CN114032328B - Huaihu PCR identification kit and application - Google Patents

Huaihu PCR identification kit and application Download PDF

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CN114032328B
CN114032328B CN202111417770.2A CN202111417770A CN114032328B CN 114032328 B CN114032328 B CN 114032328B CN 202111417770 A CN202111417770 A CN 202111417770A CN 114032328 B CN114032328 B CN 114032328B
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CN114032328A (en
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李敏
韩笑杰
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Zhejiang University of Technology ZJUT
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    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]
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Abstract

The invention discloses a feverfew PCR identification kit and application thereof, wherein specific primers in the kit are HJ856-1 and HJ856-2.HJ856-1:5'-GAACCAACGACTAGAGCCCA-3', HJ856-2:5'-AGTGTGTGTTCTGGTGTGTGAA-3'. The method for rapidly and accurately identifying the Huai chrysanthemum can be widely applied to variety identification in various links of seed selection and breeding, planting, harvesting and storing, selling and clinical application of the Huai chrysanthemum traditional Chinese medicine, and has great significance in identifying the authenticity of the traditional Chinese medicine, striking the counterfeit products, strengthening the quality supervision of the traditional Chinese medicine and promoting the modernization development of the traditional Chinese medicine.

Description

Huaihu PCR identification kit and application
Field of the art
The invention relates to a DNA molecular identification method, in particular to a traditional Chinese medicine feverfew PCR identification kit, a specific primer, a template and an identification method.
(II) background art
The flos Chrysanthemi is a dry head-like inflorescence of Chrysanthemum (Chrysanthemum morifolium Ramat.) belonging to Compositae, and the land in the road is Zhejiang tung country, henan Jiang, anhui Chuzhou, anhui Bozhou, anhui Cheng county, etc. The medicated chrysanthemum comprises flos Chrysanthemi, bo-chrysanthemum, and flos Chrysanthemi. The flos Chrysanthemi contains volatile oil, chrysanthemin, adenine, choline, amino acid, flavonoid, phenolic acid, and locust element, has antibacterial, antiviral, antiinflammatory, blood pressure lowering, wind dispelling, heat clearing away, liver suppressing, and eyesight improving effects, and can be used for treating wind-heat type common cold, headache, giddiness, conjunctival congestion, swelling and pain, dim eyesight, etc. Different chrysanthemum varieties have larger difference of effective components and drug effects. Because the chrysanthemum has more varieties, various sources and similar appearance, the appearance is difficult to distinguish from the traditional Chinese medicine detection method, and a novel identification method needs to be established.
The PCR detection method provided by the invention is a DNA molecular identification method of the Huai chrysanthemum, and can accurately identify the Huai chrysanthemum. The identification of the Huaihu by using a PCR detection method has not been reported yet. The establishment of the method has important significance for realizing modernization of traditional Chinese medicines and standardized management of planting, picking and selling of the medicinal chrysanthemums.
(III) summary of the invention
The invention aims to provide a PCR identification kit and a detection method capable of accurately identifying traditional Chinese medicine chrysanthemum, which comprise detection steps of a PCR detection key reagent PCR primer, a template and a PCR detection method, and solve the problem that the traditional Chinese medicine chrysanthemum is difficult to identify.
The technical scheme adopted by the invention is as follows:
the invention provides a feverfew PCR identification kit, wherein specific primers in the kit are HJ856-1 and HJ856-2:
HJ856-1:5’-GAACCAACGACTAGAGCCCA-3’(SEQ ID NO.2);
HJ856-2:5’-AGTGTGTGTTCTGGTGTGTGAA-3’(SEQ ID NO.3)。
further, the components of the said Huai chrysanthemum PCR identification kit are:
the invention also relates to application of the Huai chrysanthemum PCR identification kit in identifying Huai chrysanthemum, and the application method comprises the following steps:
(1) Template DNA extraction and quality identification: extracting sample DNA by a conventional extraction method to serve as a PCR template; detecting the quality of the template DNA by an electrophoresis method (the detection method is the same as the step (3)), wherein the strips are clear and non-dispersive, namely the template DNA conforming to the PCR reaction;
(2) And (3) PCR identification: PCR amplification was performed using specific primers HJ856-1 and HJ856-2 in the Huai chrysanthemum PCR identification kit:
PCR reaction solution:
ddH 2 o was added to 20. Mu.L;
the PCR reaction solution is put into a PCR amplification instrument for PCR amplification reaction, and the reaction procedure is set as follows: pre-denaturation at 93 ℃ for 3min, denaturation at 93 ℃ for 1min, renaturation at 58-62 ℃ for 1min, extension at 72 ℃ for 2min,40 cycles, and extension at 72 ℃ for 10min;
(3) And (3) carrying out electrophoresis detection on the PCR products: 10 mu L of PCR amplification product (i.e. reaction solution after the amplification reaction) is taken out and mixed with 1 mu L of sample adding buffer solution, the amplification result is detected by 1.2% agarose gel electrophoresis (containing 0.5% ethidium bromide), the amplified band appears at 459bp to be Huai chrysanthemum, and the amplified band does not appear at 459bp to be Huai chrysanthemum.
Compared with the prior art, the invention has the beneficial effects that: (1) According to gel electrophoresis, whether amplification bands exist at 459bp or not is taken as a basis for identifying the Huai chrysanthemum, a single or mixed Huai chrysanthemum DNA sample can be detected, and the detection is accurate, the sensitivity is high, the operation is simple, the time consumption is short, and the repeatability is good; (2) The feverfew PCR identification kit can be manufactured by utilizing primers HJ856-1 and HJ856-2 in the feverfew PCR identification kit and a PCR reaction solution formula; (3) The method for rapidly and accurately identifying the Huai chrysanthemum can be widely applied to variety identification in various links of seed selection and breeding, planting, harvesting and storing, selling and clinical application of the Huai chrysanthemum traditional Chinese medicine, and has great significance in identifying the authenticity of the traditional Chinese medicine, striking the counterfeit products, strengthening the quality supervision of the traditional Chinese medicine and promoting the modernization development of the traditional Chinese medicine.
(IV) description of the drawings
FIG. 1 is an electrophoresis chart of 12 samples identified by a PCR detection method, wherein a lane M is a DNA molecular weight Marker; lane 1-Hangzhou white chrysanthemum (Zhejiang tung country); 2-Huai chrysanthemum (Henan Jiang); 3-Chuzhou chrysanthemum (Anhui Chuzhou); 4-Bo chrysanthemum (Bo state of Anhui); 5-wild chrysanthemum; 6-dandelion; 7-Zhejiang atractylodes macrocephala; 8-An Baishu; 9-Zhejiang white peony root; 10-An Baique; 11-mountain white peony root; 12-Sichuan white peony root.
FIG. 2 shows the electrophoresis patterns of different concentrations of Huai-ju identified by PCR, lane M is Marker, lane 1 is 40 ng/. Mu.L, lane 2 is 20 ng/. Mu.L, lane 3 is 4 ng/. Mu.L, and lane 4 is 2 ng/. Mu.L.
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
the raw materials used in the embodiment of the invention are all collected and prepared according to the 2015 edition of Chinese pharmacopoeia.
Example 1
1. Acquisition of key reagent PCR primer for Huaihu PCR detection method
Genomic DNA was extracted from Hangzhou white chrysanthemum (Zhejiang China), huai chrysanthemum (Henan Jiang, chuzhou), chuzhou chrysanthemum (Anhui Chuzhou) and Bo chrysanthemum (Anhui Bozhou) respectively using a plant DNA rapid extraction kit (Shanghai Biotechnology Co., ltd.). Using 5000 random primers, carrying out RAPD analysis on four kinds of medicinal chrysanthemums by using a RAPD amplification method, screening to obtain a specific amplification strip of the chrysanthemums, wherein the specific amplification strip only appears in the chrysanthemums, and does not appear in other medicinal chrysanthemums, and the DNA molecules in the strip are the chrysanthemums specific DNA molecular markers. Huai Jute specific DNA molecular markers are recovered from agarose gel, cloned and sequenced, and the sequencing result is shown in SEQ ID NO.1 and named HJ856.
A pair of specific primers HJ856-1 and HJ856-2 were designed based on the HJ856 sequence, the primer sequences being
HJ856-1:5’-GAACCAACGACTAGAGCCCA -3’;
HJ856-2:5’-AGTGTGTGTTCTGGTGTGTGAA-3’。
The positions selected by the upstream primer and the downstream primer correspond to 265-284bp and 702-723bp on the position of the HJ856 sequence respectively.
SEQ ID NO.1 is:
TTATACGAAGTAATTGTTTGCATATGAGTACAATGAATACATTAAAACAAACAGAAAATAAAAGTTCATATATAACTATGAACACATGTTTAACTTTATAATTATGAGTATTGGTAAATTATGATGATATTGGAAAATTAAGTTGTAATCCGTAAAGAAAACCATATACCAATCTTCGTATTGATTAAAACATATGAAGAAATTTCTTGTAAACAATGTTGGTGGTGCAAATTGTTTAGTTACTGATAGTGTATGGGAGTTCTAGAACCAACGACTAGAGCCCATACACTCAAAACAGGCCCATAACGGGCAGAAATAATCACACTTTTCCTGGGAAGTAAGCTTTTAGGCTTGATGACAGACGAACCAAGCTCAGATAACGCATCTAGGCAAAAGCCAGACTGGGACCTTATCCCAAAAGGCCGTTACAACAGCAAACACATGTCACTACCTTATTAGCTTAGAGGTAATGTTGCGAACATATCGCTAATATATGCATGGAACATCTCATGTACTAAGCAGAGCCAAGAAGGCCACGACACATACATTTTTGTCTCCTCCTTTGGATGTCCACCAACTGGACCATCCTAAACCATCATATCGTGTTCATGCGTGACACAAAACTATAAATGTATCTCTATCAATAGAAGTTCATTTATACAAAATGGACCAAGTATGATTCAATGTGAAGCTAATTAATCTTCACACACCAGAACACACACTTCATAGTCGTATGTGAGTGTTACCCCACCTCTTAATCCTTGATCATAACTTGAGGTCCTTTCTGAATGTGGGAAATCGGACATACGATTATCCTTACCCAAATCACACCCTTACGTATCATACGTAAACTTGG。
2. PCR detection method of Huaihu
(1) Extracting sample DNA by a conventional extraction method to be used as a template of PCR; detecting the quality of the template DNA by an electrophoresis method (step (3)), wherein the bands are clear and non-dispersive, namely the template DNA conforming to the PCR reaction;
(2) Primers HJ856-1 and HJ856-2 were synthesized using an in vitro synthesizer according to the sequences of HJ856-1 and HJ856-2. PCR amplification was performed using HJ856-1 and HJ856-2 as primers.
The PCR reaction solution was prepared according to the following formulation:
ddH 2 o was added to 20. Mu.L.
The PCR reaction solution is put into a PCR amplification instrument for PCR amplification reaction, and the reaction procedure is set as follows: pre-denaturation at 93 ℃ for 3min, denaturation at 93 ℃ for 1min, renaturation at 58-62 ℃ for 1min, extension at 72 ℃ for 2min,40 cycles, and extension at 72 ℃ for 10min;
(3) And (3) carrying out electrophoresis detection on the PCR products: taking out 10 mu L of the reaction solution after PCR amplification, mixing with 1 mu L of a loading buffer solution, detecting the amplification result by 1.2% agarose gel electrophoresis (containing 0.5% ethidium bromide), wherein the sample with the amplified band at 459bp is Huai chrysanthemum, and the sample without the band at 459bp is not Huai chrysanthemum.
Example 2:
1. accuracy study of Huai chrysanthemum PCR detection method
Genomic DNA is extracted from flos Chrysanthemi (Zhejiang tung country), flos Chrysanthemi (Henan Jiang), flos Chrysanthemi (Chuzhou of Anhui), flos Chrysanthemi (Bo of Anhui), flos Chrysanthemi Indici, herba Taraxaci, atractylodis rhizoma, radix Paeoniae alba, an Baique, radix Paeoniae alba and radix Paeoniae alba. The above 12 parts of genomic DNA are used as templates, and PCR amplification is performed by using specific primers HJ856-1/HJ856-2, the PCR reaction solution, the amplification procedure and the detection method are the same as those of example 1, except that the DNA templates are replaced to verify the accuracy and the reliability of the PCR detection method of the white chrysanthemum, the results are shown in FIG. 1, lanes 1 to 12 are respectively Hangzhou white chrysanthemum (Zhejiang China), huai chrysanthemum (Henan Jiang), chuzhou chrysanthemum (Anhui Chuzhou), bo chrysanthemum (Anhui Bo), wild chrysanthemum, dandelion, zhejiang atractylodes, anzhu, zhejiang white peony, an Baique, mountain white peony root and Chuan white peony root, and lane M is marker.
FIG. 1 shows that the Huai chrysanthemum has a specific amplified band at 459bp, while other varieties have no amplified band. Experiments prove that the method for detecting the Huai chrysanthemum by PCR can be used for identifying the Huai chrysanthemum, and has the advantages of high accuracy, simplicity, high efficiency and good repeatability.
2. Sensitivity study of Huai chrysanthemum PCR detection method
The concentration of the DNA of the Huai chrysanthemum template (40 ng/. Mu.L) is diluted by four gradients, four Huai chrysanthemum genome DNA solutions with different concentrations are prepared: one portion of the solution has a concentration of 40 ng/. Mu.L; one portion was diluted to 20 ng/. Mu.L; one portion was diluted to 4 ng/. Mu.L; one portion was diluted to 2 ng/. Mu.L.
The four different concentrations of the Huai chrysanthemum genome DNA are respectively used as templates (1.0 mu L), the HJ856-1/HJ856-2 is used as a primer for PCR amplification, the PCR reaction liquid, the amplification program and the detection method are the same as those of example 1, the result is shown in the figure 2, the lane M is a Marker, the lane 1 is 40 ng/. Mu.L, the lane 2 is 20 ng/. Mu.L, the lane 3 is 4 ng/. Mu.L, and the lane 4 is 2 ng/. Mu.L.
The result shows that when the concentration of the genome DNA of the Huai chrysanthemum is 5% of the standard concentration, namely the content is 2 ng/. Mu.L, the specific band of the Huai chrysanthemum still appears at the 459bp position, and the result is stable after repeated verification, so that the Huai chrysanthemum PCR detection method provided by the invention has very high sensitivity and can be identified by the feeble Huai chrysanthemum.
Sequence listing
<110> Zhejiang university of industry
<120> Huaihu PCR identification kit and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 856
<212> DNA
<213> Chrysanthemum (Chrysanthemum morifolium Ramat)
<400> 1
ttatacgaag taattgtttg catatgagta caatgaatac attaaaacaa acagaaaata 60
aaagttcata tataactatg aacacatgtt taactttata attatgagta ttggtaaatt 120
atgatgatat tggaaaatta agttgtaatc cgtaaagaaa accatatacc aatcttcgta 180
ttgattaaaa catatgaaga aatttcttgt aaacaatgtt ggtggtgcaa attgtttagt 240
tactgatagt gtatgggagt tctagaacca acgactagag cccatacact caaaacaggc 300
ccataacggg cagaaataat cacacttttc ctgggaagta agcttttagg cttgatgaca 360
gacgaaccaa gctcagataa cgcatctagg caaaagccag actgggacct tatcccaaaa 420
ggccgttaca acagcaaaca catgtcacta ccttattagc ttagaggtaa tgttgcgaac 480
atatcgctaa tatatgcatg gaacatctca tgtactaagc agagccaaga aggccacgac 540
acatacattt ttgtctcctc ctttggatgt ccaccaactg gaccatccta aaccatcata 600
tcgtgttcat gcgtgacaca aaactataaa tgtatctcta tcaatagaag ttcatttata 660
caaaatggac caagtatgat tcaatgtgaa gctaattaat cttcacacac cagaacacac 720
acttcatagt cgtatgtgag tgttacccca cctcttaatc cttgatcata acttgaggtc 780
ctttctgaat gtgggaaatc ggacatacga ttatccttac ccaaatcaca cccttacgta 840
tcatacgtaa acttgg 856
<210> 2
<211> 20
<212> DNA
<213> Unknown (Unknown)
<400> 2
gaaccaacga ctagagccca 20
<210> 3
<211> 22
<212> DNA
<213> Unknown (Unknown)
<400> 3
agtgtgtgtt ctggtgtgtg aa 22

Claims (4)

1. The feverfew PCR identification kit is characterized in that specific primers in the kit are HJ856-1 and HJ856-2:
HJ856-1:5’-GAACCAACGACTAGAGCCCA-3’;
HJ856-2:5’-AGTGTGTGTTCTGGTGTGTGAA-3’。
2. the pocket-chrysanthemum PCR identification kit as set forth in claim 1, wherein the pocket-chrysanthemum PCR identification kit comprises:
3. use of the feverfew PCR identification kit of claim 1 for identifying feverfew.
4. The application according to claim 3, characterized in that the method of application is:
(1) Extraction of template DNA: extracting sample DNA as PCR template;
(2) And (3) PCR identification: PCR amplification was performed using specific primers HJ856-1 and HJ856-2 in the Huai chrysanthemum PCR identification kit:
PCR reaction solution:
the PCR reaction solution is put into a PCR amplification instrument for PCR amplification reaction, and the reaction procedure is set as follows: pre-denaturation at 93 ℃ for 3min, denaturation at 93 ℃ for 1min, renaturation at 58-62 ℃ for 1min, extension at 72 ℃ for 2min,40 cycles, and extension at 72 ℃ for 10min;
(3) And (3) electrophoresis detection of PCR products: 10. Mu.L of the PCR amplification product was taken out and mixed with 1. Mu.L of the loading buffer, and the amplification result was detected by 1.2% agarose gel electrophoresis, and the amplified band was found to be pocket-chrysanthemum at 459 bp.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104531838A (en) * 2014-11-27 2015-04-22 浙江工业大学 Rhizoma atractylodis macrocephalae PCR identification kit and identification method thereof
CN104774948A (en) * 2015-04-16 2015-07-15 浙江工业大学 PCR (Polymerase chain reaction) identification kit and identification method for largehead atractylode in Hebei
CN107630102A (en) * 2017-08-31 2018-01-26 浙江工业大学 Pacify root of herbaceous peony PCR identification kits and authentication method
CN108588260A (en) * 2018-05-15 2018-09-28 浙江工业大学 Osmanthus Radix Curcumae PCR identification kits and identification method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104531838A (en) * 2014-11-27 2015-04-22 浙江工业大学 Rhizoma atractylodis macrocephalae PCR identification kit and identification method thereof
CN104774948A (en) * 2015-04-16 2015-07-15 浙江工业大学 PCR (Polymerase chain reaction) identification kit and identification method for largehead atractylode in Hebei
CN107630102A (en) * 2017-08-31 2018-01-26 浙江工业大学 Pacify root of herbaceous peony PCR identification kits and authentication method
CN108588260A (en) * 2018-05-15 2018-09-28 浙江工业大学 Osmanthus Radix Curcumae PCR identification kits and identification method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
怀菊转录组中SSR位点信息分析;邢冰等;江苏农业科学;第48卷(第11期);第57-60页 *

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