CN115896260A - Primer pair for detecting goose-derived components in food and application thereof - Google Patents
Primer pair for detecting goose-derived components in food and application thereof Download PDFInfo
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention belongs to the technical field of food detection, and particularly relates to a primer pair for detecting goose-derived components in food and application thereof. The primer pair can be selected from a primer pair 1, a primer pair 2 or a primer pair 3, wherein the sequence of the primer pair 1 is shown as SEQ ID NO:1 and SEQ ID NO:2, the sequence of the primer pair 2 is shown as SEQ ID NO:3 and SEQ ID NO:4, the sequence of the primer pair 3 is shown as SEQ ID NO:5 and SEQ ID NO: and 6. The primer has extremely strong specificity to goose-origin components, and has higher accuracy and sensitivity no matter the detection is carried out by an agarose gel electrophoresis method after the ordinary PCR amplification or the direct observation is carried out by a fluorescent quantitative PCR method.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a primer pair for detecting goose-derived components in food and application thereof.
Background
In recent years, various food safety problems such as adulteration and sub-sufficiency of animal-derived foods in the market continuously occur, the phenomenon that the product identification is not completely consistent with the variety still exists, and the market order is seriously disturbed. The food safety problem examines the dining table of common people again and again, and becomes the focus of attention of common people. With the rapid increase of the consumption of meat products, people put higher requirements on the quality of meat products.
The goose meat is used as a green health food, has high nutritive value, has medical and dietary therapy functions, and has the protein content as high as 22.3 percent; the fat content is low, the texture is soft, and the fat is easy to digest and absorb by human bodies; the content of unsaturated fatty acid reaches up to 66.3 percent; the cholesterol content is low; meanwhile, the product is rich in various vitamins necessary for human growth and development. Therefore, the phenomena of swabbing goose meat by duck meat and the like in the market are frequently seen, and livestock and poultry meat adulteration events such as 'hanging goose head to sell duck meat' and 'indicating duck to be goose' occur frequently.
Therefore, a method for detecting goose-derived components in food is necessary.
Disclosure of Invention
In view of the above problems, an object of the present invention is to provide a primer pair for detecting goose-derived components in food, which is designed based on mitochondrial coi gene, has very strong specificity to goose-derived components, and has very high accuracy and sensitivity no matter whether the detection is performed by gel electrophoresis or fluorescence quantitative PCR.
In order to achieve the purpose, the following technical scheme can be adopted:
the invention provides a primer pair for detecting goose-origin components in food, which selects a primer pair 1, a primer pair 2 or a primer pair 3, wherein the sequence of the primer pair 1 is shown as SEQ ID NO:1 and SEQ ID NO:2, the sequence of the primer pair 2 is shown as SEQ ID NO:3 and SEQ ID NO:4, the sequence of the primer pair 3 is shown as SEQ ID NO:5 and SEQ ID NO: and 6.
The invention also provides a reagent for detecting goose-derived components in food, which comprises the primer pair for detecting the goose-derived components in food and a fluorescent reagent or a fluorescent probe.
The invention further provides a kit for detecting goose-derived components in food, which comprises the primer pair for detecting the goose-derived components in food or the reagent for detecting the goose-derived components in food.
The invention further provides an application of the primer pair for detecting the goose-derived component in the food or the reagent for detecting the goose-derived component in the food in detecting the goose-derived component in the food.
The invention further provides a method for detecting goose-derived components in food, which comprises the steps of carrying out PCR amplification on a DNA genome of a sample to be detected by using the primer pair for detecting the goose-derived components in the food to obtain an amplification product, carrying out gel electrophoresis on the amplification product, and detecting the goose-derived components in the food according to the gel electrophoresis condition.
The invention also provides a method for detecting goose-derived components in food, which comprises the steps of carrying out PCR amplification on a DNA genome of a sample to be detected by using the reagent for detecting the goose-derived components in the food, and judging whether the sample to be detected contains the goose-derived components or not according to a fluorescence signal in the PCR amplification process; and judging the content of the goose-derived component in the sample to be detected according to the CT value and the standard curve.
The beneficial effects of the invention include:
(1) The primer for detecting the goose-origin components in the food has extremely strong specificity to the goose-origin components, and has higher accuracy and sensitivity no matter the primer is detected by an agarose gel electrophoresis method after common PCR amplification or directly observed by a fluorescent quantitative PCR method;
(2) The method for detecting the goose-origin component based on the primer pair for detecting the goose-origin component in the food has the advantages of high speed, low cost, easy mastering and the like, can effectively detect the goose-origin component in various meat samples, and can provide a convenient, fast and accurate identification method for identifying adulterated meat in the market;
(3) The method for detecting the goose-origin components based on the primer pair for detecting the goose-origin components in the food is not limited by the state of the sample, and can be used for effectively identifying whether the sample is fresh meat or deep-processed products or whether the sample DNA is complete or seriously damaged, so that the method has higher practical value for conveniently and effectively supervising products in the market.
Drawings
FIG. 1 shows the case of gel electrophoresis for specific detection of primer set 1;
FIG. 2 shows the case of gel electrophoresis for specific detection of primer pair 2;
FIG. 3 shows the gel electrophoresis of primer pair 3;
FIG. 4 shows the case of the primer set 1 in the gel electrophoresis for sensitivity detection;
FIG. 5 shows the case of the primer pair 2 in sensitive detection gel electrophoresis;
FIG. 6 shows the case of the primer pair 3 in the sensitive detection gel electrophoresis;
FIG. 7 is a fluorescent quantitative PCR amplification graph of primer pair 1 for different animal DNAs;
FIG. 8 is a graph showing the fluorescent quantitative PCR amplification of the primer pair 2 for different animal DNAs;
FIG. 9 is a graph showing the fluorescent quantitative PCR amplification of primer pair 3 for different animal DNAs;
FIG. 10 is a graph showing the amplification of the goose DNA template by primer pair 1 at different dilution times;
FIG. 11 is a graph showing the amplification of the goose DNA template by primer pair 2 at different dilution times;
FIG. 12 is a graph showing the amplification curve of different dilution times of the primer pair 3 to goose DNA template.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context has a significantly different meaning, the singular form of expression includes the plural form of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the present invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may be present or may be added. As used herein, "/" can be interpreted as "and" or "depending on the situation.
The embodiment of the invention provides a primer pair for detecting goose-derived components in food, which selects a primer pair 1, a primer pair 2 or a primer pair 3, wherein the sequence of the primer pair 1 is shown as SEQ ID NO:1 and SEQ ID NO:2, the sequence of the primer pair 2 is shown as SEQ ID NO:3 and SEQ ID NO:4, the sequence of the primer pair 3 is shown as SEQ ID NO:5 and SEQ ID NO: and 6.
It should be noted that, the primer pair of the present invention is designed with the complete sequence of the COI gene in the mitochondrial genome of goose (the complete sequence of the Anser cygnoides mitochondrial DNA COI gene in NCBI database (GenBank: KP 026178.1) 1551bp sequence). Specifically, the animal mitochondrial genome has the characteristics of high species conservation, species specificity, corrosion resistance, high temperature resistance and the like, and is not easy to damage in the deep processing process of meat products, so that the PCR technology utilizing mitochondrial coding gene difference has unique advantages in meat and meat product identification. And the animal mitochondrial genome DNA is used as an extranuclear genetic material, has wide polymorphism both in interspecies and intraspecies, is a first-choice target for designing qualitative detection of meat components, wherein the CO I has moderate evolution rate in the mitochondrial genome, and is very suitable for qualitative detection of the source of the meat components of different types of animals. The primer pair of the invention comprises the following 3 pairs of primers:
primer pair 1:
the sequence of the upstream primer is TATGAAACACCATTTTCCTCCATCGG (SEQ ID NO: 1),
the sequence of the downstream primer is ACTTTTCGTTTGGCCGGAAA (SEQIDNO: 2);
and (3) primer pair 2:
the upstream primer sequence is CACTAACCATAAGGATTGGCACC (SEQ ID NO: 3),
the sequence of the downstream primer is TACAAGGCGTGAGCGGTA (SEQIDNO: 4);
and (3) primer pair: the upstream primer sequence is TCCCACTCACTGGTTT (SEQIDNO: 5),
the sequence of the downstream primer is GATGGAGGAAATGGTTTTCATAGT (SEQ ID NO: 6).
In some embodiments, the PCR and qPCR reactions are performed using goose-derived DNA as a positive template and 8 species of DNA such as pig, cattle, sheep, pigeon, quail, turkey, chicken, and duck as a mixed template of interfering templates. The result of the common PCR method takes an electrophoresis band appearing in an agarose gel electrophoresis map as a positive result, the result of the qPCR method takes amplification with a typical amplification curve and a Ct value of the amplification curve less than 30 as a positive result, and the method can detect DNA of a Dancer level in sensitivity. The primer pair disclosed by the invention can be simultaneously suitable for PCR and qPCR methods, has good specificity and higher sensitivity, provides a new way for identifying and exploring animal-derived components in food, and has the beneficial results of convenience in operation, stable and accurate result, low cost and the like.
The invention further provides a reagent for detecting goose-derived components in food, which comprises the primer pair for detecting the goose-derived components in food and a fluorescent reagent or a fluorescent probe.
The invention further provides a kit for detecting goose-derived components in food, which comprises the primer pair for detecting the goose-derived components in food or the reagent for detecting the goose-derived components in food. The kit may include the primer pair or the reagent. Specifically, the detection kit is composed in a conventional form known in the art, for example, the detection kit is provided with a kit instruction, for example, a reagent bottle containing various detection reagents is provided, for example, a plurality of small lattices are provided for placing the detection reagent bottle, for example, a dropper is provided for removing the detection reagent and the like.
The invention further provides an application of the primer pair for detecting the goose-derived component in the food or the reagent for detecting the goose-derived component in the food in detecting the goose-derived component in the food. When the primer pair or the reagent is used for amplifying the DNA genome of a sample to be detected, the goose-derived component genome DNA can be specifically amplified; when the detection reagent containing the fluorescent reagent or the fluorescent probe is used for amplification, stronger fluorescence can be generated in the amplification process, and detection and analysis are facilitated. In addition, the primer pair or the reagent can be combined with the micro-fluidic chip to be applied to detection of goose-derived components in food, and the characteristics of high efficiency and convenience of the micro-fluidic chip are combined, so that the great convenience is brought to the detection of the goose-derived components in food.
The invention further provides a method for detecting goose-derived components in food, which comprises the steps of carrying out PCR amplification on a DNA genome of a sample to be detected by using the primer pair for detecting the goose-derived components in the food to obtain an amplification product, carrying out gel electrophoresis on the amplification product, and detecting the goose-derived components in the food according to the condition of the gel electrophoresis.
In the method for detecting goose-derived components in food, goose-derived components in a sample to be detected can be qualitatively analyzed by detecting the PCR amplification product. Gel electrophoresis is generally selected to be performed on the PCR amplification product, and it should be understood that the amount of the PCR amplification product is an amount sufficient for gel electrophoresis, for example, 10 cycles of amplification are sufficient for gel electrophoresis, and then 10 cycles of amplification product are subjected to gel electrophoresis, for example, 20 cycles of amplification are required for gel electrophoresis, and then 20 cycles of amplification product are subjected to gel electrophoresis; it should be noted that the method for performing gel electrophoresis on the product after PCR amplification may be any method known to those skilled in the art, for example, the size and position of the electrophoretic target band are compared with the marker, so as to perform qualitative analysis on the DNA genome of the sample to be detected, and determine whether the sample to be detected contains goose-origin components; relative content of the target band can also be analyzed by using imagej, so that quantitative analysis of products after PCR amplification is realized.
The invention further provides a method for detecting goose-derived components in food, which comprises the steps of carrying out PCR amplification on a DNA genome of a sample to be detected by using the reagent for detecting the goose-derived components in the food, and judging whether the sample to be detected contains the goose-derived components or not according to a fluorescent signal in the PCR amplification process; and judging the content of the goose-derived component in the sample to be detected according to the CT value and the standard curve. Specifically, after extracting goose sample DNA, performing non-labeled fluorescence PCR amplification by using the primer pair of the invention; and (3) performing high-resolution melting curve analysis on the PCR amplification product by using fluorescence PCR instrument software to determine a Tm value melting curve map of the amplification product.
It should be noted that, besides the method of using the above primer pair to perform conventional PCR amplification detection and then using gel electrophoresis to perform quantitative and qualitative analysis on the final product of PCR amplification, the above reagents can be used to perform fluorescence PCR amplification detection, that is, a fluorescence reagent or a fluorescence probe is introduced into PCR amplification to label the formed DNA double strand during annealing and extension; thus, the PCR amplification process can be monitored in real time through the fluorescent signal emitted by the fluorescent reagent or the fluorescent probe, and the DNA template to be detected is qualitatively and quantitatively analyzed. It should be noted that, the method for qualitatively and quantitatively analyzing the goose-derived component in the sample to be detected may be a method known in the art, for example, qualitatively analyzing the goose-derived component in the sample to be detected according to whether the fluorescence-labeled amplification curve is on line, and the on line indicates that the sample to be detected contains the goose-derived component; for example, a threshold value can be set, then a CT value is obtained, and the content of the goose-origin component in the sample to be detected is obtained according to the relation between the CT value and the standard curve. It is understood that a standard curve refers to a curve that is detected by a standard of known copy number, and is within the skill of the art.
In some embodiments, in the method for detecting goose-derived component in food, the annealing temperature for PCR amplification is 58-62 deg.C, preferably 60 deg.C. It should be noted that the annealing temperature mainly depends on the base composition, length and concentration of the primer; the proper annealing temperature is especially critical to the specificity of PCR amplification reaction. Generally, the effect of the high or low annealing temperature affects the specificity and sensitivity (amplification efficiency) of the PCR reaction, and the two are inversely related, i.e., a lower annealing temperature can increase the sensitivity but has a lower specificity, while a higher annealing temperature can increase the specificity but decreases the amplification efficiency. The annealing temperature set at 58-62 deg.C, such as 59 deg.C or 60 deg.C, in the present invention, can make the above-mentioned primer pair maintain higher specificity in the above-mentioned PCR amplification reaction, and also make the PCR amplification reaction rate maintain at a higher level.
For a better understanding of the present invention, the following further illustrates the contents of the present invention with reference to specific examples, but the contents of the present invention are not limited to the following examples.
In the following implementation, the extraction of DNA is carried out according to the following steps (1) (phenol-imitation method) that raw/cooked meat samples are taken about 50mg, cut into pieces and ground, 5ml of 0.15mol/L NaCl-0.015mol/L sodium citrate solution is added and transferred into a centrifuge tube, after centrifugation for 3min at 10000r/min, the supernatant is discarded, the precipitate is washed once by repeating the steps, and the RN nucleoprotein and the DNA nucleoprotein can be separated as much as possible by repeating the washing; (2) Adding 1ml cell lysis solution (0.5% SDS-containing STE) into the precipitate, reacting for 30min, adding 10% proteinase K10 μ l, mixing, and constant temperature water bath at 55 deg.C overnight until the solution becomes viscous and slightly transparent; (3) Adding an equal volume of Tris saturated phenol (pH8.0) into the digested meat sample, repeatedly inverting the centrifuge tube for 5min, fully mixing, centrifuging at 12000rpm for 10min, and sucking the supernatant into a clean centrifuge tube. Repeating the steps once to ensure that the supernatant is clear and transparent; (4) Adding the same amount of phenol, namely chloroform (1); (5) Adding chloroform with the same volume into the supernatant, reversing the centrifuge tube for about 5min, fully and uniformly mixing, centrifuging at 12000rpm for 10min, and sucking the supernatant into a clean centrifuge tube; (6) Adding ice-cold absolute ethanol with the volume twice that of the supernatant into the supernatant, slightly shaking the centrifuge tube, stopping shaking when white vaporous precipitate appears, carefully picking out the DNA precipitate by using a pipette gun, and placing the DNA precipitate into a clean 1.5ml centrifuge tube; (7) Adding ice-cold 75% ethanol 500ul, gently shaking, carefully pouring out ethanol solution, naturally drying, sucking 100 μ l sterile double distilled water to dissolve DNA, and storing at-20 deg.C for use. And simultaneously detecting the extraction effect by agarose gel electrophoresis or detecting the concentration on a spectrophotometer.
Example 1 general PCR reaction System design
(1) Primer design
According to the complete genome sequence of 9 animal mitochondria published on NCBI database, the DNA sequences of different species COI are found out, after comparison and analysis, primers are designed by using Primer Express 3.0 program, finally goose specific primers (see table 1 below) are optimized and screened out, and PCR and qPCR amplification sequencing is carried out.
TABLE 1 primer set information Table
(2) PCR reaction system optimization
The PCR amplification system was 25. Mu.L: 2 XPCR Mix 12.5ul,10 mu mol/L forward primer and 10 mu mol/L reverse primer are respectively 0.25 mu L, template is 1 mu L, double distilled sterile water is 11 mu L; PCR amplification reaction conditions: performing pre-denaturation at 95 ℃ for 5min; then carrying out denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 45s and extension at 72 ℃ for 60s for 32 cycles; finally, extending for 10min at 72 ℃; and (3) recovering and purifying the tapping after the electrophoresis of the PCR product, and transferring the product to Shanghai Huada Gene science and technology limited for sequencing (the sequence is shown in table 2), wherein all the sequences adopt bidirectional sequencing, and the sequencing result is compared with the known sequence on GenBank.
TABLE 2 amplification sequences
(3) PCR product assay
Detecting the PCR product by adopting agarose gel electrophoresis with the mass concentration of 1.2 percent, and specifically comprising the following steps: (a) Taking 5 mu L of PCR product, adding 1 mu L of loading buffer solution, and loading; (b) electrophoresis at 120V for 15min; and (c) photographing by using a gel imaging system and storing a gel image.
EXAMPLE 2 detection of specificity of common PCR reaction System
The designed and screened primers are utilized to amplify 9 different animal muscle tissue DNA templates under the same PCR reaction system according to the method in the embodiment 1, so that the specificity of the primers is detected, the amplification results are shown in the following figures 1, 2 and 3, and as can be seen from the figures, the primer pair (the primer pair 1, the primer pair 2 and the primer pair 3) has better specificity, only has specific amplification on goose meat DNA, does not amplify other 8 species except goose, has stronger pertinence, and can be used as a characteristic primer to carry out specificity source detection on goose meat samples.
EXAMPLE 3 sensitivity detection of general PCR reaction System
And (3) detecting the concentration of all the extracted species DNA, diluting the DNA into uniform concentration, and mixing 8 animal DNAs except goose DNA in equal proportion to prepare the interference DNA Mix. Diluting the target template DNA by using the uniformly mixed DNA Mix with the dilution ratio of 10 1 Multiple to 10 times 8 The target template DNA concentrations were 10 of the original concentrations -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 . The PCR detection gel diagrams of the method according to the method of example 1 and the primer pair of the present invention were shown in FIGS. 4, 5 and 6 below. As can be seen from the figure, the concentration of DNA extracted from 50mg deep-processed meat samples is generally in microgram level, the diluted detection system can reach picogram level, and the requirement of market detection can be completely met.
Example 4 real-time fluorescent quantitative PCR System
In the embodiment of the invention, the real-time fluorescent quantitative PCR (qPCR) method is used for amplifying the DNA of all samples by using the primer pairs (primer pair 1, primer pair 2 and primer pair 3), and the whole reaction is carried out in an ABI 7500 sequence amplification instrument.
Real-time fluorescent quantitative PCR reaction system and procedure: 15ul qPCR reaction system: SYBR RealMaster Mix 7.5. Mu.L, 10 pmol/. Mu.L each of the forward and reverse primers 0.2. Mu.L, 20 ng/. Mu.L of cDNA template 1.0. Mu.L, dye 0.3. Mu.L, RNase-free double distilled water 5.8. Mu.L, set to 3 replicates per sample. qPCR reaction conditions: preheating at 50 deg.C for 2min, and pre-denaturing at 95 deg.C for 10min; then denaturation at 95 ℃ is carried out for 15s, annealing and extension at 60 ℃ are carried out for 1min, and 40 cycles are total; finally, denaturation at 95 ℃ is 15s, annealing extension at 60 ℃ is 30s, denaturation at 95 ℃ is 15s, and a melting curve is collected.
Example 5 real-time fluorescent quantitative PCR System specific detection
Using the primers designed and screened, the DNA templates of the muscle tissues of 9 different animals were amplified according to the method of example 4 in the same PCR reaction system, so as to detect the specificity of the primers, and the results are shown in FIG. 7, FIG. 8 and FIG. 9. As can be seen from the figure, the primer pair system of the invention can still obtain better specificity results in a fluorescent quantitative PCR amplification system, the 3 pairs of primers only react with goose DNA templates to form a typical amplification curve, and the Ct values of the amplification curves are all below 30, wherein the Ct value of the primer pair 1 is 22.56, the Ct value of the primer pair 2 is 25.12, and the Ct value of the primer pair 3 is 20.87. The Ct value of the DNA amplification curve of other species except geese is above 30, or no typical amplification curve. Therefore, the designed and screened primer pair system can quickly and effectively identify goose-derived samples in livestock and poultry meat.
Example 6 real-time fluorescent quantitative PCR System sensitivity detection
Detecting the concentration of all the extracted species DNA, diluting the DNA into uniform concentration, and mixing 8 animal DNAs except goose DNA in equal proportion to prepare an interference DNA Mix; diluting the target template DNA by using the uniformly mixed DNA Mix with the dilution ratio of 10 1 Multiple to 10 times 8 The target template DNA concentrations were 10 of the original concentrations -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 . The PCR detection gel diagrams of the method according to the method of example 4 and the primer set of the present invention were shown in FIGS. 10, 11 and 12 below to examine the sensitivity of the method. As can be seen, in the sensitivity detection, the concentration of the target template DNA is respectively diluted to 10 of the original concentration by the uniformly mixed DNA Mix by the same dilution method as that of the ordinary PCR -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 The amplification result shows that the dilution factor is 10 -6 (e -6 ) In this case, the 3 pairs of primers in the present invention still have a good amplification curve, and Ct values are all below 30.
Finally, the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which shall be covered by the claims of the present invention.
Claims (7)
1. A primer pair for detecting goose-derived components in food is characterized in that a primer pair 1, a primer pair 2 or a primer pair 3 is selected, wherein the sequence of the primer pair 1 is shown as SEQ ID NO:1 and SEQ ID NO:2, the sequence of the primer pair 2 is shown as SEQ ID NO:3 and SEQ ID NO:4, the sequence of the primer pair 3 is shown as SEQ ID NO:5 and SEQ ID NO: and 6.
2. A reagent for detecting goose-derived components in food, which is characterized by comprising the primer pair for detecting goose-derived components in food and a fluorescent reagent or a fluorescent probe according to claim 1.
3. A kit for detecting goose-derived components in food, which is characterized by comprising the primer pair for detecting goose-derived components in food according to claim 1 or the reagent for detecting goose-derived components in food according to claim 2.
4. The primer pair for detecting goose-derived components in food as claimed in claim 1 or the reagent for detecting goose-derived components in food as claimed in claim 2 is used for detecting goose-derived components in food.
5. A method for detecting goose-derived components in food is characterized by comprising the steps of carrying out PCR amplification on a DNA genome of a sample to be detected by using the primer pair for detecting the goose-derived components in food according to claim 1 to obtain an amplification product, carrying out gel electrophoresis on the amplification product, and detecting the goose-derived components in the food according to the gel electrophoresis condition.
6. A method for detecting goose-derived components in food, which is characterized by comprising the steps of carrying out PCR amplification on a DNA genome of a sample to be detected by using the reagent for detecting goose-derived components in food according to claim 2, and judging whether the sample to be detected contains the goose-derived components or not according to a fluorescent signal in the PCR amplification process; and judging the content of the goose-derived component in the sample to be detected according to the CT value and the standard curve.
7. The method for detecting goose-derived component in food according to claim 5 or 6, wherein the annealing temperature for PCR amplification is 58-62 ℃.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245518A (en) * | 2017-04-07 | 2017-10-13 | 北京市食品安全监控和风险评估中心 | It is a kind of while gene micro-fluid chip and its application of 26 kinds of animal derived materials of detection |
| CN108950004A (en) * | 2017-05-19 | 2018-12-07 | 英潍捷基(上海)贸易有限公司 | For detecting polynucleotides, probe, kit and the method for animal species |
-
2022
- 2022-10-10 CN CN202211235056.6A patent/CN115896260A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107245518A (en) * | 2017-04-07 | 2017-10-13 | 北京市食品安全监控和风险评估中心 | It is a kind of while gene micro-fluid chip and its application of 26 kinds of animal derived materials of detection |
| CN108950004A (en) * | 2017-05-19 | 2018-12-07 | 英潍捷基(上海)贸易有限公司 | For detecting polynucleotides, probe, kit and the method for animal species |
Non-Patent Citations (4)
| Title |
|---|
| RAYMOND M. BUCHHEIT ET AL.: "ASSESSMENT OF VARIATION IN THE DETECTION AND PREVALENCE OF BLOOD PARASITES AMONG SYMPATRICALLY BREEDING GEESE IN WESTERN ALASKA, USA", 《J WILDL DIS》, pages 20 - 00159 * |
| 励炯 等: "基于毛细管凝胶电泳与DNA条形码技术鉴别可食用动物内脏掺假方法的研究", 《食品工业科技》, pages 1002 - 0306 * |
| 王爽 等: "DNA 条形码COI 序列在常见肉类鉴别中的应用研究", 《现代食品科技》, pages 188 - 193 * |
| 穆春宇: "部分禽类mtDNA序列测定及在不同来源禽肉鉴定上的应用", 《中国优秀硕士学位论文全文数据库 农业科技辑》, pages 050 - 2 * |
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