CN114752690A - Method for rapidly identifying duck-origin components in meat products based on MIRA technology - Google Patents
Method for rapidly identifying duck-origin components in meat products based on MIRA technology Download PDFInfo
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Abstract
The invention provides a method for rapidly identifying duck-origin components in meat products based on an MIRA (micro-induced reduction amplification) technology, belonging to the field of food safety biotechnology. The invention firstly provides a primer probe combination for identifying duck-origin components in meat products, which comprises a forward primer, a reverse primer and a probe; the nucleotide sequence of the forward primer is shown as SEQ ID NO. 9; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 11; the nucleotide sequence of the probe is shown as SEQ ID NO. 13. The invention also provides a kit containing the primer probe combination and a specific identification method. The detection method for identifying the duck-origin components in the meat product has the advantages of mild conditions, simple and convenient operation, strong specificity, high sensitivity, good reliability and visualized results, and is very beneficial to field detection. The detection method provides a new choice for rapidly, simply and sensitively detecting the duck-origin ingredients in the meat product on site, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of food safety biotechnology, and particularly relates to a method for rapidly identifying duck-origin components in meat products based on an MIRA technology.
Background
With the continuous development of market globalization and trade freezation, consumers attach more and more importance to the quality and authenticity of meat products, and the safety of the meat products is improved to a new height. In recent years, meat adulteration at home and abroad occurs, so that the method causes wide social attention and causes serious harm to food safety and food trade. The meat adulteration mode is more and more, and the form is more and more complicated. At present, adulteration of meat products mainly comprises adulteration of raw meat, tissue replacement and the like, and the authenticity problems of the meat not only seriously infringe the interests of consumers, but also bring potential hazards to the edible safety of the meat products.
Therefore, the detection of animal-derived components of meat products becomes more and more important, and the nucleic acid detection can detect trace target molecules from samples, has many advantages such as sensitivity and specificity, becomes a necessary technical means in the research of food authenticity, and is widely applied in the detection field. However, the traditional nucleic acid detection technology has high requirements on laboratory environment, instruments and personnel, and cannot meet the requirements of field detection and supervision.
Some novel detection methods, such as Recombinase Polymerase Amplification (RPA), Multienzyme Isothermal Rapid Amplification (MIRA), and the like, do not require high-temperature denaturation, annealing, and other steps, so that the degree of dependence on a precision instrument is greatly reduced, and the method is an effective technical means for realizing on-site real-time detection. Meanwhile, by combining a Lateral Flow chromatography (LFD) technology, the method not only can achieve the purposes of simple, sensitive and specific detection, but also can better provide visual application service for the detection of the animal source components on site. However, the key to these methods is to find suitable primers, and the choice of primer will affect the detection speed and sensitivity. At present, finding a proper primer is a difficult point of detecting animal-derived ingredients of meat products.
A proper primer is found, a novel method for detecting duck-origin components in meat products is provided, a novel choice is provided for rapidly, simply and sensitively detecting the duck-origin components in the meat products on site, and the method has important significance for food safety.
Disclosure of Invention
The invention aims to provide a method for rapidly identifying duck-origin components in meat products based on an MIRA technology.
The invention provides a primer probe combination for identifying duck-origin components in meat products, which comprises a forward primer, a reverse primer and a probe; the nucleotide sequence of the forward primer is shown as SEQ ID NO. 9; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 11; the nucleotide sequence of the probe is shown as SEQ ID NO. 13.
The invention also provides application of the primer probe combination in identifying duck-origin components in meat products.
Further, the application is the application in the preparation of a kit for identifying duck-origin components in meat products.
Further, the method for identifying duck-origin components in meat products is a multienzyme constant-temperature rapid amplification visualization technology.
Further, the multienzyme isothermal rapid amplification visualization technology is a multienzyme isothermal rapid amplification technology combined with a lateral flow chromatography technology.
The invention also provides a kit for identifying duck-origin components in meat products, which comprises the primer probe combination.
Further, the method for identifying duck-origin components in meat products is a multienzyme constant-temperature rapid amplification visualization technology.
Further, the multienzyme isothermal rapid amplification visualization technology is a multienzyme isothermal rapid amplification technology combined with a lateral flow chromatography technology.
The invention also provides a method for identifying duck-origin components in meat products, which comprises the following steps:
(1) extracting DNA of a sample to be detected as a template;
(2) adding the primer probe combination into an amplification system containing a DNA template;
(3) detecting by using a multienzyme constant-temperature rapid amplification visualization technology;
(4) and reading the result.
Further, in the step (2), the volume ratio of the forward primer, the reverse primer, the probe and the DNA template in the amplification system is 0.1-2: 0.1-2: 0.01-0.6: 1; the volume ratio of the forward primer to the reverse primer is 1: 1;
and/or in the step (2), the reaction temperature of the amplification system is 30-45 ℃; and/or the amplification time is 1-5 min;
preferably, in the step (2), the reaction temperature of the amplification system is 37 ℃; and/or the amplification time is 5 min;
and/or, in the step (2), the concentration of the primer in the amplification system is 10 mu mol/L; and/or the concentration of the probe in the amplification system is 10 mu mol/L.
The invention successfully screens out the optimal primer probe combination of a multienzyme constant-temperature rapid amplification visualization technology detection system for duck-derived components in meat products, and provides an MIRA-LFD detection system for detecting the duck-derived components in the meat products. The MIRA-LFD detection system combines the MIRA technology and the LFD technology, has the advantages of mild detection conditions, simple and convenient operation, strong specificity, high sensitivity, good reliability, visual result and contribution to field detection. The detection method provided by the invention has good reliability, timeliness, operability and safety, provides a new choice for rapidly, simply and sensitively detecting the duck-origin ingredients of the meat product on site, and has a wide application prospect.
The research aims to improve the operability of the detection method of duck-origin ingredients in meat products, establish a detection method of animal-origin in sound meat products, establish a special multienzyme constant-temperature rapid amplification visualization technology, namely a multienzyme constant-temperature lateral flow amplification technology (MIRA-LFD) detection method aiming at duck meat products in a project group, and provide a technical means for field detection of duck-origin ingredients for supervision departments.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows an alignment of duck 12S (U59666.1) derived target sequences.
FIG. 2 shows an alignment of duck cytb (EU755252) derived target sequences.
FIG. 3 shows the result of positive control screening of duck-origin primers.
FIG. 4 shows the result of duck-origin primer probe blank control screening.
FIG. 5 shows the result of negative control screening of duck-origin primer probe.
FIG. 6 shows the result of negative control screening of duck-origin primer probe.
FIG. 7 shows the results of screening the probe volumes of the primers in the MIRA-LFD detection system.
FIG. 8 is a graph of the results of the MIRA-LFD experiments under different temperature conditions.
FIG. 9 is a graph showing the results of the MIRA-LFD experiments at different reaction times.
FIG. 10 shows the results of the detection limit of duck-origin components by fluorescent quantitative PCR.
FIG. 11 shows the detection limit of the duck-derived component MIRA-LFD detection method.
FIG. 12 shows sensitivity examination of duck-origin components.
FIG. 13 shows the specificity of duck-origin components.
FIG. 14 shows the false positive and false negative examination of duck-origin components.
Detailed Description
The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.
The samples used in the experiment comprise 10 species of pig, cattle, sheep, chicken, duck, goose, fox, mink, cat and nutria. Samples were taken from breeding plants, research institutes, farm markets, e-commerce, supermarkets, etc. And all samples are subjected to mitochondrial gene sequencing verification through synthetic primers, so that the authenticity of the samples is ensured.
Example 1, the method for rapidly identifying duck-origin ingredients in meat products
1.1DNA extraction
Extracting the genome of duck source sample according to the DNA extraction kit specification to obtain duck source nucleic acid template (DNA template), determining the DNA extraction efficiency and purity by using nucleic acid protein quantitative instrument, and performing OD260/OD280And (4) value measurement, wherein the measured value is 1.7-2.0, and the result shows that the method can be used for subsequent tests.
1.2 establishment of MIRA detection System
1.2.1MIRA detection method primer Probe design
The invention selects mitochondrial gene cytochrome oxidase b (cytb) gene and mitochondrial DNA 12S rRNA (12S) which are widely applied to species identification as target groups, and selects the gene sequences of duck 12S (U59666.1) and duck cytb (EU755252) in a Genbank database to design primers and probes.
When designing a primer, selecting a primer sequence with high base arrangement randomness and GC content of 30-70%, and marking a biotin modification group at the 5' end of a reverse primer; for the probe sequence of the MIRA method, a palindromic sequence, an internal secondary structure and continuous repeated bases are avoided, the length is generally 46nt to 52nt, a FAM label is modified at the 5 'end, a dSpacer (tetrahydrofuran) is labeled at the sequence position about 30nt away from the 5' end to serve as a recognition site of nfo (endonuclease), and in addition, the THF is about 15nt away from the 3 'end, and the 3' end is labeled with a C3-spacer modification group. The results of the alignment of duck-derived specific sequences are shown in FIGS. 1 and 2.
According to the sequence comparison result, the design principle of the MIRA primer probe is combined, the high-level structure analysis comparison is carried out on the primer and the probe sequence of the duck so as to ensure the high specificity of the duck and prevent the structures which influence the detection efficiency, such as dimer, stem loop, hairpin and the like, from occurring, 5 forward primers, 6 reverse primers and 2 probes are totally designed, and the name and the sequence information of the primer probe are shown in the table 1.
TABLE 1 primer Probe name and sequence information
1.2.2 Positive control screening study
The primers were combined according to the design in table 1, and the combination of the MIRA positive control primers totaled 15 groups, as shown in table 2.
TABLE 2MIRA Positive control primer combination Table
The MIRA electrophoresis reaction system (50 mu L) comprises: 29.4 mu L of A buffer, 1.0 mu L of duck-origin nucleic acid template and 2.5 mu L of B buffer, wherein each pair of primers (10 mu mol/L) is 0.25 mu L (shown in Table 2), and deionized water is used for supplementing 50 mu L, and a blank control group using the deionized water as a DNA template is set up; meanwhile, a positive control template unit provided by the MIRA kit is used as an experimental quality control. Positive control system formulation (provided by kit): mu.L of positive control template was added, 4. mu.L of positive control primer Mix (containing the up/down primers) was added to the primer, and the other components were formulated as above. The reaction program was 37 ℃ for 5 min. The reaction products were horizontally electrophoresed using a 3% agarose gel and the results were recorded by taking pictures using a gel imaging system.
The results are shown in FIG. 3, in which M is DNA Marker; d + number is the positive control of the corresponding primer combination in the primer pair of Table 2; d + number B is a blank control for the corresponding primer combination in the primer pair of Table 2; p is a positive control of the kit; PB is blank control corresponding to the positive control of the kit. As shown in the 15 pairs of primers and the matched positive control result of the kit in FIG. 3, the blank control results of the D3 combination and the D6 combination show obvious primer dimers, and other primer compositions and corresponding probes are screened for subsequent experiments.
1.2.3 blank control screening study
According to the experimental results in 1.2.2, the combination of MIRA positive control primer and probe is 13 pairs in total, which is shown in Table 3.
TABLE 3 MIRA blank control primer Probe combination Table
The MIRA-LFD colloidal gold reaction system (50. mu.L) comprised 29.4. mu.L of A buffer, 1.0. mu.L of deionized water and 2.5. mu.L of B buffer, 1.0. mu.L of each primer pair (10. mu. mol/L) (shown in Table 3), 0.3. mu.L of probe (10. mu. mol/L) (shown in Table 3), and made up to 50. mu.L with deionized water. The reaction program was 37 ℃ for 5 min. The colloidal gold result is shown in fig. 4, wherein line C is a control line and line T is a detection line; the numbers are the numbers in the figures in table 3; b is blank control corresponding to the positive control of the kit.
As shown in the blank results of 13 pairs of primer probes in fig. 4, no T-line appeared in the blank control of 4 pairs of primer probes, indicating that no non-specific binding appeared; the remaining 9 pairs of primer probes all showed T lines, so 4 pairs of primer probe combinations without T lines were selected for subsequent experiments.
1.2.4 negative control screening study
According to the experiment results of 1.2.3, the primer probe combination of the blank control in which the detection line (T line) does not appear is screened, and the details are shown in Table 4.
TABLE 4 MIRA-LFD negative control screening primer-probe combination table
The MIRA-LFD colloidal gold reaction system (50. mu.L) included 29.4. mu.L of A buffer, 1.0. mu.L of nucleic acid template and 2.5. mu.L of B buffer, 2.0. mu.L of each primer pair (10. mu. mol/L) (shown in Table 4), 0.6. mu.L of probe (10. mu. mol/L) (shown in Table 4), and was made up to 50. mu.L with deionized water. The nucleic acid template is divided into a negative control nucleic acid template and a positive control nucleic acid template, wherein the negative control nucleic acid template is a DNA template extracted from cattle, sheep, pigs and chicken samples, the positive control nucleic acid template is a duck source nucleic acid template, and the reaction procedure is 37 ℃ for 5 min. The colloidal gold result is shown in fig. 5, wherein line C is a control line and line T is a detection line; the numbers 1-5 correspond to DNAs extracted from beef, mutton, pork, chicken and duck-origin meat products respectively; b is blank control.
As shown in fig. 5 for the 4 pairs of primer probe negative control results, the primer probe combination BF2BR2BP showed T-line only in duck meat products, and the negative control showed no T-line, indicating that no non-specific binding occurred. While non-specific binding occurs for all other primer probe combinations.
On the basis, the number of negative control species is increased for the primer probe combination BF2BR2BP, the specificity of the primer probe combination BF2BR2BP is further examined, and a colloidal gold result graph is shown in FIG. 6, wherein a line C is a control line and a line T is a detection line; the numbers 1-10 correspond to DNAs extracted from meat products of cattle, sheep, pig, chicken, duck, goose, fox, mink, cat and nutria respectively; b is blank control.
As shown in fig. 6: the primer probe combination BF2BR2BP screened by the experiment only appears a T line in duck-origin meat products, is negative in cattle, sheep, pigs, chickens, geese, foxes, martens, cats and nutria, and does not appear obvious non-specific binding, which shows that the primer probe combination shows good specificity to duck-origin systems. Therefore, the primer probe combination BF2BR2BP is used as a primer probe combination for detecting a duck-origin system.
1.2.5MIRA-LFD detection System primer Probe volume screening
TABLE 5MIRA-LFD primer Probe volume combination Table
According to the experimental results of 1.2.4, the influence of the volumes of different primer probes on the MIRA reaction system is examined. The MIRA-LFD gold colloidal reaction system (50. mu.L) is shown in Table 5 for four combinations. Wherein, the sample is a DNA template extracted from a duck sample. The reaction program was 37 ℃ for 5 min.
The experimental result is shown in fig. 7, wherein a line C is a control line, and a line T is a detection line; d is duck-origin component detection, and DB is a blank control of a corresponding primer probe. In fig. 7, each combination C line shows a band, and each combination blank control detection line T line shows no obvious band, which indicates that compositions 1 to 4 can be used as the addition amount of the primer probe for the duck-origin meat product.
1.2.6MIRA-LFD detection System reaction temperature screening
According to the experimental results, the influence of different reaction temperatures on the MIRA reaction system is examined, and five temperature points of 30 ℃, 37 ℃, 40 ℃, 45 ℃ and 50 ℃ are selected for carrying out the experiment. The MIRA-LFD gold colloidal reaction system (50. mu.L) included 29.4. mu.L of A buffer, 1.0. mu.L of positive control nucleic acid template and 2.5. mu.L of B buffer, 0.25. mu.L of each primer pair (10. mu. mol/L), 0.075. mu.L of probe (10. mu. mol/L), and made up to 50. mu.L with deionized water. The reaction procedure was 30 deg.C, 37 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, respectively, for 5 min. Wherein the positive control sample is a DNA template extracted from a duck sample, the experimental result is shown in figure 8, a line C in the figure is a control line, and a line T is a detection line; d is duck-origin component detection, and DB is a blank control of a corresponding primer probe.
As shown in FIG. 8, T lines of animal-derived component detection results of samples at 30 ℃, 37 ℃, 40 ℃ and 45 ℃ show obvious bands, and blank control T lines of duck-derived samples show no obvious bands, and no non-specific amplification condition appears, wherein no band appears at 37 ℃. The T line band is not obvious under the condition of 50 ℃, which indicates that 50 ℃ destroys enzyme and influences MIRA reaction. The optimal temperature for the MIRA-LFD reaction of the invention was chosen to be 37 ℃ for all considerations.
1.2.7MIRA-LFD detection System reaction time screening
According to the experimental results, the influence of different reaction times on the MIRA reaction system is examined, and five reaction times of 1min, 5min, 10min, 15min and 25min are selected for carrying out the experiment. The MIRA-LFD gold colloidal reaction system (50. mu.L) included 29.4. mu.L of A buffer, 1.0. mu.L of positive control nucleic acid template and 2.5. mu.L of B buffer, 0.25. mu.L of each primer pair (10. mu. mol/L), 0.075. mu.L of probe (10. mu. mol/L), and made up to 50. mu.L with deionized water. The reaction temperature is 37 ℃, and the reaction time is 1min, 5min, 10min, 15min and 25min respectively. Wherein the positive control nucleic acid template is a DNA template extracted from a duck sample, the experimental result is shown in figure 9, a line C in the figure is a control line, and a line T is a detection line; d is duck-origin component detection, and DB is a blank control of a corresponding primer probe.
As shown in fig. 9: non-specific binding in the MIRA-LFD assay system appeared more readily with longer reaction times (white control results in the figure). Therefore, 5min is selected as the reaction time of the MIRA-LFD detection system, and false negative is easy to appear after 1min is selected.
1.2.8 detection limit test
About 200mg of duck meat tissue is weighed and transferred to a 2.0mL centrifuge tube. 400 μ L of ACL Solution (provided by the DNA extraction kit) and 20 μ L of protease K (provided by the DNA extraction kit) were then added to the centrifuge tube. Mix well by shaking, and place at 55 ℃ overnight for lysis until the sample is completely lysed.
After the sample is completely cracked, the volume (x, mu L) of a lysis solution corresponding to 1mg of tissue is calculated according to the following formula, a duck-derived tissue lysis solution is used as the sample, a chicken-derived tissue lysis solution is used as the background, and 10%, 1%, 0.1% and 0.01% (volume ratio) of simulated samples are prepared. DNA extraction was performed separately, and experiments were performed according to the fluorescent quantitative PCR method and the MIRA-LFD method.
In the formula: x is the volume of lysate corresponding to 1mg of the corresponding animal source; and a is the tissue quality of the sample.
The experimental results are shown in fig. 10: the duck source in the mixed sample is detected by fluorescent quantitative PCR, and the different dilutions form good gradients, which indicates that the sample mixing method is effective. In fig. 11, the colloidal gold test strip shows obvious color difference, which proves that whether the sample contains duck-origin components can be judged by the color depth of the colloidal gold. From fig. 10 and 11, the detection limit of the duck-origin component was 1%.
1.2.9 establishment of the MIRA-LFD detection System
The invention establishes a duck-derived component MIRA-LFD detection method through the design of a duck specific primer probe, and establishes a detection system scheme of the invention from four aspects of positive control screening, blank control screening, negative control screening and detection limit investigation respectively, as shown in Table 6.
TABLE 6 scheme for MIRA-LFD detection system
1.3 laboratory investigation
1.3.1 sensitivity investigation
The sensitivity refers to the percentage of the number of samples with positive results in the total number of positive samples when the method reaches the actual detection limit under the experimental conditions. The duck source detection system is selected to conduct sensitivity investigation of 10 detection limit samples, and the sensitivity of the method is determined, and the experimental results are shown in figure 12 and table 7. Numbers 1-10 in FIG. 12 are numbers of samples in Table 7, B represents a blank control, and P represents a positive control. The experimental result shows that the sensitivity of the detection method for detecting the duck-origin components in the meat products is good.
TABLE 7 sensitivity examination results
1.3.2 specificity Studies
The specificity of the product is reflected by the cross reaction rate of the rapid detection method and related products, namely the ratio (in percentage) of the detection limit of the target source components to the minimum concentration of the non-target source components which are detected to be positive. Mixing the target component and the non-target component according to a certain proportion, evaluating the specificity of the method, and recording the minimum concentration of the positive detection result. Specificity (cross-reactivity) was considered to be less than 1% when the concentration level of non-target source was added at a level greater than or equal to 1000 times the detection limit. The results are shown in FIG. 13 and Table 8, and the numbering meanings in FIG. 13 are shown in Table 8.
TABLE 8 specificity test results Table
1.3.3 false Positive and false negative rates
According to the reaction parameters, the system and the reaction conditions established by the invention, the method is used for investigating the false positive rate and the false negative rate. And selecting a duck detection system to investigate the false positive rate and the false negative rate of the species. The samples are all from ' 2021 year old metropolis city level risk monitoring ' samples, the information of the samples is shown in Table 9 and compared with a standard method (the standard method is shown in: Ministry of commerce of the people ' S republic of China, the real-time fluorescence PCR method for measuring animal-derived components in meat and meat products: SB/T10923-.
The false negative rate and the false positive rate are calculated according to the following formula:
false negative rate (%) — the number of negative results of positive samples × 100/total number of positive samples.
False positive rate (%) — the number of positive results of negative samples × 100/total number of negative samples.
TABLE 9 false positive and false negative rate investigation sample information table
The results are shown in FIG. 14 and Table 10.
TABLE 10 false positive and false negative rate investigation result table
The kit is established by sensitivity investigation, specificity investigation, false positive rate and false negative rate investigation on the 'animal-derived component visual detection method based on isothermal amplification technology and product development', the sensitivity of the kit is 80%, the specificity is 1%, the false positive rate is 10%, the false negative rate is 0%, and the sensitivity does not exceed 20%, so that the kit is proved to be reliable and effective.
1.4 summary of validation results
The sensitivity, specificity, false positive rate and false negative rate of duck-origin components of samples with different substrates are verified by adopting the primer probe, the colloidal gold kit and the operation instruction researched by the agricultural product safety research center, the Tianjin customs plant and food detection center and the Chengdu customs technology center of China inspection and quarantine scientific research institute. From the verification results of three units, the evaluation results are that the kit is reliable and effective.
In conclusion, the invention successfully screens out the optimal primer probe combination of the multi-enzyme constant-temperature rapid amplification visualization technology detection system for duck-derived components in meat products, and provides the MIRA-LFD detection system for detecting the duck-derived components in the meat products. The MIRA-LFD detection system combines the MIRA technology and the LFD technology, has the advantages of mild detection conditions, simple and convenient operation, strong specificity, high sensitivity, good reliability, visual result and contribution to field detection. The detection method provided by the invention has good reliability, timeliness, operability and safety, provides a new choice for rapidly, simply and sensitively detecting the duck-origin ingredients of the meat product on site, and has a wide application prospect.
SEQUENCE LISTING
<110> institute of food inspection in adult cities
<120> method for rapidly identifying duck-origin components in meat products based on MIRA technology
<130> GY392-2022P0115107CCZ
<150> 2021106808873
<151> 2021-06-18
<160> 13
<170> PatentIn version 3.5
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<400> 13
actggctatg cactacaccg cagacacatc cttgctttct cctcag 46
Claims (10)
1. A primer probe combination for identifying duck-origin ingredients in meat products is characterized in that: it comprises a forward primer, a reverse primer and a probe; the nucleotide sequence of the forward primer is shown as SEQ ID NO. 9; the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 11; the nucleotide sequence of the probe is shown as SEQ ID NO. 13.
2. Use of the primer probe combination of claim 1 for identifying duck-origin ingredients in meat products.
3. Use according to claim 2, characterized in that: the application is the application in the preparation of the kit for identifying duck-origin components in meat products.
4. Use according to claim 2 or 3, characterized in that: the method for identifying duck-origin components in meat products is a multienzyme constant-temperature rapid amplification visualization technology.
5. Use according to claim 4, characterized in that: the multienzyme isothermal rapid amplification visualization technology is a multienzyme isothermal rapid amplification technology combined with a lateral flow chromatography technology.
6. A kit for identifying duck-origin ingredients in meat products is characterized in that: the kit comprises the primer probe combination of claim 1.
7. The kit of claim 6, wherein: the method for identifying duck-origin components in meat products is a multienzyme constant-temperature rapid amplification visualization technology.
8. The kit of claim 7, wherein: the multienzyme isothermal rapid amplification visualization technology is a multienzyme isothermal rapid amplification technology combined with a lateral flow chromatography technology.
9. A method for identifying duck-origin ingredients in meat products is characterized by comprising the following steps: the method comprises the following steps:
(1) extracting DNA of a sample to be detected as a template;
(2) adding the primer probe combination of claim 1 to an amplification system comprising a DNA template;
(3) detecting by using a multienzyme constant-temperature rapid amplification visualization technology;
(4) and reading the result.
10. The method of claim 9, wherein: in the step (2), the volume ratio of the forward primer, the reverse primer, the probe and the DNA template in the amplification system is 0.1-2: 0.1-2: 0.01-0.6: 1; the volume ratio of the forward primer to the reverse primer is 1: 1;
and/or in the step (2), the reaction temperature of the amplification system is 30-45 ℃; and/or the amplification time is 1-5 min;
preferably, in the step (2), the reaction temperature of the amplification system is 37 ℃; and/or the amplification time is 5 min;
and/or, in the step (2), the concentration of the primer in the amplification system is 10 mu mol/L; and/or the concentration of the probe in the amplification system is 10 mu mol/L.
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