CN114606344A - PCR identification method for abrus cantoniensis quality product and mixed counterfeit abrus cantoniensis and abrus - Google Patents
PCR identification method for abrus cantoniensis quality product and mixed counterfeit abrus cantoniensis and abrus Download PDFInfo
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Abstract
The invention discloses a PCR identification method of abrus cantoniensis hance genuine products and mixed counterfeit products thereof, namely abrus cantoniensis hance and abrus chinensis, and specifically comprises three steps of template DNA extraction, PCR amplification reaction and agarose electrophoresis detection. The invention realizes the high-efficiency identification of the abrus herb genuine product and the false product abrus herb and abrus chinensis thereof by finding out a specific DNA fragment, designing a specific primer, amplifying the specific fragment by a PCR technology and realizing the high-efficiency identification of the abrus herb genuine product and the false product abrus herb and abrus chinensis by a Polymerase Chain Reaction (PCR) technology. The method provided by the invention is simple and feasible, stable and reliable, and by adopting the method, the authenticity of the abrus herb genuine product, the abrus herb fake product and the abrus chinensis can be quickly and accurately identified, so that the medication safety is ensured, and the healthy development of the abrus herb medicinal material market is facilitated.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine identification, and particularly relates to a PCR identification method of abrus cantoniensis hance quality products and false abrus cantoniensis hance and abrus seeds thereof.
Background
Abrus cantoniensis Hance is sweet in nature, slightly bitter and cool, has the effects of removing dampness and jaundice, clearing heat and toxic materials, and soothing liver to alleviate pain, and is commonly used for damp-heat jaundice, discomfort in hypochondrium, distending pain in stomach, and acute mastitis with swelling pain, and is distributed in Guangdong, Guangxi and other places. Abrus cantoniensis Hance is Abrus cantoniensis Hance of PapilionaceaeAbrusDried whole plant of Cantoniensis Hance, Abrus mollis of butterfly familyAbrusmollis Hance) from different plants of the same family and genus. Two species were collected simultaneously in 1990 'Guangxi Chinese medicinal material Standard', and were called as "Abrus cantoniensis". The dried whole herb of the Guangzhou acacia is collected from the Chinese pharmacopoeia 2010 edition as the abrus herb, and the abrus herb has invalid quality standard from the Guangxi Chinese medicinal material standard 1990 edition. In 2008, the dry whole herb carrying the hairy julienne is called hairy abrus herb in Guangxi Zhuang nationality autonomous region Zhuang nationality quality Standard (first volume). These two plants should be distinguished from the plant of the same genus, namely Abrus precatorius L.
According to research, because the cultivation of the garden balsam stems is easier and the yield is higher, the garden balsam stems are mainly planted in southern China and mainly concentrated in Yulin, Qinzhou, Guihong and other areas at present, while the garden balsam stems are less planted and only a few of Guihong and Qinzhou are planted. The other similar Abrus (Abrus precatorius L.) is planted less, and only in Guiping and Guigang areas, but also called Abrus herb in the areas, but farmers can not distinguish the three species, all of which are called Abrus herb (chicken bone incense and chicken bone vine). The yield per mu of the abrus herb is 200-300 kg, the yield per mu of the abrus herb is 300-500 kg, the yield is almost higher than that of the abrus herb by more than 50%, but the current price of the abrus herb is one time higher than that of the abrus herb, and the phenomenon of adulteration of the abrus herb is caused due to large price difference. Moreover, because the abrus herb and the hairy abrus herb are similar species, the abrus herb and the hairy abrus herb are planted adjacently without spacing in the planting process, and the hybridization of the abrus herb and the hairy abrus herb is easy to occur. Because the situation that the abrus cantoniensis hance and the abrus maoensis are mixed exists in Guangxi folks, two kinds of doped plants (seed source mixing) are also found in the research process, and the non-subjective adulteration situation is also caused.
Because the abrus cantoniensis hance and abrus cantoniensis hance are highly similar in appearance, the abrus cantoniensis hance and the abrus cantoniensis hance are identified only by the size of leaves and the number of hairs, the result judgment is not good, and because the leaves of leguminous plants are easy to fall off, and most of medicinal materials are only left in branches and trunks after being dried in the sun and are difficult to identify and easy to judge wrongly, the abrus cantoniensis hance and abrus are often mixed and fake as the abrus cantoniensis hance for sale. At present, the identification of abrus herb, abrus herb and abrus seed is mainly based on physicochemical identification, but the chemical components of the abrus herb, abrus herb and abrus seed are roughly the same, the abrus herb, abrus herb and abrus seed mainly contain alkaloid and flavonoid, the abrus seed mainly contains abrus alkali, schaftoside and the like, and accurate judgment on the false positive products is difficult to be made through physicochemical identification in thin-layer and content measurement items and the like. Therefore, in order to ensure the quality and the medication safety of the traditional Chinese medicine, a simple and reliable method capable of accurately, quickly and stably identifying the abrus herb genuine products and the abrus herb mixed counterfeit products is needed.
Disclosure of Invention
The invention aims to provide a PCR identification method of abrus cantoniensis hance, mixed counterfeit abrus cantoniensis hance and abrus chinensis aiming at the defects in the prior art, the identification method is simple and easy to implement, the abrus cantoniensis hance, mixed counterfeit abrus cantoniensis hance and abrus chinensis can be quickly and accurately distinguished by adopting the method, the authenticity of the abrus cantoniensis hance, the mixed counterfeit abrus cantoniensis hance and abrus chinensis is effectively identified, and the medication safety is ensured.
In order to achieve the purpose, the invention adopts the following technical scheme:
a PCR identification method for Abrus cantoniensis Hance genuine products and false products thereof, Abrus mollis Hance and Abrus precatorius comprises the following steps:
(1) extracting template DNA:
washing the sample with 75% ethanol/anhydrous ethanol for 2 times or wiping the surface, sucking excessive water with filter paper, and air drying; grinding the mixture into superfine powder for later use by a grinding instrument, weighing about 20 mg of a dry sample to be tested, putting the dry sample into a 1.5mL centrifuge tube, and extracting DNA of a sample by using a novel plant tissue genome DNA extraction kit;
(2) and (3) PCR amplification reaction:
identifying primer, which has corresponding specific primer aiming at both genuine product and two counterfeit products,
an abrus herb upstream primer: 5'-CAACATTCTTTAGTATTTTTTATTTCCTAT-3' (SEQ ID NO: 1),
a downstream primer of the abrus cantoniensis hance: 5'-CGCGCATGGTGGATTCACAATCC-3' (SEQ ID NO: 2);
abrus mollis upstream primer: 5'-CGCATTCTTTAGTATTGTTTATTTCGTTG-3' (SEQ ID NO: 3),
a hairy chicken bone herb downstream primer: 5'-ACCTTGAACCACTTGCCTACA-3' (SEQ ID NO: 4);
abrus upstream primer: 5'-GTTTTTGAAAGTAAAGGAGCAATATCAACAG-3' (SEQ ID NO: 5), Abrus downstream primer: 5'-ACTTGGTTACATCCGCCCTT-3' (SEQ ID NO: 6);
and (3) PCR reaction system: the total reaction volume is 25 muL, the reaction system comprises 12.5 muL of Adela 2X F8 Longfast PCR MasterMix premix, 0.75 muL of upstream and downstream primers (10 mumol/L), 1 muL of template DNA and 10 muL of sterile water for complement; replacing template DNA with equal volume of sterile water as a blank control;
reaction conditions are as follows: placing the centrifuge tube in a PCR instrument, and setting PCR reaction parameters: pre-denaturation at 95 ℃ for 5 min; the reaction was cycled 35 times (denaturation at 94 ℃ for 15 seconds, annealing at X ℃ for 15 seconds, and extension at 72 ℃ for 20 seconds); extension at 72 ℃ for 5 min; wherein, aiming at the abrus cantoniensis hance primer, X is 58; for Abrus cantoniensis Hance, X is 54; abrus primer, X is 62.
(3) Agarose electrophoresis detection: performing agarose gel electrophoresis (general rule 0541) to prepare 2% agarose gel electrophoresis, adding nucleic acid gel stain GelRed into the agarose gel, sampling 3ul of the product, and inspecting on a gel imager after the electrophoresis, wherein the sampling amount of the DNA molecular weight marker is 2.5 ul; in the gel electrophoresis picture of the test sample, at the position corresponding to the gel electrophoresis of the reference medicine: the abrus cantoniensis hance and the abrus tomentosa hance have obvious bands between 100 and 200bp, products amplified by the abrus cantoniensis hance primer have obvious bands between 200 and 300bp, and blank controls have no bands.
In the PCR identification method of the abrus herb genuine product and the abrus herb mixed counterfeit product and the abrus chinensis, the specific extraction steps of extracting the DNA of the sample by using the novel plant tissue genome DNA extraction kit in the step (1) are as follows:
adding 400 mu L of buffer solution FGA into a centrifugal tube filled with a test sample by adopting a Tiangen efficient plant genome DNA extraction kit, DP350, vortexing, shaking and mixing uniformly, and standing for 10 minutes at room temperature; adding 130 μ L buffer LP2, mixing well, vortexing and shaking for 1 min; centrifuge at 12,000rpm for 5 minutes, carefully transfer the supernatant to a new 1.5mL centrifuge tube;
adding a buffer solution LP3 with the volume of 1.5 times, immediately and fully shaking and uniformly mixing for 15 seconds; adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12,000rpm for 30 s, pouring out waste liquid, and placing an adsorption column CB3 into the collecting pipe; adding 600 μ L of rinsing liquid PW into adsorption column CB3, centrifuging at 12,000rpm for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collecting tube;
thirdly, repeating the rinsing process of the previous step; placing the adsorption column CB3 back into the collecting tube, centrifuging at 12,000rpm for 2 minutes, and pouring off 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 μ L of elution buffer TB (the TB is heated to 65 ℃ in a metal bath in advance) into the middle part of the adsorption membrane, standing for 5 minutes at room temperature, centrifuging at 12,000rpm for 2 minutes, and collecting the solution into the centrifuge tube;
fourthly, carefully suspending all the solution obtained in the centrifuge tube again and dropwise adding the solution to the middle part of an adsorption film of CB3, standing for 5 minutes at room temperature, centrifuging for 2 minutes at 12,000rpm, and collecting the solution into the centrifuge tube; the positive control template DNA solution was also prepared as described above.
The invention has the beneficial effects that:
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 abrus herb genuine product, the abrus herb mixed counterfeit product and the abrus herb can be distinguished, and the authenticity identification of the abrus herb genuine product, the abrus herb counterfeit product and the abrus herb can be effectively carried out. The invention overcomes the defects of the traditional abrus herb identification methods such as morphological identification, physicochemical identification and the like, can effectively identify the abrus herb genuine products, the false abrus herb mixed products and the abrus chinensis in the market without depending on the experience of medicinal material inspectors, can accurately judge the easily false products of the abrus herb and the abrus herb, effectively supplements and perfects the medicinal material standards of the abrus herb and the abrus herb, and is beneficial to realizing the healthy development of the abrus herb market.
The PCR identification method of the abrus herb genuine product and the false-mixed abrus herb and abrus seed thereof provided by the invention has the advantages of simplicity, practicability, stability, reliability, accurate result, low cost, low requirement on experimental operation of an identifier and strong objectivity, and can quickly and accurately identify the abrus herb genuine product and the false-mixed abrus herb and abrus seed thereof, thereby ensuring the safety of medication.
Drawings
FIG. 1 alignment of ITS2 fragments;
FIG. 2 alignment of psba-trnH;
FIG. 3 annealing temperature investigation of 54 ℃ and 56 ℃ glue maps;
FIG. 4 annealing temperature investigation of the glue pattern at 58 ℃ and 60 ℃;
FIG. 5 annealing temperature examine 62 deg.C, 64 deg.C glue patterns;
FIG. 6 is a diagram of enzyme species investigation of Abrus cantoniensis Hance primers and Abrus cantoniensis Hance primers;
FIG. 7 is a diagram of enzyme species investigation of the Abrus primer;
FIG. 8 reaction cycle number study;
FIG. 9 Adaptation of Abrus cantoniensis primer;
FIG. 10 is a study of specificity of Abrus cantoniensis Hance primers;
FIG. 11 Adaptation of Abrus cantoniensis Hance primer;
FIG. 12 is a study of specificity of Abrus cantoniensis Hance primers;
FIG. 13 Adaptation of the Abrus primer;
FIG. 14 Abrus primer specificity study (abrus herb sample);
FIG. 15 Abrus primer specificity study (Abrus mollis sample).
Detailed Description
Example 1
Because the NCBI does not have public genome information of the abrus cantoniensis hance, the abrus cantoniensis hance and the abrus, only sporadic DNA barcode sequences are available, and whether the corresponding basic source of the sequence uploaded to the NCBI is correct or not cannot be determined, samples of the abrus cantoniensis hance, the abrus cantoniensis hance and the abrus are collected, and the variety is determined through basic source identification. And (3) carrying out DNA extraction and quality inspection on the sample, amplifying and sequencing by using common DNA barcode primers such as ITS2, rbcL, psbA-trnH and the like, removing low-quality region data, merging sequencing results, introducing the sequencing results into Mega for comparison, searching for different fragment regions, and designing a specific primer for amplification and agarose electrophoresis detection.
1 materials
1.1 samples of three varieties are collected from Guangxi district, mainly from enterprise planting bases, scattered varieties in ordinary farmer's own fields and field collection. The samples were identified by the pharmacist of the yellow spring director of the Guangxi food and drug laboratory, and the certificate specimens were stored in the specimen room of the Guangxi food and drug laboratory, as shown in tables 1-3.
1.2 Instrument gradient PCR Amplifier (ABI veriti); gradient PCR amplificators (TA 96SG, jena, germany); electrophoresis apparatus (BIO-RAD); micro ultraviolet spectrophotometer (Nano Value PLUS); GelDoc XR + fully automated gel imaging System (BIO-RAD); ME203 electronic balance (Mettle); high speed refrigerated centrifuge model MIKRO220R (Hettich); vortex shaker (Scientific in-duration).
1.3 reagent agarose (BIOWEST); GelRed (BIOTIUM); PrimeSTAR Max DNA Polymerase (TAKARA), 2X F8 LongFast PCR Master Mix (Edley), 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 (tiangen); the primer is synthesized by Huada gene, and other reagents are all made in China and analyzed pure.
2 test method
2.1 extracting DNA from template, wiping the surface of the sample with 75% ethanol, drying, and pulverizing with ball mill to obtain about 20 mg of sample. DNA of a sample is extracted by adopting a novel Tiangen plant genome DNA extraction kit (DP 350), and the concentration and the purity of the obtained DNA are measured by a micro ultraviolet spectrophotometer (Nano Value PLUS). The specific extraction procedure for extracting DNA from the sample using the novel plant tissue genomic DNA extraction kit (DP 350) was as follows:
weighing about 20 mg of a dry test sample, placing the dry test sample into a 1.5mL centrifuge tube, adding 400 mu L of buffer solution FGA into the centrifuge tube filled with the test sample, uniformly mixing the solution by vortex oscillation, and placing the solution for 10 minutes at room temperature; adding 130 mu L of buffer solution LP2, fully and uniformly mixing, and vortexing and shaking for 1 minute; centrifuge at 12,000rpm for 5 minutes, carefully transfer the supernatant to a new 1.5mL centrifuge tube;
adding a buffer solution LP3 with the volume of 1.5 times, immediately and fully shaking and uniformly mixing for 15 seconds; adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed into a collecting pipe), centrifuging at 12,000rpm for 30 s, pouring out waste liquid, and placing an adsorption column CB3 into the collecting pipe; adding 600 μ L of rinsing liquid PW into adsorption column CB3, centrifuging at 12,000rpm for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collecting tube;
thirdly, repeating the rinsing process of the previous step; placing the adsorption column CB3 back into the collecting tube, centrifuging at 12,000rpm for 2 minutes, and pouring off 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 μ L of elution buffer TB (the TB is heated to 65 ℃ in a metal bath in advance) into the middle part of the adsorption membrane, standing for 5 minutes at room temperature, centrifuging at 12,000rpm for 2 minutes, and collecting the solution into the centrifuge tube;
fourthly, carefully suspending all the solution obtained in the centrifuge tube again and dropwise adding the solution to the middle part of an adsorption film of CB3, standing for 5 minutes at room temperature, centrifuging for 2 minutes at 12,000rpm, 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. The primer sequence information is as follows:
2.3 PCR amplification conditions the enzyme used for amplification was Edley 2X F8 Longfast PCR MasterMix premix, the PCR system was 25. mu.L, the premix was 12.5. mu.L, the upstream and downstream primers (10. mu. mol/L) were 0.75. mu.L each, the template DNA was 10-50ng, sterile water was added to 25. mu.L. The PCR reaction program is pre-denaturation at 95 ℃ for 5 minutes; denaturation at 94 ℃ for 15 seconds, annealing at X ℃ for 30 seconds, and extension at 72 ℃ for 30 seconds, at which stage 35 cycles were run; extension at 72 ℃ for 5 minutes. Wherein, ITS2 primer, X is 56; and the other two pairs of primers, X is 55. The resulting product was sent to a sequencing company for bidirectional sequencing.
And 2.4, obtaining the low-quality data of removing sequencing by using the differential sequence, and completing the splicing of the bidirectional sequence. And (3) bringing the spliced sequence into Mega for comparison, and adopting a Muscle algorithm with default parameters. J31-J34 is Abrus cantoniensis, J35 and J36 are Abrus mollis, and J37-J41 are Abrus cantoniensis.
As a result, the products obtained by the rbcL primers are not different; ITS2 has some differences from other but essentially discontinuous single base differences between abrus cantoniensis and abrus cantoniensis, as shown in figure 1. psba-trnH Abrus species have certain differences with others, but abrus cantoniensis and abrus cantoniensis are basically discontinuous single-base differences, but TTAT is formed from the 226 th base to the 229 th base of abrus cantoniensis products, and the 4 bases are deleted from the abrus cantoniensis. See fig. 2.
2.5 design of specific primers for the two differential primers, it is easier to design specific primers for abrus, but the difficulty of designing primers for distinguishing abrus cantoniensis hance and abrus cantoniensis hance by agarose electrophoresis detection is still high, and we select the psbA-trnH region for carrying out the detection, because the abrus cantoniensis hance and abrus cantoniensis hance have TTAT difference in the region. 6 pairs of primers are adjusted for the abrus cantoniensis hance, and as shown in a table 5, finally, the sixth pair of primers can specifically amplify the abrus cantoniensis hance but not amplify the abrus cantoniensis hance and the abrus; 5 pairs of primers are debugged for the carpet bugle, and finally, the fifth pair of primers can specifically amplify the carpet bugle but not the carpet bugle and the abrus; we have tuned 2 pairs of primers for abrin, see table 7, and finally the second pair of primers can specifically amplify abrin but not abrus cantoniensis and canthus love-pea vine.
3. Methodology validation
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 amount of sterile water was 25. mu.L. The PCR reaction program is pre-denaturation at 95 ℃ for 5 minutes; denaturation at 94 ℃ for 15 seconds, annealing at X ℃ for 15 seconds, and extension at 72 ℃ for 20 seconds, at which stage 35 cycles were run; extension at 72 ℃ for 5 minutes. Methodology verification test samples are J8 (abrus cantoniensis J), J26 (abrus cantoniensis M) and J30 (abrus X).
3.1 examination of annealing temperature Using the Edela 2X F8 Longfast PCR MasterMix enzyme, according to the above reaction system, the annealing temperatures were examined at 54 deg.C, 56 deg.C, 58 deg.C, 60 deg.C, 62 deg.C, 64 deg.C, and the test showed that X is 58 deg.C for abrus herb primer; aiming at the Abrus mollis primer, X is 54 ℃; for the acacia mangium, X is suitable for 62 ℃, obvious single amplification bands exist for target samples, and amplification bands do not exist in other negative samples, which is shown in the figure 3-5. It shows that the reactions have high specificity at the annealing temperatures of 58 ℃, 54 ℃ and 62 ℃, and the types of the tested samples can be determined according to the existence of bands of the corresponding identifying primers for amplifying template DNA.
3.2 type examination of enzymes
Because the difference area of the abrus cantoniensis hance and the abrus tomentosa is very small, a PCR system is sensitive to temperature and reagents. By trying different brands of enzymes including 2X F8 LongFast PCR Master Mix (Eldely), PrimeSTAR Max DNA Polymerase (TAKARA), KOD OneTM PCR Master Mix (Toyobo), platinuII Hot-Start Green PCR Master Mix (ThermoFisher), it was finally confirmed that three species could not be identified effectively at a prescribed annealing temperature except for the Eldely 2X F8 LongFast PCR Master Mix, and finally the Eldely 2X F8 Longt PCR Master Mix was assigned to meet the identification requirements. See FIGS. 6-7.
3.3 reaction cycle number investigation three varieties respectively investigate 30-35 cycles under respective reaction systems, and a single strip with obvious brightness can be obtained, which indicates that 30 cycles have reached amplification concentration. See fig. 8
3.4 examination of the brand of the apparatus because of the laboratory conditions, only 2 brands of PCR apparatuses, namely a gradient PCR amplification apparatus (ABI veriti) and a gradient PCR amplification apparatus (TA 96SG, Yena, Germany), were examined, and the results all gave good amplification effects, indicating that the method has no special requirements for the apparatus. See FIGS. 8-11. FIG. 8 shows a gradient PCR amplification apparatus (ABI veriti), and FIGS. 9 to 11 show a gradient PCR amplification apparatus (TA 96SG, Yena, Germany)
3.5 examining applicability and specificity, aiming at well-searched experimental conditions, verifying all collected samples and mixed artifacts, and displaying results that corresponding primer amplification target samples have strips, and other mixed artifacts have no amplification strips. The method has good adaptability and can achieve the purpose of identifying three varieties. See FIGS. 9-15.
4 results and analysis
4.1 because the abrus cantoniensis hance and the abrus cantoniensis hance are highly similar in appearance, the identification is only carried out according to the size and the hair of the leaves, the subjectivity is high, the judgment on the result is not good due to the fact that the leaves of leguminous plants are extremely easy to fall off, and most of medicinal materials are only left with branches and stems after being dried in the sun and are difficult to identify and easy to judge wrongly, so the abrus cantoniensis hance and the abrus hance are often sold as the abrus cantoniensis hance after being mixed with false. At present, the physical and chemical identification of the abrus herb and the abrus herb is mainly based on physical and chemical identification, but the chemical components of the three are basically the same, the three mainly contain alkaloid and flavonoid, the main components are abrine, schaftoside and the like, and the physical and chemical identification of thin layers, content measurement items and the like is difficult to accurately judge counterfeit products. In the experiment, specific DNA fragments are found out, specific primers are designed, and the specific fragments are amplified by a PCR (polymerase chain reaction) technology so as to distinguish three mixed counterfeit products and effectively identify the three mixed counterfeit products.
4.2 because abrus cantoniensis hance and hairy abrus herb are used as medicinal materials, large-area planting is carried out, and after investigation, most of operators can plant two varieties simultaneously, and in two adjacent fields, reproductive isolation cannot be achieved in the planting process, and the situation of hybridization easily occurs between kindred plant groups which are distributed in the same domain and have similar ecological characteristics. In the collected samples, the samples collected in the mixed planting base occasionally have the characteristic characteristics of the abrus cantoniensis hance and the abrus cantoniensis hance, namely, the leaves are very small but the hair quantity is large, which brings great difficulty to the identification of the foundation, for example, J12 and J14 have the appearance which is more similar to the abrus cantoniensis hance, but the leaves have sparser hairs but more hairs than the abrus cantoniensis hance and are not as dense as the abrus cantoniensis hance, the abrus cantoniensis hance primer can be used for expanding strips, and the abrus cantoniensis hance primer cannot expand the strips. The method does not need to rely on the experience of medicinal material inspectors, effectively supplements and perfects the medicinal material standards of the abrus herb and the hairy abrus herb, can accurately judge the easily mixed counterfeit products of the abrus herb and the hairy abrus herb, and is beneficial to realizing the healthy development of the abrus herb medicinal material market.
Example 2
A PCR identification method for Abrus cantoniensis Hance genuine products and false products thereof, Abrus mollis Hance and Abrus precatorius comprises the following steps:
(1) extracting template DNA:
washing the sample with anhydrous ethanol for 2 times, absorbing excessive water with filter paper, and air drying; grinding the dried sample into superfine powder for later use by a grinding instrument, weighing about 20 mg of the dried sample, placing the dried sample into a 1.5mL centrifuge tube, and extracting the DNA of the sample by using the novel plant tissue genome DNA extraction kit, wherein the specific extraction step is the same as that of the step 2.1 in the example 1 for extracting the DNA of the sample by using the novel plant tissue genome DNA extraction kit (DP 350).
(2) PCR amplification reaction:
identifying primer, which has corresponding specific primer aiming at both genuine product and two counterfeit products,
an abrus herb upstream primer: 5'-CAACATTCTTTAGTATTTTTTATTTCCTAT-3' (SEQ ID NO: 1),
a downstream primer of the abrus cantoniensis hance: 5'-CGCGCATGGTGGATTCACAATCC-3' (SEQ ID NO: 2);
abrus mollis upstream primer: 5'-CGCATTCTTTAGTATTGTTTATTTCGTTG-3' (SEQ ID NO: 3),
a hairy chicken bone herb downstream primer: 5'-ACCTTGAACCACTTGCCTACA-3' (SEQ ID NO: 4);
abrus upstream primer: 5'-GTTTTTGAAAGTAAAGGAGCAATATCAACAG-3' (SEQ ID NO: 5), Abrus downstream primer: 5'-ACTTGGTTACATCCGCCCTT-3' (SEQ ID NO: 6);
and (3) PCR reaction system: the total reaction volume is 25 muL, the reaction system comprises 12.5 muL of Adela 2X F8 Longfast PCR MasterMix premix, 0.75 muL of upstream and downstream primers (10 mumol/L), 1 muL of template DNA and 10 muL of sterile water for complement; replacing template DNA with equal volume of sterile water as a blank control;
reaction conditions are as follows: placing the centrifugal tube in a PCR instrument, and setting PCR reaction parameters: pre-denaturation at 95 ℃ for 5 min; the reaction was cycled 35 times (denaturation at 94 ℃ for 15 seconds, annealing at X ℃ for 15 seconds, and extension at 72 ℃ for 20 seconds); extension at 72 ℃ for 5 min; wherein, aiming at the abrus cantoniensis hance primer, X is 58; for Abrus cantoniensis Hance, X is 54; abrus primer, X is 62.
(3) Agarose electrophoresis detection: performing agarose gel electrophoresis (general rule 0541) to prepare 2% agarose gel electrophoresis, adding nucleic acid gel stain GelRed into the agarose gel, sampling 3ul of the product, and inspecting on a gel imager after the electrophoresis, wherein the sampling amount of the DNA molecular weight marker is 2.5 ul; in the gel electrophoresis picture of the test sample, at the position corresponding to the gel electrophoresis of the reference medicine: the abrus cantoniensis hance and the abrus cantoniensis hance have obvious bands within 100-200 bp, the products amplified by the abrus cantoniensis hance primer have obvious bands within 200-300 bp, and the blank control has no bands.
Sequence listing
<110> Guangxi Zhuang autonomous region food and drug inspection institute
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Claims (2)
1. A PCR identification method for abrus cantoniensis hance certified products and mixed counterfeit products thereof, namely abrus cantoniensis hance and abrus chinensis is characterized by comprising the following steps:
(1) extracting template DNA:
washing the sample with 75% ethanol/anhydrous ethanol for 2 times or wiping the surface, sucking excessive water with filter paper, and air drying; grinding into ultrafine powder for later use by a grinder, weighing about 20 mg of a dry sample to be tested, placing the dry sample into a 1.5mL centrifuge tube, and extracting DNA of a sample by using a novel plant tissue genome DNA extraction kit;
(2) PCR amplification reaction:
identifying primer, which has corresponding specific primer aiming at both genuine product and two counterfeit products,
an abrus herb upstream primer: 5'-CAACATTCTTTAGTATTTTTTATTTCCTAT-3' the flow of the air in the air conditioner,
a downstream primer of the abrus cantoniensis hance: 5'-CGCGCATGGTGGATTCACAATCC-3', respectively;
abrus mollis upstream primer: 5'-CGCATTCTTTAGTATTGTTTATTTCGTTG-3' the flow of the air in the air conditioner,
a hairy chicken bone herb downstream primer: 5'-ACCTTGAACCACTTGCCTACA-3';
abrus upstream primer: 5'-GTTTTTGAAAGTAAAGGAGCAATATCAACAG-3' the flow of the air in the air conditioner,
the acacia downstream primer: 5'-ACTTGGTTACATCCGCCCTT-3';
and (3) PCR reaction system: the total reaction volume is 25 muL, the reaction system comprises 12.5 muL of Adela 2X F8 Longfast PCR MasterMix premix, 0.75 muL of upstream and downstream primers (10 mumol/L), 1 muL of template DNA and 10 muL of sterile water for complement; replacing template DNA with equal volume of sterile water as a blank control;
reaction conditions are as follows: placing the centrifugal tube in a PCR instrument, and setting PCR reaction parameters: pre-denaturation at 95 ℃ for 5 min; the reaction was cycled 35 times (denaturation at 94 ℃ for 15 seconds, annealing at X ℃ for 15 seconds, and extension at 72 ℃ for 20 seconds); extension at 72 ℃ for 5 min; wherein, aiming at the abrus cantoniensis hance primer, X is 58; for Abrus cantoniensis Hance, X is 54; abrin primer, X is 62;
(3) agarose electrophoresis detection: performing 2% agarose gel electrophoresis (general rule 0541), adding nucleic acid gel stain GelRed, sampling 3ul of the product, and inspecting on a gel imager after electrophoresis; in the gel electrophoresis picture of the test sample, at the position corresponding to the gel electrophoresis of the reference medicine: the abrus cantoniensis hance and the abrus tomentosa hance have obvious bands between 100 and 200bp, products amplified by the abrus cantoniensis hance primer have obvious bands between 200 and 300bp, and blank controls have no bands.
2. The PCR identification method of abrus cantoniensis hance genuine products and mixed counterfeit products abrus cantoniensis hance and abrus chinensis as claimed in claim 1, wherein the specific extraction step of extracting DNA of the sample by using the novel plant tissue genome DNA extraction kit in step (1) is as follows:
adding 400 mu L of buffer solution FGA into a centrifugal tube filled with a test sample by adopting a Tiangen efficient plant genome DNA extraction kit, DP350, vortexing, shaking and mixing uniformly, and standing for 10 minutes at room temperature; adding 130 μ L buffer LP2, mixing well, vortexing and shaking for 1 min; centrifuge at 12,000rpm for 5 minutes, carefully transfer the supernatant to a new 1.5mL centrifuge tube;
adding a buffer solution LP3 with the volume of 1.5 times, immediately and fully shaking and uniformly mixing for 15 seconds; adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, centrifuging at 12,000rpm for 30 s, pouring out waste liquid, and placing the adsorption column CB3 into a collecting pipe; adding 600 μ L of rinsing liquid PW into adsorption column CB3, centrifuging at 12,000rpm for 30 s, pouring off waste liquid, and placing adsorption column CB3 into a collecting tube;
thirdly, repeating the rinsing process of the previous step; placing the adsorption column CB3 back into the collecting tube, centrifuging at 12,000rpm for 2 minutes, and pouring off 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 into the middle part of the adsorption membrane, heating the TB to 65 ℃ in a metal bath in advance, standing for 5 minutes at room temperature, centrifuging for 2 minutes at 12,000rpm, and collecting the solution into the centrifuge tube;
fourthly, carefully suspending all the solution obtained in the centrifuge tube again and dropwise adding the solution to the middle part of an adsorption film of CB3, standing for 5 minutes at room temperature, centrifuging for 2 minutes at 12,000rpm, and collecting the solution into the centrifuge tube; the positive control template DNA solution was also prepared as described above.
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