CN115786570B - Primer, probe, kit and method for detecting dalbergia odorifera - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- 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 gene detection, and discloses a primer, a probe, a kit and a method for detecting rosewood heart wood. The primer comprises: the nucleotide sequence of the upstream primer Do4-F is shown in SEQ ID NO:10 is shown in the figure; the nucleotide sequence of the downstream primer Do4-R is shown as SEQ ID NO: 11. The nucleotide sequence of the probe is shown as SEQ ID NO: shown at 12. The specific primer and the probe provided by the invention can effectively identify and distinguish the rosewood and 7 adulterated similar species, and have the advantages of strong specificity, high sensitivity and good reliability.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a primer, a probe, a kit and a method for detecting rosewood heart wood.
Background
Rosewood Dalbergia odorifera belongs to leguminous plants, is native to Hainan of China and is cultivated in south China. The dried heartwood of the trunk and the root of the rosewood, namely rosewood, is a rare traditional Chinese medicine, has the main functions of removing blood stasis and stopping bleeding, regulating qi and relieving pain, and is used for hematemesis, traumatic hemorrhage, liver Yu Xie pain, chest stuffiness and pain, traumatic injury pain and vomit abdominal pain. At present, pharmacological researches on rosewood heart wood show that the rosewood heart wood has the effects of increasing coronary flow, dilating blood vessels, resisting inflammation, resisting oxidation, resisting tumors and the like, and particularly has obvious performance in the aspect of treating cardiovascular and cerebrovascular diseases. Besides the medicinal value, the rosewood is extremely corrosion-resistant, attractive in texture and durable in fragrance, is commonly called Hainan yellow pear, and is an excellent material for manufacturing high-grade furniture.
The medicinal and wood values of the rosewood are high, the market demand is extremely high, the resources are in shortage, and under the drive of huge profit, mixed and fake products in the market continuously appear, such as the rosewood D.yunnanensis, the indian rosewood D.sisoo, the toe rosewood D.cocchinensis, the oblique She Huangtan D.pinnata, the two-Guangdong rosewood D.benthami, the Hainanensis D.hainanensis, the broadleaf rosewood D.latifolia and the like are often used as or mixed with the rosewood.
According to the literature, the identification modes of the rosewood pseudo product are character identification, microscopic identification, physicochemical identification, ultraviolet spectrum identification and the like, and the traditional identification methods are simple and easy to operate, but are difficult to accurately identify samples, so that the quality of the rosewood medicinal material and the clinical medication safety are seriously affected. The currently adopted molecular identification method mainly adopts a universal primer to measure the ITS fragment sequence, and the sequencing result is imported into a database for comparison and analysis to identify, however, the method has poor specificity and reliability and requires relatively long time (2-3 days).
Therefore, the invention hopes to propose a novel primer and a probe for detecting dalbergia odorifera so as to improve the accuracy and the high efficiency of dalbergia odorifera identification.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. To this end, the invention proposes a primer, a probe, a kit and a method for detecting dalbergia odorifera. The specific primer and the probe provided by the invention can effectively identify and distinguish the rosewood and 7 adulterated similar species, and have the advantages of strong specificity, high sensitivity (the sample DNA with the concentration of 0.001 ng/mu L can be detected) and good reliability.
The invention provides a primer for detecting dalbergia odorifera, which comprises the following components:
The nucleotide sequence of the upstream primer Do4-F is shown in SEQ ID NO:10 is shown in the figure;
the nucleotide sequence of the downstream primer Do4-R is shown as SEQ ID NO: 11.
The invention also provides a probe for detecting dalbergia odorifera, and the nucleotide sequence of the probe is shown as SEQ ID NO: shown at 12.
Preferably, the 5 'end of the probe is labeled with a fluorescent group, and the 3' end of the probe is labeled with a quenching group. The fluorescent group includes but is not limited to being selected from FAM, TET, HEX, CY or JOE, and the quenching group includes but is not limited to being selected from BHQ1, BHQ2, DABCYL, MGB, or Eclipse.
Preferably, at least one nucleotide in the probe is modified with a locked nucleic acid. Since Locked Nucleic Acid (LNA) can raise the annealing temperature (Tm value) of the primer/probe, it can be used to improve the detection performance of the probe.
More preferably, nucleotide 3,4,11,13,14,16,17 in the probe is modified with a locked nucleotide.
The invention also provides a kit for detecting the dalbergia odorifera, which comprises the primer for detecting the dalbergia odorifera and the probe for detecting the dalbergia odorifera.
The invention is based on NCBI database, and the design and screening of primers and probes are carried out by comparing and analyzing 8 plant genome sequences of Dalbergia, such as Dalbergia odorifera D.odontifera, dalbergia yunnanensis, dalbergia indiana D.sisoo, dalbergia tobali D.cobinnanensis, dalbergia cantonensis, dalbergia yunnanensis D.benthami, dalbergia hainanensis D.hainanensis, dalbergia latifolia and the like, and finally, the non-coding fragments between chloroplast genome cemA and petA genes are selected, so that the obtained primers and probes can well detect and distinguish Dalbergia odorifera from 7 adulteration similar species, and have the characteristics of strong specificity, high sensitivity and good reliability.
The invention also provides a method for detecting dalbergia odorifera, which comprises the following steps:
(1) Extracting DNA of a sample to be detected;
(2) Using the sample DNA as a template, and carrying out PCR reaction by adopting the primer for detecting the dalbergia odorifera and the probe for detecting the dalbergia odorifera;
(3) And collecting fluorescent signals to obtain a sample detection result.
Preferably, the method for detecting rosewood heart wood is selected from fluorescent quantitative PCR detection, microdroplet digital PCR detection or gene chip detection.
Preferably, the reaction system of the PCR reaction is as follows: PCR buffer, template, upstream primer, downstream primer, probe, ddH 2 O. Wherein the PCR buffer contains dNTPs and Mg 2+.
More preferably, the concentration of each of the upstream primer, the downstream primer and the probe is 10 to 50. Mu. Mol/L.
Preferably, the reaction conditions of the PCR reaction are: pre-denaturation at 95-96 ℃ for 10-11min; denaturation at 95-96℃for 15-16s, annealing at 60-62℃for 1-2min, and amplification at 40-50.
Compared with the prior art, the invention has the following beneficial effects:
(1) According to the sequence characteristics of 8 rosewood plants such as rosewood, the invention selects a non-coding segment between rosewood chloroplast genome cemA and petA gene, screens and designs specific primers and probes, establishes a real-time fluorescence quantitative PCR detection technology suitable for traditional Chinese medicine rosewood and products thereof, overcomes the limitations of traditional morphology and DNA bar code detection, and realizes rapid and accurate identification of rosewood. The detection technology can effectively distinguish the dalbergia odorifera from 7 adulterated similar species, has the advantages of strong specificity, high sensitivity (the sample DNA with the concentration of 0.001 ng/mu L can be detected) and good reliability, can be applied to the rapid identification and analysis of the dalbergia odorifera, and is expected to provide scientific and powerful technical support for relevant supervision departments, industry association and enterprises.
Drawings
FIG. 1 shows the design position of the Do4 primer probe for detecting rosewood heart wood on chloroplast gene.
FIG. 2 shows the specific detection results of real-time fluorescent quantitative PCR detection using a Do4 primer probe; wherein 1-15 correspond to the test materials of the corresponding serial numbers in Table 2.
FIG. 3 shows the sensitivity detection results of real-time fluorescent quantitative PCR detection using a Do4 primer probe; wherein the concentration of the DNA template in 1-6 is 100 ng/. Mu.L, 10 ng/. Mu.L, 1.0 ng/. Mu.L, 0.1 ng/. Mu.L, 0.01 ng/. Mu.L, 0.001 ng/. Mu.L, respectively.
FIG. 4 shows the specific detection results of the real-time fluorescent quantitative PCR detection of the primer, probe and detection kit for detecting rosewood heart wood in example 1 of patent application (CN 105087798A); wherein 1-15 correspond to the test materials of the corresponding serial numbers in Table 2.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited to the following embodiments, and any modifications, substitutions, and combinations made without departing from the spirit and principles of the present invention are included in the scope of the present invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Example 1: design of specific primers and probes
The study was performed by searching NCBI database (National Center of Biotechnology Information, national center for biotechnology information) for chloroplast whole genome sequences of Pterocarpus species and sequences measured by samples, performing alignment analysis by using MEGA7.0, and designing 4 pairs of specific Primer probes for non-coding genes of rosewood chloroplasts by using Primer 5.0 software and Primer-Blast in NCBI, wherein specific sequence information is shown in Table 1.
TABLE 1 primer, probe sequence information
Wherein the underlined nucleotides of Do4-P are modified with the locked nucleic acid.
FIG. 1 shows the design position of the Do4 primer probe (Do 4-F, do4-R, do-P) on the chloroplast gene.
Example 2: real-time fluorescent quantitative PCR
The real-time fluorescence quantitative PCR detection method comprises the following steps:
(1) Preparation of template DNA
The genomic DNA is extracted by using DNeasy plant nucleic acid extraction Mini kit (Qiagen GmbH in Germany), and the specific steps are as follows:
1. 200mg of wood flour to be measured is averagely packaged into 2-pipe 2-mL microcentrifuge tubes;
2. mu.L of buffer AP1, 300. Mu.L of 1% (w/v) polyvinylpyrrolidone (PVP), 200. Mu.L of proteinase K (20 mg/. Mu.L), 4. Mu.L of RNaseA were added to each tube. Vortex oscillation and uniform mixing;
3. incubating at 65 ℃ for 6-8h to lyse cells, and turning over the centrifuge tube for several times;
4. Cooling for 2min, adding 280 μL Buffer P3, mixing, and placing in a refrigerator at-20deg.C for 2 hr; centrifuging at 13000rpm for 5min, and removing impurities such as protein, polysaccharide, and detergent by precipitation;
5. sucking out the lysate, putting on a QIAshredder column sleeved with a 2mL collecting pipe, centrifuging at 13000rpm for 2min, and removing most sediment and cell debris;
6. The filtered liquid is gently transferred into a plurality of new centrifuge tubes, and the sediment is not stirred, so that the total volume of each tube of liquid is ensured to be about 450 mu L;
7. To each centrifuge tube, 1.5 volumes of AW1 buffer (675. Mu.L, i.e., 225. Mu.L AW1 and 450. Mu.L ethanol) were added and mixed well with a pipette;
8. separating with DNeasy spin column (650 μl each time, including all precipitate components) twice, centrifuging at 8000rpm for 1min, and discarding the filtrate;
9. the remaining 650. Mu.L of the mixture is put on a DNeasy spin column and centrifuged at 8000rpm for 1min, and the filtrate is discarded;
10. In order to improve the DNA yield, the DNA lysate solution of the sample of the 2 nd tube can be sequentially applied to the same rotary column, and the method is the same as that of the step 8;
11. Sleeving another 2mL collecting pipe on the DNeasy rotary column, adding 500 mu L Buffer AW2, centrifuging at 8000rpm for 1min, discarding the liquid, passing through the column (washing the column), and reusing the collecting pipe in the next step;
12. The column was washed again. Adding 500 μl Buffer AW2, centrifuging at 14000rpm for 2min to dry the DNeasy membrane, and discarding the filtrate;
13. Sleeving the DNeasy column with a sterilized 1.5mL centrifuge tube, adding 100 mu L of preheating Buffer AE at 65 ℃ to the DNeasy membrane, standing for 5min at room temperature, and centrifuging at 8000rpm for 1min;
14. adding 100 mu L Buffer AE for eluting once again, and the method is the same as that of the step 13;
15. The obtained DNA template is placed in a low-temperature refrigerator at the temperature of minus 20 ℃ for standby.
(2) Configuration of reaction System
25. Mu.L of reaction system: 12.5. Mu.L of 2 XPCR buffer (containing dNTPs and Mg 2+), 5. Mu.L of DNA template (mass concentration approximately 5 ng/. Mu.L), 0.75. Mu.L of upstream primer (10. Mu.M), 0.75. Mu.L of downstream primer (10. Mu.M), 0.5. Mu.L of probe (10. Mu.M) and ddH 2 O were added to make up the volume to 25. Mu.L.
(3) PCR reaction
Reaction conditions: pre-denaturation at 95℃for 10min; denaturation at 95 ℃, 15s, annealing extension at 60 ℃, 60s,40 cycles.
(4) And detecting the fluorescent signal, and reading the detection result according to the amplification curve.
Example 3: specific detection
1. Test materials
The experimental materials are samples collected from various places, and specific types are identified by microscopic examination of laboratory slices and stored in a plant quarantine laboratory of the Archimedes technical center. The experiment uses 15 samples of rosewood and other pterocarpus species, and the detailed information is shown in Table 2.
Table 2 test material information
| Sequence number | Name of the name | Production area | Collector/collector |
| 1 | Dalbergia odorifera (L.) Odorifera | Guangdong China | Xu Miaofeng A |
| 2 | Dalbergia odorifera (L.) Odorifera | Hainan of China | Xu Miaofeng A |
| 3 | Dalbergia odorifera (L.) Odorifera | Guangdong China | Xu Miaofeng A |
| 4 | Indian yellow sandalwood | Yunnan province of China | Gan Changtao A |
| 5 | Two Guangdong pterocarpus santalinus | Hainan of China | Wang Xianggong A |
| 6 | Oblique She Huangtan | Burmese | Wang Xianggong A |
| 7 | Two Guangdong pterocarpus santalinus | Hainan of China | Wu Hui A |
| 8 | Oblique She Huangtan | Laos | Hu Wen A |
| 9 | Pterocarpus gracilis (Willd.) DC | Yunnan province of China | Peng Bin A |
| 10 | Broad leaf pterocarpus santalinus | Malaysia (Malaysia) | Wang Xianggong A |
| 11 | Dalbergia odorifera | Malaysia (Malaysia) | Wang Xianggong A |
| 12 | Pterocarpus hainanensis (Willd.) Ohwi | Hainan of China | Wu Hui A |
| 13 | Pterocarpus gracilis (Willd.) DC | Yunnan province of China | Peng Bin A |
| 14 | Two Guangdong pterocarpus santalinus | Guangdong China | Wang Xianggong A |
| 15 | Oblique She Huangtan | Hainan of China | Wang Xianggong A |
The primers and probes of example 1 were used in combination with the real-time fluorescent quantitative PCR method of example 2 to specifically detect 15 samples of rosewood and 7 approximations thereof.
The test results of the Do1, do2 and Do3 primer probes show that other similar types are amplified at the same time except for rosewood, and the fact that the 3 primer pairs are not suitable for the authenticity identification of rosewood is indicated. The result of the specificity verification of the Do4 primer probe shows that only the rosewood sample can amplify the fluorescence curve (see figure 2), and other similar types Do not amplify the fluorescence curve, so that the pair of primer probes has good specificity.
Example 4: sensitivity detection
The Do4 primer probe with the best specific detection effect is selected, and the detection sensitivity of the PCR reaction is determined by diluting the extracted rosewood DNA with 7 DNA templates with 10-fold concentration gradients, and each group of samples is repeated three times. As shown in FIG. 3, the fluorescence curve can be amplified when the concentration of the DNA template is 0.001-100 ng/. Mu.L; when the concentration of the DNA template was 0.0001 ng/. Mu.L, the fluorescence curve could not be amplified. The above results indicate that the detection limit of the method is 0.001 ng/. Mu.L, and the optimal detection concentration is 0.01-100 ng/. Mu.L.
Comparative example 1
The test materials (SEQ ID NOS: 1-15) in Table 2 above were specifically tested using the primer, probe and test kit for detecting rosewood heart wood of example 1 of patent application (CN 105087798A), and the results are shown in FIG. 4: besides rosewood, other rosewood (yellow sandalwood of Yunnan Qian, yellow sandalwood of India) have nonspecific amplification; oblique She Huangtan, two Guangdong pterocarpus santalinus, hainan pterocarpus santalinus, broad-leaved pterocarpus santalinus and Dalbergia odorifera are not amplified.
The embodiments of the present application have been described in detail with reference to the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present application. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.
Claims (4)
1. A kit for detecting dalbergia odorifera, which is characterized by comprising a primer and a probe;
the primer comprises:
The nucleotide sequence of the upstream primer Do4-F is shown in SEQ ID NO:10 is shown in the figure;
The nucleotide sequence of the downstream primer Do4-R is shown as SEQ ID NO: 11;
the nucleotide sequence of the probe is shown as SEQ ID NO: shown at 12;
the 3,4,11,13,14,16,17 th nucleotide in the probe is modified by a locked nucleotide.
2. The kit of claim 1, wherein the 5 'end of the probe is labeled with a fluorescent group and the 3' end of the probe is labeled with a quenching group.
3. A method for detecting rosewood heart wood, comprising the steps of:
(1) Extracting DNA of a sample to be detected;
(2) Performing a fluorescent quantitative PCR reaction using the primer and probe of claim 1 using the sample DNA as a template;
(3) And collecting fluorescent signals to obtain a sample detection result.
4. The method of claim 3, wherein the reaction system of the fluorescent quantitative PCR reaction comprises: PCR buffer, template, upstream primer, downstream primer, probe and ddH 2 O.
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| Title |
|---|
| Comparative Analyses of 35 Complete Chloroplast Genomes from the Genus Dalbergia (Fabaceae) and the Identification of DNA Barcodes for Tracking Illegal Logging and Counterfeit Rosewood;Zhou Hong等;Forests;第13卷(第626期);1-16 * |
| 基于 DNA 条形码及 HPLC 对市售黄芩的种质资源鉴定和质量评价;张靖晗 等;中国中药杂志;第47卷(第7期);1-10 * |
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