CN108977571B - Fluorescent PCR detection kit for identifying four medicinal plants in Araceae and application thereof - Google Patents
Fluorescent PCR detection kit for identifying four medicinal plants in Araceae and application thereof Download PDFInfo
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Abstract
The invention discloses a fluorescence PCR detection kit for identifying four medicinal plants (rhizoma arisaematis, pinellia ternate, tuber of tiger palm and rhizoma pinelliae) in Araceae and application thereof. A detection kit for identifying four medicinal plants in Araceae comprises a rhizoma arisaematis specific primer and a specific probe, a pinellia genus universal specific primer, a pinellia ternate specific probe, a tiger palm specific probe, a rhizoma pinelliae preparata specific primer and a specific probe, an internal standard quality control specific primer and a specific probe, wherein the nucleotide sequence of the internal standard quality control specific primer and the specific probe is shown as SEQ ID No. 1-13. The invention matches the specific fluorescent probe with the template, has high specificity and specificity, high amplification efficiency, high sensitivity, high accuracy, good reproducibility and short detection period, can complete detection within 1.5 hours, can detect DNA amplification reaction in real time, has high feasibility and application prospect, and can provide a scientific and reliable method for solving the phenomenon of disordered use of the celestial family plants in the traditional Chinese medicine market.
Description
Technical Field
The invention relates to a detection method, in particular to a fluorescence PCR detection kit for simultaneously identifying four medicinal plants (rhizoma arisaematis, pinellia ternate, tuber of tiger palm and rhizoma pinelliae) in Araceae and application thereof, belonging to the technical field of molecular biology.
Background
The plant of Araceae contains more than ten effective chemical components such as alkaloids, glycosides, amino acids, sterols, flavonoids and lectins, and has important medicinal value. Rhizoma arisaematis, rhizoma Pinelliae, radix Polygoni Cuspidati, and rhizoma Pinelliae Cordatae are plants of Araceae, and their dried tubers have certain medicinal effects. Wherein the rhizoma arisaematis and the pinellia ternate are recorded in the 2015 edition of Chinese pharmacopoeia, while the tiger palm and the rhizoma pinelliae dehydrate cannot be used as the basic source plants of the rhizoma arisaematis and the rhizoma pinelliae.
Arisaema cum bile is a plant of the genus Araceae, called Arisaema cum bile ARISAEMATIS RHIZOMA as a medicine, and is a dried tuber of Arisaema erubes (Wall.) Schott, Arisaema heterophyllum Bl. or Arisaema amurense Maxim. The processed product is rhizoma arisaematis preparata. Rhizoma arisaematis has a long medicinal history, has the effects of resolving masses and relieving swelling, and is externally used for treating carbuncle swelling and snake and insect bites. Pinellia ternate PINELLIAE RHIZOMA is a dried tuber of the genus Pinellia ternate (Thunb.) breit. The main functions are drying dampness and resolving phlegm, lowering adverse qi and preventing vomiting, relieving distension and fullness and dissipating mass. According to the difference of processing, it can be divided into Fa ban Xia, Jiang ban Xia and Qing ban Xia, and its effects are also different. Fa ban Xia is good at drying dampness and strengthening spleen, Qing ban Xia is good at resolving phlegm, and Jiang ban Xia is good at checking adverse rise of qi and relieving vomiting. The rhizoma Polygoni Cuspidati is dried tuber of Pinellia Pedatisecta Schott of Pinellia, and rhizoma Pinelliae Cordatae is dried tuber of Typhonium flagelliforme (Lodd.) BI. of Balanophora Linnaeus of Absidia.
Although the four plants belong to the Araceae and have certain medicinal value, the four medicinal materials have differences in appearance, content of active ingredients and medicinal effect, so that the four medicinal materials cannot be used instead. However, in the traditional Chinese medicine market, tiger paw is often mixed with rhizoma arisaematis or pinellia ternate for seed mistake or for pattern profit. The price difference of the three is large, the drug effect is different, the rights and interests of consumers and the body health are damaged, and the market order of the traditional Chinese medicinal materials is disturbed. The rhizoma pinelliae praeparata is confused and replaced with rhizoma pinelliae in the processes of selling and using the rhizoma pinelliae praeparata from medicinal materials to decoction pieces. It is worth noting that the rhizoma arisaematis, the pinellia ternate, the rhizoma polygoni multiflori and the rhizoma pinelliae preparata have toxicity, and the toxicity of the rhizoma polygoni multiflori and the rhizoma pinelliae preparata is higher than that of the rhizoma arisaematis and the rhizoma pinelliae, so that the rhizoma polygoni multiflori and the rhizoma pinelliae preparata are mistakenly used as the rhizoma arisaematis and the rhizoma pinelliae preparata, and an accident that the existing dosage is too large and the life is threatened can be caused. Therefore, the market of the traditional Chinese medicinal materials has urgent need for standard and scientific identification of the medicinal materials in the Araceae.
The conventional medicinal material identification methods comprise tissue identification, microscopic identification, spectral identification, chromatographic identification, molecular biology technical identification and the like, but the methods have the problems of sensory detection limitation, low accuracy, high instrument matching cost, complex identification operation and the like. In recent years, the sensitivity, specificity and accuracy of real-time fluorescence PCR detection are consistently recognized in many research fields. The technology is applied to the identification of the four disordered pharmaceutical plants in the Araceae in the market, and can provide scientific basis and powerful guarantee for maintaining the rights and interests of consumers and the market order of the traditional Chinese medicinal materials.
Disclosure of Invention
Aiming at the problems, the invention provides a fluorescence PCR detection kit for identifying four medicinal plants in the Araceae and application thereof. The kit has high specificity and specificity, high amplification efficiency, high sensitivity, high accuracy, good reproducibility and short detection period, can detect the DNA amplification reaction in real time, and has high feasibility and application prospect.
In order to achieve the purpose, the invention adopts the following technical scheme: a fluorescence PCR detection kit for identifying four medicinal plants of Araceae comprises rhizoma arisaematis specific primer and specific probe, rhizoma Pinelliae Universal specific primer, rhizoma Pinelliae specific probe, radix Damnacanthi specific probe, rhizoma Pinelliae Cordatae specific primer and specific probe, internal standard quality control specific primer and specific probe, wherein,
rhizoma arisaematis specific upstream primer TNXF: 5'-AAGTGCGGGGTGAGGGAT-3' (SEQ ID NO. 1);
rhizoma arisaematis specific downstream primer TNXR: 5'-CGATCAGCCCATCCTTGC-3' (SEQ ID NO. 2);
rhizoma arisaematis specific probe TNXP: 5' -X1-CCTGCCGGGCGATTAACGGCT-Y1-3’(SEQ ID NO.3)；
Pinellia genus universal upstream primer BXUF: 5'-AGTGGTGGACGACGCTCA-3' (SEQ ID NO. 4);
pinellia genus universal downstream primer BXUR: 5'-TTTTCCTCGCTTATTTATATGCTT-3' (SEQ ID NO. 5);
pinellia ternata specific probe BXP: 5' -X2-ACCGCGAAGAACCCAGCCGT-Y2-3’(SEQ ID NO.6)；
Tiger palm specific probe HZP: 5' -X3-CCGTGAAGAACCCAGTCGTCCG-Y3-3’(SEQ ID NO.7)；
Rhizoma pinelliae preparata specific upstream primer SBXF: 5'-CCCCCACACTCGTCCTATG-3' (SEQ ID NO. 8);
rhizoma pinelliae preparata specific downstream primer SBXR: 5'-CGATGGTCGGGTTCCTCA-3' (SEQ ID NO. 9);
rhizoma typhonii flagelliformis specific probe SBXP: 5' -X4-TTGTGCACGGGCGTCATCCA-Y4-3’(SEQ ID NO.10)；
Internal standard plasmid specific upstream primer IMF: 5'-ACCGTTACCGAGTCCAGGTG-3' (SEQ ID NO. 11);
internal standard plasmid specific downstream primer IMR: 5'-TTCGGACTCGATCAGAGCAC-3' (SEQ ID NO. 12);
internal standard quality control specific probe IMP: 5' -X5-AAGTACGCTCCATTGGTGACCTCA-Y5-3’(SEQ IDNO.13)。
The 5' end of the five specific probe sequences is modified with a reporter group X1～X5The 3' end is modified with a quenching group Y1～Y5The reporter group can be any one of FAM, JOE, TAMRA, ROX, CY3 and CY5, and the quencher group can be any one of Dabcyl, BHQ1 and BHQ 2; wherein X1～X5All are different.
Preferably, the arisaema tuber specific probe TNXP: 5 '-ROX-CCTGCCGGGCGATTAACGGCT-BHQ 1-3';
preferably, the pinellia ternate specific probe BXP: 5 '-JOE-ACCGCGAAGAACCCAGCCGT-BHQ 2-3';
preferably, the tiger palm specific probe HZP: 5 '-CY 3-CCGTGAAGAACCCAGTCGTCCG-BHQ 2-3';
preferably, the rhizoma pinelliae preparata specific probe SBXP: 5 '-FAM-TTGTGCACGGGCGTCATCCA-BHQ 1-3';
internal standard quality control specific probe IMP: 5 '-CY 5-AAGTACGCTCCATTGGTGACCTCA-BHQ 2-3'.
Furthermore, the final concentration of each primer is 0.1-0.5 mu M, and the final concentration of each probe is 0.05-0.25 mu M.
Further, the fluorescence PCR detection kit of the invention also comprises: 2 XTaqMan Master Mix, internal standard plasmid CK, DNA template and double distilled water.
Further preferably, in the fluorescence PCR detection kit, the 20 μ L PCR amplification system is: 2 XTaqMan MasterMix, the final concentration of each primer is 0.1-0.5 mu M, the final concentration of each probe is 0.05-0.25 mu M, and the final concentration of internal standard plasmid CK is 10-4ng/. mu.L, 2. mu.L of DNA template of 0.5-50 ng/. mu.L, and double distilled water to make up 20. mu.L, and the preparation method is shown in Table 1.
TABLE 1 PCR reaction amplification System
Remarking: the primer set and probe composition concentrations refer to the concentration of each primer and each probe being such concentrations.
Further, the fluorescence PCR detection kit also comprises a rhizoma arisaematis positive control substance, a pinellia ternate positive control substance, a tiger palm positive control substance, a rhizoma pinelliae preparata positive control substance, a negative control substance and a blank control substance.
The invention also provides a detection method for identifying four medicinal plants in the Araceae family, which is characterized by comprising the following specific steps of:
1) extracting template DNA of a sample to be detected;
2) PCR amplification
The PCR amplification is carried out by using the fluorescent PCR detection kit, and the amplification procedure is carried out on a fluorescent quantitative PCR instrument with 5 channels or any type of more than 5 channels: at 95 ℃ for 2 min; 95 ℃ for 10 s; collecting fluorescence signals at the temperature of 58 ℃ for 35s, performing 40 cycles, and correspondingly adjusting the marked fluorescence number according to different requirements of PCR instruments of different models;
3) setting positive control, negative control and blank control, analyzing the experimental result, giving out the fluorescence increase value delta Rn and the Ct value of the amplification curve in the nth cycle, and determining which plant belongs to the Araceae family according to the fluorescence signals of different probes and the Ct value of the amplification curve.
Further, if the ROX and CY5 fluorescence modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is rhizoma arisaematis; if the JOE and CY5 fluorescence modification probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is pinellia ternate; if the CY3 and CY5 fluorescence modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is tiger palm; if the FAM and CY5 fluorescence modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is rhizoma pinelliae; if the FAM, JOE, ROX and CY3 fluorescence modified probes have no amplification curve but CY5 has an amplification curve, if Ct is less than or equal to 35, the sample to be detected is not one of the four Araceae plants.
The invention has the beneficial effects that: compared with the prior art, the invention uses the rhizoma arisaematis specific primer, the pinellia universal primer and the rhizoma typhonii specific primer to perform PCR amplification on the DNA extract of the sample to be detected, and uses the specific fluorescent probes of the four kinds of common plants of the Araceae to identify the four kinds of common plants of the Araceae by 4-color fluorescence. The invention matches the specific fluorescent probe with the template, has high specificity and specificity, high amplification efficiency, high sensitivity, high accuracy, good reproducibility and short detection period, can complete detection within 1.5 hours, can detect DNA amplification reaction in real time, has high feasibility and application prospect, and can provide a scientific and reliable method for solving the phenomenon of disordered use of the celestial family plants in the traditional Chinese medicine market.
Drawings
FIG. 1 is a graph showing the amplification of Arisaema consanguineum under different fluorescent-modified probes; as can be seen from the figure: when the ROX and CY5 fluorescent modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is rhizoma arisaematis;
FIG. 2 is a graph showing the amplification curves of pinellia ternata under different fluorescence-modified probes; as can be seen from the figure: when the JOE and CY5 fluorescent modification probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is pinellia ternate;
FIG. 3 is a graph showing the amplification curves of tiger's paw under different fluorescence-modified probes; as can be seen from the figure: when CY3 and CY5 fluorescence modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is tiger palm;
FIG. 4 is a graph showing the amplification curves of pinellia ternata under different fluorescence-modified probes; as can be seen from the figure: when the FAM and CY5 fluorescence modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is rhizoma pinelliae;
FIG. 5 is a graph showing the amplification curves of plants other than the four Araceae plants under different fluorescent-modified probes; as can be seen from the figure: when only the CY5 fluorescence modified probe has an amplification curve and Ct is less than or equal to 35, the sample to be detected is not four plants of the Araceae family;
FIG. 6 is a graph showing the sensitivity amplification curves of the template of Arisaema cum bile at 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng and 0.00064ng, respectively; as can be seen from the figure: the lowest detection limit is 0.0032 ng;
FIG. 7 is a graph showing the sensitivity amplification curves of pinellia ternate templates at 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng and 0.00064ng, respectively; as can be seen from the figure: the lowest detection limit is 0.0032 ng;
FIG. 8 is a graph showing the sensitivity amplification curves of 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng and 0.00064ng of tiger palm template; as can be seen from the figure: the lowest detection limit is 0.0032 ng;
FIG. 9 is a graph showing the sensitivity amplification curves of the template of pinellia ternata (processed) at 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng and 0.00064ng, respectively; as can be seen from the figure: the lowest detection limit is 0.0032 ng;
Detailed Description
The present invention will be further described with reference to the accompanying drawings and examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.
The experimental materials, reagents and instruments used in the present invention are as follows:
experimental materials: rhizoma arisaematis, rhizoma Pinelliae, radix Polygoni Cuspidati, rhizoma Pinelliae Preparatum, Bulbus Fritillariae Ussuriensis, caulis Spatholobi, bupleuri radix, semen Papaveris, stigma croci Sativi, flos Lonicerae, herba Dendrobii, Ginseng radix, radix Codonopsis and Atractylodis rhizoma.
The reagents used were: the plant DNA extraction kit, DNA molecular weight MakerDL2000, electrophoresis sample buffer solution and other PCR reaction reagents are purchased from Bao bioengineering (Dalian) Co., Ltd. The primers and probes were synthesized by Biotechnology engineering (Shanghai) Ltd. 2 × TaqMan Master Mix is DBI Bioscience brand. DNA sequencing was performed by the Biotechnology center, institute of agricultural sciences, Shandong province.
The apparatus used was: the ABI 7500 fluorescent quantitative PCR instrument is a product of ABI company, and the Takara PCR instrument is a product of Bao bioengineering (Dalian) company Limited. Model 5424D high speed centrifuge is a product of Eppendorf corporation.
Example 1
1. Extracting DNA of a rhizoma arisaematis sample, a pinellia ternate sample, a tiger palm sample, a rhizoma pinelliae preparata sample and other traditional Chinese medicine samples:
the plant DNA extraction kit is adopted for extraction, and the specific operation steps are shown in the kit specification. The purity and concentration of the extracted genomic DNA are measured by an ultraviolet spectrophotometer. The measured OD260/OD280 values are all about 1.8-1.9, and the concentration is more than 10 ng/muL, which shows that the DNA has high purity and moderate concentration and meets the PCR amplification requirement.
2. Selection of target genes and design of primers: based on the DNA barcode technology, ITS2 gene was selected as the target gene, and specific primers and specific probes were designed. The nucleotide sequences of the primers and probes are shown in Table 2.
TABLE 2 nucleotide sequences of primers and probes
PCR fluorescence detection:
a20. mu.L real-time fluorescent PCR amplification system was used, and the reaction system is shown in Table 1.
4. The PCR amplification conditions were: 2min at 95 ℃; fluorescence signals were collected at 95 ℃ for 10s, 58 ℃ for 35s, 40 cycles.
5. And (4) analyzing results: and (3) setting a rhizoma arisaematis positive control substance, a pinellia ternate positive control substance, a tiger palm positive control substance, a rhizoma typhonii positive control substance, a rhizoma pinelliae preparata positive control substance, a negative control and a blank control in each test, opening analysis software after the test is finished, analyzing the test result, giving out delta Rn (fluorescence increase value in the nth cycle) and an amplification curve Ct value, and judging whether the sample to be tested is four common plants in the Araceae according to the probe fluorescence signal and the amplification curve Ct value. The result is shown in figure 1, when the ROX and CY5 fluorescent modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is rhizoma arisaematis; FIG. 2 shows that when the JOE and CY5 fluorescence modification probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is pinellia ternate; FIG. 3 shows that when CY3 and CY5 fluorescence-modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is tiger palm; FIG. 4 shows that when the FAM and CY5 fluorescence-modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is rhizoma Pinelliae Cordatae; FIG. 5 shows that when only CY5 fluorescence modified probe has an amplification curve and Ct ≦ 35 is satisfied, it indicates that the sample to be tested is not four plants of Araceae.
Example 2 specificity verification
By using the primers and the probes designed by the invention, the total genome DNA of the plants including rhizoma arisaematis, pinellia ternate, tuber of tiger palm, rhizoma pinellinae praeparata, fritillaria ussuriensis, caulis spatholobi, radix bupleuri, poppy, saffron crocus, saffron, honeysuckle, dendrobium, ginseng, codonopsis pilosula and rhizoma atractylodis macrocephalae are respectively used as templates to perform real-time fluorescence PCR detection, and the specificity of the primers and the probes is verified. The results are shown in Table 3 and FIGS. 1-5, which indicate that the probes and primers designed in this study have strong specificity.
TABLE 3 specificity verification test
Example 3 sensitivity test
Genomic DNA of rhizoma arisaematis, pinellia ternate, rhizoma anemones Rivularis and rhizoma typhonii flagelliformis is quantified to 5 ng/mu L respectively, and diluted according to 5 multiplied by gradient, 2.0 mu L of each gradient is taken as a template amount (namely, 10ng, 2ng, 0.4ng, 0.08ng, 0.016ng, 0.0032ng and 0.00064ng) to carry out real-time fluorescence quantitative PCR detection, and the detection limit of the invention is evaluated. The results, shown in FIGS. 6-9, indicate that the quantitative detection limit of the method is 0.0032ng, which indicates that the method provided by the present invention has high sensitivity.
EXAMPLE 4 actual sample testing
20 samples are purchased from large Chinese medicinal shops and Chinese medicinal market in the south of the Jinan and are classified according to appearance forms. The multiple real-time fluorescence PCR detection method after the optimization of the kit provided by the invention is used for detecting 20 samples, wherein 8 parts of rhizoma arisaematis are detected to obtain 4 tiger palm-derived components, 4 parts of tiger palm samples are detected to obtain 2 parts of non-four plant components, and the rest parts are consistent with morphological classification results. Compared with a sequencing result, the method is verified, and the result shows that the method is completely consistent with the sequencing result, and is more accurate, reliable, sensitive and quick compared with a morphology and a liquid phase method. The statistics are shown in Table 4.
TABLE 4 actual sample test results
SEQUENCE LISTING
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Claims (8)
1. A fluorescence PCR detection kit for identifying four medicinal plants of Araceae is characterized in that the four medicinal plants of Araceae are rhizoma arisaematis, rhizoma Pinelliae, radix Damnacanthi and rhizoma Pinelliae Cordatae; the fluorescence PCR detection kit comprises a rhizoma arisaematis specific primer and a specific probe, a pinellia genus universal specific primer, a pinellia ternate specific probe, a tiger palm specific probe, a rhizoma typhonii flagelliformis specific primer and a specific probe, and an internal standard quality control specific primer and a specific probe, wherein,
rhizoma arisaematis specific upstream primer TNXF: 5'-AAGTGCGGGGTGAGGGAT-3', respectively;
rhizoma arisaematis specific downstream primer TNXR: 5'-CGATCAGCCCATCCTTGC-3', respectively;
rhizoma arisaematis specific probe TNXP: 5 '-ROX-CCTGCCGGGCGATTAACGGCT-BHQ 1-3'; pinellia genus universal upstream primer BXUF: 5'-AGTGGTGGACGACGCTCA-3', respectively;
pinellia genus universal downstream primer BXUR: 5'-TTTTCCTCGCTTATTTATATGCTT-3', respectively;
pinellia ternata specific probe BXP: 5 '-JOE-ACCGCGAAGAACCCAGCCGT-BHQ 2-3';
tiger palm specific probe HZP: 5 '-CY 3-CCGTGAAGAACCCAGTCGTCCG-BHQ 2-3';
rhizoma pinelliae preparata specific upstream primer SBXF: 5'-CCCCCACACTCGTCCTATG-3', respectively;
rhizoma pinelliae preparata specific downstream primer SBXR: 5'-CGATGGTCGGGTTCCTCA-3', respectively;
rhizoma typhonii flagelliformis specific probe SBXP: 5 '-FAM-TTGTGCACGGGCGTCATCCA-BHQ 1-3';
internal standard plasmid specific upstream primer IMF: 5'-ACCGTTACCGAGTCCAGGTG-3', respectively;
internal standard plasmid specific downstream primer IMR: 5'-TTCGGACTCGATCAGAGCAC-3', respectively;
internal standard quality control specific probe IMP: 5 '-CY 5-AAGTACGCTCCATTGGTGACCTCA-BHQ 2-3'.
2. The fluorescence PCR assay kit for identifying four pharmaceutical plants of Araceae as claimed in claim 1, wherein the final concentration of each primer is 0.1-0.5 μ M and the final concentration of each probe is 0.05-0.25 μ M.
3. The fluorescence PCR assay kit for identifying four pharmaceutical plants of Araceae as claimed in claim 2, wherein the fluorescence PCR assay kit further comprises: 2 XTaqMan Master Mix, internal standard plasmid CK, DNA template and double distilled water.
4. The fluorescence PCR assay kit for identifying four medicinal plants of Araceae as claimed in claim 3, wherein the 20 μ L PCR amplification system of the fluorescence PCR assay kit is: 2 xTaqMan Master Mix, the final concentration of each primer is 0.1-0.5 muM, the final concentration of each probe is 0.05-0.25 muM, the final concentration of internal standard plasmid CK is 10-4 ng/muL, 2 muL of DNA template of 0.5-50 ng/muL, and double distilled water is used for complementing 20 muL.
5. The fluorescence PCR detection kit for identifying four pharmaceutical plants of Araceae as claimed in any one of claims 1-4, wherein the fluorescence PCR detection kit further comprises rhizoma arisaematis positive control, rhizoma Pinelliae positive control, rhizoma anemones Rigidae positive control, rhizoma Pinelliae Cordatae positive control, negative control and blank control.
6. A detection method for identifying four medicinal plants in Araceae is characterized by comprising the following steps:
1) extracting template DNA of a sample to be detected;
2) PCR amplification
Performing PCR amplification using the fluorescent PCR detection kit of any one of claims 1-4;
3) setting positive control, negative control and blank control, analyzing the experimental result, giving out the fluorescence increase value delta Rn and the Ct value of the amplification curve in the nth cycle, and judging according to the fluorescence signals of different probes and the Ct value of the amplification curve.
7. The assay method according to claim 6, wherein the four medicinal plants of Araceae are selected from the group consisting of,
if the ROX and CY5 fluorescent modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is rhizoma arisaematis; if the JOE and CY5 fluorescence modification probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is pinellia ternate; if the CY3 and CY5 fluorescence modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is tiger palm; if the FAM and CY5 fluorescence modified probes have amplification curves and Ct is less than or equal to 35, the sample to be detected is rhizoma pinelliae; if the FAM, JOE, ROX and CY3 fluorescence modified probes have no amplification curve but CY5 has an amplification curve, if Ct is less than or equal to 35, the sample to be detected is not one of the four Araceae plants.
8. The assay method according to claim 6, wherein the PCR of step (2) is performed in a 5-channel or more fluorescence quantitative PCR instrument, and the amplification procedure comprises: at 95 ℃ for 2 min; 95 ℃ for 10 s; fluorescence signals were collected at 58 ℃ for 35s, 40 cycles.
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| CN201811025730.1A CN108977571B (en) | 2018-09-04 | 2018-09-04 | Fluorescent PCR detection kit for identifying four medicinal plants in Araceae and application thereof |
| PCT/CN2019/088465 WO2020048158A1 (en) | 2018-09-04 | 2019-05-27 | Fluorescent pcr detection kit for identifying four araceae medicinal plants and use thereof |
| ZA2020/03907A ZA202003907B (en) | 2018-09-04 | 2020-06-26 | Fluorescent pcr detection kit for identifying four araceae medicinal plants and use thereof |
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| CN110964844B (en) * | 2019-12-31 | 2023-01-03 | 星阵（广州）基因科技有限公司 | Primer, kit and method for qualitative determination of ginseng, poria cocos and bighead atractylodes rhizome powder |
| CN112626251A (en) * | 2020-12-25 | 2021-04-09 | 红塔烟草(集团)有限责任公司 | Specific sequence for identifying variety of Yunyan 87 roasted tobacco, kit and using method thereof |
| CN113493824A (en) * | 2020-12-30 | 2021-10-12 | 锦州奥鸿药业有限责任公司 | Real-time fluorescent PCR method for calf serum animal-derived component species detection |
| CN114540346B (en) * | 2022-02-28 | 2024-04-16 | 中国食品药品检定研究院 | Probe primer, fluorescence quantitative PCR detection method for fritillary bulb specificity detection and application thereof |
| CN115058530A (en) * | 2022-05-26 | 2022-09-16 | 中国疾病预防控制中心职业卫生与中毒控制所 | Nucleic acid reagent, kit and detection method for detecting celery |
| CN116790791A (en) * | 2023-06-28 | 2023-09-22 | 中国中医科学院中药研究所 | SNP molecular marker for identifying pinellia ternate and common adulterated species thereof and application thereof |
| CN117164671B (en) * | 2023-11-02 | 2024-01-02 | 山东省食品药品检验研究院 | Arisaema tuber characteristic peptide fragment and detection method and application thereof |
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