CN116287148A - Method for identifying Panax schinseng, primer, probe and application thereof - Google Patents
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Abstract
The invention discloses a method for identifying a ginseng, a primer, a probe and application thereof, belonging to the technical field of identification of kindred species. The method comprises the following steps: taking genomic DNA of a sample to be identified as a template, carrying out real-time fluorescent quantitative PCR amplification reaction by adopting primers TZF2 and TZR2 and a probe-2, and identifying the sample as a ginseng bead if the CT value is less than 40; the sequence of the primer TZF2 is shown as SEQ ID No.1, the sequence of the primer TZR2 is shown as SEQ ID No.2, and the sequence of the probe-2 is shown as SEQ ID No. 3. The invention has excellent specificity and sensitivity, and can rapidly and reliably identify the ginseng.
Description
Technical Field
The invention relates to a method for identifying a ginseng, a primer, a probe and application thereof, belonging to the technical field of identification of kindred species.
Background
Radix seu herba PhysochlainaePanax major) Is dry rhizome of Panax ginseng or Panax pseudoginseng C.A. Meyer of Panax of Araliaceae, mainly produced in Yunnan, and is a traditional medicine for minority nationality such as white, naxi, tibetan, and Yi nationality, with bitter taste, sweet taste, and slightly sexual propertyCold, has effects of nourishing liver, lung and stomach channel, nourishing lung yin, removing blood stasis, relieving pain, stopping bleeding, and can be used for treating symptoms such as deficiency of both qi and yin, cough due to asthenia, traumatic injury, traumatic hemorrhage, gastralgia, and laryngopharynx swelling and pain. The rhizoma panacis majoris contains various saponins: oleanolic acid-28-O-beta-D-glucopyranoside, ginsenoside IVa, 20 (S) -protopanaxadiol type saponin, 20 (S) -protopanaxatriol type saponin, etc., are similar to other plants of the genus Panax in morphological characteristics and chemical components are partially the same. However, in the pharmaceutical use, the differences in the specific types and contents of the active chemical components such as saponins can cause significant differences in the efficacy, and if the plant rootstock is confused with the rootstock of other related plants, the efficacy of the formulation can be affected, thus delaying the treatment of patients. Therefore, in the growing area of the ginseng plant, especially in the area with abundant wild resources of the ginseng plant, the reliable identification method of the medicinal materials from the related species is particularly important.
At present, the traditional identification method mainly comprises basic source identification, character identification, microscopic identification and physicochemical identification, but the theoretical basis of the traditional identification method is established on character characteristic analysis of a taxonomic group, wherein the characters are phenotypes closely related to the environment, are influenced by the growth age, physiological state and growth environment of plants, and are difficult to ensure the reliability of identification results. In recent years, with the development of DNA molecular technology, molecular biology identification methods have been developed, and the DNA molecular marker technology is applied to the identification of plants, and the genomic sequence of the plants is directly used as the identification basis, so that the reliability of the identification result is greatly improved. However, the technology applied to the identification of the ginseng medicinal materials, such as RAPD, SSR, RFLP, has the defects of complex operation steps, large workload, poor repeatability of experimental results and the like. When the real-time fluorescent quantitative PCR technology is applied to species identification, the identification purpose is achieved by detecting the change of fluorescence intensity by using one or more groups of specific primer probes, and the method has the advantages of strong specificity, high sensitivity, simplicity in operation and the like, is high in detection efficiency, and has good market application prospect and popularization and application value.
Disclosure of Invention
The invention aims to overcome the defects of poor reliability, complex operation, large workload, poor repeatability of experimental results and the like of an identification method in the prior art, and provides a method for identifying a ginseng bead, a primer, a probe and application thereof.
A first aspect of the present invention provides a method of identifying a ginseng bead comprising the steps of: taking genomic DNA of a sample to be identified as a template, carrying out real-time fluorescent quantitative PCR amplification reaction by adopting primers TZF2 and TZR2 and a probe-2, and identifying the sample as a ginseng bead if the CT value is less than 40; the sequence of the primer TZF2 is shown as SEQ ID No.1, the sequence of the primer TZR2 is shown as SEQ ID No.2, and the sequence of the probe-2 is shown as SEQ ID No. 3.
Preferably, FAM is modified at the 5 'end of the probe-2, and MGB is modified at the 3' end of the probe-2.
Preferably, the lowest detection limit of the template is 0.001 ng/. Mu.L.
Preferably, the concentrations of the primers TZF2 and TZR2 are each 0.45 to 0.55. Mu. Mol/. Mu.L.
Preferably, the concentration of the probe-2 is 0.45 to 0.55. Mu. Mol/. Mu.L.
In a second aspect, the invention provides a primer pair for identifying a bead reference, wherein the primer pair consists of primers TZF2 and TZR2, the sequence of the primer TZF2 is shown as SEQ ID No.1, and the sequence of the primer TZR2 is shown as SEQ ID No. 2.
In a third aspect of the present invention there is provided a probe for identifying Panax ginseng, the probe-2 having the sequence shown in SEQ ID No. 3.
In a fourth aspect, the present invention provides a reagent for identifying Panax ginseng, comprising the above primer pair and the above probe-2.
In a fifth aspect the invention provides the use of a primer pair as defined above, a probe as defined above or a reagent as defined above for identifying a ginseng bead.
The beneficial effects of the invention are as follows:
the identification method, the primer pair, the probe and the reagent have excellent specificity and sensitivity, can accurately identify or detect the species of the Panax ginseng, and realize the rapid identification of the Panax ginseng.
Drawings
FIG. 1 is a standard curve of a second embodiment of the present invention.
FIG. 2 is an amplification curve of a second embodiment of the present invention, wherein the detection of this embodiment uses 96-well plates, column: 1-12, row: A-H, the curve of A-H in the figure shows the detection result of each row of holes.
FIG. 3 is an amplification curve of a third embodiment of the present invention, wherein the detection of this embodiment uses 96-well plates, column: 1-12, row: A-H, the curve of A-H in the figure shows the detection result of each row of holes.
FIG. 4 shows an amplification curve of a fourth embodiment of the present invention, wherein the detection of this embodiment uses 96-well plates, column: 1-12, row: A-H, the curve of A-H in the figure shows the detection result of each row of holes.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by a person skilled in the art without any inventive effort, are intended to be within the scope of the present invention, based on the embodiments of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example one PCR amplification and identification of target plant samples
The extracted genome DNA of the rhizoma panacis majoris is used as a template, primers TZF2 and TZR2 are used for carrying out PCR amplification, a probe-2 is used for carrying out real-time fluorescence quantitative PCR detection, the sequence of the primer TZF2 is shown as SEQ ID No.1, the sequence of the primer TZR is shown as SEQ ID No.2, the sequence of the probe-2 is shown as SEQ ID No.3, FAM is modified at the 5 'end of the probe-2, and MGB is modified at the 3' end of the probe-2. The reaction system of the amplification is shown in table 1, the amplification procedure is shown in table 2, and the identification result and the PCR product are obtained after the amplification.
TABLE 1 reaction system
TABLE 2 amplification procedure
Example preparation of a two-bead reference Standard plasmid and creation of a Probe Standard Curve
Positive clone samples were prepared according to the PCR product obtained in example one, then sequenced, positive clone samples with the complete correct sequence in the sequencing result were selected, and plasmid extraction was completed using a plasmid extraction kit.
Plasmid concentration copy number was calculated as follows:
C=A·B -1 ×6.02×10 14 ，
wherein A is plasmid concentration (ng/. Mu.L), B is plasmid DNA molecular weight, and C is copies/. Mu.L -1 . Calculating to obtain the concentration of the mother solution: standard plasmid of the bead (3.23 x 10) 10 copies·μL -1 ) The plasmid was used as a standard for experimental plasmids.
The plasmid standard of the plasmid of the bead is diluted to 3.23 x 10 using a solvent 10 copies·μL -1 Diluting to 3.23 x 10 8 copies·μL -1 Then diluting the plasmid into 5 gradients according to 10 times concentration, then respectively taking diluted plasmid standards of each gradient as templates, carrying out real-time fluorescence quantitative PCR detection under the reaction condition of the first embodiment, repeating each group of experiments for 3 times, and making a standard curve. The results of the detection of the plasmid reference beads are shown in Table 3, the standard curve is shown in FIG. 1, and the relevant amplification curve is shown in FIG. 2.
Standard curve equation for bead parameters: y= -3.8259 x+50.819, r 2 =1，R 2 Reaching more than 0.99 and meeting the standard requirements of real-time fluorescence quantitative PCR.
TABLE 3 detection results of plasmid Standard of Panax ginseng
EXAMPLE three identification of specificity verification of Panax schinseng
The embodiment provides a method for identifying a ginseng bead, which comprises the following steps: genomic DNA of seven kindred species of Panax ginseng, panax quinquefolium, panax ginger-like Notoginseng, panax angustifolium, panax notoginseng and Panax schinseng off-line were used as templates, respectively, and a negative control group (ddH) was established 2 O) the template was amplified using the primer set, probe-2, reaction system and amplification procedure of example I, each sample was repeated 3 times, the detection results are shown in Table 4, and the amplification curves are shown in FIG. 3.
TABLE 4 specificity test results
As can be seen from Table 4, among the seven closely related species of Panax, the primer set and probe-2 have amplification reaction and fluorescence phenomenon only with the Panax ginseng, and have good specificity, similar CT values of 3 repetitions, and good repeatability.
Example sensitivity verification of four primer probes
The DNA concentrations of the bead samples used in example three were set to six concentration gradients of 1 ng/. Mu.L, 0.1 ng/. Mu.L, 0.01 ng/. Mu.L, 0.001 ng/. Mu.L, 0.0001 ng/. Mu.L, and 0.00001 ng/. Mu.L, respectively, and the samples were amplified using the primer set, probe-2, reaction system, and amplification program of example one to test the sensitivity of the primer set and probe-2, and each sample was repeated three times and assayed using a real-time fluorescent quantitative PCR apparatus, and the detection results are shown in Table 5 and FIG. 4.
TABLE 5 detection results of sensitivity of bead reference probes
As can be seen from Table 5, the sensitivity of the primer set and the probe-2 of the present invention to the ginseng bead was high, the lowest detection limit was 0.001 ng/. Mu.L, and the average CT value was 34.452.
Example five repeatability verification
1. Repeated verification of enzymes in different batches by the same experimenter and the same real-time fluorescence quantitative PCR instrument;
2. and (5) repeatedly verifying the operation of different personnel of the same batch enzyme and the real-time fluorescence quantitative PCR instrument.
Samples were obtained in the field and morphologically and chloroplast genome comparison were used to confirm two known samples of the Panax schinseng beads, each sample was repeated 3 times using the primer pair of example one, probe-2, reaction system and amplification procedure, and the repeatability evaluation was performed by calculating the coefficient of variation (CV%) value. The coefficient of variation value is calculated as:
cv=sd (standard deviation)/Mean (Mean) ×100%.
The results of the verification of the enzymes of different batches from the same experimenter and the QPCR instrument are shown in tables 6.1 and 6.2, and the results of the verification of the operations of the enzymes of the same batch and the different personnel of the QPCR instrument are shown in tables 7.1 and 7.2.
TABLE 6.1 verification results of the same laboratory technician and QPCR apparatus for the first batch of enzyme
TABLE 6.2 verification results of the same experimenter as the QPCR apparatus for the second batch of enzyme
TABLE 7.1 results of verification of same batch enzyme and QPCR instrument laboratory technician A operation
TABLE 7.2 results of verification of same batch enzyme and QPCR instrument laboratory technician B operation
As can be seen from tables 6.1, 6.2, 7.1 and 7.2, the repeatability verification result variation coefficient value is less than 3% no matter between different batches of enzymes of the same experimenter and the same QPCR instrument or between the same batches of enzymes and different personnel of the same QPCR instrument, the experimental repeatability is good, the stability of the polymerase in different batches is excellent, and the reaction system is stable.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (9)
1. A method of identifying a ginseng bead comprising the steps of: taking genomic DNA of a sample to be identified as a template, carrying out real-time fluorescent quantitative PCR amplification reaction by adopting primers TZF2 and TZR2 and a probe-2, and identifying the sample as a ginseng bead if the CT value is less than 40; the sequence of the primer TZF2 is shown as SEQ ID No.1, the sequence of the primer TZR2 is shown as SEQ ID No.2, and the sequence of the probe-2 is shown as SEQ ID No. 3.
2. The method of identifying a ginseng bead according to claim 1, wherein: FAM is modified at the 5 'end of the probe-2, and MGB is modified at the 3' end of the probe-2.
3. The method of identifying a ginseng bead according to claim 1, wherein: the lowest detection limit of the template is 0.001 ng/. Mu.L.
4. The method of identifying a ginseng bead according to claim 1, wherein: the concentrations of the primers TZF2 and TZR2 are 0.45-0.55 mu mol/mu L.
5. The method of identifying a ginseng bead according to claim 1, wherein: the concentration of the probe-2 is 0.45-0.55 mu mol/mu L.
6. A primer pair for identifying a bead reference, characterized by: the primer pair consists of primers TZF2 and TZR2, the sequence of the primer TZF2 is shown as SEQ ID No.1, and the sequence of the primer TZR2 is shown as SEQ ID No. 2.
7. A probe for identifying a ginseng bead, characterized in that: the sequence of the probe-2 is shown as SEQ ID No. 3.
8. A reagent for identifying a ginseng bead, characterized in that: comprising the primer set according to claim 6 and the probe-2 according to claim 7.
9. Use of a primer pair according to claim 6, a probe according to claim 7 or a reagent according to claim 8 for identifying a ginseng bead.
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| CN116287412A (en) * | 2023-03-29 | 2023-06-23 | 中国科学院昆明植物研究所 | Primer, probe and detection method for pseudo-ginseng medicinal material authenticity detection and identification based on second-generation sequencing development |
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