CN116814797A - Primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles, application thereof and identification method - Google Patents
Primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles, application thereof and identification method Download PDFInfo
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Abstract
The invention discloses a primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles, which comprises a primer I and a primer II, wherein the sequence of the primer I is shown as SEQ ID NO:6, the sequence of the primer II is shown as SEQ ID NO: shown at 7. The invention also discloses application of the primer combination and an identification method based on the primer combination. By implementing the invention, ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles can be effectively identified.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicine identification, in particular to a primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles, application thereof and an identification method.
Background
The Chinese pharmacopoeia 2020 edition specifies that the Eupolyphaga Seu Steleophaga source is dried female Eupolyphaga of Eupolyphaga Eupolyphaga sinensis Walker or Eupolyphaga Steleophaga plancyi (Boleny) of Eupolyphaga Seu Steleophaga. Eupolyphaga Seu Steleophaga was originally recorded in Shen nong Ben Cao Jing (Shen nong's herbal), which is listed as a middle-grade product, and all the generation of herbal is recorded. The female worms have the effects of dissipating blood stasis, eliminating stagnation, resolving coagulation, activating blood, promoting reunion of fractured bones, detumescence, relieving pain, promoting lactation and dredging channels and the like, and are used for traumatic injury, muscle injury and fracture, blood stasis amenorrhea, postpartum abdominal pain due to stasis, mass and trace lump and the like. Recent pharmacological studies show that ground beetle has the activities of anticoagulation, anti-tumor, blood lipid regulation, pain relief, anti-inflammatory and sterilization, etc.
The ground beetles are used as common traditional Chinese medicines, mixed and fake ground beetles are frequently appeared in the market, the common mixed and fake ground beetles comprise golden-edged ground beetles (Opisthoplatia orientalis (B.)), golden-edged cockroaches (Hydrophilus acuminatus (M.)), american cockroaches (Periplaneta americana (L.)) and the like, the characteristics of the common mixed and fake ground beetles are close to each other, the common mixed and fake ground beetles are similar in shape, the common mixed and fake ground beetles are difficult to identify by means of the traditional identification method, the accuracy is low, and particularly after the common mixed and fake ground beetles are prepared into standard decoction or other dosage forms through water extraction, the effective identification is more difficult to perform, and particularly the doping detection of the similar shape is less in the prior related reports. In order to ensure the quality and curative effect of medicinal materials, decoction pieces, water extracts, formula particles and the like of ground beetles (ground beetles), a method for accurately and rapidly identifying the qualified products and the counterfeit products of the ground beetles is urgently needed.
Disclosure of Invention
The invention aims to solve the technical problem of providing a primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles, which has good specificity and high sensitivity.
The invention also solves the technical problem of providing application of the primer combination.
The invention also solves the technical problem of providing a primer combination identification method for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles.
In order to solve the technical problems, the invention provides a primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles, which comprises a primer I and a primer II, wherein the sequence of the primer I is shown as SEQ ID NO:6 is shown in the figure; the sequence of the primer II is shown as SEQ ID NO: shown at 7.
Correspondingly, the invention also discloses application of the primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles in any one of (1) to (4):
(1) Identifying ground beetle medicinal materials, ground beetle standard decoction and/or ground beetle traditional Chinese medicine formula granules;
(2) Kit for identifying Eupolyphaga Seu Steleophaga medicinal material, eupolyphaga Seu Steleophaga standard decoction, and/or Eupolyphaga Seu Steleophaga Chinese medicinal granule;
(3) Identifying whether the sample to be detected contains ground beetle medicinal materials, ground beetle standard decoction and/or ground beetle traditional Chinese medicine formula particles;
(4) And (3) identifying whether the sample to be detected contains ground beetle medicinal materials, ground beetle standard decoction and/or ground beetle traditional Chinese medicine formula particles.
Correspondingly, the invention also discloses a kit comprising the primer combination.
Correspondingly, the invention also discloses a method for identifying the primer combination based on the PCR identification of the ground beetle medicinal material, the standard decoction and the traditional Chinese medicine formula particles, which comprises the following steps:
extracting genome DNA of a sample to be detected;
the genome DNA is used as a template, the primer combination for PCR identification of the ground beetle medicinal material, the standard decoction and the traditional Chinese medicine formula particles is adopted for PCR amplification, and if the amplification product contains 237bp DNA fragments, the sample to be detected is the ground beetle medicinal material, the ground beetle standard decoction and/or the ground beetle traditional Chinese medicine formula particles.
As an improvement of the technical scheme, when the sample to be detected is a medicinal material, the method for extracting the genomic DNA of the sample to be detected is as follows:
adding SDS lysate and proteinase K to precipitate and extract a sample to be detected, then adding chloroform-isoamyl alcohol to extract for 2-3 times, taking supernatant after extraction, adding isopropanol or isopropanol-sodium acetate to precipitate and extract, washing and incubating the precipitate, and then dissolving the precipitate with water to obtain genome DNA of the sample to be detected;
when the sample to be detected is a standard decoction or traditional Chinese medicine formula particle, the method for extracting the genome DNA of the sample to be detected comprises the following steps:
taking a sample to be detected, and adding CTAB precipitation liquid to precipitate for 1-2 times; adding CTAB extract, proteinase K and beta-mercaptoethanol into the precipitate for extraction, and then adding chloroform-isoamyl alcohol for extraction for 2-5 times; and taking supernatant after extraction, adding isopropanol or isopropanol-sodium acetate for precipitation, washing and incubating the precipitate, and dissolving with water to obtain the genomic DNA of the sample to be detected.
As an improvement of the technical scheme, a template, a primer combination, a PCR buffer solution, dNTP, speedSTAR and water are uniformly mixed to obtain a PCR amplification system;
amplifying the PCR amplification system according to a preset amplification program to obtain an amplification product;
and (3) carrying out electrophoresis analysis on the amplified product, recording an electrophoresis pattern of the amplified product, and if the pattern contains 237bp DNA fragments, taking the sample to be detected as ground beetle medicinal materials, ground beetle standard decoction and/or ground beetle traditional Chinese medicine formula particles.
As an improvement of the above technical scheme, the PCR amplification system consists of:
10×Fast BufferⅠ(Mg 2+ plus) 2. Mu.L, dNTP mix 0.6. Mu.L, primer one 0.2. Mu.L, primer two 0.2. Mu.L, speedSTAR 0.2. Mu.L, DNA template 1. Mu.L, deionized water 15.8. Mu.L.
As an improvement of the above technical scheme, the PCR amplification procedure is:
pre-denaturing the amplification system at 90-95 ℃ for 2-5 min, circulating 39-41 times under a preset program, and extending at 70-75 ℃ for 3-7 min;
wherein, the preset program is as follows: the amplification system is denatured at 90-95 ℃ for 15-25 s, then annealed at 55-61 ℃ for 15-25 s, and then extended at 70-75 ℃ for 25-35 s.
As an improvement of the above technical scheme, the PCR amplification procedure is:
pre-denaturing the amplification system at 95 ℃ for 3min, then cycling 39 times under a preset program, and finally extending at 72 ℃ for 5min;
wherein, the preset program is as follows: the amplification system was denatured at 95℃for 20s, then annealed at 55-59℃for 20s, and then extended at 72℃for 30s.
As an improvement of the above technical solution, the electrophoresis analysis method is as follows: and diluting the amplified product, spotting on 1.5% agarose gel, carrying out electrophoresis for 15-30 min under the condition of 170-180V voltage, and carrying out Gelred color development to obtain an electrophoresis pattern.
The implementation of the invention has the following beneficial effects:
the invention designs the primer combination aiming at the ground beetle medicinal materials, the standard decoction and the traditional Chinese medicine formula particles, adopts a specific PCR amplification method, realizes the rapid identification of the ground beetle medicinal materials, the standard decoction and the traditional Chinese medicine formula particles, has strong specificity and high sensitivity, and can effectively solve the difficult problem that the standard decoction and the traditional Chinese medicine formula particles are difficult to identify after losing form.
Drawings
FIG. 1 is a diagram showing the result of electrophoresis detection after PCR amplification of the primer set DB-01F/R in example 1; wherein M: DNA Maker DL1000,1-2 ground beetle medicinal materials and decoction pieces, 3-4 ground beetle standard decoction, 5-6 ground beetle traditional Chinese medicine formula granules, 7-8 American cockroach medicinal materials, 9-10 Jinbian tara medicinal materials, 11-12 Jinbian ground beetle medicinal materials, and N blank (ddH) 2 O)；
FIG. 2 is a diagram showing the result of electrophoresis detection after PCR amplification of the primer set DB-02F/R in example 1; wherein M: DNA Maker DL1000,1-2 ground beetle medicinal materials and decoction pieces, 3-4 ground beetle standard decoction, 5-6 ground beetle traditional Chinese medicine formula particles, 7-8 is periplaneta americana medicinal material, 9-10 is golden edge dragon lice medicinal material, 11-12 is golden edge ground beetle medicinal material, and N is blank (ddH 2 O)；
FIG. 3 is a diagram showing the result of electrophoresis detection after PCR amplification of the primer set DB-03F/R in example 1; wherein M is DNA Maker DL1000,1-2 is Eupolyphaga Seu Steleophaga medicinal material and decoction pieces, 3-4 is Eupolyphaga Seu Steleophaga standard decoction, 5-6 is Eupolyphaga Seu Steleophaga Chinese medicinal granule, 7-8 is Periplaneta americana medicinal material, 9-10 is Larimonia aurea medicinal material, 11-12 is Eupolyphaga Seu Steleophaga medicinal material, and N is blank (ddH) 2 O)；
FIG. 4 is a diagram showing the result of electrophoresis detection after PCR amplification of the primer set DB-04F/R in example 1; wherein M: DNA Maker DL1000,1-2 ground beetle medicinal materials and decoction pieces, 3-4 ground beetle standard decoction, 5-6 ground beetle traditional Chinese medicine formula granules, 7-8 American cockroach medicinal materials, 9-10 Jinbian tara medicinal materials, 11-12 Jinbian ground beetle medicinal materials, and N blank (ddH) 2 O)；
FIG. 5 is a graph showing the result of electrophoresis after PCR amplification of each sample in example 3; wherein, in the ground beetle formula granule row: m is DNA maker DL1000,1 is Eupolyphaga Seu Steleophaga (Eupolyphaga Seu Steleophaga), 2-20 is Eupolyphaga Seu Steleophaga (Eupolyphaga Seu Steleophaga) Chinese medicinal granule, and N is blank (ddH) 2 O); in Eupolyphaga Seu Steleophaga decoction piece row, M is DNA maker DL1000,1 is Eupolyphaga Seu Steleophaga (Eupolyphaga Seu Steleophaga) medicinal material, 2-22 is Eupolyphaga Seu Steleophaga (Eupolyphaga Seu Steleophaga) Chinese medicinal granule, and N is blank (ddH) 2 O)；
FIG. 6 is a graph showing the result of electrophoresis after PCR amplification of each sample in example 3; wherein, in the ground beetle standard decoction row: m is DNA maker DL1000,1 is Eupolyphaga Seu Steleophaga (Eupolyphaga Seu Steleophaga), 2-22 is Eupolyphaga Seu Steleophaga (Eupolyphaga Seu Steleophaga) standard decoction, and N is blank (ddH) 2 O); in the pseudo-line, M is DNA maker DL1000,1 is ground beetle (ground beetle) medicinal material, 2-4 is American cockroach medicinal material, 5-6 is Jinbian dragon lice medicinal material, 7-8 is Jinbian ground beetle medicinal material, 9-11 is American cockroach standard decoction, 12-13 is Jinbian dragon lice standard decoction, 14-15 is Jinbian ground beetle standard decoction, and N is blank (ddH 2 O)；
FIG. 7 is a graph showing the results of electrophoresis detection after PCR amplification of each Eupolyphaga Seu Steleophaga formulation in example 4; wherein M is DNA maker DL1000, 1: DNA amount 15ng,2: DNA amount 1.5ng,3: DNA amount 0.15ng,4: DNA (deoxyribonucleic acid)Amounts 0.015ng,5: DNA amount 0.0015ng,6: DNA amount 0.00015ng, N: blank (ddH) 2 O)；
FIG. 8 is a graph showing the result of electrophoresis detection after PCR amplification of each Eupolyphaga Seu Steleophaga in example 4; wherein M is DNA maker DL1000, 1: DNA amount 90ng,2: DNA amount 9ng,3: the amount of DNA was 0.9ng,
4: DNA amount 0.09ng,5: DNA amount 0.009ng,6: DNA amount 0.0009ng, N: blank (ddH) 2 O)；
FIG. 9 is a graph showing the results of detection in example 5 using a2 XM 5 enzyme; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 10 is a graph showing the results of detection using rTaq enzyme in example 5; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 11 is a graph showing the results of detection using Ex Taq enzyme in example 5; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 12 is a graph showing the detection results when the Mighty AMP enzyme is used in example 5; wherein M is DNA maker DL1000,1-2 is Eupolyphaga Seu Steleophaga (Eupolyphaga Seu Steleophaga) granule, 3-4 is Eupolyphaga Seu Steleophaga (Eupolyphaga Seu Steleophaga) standard decoction, 5 is Eupolyphaga Seu Steleophaga (Eupolyphaga Seu Steleophaga) pairAccording to the medicinal materials, 6 is ground beetle (ground beetle) medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, and N is blank (ddH 2 O)；
FIG. 13 is a graph showing the results of the detection in example 5 using the speedstar enzyme; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 14 is a graph showing the results of the measurement in example 6 using an annealing temperature of 53 ℃; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 15 is a graph showing the results of the annealing at 55℃in example 6; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 16 is a graph showing the results of the test in example 6 using an annealing temperature of 57 ℃; wherein M is DNA maker DL1000,1-2 is Eupolyphaga (Eupolyphaga sinensis) formulation granule, 3-4 is Eupolyphaga (Eupolyphaga sinensis) standard decoction, 5 is Eupolyphaga (Eupolyphaga sinensis) reference medicinal material, 6 is Eupolyphaga (Eupolyphaga sinensis) medicinal material, 7 is Periplaneta americana medicinal material, 8 is Periplaneta americana standard decoction, 9 is Auricularia auricula medicinal material, 10 is Auricularia auriculaThe psyllid standard decoction, 11 is a golden-edge ground beetle medicinal material, 12 is a golden-edge ground beetle standard decoction, and N is blank (ddH 2 O)；
FIG. 17 is a graph showing the results of the measurement in example 6 using an annealing temperature of 59 ℃; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 18 is a graph showing the results of the annealing at 61℃in example 6; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 19 is a graph showing the results of the test in example 7 with a cycle count of 33; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 20 is a graph showing the results of the test in example 7 with 35 cycles; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 21 is a graph showing the results of the test in example 7 with the number of cycles being 37; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 22 is a graph showing the results of the test in example 7 with 39 cycles; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
FIG. 23 is a graph showing the results of the test in example 7 with the number of cycles being 41; wherein M is DNA maker DL1000,1-2 is ground beetle formula granule, 3-4 is ground beetle standard decoction, 5 is ground beetle control medicinal material, 6 is ground beetle medicinal material, 7 is American cockroach medicinal material, 8 is American cockroach standard decoction, 9 is Jinbian taro medicinal material, 10 is Jinbian taro standard decoction, 11 is Jinbian ground beetle medicinal material, 12 is Jinbian ground beetle standard decoction, N is blank (ddH 2 O)；
Detailed Description
The present invention will be described in further detail with reference to the drawings and the detailed description, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.
The invention provides a primer combination for detecting ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles, which specifically comprises the following steps: primer one (forward primer) and primer two (reverse primer), wherein primer one is a nucleotide sequence as set forth in SEQ ID NO: 6; the primer II is a nucleotide sequence shown as SEQ ID NO: 7.
Determining SNP sites of ground beetles by homologous comparison and analysis of Cytochrome Oxidase I sequences of ground beetles (ground beetles), american cockroaches, jinbian dragon lice, golden bian ground beetles and the like, designing primers according to base sequences containing the SNP sites, and comprehensively considering information such as damage effect of high-temperature extraction on DNA base sequences, product strips, primer scores and the like to obtain the primer combination. According to the primer combination, PCR amplification is carried out on ground beetle medicinal materials, ground beetle standard decoction and ground beetle traditional Chinese medicine formula particles, and a specific fragment with the size of 237bp can be obtained.
Based on the above characteristics, the primer of the present invention can be used in the following applications:
(1) Identifying whether the sample to be detected is medicinal materials of ground beetles, standard decoction and/or traditional Chinese medicine formula particles;
(2) Identifying whether the sample to be detected contains ground beetle medicinal materials, standard decoction and/or compound traditional Chinese medicines of traditional Chinese medicine formula granules.
(3) Preparing medicinal materials, standard decoction and/or traditional Chinese medicine formula particles for identifying whether a sample to be detected is ground beetle; is provided.
(4) Preparing a kit for identifying whether a sample to be detected contains a ground beetle medicinal material, a standard decoction and/or a compound traditional Chinese medicine of traditional Chinese medicine formula granules.
Accordingly, the invention discloses a kit comprising the primer combination, and further comprising PCR buffer, dNTP, speedSTAR and water, but not limited thereto.
The invention also discloses an identification method based on the primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles, which comprises the following steps:
(1) Extracting genome DNA of a sample to be detected;
wherein, the sample to be detected is provided in a solid state, and the standard decoction is provided in a freeze-dried powder form (namely, the freeze-dried powder is obtained after the standard decoction is freeze-dried).
Specifically, when the sample is a medicinal material, the extraction method is as follows:
a1: taking a sample to be detected, adding SDS lysate and proteinase K to precipitate and extract, then adding chloroform-isoamyl alcohol to extract for 2-3 times, and taking supernatant after extraction;
specifically, 0.01-0.1 g of sample to be detected is taken, ground into powder, placed into a centrifuge tube, added with 1-1.5 mL of SDS lysate and 10 mu L of proteinase K, mixed uniformly and incubated for 4-6h at 50-60 ℃; cooling to room temperature, adding equal volume of chloroform-isoamyl alcohol mixed solution, fully mixing, centrifuging, absorbing 800-1000 mu L of supernatant after centrifuging, adding equal volume of chloroform-isoamyl alcohol mixed solution, fully mixing, centrifuging, absorbing 600-800 mu L of supernatant after centrifuging, adding new centrifuge tube, adding equal volume of chloroform-isoamyl alcohol mixed solution, fully mixing, centrifuging, absorbing 400-600 mu L of supernatant after centrifuging, and adding the new centrifuge tube.
A2: adding isopropanol or isopropanol-sodium acetate into the supernatant after extraction for precipitation extraction, washing and incubating the precipitate, and dissolving the precipitate with water to obtain the genomic DNA of the sample to be detected.
Specifically, adding an equal volume of chloroform-isoamyl alcohol mixed solution into the supernatant after extraction, fully and uniformly mixing, centrifuging, absorbing 800-1000 mu L of the supernatant after centrifugation, adding a new centrifuge tube, fully and uniformly mixing, centrifuging, absorbing 600-800 mu L of the supernatant after centrifugation, adding a new centrifuge tube, adding an equal volume of chloroform-isoamyl alcohol mixed solution, fully and uniformly mixing, centrifuging, absorbing 400-600 mu L of the supernatant after centrifugation, adding a new centrifuge tube, adding an equal volume of isopropanol or isopropanol-sodium acetate mixed solution, and standing at-25 to-15 ℃ for 60-90 min; standing and centrifuging, washing the obtained precipitate with 75% ethanol for 2-4 times, washing with absolute ethanol for 1 time, incubating for 20-40 min at 30-40 ℃, adding 30-50 mu L of sterilized water for dissolving after the ethanol volatilizes, and obtaining the genome DNA of the sample to be detected.
Specifically, when the sample is a standard decoction or a traditional Chinese medicine formula granule, the extraction method is as follows:
b1: taking a sample to be detected, adding CTAB precipitation liquid to precipitate for 1-2 times to obtain a precipitate;
taking 0.01-0.1 g of sample to be detected, grinding into powder, placing into a centrifuge tube, adding 1-2 mL of CTAB precipitate, uniformly mixing, incubating for 60-80 min at 60-70 ℃, centrifuging and discarding supernatant; adding 1-1.5 mL CTAB precipitation liquid into the precipitate obtained by centrifugation, uniformly mixing, incubating for 20-40 min at 60-70 ℃, centrifuging and discarding supernatant; obtaining the product;
wherein, the composition of CTAB precipitation liquid is: 1-3% (w/v) cetyl trimethylammonium bromide (CTAB), 80-120 mmol/L Tris-HCl (Tris-HCl, pH 8.0), 10-30 mmol/L disodium ethylenediamine tetraacetate (EDTA, pH=8.0).
B2: adding CTAB extract, proteinase K and beta-mercaptoethanol into the precipitate for extraction, and then adding chloroform-isoamyl alcohol for extraction for 2-5 times;
specifically, adding 1-1.5 mL of CTAB precipitation liquid into the precipitate obtained by centrifugation, uniformly mixing, incubating for 20-40 min at 60-70 ℃, centrifuging and discarding supernatant; adding 800-1000 mu L of CTAB extract, 5-15 mu L of proteinase K and 5-15 mu L of beta-mercaptoethanol into the precipitate obtained by centrifugation, uniformly mixing, incubating at 60-70 ℃ for 1-2.5 h, cooling to room temperature, adding an equal volume of chloroform-isoamyl alcohol (24:1) mixed solution, vibrating and uniformly mixing, centrifuging, taking 700-800 mu L of supernatant, adding an equal volume of chloroform-isoamyl alcohol mixed solution, vibrating and uniformly mixing, centrifuging, and repeating for 2-4 times; taking 400-600 mu L of supernatant fluid.
Wherein, the composition of CTAB extract is: 1 to 3% (w/v) CTAB,80 to 120mmol/L Tris-HCl (pH=8.0), 10 to 30mmol/L EDTA (pH=8.0), 1 to 3mol/LNaCl,1 to 3% (w/v) PVP40.
B3: and adding isopropanol or isopropanol-sodium acetate into the extracting solution to precipitate, washing and incubating the precipitate, and dissolving the precipitate with water to obtain the genomic DNA of the sample to be detected.
Specifically, taking 400-600 mu L of supernatant, adding isopropanol or isopropanol-3 mol/L sodium acetate mixed solution with equal volume, standing for 60-90 min at-30 to-20 ℃, centrifuging, removing the supernatant, washing the precipitate with 75% ethanol for 2-4 times, washing with absolute ethanol for 1 time, removing the supernatant, incubating the precipitate at 35-38 ℃ for 20-40 min, adding 30-50 mu L of sterilized water for dissolution after the ethanol volatilizes, and obtaining the genome DNA of the sample to be detected.
Based on the extraction method, the genome DNA information in the ground beetle standard decoction and the ground beetle traditional Chinese medicine formula granule can be reserved as much as possible, and the problem of auxiliary material interference can be overcome.
(2) Forming a PCR amplification system, and amplifying to obtain an amplification product;
specifically, uniformly mixing a DNA template, a primer combination, a PCR buffer solution, dNTPs, polymerase (speedSTAR) and water to obtain a PCR amplification system;
more specifically, the total volume of the PCR amplification system was 20. Mu.L, which comprises: 10 xFast Buffer I (Mg) 2+ plus) 2. Mu.L, dNTP mix 0.6. Mu.L, primer one 0.2. Mu.L, primer two 0.2. Mu.L, speedSTAR 0.2. Mu.L, DNA template 1. Mu.L, deionized water 15.8. Mu.L.
The amplification procedure was: pre-denaturation at 90-95 ℃ for 2-5 min; denaturation at 90-95 ℃ for 15-25 s, annealing at 55-61 ℃ for 15-25 s, extension at 70-75 ℃ for 25-35 s, and circulation for 39-41 times; extending for 3-7 min at 70-75 ℃.
Preferably, the amplification procedure is: pre-denaturation at 95℃for 3min; denaturation at 95 ℃ for 20s, annealing at 55-59 ℃ for 20s and extension at 72 ℃ for 30s, 39 cycles in total; finally, the mixture was extended at 72℃for 5min.
(3) Analyzing the amplified product;
specifically, the amplification product may be analyzed by electrophoresis or fluorescent staining, but is not limited thereto.
Preferably, the amplified product is analyzed by electrophoresis, specifically as follows:
adding 4 mu L of 6×loading buffer into the amplified product, mixing, taking 5-10 mu L of the mixed product, spotting on 1.5% agarose gel, electrophoresis for 15-30 min under 170-180V voltage, and Gelred developing to obtain an electrophoresis pattern. If the electrophoresis pattern contains 237bp DNA fragment, the sample to be detected is Eupolyphaga Seu Steleophaga medicinal material, eupolyphaga Seu Steleophaga standard decoction or Eupolyphaga Seu Steleophaga Chinese medicinal granule.
The invention is illustrated below by means of specific examples.
Example 1 primer design
The method comprises the steps of carrying out homologous alignment on Cytochrome Oxidase I sequences of ground beetles (ground beetles), american cockroaches, jinbian dragon lice, jinbian ground beetles and the like in a GenBank database by using BioEdit software, analyzing specific SNP sites of the ground beetles (ground beetles) after alignment, introducing base sequences containing the SNP sites into Primer Premier5 software, and carrying out Primer design.
Through sequence comparison, the specific locus of ground beetle (ground beetle) is C, the other is A or T, after the SNP locus is determined, the SNP locus is close to the 3' end of the forward primer, the score and GC content of the primer are regulated by moving the position of the upstream primer, and the damage to the DNA clipping sequence by a high-temperature extraction process is fully considered. Primer information shown in the following table was obtained. Furthermore, through an actual identification experiment (specific method is referred to in example 2), when the primer DB-04F/R is adopted for amplification, target bands (237 bp) can be obtained from the genuine products, and no target bands are obtained from the counterfeit products, so that the optimal primer for specific identification of the ground beetles is DB-04F/R. The primer screening results are shown in FIGS. 1 to 4.
Example 2 authentication method establishment
(1) Template extraction
Ground beetle (ground beetle) and common pseudo medicinal materials 0.05g are ground into powder, placed in a 2mL centrifuge tube, added with 1.0mL SDS lysate (50 mmol/L Tris-HCl,200mmol/L NaCl,250mmol/LEDTA,1% SDS) and 10 mu L proteinase K, and incubated for 4-6 hours at 56 ℃ after vortex shaking and mixing. Taking out, cooling to room temperature, adding equal volume of chloroform-isoamyl alcohol (24:1), fully mixing to be milky white, centrifuging at 4 ℃ for 10 minutes (the rotating speed is 12000 rpm), sucking 800-1000 mu L of supernatant into a new 2mL centrifuge tube, adding equal volume of chloroform-isoamyl alcohol (24:1), fully mixing to be milky white, and centrifuging at 4 ℃ for 10 minutes (the rotating speed is 12000 rpm). The supernatant was aspirated at 600-800. Mu.L, an equal volume of chloroform-isoamyl alcohol (24:1) was added, thoroughly mixed to cream, and centrifuged at 4℃for 10 min (at 12000 rpm). 400-600 mu L of supernatant is sucked into a 1.5mL centrifuge tube, added with equal volume of isopropanol or isopropanol-3 mol/L sodium acetate and kept stand for 60-90 minutes at minus 20 ℃. Taking out, centrifuging (rotation speed is 12000 r/min) for 5min, discarding supernatant, washing precipitate with 75% ethanol for 3 times, washing precipitate with anhydrous ethanol for 1 time, incubating at 37deg.C for 30min, adding sterilized water 50 μl after ethanol volatilizes, and standing at 4deg.C or at 20deg.C for long term.
Taking ground beetle (ground beetle) standard decoction freeze-dried powder and 0.05g of formula granule sample, grinding into powder, placing into a 2mL centrifuge tube, adding 1.2mLCTAB precipitate (2%CTAB,100mM Tris-HCl,20mM EDTA (pH=8.0)), stirring, mixing uniformly, incubating at 65 ℃ for 1 hour, and turning over and mixing uniformly for 2-3 times. Centrifuging, removing supernatant, adding 1.2mL of CTAB precipitate, stirring, mixing, incubating at 65 ℃ for 30 minutes, and turning over for 3-5 times. Centrifuging, discarding supernatant, adding 900 μLCTAB extractive solution (2%CTAB,100mM Tris-HCl,20mM EDTA (pH=8.0), 2.5mol/L NaCl,2% PVP 40), 10 μL proteinase K and 10 μL beta-mercaptoethanol, mixing by vortex shaking, incubating at 65deg.C for 2 hr, and tumbling and mixing for 2-3 times. Taking out, cooling to room temperature, adding equal volume of chloroform-isoamyl alcohol (24:1), fully mixing to be milky white, centrifuging at 4 ℃ for 10 minutes (the rotating speed is 12000 rpm), sucking about 800 mu L of supernatant into a new 2mL centrifuge tube, adding equal volume of chloroform-isoamyl alcohol (24:1), fully mixing to be milky white, centrifuging at 4 ℃ for 10 minutes (the rotating speed is 12000 rpm), and repeating the operation for 2-4 times until the middle emulsion layer is free of white impurities. 400-600 mu L of supernatant is sucked into a 1.5mL centrifuge tube, added with equal volume of isopropanol or isopropanol-3 mol/L sodium acetate and kept stand for 60-90 minutes or overnight at minus 20 ℃. Taking out, centrifuging (rotation speed is 12000 r/min) for 5min, discarding supernatant, washing precipitate with 75% ethanol for 3 times, washing precipitate with anhydrous ethanol for 1 time, incubating at 37deg.C for 30min, adding sterilized water 30 μl after ethanol is completely volatilized, dissolving, and storing at 4deg.C or at 20deg.C for long term.
The DNA samples were taken and the DNA concentration was measured using a BioSpec-nano micro-UV spectrophotometer while recording OD260/OD230, OD260/OD280 and adjusting the concentration to 50-100 ng/. Mu.L.
(2) Designing primer combinations
The sequence of the primer combination is as follows: primer one (forward primer) is shown as SEQ ID NO:6 is shown in the figure; primer two (reverse primer) is shown as SEQ ID NO: shown at 7.
(3) PCR amplification
PCR amplification system: a total of 20 μl, comprising: 10 xFast Buffer I (Mg) 2+ plus) 2. Mu.L, dNTP mix (2.5 mmol/L each) 0.6. Mu.L, primer one/primer two (10. Mu. Mol/L) 0.2. Mu.L, speedSTAR (5U/. Mu.L) 0.2. Mu.L, DNA template (10-300 ng) 1. Mu.L, deionized water 15.8. Mu.L.
PCR amplification procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 20s; annealing at 55-59 ℃ for 20s, and extending at 72 ℃ for 30s, wherein the total number of the annealing is 39 cycles; finally, the extension is carried out for 5min at 72 ℃.
(4) Electrophoresis detection
Adding 4. Mu.L of 6×loading buffer into the amplified product, mixing, spotting 8. Mu.L of the mixed product on 1.5% agarose gel, electrophoresis under 170V for 25min, gelred developing, and finally observing and recording the result in a gel imager.
If the electrophoresis pattern contains 237bp DNA fragment, the sample to be detected is Eupolyphaga Seu Steleophaga medicinal material, eupolyphaga Seu Steleophaga standard decoction or Eupolyphaga Seu Steleophaga Chinese medicinal granule.
Example 3 specificity verification
Samples were identified using the samples in the following table, with the identification method established in example 2. Wherein, in the PCR system, the amount of the DNA template is 50ng.
The identification results are shown in fig. 5 and 6, and the identification results show that the identification rate of the ground beetle medicinal material, ground beetle standard decoction (freeze-dried powder) and ground beetle traditional Chinese medicine formula particles are all 237bp bands, the true positive rate is 100%, the true negative rate is 100%, the false positive rate is 0% and the false negative rate is 0%; while none of the other samples gave the strip. The result shows that the identification method has good specificity.
Example 4 sensitivity test
Respectively taking ground beetle traditional Chinese medicine formula granules and medicinal materials produced by different manufacturers. Extracting genes of a sample to be testedGroup DNA and use ddH 2 O dilutes the genome DNA, and then tests the genome DNA by adopting the method of the steps 3-4 in the example 2, and the results are shown in fig. 7 and 8, and the results show that the specific identification detection limit of the ground beetle (ground beetle) formula particle is 1.5ng and the specific identification detection limit of the ground beetle (ground beetle) medicinal material is 0.9ng.
EXAMPLE 5 methodological validation-investigation of different polymerases
The influence of different DNA polymerases on PCR amplification is examined by a single factor, wherein the adopted DNA polymerase is as follows: 2 XM 5 supplier FastTaq (lot number: 21CB0808, manufacturer: beijing polymerase Biotechnology Co., ltd.); rTaq (TaKaRa Taq, lot number: AJ 92482A), ex Taq (lot number: AK 91785A), mighty AMP (lot number: AKE 2435A), and Speed STAR (lot number: AK 42696A) are all manufactured by Takara doctor materials technology (Beijing) Inc. The remaining conditions are as described in example 2. Specifically, the PCR reaction system corresponding to each polymerase is as follows:
2×m5 Supper FastTaq PCR MasterMix polymerase system: comprises 10. Mu.L of 2 XM 5 Supper FastTaq PCR MasterMix, 0.4. Mu.L of each of primer one and primer two, 1. Mu.L of DNA template, and 20. Mu.L of sterilized water.
TaKaRa Taq polymerase System: comprises 2. Mu.L of 10 XBuffer, 0.4. Mu.L of dNTP, 0.2. Mu.L of TaKaRa Taq polymerase, 0.2. Mu.L of each of primer one and primer two, 1. Mu.L of DNA template, and 20. Mu.L of sterilized water.
Ex Taq polymerase System: comprises 2. Mu.L of 10 XEx Taq Buffer, 0.4. Mu.L of dNTP, 0.2. Mu.L of Ex Taq polymerase, 0.2. Mu.L of each of primer one and primer two, 1. Mu.L of DNA template, and 20. Mu.L of sterilized water.
Mighty AMP polymerase system: the kit comprises 10 mu L of 2 XmightyAmp Buffer Ver.2,0.2 mu LMhtyAmp polymerase, 0.2 mu L of each of primer one and primer two, 1 mu L of DNA template, and 20 mu L of sterilized water.
SpeedSTAR HS DNA polymerase system: comprises 2. Mu.L of 10 XFast Buffer I (Mg2+puls), 0.6. Mu.L of dNTP, 0.2. Mu. L SpeedSTAR HS DNA polymerase, 0.2. Mu.L of each of primer one and primer two, 1. Mu.L of DNA template, and sterilized water to 20. Mu.L.
The experimental results are shown in fig. 9-13, and the results show that amplified bands of ground beetle (ground beetle) DNA samples can be obtained by using ExTaq, speedSTAR polymerase, but ex taq can not obtain target bands of ground beetle (ground beetle) standard decoction and formula particles, so that SpeedSTAR polymerase is used as DNA polymerase to ensure the quality of the target bands.
Example 6 methodological verification-investigation of different annealing temperatures
The effect of the annealing temperatures of 53℃at 55℃at 57℃at 59℃and 61℃on PCR amplification was examined by single factor, and the other conditions were as described in example 2.
The experimental results are shown in fig. 14 to 18, and it can be seen from the figures: the result shows that false positive exists in the false PCR amplified band when the annealing temperature is 53 ℃; when the annealing temperature is 55 ℃, 57 ℃, 59 ℃ and 61 ℃, the difference of PCR amplified bands is small and no false positive appears in blank control, but the brightness of ground beetle (ground beetle) formula particles, standard decoction and medicinal material bands gradually decreases along with the increase of temperature, so that the annealing temperature is 55-59 ℃ for ensuring the band brightness.
Example 7 methodological verification-investigation of different cycle times
The effects on PCR amplification were examined by single factor for the number of cycles of 33, 35, 37, 39 and 41, respectively, and the remaining conditions were as described in example 2.
As shown in FIGS. 19 to 23, the PCR amplified bands were brighter and less different when the cycle numbers were 39 and 41, so that the cycle number was 39 in order to secure the band brightness and save the experiment time.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, such changes and modifications are also intended to be within the scope of the invention.
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Sequence listing
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Claims (10)
1. The primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles is characterized by comprising a primer I and a primer II, wherein the sequence of the primer I is shown as SEQ ID NO:6 is shown in the figure; the sequence of the primer II is shown as SEQ ID NO: shown at 7.
2. The use of the primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles according to claim 1 in any one of (1) to (4):
(1) Identifying ground beetle medicinal materials, ground beetle standard decoction and/or ground beetle traditional Chinese medicine formula granules;
(2) Kit for identifying Eupolyphaga Seu Steleophaga medicinal material, eupolyphaga Seu Steleophaga standard decoction, and/or Eupolyphaga Seu Steleophaga Chinese medicinal granule;
(3) Identifying whether the sample to be detected contains ground beetle medicinal materials, ground beetle standard decoction and/or ground beetle traditional Chinese medicine formula particles;
(4) And (3) identifying whether the sample to be detected contains ground beetle medicinal materials, ground beetle standard decoction and/or ground beetle traditional Chinese medicine formula particles.
3. A kit comprising the primer combination of claim 1.
4. An identification method based on the primer combination for PCR identification of ground beetle medicinal materials, standard decoction and traditional Chinese medicine formula particles as claimed in claim 1, which is characterized by comprising the following steps:
extracting genome DNA of a sample to be detected;
the genome DNA is used as a template, the primer combination for PCR identification of the ground beetle medicinal material, the standard decoction and the traditional Chinese medicine formula particles is adopted for PCR amplification, and if the amplification product contains 237bp DNA fragments, the sample to be detected is the ground beetle medicinal material, the ground beetle standard decoction and/or the ground beetle traditional Chinese medicine formula particles.
5. The method according to claim 4, wherein when the sample to be detected is a medicinal material, the method for extracting genomic DNA of the sample to be detected comprises the following steps:
adding SDS lysate and proteinase K to precipitate and extract a sample to be detected, then adding chloroform-isoamyl alcohol to extract for 2-3 times, taking supernatant after extraction, adding isopropanol or isopropanol-sodium acetate to precipitate and extract, washing and incubating the precipitate, and then dissolving the precipitate with water to obtain genome DNA of the sample to be detected;
when the sample to be detected is a standard decoction or traditional Chinese medicine formula particle, the method for extracting the genome DNA of the sample to be detected comprises the following steps:
taking a sample to be detected, and adding CTAB precipitation liquid to precipitate for 1-2 times; adding CTAB extract, proteinase K and beta-mercaptoethanol into the precipitate for extraction, and then adding chloroform-isoamyl alcohol for extraction for 2-5 times; and taking supernatant after extraction, adding isopropanol or isopropanol-sodium acetate for precipitation, washing and incubating the precipitate, and dissolving with water to obtain the genomic DNA of the sample to be detected.
6. The method of claim 4, wherein the template, the primer combination, the PCR buffer, dNTP, speedSTAR and water are mixed uniformly to obtain a PCR amplification system;
amplifying the PCR amplification system according to a preset amplification program to obtain an amplification product;
and (3) carrying out electrophoresis analysis on the amplified product, recording an electrophoresis pattern of the amplified product, and if the pattern contains 237bp DNA fragments, taking the sample to be detected as ground beetle medicinal materials, ground beetle standard decoction and/or ground beetle traditional Chinese medicine formula particles.
7. The identification method of claim 6, wherein the PCR amplification system consists of:
10×Fast BufferⅠ(Mg 2+ plus) 2. Mu.L, dNTP mix 0.6. Mu.L, primer one 0.2. Mu.L, primer two 0.2. Mu.L, speedSTAR 0.2. Mu.L, DNA template 1. Mu.L, deionized water 15.8. Mu.L.
8. The method of claim 6, wherein the PCR amplification procedure is:
pre-denaturing the amplification system at 90-95 ℃ for 2-5 min, circulating 39-41 times under a preset program, and extending at 70-75 ℃ for 3-7 min;
wherein, the preset program is as follows: the amplification system is denatured at 90-95 ℃ for 15-25 s, then annealed at 55-61 ℃ for 15-25 s, and then extended at 70-75 ℃ for 25-35 s.
9. The identification method of claim 8, wherein the PCR amplification procedure is:
pre-denaturing the amplification system at 95 ℃ for 3min, then cycling 39 times under a preset program, and finally extending at 72 ℃ for 5min;
wherein, the preset program is as follows: the amplification system was denatured at 95℃for 20s, then annealed at 55-59℃for 20s, and then extended at 72℃for 30s.
10. The identification method of claim 6, wherein the electrophoretic analysis method is: and diluting the amplified product, spotting on 1.5% agarose gel, carrying out electrophoresis for 15-30 min under the condition of 170-180V voltage, and carrying out Gelred color development to obtain an electrophoresis pattern.
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