CN113151556A - Method for detecting botanical ingredients of radix aconiti kusnezoffii - Google Patents
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Abstract
The invention provides a primer and a probe for detecting the wild aconite root plant source components by using a real-time fluorescence PCR method and a specific detection method, which have good sensitivity and specificity and can be used for quickly detecting the wild aconite root plant source components.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for detecting wild aconite root plant source components, in particular to primers, probes and a method for detecting the wild aconite root plant source components.
Background
Radix Aconiti Kusnezoffii is Aconitum kusnezoffii (Aconiti Kusnezoffii) of RanunculaceaeAconitum kusnezoffii Reichb.) The dry root tuber is pungent, bitter and hot in taste, has the effects of dispelling wind and eliminating dampness, warming channels and relieving pain, and is used for treating anemofrigid-damp arthralgia, psychroalgia of heart and abdomen, cold hernia pain and anesthesia pain. Although kusnezoff monkshood root has medicinal value, poisoning easily occurs due to difficulty in mastering poisoning dose and treatment dose when in use. The main component causing the aconitine to cause the aconite root poisoning is the aconitine, the toxicity of the aconite root poisoning to people is extremely strong, and usual people can have palpitation, arrhythmia and even sudden cardiac arrest by only taking 3-4 mg of the aconitine.
At present, the detection of the kusnezoff monkshood root is mainly carried out through appearance identification, but the appearance identification is not suitable for the detection of processed products, particularly powder medicines. In addition, the primary detection of the kusnezoff monkshood root is carried out by a spectrophotometry method, an indicator method and the like, but the sensitivity and the specificity are poor.
The real-time fluorescence PCR method can carry out species detection from a gene level, and specifically comprises the steps of adding a specific fluorescence probe into a PCR amplification reaction system, and utilizing fluorescence signal accumulation to monitor the whole PCR process in real time, namely detecting the amplification state of a target nucleotide sequence so as to judge the result. Its main advantage is: (1) the detection of the amplification product is carried out in a closed tube state, and the post-treatment of a PCR product is not needed, so that false positive caused by the pollution of the amplification product is avoided, and the reliability of the result is ensured; (2) the probe has stronger hybridization specificity and higher sensitivity, a fluorescent probe which can be complementarily matched with the template is added on the basis of the common PCR amplification, the specificity is improved, and the fluorescent signal is automatically collected by an instrument, so that the sensitivity is further improved; (3) the existence of enzyme cutting sites is not needed, enzyme cutting is not needed, electrophoresis is carried out, the time is saved, and subsequent treatment is not needed after PCR; (4) the whole experiment consumes short time, the operation is simple and quick, and the data analysis is simple.
At present, a real-time fluorescent PCR detection method for the wild aconite root plant source components is not available, and the development of a rapid and simple technology for detecting the wild aconite root plant source components is urgently needed.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a primer, a probe and a detection method for detecting the wild aconite root plant source components by using a real-time fluorescence PCR method.
By homology analysis of sequences of the aconitum kusnezoffii rbc L gene, MATK gene, ITS2 gene, psbA-trnH gene and the like, aconitum (Aconitum species) cannot be excludedAconitum.) Finally, repeatedly comparing the interference of other species on the detection of the kusnezoff monkshood root, confirming that partial sequences of the NADH dehydrogenase 3 subunit (NADH dehydrogenase subunit 3) of the kusnezoff monkshood root have better specificity, and designing a specific detection primer and a probe after comparing homologous sequences:
the primers for real-time fluorescent PCR detection of the wild aconite root plant source components are as follows:
an upstream primer: 5'-ATGTCAGAATTTGCACCTATTTGTAT-3' the flow of the air in the air conditioner,
a downstream primer: 5'-CCTATCCAGAAAAATTGTCGGC-3' the flow of the air in the air conditioner,
the fluorescent probe is as follows: 5'-TATTTAGTGATCAGTCCGCTA-3' are provided.
The above fluorescent probe, 5‘End is FAM fluorescent label, 3’The end is MGB fluorescent label.
The real-time fluorescence PCR detection method for the wild aconite root plant source components comprises the following steps:
1. extracting the genomic DNA of a sample to be detected
2. Respectively diluting an upstream primer, a downstream primer and a probe for detecting the plant source components of the kusnezoff monkshood root to 10 [ mu ] mol/L, taking the extracted DNA as a template, adding the primer pair, the fluorescent dye-labeled probe and an enzyme reaction solution for carrying out real-time fluorescent PCR reaction, and recording the Ct value of the sample reaction;
the real-time fluorescent PCR reaction system is as follows:
2 Xreal-time PCR premixed solution 10 mu L
Upstream primer (10 mu mol/L) 1 mu L
Downstream primer (10 mu mol/L) 1 mu L
Fluorescent probe (10 mu mol/L) 1 mu L
Sample DNA (10-50 ng/muL) 2 muL
ddH2O 5µL
3. The real-time fluorescent PCR reaction condition is 95 ℃ for 10min, 1 cycle; the FAM channel is set up at 95 ℃ for 15s, 56 ℃ for 30s and 40 cycles.
The real-time fluorescence PCR method is also provided with a blank control, a negative control and a positive control, and the judgment standard is as follows:
blank control: by ddH2Performing real-time fluorescence PCR reaction by using O as a template under the conditions of 2 and 3, wherein the Ct value detected by an FAM channel is more than or equal to 40;
negative control: carrying out real-time fluorescence PCR reaction by using non-kusnezoff monkshood root genomic DNA as a template under the conditions of 2 and 3, wherein the Ct value detected by an FAM channel is more than or equal to 40;
positive control: carrying out real-time fluorescence PCR reaction by using genomic DNA containing kusnezoff monkshood as a template under the conditions of 2 and 3, wherein the Ct value detected by an FAM channel is less than or equal to 30;
the FAM channel detection Ct value of the sample to be detected is more than or equal to 40, and if the negative control result, the positive control result and the blank control result are normal, the sample is judged to have no detected wild aconite root plant source component;
the FAM channel detection Ct value of the sample to be detected is less than 36, and the negative control, the positive control and the blank control result are normal, and the detected wild aconite root plant source component of the sample is judged;
the Ct value of FAM channel detection of a sample to be detected is 36-40, real-time fluorescence PCR amplification is repeated, the Ct value of the result after the secondary amplification is not less than 40, negative control is performed, the positive control and blank control result are normal, and the fact that the kusnezoff monkshood root plant source component is not detected in the sample is judged; and (5) after the secondary amplification, determining that the detected wild aconite root plant source component of the sample is still less than 40, and judging that the Ct value of the result is less than 40, the negative control result and the positive control result and the blank control result are normal.
The invention provides a primer and a probe for detecting the wild aconite root plant source components by using a real-time fluorescence PCR method and a specific detection method, which have good sensitivity and specificity and can be used for quickly detecting the wild aconite root plant source components.
Drawings
FIG. 1 is a real-time fluorescence PCR amplification chart for detecting a positive sample of kusnezoff monkshood root in example 1 of the present invention; wherein, 1 is 50ng kusnezoff monkshood root DNA sample, and 2 is 0.05ng kusnezoff monkshood root DNA sample;
FIG. 2 is a real-time fluorescence PCR amplification chart for detecting samples of radix Aconiti Kusnezoffii, and radix Aconiti Kusnezoffii, in example 2 of the present invention; wherein 1 is radix Aconiti Kusnezoffii; 2 is prepared from Bulleyaconitum vilmorinianum (Bulley aconitum kusnezoffii) (Bulley root)Aconitum vilmorinianum) (ii) a 3 is prepared from radix Aconiti KusnezoffiiAconitum episcopale) (ii) a 4 is prepared from Aconitum carmichaeli Debx (radix Aconiti Kusnezoffii)Aconitum hemsleyanum）。
The specific implementation mode is as follows:
the present invention is described in further detail below.
Example 1
1. Primers and probes used:
designing and synthesizing a real-time fluorescent PCR specific amplification primer and a probe according to the gene sequence of the NADH dehydrogenase subunit 3 (NADH dehydrogenase subunit 3) of the kusnezoff monkshood root:
an upstream primer: 5'-ATGTCAGAATTTGCACCTATTTGTAT-3' the flow of the air in the air conditioner,
a downstream primer: 5'-CCTATCCAGAAAAATTGTCGGC-3' the flow of the air in the air conditioner,
the fluorescent probe is as follows: 5'-TATTTAGTGATCAGTCCGCTA-3' are provided.
The fluorescent probe has FAM fluorescent label at the 5 'end and MGB fluorescent label at the 3' end.
2. The extraction and determination of the sample DNA are carried out according to the following steps:
(1) weighing 0.5g of crushed kusnezoff monkshood root, and putting the crushed material into a 50mL centrifuge tube;
(2) adding a preheated 10mLCTAB extraction lysis buffer (adding 20 muL protease K, 20 muL RNA and 200 muL beta-mercaptoethanol before use), carrying out warm bath at 65 ℃ for 1h after oscillating suspension, continuously shaking the solution, cooling the solution to room temperature, and centrifuging the solution for 10min at 5000 g;
(3) adding 950 mu L of supernatant into a 2mL centrifuge tube, adding isovolumetric chloroform isoamylol (24: 1), gently mixing uniformly, standing for 5min, and centrifuging at 12000rpm for 15 min;
(4) adding 750 mu L of supernatant into a 2mL centrifuge tube, adding isovolumetric chloroform isoamylol (24: 1), gently mixing uniformly, standing for 5min, and centrifuging at 12000rpm for 15 min;
(5) adding 500 muL of supernatant into a 1.5mL centrifuge tube, adding 300 muL of isopropanol and 50 muL of potassium acetate solution, slightly inverting and uniformly mixing, standing at room temperature for 30min, centrifuging at 12000rpm for 15min, and discarding the supernatant;
(6) adding 1.0mL of 70% ethanol solution, suspending and soaking the precipitate, inclining a centrifugal tube, slightly rotating for several circles, standing at room temperature for 2min, centrifuging at 12000rpm for 10min, and discarding the supernatant, wherein the step is repeated for more times if the precipitate exists;
(7) carefully discard the supernatant, dry the DNA pellet, add 50-100 μ L ddH2O, DNA was dissolved at 65 ℃.
(8) The extracted DNA solution was checked for concentration and purity using a micro-spectrophotometer, diluted to 10-50 ng/. mu.L, and then subjected to 10-fold serial gradient dilution.
3. Establishing a real-time fluorescent PCR amplification reaction system
Respectively diluting an upstream primer, a downstream primer and a probe for detecting the kusnezoff monkshood root to 10 mu mol/L, wherein a real-time fluorescence PCR reaction system is as follows:
2 Xreal-time PCR premixed solution 10 mu L
Upstream primer (10 mu mol/L) 1 mu L
Downstream primer (10 mu mol/L) 1 mu L
Fluorescent probe (10 mu mol/L) 1 mu L
Sample DNA (10-50 ng/muL) 2 muL
ddH2O 5µL
50ng of kusnezoff monkshood root DNA and 0.05ng of kusnezoff monkshood root DNA are respectively added into each set real-time fluorescent PCR reaction tube, the tube is tightly covered, and the instantaneous separation is carried out for 5 s.
And (3) placing the reaction tube added with the sample into a real-time fluorescence PCR detection system, and recording the placing sequence of the sample.
Setting circulation conditions: 10min at 95 ℃ for 1 cycle; and (3) carrying out 40 cycles at 95 ℃ for 15s and 56 ℃ for 30s, and judging the result according to the amplification curve and the Ct value after the detection is finished.
4. And (3) detection results:
the pore canal containing 50ng kusnezoff monkshood root DNA sample and the pore canal containing 0.05ng kusnezoff monkshood root DNA sample show positive amplification curves as shown in figure 1, and Ct values of the positive amplification curves are 25.34 and 35.93 respectively, which shows that the primer probe can well amplify kusnezoff monkshood root plant source components, has good sensitivity, and the detection limit can reach 0.05ng DNA.
Example 2
1. Primers and probes used:
designing and synthesizing a real-time fluorescent PCR specific amplification primer and a probe according to the gene sequence of the NADH dehydrogenase subunit 3 (NADH dehydrogenase subunit 3) of the kusnezoff monkshood root:
an upstream primer: 5'-ATGTCAGAATTTGCACCTATTTGTAT-3' the flow of the air in the air conditioner,
a downstream primer: 5'-CCTATCCAGAAAAATTGTCGGC-3' the flow of the air in the air conditioner,
the fluorescent probe is as follows: 5'-TATTTAGTGATCAGTCCGCTA-3' are provided.
The fluorescent probe has FAM fluorescent label at the 5 'end and MGB fluorescent label at the 3' end.
2. The extraction and determination of the sample DNA are carried out according to the following steps:
(1) respectively weighing 0.5g of aconitum kusnezoffii, aconitum sinense kusnezoffii, aconitum kusnezoffii, cucurbita pepo and other samples, crushing, and putting the crushed materials into different 50mL centrifuge tubes;
(2) adding a preheated 10mLCTAB extraction lysis buffer (adding 20 muL protease K, 20 muL RNA and 200 muL beta-mercaptoethanol before use), carrying out warm bath at 65 ℃ for 1h after oscillating suspension, continuously shaking the solution, cooling the solution to room temperature, and centrifuging the solution for 10min at 5000 g;
(3) adding 950 mu L of supernatant into a 2mL centrifuge tube, adding isovolumetric chloroform isoamylol (24: 1), gently mixing uniformly, standing for 5min, and centrifuging at 12000rpm for 15 min;
(4) adding 750 mu L of supernatant into a 2mL centrifuge tube, adding isovolumetric chloroform isoamylol (24: 1), gently mixing uniformly, standing for 5min, and centrifuging at 12000rpm for 15 min;
(5) adding 500 muL of supernatant into a 1.5mL centrifuge tube, adding 300 muL of isopropanol and 50 muL of potassium acetate solution, slightly inverting and uniformly mixing, standing at room temperature for 30min, centrifuging at 12000rpm for 15min, and discarding the supernatant;
(6) adding 1.0mL of 70% ethanol solution, suspending and soaking the precipitate, inclining a centrifugal tube, slightly rotating for several circles, standing at room temperature for 2min, centrifuging at 12000rpm for 10min, and discarding the supernatant, wherein the step is repeated for more times if the precipitate exists;
(7) carefully discard the supernatant, dry the DNA pellet, add 50-100 μ L ddH2O, DNA was dissolved at 65 ℃.
(8) The extracted DNA solution was checked for concentration and purity using a micro-spectrophotometer and diluted to 10-50 ng/. mu.L.
3. Establishing a real-time fluorescent PCR amplification reaction system
Respectively diluting an upstream primer, a downstream primer and a probe for detecting the kusnezoff monkshood root to 10 mu mol/L, wherein a real-time fluorescence PCR reaction system is as follows:
2 Xreal-time PCR premixed solution 10 mu L
Upstream primer (10 mu mol/L) 1 mu L
Downstream primer (10 mu mol/L) 1 mu L
Fluorescent probe (10 mu mol/L) 1 mu L
Sample DNA (10-50 ng/muL) 2 muL
ddH2O 5µL
The prepared template DNA solution is added into each set real-time fluorescent PCR reaction tube, the tube is tightly covered, and the instant separation is carried out for 5 s.
And (3) placing the reaction tube added with the sample into a real-time fluorescence PCR detection system, and recording the placing sequence of the sample.
Setting circulation conditions: 10min at 95 ℃ for 1 cycle; and (3) carrying out 40 cycles at 95 ℃ for 15s and 56 ℃ for 30s, and judging the result according to the amplification curve and the Ct value after the detection is finished.
4. And (3) detection results:
the Ct values of the amplified samples of aconitum vilmorinianum kusnezoffii, aconitum sinense and aconitum kusnezoffii of aconitum are all more than 40, and the Ct value of the samples of aconitum kusnezoffii is 25.09, which shows that the primers and the probes have good specificity.
The above examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention; it should be noted that various changes and modifications can be made by those skilled in the art without departing from the scope of the inventive concept, which falls within the scope of the invention; therefore, all equivalent changes and modifications within the scope of the claims of the present invention should be covered by the claims of the present invention.
Claims (4)
1. A method for detecting the botanical ingredients of radix aconiti kusnezoffii is characterized by comprising the following steps: the real-time fluorescence PCR method for detecting the botanical ingredients of the kusnezoff monkshood root comprises the following primers and a fluorescent probe respectively:
an upstream primer: 5’-ATGTCAGAATTTGCACCTATTTGTAT-3’，
A downstream primer: 5’-CCTATCCAGAAAAATTGTCGGC-3’，
The fluorescent probe is as follows: 5’-TATTTAGTGATCAGTCCGCTA-3’。
2. The method for detecting the botanical ingredient of radix aconiti kusnezoffii as claimed in claim 1, wherein: fluorescent probe 5’End is FAM fluorescent label, 3’The end is MGB fluorescent label.
3. The method for detecting the botanical ingredient of radix aconiti kusnezoffii as claimed in claim 1, wherein: respectively diluting an upstream primer, a downstream primer and a fluorescent probe for detecting the wild aconite root plant source components to 10 mu mol/L, wherein the real-time fluorescent PCR reaction system is as follows:
2 Xreal-time PCR premixed solution 10 mu L
Upstream primer (10 mu mol/L) 1 mu L
Downstream primer (10 mu mol/L) 1 mu L
Fluorescent probe (10 mu mol/L) 1 mu L
Sample DNA (10-50 ng/muL) 2 muL
ddH2O 5µL。
4. The method for detecting the botanical ingredient of radix aconiti kusnezoffii as claimed in claim 1, wherein: the real-time fluorescent PCR reaction condition is 95 ℃ for 10min, 1 cycle; 95 ℃ for 15s, 56 ℃ for 30s, 40 cycles.
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| CN110452959A (en) * | 2019-07-01 | 2019-11-15 | 昆明理工大学 | A kind of screening technique of yellow grass crow real-time quantitative PCR reference gene |
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| CN110452959A (en) * | 2019-07-01 | 2019-11-15 | 昆明理工大学 | A kind of screening technique of yellow grass crow real-time quantitative PCR reference gene |
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| 朱高倩等: "乌头属5种植物ITS2和psbA-trnH鉴别适用性研究", 《广州中医药大学学报》 * |
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Application publication date: 20210723 |