CN117265177B - Primer probe set for identifying holly spring and real-time fluorescence PCR identification method - Google Patents
Primer probe set for identifying holly spring 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 set for identifying hollyhock springs and a real-time fluorescence PCR identification method. The invention takes the solanum dulcamara as a research object, takes chloroplast gene sequences of the solanum dulcamara, the solanum dulcamara and common confusion products as the basis, finds out the specific primer and the probe of the solanum dulcamara through research, and uses the primer, the probe and a fluorescent PCR method to identify the solanum dulcamara medicinal material from the DNA level.
Description
Technical Field
The invention belongs to the field of medicinal material identification, and particularly relates to a primer probe set for identifying hollyhock springs and a real-time fluorescence PCR identification method.
Background
Solanum dulcis is a plant of Solanaceae, namely, qinghui wolfberrySolanum SptemlobumBunge. Whole grass, originally carried in Shen nong Ben Cao Jing, is listed as a middle-grade product. Has effects of clearing heat and detoxicating, and can be used for treating sore and carbuncle due to heat toxin, sore throat, skin prurigo, etc. Solanum dulcamara is Solanum dulcamara of SolanaceaeSolanum lyratumThe dried whole herb of Thunb has effects of clearing heat, promoting diuresis, removing toxic substance and detumescence, and can be used for treating wind-heat type common cold, cough, etc. Both are called as "Baimao vine", and modern medicine is confusing.
In the traditional Chinese medicine standard or traditional Chinese medicine decoction piece processing standard of each province, the hollyhock spring detection method is microscopic identification, the solanum dulcamara and the hollyhock spring are all administered by whole herbs, the medicinal materials are all dry whole herb fragments in the market, after processing, the appearance and microscopic characteristics are damaged to a certain extent, and the characteristic points of a plurality of medicinal materials disappear or coincide, so that the accurate foundation cannot be identified from the aspects of properties and microscopy. 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.
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 dulcis thunb.
The invention also provides a fluorescence PCR method for identifying the holly spring 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 holly spring 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 hollyhock spring for a mixed sample.
The technical scheme adopted by the invention for achieving the purpose is as follows:
the invention provides a primer probe group for identifying holly spring, which comprises the following components:
SYQ-F：5'- TATAAAATCAAACTCGAATACTCAA -3'；
SYQ-R2：5'- AGTATTTTATTGACATAGACTTTTT -3'；
SYQ-P：5'FAM- AAATGCAGGCAAATACCCTCTCC -3'BHQ1。
further, the primer probe group specifically recognizes the gene sequence of the solanum dulcamara:
CACTGCCTTGATCCACTTGGCTACATCCGCCCCCTCGTCTACTTACAGTCCATTTTTACATTATTTAAATTAGAAAACAAAAGAGTAAAGTTCGAATATTTCTCTTCTTTCTTATTTCAATGATATTATTATTTCGATTATTATTTCAAAGAATAAGAATATGAAGTAAAAATTCTATTTTTTGTGAAATGAAATAAAAAAGATATAGTAACATTAGCAACAAGAGGAACAAGTTATATTTCTACAATTTTCAATAAATATAAAATCAAACTCGAATACTCAATCATGAATAAATGCAGGCAAATACCCTCTCCTTCTTTTTCTATAATGTAAACAAAAAAGTCTATGTCAATAAAATACTAGGAAATTAATAAAGAAAAAGAAAAAAAGAAAGGAGCAATAGCACCCTCTTGATAGAACAAGAAAATGGTTATTGCTCCTTTCTTTTCAAAACCTCCTATAGACTAGGCTCGGATCTTATCCATTTGTAGATGGAGCTTCGATAGCAGCTAGGTCTAGAGGGAAG。
the invention also provides a detection kit containing the primer probe set for identifying the hollyhock springs.
Another object of the present invention is to provide a fluorescent PCR method for identifying spa in real time using the primer probe set for identifying spa as described above, 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 (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 (2.5 mu mol/L) 1 mu L, each 1 mu L of upstream and downstream primer (5 mu mol/L), sample solution 1 mu L and sterilized ultrapure water 6 mu L are respectively added.
Further, in step (3), the control reaction system includes a positive control and a negative control; the positive control is herba Solani Lyrati; the negative control is a white-leaved quartz control medicinal material.
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, 62 ℃,35 seconds, 40 cycles.
Further, the judgment principle in the authentication process is as follows: the white nightshade has no fluorescence logarithmic growth, the hollyhock spring has fluorescence logarithmic growth, and the corresponding CT value is less than 32.0.
The beneficial effects of the invention are as follows: the invention takes the solanum dulcamara as a research object, takes the chloroplast gene sequences of the solanum dulcamara and the solanum dulcamara as the basis, finds out the specific primer and the probe of the solanum dulcamara by research, and applies the primer, the probe and the fluorescence PCR method to the solanum dulcamara medicinal material.
Drawings
FIG. 1 shows the amplification curves of the Sichuan springs for group A primers and probes;
FIG. 2 shows the amplification curves of Solanum dulcamara for group A primers and probes;
FIG. 3 shows the amplification curve of the Sichuan spring for group B primers and probes;
FIG. 4 shows the amplification curves of Solanum dulcamara for the primers and probes of group B;
FIG. 5 is a graph of repeatability test results;
FIG. 6 is a graph showing the results of the precision test;
FIG. 7 is a graph showing the results of an amplification efficiency test;
FIG. 8 is a diagram of a detection limit result;
FIG. 9 is a sample real-time fluorescent amplification curve.
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 the hollyhock medicinal materials 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 (synthesized by Shanghai Biotechnology Co., ltd.), chloroform and absolute ethyl alcohol as analytically pure, solanum dulcamara (China medicine Biotechnology institute, batch No. 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 (number: SYQ) into superfine powder, and preparing sample template DNA solution of about 30mg into positive medicinal solution.
Negative control: grinding herba Solani Lyrati reference material into superfine powder, and preparing negative reference material solution with about 30mg sample template DNA solution.
2.3 Real-time fluorescent PCR method establishment
2.3.1 Probe and primer design
The psb sequences of Solanum dulcamara and Solanum dulcamara are downloaded from a GenBank database, and are subjected to homologous alignment together with sequences obtained by sequencing by using BioEdit software, different SNP sites are found after manual correction, primers and probes are designed in regions of conservation and interspecific differences in the seeds by software analysis, wherein the 5 '-end of each probe is marked with a fluorescein group FAM, the 3' -end of each probe is marked with a quenching group BHQ1, and Primer Premier 5.0 software is used for designing the specific identification primers and probes of the Solanum dulcamara.
The origin of the sequences of the primer and probe of the hollyhock spring (shown as SEQ ID NO. 1):
CACTGCCTTGATCCACTTGGCTACATCCGCCCCCTCGTCTACTTACAGTCCATTTTTACATTATTTAAATTAGAAAACAAAAGAGTAAAGTTCGAATATTTCTCTTCTTTCTTATTTCAATGATATTATTATTTCGATTATTATTTCAAAGAATAAGAATATGAAGTAAAAATTCTATTTTTTGTGAAATGAAATAAAAAAGATATAGTAACATTAGCAACAAGAGGAACAAGTTATATTTCTACAATTTTCAATAAATATAAAATCAAACTCGAATACTCAATCATGAATAAATGCAGGCAAATACCCTCTCCTTCTTTTTCTATAATGTAAACAAAAAAGTCTATGTCAATAAAATACTAGGAAATTAATAAAGAAAAAGAAAAAAAGAAAGGAGCAATAGCACCCTCTTGATAGAACAAGAAAATGGTTATTGCTCCTTTCTTTTCAAAACCTCCTATAGACTAGGCTCGGATCTTATCCATTTGTAGATGGAGCTTCGATAGCAGCTAGGTCTAGAGGGAAG。
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 is 20 mu L in the preparation of the PCR reaction system, 10 mu L of fluorescence PCR reaction mixed solution (2X) is added respectively, 1 mu L of probes (2.5 mu mol/L), 1 mu L of upstream and downstream primers (5 mu mol/L) are added respectively, 1 mu L of sample solution is tested, and 6 mu L of sterilized ultrapure water is added. The positive control reaction system is a reaction system which uses an equal volume of positive medicinal material solution to replace a sample solution; the negative control reaction system is a reaction system which uses an equal volume of negative control 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, 62 ℃,35 seconds, 40 cycles.
As a result, the primer and the probe (SYQ-F, SYQ-R1, SYQ-P) of the group A have S-type fluorescence amplification curves, while the Solanum dulcamara (numbered SYQ) control medicinal material (numbered BYDZ) and the Solanum dulcamara medicinal material (numbered BY-01) have fluorescence amplification curves, so that the primer and the probe have poor specificity. The primers and probes (SYQ-F, SYQ-R2 and SYQ-P) of the B group have S-type fluorescence amplification curves, the solanum dulcamara (numbered SYQ) is free of fluorescence amplification curves of the solanum dulcamara control medicinal material (numbered BYDZ) and the solanum dulcamara medicinal material (numbered BY-01), and the specificity and the effectiveness are good, so the primers and probes of the B group 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 (SYQ-F, SYQ-R2, SYQ-P) of the group B, 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 (2.5 mu mol/L) 1 mu L, each 1 mu L of upstream and downstream primer (5 mu mol/L), sample solution 1 mu L and sterilized ultrapure water 6 mu L are respectively added. The positive control reaction system is a reaction system which uses an equal volume of positive medicinal material solution to replace a sample solution; the negative control reaction system is a reaction system which uses an equal volume of negative control 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, 62 ℃,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 < 32.0.
And (3) result judgment: the white nightshade has no fluorescence logarithmic growth, the hollyhock spring has fluorescence logarithmic growth, and the corresponding CT value is less than 32.0. The amplification curves are shown in FIGS. 3-4.
2.3.3 methodological testing
2.3.3.1 repeatability test
Collecting herba Solani Lyrati (SYQ), 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 about 30mg each part in 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 25.130 and the RSD is 1.8, which shows that the method has good repeatability. The results are shown in Table 3 and FIG. 5.
TABLE 3 results of repeatability investigation
2.3.3.2 Precision of
Grinding herba Solani Lyrati (SYQ) 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.
Six complex wells were set according to the determined method and conditions, and the average CT value was 24.903 and the RSD was 0.32, indicating good precision. The results are shown in Table 4 and FIG. 6.
Table 4 results of precision investigation
2.3.3.3 Amplification efficiency
Grinding herba Solani Lyrati (SYQ) 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 was 93.1% by measurement according to the method and conditions, R 2 0.993. The results are shown in FIG. 7.
2.3.3.4 Detection limit
In order to examine the detection sensitivity of the method, experiments were carried out by mixing Solanum dulcamara (code SYQ) powder with different proportions of Solanum dulcamara control powder, wherein the proportions of Solanum dulcamara powder are 1%, 3%, 5%, 10%, 30% and 50%, respectively. Placing the mixture into a 1.5mL centrifuge tube, and extracting the mixture by using a plant genome DNA extraction kit to obtain a template DNA solution.
The results showed a logarithmic increase in fluorescence and a CT value of 31.572<32 at an incorporation ratio of 5%, which was considered to be a detection of spa. Considering errors caused by factors such as market current situation of medicinal materials and decoction pieces, experimental environment and the like, the detection limit is determined when the blending proportion is 5%. The results are shown in Table 5 and FIG. 8.
TABLE 5 investigation of the incorporation ratio
2.4 sample measurement
And carrying out real-time fluorescence PCR identification on the collected solanum dulcamara by adopting an established method. For the Shuyangquan sample, a fluorescent amplification curve exists as a result, the corresponding CT value is smaller than 32, and the detection result is positive; and the white nightshade has no fluorescence amplification curve, and the detection result is negative. The specific experimental results are shown in fig. 9 and table 6.
TABLE 6 sample measurement results
Note that: "-" represents a non-fluorescent amplification curve.
Claims (6)
1. The primer probe group for identifying the hollyhock springs is characterized by comprising the following components in percentage by weight:
SYQ-F：5'- TATAAAATCAAACTCGAATACTCAA -3'；
SYQ-R2：5'- AGTATTTTATTGACATAGACTTTTT -3'；
SYQ-P：5'FAM- AAATGCAGGCAAATACCCTCTCC -3'BHQ1；
the primer probe group specifically recognizes the gene sequence of the solanum dulcamara:
CACTGCCTTGATCCACTTGGCTACATCCGCCCCCTCGTCTACTTACAGTCCATTTTTACATTATTTAAATTAGAAAACAAAAGAGTAAAGTTCGAATATTTCTCTTCTTTCTTATTTCAATGATATTATTATTTCGATTATTATTTCAAAGAATAAGAATATGAAGTAAAAATTCTATTTTTTGTGAAATGAAATAAAAAAGATATAGTAACATTAGCAACAAGAGGAACAAGTTATATTTCTACAATTTTCAATAAATATAAAATCAAACTCGAATACTCAATCATGAATAAATGCAGGCAAATACCCTCTCCTTCTTTTTCTATAATGTAAACAAAAAAGTCTATGTCAATAAAATACTAGGAAATTAATAAAGAAAAAGAAAAAAAGAAAGGAGCAATAGCACCCTCTTGATAGAACAAGAAAATGGTTATTGCTCCTTTCTTTTCAAAACCTCCTATAGACTAGGCTCGGATCTTATCCATTTGTAGATGGAGCTTCGATAGCAGCTAGGTCTAGAGGGAAG。
2. a test kit comprising the primer probe set for identifying solanum dulcamara according to claim 1.
3. A fluorescent PCR method for identifying spa according to 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 (3) performing PCR reaction and performing qualitative detection.
4. The fluorescent PCR method for real-time identification of spa 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 2.5 mu mol/L, and each 1 mu L of upstream and downstream primers with concentration of 5 mu mol/L are respectively added, sample solution 1 mu L, and sterilized ultrapure water 6 mu L.
5. The fluorescent PCR method for real-time identification of spa according to claim 3, wherein the PCR reaction parameters are: stage 1:95 ℃ for 3 minutes; stage 2:95 ℃,10 seconds, 62 ℃,35 seconds, 40 cycles.
6. The fluorescent PCR method for real-time identification of spa according to any one of claims 3 to 5, wherein the identification criteria are: the white nightshade has no fluorescence logarithmic growth, the hollyhock spring has fluorescence logarithmic growth, and the corresponding CT value is less than 32.0.
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| CN116814812A (en) * | 2023-08-14 | 2023-09-29 | 山东省食品药品检验研究院 | Primer probe set for identifying scorpion of Buthus martensii Karsch and real-time fluorescence PCR identification method |
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| CN116814812A (en) * | 2023-08-14 | 2023-09-29 | 山东省食品药品检验研究院 | Primer probe set for identifying scorpion of Buthus martensii Karsch and real-time fluorescence PCR identification method |
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