CN116949191A - Composition for meat adulteration identification, kit containing composition and application of kit - Google Patents
Composition for meat adulteration identification, kit containing composition and application of kit Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The application belongs to the technical field of gene detection, and particularly relates to a composition for identifying meat adulteration, a kit containing the composition and application of the kit. The present application provides a composition for meat adulteration identification comprising a first nucleic acid composition, a second nucleic acid composition and a fluorescent probe set. 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 identifying adulterated meat.
Description
Technical Field
The application belongs to the technical field of gene detection, and particularly relates to a composition for identifying meat adulteration, a kit containing the composition and application of the kit.
Background
The cytochrome b (cytb) gene is one of the most commonly used species-specific genes at present, and one subunit of the gene encoding cytochrome b oxidase on the inner mitochondrial membrane is encoded by the H chain of mtDNA. The coding sequence region of the gene has slow evolution and relatively conservation, but the variable region in the conservation region has fast evolution and large difference among different species, so that the species can be distinguished according to the sequence difference. Most related studies have shown that cytb gene detection can be an important tool in species identification.
The meat adulteration identification method comprises detection methods based on sensory technology, physical technology, spectroscopic technology, immunological technology, DNA analysis and the like. Wherein, the identification means based on the traditional meat morphology relying on sense and experience can not accurately identify the processed meat products. The identification method based on the spectrometry, such as Raman spectroscopy and infrared spectroscopy, is carried out by means of the content and composition of metabolites such as amino acid, phenols and saccharides in meat, and has high instrument price and difficult popularization. Immunological discrimination methods, such as enzyme-linked immunosorbent assay, utilize antigen-antibody interactions to detect species-specific proteins, which are susceptible to high temperature damage, poor stability, and low sensitivity and specificity.
DNA molecules exist in all tissues, have high-temperature thermal stability, and the DNA analysis method detects by analyzing the difference of mitochondrial or nuclear DNA sequences among species, has high sensitivity and high specificity, and is a core method for identifying meat adulteration. In the actual meat identification process, PCR (polymerase chain reaction) is most widely applied, and has the advantages of higher detection sensitivity, good result repeatability and the like, but the experimental procedure is more complex, and the operation is tedious and time-consuming. Real-time fluorescent quantitative PCR (RT-PCR) can be divided into a dye method and a probe method, wherein the probe method combines a template with a probe and hydrolyzes to ensure that the specificity is stronger, electrophoresis is not needed, the flow is simplified, and the aim of simultaneously identifying multiple meats by a single reaction can be fulfilled. Whether PCR or RT-PCR is a PCR-based detection method, the sensitivity and specificity of the method are limited, and false detection is likely to be caused for trace target genes in samples.
The LAMP technology is a high-specificity and high-sensitivity isothermal amplification means. The multiplex LAMP system can realize detection of a plurality of targets in a monomer system, but the DNA polymerase used in the LAMP system lacks 5'-3' exonuclease activity and can not hydrolyze probes to release fluorescence, so that a certain technical bottleneck appears in quantitative and real-time detection of multiplex LAMP amplification. Thus, the existing research reports that the established multiplex LAMP system based on the fluorescent probe is mainly activated by strand displacement, but the specificity is still insufficient. And this technique does not distinguish SNPs with highly similar sequences, for example, chinese patent application 201180040896.0 discloses a gene-based diagnostic method capable of rapidly distinguishing selected strains of a selected pathogen from other populations within the same species, by performing sequence-specific real-time monitoring of LAMP of DNA using oligonucleotide probes, termed "assimilating probes", comprising two oligonucleotide strands, one of which comprises a quencher (termed quenching probe) and the other of which comprises a fluorophore (termed fluorescent probe), which, when replaced with each other, generates a fluorescent signal during the LAMP reaction; chinese patent application 202110824236.7 discloses a primer probe combination, a kit and a detection method for detecting a novel coronavirus based on the LAMP technology, wherein the primer probe combination comprises an N-gene primer probe combination and/or an E-gene primer probe combination, the probe comprises two oligonucleotide chains, wherein a first oligonucleotide chain comprises a quencher at the 3' end and a second oligonucleotide chain comprises a fluorescent group at the 5' end and is complementary to the first oligonucleotide chain at the 5' portion thereof, and a fluorescent signal is generated when the two oligonucleotide chains are replaced with each other during a loop-mediated isothermal amplification reaction.
Therefore, a simple, quick, sensitive and specific technical scheme needs to be established to realize quick detection of meat and meat product sources.
Disclosure of Invention
To overcome the defects of the prior art, the application provides a composition for identifying meat adulteration and a kit containing the composition.
According to the application, a loop-mediated isothermal amplification (LAMP) technology is introduced to perform amplification of target genes, so that more convenient and efficient nucleic acid amplification is realized. However, the conventional LAMP detection method cannot detect multiple targets at the same time, so that in order to further simplify the operation steps and realize the simultaneous detection of multiple targets in a single sample, a monomer system multiplex LAMP strategy based on DNA glycosylase activated probe fluorescence is designed, and the simultaneous and rapid detection of beef and pork is realized. The nucleotide containing modified base is introduced into the probe, and can be specifically identified and activated by DNA glycosylase, so that compared with the existing fluorescent probe based on the strand displacement reaction, the specificity of the reaction is further improved. On this basis, since fluorescence generation is not affected by primer dimer formation, sensitivity can be improved by increasing primer concentration.
In addition, the species-specific gene cytb selected by the application is positioned on the circular mitochondrial DNA with high copy number, is more resistant to degradation than nuclear DNA, and can be used for detecting target DNA with higher abundance, thereby being more suitable for degradation and trace detection materials.
The technical scheme provided by the application is as follows:
in a first aspect, the present application provides a composition for meat adulteration identification comprising a first nucleic acid composition, a second nucleic acid composition and a set of fluorescent probes;
wherein the first nucleic acid composition comprises:
cytb-F3-1，cytb-B3-1，cytb-FIP-1，cytb-BIP-1，cytb-LF-1，cytb-LB-1；
the second nucleic acid composition comprises:
cytb-F3-2，cytb-B3-2，cytb-FIP-2，cytb-BIP-2，cytb-LF-2，cytb-LB-2；
the nucleotide sequence of cytb-FIP-1 is shown as SEQ ID NO. 1;
the nucleotide sequence of cytb-BIP-1 is shown as SEQ ID NO. 2;
the nucleotide sequence of cytb-F3-1 is shown as SEQ ID NO. 3;
the nucleotide sequence of cytb-B3-1 is shown as SEQ ID NO. 4;
the nucleotide sequence of cytb-LF-1 is shown as SEQ ID NO. 5;
the nucleotide sequence of cytb-LB-1 is shown as SEQ ID NO. 6;
the nucleotide sequence of cytb-FIP-2 is shown as SEQ ID NO. 8;
the nucleotide sequence of cytb-BIP-2 is shown as SEQ ID NO. 9;
the nucleotide sequence of cytb-F3-2 is shown as SEQ ID NO. 10;
the nucleotide sequence of cytb-B3-2 is shown as SEQ ID NO. 11;
the nucleotide sequence of cytb-LF-2 is shown as SEQ ID NO. 12;
the nucleotide sequence of cytb-LB-2 is shown as SEQ ID NO. 13.
In one embodiment, the GC content of the first and second nucleic acid compositions is between 30% -70%.
In one embodiment, the Tm values of cytb-FIP-1, cytb-BIP-1, cytb-FIP-2 and cytb-BIP-2 are between 55℃and 70 ℃.
In one embodiment, the T of cytb-F3-1, cytb-B3-1, cytb-F3-2 and cytb-B3-2 m The values are between 50℃and 65 ℃.
In one embodiment, the T of the cytb-LF-1, cytb-LB-1, cytb-LF-2 and cytb-LB-2 m The values are between 50℃and 65 ℃.
In one embodiment, the set of fluorescent probes comprises at least two fluorescent probes:
at least one first fluorescent probe for the bovine cytb gene and at least one second fluorescent probe for the porcine cytb gene;
wherein a first fluorescent probe is used in combination with a first nucleic acid composition and a second fluorescent probe is used in combination with a second nucleic acid composition.
In one embodiment, the fluorescent probe binds to the Loop region of the LAMP amplicon.
In one embodiment, the fluorescent probe is T m The values are between 55℃and 70 ℃.
In one embodiment, the GC content of the fluorescent probe is between 30% -70%.
In one embodiment, the fluorescent probe is between 20 and 60 bases in length.
In one embodiment, the fluorescent probe comprises deoxyribonucleotides with one base replaced with a modified base.
In one embodiment, the deoxyribonucleotide has a fluorescent reporter group attached to both sides thereof and a quencher group.
In one embodiment, the modified base is selected from one of 7, 8-dihydroxy-8-oxoguanine, 8-hydroxy adenine, famy-guanine, methyl-famy-guanine, famy-adenine, aflatoxin B1-famy-guanine, 5-hydroxy-cytosine, and 5-hydroxy-uracil.
In one embodiment, the fluorescent reporter group is selected from at least one of FAM, SIMA, HEX, ROX, TAMRA, texas Red and CalFluor 610.
In one embodiment, the quenching group is selected from at least one of BHQ1, BHQ2, BHQ3, MGB, dabcyl and Eclipse.
In one embodiment, the fluorescent reporter group is 1-5 bases apart from the quencher group.
In one embodiment, the 3' end of the fluorescent probe is modified with a blocking group selected from at least one of a C3Spacer, a C6 Spacer, a phosphate group, an amino group, biotin-TEG, polyhexamethylene glycol, inverted dT, inverted dG, or dideoxynucleotide.
In one embodiment, the nucleotide sequence of the first fluorescent probe is shown as SEQ ID NO. 7, and the nucleotide sequence of the second fluorescent probe is shown as SEQ ID NO. 14.
In a second aspect, the present application provides a kit for meat adulteration identification comprising the composition of the first aspect.
In one embodiment, the kit further comprises a sample processing fluid, mg 2+ At least one of dNTPs, DNA polymerase, DNA glycosylase and UDG enzyme.
In one embodiment, the dNTPs may comprise 0.1mM-10mM dATP, 0.1mM-10mM dTTP, 0.1mM-10mM dCTP, 0.1mM-10mM dGTP, and 0.1mM-10mM dUTP.
In one embodiment, the sample processing fluid may be a 1mM-50M inorganic salt solution and/or a 5% -95% organic solution.
In one embodiment, the inorganic salt solution may be selected from at least one of a NaCl solution, a KCl solution, a LiCl solution, a NaOH solution, a KOH solution, and a CaOH solution.
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.
In a preferred embodiment, the DNA polymerase may be Bst DNA Polymerase in the range of 1U to 50U.
In a preferred embodiment, the DNA glycosylase may be a 0.1U-80U 8-oxoguanine DNA glycosylase having two enzymatic activities: DNA N-glycosylase activity and AP lyase activity.
In a preferred embodiment, the UDG enzyme may be 0.1U-50U uracil-DNA glycosylase.
In a third aspect, the application provides the use of said composition or said kit in the identification of meat adulteration.
In a fourth aspect, the present application provides a method of identifying meat adulteration, the method comprising the steps of:
(1) Adding a sample treatment liquid into a sample to be detected to extract or release nucleic acid;
(2) Amplifying the nucleic acid obtained in step (1) using the composition of the first aspect or the kit of the second aspect;
(3) And judging the result.
In one embodiment, the sample to be tested comprises a DNA sample, a tissue sample, a blood sample, a bone sample, or the like.
In one embodiment, the amplification may be loop-mediated isothermal amplification.
In a preferred embodiment, the amplification system is 0.1mM-200mM Tris-HCl,1mM-400mM KCl,1mmol-80mmol NaCl,0.5mM-50mM MgSO 4 ，5mM-300mM MgCl 2 ，0.1％-10％Tween-20，0.2mM-200mM(NH 4 ) 2 SO 4 0.2mM-20mM dNTP mix,0.1-50U Bst DNA Polymerase,0.1U-120U of DNA glycosylase and 0.2-60U of UDG enzyme.
In one embodiment, the reaction conditions for the amplification are constant temperature 58 ℃ -68 ℃.
Compared with the prior art, the composition and/or the kit provided by the application have at least the following beneficial effects:
(1) The sensitivity is high, and target DNA as low as 100 copies can be detected;
(2) The detection efficiency is high, and a direct amplification technology is adopted for the sample;
(3) The specificity is high, and the amplification kinetic curve corresponding to each probe is monitored in real time through fluorescent probes, or the final fluorescent signal of the reaction is directly detected;
(4) Preventing aerosol pollution.
Drawings
FIG. 1 shows amplification kinetics curves for bovine species-specific primer probe set I.
FIG. 2 shows amplification kinetics curves for bovine species-specific primer probe set II.
FIG. 3 shows the amplification kinetics of porcine species-specific primer probe set I.
FIG. 4 shows the amplification kinetics of porcine species-specific primer probe set II.
FIG. 5 shows the results of the specificity verification of the bovine species-specific primer probe set of the present application.
FIG. 6 shows the results of the specificity verification of the porcine species-specific primer probe set of the present application.
FIG. 7 shows the results of the specificity verification of the dye method without fluorescent probe for bovine cytb target genes.
FIG. 8 shows the results of the specificity verification of the dye method without fluorescent probe for the pig cytb target gene.
FIG. 9 shows the results of sensitivity verification of the bovine species-specific primer probe set of the present application.
FIG. 10 shows the results of sensitivity verification of the porcine species-specific primer probe set of the present application.
FIG. 11 shows the results of the sensitivity verification of the dye method without fluorescent probe to bovine cytb target gene.
FIG. 12 shows the results of the sensitivity verification of the dye method without fluorescent probe to the pig cytb target gene.
FIG. 13 shows the amplification kinetics of a primer probe set of the present application to a beef sample.
FIG. 14 shows the amplification kinetics of a pork sample by a primer probe set of the present application.
FIG. 15 shows the amplification kinetics of the primer probe set of the present application against a simulated sample of adulterated beef as a template.
FIG. 16 shows the amplification kinetics of a chicken sample as a template by a primer probe set of the present application.
FIG. 17 shows the amplification kinetics of the primer probe set of the present application against a negative control as a template.
Detailed Description
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this application belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.
The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.
The present application will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present application more apparent. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the application. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the application in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present application. Such structures and techniques are also described in a number of publications.
The application provides a multiplex LAMP fluorescent probe based on DNA glycosylase activation and a rapid detection method for meat adulteration identification, comprising the following steps:
step one: taking a small amount of sample, adding 10 mu L-100 mu L of sample treatment liquid, standing for 1min-5min, and directly taking the sample as a template for the next reaction;
step two: for cytb used for species identification, respectively designing a bovine species-specific primer probe group (three pairs of primers, namely an outer primer F3/B3, an inner primer FIP/BIP and a Loop primer LF/LB, and a probe complementary to the Loop region of the amplicon), a porcine species-specific primer probe group (three pairs of primers, namely an outer primer F3/B3, an inner primer FIP/BIP, a Loop primer LF/LB and a probe complementary to the Loop region of the amplicon), and identifying and amplifying target genes of the cattle and the pig, wherein the two probes are respectively marked with different fluorescent reporter groups, quenching groups and a modified base site;
step three: performing loop-mediated isothermal amplification (LAMP) reaction in a tube for meat species identification of each sample, adding a bovine species-specific primer probe group and a porcine species-specific primer probe group, placing the mixture on a real-time fluorescent quantitative PCR (polymerase chain reaction) instrument for incubation, or placing the mixture in a water bath for incubation and then detecting by an enzyme-labeled instrument, wherein the amplification reaction system is shown in table 1;
step four: judging meat species identification results according to real-time detection of a real-time fluorescent quantitative PCR instrument or generation of amplification products of end-point fluorescent detection, and if bovine species specific probes are excited in a fluorescent manner and no porcine species specific probes are excited in a fluorescent manner, indicating that the sample to be detected contains bovine-derived components; if the pig species specific probe is excited by fluorescence, but the cow species specific probe is not excited by fluorescence, the sample to be detected contains pig source components; if the fluorescence of the two probes is excited, the sample to be tested contains bovine-derived components and porcine-derived components; if no probe is excited by fluorescence, the sample to be tested does not contain bovine-derived components or porcine-derived components.
TABLE 1 amplification reaction System of the application
The sample type in the first step can be a DNA sample, a tissue sample, a blood sample or a bone sample.
The sample processing liquid in the first step comprises 0.1mM-100mM inorganic salt solution and/or 1% -99% organic solution.
Example 1 design and optimization of primers
Designing a primer probe group aiming at the species specific gene cytb: the cytb gene sequences of 10 animals including cattle, pigs, horses, donkeys, sheep, dogs, rabbits, chickens, ducks and geese are confirmed through NCBI database, and the bovine species specific primer probe group and the porcine species specific primer probe group are designed and synthesized according to the cytb gene sequences. For the gene cytb to be detected, a target gene is identified and amplified by a bovine species specific primer probe group and a porcine species specific primer probe group, and each primer probe group is complementary with a corresponding template sequence. To facilitate subsequent multiplex LAMP amplification, labeling of fluorescent reporter groups and quencher groups and substitution of modified bases are performed for the two sets of probes designed. To increase amplification specificity and detection resolution, multiple sets of primers were designed for assay screening.
Screening an optimal primer probe group according to the following targets: (1) high amplification specificity: the bovine species specific primer probe set and the porcine species specific primer probe set can only amplify specific target strips for the corresponding templates. (2) The amplification sensitivity is high, and the target fragment can be effectively amplified under the condition that target sequences as low as one ten thousandth exist in a sample. (3) The sample type has high adaptability, is suitable for common genome DNA samples, and can effectively and specifically amplify meat samples, blood samples and bone samples.
For amplification accuracy and specificity, screening was performed as follows: genomic DNAs of cattle (cattle), pig (pig), horse (horse), donkey (ass), sheep (dog), rabbit (rabbit), chicken (chicken), duck (duck), goose (goose) are used as templates, and the templates are amplified respectively by a multiplex LAMP method by using a plurality of sets of designed alternative primer probe sets, and negative quality control is set. The accuracy and the specificity of the designed primers and probes are determined by detecting the amplification kinetic curve of the fluorescent excitation of the probes in real time.
The primer probe group I and the primer probe group II are respectively designed into 2 sets of alternatives, the primer probe group I and the primer probe group II are used for amplification by taking the genomic DNAs of the 10 animals as templates, and the result shows that the primer probe group I has nonspecific amplification when taking the genomic DNAs of horses, dogs and rabbits as templates (figure 1), the primer probe group II has specific amplification when taking the genomic DNAs of the cows as templates, and has no nonspecific amplification when taking the genomic DNAs of other 9 animals as templates (figure 2). 2 sets of alternatives are designed for the pig species specific primer probe group, namely a primer probe group III and a primer probe group IV, and the primer probe group III and the primer probe group IV are respectively amplified by taking the genomic DNAs of the 10 animals as templates, and the result shows that the primer probe group III has nonspecific amplification when the genomic DNAs of donkeys and rabbits are taken as templates (figure 3), the primer probe group IV has specific amplification when the genomic DNAs of pigs are taken as templates, and has no nonspecific amplification when the genomic DNAs of other 9 animals are taken as templates (figure 4).
Experimental results show that after the primer probe groups II and IV amplify samples, samples from different species can be distinguished from samples with bovine species cytb targeted genes or porcine species cytb targeted genes. In addition, no false positive and false negative results appear, consistent with the expected results.
Therefore, the final determination is carried out to obtain the primer probe groups II and IV by screening as the bovine species specific primer probe group and the porcine species specific primer probe group respectively. Wherein the nucleotide sequences of the bovine species-specific primer group (first nucleic acid composition) are shown in SEQ ID NO. 1-SEQ ID NO. 7, respectively:
the nucleotide sequence of cytb-FIP-1 is shown as SEQ ID NO: 1:
5’-AGCTCCGTTTGCGTGTATGTATCGATACACTACACATCCGACACAACAA-3’；
the nucleotide sequence of cytb-BIP-1 is shown as SEQ ID NO. 2:
5’-ATATGCACGTAGGACGAGGCTTATAAGCAGAAGGATTACTCCAATATTTC-3’；
the nucleotide sequence of cytb-F3-1 is shown as SEQ ID NO. 3;
5’-ATCCTCACAGGCCTATTCCTAG-3’；
the nucleotide sequence of cytb-B3-1 is shown as SEQ ID NO. 4:
5’-TCCTATAAATGCTGTGGCTATTAC-3’；
the nucleotide sequence of cytb-LF-1 is shown as SEQ ID NO. 5:
5’-ATTCAGCCGTAGTTCACGTCTC-3’；
the nucleotide sequence of cytb-LB-1 is shown as SEQ ID NO: 6:
5’-TACGGGTCTTACACTTTTCTAGA-3’。
the nucleotide sequence of the bovine species-specific probe (first fluorescent probe) is shown in SEQ ID NO: 7:
5’-AGCATTCTCCTC[dT-ROX][8-oxoG][BHQ2-dT]TACCCATATCTG-[3’-block]。
the nucleotide sequences of the finally determined pig species-specific primer set (second nucleic acid composition) are shown in SEQ ID NO. 8-SEQ ID NO. 14, respectively:
the nucleotide sequence of cytb-FIP-2 is shown in SEQ ID NO. 8:
5’-TGCTCCGTTTGCATGTAGATAGCGATACACTACACATCCGACACAACAA-3’；
the nucleotide sequence of cytb-BIP-2 is shown as SEQ ID NO: 9:
5’-TCATCCACGTAGGCCGAGGTCTAGCAGAAGGATTACTCCAATATTTC-3’
the nucleotide sequence of cytb-F3-2 is shown as SEQ ID NO. 10:
5’-ATCCTCACAGGCCTATTCCTAG-3’
the nucleotide sequence of cytb-B3-2 is shown as SEQ ID NO. 11:
5’-TCCTATAAATGCTGTGGCTATTAC-3’
the nucleotide sequence of cytb-LF-2 is shown as SEQ ID NO. 12:
5’-ATTCAGCCGTAGTTCACGTCTC-3’
the nucleotide sequence of cytb-LB-2 is shown as SEQ ID NO: 13:
5’-TACGGGTCTTACACTTTTCTAGA-3’。
the pig species specific probe (second fluorescent probe) is shown in SEQ ID NO. 14:
5’-GCTTTCTCA[dT-FAM]CA[8-oxoG][BHQ1-dT]TACACACATTTGTC-[3’-block]。
example 2 specificity verification of the detection method
Constructing plasmids containing target fragments of cytb genes of 10 animals including cattle, pigs, horses, donkeys, sheep, dogs, rabbits, chickens, ducks and geese. Bovine and porcine cytb plasmids were mixed with the cytb plasmid mixture of the other 9 animals, respectively, and the total DNA content was 0.1ng, so that the bovine cytb plasmids respectively accounted for 10%,1%,0.1%,0.01%,0%, as templates, were added to the following table, LAMP amplification was performed on the templates, respectively, and negative quality control was set.
TABLE 2 amplification System of this example
After the reagent is added, the mixture is placed on a real-time fluorescence quantitative PCR instrument, incubated at the constant temperature of 65 ℃ for 30min, fluorescence signals are monitored every 30s, and the types and the intensities of the fluorescence signals are recorded.
Because the first fluorescent probe aiming at the bovine cytb gene and the second fluorescent probe aiming at the porcine cytb gene are different in fluorescence reporter group, after the reaction is finished, whether the bovine-derived target gene and the porcine-derived target gene are successfully amplified can be judged through different fluorescent colors, so that the specificity of the designed LAMP method for single-target amplification is determined.
The results showed that the LAMP reactions with 10% to 0.01% of bovine or porcine cytb plasmid as template all had fluorescent probe excitation, while the amplification with animal plasmid mixture as template (0%) had no fluorescent probe excitation, indicating that the detection method can amplify bovine cytb target gene containing 0.01% (ten-thousandth) or porcine cytb target gene containing 0.01% (ten-thousandth), and the reaction system has good specificity (FIGS. 5 and 6).
In addition, the detection method established by the application is compared with the dye method which is not introduced with a specific fluorescent probe and is commonly used in the past. The amplification system of the dye method is not added with a corresponding fluorescent probe, and the rest steps are the same as the above. The results showed that the detection specificity was lower compared to the present detection method by using the dye method to amplify the cytb plasmid containing 0.1% bovine and the cytb plasmid containing 0.1% porcine, respectively (FIGS. 7 and 8).
Example 3 sensitivity verification of detection method
The sensitivity of the assay was evaluated by a series of fold ratio dilutions of the recombinant DNA plasmid: 10000 copies, 1000 copies, 500 copies, 100 copies, 50 copies of the bovine cytb plasmid or the porcine cytb plasmid constructed in example 2 were used as templates, and the substances in the following table were added to perform multiplex LAMP amplification of the above templates, respectively, while setting negative quality control.
TABLE 3 amplification System of this example
After the reagent is added, the mixture is placed on a real-time fluorescence quantitative PCR instrument, incubated at the constant temperature of 65 ℃ for 30min, fluorescence signals are monitored every 30s, and the types and the intensities of the fluorescence signals are recorded.
Because the first fluorescent probe aiming at the bovine cytb gene and the second fluorescent probe aiming at the porcine cytb gene are different in fluorescence reporter group, after the reaction is finished, whether the bovine-derived target gene and the porcine-derived target gene are successfully amplified can be judged through different fluorescent colors, so that the sensitivity of the designed LAMP method to single-target amplification is determined.
The results showed that amplification curves for either the LAMP reactions using either the bovine cytb plasmid or the porcine cytb plasmid as template, which were diluted in a gradient (from 10000 copies to 50 copies), showed that the sensitivity of the detection method reached 50 copies/reaction (FIGS. 9 and 10).
In addition, the composition provided by the application is compared with the dye which is not introduced with a specific fluorescent probe and is commonly used in the past. The amplification system of the dye method is not added with a corresponding fluorescent probe, and the rest steps are the same as the above. The amplification results showed that the detection sensitivity was lower than that of the present detection method by using the dye method to amplify 100 copies of bovine cytb plasmid or 500 copies of porcine cytb plasmid, respectively (FIGS. 11 and 12).
Example 4 detection of genomic DNA samples
Purchasing beef, pork, chicken and duck samples from a Wolmar shopping square in elm secondary region of jin in, and taking 5 cases for later use; pork is added into beef according to the proportion of 50%,20%,10%,1% and 0.1%, so as to prepare simulation samples of the adulterated beef, and 3 cases of adulterated beef with different proportions are respectively taken for standby.
Extracting genome DNA from the beef, pork, chicken and duck meat samples and 5 proportion adulterated beef analog samples to obtain respective purified genome DNA samples, and detecting genes related to the application, wherein the specific operation steps are as follows: the centrifuge tube was taken and added with the following substances (each tube was added with the bovine species-specific primer probe set and the porcine species-specific primer probe set), and the amplification system was as shown in Table 4:
TABLE 4 amplification System of this example
The centrifuge tube is placed in a real-time fluorescent quantitative PCR instrument or a water bath kettle and incubated for 30min at a constant temperature of 65 ℃. The fluorescent signal was monitored every 30s and the fluorescent signal type and intensity were recorded.
And judging the meat identification result according to the real-time detection of the real-time fluorescent quantitative PCR instrument or the generation of amplified products of the end-point fluorescent detection. Because the first fluorescent probe aiming at the bovine cytb gene and the second fluorescent probe aiming at the porcine cytb gene are different in labeled fluorescent reporter groups, whether the sample contains the bovine-derived target gene and the porcine-derived target gene can be judged by different fluorescent colors.
The experimental results show that the amplification kinetic curve using the beef sample as a template has bovine species-specific probe fluorescence (ROX) excitation and no porcine species-specific probe Fluorescence (FAM) excitation, so that the beef sample is judged to contain beef-derived components and does not contain pork-derived components, and the result is consistent with the true source species of the sample (figure 13); amplification with pork sample as template, with excitation of pig species specific probe Fluorescence (FAM) and no excitation of bovine species specific probe fluorescence (ROX), thus discriminating as containing pork-derived component, not beef-derived component, and the discrimination result being consistent with the true source species of the sample (fig. 14); amplification with the adulterated beef analog sample as a template, and excitation of bovine species-specific probe fluorescence (ROX) and porcine species-specific probe Fluorescence (FAM) simultaneously, so as to judge that the beef-derived component and the pork-derived component are contained, wherein the judgment result is consistent with the real source of the sample (figure 15); amplification with chicken samples as templates, without excitation of bovine species-specific probe fluorescence (ROX) and porcine species-specific probe Fluorescence (FAM), resulted in discrimination of being free of beef-derived and pork-derived components, consistent with the true source of the samples (fig. 16); amplification with negative control as template, without bovine species-specific probe fluorescence (ROX) and porcine species-specific probe Fluorescence (FAM) excitation, precluded false positives (fig. 17).
The experimental statistical results are shown in table 5, and the detection results of 35 samples are consistent with the corresponding real sources, so that the method is reliable in application to purifying the genomic DNA samples.
TABLE 5 sample amplification results of this example
Example 5 detection of meat samples
The genes according to the present application were tested using the composition according to the present application using the meat sample prepared in example 4, and the specific procedure was as follows: adding a small amount of meat sample into a centrifuge tube, sucking 100 mu L of sample treatment liquid into the centrifuge tube by using a liquid transfer pipette, and standing for 5min for later use; the centrifuge tube was taken and added with the following substances (each tube was added with the bovine species-specific primer probe set and the porcine species-specific primer probe set), and the amplification system was as shown in Table 6:
TABLE 6 amplification System of this example
The centrifuge tube is placed in a real-time fluorescent quantitative PCR instrument or a water bath kettle and incubated for 30min at a constant temperature of 65 ℃. The fluorescent signal was monitored every 30s and the fluorescent signal type and intensity were recorded.
And judging the meat identification result according to the real-time detection of the real-time fluorescent quantitative PCR instrument or the generation of amplified products of the end-point fluorescent detection. Because the first fluorescent probe aiming at the bovine cytb gene and the fluorescent reporter group marked by the second fluorescent probe aiming at the porcine cytb gene are different, whether the sample contains the bovine-derived target gene and the porcine-derived target gene can be judged by different fluorescent colors.
The experimental statistical results are shown in table 7, and the detection results of 35 samples are consistent with the corresponding real sources, so that the method is reliable when applied to meat samples.
TABLE 7 sample amplification results of this example
Example 6 detection of blood samples
Purchasing bovine blood, pig blood, chicken blood and duck blood samples from a Wolmart shopping square in elm secondary region of Jinzhong, and taking 5 cases for later use; pig blood is added into cow blood according to the proportion of 50%,20%,10%,1% and 0.1%, so as to prepare simulation samples of the adulterated cow blood, and 3 cases of adulterated cow blood with different proportions are taken for standby.
The blood sample is used as a template to detect the gene related to the application, and the specific operation steps are as follows: adding 2 mu L of blood sample into a centrifuge tube, sucking 20 mu L of sample treatment liquid into the centrifuge tube by using a pipette, and standing for 5min for standby; the centrifuge tube was taken and added with the following substances (each tube was added with bovine species-specific primer probe set and porcine species-specific primer probe set), and the amplification system was as shown in Table 8:
TABLE 8 amplification System of this example
The centrifuge tube is placed in a real-time fluorescent quantitative PCR instrument or a water bath kettle and incubated for 30min at a constant temperature of 65 ℃. The fluorescent signal was monitored every 30s and the fluorescent signal type and intensity were recorded.
And judging and reading the blood sample identification result according to the real-time detection of the real-time fluorescent quantitative PCR instrument or the generation of the amplification product of the end-point fluorescent detection. Because the first fluorescent probe aiming at the bovine cytb gene and the fluorescent reporter group marked by the second fluorescent probe aiming at the porcine cytb gene are different, whether the sample contains the bovine-derived target gene and the porcine-derived target gene can be judged by different fluorescent colors.
The experimental statistical results are shown in table 9, and the detection results of 35 samples are consistent with the corresponding real sources, so that the method is reliable when applied to blood samples.
TABLE 9 sample amplification results of this example
Finally, it should be noted that the above description is only for illustrating the technical solution of the present application, and not for limiting the scope of the present application, and that the simple modification and equivalent substitution of the technical solution of the present application can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present application.
Claims (10)
1. A composition for meat adulteration identification, wherein said composition comprises a first nucleic acid composition, a second nucleic acid composition and a fluorescent probe set;
wherein the first nucleic acid composition comprises:
cytb-F3-1，cytb-B3-1，cytb-FIP-1，cytb-BIP-1，cytb-LF-1，cytb-LB-1；
the second nucleic acid composition comprises:
cytb-F3-2，cytb-B3-2，cytb-FIP-2，cytb-BIP-2，cytb-LF-2，cytb-LB-2；
the nucleotide sequence of cytb-FIP-1 is shown as SEQ ID NO. 1;
the nucleotide sequence of cytb-BIP-1 is shown as SEQ ID NO. 2;
the nucleotide sequence of cytb-F3-1 is shown as SEQ ID NO. 3;
the nucleotide sequence of cytb-B3-1 is shown as SEQ ID NO. 4;
the nucleotide sequence of cytb-LF-1 is shown as SEQ ID NO. 5;
the nucleotide sequence of cytb-LB-1 is shown as SEQ ID NO. 6;
the nucleotide sequence of cytb-FIP-2 is shown as SEQ ID NO. 8;
the nucleotide sequence of cytb-BIP-2 is shown as SEQ ID NO. 9;
the nucleotide sequence of cytb-F3-2 is shown as SEQ ID NO. 10;
the nucleotide sequence of cytb-B3-2 is shown as SEQ ID NO. 11;
the nucleotide sequence of cytb-LF-2 is shown as SEQ ID NO. 12;
the nucleotide sequence of cytb-LB-2 is shown as SEQ ID NO. 13.
2. The composition of claim 1, wherein the GC content of the first and second nucleic acid compositions is between 30% -70%; and/or
T of the cytb-FIP-1, cytb-BIP-1, cytb-FIP-2 and cytb-BIP-2 m Values between 55 ℃ and 70 ℃; and/or
T of cytb-F3-1, cytb-B3-1, cytb-F3-2 and cytb-B3-2 m Values between 50 ℃ and 65 ℃; and/or
T of the cytb-LF-1, cytb-LB-1, cytb-LF-2 and cytb-LB-2 m The values are between 50℃and 65 ℃.
3. The composition of claim 1 or 2, wherein the set of fluorescent probes comprises at least two fluorescent probes:
at least one first fluorescent probe for the bovine cytb gene and at least one second fluorescent probe for the porcine cytb gene;
wherein a first fluorescent probe is used in combination with a first nucleic acid composition and a second fluorescent probe is used in combination with a second nucleic acid composition;
preferably, the fluorescent probe binds to the Loop region of the LAMP amplicon;
preferably, T of the fluorescent probe m Has a value between 55℃and 70℃and/or
The GC content of the fluorescent probe is between 30% and 70%, and/or
The length of the fluorescent probe is between 20 and 60 bases.
4. The composition of claim 3, wherein the fluorescent probe comprises deoxyribonucleotides wherein one base is replaced with a modified base;
preferably, both sides of the deoxyribonucleotide are connected with a fluorescent reporter group and a quenching group;
preferably, the modified base is selected from one of 7, 8-dihydroxy-8-oxoguanine, 8-hydroxy adenine, fame-guanine, methyl-fame-guanine, fame-adenine, aflatoxin B1-fame-guanine, 5-hydroxy-cytosine and 5-hydroxy-uracil.
5. The composition of claim 4, wherein the fluorescent reporter group is selected from at least one of FAM, SIMA, HEX, ROX, TAMRA, texas Red and CalFluor 610;
and/or the quenching group is selected from at least one of BHQ1, BHQ2, BHQ3, MGB, dabcyl and Eclipse;
and/or, the fluorescent reporter group is 1-5 bases apart from the quencher group.
6. The composition of claim 4, wherein 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, polyhexamethylene glycol, inverted dT, inverted dG, and dideoxynucleotide.
7. The composition of claim 5, wherein 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.
8. A kit for meat adulteration identification, characterized in that the kit comprises a composition according to any of claims 1-7.
9. Use of a composition according to any one of claims 1-6 or a kit according to claim 8 in meat adulteration identification.
10. A method of identifying meat adulteration, said method comprising the steps of:
(1) Adding a sample treatment liquid into a sample to be detected to extract or release nucleic acid;
(2) Amplifying the nucleic acid obtained in step (1) using the composition of any one of claims 1-6 or the kit of claim 8;
(3) And judging the result.
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