CN117025791A - Composition for detecting food meat origin, kit containing same and application - Google Patents
Composition for detecting food meat origin, kit containing same and application Download PDFInfo
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- CN117025791A CN117025791A CN202311125109.3A CN202311125109A CN117025791A CN 117025791 A CN117025791 A CN 117025791A CN 202311125109 A CN202311125109 A CN 202311125109A CN 117025791 A CN117025791 A CN 117025791A
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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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- 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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Abstract
The invention belongs to the technical field of gene detection, and particularly relates to a composition for detecting food meat origin, a kit containing the composition and application of the kit. The invention provides a composition for detecting food meat origin, which comprises a first nucleic acid composition, a second nucleic acid composition and a fluorescent probe set, wherein the nucleotide sequence of the first nucleic acid composition is shown as SEQ ID NO. 1-6; the nucleotide sequence of the second nucleic acid composition is shown in SEQ ID NO. 7-12. The composition has high sensitivity, high detection efficiency and high specificity, and can be used for rapidly detecting the meat origin of food.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a composition for detecting food meat origin, 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 traditional method for identifying the meat origin of the food comprises the following steps: morphological methods, immunological methods, protein identification methods based on mass spectrometry and chromatography, and nucleic acid-based molecular biological methods. The most commonly used molecular biology method has higher sensitivity, specificity and accuracy compared with other methods; common molecular biology techniques mainly include Polymerase Chain Reaction (PCR), multiplex PCR, real-time fluorescent quantitative PCR techniques, digital PCR techniques, sequencing techniques, and the like. However, these methods have generally had certain drawbacks such as long time consumption, cumbersome operation, and reliance on sophisticated instruments and the like.
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, rapid, sensitive and specific technical scheme needs to be established to realize rapid detection of meat and meat product sources.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a composition for detecting the meat origin of food and a kit containing the composition.
According to the invention, on the basis of adding a plurality of pairs of primers, a specific probe is added, and an abasic site is added in the probe, and the site is specifically identified and fluorescence is activated by using a DNA repair enzyme, so that compared with the existing fluorescent probe based on the strand displacement reaction, the specificity and the signal to noise ratio of the reaction are further improved, and the rapid amplification of nucleic acid and the detection of multiple LAMP (loop-mediated isothermal amplification) on multiple targets in a monomer system can be realized.
The technical scheme provided by the invention is as follows:
in a first aspect, the present invention provides a composition for detecting the meat origin of a food product,
the composition comprises 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-F3-1 is shown as SEQ ID NO. 1;
the nucleotide sequence of cytb-B3-1 is shown as SEQ ID NO. 2;
the nucleotide sequence of cytb-FIP-1 is shown as SEQ ID NO. 3;
the nucleotide sequence of cytb-BIP-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-F3-2 is shown as SEQ ID NO. 7;
the nucleotide sequence of cytb-B3-2 is shown as SEQ ID NO. 8;
the nucleotide sequence of cytb-FIP-2 is shown as SEQ ID NO. 9;
the nucleotide sequence of cytb-BIP-2 is shown as SEQ ID NO. 10;
the nucleotide sequence of cytb-LF-2 is shown as SEQ ID NO. 11;
the nucleotide sequence of cytb-LB-2 is shown as SEQ ID NO. 12.
In one embodiment, the set of fluorescent probes comprises at least two fluorescent probes:
at least one first fluorescent probe directed against the porcine CYTB gene and at least one second fluorescent probe directed against the bovine CYTB gene.
In one embodiment, the first fluorescent probe is positioned between cytb-FIP-1 and cytb-BIP-1 and the second fluorescent probe is positioned between cytb-FIP-2 and cytb-BIP-2.
In one embodiment, the first T base and the second T base in the fluorescent probe are each replaced with a recombinant T base to which a fluorescent group and a quenching group are attached.
In one embodiment, the distance between the first T base and the second T base is 1-5 bases.
In a preferred embodiment, the distance between the first T base and the second T base may be 1, 2, 3, 4, 5 bases.
In one embodiment, the fluorophore is selected from FAM, HEX, cy, VIC, ROX, NED or Cy5;
and/or the quenching group is selected from BHQ1, BHQ2 or BHQ650.
In one embodiment, any deoxyribonucleotide between the first T base and the second T base is replaced with a tetrahydrofuran.
In one embodiment, the third T base between the recombinant first T base and the second T base is replaced with 5, 6-dihydroxythymine or thymine ethylene glycol.
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, biotin-TEG, and dideoxycytidine.
In a preferred embodiment, the nucleotide sequence of the first fluorescent probe is shown as SEQ ID NO. 13 and the nucleotide sequence of the second fluorescent probe is shown as SEQ ID NO. 14.
In a second aspect, the invention provides a kit for detection of food meat origin, the kit comprising the kit of the first aspect.
In one embodiment, the kit further comprises at least one of a sample processing fluid, dNTPs, a DNA polymerase, a UDG enzyme, an endonuclease, glycerol, betaine, and bovine serum albumin.
In a preferred embodiment, the endonuclease comprises endonuclease III and/or endonuclease IV.
In one embodiment, the sample processing fluid comprises a nonionic surfactant and/or an ionic surfactant.
In a preferred embodiment, the sample treatment fluid comprises 1mM-200mM KOH,1mM-500mM NaOH,10% -90% PEG-400,0.1M-5M NaCl,0.001% -5% Tween-80,0.1mM-30mM EDTA,0.1% -20% Triton X-100,0.1% -10% ethylphenyl polyethylene glycol, and 1% -10% SDS solution.
In a preferred embodiment, the dNTPs comprise 0.1mM-4mM dATP,0.1mM-4mM dTTP,0.1mM-4mM dCTP,0.1mM-4mM dGTP and 0.1mM-4mM dUTP.
In a third aspect, the present invention provides a method of detecting the meat origin of a food product, 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 system for amplifying of step (2) comprises 1mM-100mM Tris-HCl,10mM-800mM KCl,0.1mM-60mM MgSO 4 ，1mM-70mM NaCl，0.1mM-100mM(NH 4 ) 2 SO 4 0.1mM-5mM dNTP mix,0.1U-20U Bst DNA polymerase, 0.01U-20U uracil-DNA glycosylase (UDG enzyme), 1U-50U endonuclease III or endonuclease IV,0.1% -10% glycerol, 0.1M-15M betaine and 0.1mg/mL-10mg/mL bovine serum albumin.
Compared with the prior art, the composition and/or the kit provided by the invention have at least the following beneficial effects:
(1) The sensitivity is high, and target DNA as low as 100copies can be detected;
(2) The detection efficiency is high, and the nucleic acid taking DNA as a template is directly amplified;
(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 the results of the specificity verification of the optimal primer probe set targeting the porcine cytb gene.
FIG. 2 shows the results of the specificity verification of the optimal primer probe set targeting the bovine cytb gene.
FIGS. 3 and 4 show amplification kinetics curves for 2 non-optimal primer probe sets for the pig cytb gene.
FIGS. 5 and 6 show amplification kinetics curves for 2 non-optimal primer probe sets for the bovine cytb gene.
Fig. 7 and 8 show the results of sensitivity verification of the composition of the present invention.
Fig. 9 and 10 show the results of verification of the specificity of the composition of the present invention.
Fig. 11 and 12 show the results of sensitivity verification of the fluorescent dye assay.
FIGS. 13 and 14 show the results of the specificity verification of the fluorescent dye assay.
FIG. 15 shows the amplification kinetics of the test samples from a mixture of pigs and cattle.
FIG. 16 shows the amplification kinetics curves when the sample to be tested is from a pig.
FIG. 17 shows the amplification kinetics curves when the sample to be tested is from cattle.
FIG. 18 shows amplification kinetics curves for samples to be tested that are non-porcine and non-bovine samples.
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 invention 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 invention 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 invention 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 invention. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention 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 invention. Such structures and techniques are also described in a number of publications.
The invention provides a nucleic acid fluorescent probe based on DNA repair enzyme activation and a method for rapidly detecting food meat origin by using the same, which comprises the following steps:
step 1: sample acquisition: the sample type may be a tissue sample, a blood sample, a bone sample, etc.
Step 2: sample processing: taking the sample to be tested in the step 1, adding 1 mu L-100 mu L of sample treatment liquid, standing for 1min-10min, and directly taking the sample as a template to perform the next reaction;
step 3: aiming at cytb genes of samples to be detected, a 2-tube LAMP reaction system is configured, two tubes are the same in reaction condition, wherein one tube is subjected to LAMP reaction with ddH 2 O is used as a template and is used as a negative control, two reaction tubes are simultaneously placed under a constant temperature condition (59 ℃ to 66 ℃) to obtain an amplification product, and an amplification reaction system is shown in table 1;
step 4: and (3) carrying out real-time fluorescence detection on the reaction solution in the step (3) in a real-time fluorescence PCR instrument. And judging whether the detected sample has a corresponding pig or beef sample according to the amplification kinetic curve or the final fluorescence signal intensity after the amplification is finished.
TABLE 1 amplification System according to the invention
If only amplification kinetic curves or final fluorescent signals representing swine-derived or bovine-derived cytb target genes appear, and the negative control has no fluorescent signals, the corresponding swine-derived or bovine-derived sample is obtained; if the amplification kinetic curves or the final fluorescence signals representing the swine-derived and bovine-derived cytb target genes are monitored and the negative control has no fluorescence signal, the mixed meat product of the swine-derived and bovine-derived cytb target genes is obtained; if neither amplification kinetics curves nor terminal fluorescent signals occur, then neither a porcine-nor bovine-derived sample is obtained.
Example 1 design and optimization of probes and primers for porcine cytb Gene and bovine cytb Gene
Primers were designed for cytb genes, and the sequences of 13 species (horse, cow, donkey, sheep, goat, dog, cat, rabbit, chicken, duck, goose, carp, grass carp, and crucian carp) were analyzed by NCBI to determine the sequences of the species, and specific probes and primers were designed based on the results. 3 pairs of primers (inner primer, outer primer, loop primer) and a specific probe are designed for each gene sequence, and 9 independent areas on the target sequence are respectively and specifically identified. To increase amplification specificity and detection resolution, multiple sets of probes and primer pairs are designed for each sequence for experimental screening.
Screening the optimal probes and primers according to the following targets: (1) high amplification specificity: the pig, cow and other animal samples can be distinguished, if the species specific gene cytb targeting genes from the pig or cow exist in the sample to be detected, an amplification kinetic curve or a final fluorescent signal can be observed within 30min, and the other animal samples are negative amplification; (2) high amplification sensitivity: fragments of interest as low as 100copies can be detected; (3) The method has high adaptability to sample types, is suitable for common DNA samples, and can effectively and specifically amplify tissue samples, blood samples and the like which are taken by hands.
The screening is carried out according to the following steps: the DNA templates of the above species are amplified by LAMP method by using known and determined pork or beef genome DNA and DNA of other species as templates and using a plurality of sets of designed alternative probes and primer groups, and negative quality control is set. The accuracy and specificity of the designed probes and primers were determined by detecting the amplification kinetics curves or the final fluorescent signals, and the amplification system is shown in Table 2:
TABLE 2 amplification System of this example
The finally designed pig cytb gene primer group (first nucleic acid composition) is shown in SEQ ID NO. 1-SEQ ID NO. 6, specifically:
the nucleotide sequence of cytb-F3-1 is shown as SEQ ID NO: 1:
5’-AGGAGCTACGGTCATCACAA-3’；
the nucleotide sequence of cytb-B3-1 is shown as SEQ ID NO. 2:
5’-GTATGGGTGAAATGGAATTTTGTCT-3’；
the nucleotide sequence of cytb-FIP-1 is shown as SEQ ID NO: 3:
5’-GGTTGCTTTGTCGACGGAAAAGCATCAGCTATCCCTTATATCGGAACA-3’；
the nucleotide sequence of cytb-BIP-1 is shown as SEQ ID NO. 4:
5’-TATCCTGCCATTCATCATTACCGCCTCCGGTAGGGTTGTTGGATC-3’；
the nucleotide sequence of cytb-LF-1 is shown as SEQ ID NO. 5:
5’-CCCTCAGATTCATTCTACGAGGTC-3’；
the nucleotide sequence of cytb-LB-1 is shown as SEQ ID NO: 6:
5’-CTCGCAGCCGTACATCTCA-3’；
the sequence of the pig cytb gene specific probe (first fluorescent probe) is shown in SEQ ID NO. 13:
5’-CTCACACGA[FAM-dT]T[THF][BHQ1-dT]TCGCCTTCCACTT[3’-block]-3’。
the nucleotide sequence of the finally designed bovine cytb gene specific primer group (second nucleic acid composition) is shown in SEQ ID NO 7-SEQ ID NO 12, specifically:
the nucleotide sequence of cytb-F3-2 is shown in SEQ ID NO: 7:
5’-GGCCCTCTTACTAATTCTAGCTCTA-3’；
the nucleotide sequence of cytb-B3-2 is shown as SEQ ID NO. 8:
5’-TGCTTCGTTGTTTGGAGGT-3’；
the nucleotide sequence of cytb-FIP-2 is shown as SEQ ID NO. 9:
5’-GAGGGGGTGTGTTGAGTGGATTTACTATTCGCACCCGACCTC-3’；
the nucleotide sequence of cytb-BIP-2 is shown as SEQ ID NO. 10:
5’-TACGCAATCTTACGATCAATCCCCAAGCAAGAATTAGGATAGAGAAGGC-3’；
the nucleotide sequence of cytb-LF-2 is shown as SEQ ID NO. 11:
5’-TGTAGTTATCTGGGTCTCCGAG-3’；
the nucleotide sequence of cytb-LB-2 is shown as SEQ ID NO. 12:
5’-ACAAACTAGGAGGAGTACTAGCC-3’；
the nucleotide sequence of the bovine cytb gene specific probe (second fluorescent probe) is shown in SEQ ID NO. 14:
5’-ATCAAACCCGAG[ROX-dT][THF]A[BHQ2-dT]ACTTCTTATTTGC[3’-block]-3’。
experimental results show that after the optimized probes and primers amplify the samples, the species specific genes cytb of pigs or cattle can be judged and distinguished according to amplification dynamics curves or final fluorescent signals; in addition, no false positives occurred and were consistent with the expected results (fig. 1 and 2). The probe primer groups A and B of the target pig cytb specific sequence are subjected to nonspecific amplification, and the amplification efficiency is low (shown in figures 3 and 4), so that the target pig cytb specific sequence is eliminated in the screening process; neither the probe primer set C nor the probe primer set D, which target bovine cytb specific sequences, showed amplification curves (FIGS. 5 and 6), which were rejected during the screening process.
Example 2 detection sensitivity and specificity test
Recombinant DNA containing 15 animal cytb gene sequences as described in example 1 was constructed, and 13 plasmid DNAs of porcine origin and bovine origin were mixed to obtain a mixed plasmid (30000 copies/. Mu.L). A series of concentrations of mixed plasmids of porcine and bovine origin plasmids (10000 copies/. Mu.L, 5000 copies/. Mu.L, 1000 copies/. Mu.L, 500 copies/. Mu.L, 100 copies/. Mu.L) and other species were single-target amplified to ddH using the optimal probe primer set of example 1 2 O served as a blank control and the amplification system is shown in table 3:
TABLE 3 amplification System of this example
The centrifuge tube is placed in a real-time fluorescence PCR instrument, and is incubated for 30min at a constant temperature of 65 ℃.
The amplification curve was obtained from the fluorescence signal detected by the instrument, and the result is shown in FIG. 5.
The experimental results show that the detection lower line of the system is 100copies (figures 7 and 8); no amplification curves were obtained for the mixed plasmids of the other species (FIGS. 9 and 10), and the specificity of the reaction system was good.
Example 3 fluorescent dye detection
LAMP amplification of the plasmid templates described in example 2 was performed using fluorescent dyes and the optimal primer set described in example 1, the amplification system being as shown in Table 4:
TABLE 4 amplification System of this example
An amplification curve was obtained from the fluorescence signal detected by the instrument in the same manner as in example 2.
Experimental results show that under the system, the detection lower line of the two systems is 500copies (figures 11 and 12); nonspecific amplification occurred for the mixed plasmids of other species (FIGS. 13 and 14), and the sensitivity and specificity of the reaction system were reduced.
Example 4 detection of blood samples
The present embodiment describes, as an example, the detection of a blood sample:
taking 5 blood samples (A1-A5) from pork, 5 blood samples (A6-A10) from beef, 1 blood sample (A11) from mutton and 1 blood sample (A12) from chicken; in addition, according to 1:1, respectively preparing the mixed samples of the blood from different sources (B1: pork and beef, B2: pork and mutton, B3: pork and chicken, B4: beef and mutton, and B5: beef and chicken). The detection by using the composition provided by the invention comprises the following specific operation steps: for each sample, 2 mu L of blood sample is added into a 0.2mL centrifuge tube, 10 mu L of sample treatment liquid is sucked by a liquid mover, added into the centrifuge tube, and kept stand for 1min for standby; two 0.2mL centrifuge tubes were taken and added with the following substances (ddH as template in tube B) 2 O, as a blank negative control), the amplification system is shown in table 5:
TABLE 5 amplification System of this example
The centrifuge tube was placed in a fluorescence detector and incubated at a constant temperature of 64℃for 30min.
Monitoring amplification kinetic curves generated in the amplification process of the A, B two tubes and final fluorescent signals after amplification are finished, wherein the fluorescent signals of FAM and ROX are amplified, and the sample to be detected is marked as a mixed blood sample of pigs and cattle (figure 15); the FAM fluorescent signal showed an amplification curve, noted blood from pigs (fig. 16); the amplification curve of the ROX fluorescent signal appears as blood from cattle (FIG. 17); the no amplification curve was noted as a negative sample (fig. 18), which was not derived from pigs nor cattle.
The experimental results show that the detection results of the 17 samples are the same as the corresponding real results, no false positive results appear, and the results are shown in table 6. Indicating that the method is reliable for application to blood samples.
TABLE 6 detection results of this example
| Sample of | Fluorescent signal | Interpretation of results (sample Source) | Accuracy of |
| A1-A5 | FAM | Pig | Accurate and accurate |
| A6-A10 | ROX | Cattle | Accurate and accurate |
| A11 | / | Others | Accurate and accurate |
| A12 | / | Others | Accurate and accurate |
| B1 | FAM/ROX | Mixed blood sample of pig and cattle | Accurate and accurate |
| B2 | FAM | Pig | Accurate and accurate |
| B3 | FAM | Pig | Accurate and accurate |
| B4 | ROX | Cattle | Accurate and accurate |
| B5 | ROX | Cattle | Accurate and accurate |
Example 5 detection of tissue samples
The present embodiment is described by taking tissue sample detection as an example:
using the same tissue samples as in example 4, assays were performed using the compositions provided by the present invention, and the following procedure was followed: sampling tissueThe method comprises the steps of grinding a sample by using a disposable grinding rod, adding a small amount of grinding sample into a 1.5mL centrifuge tube, sucking 100 mu L of sample treatment liquid by using a liquid transfer pipette, adding the sample treatment liquid into the centrifuge tube, and standing for 5min for later use; two 0.2mL centrifuge tubes were taken and added with the following substances (ddH as template in tube B) 2 O, as a blank negative control), the amplification system is shown in table 7:
TABLE 7 amplification System of this example
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The centrifuge tube was placed in a metal bath and incubated at a constant temperature of 64℃for 30min.
The amplification kinetics curves generated during the amplification of the A, B two tubes and the final fluorescent signal after the amplification was monitored and analyzed in the same manner as in example 4.
The experimental results show that the detection results of the 12 samples are the same as the corresponding real results, no false positive results appear, and the results are shown in table 8. Indicating that the method is reliable for application to tissue samples.
TABLE 8 detection results of this example
| Sample of | Fluorescent signal | Interpretation of results (sample Source) | Accuracy of |
| A1-A5 | FAM | Pig | Accurate and accurate |
| A6-A10 | ROX | Cattle | Accurate and accurate |
| A11 | / | Others | Accurate and accurate |
| A12 | / | Others | Accurate and accurate |
Example 4 detection of bone samples
The present embodiment is described by taking tissue sample detection as an example:
the same sample sources as in example 2 were used for detection using the genes according to the invention, and the specific procedure was as follows: taking a bone sample, grinding the bone sample, and extracting DNA; two 0.2mL centrifuge tubes were taken and added with the following substances (ddH as template in tube B) 2 O, as a blank negative control), the amplification reaction system is shown in table 9:
TABLE 9 amplification reaction System of this example
The centrifuge tube was placed in a metal bath and incubated at a constant temperature of 64℃for 30min.
The amplification kinetics curves generated during the amplification of the A, B two tubes and the final fluorescent signal after the amplification was monitored and analyzed in the same manner as in example 4.
The experimental results show that the detection results of the 11 samples are the same as the corresponding real results, no false positive results appear, and the results are shown in table 10. Indicating that the method is reliable for application to bone samples.
Table 10 the test results of this example
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Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A composition for use in food meat origin detection, the composition 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-F3-1 is shown as SEQ ID NO. 1;
the nucleotide sequence of cytb-B3-1 is shown as SEQ ID NO. 2;
the nucleotide sequence of cytb-FIP-1 is shown as SEQ ID NO. 3;
the nucleotide sequence of cytb-BIP-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-F3-2 is shown as SEQ ID NO. 7;
the nucleotide sequence of cytb-B3-2 is shown as SEQ ID NO. 8;
the nucleotide sequence of cytb-FIP-2 is shown as SEQ ID NO. 9;
the nucleotide sequence of cytb-BIP-2 is shown as SEQ ID NO. 10;
the nucleotide sequence of cytb-LF-2 is shown as SEQ ID NO. 11;
the nucleotide sequence of cytb-LB-2 is shown as SEQ ID NO. 12.
2. The composition of claim 1, wherein the set of fluorescent probes comprises at least two fluorescent probes:
at least one first fluorescent probe for a porcine cytb gene and at least one second fluorescent probe for a bovine cytb gene;
preferably, the first fluorescent probe is located between cytb-FIP-1 and cytb-BIP-1 and the second fluorescent probe is located between cytb-FIP-2 and cytb-BIP-2;
preferably, T of the fluorescent probe m The values are 66℃to 71 ℃.
3. The composition of claim 1 or 2, wherein the first T base and the second T base in the fluorescent probe are replaced with a recombinant T base to which a fluorescent group and a quenching group are attached, respectively;
preferably, the distance between the first T base and the second T base is 1-5 bases.
4. A composition according to claim 3, wherein the fluorophore is selected from FAM, HEX, cy3, VIC, ROX, NED or Cy5;
and/or the quenching group is selected from BHQ1, BHQ2 or BHQ650.
5. The composition of claim 4, wherein any deoxyribonucleotide between the first T base and the second T base is replaced with a tetrahydrofuran;
and/or a third T base between the first T base and the second T base is replaced with 5, 6-dihydroxythymine or thymine ethylene glycol;
and/or, the 3' end of the fluorescent probe is modified with a blocking group, and the blocking group is at least one selected from the group consisting of C3Spacer, C6 Spacer, phosphate group, biotin-TEG and dideoxycytidine.
6. The composition of claim 5, wherein the nucleotide sequence of the first fluorescent probe is shown in SEQ ID NO. 13 and the nucleotide sequence of the second fluorescent probe is shown in SEQ ID NO. 14.
7. A kit for detection of food meat origin, characterized in that it comprises a composition according to any one of claims 1-6.
8. The kit of claim 7, further comprising at least one of a sample processing fluid, dNTPs, a DNA polymerase, a UDG enzyme, an endonuclease, glycerol, betaine, and bovine serum albumin;
preferably, the endonuclease comprises endonuclease III or endonuclease IV;
preferably, the sample processing liquid comprises a nonionic surfactant or an ionic surfactant.
9. Use of a composition according to any one of claims 1-6 or a kit according to any one of claims 7-8 in the detection of food meat origin.
10. A method for detecting the meat origin of a food product, 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 any one of claims 1-6 or the kit of any one of claims 7-8;
(3) And judging the result.
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