sequence of cytb-LF-1 is shown in SEQ ID NO:5:

[0116] 5'-ATTCAGCCGTAGTTCACGTCTC-3';

[0117] The nucleotide sequence of cytb-LB-1 is shown in SEQ ID NO:6:

[0118] 5'-TACGGGTCTTACACTTTTCTAGA-3'.

[0119] The nucleotide sequence of the bovine species-specific probe (first fluorescent probe) is shown in SEQ ID NO:7:

[0120] 5'-AGCATTCTCCTC[dT-ROX][8-oxoG][BHQ2-dT]TACCCATATCTG-[3'-block].

[0121] The final determined nucleotide sequences of the pig species-specific primer set (second nucleic acid composition) are shown in SEQ ID NO:8-SEQ ID NO:14, respectively:

[0122] The nucleotide sequence of cytb-FIP-2 is shown in SEQ ID NO:8:

[0123] 5'-TGCTCCGTTTGCATGTAGATAGCGATACACTACACATCCGACACAACAA-3';

[0124] The nucleotide sequence of cytb-BIP-2 is shown in SEQ ID NO:9:

[0125] 5'-TCATCCACGTAGGCCGAGGTCTAGCAGAAGGATTACTCCAATATTTC-3'

[0126] The nucleotide sequence of cytb-F3-2 is shown in SEQ ID NO:10:

[0127] 5'-ATCCTCACAGGCCTATTCCTAG-3'

[0128] The nucleotide sequence of cytb-B3-2 is shown in SEQ ID NO:11:

[0129] 5'-TCCTATAAATGCTGTGGCTATTAC-3'

[0130] The nucleotide sequence of cytb-LF-2 is shown in SEQ ID NO:12:

[0131] 5'-ATTCAGCCGTAGTTCACGTCTC-3'

[0132] The nucleotide sequence of cytb-LB-2 is shown in SEQ ID NO:13:

[0133] 5'-TACGGGTCTTACACTTTTCTAGA-3'.

[0134] The pig species-specific probe (second fluorescent probe) is shown in SEQ ID NO:14:

[0135] 5'-GCTTTCTCA[dT-FAM]CA[8-oxoG][BHQ1-dT]TACACACATTTGTC-[3'-block].

[0136] Example 2: Validation of the Specificity of the Detection Method

[0137] Plasmids containing target fragments of the cytb gene from 10 animals (cattle, pigs, horses, donkeys, sheep, dogs, rabbits, chickens, ducks, and geese) were constructed. The cytb plasmids from cattle and pigs were mixed with cytb plasmids from the other nine animals, respectively, to a total DNA content of 0.1 ng, resulting in the cattle cytb plasmid comprising 10%, 1%, 0.1%, 0.01%, and 0% of the total DNA, respectively. Using this plasmid as a template, substances listed in the table below were added, and LAMP amplification was performed on each template, with negative controls set.

[0138] Table 2. Amplification system of this embodiment

[0139]

[0140] After adding the reagents, place the sample on a real-time quantitative PCR instrument and incubate at 65°C for 30 minutes. Monitor the fluorescence signal every 30 seconds and record the type and intensity of the fluorescence signal.

[0141] Since the first fluorescent probe targeting the bovine cytb gene and the second fluorescent probe targeting the porcine cytb gene are labeled with different fluorescent reporter groups, after the reaction, different fluorescence colors can be used to determine whether the bovine and porcine target genes have been successfully amplified, thereby determining the specificity of the designed LAMP method for single-target amplification.

[0142] The results showed that LAMP reactions using bovine or porcine cytb plasmids as templates, ranging from 10% to 0.01%, were excited by fluorescent probes, while amplification using a mixture of animal plasmids as templates (0%) did not elicit fluorescent probe activation. This indicates that the detection method can amplify bovine cytb target genes containing 0.01% (one ten-thousandth) or porcine cytb target genes, and the reaction system exhibits good specificity. Figure 5 and Figure 6 ).

[0143] Furthermore, the detection method established in this application was compared with the commonly used dye method without the introduction of specific fluorescent probes. The amplification system of the dye method does not contain the corresponding fluorescent probe, and the remaining steps are the same as above. The results showed that the detection specificity of the dye method for amplifying bovine cytb plasmids containing 0.1% and porcine cytb plasmids containing 0.1% was lower than that of this detection method. Figure 7 and Figure 8 ).

[0144] Example 3: Sensitivity Verification of the Detection Method

[0145] The sensitivity of the detection method was evaluated by serially diluting recombinant DNA plasmids: using bovine cytb plasmids or porcine cytb plasmids constructed in Example 2 at concentrations of 10,000 copies, 1,000 copies, 500 copies, 100 copies, and 50 copies as templates, the substances listed in the table below were added, and multiplex LAMP amplification was performed on the templates, with negative controls set.

[0146] Table 3. Amplification system of this embodiment

[0147]

[0148] After adding the reagents, place the sample on a real-time quantitative PCR instrument and incubate at 65°C for 30 minutes. Monitor the fluorescence signal every 30 seconds and record the type and intensity of the fluorescence signal.

[0149] Since the first fluorescent probe targeting the bovine cytb gene and the second fluorescent probe targeting the porcine cytb gene are labeled with different fluorescent reporter groups, after the reaction, different fluorescence colors can be used to determine whether the bovine and porcine target genes have been successfully amplified, thereby determining the sensitivity of the designed LAMP method for single-target amplification.

[0150] The results showed that LAMP reactions using serially diluted bovine or porcine cytb plasmids (from 10,000 copies to 50 copies) as templates all produced amplification curves, indicating that the sensitivity of this detection method reached 50 copies / reaction. Figure 9 and Figure 10 ).

[0151] Furthermore, the composition provided in this application was compared with commonly used dyes that do not incorporate specific fluorescent probes. The amplification system of the dye method did not include the corresponding fluorescent probe, and the remaining steps were the same as above. Amplification results showed that the detection sensitivity of amplifying bovine cytb plasmids containing 100 copies or porcine cytb plasmids containing 500 copies using the dye method was lower than that of the detection method described above. Figure 11 and Figure 12 ).

[0152] Example 4: Detection of genomic DNA samples

[0153] Five samples of beef, pork, chicken, and duck were purchased from Walmart Shopping Plaza in Yuci District, Jinzhong City, for later use. Pork was added to the beef at proportions of 50%, 20%, 10%, 1%, and 0.1% to create simulated adulterated beef samples. Three samples of each adulterated beef were taken for later use.

[0154] Genomic DNA was extracted from the above-mentioned beef, pork, chicken, and duck samples, as well as five proportions of simulated beef adulteration samples, to obtain purified genomic DNA samples. These samples were then used to detect the genes involved in this invention. The specific operational steps are as follows: Centrifuge tubes were taken, and the following substances were added (bovine species-specific primer and probe sets and swine species-specific primer and probe sets were added to each tube). The amplification system is shown in Table 4.

[0155] Table 4. Amplification system of this embodiment

[0156]

[0157]

[0158] Place the centrifuge tubes in a real-time quantitative PCR instrument or a water bath and incubate at 65°C for 30 minutes. Monitor the fluorescence signal every 30 seconds and record the type and intensity of the fluorescence signal.

[0159] Meat identification results are interpreted based on the real-time detection or endpoint fluorescence detection of amplified products using a real-time fluorescence quantitative PCR instrument. Because the first fluorescent probe targeting the bovine cytb gene and the second fluorescent probe targeting the porcine cytb gene are labeled with different fluorescent reporter groups, the presence of bovine and porcine target genes in the sample can be determined by their different fluorescence colors.

[0160] Experimental results showed that the amplification kinetics curve using beef samples as templates exhibited bovine species-specific probe fluorescence (ROX) excitation but not porcine species-specific probe fluorescence (FAM) excitation, thus indicating the presence of beef-derived components and the absence of pork-derived components, consistent with the true species origin of the samples. Figure 13 Amplification using pork samples as templates showed excitation by pig species-specific probe fluorescence (FAM) but not by bovine species-specific probe fluorescence (ROX), thus indicating the presence of pork-derived components and the absence of beef-derived components. This determination was consistent with the true species origin of the sample. Figure 14 Amplification using a simulated sample of adulterated beef as a template, simultaneously excited by bovine species-specific probe fluorescence (ROX) and swine species-specific probe fluorescence (FAM), was thus identified as containing both beef and pork-derived components. The identification results were consistent with the true origin of the sample. Figure 15 Amplification using chicken samples as templates did not elicit bovine species-specific probe fluorescence (ROX) or swine species-specific probe fluorescence (FAM) excitation, thus indicating that the samples did not contain beef or pork-derived components. This determination was consistent with the true origin of the samples. Figure 16 Using a negative control as a template for amplification, no bovine species-specific probe fluorescence (ROX) or swine species-specific probe fluorescence (FAM) was detected, thus ruling out false positives. Figure 17 ).

[0161] The experimental results are shown in Table 5. The detection results of 35 samples were consistent with their corresponding real sources, indicating that the method is reliable for purifying genomic DNA samples.

[0162] Table 5. Sample amplification results of this embodiment.

[0163]

[0164] Example 5: Detection of meat samples

[0165] Using the meat sample prepared in Example 4, the composition of the present invention was used to detect the gene involved in the present invention. The specific operation steps are as follows: Take a small amount of meat sample and add it to a centrifuge tube. Use a pipette to add 100 μL of sample processing solution to the centrifuge tube and let it stand for 5 min. Take the centrifuge tube and add the following substances (each tube contains bovine species-specific primer and probe sets and swine species-specific primer and probe sets). The amplification system is shown in Table 6:

[0166] Table 6. Amplification system of this embodiment

[0167]

[0168] Place the centrifuge tubes in a real-time quantitative PCR instrument or a water bath and incubate at 65°C for 30 minutes. Monitor the fluorescence signal every 30 seconds and record the type and intensity of the fluorescence signal.

[0169] Meat identification results are interpreted based on the real-time detection or endpoint fluorescence detection of amplified products using a real-time fluorescence quantitative PCR instrument. Because the first fluorescent probe targeting the bovine cytb gene and the second fluorescent probe targeting the porcine cytb gene are labeled with different fluorescent reporter groups, the presence of bovine and porcine target genes in the sample can be determined by the different fluorescence colors.

[0170] The experimental results are shown in Table 7. The test results of 35 samples were consistent with their corresponding real sources, indicating that the method is reliable when applied to meat samples.

[0171] Table 7. Sample amplification results of this embodiment.

[0172]

[0173]

[0174] Example 6: Detection of blood samples

[0175] Five samples of cow blood, pig blood, chicken blood, and duck blood were purchased from Walmart Shopping Plaza in Yuci District, Jinzhong City, for later use. Pig blood was added to cow blood at proportions of 50%, 20%, 10%, 1%, and 0.1% to create simulated adulterated cow blood samples. Three samples of each adulterated cow blood were taken for later use.

[0176] Using the aforementioned blood samples as templates, the genes involved in this invention were detected. The specific operating steps are as follows: Add 2 μL of blood sample to a centrifuge tube, pipette 20 μL of sample processing solution into the centrifuge tube, and let stand for 5 minutes; take the centrifuge tube and add the following substances (each tube contains bovine species-specific primer and probe sets and swine species-specific primer and probe sets). The amplification system is shown in Table 8.

[0177] Table 8. Amplification system of this embodiment

[0178]

[0179] Place the centrifuge tubes in a real-time quantitative PCR instrument or a water bath and incubate at 65°C for 30 minutes. Monitor the fluorescence signal every 30 seconds and record the type and intensity of the fluorescence signal.

[0180] The identification results of blood samples are interpreted based on the real-time detection or endpoint fluorescence detection of amplification products using a real-time fluorescence quantitative PCR instrument. Because the first fluorescent probe targeting the bovine cytb gene and the second fluorescent probe targeting the porcine cytb gene are labeled with different fluorescent reporter groups, the presence of bovine and porcine target genes in the sample can be determined by the different fluorescence colors.

[0181] The experimental results are shown in Table 9. The test results of 35 samples were consistent with their corresponding real sources, indicating that the method is reliable when applied to blood samples.

[0182] Table 9. Sample amplification results of this embodiment.

[0183]

[0184] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A composition for identifying adulterated meat, characterized in that, The composition comprises a first nucleic acid composition, a second nucleic acid composition, and a fluorescent probe group; The first nucleic acid composition consists of: cytb -F3-1, cytb -B3-1, cytb -FIP-1, cytb -BIP-1, cytb -LF-1, cytb -LB-1 composition; The second nucleic acid composition consists of: cytb -F3-2, cytb -B3-2, cytb -FIP-2, cytb -BIP-2, cytb -LF-2, cytb -LB-2 composition; The cytb The nucleotide sequence of -FIP-1 is shown in SEQ ID NO:1; The cytb The nucleotide sequence of -BIP-1 is shown in SEQ ID NO:2; The cytb The nucleotide sequence of -F3-1 is shown in SEQ ID NO:3; The cytb The nucleotide sequence of -B3-1 is shown in SEQ ID NO:4; The cytb The nucleotide sequence of -LF-1 is shown in SEQ ID NO:5; The cytb The nucleotide sequence of -LB-1 is shown in SEQ ID NO:6; The cytb The nucleotide sequence of -FIP-2 is shown in SEQ ID NO:8; The cytb The nucleotide sequence of -BIP-2 is shown in SEQ ID NO:9; The cytb The nucleotide sequence of -F3-2 is shown in SEQ ID NO:10; The cytb The nucleotide sequence of -B3-2 is shown in SEQ ID NO:11; The cytb The nucleotide sequence of -LF-2 is shown in SEQ ID NO:12; The cytb The nucleotide sequence of -LB-2 is shown in SEQ ID NO:13; The fluorescent probe set includes two fluorescent probes: A line targeting cattle cytb The first fluorescent probe for a gene, and a pig cytb The second fluorescent probe for the gene; The first fluorescent probe is used in combination with the first nucleic acid composition, and the second fluorescent probe is used in combination with the second nucleic acid composition; The fluorescent probe binds to the loop region of the LAMP amplicon; The nucleotide sequence of the first fluorescent probe is shown in SEQ ID NO:7, and the nucleotide sequence of the second fluorescent probe is shown in SEQ ID NO:

14.

2. The composition according to claim 1, characterized in that, The fluorescent probe contains a base that has been replaced with a modified deoxyribonucleotide. The deoxyribonucleotide is flanked by a fluorescent reporter group and a quencher group.

3. The composition according to claim 2, characterized in that, The fluorescent reporter group is selected from at least one of SIMA, HEX, ROX, TAMRA, Texas Red, and CalFluor610; And / or, the quenching group is selected from at least one of BHQ1, BHQ3, MGB, Dabcyl and Eclipse; And / or, the fluorescent reporter group and the quencher group are spaced 1-5 bases apart.

4. The composition according to claim 1, characterized in that, The 3' end of the fluorescent probe is modified with a blocking group, which is selected from at least one of C6 spacer, phosphate group, amino group, biotin-TEG, polyhexaethylene glycol, reverse dT, reverse dG and dideoxynucleotide.

5. A reagent kit for identifying adulterated meat, characterized in that, The kit comprises the composition according to any one of claims 1-4.

6. The application of the composition according to any one of claims 1-4 or the kit according to claim 5 in the identification of adulterated meat.

7. A method for identifying adulterated meat, characterized in that, The method includes the following steps: (1) Add sample processing solution to the sample to be tested to extract or release nucleic acids; (2) Amplify the nucleic acid obtained in step (1) using any of the compositions described in claims 1-4 or the kit described in claim 5; (3) Result interpretation.

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