CN110885898A - Molecular specific marker primer and method for identifying amaranthus rugosus and 2 common confused products - Google Patents
Molecular specific marker primer and method for identifying amaranthus rugosus and 2 common confused products Download PDFInfo
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Abstract
The invention discloses a molecular specificity marker primer and a method for identifying amaranthus rugosus and 2 common confused products, belonging to the technical field of biology. Designing an upstream specificity forward primer according to the matK gene sequences of the amaranthus rugosus, the amaranthus caudatus and the amaranthus retroflexus: AvF: 5'-CTTTTTGTTCCTATATAATCTAG-3', AaF: 5'-CTAGTAAAAGTCAAAGTTAAGCG-3', ArF: 5'-GACTCAATTCTTCAGTAATACCA-3' and downstream universal reverse primer matk-R: 5'-TATAATAATGAGAAAGATTTCGG-3' are provided. The specific primers are utilized to respectively construct a multiplex PCR system and a fluorescent quantitative PCR reaction system, so that the rapid and accurate qualitative and quantitative identification of the amaranthus rugosus, the amaranthus caudatum and the amaranthus retroflexus is realized.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a molecular specificity marker primer and a method for identifying amaranthus rugosus and 2 common confused products.
Background
(iii) herba Amaranthi Tricoloris (a medicinal plant)Amaranthus viridisL.) herb has the actions of clearing heat and removing toxicity, inducing diuresis and alleviating painThe efficacy of (1). The amaranthus rugosus is also commonly used as a wild vegetable in folks due to the fact that the amaranthus rugosus contains rich nutritional ingredients such as protein, riboflavin, magnesium, fat, vitamin C, dietary fiber and the like. The wild plant germplasm resources are widely distributed, and no cultivated variety is formed at present. Due to Amaranthus mangostanus (A. Amaranthus mangostanus L.)Amaranthus albus L.) Amaranthus retroflexus (A. reflexus)Amaranthus retroflexusL.) has a similar shape and distribution to the amaranthus rugosus, and is often accompanied by confusion and misuse. The amaranthus Amaranthus and Amaranthus retroflexus are common confused products of amaranthus rugosus, but the amaranthus retroflexus have different attributes and medicinal values, wherein the Amaranthus retroflexus is mainly used for treating diarrhea, dysentery, swelling, pain and bleeding of hemorrhoids and the like. The Amaranthus mangostanus has the effects of clearing heat and removing toxicity, and can be used for treating sore swelling, ulcerative gingivitis and insect bite by external application. Especially white amaranth is more distinguished and often also distinguished as "wrinkled amaranth" in folk, which are easily mixed with each other. Therefore, the three are required to be accurately identified to ensure accurate medication.
Although fresh leaves of amaranthus rugosus were identified from their confounds by microscopic identification differences between the non-glandular vascular bundles of veins, appendages and leaves, amaranthus rugosus in the form of powder or crushed pieces was difficult to identify with 2 common plants by morphological and histological techniques. Over the last few years, identification of Chinese medicinal materials has been carried out by chemical analysis techniques such as TLC, HPLC, MS and the like. Although these methods may to some extent complement the limitations of morphological or histological identification, chemical fingerprinting detects only a fraction of the compounds, provides only limited species composition information, and does not allow easy and accurate identification, particularly in species identification that closely resembles it. Therefore, the selection of the proper method for identifying the purslane product is critical to monitoring quality.
With the development of molecular biology, DNA barcode technologies such as DNA molecular markers such as RAPD, ISSR, and SSR, its (internal transcribed spacer) and the like, provide reliable methods for identifying medicinal materials. However, these molecular marker techniques based on genomic DNA are susceptible to degradation of genomic DNA, and since nuclear genomic DNA has a smaller number of copies than chloroplast DNA, it is more easily destroyed in processed medicinal materials, and thus cannot be identified by their molecular marker techniques. The chloroplast matK gene sequence is used as a DNA barcode, can be easily amplified by using a universal primer, is proved to be used for identifying various plants at a variety of levels, and is widely used for detecting plant products. In earlier studies, the subject group also found that identification technology based on chloroplast DNA matK gene sequence is more stable and effective than molecular marker technology based on nuclear genome DNA for dry or processed medicinal materials.
The research is used for sequencing the matK gene sequences of the amaranthus rugosus, the amaranthus alba and the amaranthus retroflexus, analyzing the sequence structures and the characteristics of the sequences, searching for Single Nucleotide Polymorphism (SNP) molecular markers, designing specific identification primers for respectively identifying the amaranthus rugosus, the amaranthus alba and the amaranthus retroflexus, establishing a multiplex PCR (polymerase chain reaction) technology and a fluorescent quantitative PCR (polymerase chain reaction), and quickly and simply identifying the amaranthus rugosus, the amaranthus alba and the amaranthus retroflexus. The invention provides an effective method for qualitative and quantitative molecular identification of the amaranthus rugosus and 2 common confused products, and has very important significance for effectively identifying and protecting 3 traditional Chinese medicine germplasm resources.
Disclosure of Invention
The invention aims to provide a molecular specific marker primer and a method for identifying amaranthus rugosus and 2 common confused products, and the method can be used for quickly, simply and conveniently identifying the amaranthus rugosus, the amaranthus alba and the amaranthus retroflexus.
In order to achieve the purpose, the invention adopts the following technical scheme:
analyzing specific SNP sites of the matK gene sequences of the amaranthus rugosus, the amaranthus alba and the amaranthus retroflexus obtained by the early-stage sequencing experiment, and respectively designing upstream specific forward primers AvF for specifically recognizing the amaranthus rugosus: 5'-CTTTTTGTTCCTATATAATCTAG-3', respectively; an upstream specific forward primer AaF for specifically recognizing the amaranthus tricolor: 5'-CTAGTAAAAGTCAAAGTTAAGCG-3', respectively; an upstream specific forward primer ArF for specifically recognizing the amaranthus retroflexus: 5'-GACTCAATTCTTCAGTAATACCA-3', downstream universal reverse primer matk-R: 5'-TATAATAATGAGAAAGATTTCGG-3' are provided.
And (3) establishing a multiple PCR reaction system by using the specific forward primers, and qualitatively identifying and analyzing the amaranthus rugosus, the amaranthus caudatus and the amaranthus retroflexus.
The construction of the multiplex PCR system comprises the following steps: the total amount was 20. mu.L, 10 ng of genomic DNA was extracted from the plant as an amplification template, and 1. mu.L of 1 XStart TopTaq DNA polymerase (TransGen), 2. mu.L of 10 XStart TopTaq Buffer, dNTP 2.5mM, 0.2. mu.M of downstream primer matk-R (5'-TATAATAATGAGAAAGATTTCGG-3'), and 0.1. mu.M of each of the 3 specific primers AvF, AaF and ArF at the upstream, and the balance was sterile water, were added. Constructing a multiplex PCR system, and carrying out the following reaction procedures: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 30s, primer annealing at 51 ℃ for 30s and extension at 72 ℃ for 2 min, 32 cycles; the last cycle was extended for 7 min at 72 ℃ to ensure a complete extension PCR product. The amaranthus rugosus generates 629 bp specific fragments, the amaranthus alba generates 490bp specific fragments, and the amaranthus retroflexus generates 237bp specific fragments.
And establishing a fluorescent quantitative PCR reaction system by using the specific forward primers to quantitatively identify the amaranthus rugosus, the amaranthus caudatus and the amaranthus retroflexus.
The fluorescent quantitative PCR:
an upstream specific forward primer AvF and a downstream reverse primer AvR are used for specifically recognizing the amaranthus rugosus: 5'-GATATTTTAGCTTTCCGTAA-3', respectively; an upstream specific forward primer AaF and a downstream reverse primer AaR for specifically recognizing the amaranthus tricolor: 5'-GCCAGAAAGCGATAAAGTAA-3', respectively; the upstream specific forward primer ArF and the downstream reverse primer matk-R for specifically recognizing the amaranthus retroflexus are subjected to fluorescent quantitative PCR differential analysis, and the fluorescent quantitative PCR is constructed for 40 cycles, wherein the reaction system is as follows: the total amount is 20 mu L, 10 ng of genome DNA extracted from the plant is used as an amplification template, 10 mu L of 2 XSSYBR Green Real-timePCR Master Mix (TaKaRa), 0.2 mu M of upstream specific primers, 0.2 mu M of downstream primers and the balance of sterile water; the reaction procedure was as follows: in a real-time quantitative PCR instrument (ABI 7900), 50 ℃ pre-denaturation for 2 min, 95 ℃ pre-denaturation for 10 min, 95 ℃ denaturation for 15s, primer annealing for 58 ℃ for 15s and extension at 72 ℃ for 30s, 40 cycles.
Primers AvF and AvR were used for quantitative analysis of amaranthus rugosus in the mixed product; primers AaF and AaR were used for quantitative analysis of amaranth in the mixed product. Primers ArF and matk-R were used for quantitative analysis of Amaranthus retroflexus in the mixed product. All primers were at a concentration of 0.2. mu.M. For real-time quantitative PCR methodThe quantitative analysis method can be performed by a relative quantitative analysis method R =2-△△CtIt is deduced that because the total DNA of the same DNA sample is used as a template for amplification, the Ct (total) value corresponding to the total template DNA of the mixed sample is constant, i.e., the Ct (total) value in a specific mixed sample is constant. The hybridization ratio of amaranthus rugosus (Av) to amaranthus alba (Aa) in the same sample can be calculated by this formula:
RAv/Aa=2-△△Ct=2-{[Ct(Av)- Ct（total）]-[Ct(Aa)-Ct（total）]}=2Ct(Aa)-Ct(Av)
ct (total)) is the Ct value of the total mixed sample, Ct (Av) is the Ct value of the primers AvF and AvR, namely the Ct value representing the content of the amaranth, and Ct (aa) is the Ct value of the primers AaF and AaR, namely the Ct value representing the content of the amaranth. By analogy, the promiscuous ratio between the other two can be calculated.
The invention has the advantages that:
because the identification is carried out only according to the existence of certain characteristic active chemical components, the identification is easily influenced by external conditions such as plant growth environment and the like, and the accurate identification is difficult to carry out, particularly the identification of mixed samples. The method is based on the genetic background to carry out qualitative and quantitative analysis of molecules, is not influenced by external factors such as the production area of medicinal materials, and is more accurate, simpler and more convenient compared with a chemical method.
Since the chloroplast DNA has a larger copy number than the genome DNA and is more stable in the dried and processed product, the method uses the chloroplast DNA as a detection object and is more suitable for the identification of the dried and processed product than the genome DNA.
The method can perform molecular qualitative and quantitative identification on the amaranthus rugosus and 2 common confusions simultaneously, accurately determine the existence and mixing ratio of the 3 confusions, and has higher efficiency and more convenience.
Drawings
FIG. 1 shows the design of primers for identifying amaranthus rugosus, amaranthus alba and amaranthus retroflexus mutually.
FIG. 2 gel electrophoresis of products of multiplex PCR using primers matk-R, AaF, AaF, ArF. M is DNA ladder; 1-6: amaranthus rugosus; 7-12: white amaranth; 13-18: amaranthus retroflexus.
FIG. 3 Ct/x plot for primer pairs AvF, AvR.
FIG. 4 is a graph showing Ct/x curves of the primer pairs AaF and AaR.
FIG. 5 Ct/x plot for primer pairs ArF, matk-R.
Detailed Description
1 apparatus
PCR instrument (Eppendorf, model 5332), electrophoresis system (six instruments, model DYY-12, Beijing), cryocentrifuge (Eppendorf, model 5810R), gel imaging analyzer (BIO-RAD ChemiDoc XRS), micropipette (Eppendorf).
2 reagent
2 xCTAB extracting solution, 1 xTAE buffer solution, agarose (Promega company), ethidium bromide (Fluka company), TransStart TopTaq DNA Polymerase (Beijing all-purpose gold company), trichloromethane, absolute ethyl alcohol and isopropyl alcohol which are all domestic analytical purity.
3 materials
The research experiment samples are samples collected from different producing areas and identified and collected by the smart of welfare art of the Fujian traditional Chinese medicine university: 10 amaranthus rugosus, amaranthus alba and amaranthus retroflexus samples are collected from different Chinese producing areas respectively.
Method of producing a composite material
4.1 extraction of genomic DNA:
⑴ cutting 5 g of fresh wrinkle Amaranthus mangostanus, white Amaranthus mangostanus and Amaranthus retroflexus leaf, placing in a sterilized mortar, adding 0.5 g of PVP powder, adding liquid nitrogen, rapidly grinding, collecting the powder in a bag, storing at-20 deg.C, and storing at-80 deg.C for a long time;
⑵ preheating 10 mL of CTAB solution in 65 deg.C water, adding 0.2 mL (200 μ L) mercaptoethanol to the preheated CTAB (mercaptoethanol: CTAB solution =1: 50);
⑶ putting 0.2 g of the powder obtained in step (1) into a 2mL LEP tube (centrifuge tube), quickly adding 1 mL of preheated CTAB solution containing mercaptoethanol (added when the sample is at low temperature), shaking for about 2 minutes, putting into a 65 ℃ water bath kettle, water bathing for about 1 hour, (shaking once every 20 minutes, and shaking for several times if appropriate)
⑷ mu.L of chloroform-isoamyl alcohol mixture (chloroform: isoamyl alcohol volume ratio =24:1, which must be handled in a fume hood) is added to the tube, shaken for 5 minutes, and then centrifuged at 12000rpm for 10 minutes at 15-20 ℃;
⑸ collecting 600 μ L supernatant, adding 1.2mL (2 times) precooled anhydrous ethanol or 360 μ L (0.6 times) isopropanol, and standing at-20 deg.C for about 3 hr or overnight;
⑹ taking out EP tube, centrifuging at 4 deg.C at 12000 rmp for 10 min, removing supernatant, and placing on absorbent paper for several minutes;
⑺ adding 700 μ L70% ethanol, reversing the mixture several times, washing the precipitate, centrifuging at 4 deg.C at 12000 rmp for 10 min, removing supernatant, repeating the above steps, and blow-drying in a clean bench (note that excessive blow-drying cannot be achieved)
⑻ adding 30-50 μ L deionized water (ddH 2O) or 1 × TE solution into EP tube, standing at 4 deg.C for 3 hr or overnight for dissolving completely, and mixing with mixer to promote dissolution if it is difficult to dissolve by hand shaking;
⑼ it can be detected by agarose gel to determine if it contains genomic DNA, and then purified.
4.2 specific primer design:
applying ClustalX software to analyze specific SNP sites of matK gene sequences of Amaranthus rugosus (Amaranthus _ viridis, Av), Amaranthus alba (Amaranthus _ albus, Aa) and Amaranthus retroflexus (Ar) obtained by a preliminary sequencing experiment, selecting a Amaranthus rugosus specific SNP site G at the 48 th site of the sequences as an identification site, and replacing a base at the 47 th site from T to A to improve the identification capability to design a primer AvF for specifically identifying the Amaranthus rugosus; using a amaranthus specific SNP site G at the 187 th site as a discrimination site, and replacing the base at the 186 th site by C from G to design a primer AaF for specifically recognizing the amaranthus; the 440 th amaranthus retroflexus specific SNP site A is used as an identification site, and the 439 th site base is replaced by C from G, so that a primer ArF for specifically identifying the amaranthus retroflexus is designed; and designing a common reverse primer matk-R, wherein a primer design schematic diagram and the corresponding PCR amplification fragment size are shown in figure 1. 629 bp amaranthus rugosus specific fragment, 490bp amaranthus alba specific fragment and 237bp amaranthus retroflexus specific fragment are obtained by PCR amplification.
To ensure the accuracy of molecular quantitative analysis, the PCR product of fluorescent quantitative PCR must be a short fragment. Therefore, the downstream reverse primer AvR is designed to be matched with the upstream specific forward primer AvF for quantitatively analyzing the amaranthus rugosus sample, and the PCR product of the amaranthus rugosus sample is 140 bp; designing a downstream reverse primer AaR to be matched with an upstream specific forward primer AaF for quantitatively analyzing the amaranth sample, wherein the PCR product is 157 bp; the upstream specific forward primer ArF and the downstream reverse primer matk-R for specifically recognizing the amaranthus retroflexus can be directly subjected to fluorescence quantitative PCR analysis, and the PCR product is 237 bp.
The forward and reverse primers for identifying amaranthus rugosus, amaranthus alba and amaranthus retroflexus are shown in Table 1.
TABLE 1 primers
4.3 molecular qualitative identification System for establishing multiplex PCR
Constructing a multiple PCR system by using specific primers AvF, AaF and ArF of the amaranthus rugosus, the amaranthus caudatus and the amaranthus retroflexus and a general primer matk-R respectively: the total volume was 20. mu.L, 10 ng of genomic DNA was extracted from the plants as an amplification template, and 1 XStart TopTaq DNA polymerase (1. mu.L) (TransGen Biotechnology), 2. mu.L of 10 XStart TopTaqBuffer, dNTP 2.5mM, 0.2. mu.M reverse universal primer matk-R and three forward specific primers AvF, AaF, ArF each 0.1. mu.M, the remainder being sterile water were added. The reaction procedure was as follows: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 30s, primer annealing at 51 ℃ for 30s and extension at 72 ℃ for 2 min, 32 cycles; the final cycle was extended at 72 ℃ for 7 minutes to ensure complete extension PCR product.
The PCR products are shown in FIG. 2, from which it is clear that: the amaranthus rugosus produces a specific strip of 629 bp, the amaranthus rugosus produces a specific strip of 490bp, and the amaranthus retroflexus produces a specific strip of 237bp, which shows that the specific primer designed by the invention can carry out high-specificity, accurate and rapid identification on the amaranthus rugosus, the amaranthus rugosus and the amaranthus retroflexus.
4.4 molecular quantitative identification System for establishing fluorescent quantitative PCR
To determine whether this particular primer is suitable for molecular quantification of mixed samples, we performed real-time PCR assays with mixed DNA samples of amaranthus rugosus, amaranthus alba, and amaranthus retroflexus diluted in a gradient. Taking 77.5 mu g/mu L of the sample basic genome DNA to carry out continuous ten-fold dilution (1/10/100/1000/10000/100000-fold dilution), adding 2 mu L of sample DNA as a template into a reaction system with the total volume of 20 mu L, adding 10 mu L of 2 XSSYBR Green Real-time PCR Master Mix (TaKaRa), 0.2 mu M of upstream specific primers, 0.2 mu M of downstream primers and the balance of sterile water; the reaction procedure was as follows: in a real-time quantitative PCR instrument (ABI 7900), 50 ℃ pre-denaturation for 2 min, 95 ℃ pre-denaturation for 10 min, 95 ℃ denaturation for 15s, primer annealing for 58 ℃ for 15s and extension at 72 ℃ for 30s, 40 cycles. A curve is constructed relative to the Ct value of the specific DNA and is shown in figures 3, 4 and 5, and the result shows that the Ct value of the 3 pairs of specific primers has a linear relation with the logarithm of the initial template, and R2>0.99, which shows that the 3 pairs of primers can be designed to perform quantitative determination on unknown samples, therefore, the designed real-time fluorescence quantitative PCR is an effective method for performing molecular quantification on the mixed samples. The Ct values of the amplified AvF and AvR primer pairs and the logarithm values of the relative amount of the sample form a regression curve:
y = -3.1361x + 34.71，R2= 0.9993
the Ct value of the primer pair AaF and AaR amplification and the logarithm value of the relative amount of the sample form a regression curve:
y = -3.3382x + 35.761，R2= 0.9995
the Ct value of the primer pair ArF and matk-R amplification and the logarithm value of the sample relative quantity form a regression curve:
y = -3.1058x + 32.7，R2= 0.9971
since the Ct values of the 2 pairs of primers in the mixed sample are linearly related to the 5 dilution concentrations (1/10/100/100/10000/100000-fold dilution) of the DNA template, accurate quantification can be achieved within the dilution concentration range. The content proportion of chloroplast DNA of amaranthus rugosus, amaranthus alba and amaranthus retroflexus in the miscible plant of the same sample can be calculated by the method, namely the mixing ratio of the amaranthus rugosus (Av) and the amaranthus albus (Aa) in the same sample can be calculated by the formula:
RAv/Aa=2-△△Ct=2-{[Ct(Av)- Ct（total）]-[Ct(Aa)-Ct（total）]}=2Ct(Aa)-Ct(Av)the other mixing ratio between the two is analogized.
The result shows that the real-time PCR is a sufficiently sensitive and accurate method for evaluating the proportion of the chloroplast DNA content of the amaranthus rugosus, the amaranthus alba and the amaranthus retroflexus in the mixed sample, and the proportion of the amaranthus rugosus, the amaranthus alba and the amaranthus retroflexus mixed sample is evaluated according to the content. Because the degradation rate of chloroplast DNA in a specific mixed sample is the same relative to the amaranthus rugosus, the amaranthus alba and the amaranthus retroflexus, the method can effectively perform molecular quantitative analysis on the mixed sample of the amaranthus rugosus, the amaranthus alba and the amaranthus retroflexus.
TABLE 1 Ct values relative amounts of samples
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian university of traditional Chinese medicine
<120> molecular specificity marker primer and method for identifying amaranthus rugosus and 2 common confused products
<130>9
<160>9
<170>PatentIn version 3.3
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acgaatccat ttttgttatt ctacgcaagc aatcctctta tttacgatca acgtcttttg 120
gagcccttct tgaacgaatc catttttacg gaaagctaaa atatctagta aaagtcaaag 180
ttaaggtttt tggggttatc ctatggcttt tcaaagaacc ttttctgcat tatgttcggt 240
atcaaggaaa atgccttctg gcttcaaaag ggacatcctt tctgatgtat aaatggaaat 300
attactttat cgctttctgg caatgtcatt tttctgtgtg gtctcaacca agaagaatct 360
atatcaatca attatcaaac tattccctcg actttatggg ttttatttca aatgtgggac 420
tcaattcttc agtaatacga agtcaaatgt tagaaaattc atttctagta gataatatta 480
ttaagaagtt tgataccata gttccaatta ttcctctggt tggctcgttg gctaaagcga 540
aattttgtaa tggattaggt catcccatta gtaagtcggt ctggaccgat ttatccgatg 600
ctgatattat tgaccgattt ggacgtatat gccgaaatct ttctcattat tata 654
<210>4
<211>23
<212>DNA
<213>AvF
<400>4
ctttttgttc ctatataatc tag 23
<210>5
<211>23
<212>DNA
<213>AaF
<400>5
ctagtaaaag tcaaagttaa gcg 23
<210>6
<211>23
<212>DNA
<213>ArF
<400>6
gactcaattc ttcagtaata cca 23
<210>7
<211>23
<212>DNA
<213>matk-R
<400>7
tataataatg agaaagattt cgg 23
<210>8
<211>20
<212>DNA
<213>AvR
<400>8
<210>9
<211>20
<212>DNA
<213>AaR
<400>9
Claims (3)
1. The molecular specific marker primer for identifying the amaranthus rugosus and 2 common confounding products is characterized by comprising the following steps of: the primers comprise the following components:
identifying the upstream specific forward primer AvF of the amaranthus rugosus: 5'-CTTTTTGTTCCTATATAATCTAG-3', respectively;
identifying the upstream specific forward primer AaF of the amaranthus tricolor: 5'-CTAGTAAAAGTCAAAGTTAAGCG-3', respectively;
identifying an upstream specific forward primer ArF of the amaranthus retroflexus: 5'-GACTCAATTCTTCAGTAATACCA-3', respectively;
the downstream universal reverse primer matk-R: 5'-TATAATAATGAGAAAGATTTCGG-3' are provided.
2. A multiplex PCR method for identifying amaranthus rugosus and 2 common confounders by using the molecular specific marker primer of claim 1, characterized in that: the total volume of the multiplex PCR system is 20 mu L, 10 ng of genome DNA is extracted from amaranthus rugosus, amaranthus alba and amaranthus retroflexus plants and is used as an amplification template, 1 mu L of 1 × TransStart TopTaq DNA polymerase, 2 mu L of 10 × TransStart TopTaq Buffer, 2.5mM of dNTP, 0.2 mu M of downstream universal reverse primer matk-R, 3 specific upstream forward primers AvF, AaF and ArF which are 0.1 mu M respectively and the balance of sterile water are added; the reaction procedure was as follows: in a PCR instrument, pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 30s, primer annealing at 51 ℃ for 30s and extension at 72 ℃ for 2 min for 32 cycles; the final cycle was extended for 7 minutes at 72 ℃.
3. A fluorescent quantitative PCR method for identifying amaranthus rugosus and 2 common confounders by using the molecular specific marker primer of claim 1, which is characterized in that: an upstream specific forward primer AvF and a downstream reverse primer AvR which specifically recognize the amaranthus rugosus are used: 5'-GATATTTTAGCTTTCCGTAA-3', respectively; an upstream specific forward primer AaF and a downstream reverse primer AaR for specifically recognizing the amaranthus tricolor: 5'-GCCAGAAAGCGATAAAGTAA-3', respectively; performing fluorescent quantitative PCR identification analysis on an upstream specific forward primer ArF and a downstream reverse primer matk-R for specifically identifying the amaranthus retroflexus to construct fluorescent quantitative PCR;
the reaction system is as follows: the total volume is 20 mu L, 10 ng of genomic DNA extracted from plants is used as an amplification template, 10 mu L of 2 × SYBR Green Real-time PCR Master Mix, 0.2 mu M of upstream specific forward primer AvF or AaF or ArF, 0.2 mu M of downstream general reverse primer AvR or AaR or mat-R, and the balance of sterile water; the reaction procedure was as follows: in a real-time quantitative PCR instrument, pre-denaturation is carried out for 2 minutes at 50 ℃ and for 10 minutes at 95 ℃; denaturation at 95 ℃ for 15s, primer annealing at 58 ℃ for 15s and extension at 72 ℃ for 30s, 40 cycles.
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