CN117778615A - Primer group, kit and detection method for detecting lycium ruthenicum mill source components - Google Patents
Primer group, kit and detection method for detecting lycium ruthenicum mill source components Download PDFInfo
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Abstract
The invention discloses a primer group, a kit and a detection method for detecting a lycium ruthenicum murr-derived component, wherein the primer group is a real-time fluorescent quantitative PCR primer group. The primer group, the kit and the method for detecting the black wolfberry source component provide reliable basis for verifying authenticity of the black wolfberry product, theoretical basis for market supervision, and powerful protection for guaranteeing quality of the black wolfberry product and guaranteeing benefits of manufacturers and consumers.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group for detecting lycium ruthenicum source components and application thereof.
Background
Lycium ruthenicum (Lycium ruthenicum Murr.) is named "Qiao Nuoying-Harmat" and Tibetan medicine name "side Mary", belonging to the genus Lycium of Solanaceae. The black fruit medlar is spiny shrub, multi-branched, and hard in branches, is bent in a zigzag shape and is white, and is distributed in Shanxi province, gansu province, qinghai province, ningxia Hui autonomous region, xinjiang Uygur autonomous region and Tibet autonomous region in Europe.
Lycium ruthenicum Murr is sweet and neutral in taste and is rich in various nutritional ingredients such as proteins, lycium ruthenicum Murr polysaccharide, amino acids, vitamins, minerals, microelements and the like. The Lycium ruthenicum Murr primary pulp is prepared by soaking the original fruit of Lycium ruthenicum Murr, extracting fruit juice and pulp, and then performing biological fermentation processing. Compared with the lycium ruthenicum mill, the lycium ruthenicum mill magma is used as a liquid drink, and is rich in taste and fresh in taste. In addition, the lycium ruthenicum mill primary pulp is rich in various polyphenol compounds, including jujube glycoside, carotenoid, anthocyanin, flavone and the like, and has the functions of resisting oxidation, improving immunity, relieving fatigue and the like. The black fruit is rich in black fruit pigment which is not contained in the red fruit, the black fruit pigment is natural procyanidine (OPC), the black fruit is a natural wild plant with highest OPC content discovered so far, the black fruit pigment contains 3690 milligrams of OPC per 100 grams of black fruit, and the black fruit pigment contains only 330-3380 milligrams of OPC per 100 grams of blueberries. OPC is a very effective natural water-soluble free radical scavenger with an efficacy equivalent to 20 times that of Vc and 50 times that of VE.
Thus, beverages containing Lycium ruthenicum juice or Lycium ruthenicum puree have become a major nutritional product in modern times, and due to the high price of Lycium ruthenicum products due to the far more demanding supply, there are frequent incidents of Lycium ruthenicum product counterfeiting in the market, such as the use of white thorn fruit, blueberry fruit or other similar fruit stains to impersonate the Lycium ruthenicum product. The adulteration of the lycium ruthenicum product becomes an important problem of food supervision, however, the detection and identification of the lycium ruthenicum components at present mostly adopt liquid chromatography, the detection process is complicated, the detection period is long, and the rapid identification of the original components of the lycium ruthenicum product is not facilitated.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a primer group, a kit and a detection method for detecting lycium ruthenicum source components.
The first object of the invention is to provide a set of real-time fluorescent quantitative PCR primer sets for detecting the components of origin of lycium ruthenicum.
The second object of the invention is to provide a kit for detecting a lycium ruthenicum mill source component.
The third object of the invention is to provide a qualitative detection method for the components of origin of lycium ruthenicum.
In order to achieve the above object, the present invention is realized by the following technical scheme:
a real-time fluorescent quantitative PCR primer group for detecting the lycium ruthenicum mill source component comprises a forward primer with a nucleotide sequence shown in SEQ ID NO:1 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 2.
Preferably, the real-time fluorescent quantitative PCR primer set further comprises a nucleotide sequence shown in SEQ ID NO:3, and a probe sequence shown in 3.
More preferably, the probe sequence has a quenching group attached to the 3 'end and a fluorescent group attached to the 5' end.
Most preferably, the quenching group is TAMRA; the fluorescent group is FAM.
The application of the real-time fluorescence quantitative PCR primer group in preparing a detection kit for detecting the lycium ruthenicum source component is also in the protection scope of the invention.
A kit for detecting a lycium ruthenicum source component, wherein the kit comprises the real-time fluorescent quantitative PCR primer group.
Preferably, the forward primer nucleotide sequence of the real-time fluorescent quantitative PCR primer set is shown as SEQ ID NO:1, the nucleotide sequence of the reverse primer is shown as SEQ ID NO:2, the nucleotide sequence of the probe sequence is shown as SEQ ID NO: 3.
More preferably, the 3 'end of the probe sequence is connected with a fluorescence quenching group TAMRA, and the 5' end is connected with a fluorescence reporting group FAM.
Preferably, the reaction system of the detection kit comprises a positive control, a negative control, a real-time fluorescence PCR reaction mixed solution and sterile water.
More preferably, the positive control contains lycium ruthenicum DNA, and the negative control contains one or more of lycium ruthenicum DNA, blueberry DNA, nitraria tangutorum DNA, sea buckthorn DNA, blackcurrant DNA, summer black grape DNA or black raspberry DNA.
Most preferably, the negative control contains blueberry DNA.
Most preferably, the reaction system of the detection kit comprises a real-time fluorescence PCR reaction mixed solution and 10 mu mol/L nucleotide sequence shown in SEQ ID NO:1, and the nucleotide sequence of 10 mu mol/L is shown as SEQ ID NO:2 and 10 mu mol/L nucleotide sequence as shown in SEQ ID NO:3, and a probe sequence shown in 3.
As a specific reference scheme, the single reaction of the detection kit comprises: 5 mu L of sample DNA to be detected, 12.5 mu L of real-time fluorescence PCR reaction mixed solution and 10 mu mol/L of nucleotide sequence shown as SEQ ID NO:1, and the nucleotide sequence of the forward primer shown in the formula 1 is 0.5 mu L and 10 mu mol/L and is shown as SEQ ID NO:2, and the nucleotide sequence of the reverse primer is 0.5 mu L and 10 mu mol/L as shown in SEQ ID NO:3 and 6. Mu.L of sterile water.
A qualitative detection method of a lycium ruthenicum source component, comprising the following steps:
s1, extracting DNA of a sample to be detected;
s2, taking the DNA of the sample to be detected obtained in the step S1 as a template, and performing fluorescent quantitative PCR reaction by using the real-time fluorescent quantitative PCR primer group;
s3, if the sample to be detected does not have an S-shaped amplification curve, the sample to be detected does not contain lycium ruthenicum source components; if the sample to be detected has an S-shaped amplification curve, the sample to be detected contains the lycium ruthenicum source component.
Preferably, the reaction system of the real-time fluorescence quantitative PCR in the step S2 further comprises a real-time fluorescence PCR reaction mixed solution and sterile water.
Specifically, the real-time fluorescent quantitative PCR reaction system in the step S2 comprises a forward primer nucleotide sequence shown in SEQ ID NO:1, the nucleotide sequence of the reverse primer is shown as SEQ ID NO:2, the nucleotide sequence of the probe sequence is shown as SEQ ID NO:3, and one or more of real-time fluorescence PCR reaction mixed solution and/or sterile water.
More preferably, the real-time fluorescent quantitative PCR reaction system in the step S2 comprises a real-time fluorescent PCR reaction mixture and a nucleotide sequence of 10 mu mol/L as shown in SEQ ID NO:1, and the nucleotide sequence of 10 mu mol/L is shown as SEQ ID NO:2 and 10 mu mol/L nucleotide sequence as shown in SEQ ID NO:3, and a probe sequence shown in 3.
As a specific reference scheme, the single reaction of the real-time fluorescent quantitative PCR reaction system in step S2 includes: 5 mu L of sample DNA to be detected, 12.5 mu L of real-time fluorescence PCR reaction mixed solution and 10 mu mol/L of nucleotide sequence shown as SEQ ID NO:1, and the nucleotide sequence of the forward primer shown in the formula 1 is 0.5 mu L and 10 mu mol/L and is shown as SEQ ID NO:2, and the nucleotide sequence of the reverse primer is 0.5 mu L and 10 mu mol/L as shown in SEQ ID NO:3 and 6. Mu.L of sterile water.
Preferably, the conditions of the real-time fluorescent quantitative PCR reaction in step S2 are: 50 ℃ for 2min; pre-denaturation at 95 ℃ for 5-10 min; 15s at 95 ℃, 1min at 55-60 ℃ and 40-50 cycles.
More preferably, the conditions of the real-time fluorescent quantitative PCR reaction in step S2 are: 50 ℃ for 2min;95 ℃ for 10min; 15s at 95℃and 1min at 60℃for a total of 40 cycles.
Preferably, in step S2, a positive real-time fluorescent quantitative PCR reaction system is prepared by using lycium ruthenicum DNA as a positive template, a blank real-time fluorescent quantitative PCR reaction system is prepared by using sterile water as a blank template, and a negative real-time fluorescent quantitative PCR reaction system is prepared by using blueberry DNA as a negative template;
performing real-time fluorescent quantitative PCR reaction by using the prepared positive real-time fluorescent quantitative PCR reaction system, the prepared blank real-time fluorescent quantitative PCR reaction system and the prepared negative real-time fluorescent quantitative PCR reaction system;
in step S3, after the real-time fluorescent quantitative PCR reaction in step S2, determining whether the sample to be tested contains the lycium ruthenicum source component according to the amplification curve of the sample to be tested, and if one of the following conditions is not satisfied, the experiment is regarded as invalid:
i. the positive real-time fluorescent quantitative PCR reaction system has an S-shaped amplification curve;
ii, blank real-time fluorescent quantitative PCR reaction system has no S-shaped amplification curve;
negative real-time fluorescent quantitative PCR reaction system has no S-shaped amplification curve;
under the condition that the amplification curve of the sample to be detected does not appear, the sample to be detected is proved to contain no lycium ruthenicum source component, and if the amplification curve of the sample to be detected appears, the sample to be detected is proved to contain the lycium ruthenicum source component.
Compared with the prior art, the invention has the following beneficial effects:
real-time fluorescent PCR techniques enable analysis of species sources from the gene level with higher sensitivity and specificity and shorter identification times. The invention skillfully utilizes the high-efficiency amplification of the PCR technology, the specificity of nucleic acid hybridization and the rapid and sensitive performance of the fluorescence detection technology, the invention can identify the template through the specific hybridization of the primer or the probe and the template, can adopt the complete closed-tube detection without the post-treatment of PCR, thereby avoiding cross contamination and false positive and shortening the detection time.
The method for detecting the lycium ruthenicum mill source components disclosed by the invention can be used for simply, rapidly, specifically and sensitively qualitatively detecting the lycium ruthenicum mill source components in the lycium ruthenicum mill products in the markets at home and abroad. The method provides a reliable basis for verifying the authenticity of the lycium ruthenicum mill magma, a theoretical basis for market supervision, a powerful protection for guaranteeing the quality of the lycium ruthenicum mill magma product and the benefits of manufacturers and consumers, a support for standardizing the health-care food market, and good development prospect and application value are provided.
Drawings
FIG. 1 is a graph showing the results of alignment analysis of the sequences of the matK, ITS, and CHS genes.
FIG. 2 is a graph of amplification results of primer sets designed for the sequences of the matK, ITS, and CHS genes, wherein A in FIG. 2 is the result of amplification of the primer set designed for the matK gene; b in FIG. 2 is the result of amplification of the primer set designed for the CHS gene; c in FIG. 2 is the result of amplification of the primer set designed for the ITS gene.
Fig. 3 shows the detection result of probe-specific real-time fluorescence PCR of lycium ruthenicum primer, 1 in fig. 3 is a lycium ruthenicum sample, and 2-8 in fig. 3 are sequentially medlar, blueberry, nitraria tangutica, sea buckthorn, blackcurrant, summer black grape, black raspberry and sterile water samples.
FIG. 4 shows the real-time fluorescence PCR absolute sensitivity detection result of the Lycium ruthenicum primer probe, wherein 1 in FIG. 4 is a sample with the DNA concentration of 50 ng/. Mu.L; FIG. 4 is a graph showing a sample of Lycium ruthenicum DNA at a concentration of 5 ng/. Mu.L; FIG. 4 shows a sample of Lycium ruthenicum DNA at a concentration of 0.5 ng/. Mu.L; FIG. 4 is a sample of Lycium ruthenicum DNA at a concentration of 0.05 ng/. Mu.L; FIG. 4 shows a sample of Lycium ruthenicum DNA at a concentration of 0.005 ng/. Mu.L; CK represents a blank.
FIG. 5 shows the real-time fluorescence PCR relative sensitivity detection result of the Lycium ruthenicum primer probe, wherein 1 in FIG. 5 is a sample with the weight ratio of Lycium ruthenicum juice being 100%; FIG. 5 is a graph of 50% by weight of Lycium ruthenicum fruit juice/Sucus Vitis vinifera; FIG. 5 shows a sample of Lycium ruthenicum juice/Sucus Vitis viniferae at a weight ratio of 10%; FIG. 5 shows a sample of Lycium ruthenicum juice/Sucus Vitis viniferae at a weight ratio of 1%; FIG. 5 is a sample of Lycium ruthenicum juice/Charpy juice at a weight ratio of 0.1%; FIG. 5 is a graph of a sample of Lycium ruthenicum juice/Charpy juice at a weight ratio of 0.01%; CK represents a blank.
Detailed Description
The invention will be further elaborated in connection with the drawings and the specific embodiments described below, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 primer design and screening in the method for real-time fluorescent quantitative PCR detection of Lycium ruthenicum Murr-derived components
1. Design method
(1) Real-time fluorescent quantitative PCR primer design
According to the matK, ITS and CHS gene sequences on NCBI GenBank, a conserved region suitable for primer design is searched, and as the Lycium ruthenicum Murr and ITS products are subjected to sun-drying, baking, microwave, far infrared and other modes in the processing process, DNA is possibly broken or degraded, the target sequence needs to be controlled within 200bp to ensure the detection requirements of the Lycium ruthenicum Murr products with different processing degrees.
By comparison analysis of the sequences of the matK, ITS and CHS genes, the comparison analysis results are shown in FIG. 1, and 3 sets of real-time fluorescent quantitative PCR primer sets are designed according to the conserved regions, as shown in Table 1, wherein A-P, B-P and C-P are both probe sequences.
TABLE 1 fluorescent quantitative PCR primer set sequence information
(2) Test set of real-time fluorescent quantitative PCR primers
The real-time fluorescent quantitative PCR reaction system for testing the designed 3 sets of real-time fluorescent quantitative PCR primers comprises the following steps: 12.5. Mu.L of the real-time fluorescent PCR reaction mixture, 5. Mu.L of the template DNA, 10. Mu. Mol/L of the upstream primer F and 10. Mu. Mol/L of the downstream primer R each 0.5. Mu.L, 10. Mu. Mol/L of the probe P0.5. Mu.L, and finally, the use of sterilized water (dd 2 O) was made up to a total volume of 25. Mu.L.
The real-time fluorescent quantitative PCR amplification conditions are as follows: 50 ℃ for 2min; pre-denaturation at 95℃for 10min; 15s at 95℃and 1min at 60℃for 40 cycles.
2. Experimental results
The amplification result is shown in figure 2, wherein A in figure 2 is the amplification result of a primer set designed for the matK gene, the primer set can detect non-lycium ruthenicum species according to A in figure 2, and the specificity of the primer set of B is not in accordance with the requirement; b in FIG. 2 is the result of amplification of a primer set designed for the CHS gene, and it can be seen from B in FIG. 2 that the primer set can detect non-Lycium ruthenicum species, and that the specificity of the primer set of C is not satisfactory; c in FIG. 2 is the amplification result of a primer set designed for the ITS gene, the primer set designed for the ITS gene has the advantage of high specificity, and the Ct value of the primer set amplification is between 18 and 24, the amplification efficiency is high, namely A-F, A-R and A-P are more suitable for a fluorescent quantitative PCR amplification system in the invention, wherein the 3 'end of the probe A-P is connected with a fluorescent quenching group TAMRA, the 5' end of the probe A-P is connected with a fluorescent reporting group FAM, and the nucleotide sequence of the specific amplification target sequence of the primer of the A group is as shown in SEQ ID NO: 4.
Example 2 real-time fluorescence PCR detection method for Lycium ruthenicum Murr source component
A real-time fluorescence quantitative PCR detection method for Lycium ruthenicum Murr source components comprises the following specific steps:
s1, extracting DNA of a sample to be detected;
s2, preparing a real-time fluorescent quantitative PCR reaction system by taking the DNA of the sample to be detected obtained in the step S1 as a template, wherein the real-time fluorescent quantitative PCR reaction system is as follows: 12.5 mu L of real-time fluorescence PCR reaction mixed solution, 5 mu L of template DNA and 10 mu mol/L of nucleotide sequence shown in SEQ ID NO:1 and 10 mu mol/L nucleotide sequences shown in SEQ ID NO:2, 0.5. Mu.L each of A-R shown in FIG. 2, 10. Mu. Mol/L nucleotide sequenceAs set forth in SEQ ID NO: 3A-P0.5. Mu.L, sterilized water (dd 2 O) make up to a total volume of 25. Mu.L;
meanwhile, taking lycium ruthenicum DNA as a positive template to prepare a positive real-time fluorescent quantitative PCR reaction system, wherein the positive real-time fluorescent quantitative PCR reaction system is the same as the real-time fluorescent quantitative PCR reaction system, and the difference is that the DNA of a sample to be detected is replaced by lycium ruthenicum DNA;
to sterilize water (dd) 2 O) is a blank template, a blank real-time fluorescent quantitative PCR reaction system is prepared, and the blank real-time fluorescent quantitative PCR reaction system is identical to the real-time fluorescent quantitative PCR reaction system, except that DNA of a sample to be detected is replaced by sterilized water;
preparing a negative real-time fluorescent quantitative PCR reaction system by taking blueberry DNA as a negative template, wherein the negative real-time fluorescent quantitative PCR reaction system is the same as the real-time fluorescent quantitative PCR reaction system, and the difference is that the DNA of a sample to be detected is replaced by blueberry DNA;
s3, placing the real-time fluorescent quantitative PCR reaction system, the positive real-time fluorescent quantitative PCR reaction system, the blank real-time fluorescent quantitative PCR reaction system and the negative real-time fluorescent quantitative PCR reaction system obtained in the step S2 into a real-time fluorescent quantitative PCR reaction instrument together, and running the real-time fluorescent quantitative PCR reaction under the following real-time fluorescent quantitative PCR amplification conditions: 50 ℃ for 2min;95 ℃ for 10min; 15s at 95 ℃ and 1min at 60 ℃ for 40 cycles;
s4, directly judging whether the lycium ruthenicum source component exists in the sample to be detected according to whether an amplification curve exists in the sample to be detected after the real-time fluorescence quantitative PCR reaction is finished, and considering that the experiment is invalid when one of the following conditions is not met:
i. the positive real-time fluorescent quantitative PCR reaction system has an S-shaped amplification curve;
a negative real-time fluorescent quantitative PCR reaction system has no S-shaped amplification curve;
negative real-time fluorescent quantitative PCR reaction system has no S-shaped amplification curve;
under the condition that the amplification curve of the sample to be detected does not appear, the sample to be detected is proved to contain no lycium ruthenicum source component, and if the amplification curve of the sample to be detected appears, the sample to be detected is proved to contain the lycium ruthenicum source component.
Example 3A real-time fluorescent quantitative PCR detection kit for Lycium ruthenicum Murr-derived components
A real-time fluorescent quantitative PCR detection kit for lycium ruthenicum source components comprises a nucleotide sequence shown in SEQ ID NO: 1-3, and also contains a real-time fluorescence PCR reaction mixed solution and sterile water.
The detection method of the detection kit is the same as that of the real-time fluorescence quantitative PCR detection method of the lycium ruthenicum source component in the embodiment 2.
Example 4 specificity test of method for real-time fluorescent quantitative PCR detection of Lycium ruthenicum Murr-derived Components
1. Test method
The invention utilizes DNA extraction kitFood and DNeasy Mericon Food Kit), DNA samples of lycium ruthenicum, lycium barbarum, blueberry, nitraria tangutica, hippophae rhamnoides, blackcurrant, black grape in summer and black raspberry were extracted, and the obtained DNA samples were detected by a real-time fluorescent quantitative PCR detection method for lycium ruthenicum source ingredient of example 2, using sterile water as a blank control group (denoted CK), 2 replicates per DNA sample, and the detection results are shown in fig. 3.
2. Test results
As can be seen from FIG. 3, only the DNA corresponding to Lycium ruthenicum showed amplification curve (1 in FIG. 3), and the DNA corresponding to other fruits did not show amplification curve. Therefore, the real-time fluorescence quantitative PCR detection method for the lycium ruthenicum mill source component in the embodiment 2 has the advantage of high specificity, and can be used for distinguishing common adulterated species such as nitraria tangutica and blueberries and the like and also distinguishing lycium.
Example 5 absolute sensitivity test of method for real-time fluorescent quantitative PCR detection of Lycium ruthenicum Source
1. Test method
The nucleic acid concentration of the lycium ruthenicum DNA obtained in example 2 was measured, and the extracted lycium ruthenicum DNA was diluted with sterilized water to a DNA concentration of 50 ng/. Mu.L, 5 ng/. Mu.L, 0.5 ng/. Mu.L, 0.05 ng/. Mu.L and 0.005 ng/. Mu.L of lycium ruthenicum DNA dilution.
Absolute sensitivity testing of the real-time fluorescent quantitative PCR assay for lycium ruthenicum source components was performed using the lycium ruthenicum DNA dilutions, using sterile water as a blank (noted CK), 3 replicates per dilution. The detection result is shown in FIG. 4 by using the real-time fluorescence quantitative PCR detection method of the Lycium ruthenicum source component of example 2.
2. Test results
As can be seen from FIG. 4, the DNA concentrations of 50 ng/. Mu.L, 5 ng/. Mu.L, 0.5 ng/. Mu.L, 0.05 ng/. Mu.L and 0.005 ng/. Mu.L of the Lycium ruthenicum DNA dilutions all showed amplification curves, so that the minimum detection limit of the real-time fluorescent quantitative PCR detection method of the Lycium ruthenicum source component of example 2 was 5 pg/. Mu.L.
Example 6 relative sensitivity test of method for real-time fluorescent quantitative PCR detection of Lycium ruthenicum Murr-derived Components
1. Test method
Crushing and homogenizing fresh lycium ruthenicum fruits by using a juicer to prepare lycium ruthenicum juice; in order to avoid cross contamination of samples, another juicer is used for crushing and homogenizing the fresh summer black grape fruits to prepare the summer black grape juice.
The prepared black matrimony vine juice is doped with summer black grape juice with different weights, and the mixed sample of the black matrimony vine juice and the summer black grape juice with the weight ratio of the black matrimony vine juice to the summer black grape juice of 100%, 50%, 10%, 1%, 0.1% and 0.01% is prepared.
DNA extraction kitFood DNA extraction kit), DNA of the mixed sample of lycium ruthenicum juice and summer black grape juice was extracted, and the obtained DNA was detected by a real-time fluorescent quantitative PCR detection method of lycium ruthenicum source component as described in example 2, using sterile water as a blank control group (denoted CK), 3 replicates per DNA sample, and the detection results are shown in fig. 5.
2. Test results
As can be seen from FIG. 5, the mixture of Lycium ruthenicum juice and Sucus Vitis vinifera at the weight ratio of 100%, 50%, 10%, 1%, 0.1% and 0.01% showed amplification curves, and the higher the weight ratio of Lycium ruthenicum juice and Sucus Vitis vinifera, the earlier the amplification curves were. Therefore, the real-time fluorescence quantitative PCR detection method for the Lycium ruthenicum Murr-derived components described in example 2 can detect the Lycium ruthenicum Murr fruit juice mixed sample with the lowest weight ratio of 0.01%, and even if the Lycium ruthenicum Murr fruit juice only occupies a very small weight ratio of the mixed sample, the detection method can still accurately detect the Lycium ruthenicum Murr-derived components.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. A real-time fluorescent quantitative PCR primer group for detecting lycium ruthenicum source components is characterized by comprising a forward primer nucleotide sequence shown in SEQ ID NO:1 and the nucleotide sequence of the forward primer is shown as SEQ ID NO: 2.
2. The real-time fluorescent quantitative PCR primer set of claim 1, further comprising a nucleotide sequence set forth in SEQ ID NO:3, and a probe sequence shown in 3.
3. The real-time fluorescent quantitative PCR primer set of claim 2, wherein the probe sequence has a quenching group attached to the 3 'end and a fluorescent group attached to the 5' end.
4. Use of the real-time fluorescent quantitative PCR primer set according to any one of claims 1 to 3 for preparing a kit for detecting lycium ruthenicum source components.
5. A kit for detecting a lycium ruthenicum source component, which is characterized by comprising the real-time fluorescent quantitative PCR primer set according to any one of claims 1 to 3.
6. The detection kit according to claim 5, wherein the reaction system of the detection kit comprises one or more of a positive control, a negative control, a real-time fluorescent PCR reaction mixture and/or sterile water.
7. The test kit of claim 6, wherein the positive control comprises lycium ruthenicum DNA and the negative control comprises one or more of lycium ruthenicum DNA, blueberry DNA, nitraria tangutica DNA, hippophae rhamnoides DNA, blackcurrant DNA, summer black grape DNA, or blackberry DNA.
8. The qualitative detection method for the lycium ruthenicum source component is characterized by comprising the following steps of:
s1, extracting DNA of a sample to be detected;
s2, carrying out a real-time fluorescent quantitative PCR reaction by using the DNA of the sample to be detected obtained in the step S1 as a template and using the real-time fluorescent quantitative PCR primer set according to any one of claims 1 to 3;
s3, if the sample to be detected does not have an S-shaped amplification curve, the sample to be detected does not contain lycium ruthenicum source components; if the sample to be detected has an S-shaped amplification curve, the sample to be detected contains the lycium ruthenicum source component.
9. The qualitative detection method according to claim 8, wherein the reaction system of the real-time fluorescent quantitative PCR in step S2 further comprises a real-time fluorescent PCR reaction mixture and sterile water.
10. The qualitative detection method according to claim 8, wherein the real-time fluorescent quantitative PCR reaction in step S3 is performed under the following conditions: 50 ℃ for 2min;95 ℃ for 5-10 min; 15s at 95 ℃, 1min at 55-60 ℃ and 40-50 cycles.
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