CN114606344B - Method for PCR identification of boneset standard and mixed false boneset and acacia - Google Patents
Method for PCR identification of boneset standard and mixed false boneset and acacia Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/686—Polymerase chain reaction [PCR]
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Abstract
The invention discloses a PCR identification method of a boneset genuine product and a mixed fake product of the boneset genuine product, namely the boneset and the acacia, which comprises three steps of template DNA extraction, PCR amplification reaction and agarose electrophoresis detection. The invention designs a specific primer by finding out a specific DNA fragment, amplifies the specific fragment by a PCR technology, and realizes the efficient identification of the boneset genuine product and the mixed and fake products of the boneset and the acacia by a Polymerase Chain Reaction (PCR) technology. The method provided by the invention is simple and feasible, stable and reliable, and can be used for rapidly and accurately identifying the authenticity of the boneset herb genuine product and the mixed fake product of the boneset herb, namely the boneset herb and the acacia roots, ensuring the medication safety and being beneficial to realizing the healthy development of the boneset herb market.
Description
Technical Field
The invention belongs to the technical field of Chinese herbal medicine identification, and particularly relates to a PCR identification method of a boneset genuine product and a mixed fake product of the boneset and acacia.
Background
Herba abri is sweet in nature, slightly bitter in taste, cool, good in effects of promoting diuresis and removing jaundice, clearing heat and removing toxicity, soothing liver and relieving pain, and is often used for damp-heat jaundice, hypochondriac discomfort, epigastric distending pain, acute mastitis and swelling pain, and is distributed in Guangdong, guangxi and other places. Herba abri is Abrus cantoniensis of PapilionaceaeAbruscantoniensis Hance) dried whole plant of Papilionaceae Mao Xiangsai seedAbrusmollis Hance) from different species of plants of the same genus. Two seeds are simultaneously collected in 1990 edition of Guangxi Chinese medicinal material standard, namely "Jigucao". The dried whole herb of the Abrus cantoniensis hance of the year 2010 edition of the Chinese pharmacopoeia is "Abrus cantoniensis hance", and the quality standard of "Abrus cantoniensis hance" of the year 1990 edition of the Guangxi Chinese medicinal material standard fails. In 2008, the dried whole grass of the Guangxi Zhuang nationality Zhuang medicine quality Standard (first volume) on which Mao Xiangsai seeds are collected is "Mujigu grass". These two plants should also be distinguished from the same genus plant acacia (academic name: abrus precatorius L.).
According to investigation, the cultivation of the herba abri is easier, the yield is higher, the current Guangxi cultivation is mainly carried out on the herba abri, and the herba abri is mainly concentrated in areas such as Yulin, qinzhou, guigang and the like, but the cultivation of the herba abri is less, and only a few Guigang and Qinzhou are planted. Another approximation, abrus cantoniensis (academic name: abrus precatorius L.) is less planted, only in Gui Ping, guigang areas, but the folk is called herba abri in the area, and farmers cannot separate the three species, all called herba abri (herba abri ). The yield of the grass planted per mu is 200-300 kg, the yield of the grass planted per mu is 300-500 kg, the yield is almost 50% higher than that of the grass, but the current price of the grass is doubled compared with that of the grass, and the adulteration phenomenon of the grass is caused by the large price difference. In addition, because the abrus herb and the abrus herb are similar species, in the planting process, adjacent planting is not spaced, and the situation that the abrus herb and the abrus herb are hybridized easily occurs. Because the Guangxi folks have the mixed use condition of the abrus herb and the pubescent abrus herb, two species doping planting (species source mixing) is found in the research process, and the non-subjective adulteration condition is also caused.
The appearance of the abrus herb and the appearance of the abrus herb are similar, the abrus herb and the abrus herb are identified only by the size of leaves and Mao Duomao, the subjectivity is realized, the experience of a tester is highly relied, the judgment on the result is unfavorable, and most of medicinal materials are left to branch after being dried in the sun and are more difficult to identify and easily judge by mistake because the leaves of leguminous plants are easy to fall off, so the abrus herb and the abrus herb are often used as the abrus herb to be mixed for selling. At present, the identification of the abrus herb, the abrus herb and the abrus herb mainly takes physical and chemical identification, but the chemical components of the three are basically the same, mainly contain alkaloids and flavonoids, and mainly contain abrus herb alkali, schaftoside and the like, and the accurate judgment of the false and correct products is difficult to be carried out in the physical and chemical identification of thin layers, content measurement projects and the like. Therefore, in order to ensure the quality of the traditional Chinese medicine materials and the medication safety, a simple and reliable method for accurately, quickly and stably identifying the genuine and counterfeit herba abri products and the mixed and counterfeit products thereof is needed.
Disclosure of Invention
The invention aims to provide a PCR identification method for a boneset genuine product and a mixed and fake product of a boneset and an acacia son, which aims at the defects in the prior art, and the identification method is simple and easy to implement.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a PCR identification method of a boneset genuine product and a mixed fake product of the boneset and the acacia comprises the following steps:
(1) Template DNA extraction:
taking a sample, washing the sample with 75% ethanol/absolute ethanol for 2 times or wiping the surface, sucking excessive water by filter paper, and airing; grinding into superfine powder by a grinder for later use, weighing about 20. 20 mg of a dry sample to be tested, placing the dry sample into a 1.5mL centrifuge tube, and extracting DNA of the sample by using a novel plant tissue genome DNA extraction kit;
(2) PCR amplification reaction:
identifying primers, and aiming at the genuine product and the two counterfeit products, having corresponding specific primers,
the abrus cantoniensis upstream primer: 5'-CAACATTCTTTAGTATTTTTTATTTCCTAT-3' (SEQ ID NO: 1),
the abrus herb downstream primer: 5'-CGCGCATGGTGGATTCACAATCC-3' (SEQ ID NO: 2);
the upstream primer of the herba abri: 5'-CGCATTCTTTAGTATTGTTTATTTCGTTG-3' (SEQ ID NO: 3),
the downstream primer of the herba abri: 5'-ACCTTGAACCACTTGCCTACA-3' (SEQ ID NO: 4);
an upstream primer of acacia catechu: 5'-GTTTTTGAAAGTAAAGGAGCAATATCAACAG-3' (SEQ ID NO: 5), abrus downstream primer: 5'-ACTTGGTTACATCCGCCCTT-3' (SEQ ID NO: 6);
PCR reaction system: the total reaction volume is 25 mu L, the reaction system comprises 12.5 mu L of Aidelai 2X F8 LongFast PCR MasterMix premix, 0.75 mu L of each of the upstream primer and the downstream primer (10 mu mol/L), 1 mu L of template DNA and 10 mu L of sterile water; an equal volume of sterile water is taken to replace the template DNA and is used as a blank control;
reaction conditions: centrifuge tube was placed in PCR instrument, PCR reaction parameters: pre-denaturation at 95 ℃ for 5 min; cycling for 35 times (denaturation at 94℃for 15 seconds, annealing at X℃for 15 seconds, extension at 72℃for 20 seconds); extending at 72 ℃ for 5 minutes; wherein, aiming at the abrus herb primer, X is 58; for herba abri, X is 54; the acacia primer and X is 62.
(3) Agarose electrophoresis detection: preparing 2% agarose electrophoresis according to agarose gel electrophoresis method (general rule 0541), adding nucleic acid gel stain GelRed into gel, sampling 3ul of product, loading DNA molecular weight marker with sample amount of 2.5 μl, and inspecting on gel imager after electrophoresis; in the gel electrophoresis chart of the test sample, the position corresponding to the gel electrophoresis of the control medicinal material is: the abrus herb and the abrus herb have obvious bands between 100 and 200bp, the amplified product of the abrus primer has obvious bands between 200 and 300bp, and the blank control has no bands.
The PCR identification method of the bonesetting quality product and the mixed quality product of the bonesetting and the acacia, the specific extraction steps of extracting the DNA of the sample in the step (1) by using the novel plant tissue genome DNA extraction kit are as follows:
(1) adopting a high-efficiency plant genome DNA extraction kit of the radix angelicae, adding 400 mu L of buffer FGA into a centrifuge tube filled with a test sample, carrying out vortex vibration and mixing uniformly, and standing for 10 minutes at room temperature; 130 mu L of buffer LP2 is added, fully and uniformly mixed, and vortex oscillation is carried out for 1 minute; centrifugation at 12,000rpm for 5 minutes, carefully transferring the supernatant to a new 1.5mL centrifuge tube;
(2) adding 1.5 times of buffer solution LP3, immediately and fully shaking and uniformly mixing for 15 seconds; adding the solution obtained in the last step and flocculent precipitate into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12,000rpm for 30 seconds, pouring out waste liquid, and placing the adsorption column CB3 into the collecting pipe; adding 600 μl of rinse solution PW to the adsorption column CB3, centrifuging at 12,000rpm for 30 seconds, pouring out the waste liquid, and placing the adsorption column CB3 into a collection tube;
(3) repeating the rinsing process of the previous step; placing the adsorption column CB3 back into a collecting pipe, centrifuging at 12,000rpm for 2 minutes, and pouring out waste liquid; placing the adsorption column CB3 at room temperature for 2 minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean 1.5mL centrifuge tube, suspending and dripping 50 mu L of elution buffer TB (TB is preheated to 65 ℃ in a metal bath in advance) into the middle part of the adsorption film, standing for 5 minutes at room temperature, centrifuging at 12,000rpm for 2 minutes, and collecting the solution into the centrifuge tube;
(4) carefully suspending all the solution obtained in the centrifuge tube, dripping the solution to the middle part of the adsorption film of CB3 in a suspending way, standing for 5 minutes at room temperature, centrifuging at 12,000rpm for 2 minutes, and collecting the solution into the centrifuge tube; the positive control template DNA solution was also prepared as described above.
The beneficial effects of the invention are as follows:
according to the invention, the specific DNA fragment is found out, the specific primer is designed, and the specific fragment is amplified by the PCR technology, so that the genuine herba abri and the mixed and fake herba abri and acacia can be distinguished, and the three can be effectively identified. The invention overcomes the defects of the traditional methods for identifying the abrus herb, such as morphological identification, physicochemical identification and the like, can effectively identify the abrus herb genuine product and the abrus herb mixed with the genuine product in the market without depending on the experience of medicinal material inspection staff, accurately judges the abrus herb and the abrus herb easily mixed with the genuine product, effectively supplements and perfects the medicinal material standard of the abrus herb and the abrus herb, and is beneficial to realizing the healthy development of the market of the abrus herb.
The PCR identification method of the boneset standard and the mixed and fake boneset and the acacia can be used for rapidly and accurately identifying the boneset standard and the mixed and fake boneset and the acacia, and the medication safety is ensured.
Drawings
FIG. 1 ITS2 fragment alignment;
FIG. 2 psba-trnH alignment;
FIG. 3 annealing temperature investigation 54℃and 56℃glue pattern;
FIG. 4 annealing temperature investigation is 58 ℃ and 60 ℃ glue graph;
FIG. 5 annealing temperature investigation is carried out at 62 ℃ and 64 ℃ for a glue graph;
FIG. 6 is a view of a picture of enzyme class investigation of the abrus cantoniensis primer;
FIG. 7 is an enzyme class examination chart of the acacia primer;
FIG. 8 number of reaction cycles;
FIG. 9 Abrus cantoniensis primer suitability investigation;
FIG. 10 Abrus cantoniensis primer specificity study;
FIG. 11A-C primer adaptation study;
FIG. 12 specific investigation of the Tinospora cordifolia primers;
FIG. 13 Abrus primer suitability investigation;
FIG. 14 Abrus primer specificity study (Abrus cantoniensis sample);
FIG. 15 Abrus primer specificity investigation (Abrus pubescens sample).
Detailed Description
Example 1
Since there is no public genomic information of abrus herb, abrus herb and abrus herb on NCBI, only sporadic DNA barcode sequences, and whether the basic sources corresponding to the sequences uploaded to NCBI are correct or not cannot be determined, samples of abrus herb, abrus herb and abrus herb are collected, and varieties are determined through basic source identification. Extracting DNA and quality inspection are carried out on the sample, common DNA bar code primers such as ITS2, rbcL, psbA-trnH and the like are used for amplification and sequencing, low-quality area data are removed, sequencing results are combined and are imported into Mega for comparison, fragment areas with differences are searched, and specific primers are designed for amplification and agarose electrophoresis detection.
1 Material
1.1 Three variety samples are collected from Guangxi region, mainly from enterprise planting base, common farmer's own home field seed dispersion and field collection. The samples were identified by the main pharmacist of the Guangxi food and drug institute Huang Qingquan, and the voucher specimens were stored in the specimen room of the Guangxi food and drug institute, see tables 1-3.
1.2 An instrument gradient PCR amplification instrument (ABI veriti); gradient PCR amplicons (TA 96SG, yena, germany); electrophoresis apparatus (BIO-RAD); a micro ultraviolet spectrophotometer (Nano Value PLUS); geldoc XR+ fully automated gel imaging System (BIO-RAD); ME203 electronic balance (Mettle); MIKRO220R high speed refrigerated centrifuge (Hettich); vortex shaking instrument (Scientific in-conservation).
1.3 Reagent agarose (bisquest); gelRed (BIOTIUM); primeSTAR Max DNA Polymerase (TAKARA), 2X F8 LongFast PCR MasterMix (Aidelai), KOD OneTM PCR Master Mix (Toyobo), platinu II Hot-Start Green PCR Master Mix (ThermoFisher), DNA Marker (TaKaRa); Q5 enzyme (MEW ENGLEND); novel plant tissue genomic DNA extraction kit (tenna); the primer is synthesized by Huada genes, and other reagents are all of domestic analytical purity.
2. Test method
2.1 Extracting template DNA, wiping the surface of the sample with 75% ethanol, air drying, pulverizing with a ball mill, and collecting about 20 mg of the sample. DNA of the sample was extracted using a novel plant genomic DNA extraction kit (DP 350), and the obtained DNA was measured for concentration and purity by a micro ultraviolet spectrophotometer (Nano Value PLUS). The specific extraction procedure for extracting DNA from samples using the novel plant tissue genomic DNA extraction kit (DP 350) is as follows:
(1) weighing about 20. 20 mg of a dry test sample, placing the dry test sample into a 1.5mL centrifuge tube, adding 400 mu L of buffer FGA into the centrifuge tube filled with the test sample, uniformly mixing by vortex vibration, and standing at room temperature for 10 minutes; 130 mu L of buffer LP2 is added, fully and uniformly mixed, and vortex oscillation is carried out for 1 minute; centrifugation at 12,000rpm for 5 minutes, carefully transferring the supernatant to a new 1.5mL centrifuge tube;
(2) adding 1.5 times of buffer solution LP3, immediately and fully shaking and uniformly mixing for 15 seconds; adding the solution obtained in the last step and flocculent precipitate into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12,000rpm for 30 seconds, pouring out waste liquid, and placing the adsorption column CB3 into the collecting pipe; adding 600 μl of rinse solution PW to the adsorption column CB3, centrifuging at 12,000rpm for 30 seconds, pouring out the waste liquid, and placing the adsorption column CB3 into a collection tube;
(3) repeating the rinsing process of the previous step; placing the adsorption column CB3 back into a collecting pipe, centrifuging at 12,000rpm for 2 minutes, and pouring out waste liquid; placing the adsorption column CB3 at room temperature for 2 minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean 1.5mL centrifuge tube, suspending and dripping 50 mu L of elution buffer TB (TB is preheated to 65 ℃ in a metal bath in advance) into the middle part of the adsorption film, standing for 5 minutes at room temperature, centrifuging at 12,000rpm for 2 minutes, and collecting the solution into the centrifuge tube;
(4) carefully suspending all the solution obtained in the centrifuge tube, dripping the solution to the middle part of the adsorption film of CB3 in a suspending way, standing for 5 minutes at room temperature, centrifuging at 12,000rpm for 2 minutes, and collecting the solution into the centrifuge tube; the positive control template DNA solution was also prepared as described above.
2.2 Amplification and sequencing of common barcode primers common DNA barcode primers such as ITS2, rbcL, and psbA-trnH were used for amplification and sequencing. Primer sequence information is as follows:
2.3 The enzyme for PCR amplification condition amplification is Edley 2X F8 LongFast PCR MasterMix premix, the PCR system is 25 mu L, the premix is 12.5 mu L, the upstream and downstream primers (10 mu mol/L) are 0.75 mu L respectively, the template DNA is 10-50ng, and the sterile water is added to 25 mu L. The PCR reaction procedure was 95℃for 5 minutes of pre-denaturation; denaturation at 94℃for 15 seconds, annealing at X℃for 30 seconds, elongation at 72℃for 30 seconds, 35 cycles at this stage were run; extension was carried out at 72℃for 5 minutes. Wherein, ITS2 primer, X is 56; the other two pairs of primers, X is 55. The resulting product was sent to a sequencing company for bidirectional sequencing.
2.4 differential sequences obtain low quality data from the removal sequencing and complete the splicing of the bi-directional sequences. And (3) carrying the spliced sequences into Mega for comparison, and adopting a Musdle algorithm to default parameters. J31-J34 are abrus, J35 and J36 are abrus herb, and J37-J41 are abrus herb.
As a result, rbcL primers gave no difference in the products; ITS2 acacia has some difference from others, but the abrus herb and abrus herb are basically discontinuous single base differences, see FIG. 1. The psba-trnH abri is different from others to some extent, but the abrus cantoniensis and abrus cantoniensis are basically discontinuous single base differences, but the 4 bases are deleted at the positions from 226 th base to 229 th base of abrus cantoniensis product, which are TTAT. See fig. 2.
2.5 Specific primer design for the two different primers, it is easier to design specific primers of abri, but the difficulty of designing primers for distinguishing abrus herb from abrus herb by agarose electrophoresis detection is still high, and we select the region of psbA-trnH for carrying out because abrus herb and abrus herb have TTAT difference in the region. For the abrus herb, 6 pairs of primers are debugged, and the final sixth pair of primers can specifically amplify abrus herb but not abrus herb and abrus herb; for the herba abri, 5 pairs of primers are debugged, and the fifth pair of primers can specifically amplify the herba abri but not the herba abri and the acacia catechu finally, as shown in table 6; for Abrus, we have debugged 2 pairs of primers, see Table 7, and finally the second pair of primers can specifically amplify Abrus and not amplify Abrus cantoniensis and Abrus cantoniensis.
3. Methodological verification
The PCR system was 25. Mu.L, the premix was 12.5. Mu.L, the upstream and downstream primers (10. Mu. Mol/L) were each 0.75. Mu.L, the template DNA was 10-50ng, and the sterile water was made up to 25. Mu.L. The PCR reaction procedure was 95℃for 5 minutes of pre-denaturation; denaturation at 94℃for 15 seconds, annealing at X℃for 15 seconds, elongation at 72℃for 20 seconds, 35 cycles at this stage were run; extension was carried out at 72℃for 5 minutes. The methodological verification test samples are J8 (abrus herb J), J26 (abrus herb M) and J30 (abrus herb X), respectively.
3.1 The annealing temperature is examined by using Edley 2X F8 LongFast PCR MasterMix enzyme, 54 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃ and 64 ℃ are respectively examined according to the reaction system, and the annealing temperature is tested, and the X is 58 ℃ aiming at the abrus herb primer; aiming at the herba abri primers, X is 54 ℃; aiming at acacia, X is relatively suitable at 62 ℃, and has obvious single amplified bands for target samples, and all other negative samples have no amplified bands, as shown in figures 3-5. The reaction is highly specific at 58 ℃, 54 ℃ and 62 ℃ at the annealing temperature, and the type of the sample can be determined according to whether the corresponding discrimination primer amplifies the template DNA.
3.2 Class investigation of enzymes
Because the difference area of the abrus herb and the abrus herb is few, the PCR system is sensitive to temperature and reagent. By trying different brands of enzymes including 2X F8 LongFast PCR MasterMix (Edley), primeSTAR Max DNA Polymerase (TAKARA), KOD OneTM PCR Master Mix (Toyobo), platinu II Hot-Start Green PCR Master Mix (ThermoFisher), it was finally confirmed that three varieties could not be effectively identified except Edley 2X F8 LongFast PCR MasterMix at the specified annealing temperature, so that the identification requirement could be met by finally specifying Edley 2X F8 LongFast PCR MasterMix. See fig. 6-7.
3.3 The three varieties are inspected for 30-35 cycles under the respective reaction systems, and a single strip with obvious brightness can be obtained, which shows that 30 cycles reach the amplification concentration. See FIG. 8
3.4 The brand inspection of the instrument is limited by laboratory conditions, only 2 brands of PCR instruments, namely a gradient PCR amplification instrument (ABI veriti) and a gradient PCR amplification instrument (TA 96SG, yes in Germany), can obtain good amplification effects, and the method has no special requirement on the instruments. See fig. 8-11. FIG. 8 shows a gradient PCR amplification unit (ABI veriti), and FIGS. 9-11 show a gradient PCR amplification unit (TA 96SG, yes Germany)
3.5 The suitability and specificity inspection aims at the well-searched experimental conditions, all the collected samples and mixed and false products are verified, and the results show that the corresponding primer amplified target samples are all provided with strips, and the other mixed and false products are not provided with amplified strips. The adaptability is good, and the three varieties can be identified. See fig. 9-15.
4. Results and analysis
4.1 because the appearance of the abrus herb and the abrus herb is highly similar, the abrus herb and the abrus herb are identified only by the size of the leaf and less Mao Duomao, the abrus herb and the abrus herb are subjective and highly dependent on the experience of an inspector, and are unfavorable to the judgment of the result, most of medicinal materials are easy to fall off, and only branch is left after the medicinal materials are dried in the sun, so that the abrus herb and the abrus herb are more difficult to identify and easily judge by mistake, so the abrus herb and the abrus herb are often used as the abrus herb to be sold in a blending way. At present, the identification of the herb materials of the abrus herb and the pubescent abrus herb mainly takes physical and chemical identification, but the three herb materials have the same chemical components, mainly contain alkaloids and flavonoids, mainly contain abrine, schaftoside and the like, and the physical and chemical identification in thin layers, content measurement projects and the like is difficult to accurately judge the false products. The experiment is to find out specific DNA fragments, design specific primers, amplify specific fragments by PCR technology to distinguish three mixed and false products, and can effectively identify the authenticity of the three mixed and false products.
4.2 Because the abrus herb and the abrus herb are used as medicinal materials, large-area planting is carried out, most operators plant two varieties simultaneously through investigation, and in two adjacent lands, the reproduction isolation cannot be achieved in the planting process, and hybridization is easy to occur between closely related plant groups with similar domain distribution and ecological characteristics. In the collected samples, the samples collected at the mixed-seed base have the characteristics of the abrus herb and the abrus herb at the same time, namely, the leaves are small but Mao Liang are more, which brings great difficulty to the basic source identification, for example, J12 and J14 have appearance more similar to the abrus herb, but leaves are sparse but more than the abrus herb and not as dense as the abrus herb, the abrus herb is obtained through a DNA sequencing result, the abrus herb primer can be used for expanding the strip, and the abrus herb primer can not be used for expanding the strip. The method does not depend on experience of medicinal material inspectors, effectively supplements and perfects medicinal material standards of the abrus herb and the abrus herb, can accurately judge the easily-mixed counterfeits of the abrus herb and the abrus herb, and is beneficial to realizing healthy development of the abrus herb market.
Example 2
A PCR identification method of a boneset genuine product and a mixed fake product of the boneset and the acacia comprises the following steps:
(1) Template DNA extraction:
washing the sample with absolute ethanol for 2 times, absorbing excessive water with filter paper, and air drying; grinding into superfine powder by a grinder for later use, weighing about 20. 20 mg of a dry sample to be tested, placing the dry sample into a 1.5mL centrifuge tube, and extracting DNA of the sample by using a novel plant tissue genome DNA extraction kit, wherein the specific extraction step is the same as the specific extraction step of extracting DNA of the sample by using the novel plant tissue genome DNA extraction kit (DP 350) in the 2.1 part of the example 1.
(2) PCR amplification reaction:
identifying primers, and aiming at the genuine product and the two counterfeit products, having corresponding specific primers,
the abrus cantoniensis upstream primer: 5'-CAACATTCTTTAGTATTTTTTATTTCCTAT-3' (SEQ ID NO: 1),
the abrus herb downstream primer: 5'-CGCGCATGGTGGATTCACAATCC-3' (SEQ ID NO: 2);
the upstream primer of the herba abri: 5'-CGCATTCTTTAGTATTGTTTATTTCGTTG-3' (SEQ ID NO: 3),
the downstream primer of the herba abri: 5'-ACCTTGAACCACTTGCCTACA-3' (SEQ ID NO: 4);
an upstream primer of acacia catechu: 5'-GTTTTTGAAAGTAAAGGAGCAATATCAACAG-3' (SEQ ID NO: 5), abrus downstream primer: 5'-ACTTGGTTACATCCGCCCTT-3' (SEQ ID NO: 6);
PCR reaction system: the total reaction volume is 25 mu L, the reaction system comprises 12.5 mu L of Aidelai 2X F8 LongFast PCR MasterMix premix, 0.75 mu L of each of the upstream primer and the downstream primer (10 mu mol/L), 1 mu L of template DNA and 10 mu L of sterile water; an equal volume of sterile water is taken to replace the template DNA and is used as a blank control;
reaction conditions: centrifuge tube was placed in PCR instrument, PCR reaction parameters: pre-denaturation at 95 ℃ for 5 min; cycling for 35 times (denaturation at 94℃for 15 seconds, annealing at X℃for 15 seconds, extension at 72℃for 20 seconds); extending at 72 ℃ for 5 minutes; wherein, aiming at the abrus herb primer, X is 58; for herba abri, X is 54; the acacia primer and X is 62.
(3) Agarose electrophoresis detection: preparing 2% agarose electrophoresis according to agarose gel electrophoresis method (general rule 0541), adding nucleic acid gel stain GelRed into gel, sampling 3ul of product, loading DNA molecular weight marker with sample amount of 2.5 μl, and inspecting on gel imager after electrophoresis; in the gel electrophoresis chart of the test sample, the position corresponding to the gel electrophoresis of the control medicinal material is: the abrus herb and the abrus herb have obvious bands between 100 and 200bp, the amplified product of the abrus primer has obvious bands between 200 and 300bp, and the blank control has no bands.
Sequence listing
<110> Guangxi Zhuang nationality food and drug institute
<120> PCR identification method of genuine chicken bone herb and its mixed fake chicken bone herb and acacia
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cgcgcatggt ggattcacaa tcc 23
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cgcattcttt agtattgttt atttcgttg 29
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accttgaacc acttgcctac a 21
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gtttttgaaa gtaaaggagc aatatcaaca g 31
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acttggttac atccgccctt 20
Claims (2)
1. The PCR identification method of the boneset genuine product and the mixed fake product of the boneset and the acacia is characterized by comprising the following steps:
(1) Template DNA extraction:
taking a sample, washing the sample with 75% ethanol or absolute ethanol for 2 times or wiping the surface, sucking excessive water by filter paper, and airing; grinding into superfine powder with a grinding instrument for standby; weighing the dried sample powder 20 and mg, placing the dried sample powder into a 1.5mL centrifuge tube, and extracting DNA of the dried sample powder by using a novel plant tissue genome DNA extraction kit;
(2) PCR amplification reaction:
identifying primers, wherein the primers are specific primers corresponding to the abrus herb, the abrus herb and the acacia,
amplification of the abrus cantoniensis hance upstream primer: 5'-CAACATTCTTTAGTATTTTTTATTTCCTAT-3' the number of the individual pieces of the plastic,
amplification of the abrus cantoniensis hance downstream primer: 5'-CGCGCATGGTGGATTCACAATCC-3';
amplification of the upstream primer by the herba abri: 5'-CGCATTCTTTAGTATTGTTTATTTCGTTG-3' the number of the individual pieces of the plastic,
amplification of the downstream primer by the herba abri: 5'-ACCTTGAACCACTTGCCTACA-3';
abrus amplification upstream primer: 5'-GTTTTTGAAAGTAAAGGAGCAATATCAACAG-3' the number of the individual pieces of the plastic,
amplification of acacia catechu downstream primer: 5'-ACTTGGTTACATCCGCCCTT-3';
PCR reaction system: the total reaction volume was 25. Mu.L, 12.5. Mu.L of Edley 2X F8 LongFast PCR MasterMix premix, 0.75. Mu.L of each of the upstream and downstream primers, 1. Mu.L of template DNA, and 10. Mu.L of sterile water; an equal volume of sterile water is taken to replace the template DNA and is used as a blank control;
reaction conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 94℃for 15 seconds, annealing at X℃for 15 seconds, elongation at 72℃for 20 seconds, and cyclic reaction for 35 times; extending at 72 ℃ for 5 minutes; wherein, aiming at the abrus herb primer, X is 58; aiming at the herba abri primers, X is 54; x is 62 for the acacia primer;
(3) Agarose electrophoresis detection: preparing 2% agarose gel according to an agarose gel electrophoresis method, adding a nucleic acid gel coloring agent GelRed into the gel, taking 3 mu L of a sample product obtained after PCR amplification reaction for sample loading, wherein the sample loading amount of a DNA molecular weight mark is 2.5 mu L, and inspecting on a gel imager after electrophoresis is finished; in the gel electrophoresis chart of the test sample, the position corresponding to the gel electrophoresis of the control medicinal material is: the abrus herb and the abrus herb have obvious bands between 100 and 200bp, the amplified product of the abrus primer has obvious bands between 200 and 300bp, and the blank control has no bands.
2. The PCR identification method of the genuine chicken bone herb and the mixed fake chicken bone herb and the acacia catechu according to claim 1, wherein the specific extraction step of extracting the DNA of the dry test sample powder by using the novel plant tissue genome DNA extraction kit in the step (1) is as follows:
(1) adopting a high-efficiency plant genome DNA extraction kit of the radix angelicae, DP350, adding 400 mu L of buffer FGA into a centrifuge tube filled with test sample powder, vortex shaking and mixing uniformly, and standing for 10 minutes at room temperature; 130 mu L of buffer LP2 is added, fully and uniformly mixed, and vortex oscillation is carried out for 1 minute; centrifugation at 12,000rpm for 5 minutes, carefully transferring the supernatant to a new 1.5mL centrifuge tube;
(2) adding 1.5 times of buffer solution LP3, immediately and fully shaking and uniformly mixing for 15 seconds; adding the obtained solution and flocculent precipitate into an adsorption column CB3, centrifuging at 12,000rpm for 30 seconds, pouring out waste liquid, and placing the adsorption column CB3 into a collecting pipe; adding 600 μl of rinse solution PW to the adsorption column CB3, centrifuging at 12,000rpm for 30 seconds, pouring out the waste liquid, and placing the adsorption column CB3 into a collection tube;
(3) repeating the rinsing process of the previous step; placing the adsorption column CB3 back into a collecting pipe, centrifuging at 12,000rpm for 2 minutes, and pouring out waste liquid; placing the adsorption column CB3 at room temperature for 2 minutes to thoroughly dry the residual rinsing liquid in the adsorption material; transferring the adsorption column CB3 into a clean 1.5mL centrifuge tube, and suspending and dropwise adding 50 mu L of elution buffer solution TB into the middle part of the adsorption film, wherein the TB is preheated to 65 ℃ in a metal bath in advance; standing at room temperature for 5 minutes, centrifuging at 12,000rpm for 2 minutes, and collecting the solution into a centrifuge tube;
(4) the whole solution obtained in the centrifuge tube was carefully suspended again and dropped onto the middle part of the adsorption film of CB3, left at room temperature for 5 minutes and centrifuged at 12,000rpm for 2 minutes, and the solution was collected in the centrifuge tube.
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| CN103898204A (en) * | 2012-12-28 | 2014-07-02 | 财团法人工业技术研究院 | Method, database and primer pair for identifying herbal medicinal materials |
| CN108060208A (en) * | 2017-11-30 | 2018-05-22 | 广西壮族自治区食品药品检验所 | A kind of discrimination method of snake bile |
| CN109971868A (en) * | 2019-05-07 | 2019-07-05 | 镇江市食品药品监督检验中心 | One group for identifying the specific primer and deer horn authenticity identification method of deer horn |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103898204A (en) * | 2012-12-28 | 2014-07-02 | 财团法人工业技术研究院 | Method, database and primer pair for identifying herbal medicinal materials |
| CN108060208A (en) * | 2017-11-30 | 2018-05-22 | 广西壮族自治区食品药品检验所 | A kind of discrimination method of snake bile |
| CN109971868A (en) * | 2019-05-07 | 2019-07-05 | 镇江市食品药品监督检验中心 | One group for identifying the specific primer and deer horn authenticity identification method of deer horn |
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| ITS，rbcL和matK序列作为中药鸡骨草条形码的研究;王晓明;中国优秀硕士学位论文全文数据库 农业科技辑;D047-85 * |
| 不同地区毛鸡骨草与鸡骨草rbcL序列差异分析;魏妮娜等;广东农业科学;第40卷(第17期);第137-139页 * |
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