CN117265178B - Primer probe group for identifying solanum dulcamara and formula particles thereof and real-time fluorescence PCR identification method - Google Patents
Primer probe group for identifying solanum dulcamara and formula particles thereof and real-time fluorescence PCR identification method Download PDFInfo
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Abstract
The invention belongs to the field of medicinal material identification, and particularly relates to a primer probe group for identifying solanum dulcamara and formulation particles thereof and a real-time fluorescence PCR identification method. The invention takes the white nightshade as a research object, takes the ITS2 gene sequence of the white nightshade and common confusion of hollyhock spring as a basis, researches and finds out the specific primer and probe of the white nightshade, and applies the primer, probe and fluorescent PCR method to the white nightshade medicinal materials and the prescription granules thereof, and the method identifies the white nightshade medicinal materials and the prescription granules thereof from the DNA level, thereby being quick, accurate, effective and safe and being capable of realizing qualitative detection of the white nightshade medicinal materials and the white nightshade prescription granules.
Description
Technical Field
The invention belongs to the field of medicinal material identification, and particularly relates to a primer probe group for identifying solanum dulcamara and formulation particles thereof and a real-time fluorescence PCR identification method.
Background
The medicinal Solanum dulcamara has long history, is a dried whole herb of Solanum dulcamara Thunb. Of Solanaceae, is listed as the top grade in Shennong Ben Cao Jing, and has the effects of clearing heat, promoting diuresis, removing toxic substance and relieving swelling. The Chinese medicinal composition is used for treating wind-heat type common cold, cough, icteric hepatitis, cholecystitis and the like, and modern pharmacological effects indicate that the solanum dulcamara has an anticancer effect. Shu sheep spring is the whole herb of the Solanaceae plant Qing Qi Solanum Sptemlobum Bunge, also listed as the middle-grade product in Shen nong Ben Cao Jing. The modern pharmaceutical special name is solanum dulcamara or solanum lyratum thunb, the fresh plants of the solanum dulcamara and the solanum lyratum thunb are obviously different, but the commercial products are all cut-off products after sun drying, and in the traditional Chinese medicine standard or traditional Chinese medicine decoction piece processing specification of each province, the detection is only carried out from microscopic identification and thin-layer chromatography identification, so that the mixed sample of the solanum dulcamara and the solanum lyratum thunb cannot identify an accurate foundation. DNA bar code technology using ribosomal DNA second internal transcription spacer TIS2-ITS3 or chloroplast psbA-trnH as primer under the guiding principle of DNA bar code molecular identification method of traditional Chinese medicine in four general rules of Chinese pharmacopoeia of 2020 edition can identify Solanum dulcamara and Solanum dulcamara medicinal materials from the DNA level, but is not applicable to mixed samples.
The solanum dulcamara prescription granule is prepared by processing dried whole herb of the solanum dulcamara according to the main quality index of standard decoction, and the detection method of the current quality standard comprises thin layer chromatography identification, characteristic spectrum, content measurement and the like, so that DNA degradation is difficult to extract after processing, and a method for identifying an accurate basic source from a gene level is lacking at present.
Disclosure of Invention
Aiming at the problems that the authenticity cannot be accurately identified in the prior art, the invention provides a primer probe group for identifying the solanum dulcamara and the prescription particles thereof.
The invention also provides a fluorescence PCR method for identifying the solanum dulcamara and the prescription particles thereof in real time by using the primer probe set, which fills up the technical blank in the field and standardizes the market order of the solanum dulcamara medicinal materials; compared with the DNA bar code technology, the real-time fluorescence PCR method provided by the invention has higher accuracy, is quicker, does not need a generation of sequencing, saves test cost, and can rapidly and accurately identify the solanum dulcamara or related preparations containing the same for mixed samples.
The technical scheme adopted by the invention for achieving the purpose is as follows:
the invention provides a primer probe group for identifying solanum dulcamara and formula particles thereof, which comprises the following components:
BQ-01-F1：5'-CTTAAGCGCGAACAGGGTC-3'；
BQ-01-R：5'-GCTCAACTCTCTCTGTTGTCGC-3'；
BQ-01-P：5'FAM-TCTGGGAGTCCGAGCGCGCGAT-3'BHQ1。
further, the gene sequence of the solanum dulcamara specifically recognized by the primer probe group is as follows:
ACGACGGAGCCGCCCGATTCTAAGGCTGGGCTGTTCCCGGTTCGCTCGCCGTTAATAGGGGAATCCTTGTAAGTTTCTTTTCCTCCGCTTATTGATATGCTTAAACTCAGCGGGTAATCCCACCTGACCTGGAGTCGCGGTCAGAGCGCCTTAAGCGCGAACAGGGTCTGGGAGTCCGAGCGCGCGATGGGCTGTGGCCGCGACAACAGAGAGAGTTGAGCTTTCAACCACCACTTGCCGCGACGTCCGTCGACGTGGACTCGCATTTAGKACGGCCGTGGGCTCGAGGCGCACGGGAGGCCAGTATCCGCACCATGACACCCTGCGGCTTGCGGGGGGCGACGTTACGCGTGACGCCCAGGCAGACGTGCCCTCGGCCTAATGGCTTCGGGCGCAACTTGCGTTCAAAGACTCGATGGTTCACAGGATTCTGCAATTCACA。
the invention also provides a detection kit containing the primer probe group for identifying the Bai Yingji source in the solanum dulcamara and the prescription particles thereof.
The invention also provides a fluorescence PCR method for identifying the solanum dulcamara and the prescription particles thereof in real time by using the primer probe group for identifying the solanum dulcamara and the prescription particles thereof, which comprises the following steps:
(1) Extracting template DNA of a sample to be detected;
(2) Preparing a PCR reaction system: fluorescent PCR reaction mixed solution, a probe, an upstream primer, a downstream primer, a sample solution and sterilized ultrapure water;
(3) The control reaction system is a reaction system which uses an equal volume of control medicinal material solution to replace a sample solution; the blank control reaction system is a reaction system which uses equal volume of sterilized ultrapure water to replace a sample solution; the sample and the contrast are provided with two repetitions, and the CT value takes the average value of the two repetitions as the final result;
(4) And (3) performing PCR reaction and performing qualitative detection.
Further, in the step (2), the PCR reaction system specifically includes: the total volume is 20 mu L, and fluorescence PCR reaction mixed solution (2X) 10 mu L, probe (0.5 mu mol/L) 1 mu L, each 1 mu L of upstream and downstream primer (1 mu mol/L), sample solution 0.5 mu L and sterilized ultrapure water 6.5 mu L are respectively added.
The identification method provided by the invention comprises the following parameters of PCR reaction: stage 1:95 ℃ for 3 minutes; stage 2:95 ℃,10 seconds, 66 ℃,35 seconds, 40 cycles.
Further, in the step (3), the control reaction system is a positive control and a negative control; the positive control is a white-leaved quartz control medicinal material; the negative control is herba Solani Lyrati.
Further, the judgment principle in the authentication process is as follows: the positive control has fluorescence logarithmic growth, and the corresponding CT value is less than 30.0; the fluorescence logarithm is increased, and the absolute value of the difference between CT values of the sample and the positive control is less than or equal to 4, so that the white quartz is detected; there is no fluorescence log increase in hollyhock springs.
The related preparation of the solanum dulcamara detected by the invention is preferably a solanum dulcamara formula particle, and the primer probe group provided by the invention can detect whether Bai Yingji of the solanum dulcamara formula particle is accurate or not.
The beneficial effects of the invention are as follows: the invention takes the solanum dulcamara as a research object, takes the ITS2 gene sequences of the solanum dulcamara and the hollyhock spring as the basis, researches and finds out the specific primer and the probe of the solanum dulcamara, and applies the primer, the probe and the fluorescence PCR method to the solanum dulcamara medicinal materials and the prescription granules thereof.
Drawings
FIG. 1 shows amplification curves of Solanum dulcamara for primers and probes of group A;
FIG. 2 is an amplification curve of the primer and probe of group A in Shuquan;
FIG. 3 shows amplification curves of Solanum dulcamara for primers and probes of group B;
FIG. 4 is an amplification curve of the primer and probe of group B in Shuquan;
FIG. 5 is a specific amplification curve of Solanum dulcamara in the identification method of the present invention;
FIG. 6 is a graph showing the specific amplification of Sichuan spring by the identification method of the present invention;
FIG. 7 is a graph of repeatability test results;
FIG. 8 is a graph showing the results of the precision test;
FIG. 9 is a graph showing the results of amplification efficiency experiments;
FIG. 10 is a graph of recovery rate test results;
FIG. 11 is a graph of actual sample measurement results;
FIG. 12 is a graph showing amplification of the extract of Solanum dulcamara formula particles using the plant genomic DNA extraction kit;
FIG. 13 is a graph showing amplification of Wizard SV Genomic DNA Purification System kit extract Solanum dulcamara formula particles;
FIG. 14 is a DNeasy three Food Kit (50) Kit extract Solanum dulcamara particle amplification plot.
Detailed Description
The technical scheme of the invention is further explained and illustrated by specific examples.
The detailed information table of the solanum dulcamara and solanum dulcamara prescription granule is shown in the table 1.
TABLE 1 sample collection case
Example 1
Fluorescent quantitative PCR method establishment
1. Instrument and reagent
An electronic analytical balance (Sartorius CP 225D), a pure water meter, a centrifuge (Eppendorf 2545), a mini centrifuge, a thermostatic mixer (Rui Cheng instruments Co., ltd., TS 100), real-time quantitative PCR instrument (ABI Co., quantum studio 5), plant genomic DNA extraction kit (TIANGEN, DP 305-03), bestar TM qPCR Master Mix (DBI, P-2025259), primer (Shanghai Biotechnology Co., ltd.) and absolute ethanol as analytically pure, solanum dulcamara (China pharmaceutical Biotechnology institute, lot number 121316-200402).
2. Sample processing and DNA extraction
2.1 Sample processing
Taking the medicinal materials, wiping the surfaces of the medicinal materials with 75% ethanol, airing the surface moisture, and grinding the medicinal materials into superfine powder in a mortar for later use.
2.2 DNA extraction
2.2.1 Template DNA extraction
Sample template DNA extraction: about 30mg of the sample powder was taken, placed in a 1.5mL centrifuge tube, and DNA was extracted with a plant genomic DNA extraction kit (DP 305, TIANGEN), and the following steps were performed: adding 700 mu L of a preheating buffer solution GP1 at 65 ℃, rapidly reversing and uniformly mixing, standing for 20min at 65 ℃, and oscillating for several times in the standing process; adding 700 mu L of chloroform, fully and uniformly mixing, and centrifuging at 12000rpm (13400 Xg) for 5min; carefully transferring the upper water phase obtained in the previous step into a new centrifuge tube, adding 700 mu L of buffer solution GP2, and fully and uniformly mixing; transferring the uniformly mixed liquid into an adsorption column CB3 (the adsorption column is placed in a collecting pipe), centrifuging at 12000rpm (13400 Xg) for 30sec, and discarding the waste liquid; adding 500 mu L buffer solution GD into an adsorption column CB3, centrifuging at 12000rpm (13400 Xg) for 30sec, pouring out waste liquid, and placing the adsorption column GB3 back into a collecting pipe; adding 600 mu L buffer PW into an adsorption column CB3, centrifuging at 12000rpm (13400 Xg) for 30sec, pouring out waste liquid, and placing the adsorption column GB3 back into a collecting pipe; adding 600 mu L buffer PW into an adsorption column CB3, centrifuging at 12000rpm (13400 Xg) for 30sec, pouring out waste liquid, and placing the adsorption column GB3 back into a collecting pipe; centrifuging at 12000rpm (13400 Xg) for 2min, pouring out the waste liquid, and standing the adsorption column GB3 at room temperature for several minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping 50 mu l of elution buffer TE into the middle part of the adsorption film, standing for 5min at room temperature, centrifuging at 12000rpm (13400 Xg) for 2min, and collecting the solution into the centrifuge tube to obtain a DNA solution, namely a sample solution.
Positive control: grinding herba Solani Lyrati reference material (number: BYDZ) into superfine powder, and preparing sample template DNA solution of about 30mg into positive reference material solution.
Negative control: grinding herba Solani Lyrati into superfine powder, and preparing negative medicinal solution with sample template DNA solution of about 30 mg.
2.3 Real-time fluorescent PCR method establishment
2.3.1 Probe and primer design
The GenBank database downloads ITS2 sequences of Solanum dulcamara and Solanum dulcamara, uses BioEdit software to carry out homologous alignment together with sequences obtained by sequencing, searches for differential SNP sites after manual correction, designs primers and probes in a conserved and inter-species difference region by software analysis, wherein the 5 '-end of the probes is marked with a fluorescein group FAM, the 3' -end of the probes is marked with a quenching group BHQ1, and the Primer Premier 5.0 software is used for designing the Solanum specificity identification primers and probes.
The source of the solanum dulcamara primer and probe sequences (shown in SEQ ID NO. 1):
ACGACGGAGCCGCCCGATTCTAAGGCTGGGCTGTTCCCGGTTCGCTCGCCGTTAATAGGGGAATCCTTGTAAGTTTCTTTTCCTCCGCTTATTGATATGCTTAAACTCAGCGGGTAATCCCACCTGACCTGGAGTCGCGGTCAGAGCGCCTTAAGCGCGAACAGGGTCTGGGAGTCCGAGCGCGCGATGGGCTGTGGCCGCGACAACAGAGAGAGTTGAGCTTTCAACCACCACTTGCCGCGACGTCCGTCGACGTGGACTCGCATTTAGKACGGCCGTGGGCTCGAGGCGCACGGGAGGCCAGTATCCGCACCATGACACCCTGCGGCTTGCGGGGGGCGACGTTACGCGTGACGCCCAGGCAGACGTGCCCTCGGCCTAATGGCTTCGGGCGCAACTTGCGTTCAAAGACTCGATGGTTCACAGGATTCTGCAATTCACA。
two groups of probes and primers were designed based on the sequences, and the sequences of the identified probes and primers are shown in Table 2.
TABLE 2 probes and primer sequence listing
2.3.2 Probe and primer screening
The total volume of the PCR reaction system is 20 mu L, 10 mu L of fluorescent PCR reaction mixed solution (2X) is respectively added, 1 mu L of probes (0.5 mu mol/L), 1 mu L of upstream and downstream primers (1 mu mol/L) are respectively added, 0.5 mu L of sample solution is tested, and 6.5 mu L of sterilized ultrapure water is added. The positive control reaction system is a reaction system which uses an equal volume of positive control medicinal material solution to replace a test sample solution; the negative control reaction system is a reaction system which uses an equal volume of negative medicinal material solution to replace a sample solution; the blank control reaction system is to replace the test sample solution with an equal volume of sterilized ultrapure water. The test sample and the control are provided with two repetitions, and the CT value takes the average value of the two repetitions as the final result.
And (3) PCR reaction: the reaction system is placed in a fluorescence quantitative PCR instrument for carrying out, and the following parameters are set: stage 1:95 ℃ for 3 minutes; stage 2:95 ℃,10 seconds, 66 ℃,35 seconds, 40 cycles.
As a result, the primers and probes (BQ-01-F1, BQ-01-R, BQ-01-P) of the group A have S-type fluorescent amplification curves, while the Solanum dulcamara (BY-01, BYDZ) has no fluorescent amplification curves, and the specificity and the effectiveness are good. The primers and probes of group B (BQ-02-F, BQ-02-R, BQ-02-P) have fluorescent amplification curves, while the Solanum dulcamara (BY-01, BYDZ) has fluorescent amplification curves, and the Shuquan (SYQ) has fluorescent amplification curves, so that the specificity of the primers and probes of group A is poor, and therefore, the primers and probes of group A are selected for testing. The specific results are shown in FIGS. 1-4.
By examining a series of test parameters such as the concentration of the primer and the probe (BQ-01-F1, BQ-01-R, BQ-01-P) of the group A, the amplification cycle number and the like, the system for determining the fluorescent PCR reaction according to the test result is as follows:
the total volume is 20 mu L, and fluorescence PCR reaction mixed solution (2X) 10 mu L, probe (0.5 mu mol/L) 1 mu L, each 1 mu L of upstream and downstream primer (1 mu mol/L), sample solution 0.5 mu L and sterilized ultrapure water 6.5 mu L are respectively added. The positive control reaction system is a reaction system which uses an equal volume of positive control medicinal material solution to replace a test sample solution; the negative control reaction system is a reaction system which uses an equal volume of negative medicinal material solution to replace a sample solution; the blank control reaction system is to replace the test sample solution with an equal volume of sterilized ultrapure water. The test sample and the control are provided with two repetitions, and the CT value takes the average value of the two repetitions as the final result.
PCR reaction conditions: the reaction system is placed in a fluorescence quantitative PCR instrument for carrying out, and the following parameters are set: stage 1:95 ℃ for 3 minutes; stage 2:95 ℃,10 seconds, 66 ℃,35 seconds, 40 cycles.
The quality requirements are as follows: the experiment should simultaneously meet the following conditions: (1) The blank control has no fluorescence logarithmic growth or corresponding CT value > 40.0; (2) Negative control no fluorescence logarithmic growth or corresponding CT value > 40.0; (3) The positive control had a logarithmic increase in fluorescence and the corresponding CT value was < 30.0.
And (3) result judgment: the fluorescence logarithm is increased, and the absolute value of the difference between CT values of the sample and the positive control is less than or equal to 4, so that the white quartz is detected. The amplification curves are shown in FIGS. 5-6.
2.3.3 methodological testing
2.3.3.1 repeatability test
Collecting herba Solani Lyrati (BY-01), wiping the surface of the medicinal material with 75% ethanol, air drying, grinding into superfine powder in a mortar, sampling six parts in parallel, placing each part in about 30mg, placing into a 1.5mL centrifuge tube, and extracting with plant genome DNA extraction kit to obtain template DNA solution.
The measurement is carried out according to the determined method and conditions, and the average CT value of 6 parts of the solanum dulcamara medicinal material is 24.917, and the RSD is 0.59%, which shows that the method has good repeatability. The results are shown in Table 3 and FIG. 7.
TABLE 3 results of repeatability investigation
2.3.3.2 Precision of
Grinding herba Solani Lyrati (number BY-01) into superfine powder in a mortar, placing about 30mg in a 1.5mL centrifuge tube, and extracting with plant genome DNA extraction kit to obtain template DNA solution.
Six complex wells were set according to the determined method and conditions, with an average CT value of 23.408 and an RSD of 0.35, indicating good precision. The results are shown in Table 4 and FIG. 8.
Table 4 results of precision investigation
2.3.3.3 Amplification efficiency
Grinding herba Solani Lyrati reference material (BYDZ) into superfine powder in a mortar, placing about 30mg into a 1.5mL centrifuge tube, and extracting with plant genome DNA extraction kit to obtain template DNA solution. Stepwise 5-fold gradient dilution with sterilized ultrapure water was carried out, and total dilution was carried out to 5 concentration gradients.
The amplification efficiency obtained by the measurement according to the determined method and conditions was 99.1%, R 2 0.991. The results are shown in FIG. 9.
2.3.3.4 recovery rate
In order to examine the detection sensitivity of the method, the solanum dulcamara control medicinal material (number BYDZ) powder with different proportions is mixed into the Sichuan spring medicinal material powder for experiment, and the adulteration proportion and the recovery rate are calculated by a CT difference value method.
The recovery rate of the quantitative detection of the white-English adulteration is 70.83% -109.01%, and the results are shown in Table 5 and FIG. 10.
TABLE 5 investigation of the incorporation ratio
2.3.3.5 detection limit
The PCR reaction is exponentially amplified, the cycle number when the threshold value is reached is CT value, the difference between CT values of the control (S) and the test sample (T) is represented by delta CT value, and the adulteration ratio is calculated:
test (T)/control (S)% =2 ΔCT ╳100%
When the adulteration proportion is 10%, the absolute value of delta CT is 3-4, and the absolute value of delta CT is less than or equal to 4 as a detection basis in consideration of errors caused by factors such as market current situation of medicinal materials and decoction pieces, experimental environment and the like.
TABLE 6 absolute value of DeltaCT for different incorporation ratios
2.4 sample measurement
And carrying out real-time fluorescence PCR identification on the collected solanum dulcamara by adopting an established method. For the solanum dulcamara samples, the result 10 batches of the solanum dulcamara medicinal materials have fluorescence amplification curves, compared with the positive control medicinal materials, the absolute value of the difference between the corresponding CT values is less than or equal to 4, and the detection result is positive; and the detection result is negative due to the non-fluorescent amplification curve of the hollyhock spring. The specific experimental results are shown in fig. 11 and table 7.
TABLE 7 sample measurement results
Note that: "-" represents a non-fluorescent amplification curve.
Example 2
Real-time fluorescence PCR method for detecting Solanum dulcamara prescription granule (extraction of DNA in prescription granule)
1. Reagent
Plant genome DNA extraction Kit (TIANGEN, DP 305-03), wizard SV Genomic DNA Purification System Kit (Promega, 0000576156), DNeasy three mericon Food Kit (50) Kit (QIAGEN, 172011941)
2. Sample processing and DNA extraction
2.1 Sample processing
Grinding herba Solani Lyrati granule into superfine powder in a mortar for use.
2.2 DNA extraction
2.2.1 Extraction of plant genome DNA extraction kit
400mg of the powder of the Dioscorea dulcis formula particles were placed in a 2mL centrifuge tube, 700. Mu.L of prewashing buffer (700mM NaCl;20mM EDTA;100mM Tris;2% PVP-40;0.4% DTT) was added, vortexed for 5 minutes, centrifuged at 7500rpm for 3 minutes, the supernatant was removed, and the above steps were repeated until the supernatant was almost colorless. The extraction was performed according to the DNA extraction instructions.
2.2.2 Wizard cube SV Genomic DNA Purification System kit extraction
40mg of the solanum dulcamara formula particle powder is taken and placed in a 2mL centrifuge tube, and extraction is carried out according to a DNA extraction instruction.
2.2.3 DNeasy three Food Kit (50) Kit extraction of the powder 400mg of the solanum dulcamara prescription granule, placing in a 2mL centrifuge tube, and extracting according to the DNA extraction instruction.
2.3 Real-time fluorescence PCR method
The PCR reaction system is as follows: the total volume is 20 mu L, and fluorescence PCR reaction mixed solution (2X) 10 mu L, probe (0.5 mu mol/L) 1 mu L, each 1 mu L of upstream and downstream primer (1 mu mol/L), sample solution 0.5 mu L and sterilized ultrapure water 6.5 mu L are respectively added. The positive control reaction system is a reaction system which uses an equal volume of positive control medicinal material solution to replace a test sample solution. The test sample and the control are provided with two repetitions, and the CT value takes the average value of the two repetitions as the final result.
PCR reaction conditions: the reaction system is placed in a fluorescence quantitative PCR instrument for carrying out, and the following parameters are set: stage 1:95 ℃ for 3 minutes; stage 2:95 ℃,10 seconds, 66 ℃,35 seconds, 40 cycles.
And (3) result judgment: the fluorescence logarithm is increased, and the absolute value of the difference between CT values of the sample and the positive control is less than or equal to 4, so that the white quartz is detected.
2.4 Sample detection
And carrying out real-time fluorescence PCR identification on the solanum dulcamara formula particles extracted by the three kits by adopting an established method. Compared with a positive control medicinal material, the DNA extracted by the DNeasy cube Food Kit (50) Kit has absolute values of differences between corresponding CT values of less than or equal to 4, and the detection result is positive; the other two kits extract DNA without fluorescence amplification curve. The specific experimental results are shown in fig. 12-14 and table 8.
Table 8 determination of the Solanum dulcamara formulation granules
Note that: "-" represents a non-fluorescent amplification curve.
Claims (7)
1. The primer probe group for identifying the solanum dulcamara and the formula particles thereof is characterized in that the primer probe group is as follows:
BQ-01-F1：5'-CTTAAGCGCGAACAGGGTC-3'；
BQ-01-R：5'-GCTCAACTCTCTCTGTTGTCGC-3'；
BQ-01-P：5'FAM-TCTGGGAGTCCGAGCGCGCGAT-3'BHQ1，
the gene sequence of the solanum dulcamara specifically recognized by the primer probe group is as follows:
ACGACGGAGCCGCCCGATTCTAAGGCTGGGCTGTTCCCGGTTCGCTCGCCGTTAATAGGGGAATCCTTGTAAGTTTCTTTTCCTCCGCTTATTGATATGCTTAAACTCAGCGGGTAATCCCACCTGACCTGGAGTCGCGGTCAGAGCGCCTTAAGCGCGAACAGGGTCTGGGAGTCCGAGCGCGCGATGGGCTGTGGCCGCGACAACAGAGAGAGTTGAGCTTTCAACCACCACTTGCCGCGACGTCCGTCGACGTGGACTCGCATTTAGKACGGCCGTGGGCTCGAGGCGCACGGGAGGCCAGTATCCGCACCATGACACCCTGCGGCTTGCGGGGGGCGACGTTACGCGTGACGCCCAGGCAGACGTGCCCTCGGCCTAATGGCTTCGGGCGCAACTTGCGTTCAAAGACTCGATGGTTCACAGGATTCTGCAATTCACA。
2. a test kit comprising a primer probe set according to claim 1 for identifying the source of Bai Yingji in solanum dulcamara and its formulation.
3. A fluorescent PCR method for real-time identification of solanum dulcamara and its formulation particles using the primer probe set for identification of solanum dulcamara and its formulation particles as claimed in claim 1, comprising the steps of:
(1) Extracting template DNA of a sample to be detected;
(2) Preparing a PCR reaction system: fluorescent PCR reaction mixed solution, a probe, an upstream primer, a downstream primer, a sample solution and sterilized ultrapure water;
(3) The control reaction system is a reaction system which uses an equal volume of control medicinal material solution to replace a sample solution; the blank control reaction system is a reaction system which uses equal volume of sterilized ultrapure water to replace a sample solution; the sample and the contrast are provided with two repetitions, and the CT value takes the average value of the two repetitions as the final result;
(4) And (5) performing PCR reaction and qualitative detection.
4. The fluorescence PCR method for real-time identification of Solanum dulcamara and its formulation according to claim 3, wherein in step (2), the PCR reaction system is specifically: the total volume is 20 mu L, and fluorescence PCR reaction mixed solution 10 mu L, probe 1 mu L with concentration of 0.5 mu mol/L, and each 1 mu L of upstream and downstream primers with concentration of 1 mu mol/L are added respectively, and sample solution 0.5 mu L and sterilized ultrapure water 6.5 mu L are tested.
5. The fluorescent PCR method for real-time identification of solanum dulcamara and its formulation according to claim 3, wherein in step (3), said control reaction system is a positive control and a negative control; the positive control is a white-leaved quartz control medicinal material; the negative control is herba Solani Lyrati.
6. The fluorescent PCR method for real-time identification of solanum dulcamara and its formulation according to claim 3, wherein said PCR reaction parameters are: stage 1:95 ℃ for 3 minutes; stage 2:95 ℃,10 seconds, 66 ℃,35 seconds, 40 cycles.
7. The fluorescence PCR method for identifying the solanum dulcamara and the formula particles thereof according to any one of claims 3 to 6, wherein the judgment principle in the identification process is as follows: the positive control has fluorescence logarithmic growth, and the corresponding CT value is less than 30.0; the fluorescence logarithm is increased, and the absolute value of the difference between CT values of the sample and the positive control is less than or equal to 4, so that the white quartz is detected; there is no fluorescence log increase in hollyhock springs.
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| CN114807419A (en) * | 2022-05-09 | 2022-07-29 | 山东省食品药品检验研究院 | Method for identifying lonicera confusa and preparation thereof by using TaqMan probe and specific primer |
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| CN102191318A (en) * | 2011-03-04 | 2011-09-21 | 广州中医药大学 | Application of nucleotide sequence of rDNA (recombinant deoxyribonucleic acid) ITS (internal transcribed spacer)-D3 region in establishment of DNA (deoxyribonucleic acid) bar code identification system for medicinal plants |
| CN114807419A (en) * | 2022-05-09 | 2022-07-29 | 山东省食品药品检验研究院 | Method for identifying lonicera confusa and preparation thereof by using TaqMan probe and specific primer |
Non-Patent Citations (2)
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|---|
| ITS2二级结构系统发育信息在茄属药用植物DNA条形码鉴定中的应用价值;杨烁;薛渊元;李美慧;赵奉熙;赵宏;张伟;;中国中药杂志(第03期);第456-464页 * |
| 基于DNA 条形码鉴定龙葵及其近缘种;陈小露;中药材;第10卷(第2期);第290-292页 * |
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