CN111363845A

CN111363845A - Special primer for specific identification of Chinese Huangguo ginseng and PCR method

Info

Publication number: CN111363845A
Application number: CN202010384567.9A
Authority: CN
Inventors: 王洪涛; 李桂生; 王英平; 王戎博; 陈玉春
Original assignee: Yantai University
Current assignee: Yantai University
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2020-07-03
Anticipated expiration: 2040-05-08
Also published as: CN111363845B

Abstract

The invention relates to a short single copy region of ginseng chloroplastccsANon-synonymous mutation SNP locus of Chinese Huangguo ginseng is explored in the gene, and a conventional and fluorescent quantitative PCR method for specifically identifying the Chinese Huangguo ginseng is provided according to the locus. The conventional PCR method established by the invention can complete detection in a general molecular biology laboratory, and the established fluorescent quantitative PCR method does not need to mark a primer, can analyze a large number of samples in a short time, and can effectively realize large-scale rapid identification and screening in the field of the radix polygonati officinalis. Therefore, the invention provides an effective DNA molecular marker technical means for variety identification of the yellow fruit ginseng, and has important significance for germplasm resource protection, variety management and ginseng product quality optimization of the yellow fruit ginseng in China.

Description

Special primer for specific identification of Chinese Huangguo ginseng and PCR method

Technical Field

The invention relates to the technical field of molecular identification of traditional Chinese medicinal materials, in particular to a special primer and a PCR (polymerase chain reaction) method for specifically identifying Chinese yellow fruit ginseng.

Background

Ginseng (Panax ginseng c.a. meyer) is a perennial herb plant of the family araliaceae, is a traditional famous and precious Chinese medicinal material, has rich medicinal and edible values, and has the reputation of 'king of herbaceous'. The main effective component of Ginseng radix is ginsenoside, and has anti-aging, anti-fatigue, and anti-inflammatory effects. After the artificially planted ginseng is officially approved as a new resource food by the Ministry of health, the development of the ginseng industry is greatly promoted. The cultivated ginseng has more than two thousand years of ginseng cultivation history, and is continuously subjected to genetic variation and differentiation in the artificial and natural selection processes, and the cultivated ginseng mainly comprises farmer varieties such as big horse teeth, two horse teeth, long neck, round winged reed rhizome, yellow fruit ginseng and the like. The Huangguo ginseng is a new ginseng cultivation variety bred by a systematic breeding method in China, and has the characteristic of high content of active ingredients such as ginsenoside, volatile oil, amino acid and the like. The successful breeding of the Huangguo ginseng increases new precious germplasm resources for the cultivation and breeding of the ginseng in China. However, the morphological characteristics of different ginseng varieties are very similar in the early growth and development stages, and moreover, the rapid and accurate ginseng variety identification method is lacked in China, so that different ginseng varieties are mixed and distributed in cultivation groups in various regions, and the varieties are disordered and production seeds are mixed. The hybrid distribution of the cultivated population seriously affects the germplasm purity and the yield of the xanthoceras sorbifolia bunge, so that the establishment of a molecular identification method of the xanthoceras sorbifolia bunge is urgently needed to protect the germplasm resources of the xanthoceras sorbifolia bunge and maintain the quality stability of the xanthoceras sorbifolia bunge.

Since the morphological markers of ginseng are easily affected by the growth and development stages and the environment, various DNA molecular marker techniques have been widely used in recent years for genetic diversity analysis, germplasm resource identification and evaluation in ginseng species. At present, researches on the xanthoceras sorbifolia bunge mainly focus on aspects of genetic diversity, genetic structure analysis and the like, for example, rapd (random Amplified Polymorphic DNA) and a cluster analysis technology are utilized by any of the Elsholtzia splendens and the like to research the genetic relationship between the xanthoceras sorbifolia bunge and the red fruit and the American ginseng, and molecular markers of RAMP (random Amplified microscopic molecular marker) and ISSR (Inter-simple Sequence Repeat) are utilized by the Wangzqing and the like to research the genetic difference between the "Formica Fuscai 01" ginseng and the farmhouse ginseng such as the xanthoceras sorbifolia bunge, but a molecular marker method for quickly and accurately identifying the xanthoceras sorbifolia bunge is not found so far.

Disclosure of Invention

The invention aims to solve the technical problem of providing a special primer and a PCR method for specifically identifying Chinese yellow fruit ginseng. The first problem to be solved is: provides a PCR special primer for specifically identifying the yellow fruit ginseng. The second technical problem to be solved by the invention is: providing a conventional PCR method for specifically identifying Chinese Huangguo ginseng; the third technical problem to be solved by the invention is: provides a fluorescent quantitative PCR method for specifically identifying Chinese Huangguo ginseng.

The PCR special primer for specifically identifying the xanthoceras sorbifolia bunge comprises 1 forward primer HgF (SEQ ID No: 1 in a sequence table) and 1 reverse primer HgR (SEQ ID No: 2 in the sequence table).

The sequence of the Huangguo ginseng chloroplast ccsA gene is shown as SEQ ID No: 3, the sequence of chloroplast ccsA gene of other ginseng varieties is shown as SEQ ID No: 4, respectively.

The base of the chloroplast ccsA gene sequence of the yellow fruit ginseng is G at 635bp, the base of other ginseng varieties at the position is A, and as shown in figure 1, a PCR special primer HgR for specifically identifying the yellow fruit ginseng is designed according to the SNP site of the yellow fruit at 635 bp. The derivative sequence of the primer sequence special for the yellow fruit ginseng also belongs to the invention. The derivative sequence is a sequence obtained by adding and reducing one or more bases to the 5 'end and/or the 3' end of the sequence based on the primer of the present invention.

The conventional PCR method for specifically identifying Chinese Huangguo ginseng provided by the invention is to carry out agarose gel electrophoresis analysis on a PCR amplification product after carrying out PCR amplification on the genome DNA of a ginseng sample to be detected by using the primers. Since the primers HgF and HgR only have specificity to the yellow fruit ginseng, only the yellow fruit ginseng can amplify a specific band (SEQ ID No: 5 in the sequence table) with the length of 217bp, and other ginseng varieties cannot detect PCR amplification products.

The total volume of the conventional PCR reaction was 20. mu.L, including 1. mu.L (10ng) of the genomic DNA of the sample to be tested, 2. mu.L (0.5. mu.M) of the primer HgF, 2. mu.L (0.5. mu.M) of the primer HgR, 5. mu.L of sterilized ultrapure water, and 10. mu.L of 2 × Easy Taq Supermix DNApolymerase.

The reaction conditions of the conventional PCR are as follows: pre-denaturation at 94 ℃ for 4 min; the 33 thermal cycle parameters comprise denaturation at 94 ℃ for 30s, annealing at 62 ℃ for 30s and extension at 72 ℃ for 30s, and extension at 72 ℃ is continued for 5min after the cycle is finished.

The invention provides a fluorescent quantitative PCR method for specifically identifying Chinese Huangguo ginseng, which is characterized in that after the primer is used for carrying out fluorescent quantitative PCR amplification on the genome DNA of a ginseng sample to be detected, EndPoint end point analysis is carried out on a PCR amplification product. Since the primers HgF and HgR are specific to only Huangguo ginseng, only Huangguo ginseng produces a fluorescence signal during the amplification process, and other ginseng species cannot be amplified by the primers and cannot detect the fluorescence signal, so that the Huangguo ginseng can be distinguished from other species by the level of the fluorescence signal.

The fluorescent quantitative PCR reaction volume is 10 muL, which comprises 1 muL (10ng) of genome DNA of a sample to be detected, 1 muL (0.5 muM) of primer HgF, 1 muL (0.5 muM) of primer HgR, 2 muL of sterilized ultrapure water, and 5 muL of 2 × SYBR Green I Mastermix.

The fluorescent quantitative PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; the 40 thermal cycle parameters included denaturation at 95 ℃ for 10s, annealing at 62 ℃ for 15s and extension at 72 ℃ for 20s, and the melting curve conditions increased from 72 ℃ to 95 ℃ after the cycle ended, with an increase of 1 ℃ per step. After the melting step was completed, the experimental results were analyzed using end point analysis.

The invention discovers the SNP locus of the yellow fruit ginseng from the gene of the short single copy region ccsA (c-type cytochrom synthesis gene) of the ginseng chloroplast, designs the variety specific primer of the yellow fruit ginseng according to the locus, and establishes the PCR method for specifically identifying the Chinese yellow fruit ginseng.

The invention discovers nonsynonymous mutation SNP sites of Chinese Huangguo ginseng from the ccsA gene of the ginseng chloroplast short single copy region, and provides a conventional and fluorescent quantitative PCR method for specifically identifying the Chinese Huangguo ginseng. The conventional PCR method can complete detection in a general molecular biology laboratory, fluorescent quantitative PCR removes bottlenecks in the rapid identification process of two ginseng varieties, namely gel preparation and electrophoretic analysis in the conventional PCR method, improves the detection speed and efficiency, can rapidly identify and analyze a large number of samples in a short time, and has stable and reliable identification results. Therefore, the invention provides an effective DNA molecular marker technical means for variety identification of the yellow fruit ginseng, and has important significance for germplasm resource protection, variety management and ginseng product quality optimization of the yellow fruit ginseng in China.

Drawings

FIG. 1 is a diagram showing the relative positions of SNP sites and primers of Huangguo ginseng in the cccA gene sequence.

FIG. 2 is a diagram showing the results of gel electrophoresis identification of conventional PCR products of Cucumis metuliferus.

FIG. 3 is a diagram of the result of fluorescent quantitative PCR identification of Huanggugen ginseng.

Detailed Description

The present invention will be described in detail with reference to specific examples.

The invention is made based on the discovery of specific SNP sites of the yellow fruit ginseng in the CCsA gene of the chloroplast short single copy region of the ginseng by the inventor. The identification of Huangguo ginseng using fluorescence quantitative PCR technique is described in detail in the following examples.

The methods used in the examples are conventional methods unless otherwise specified, and the various biomaterials described in the examples are obtained by a route that provides only experimental access for the purposes of this disclosure and should not be limiting as to the source of the biomaterials of the present invention. All primer synthesis and sequencing work was done by Saimer Feishale (China) science and technology Limited.

The embodiments are provided in order to provide detailed embodiments and specific procedures, which will help understanding of the present invention, but the scope of the present invention is not limited to the following embodiments.

Example 1 PCR Special primer designed for specific identification of Panaxanthus gracilis

Amplification and comparison of CCsA gene sequences of chloroplast short single-copy regions of different ginseng varieties

1.1 extraction of genomic DNA of different Ginseng species

Collecting multiple samples of different ginseng varieties from different producing areas, cleaning collected ginseng roots or leaves with deionized water, placing a proper amount of samples in a mortar, adding liquid nitrogen and grinding into fine powder, placing about 20mg of fine powder in a centrifuge tube, extracting ginseng DNA according to the operation instruction of a DNA extraction kit, and storing the extracted DNA in a refrigerator at-20 ℃ for later use.

Table 1 collected Ginseng samples of different varieties

1.2 PCR amplification of chloroplast ccsA Gene sequences of different Ginseng species

20 μ L of PCR reaction system was 1 μ L (10ng) of genomic DNA of ginseng, 2 μ L (0.5 μ M) of primer ccsF (5'-AGACACGCTGCTCTTAGGAAG-3'), 2 μ L (0.5 μ M) of primer ccsR (5'-ACCCGATATGTTTGGTCTTTG-3'), 5 μ L of sterilized ultrapure water, 10 μ L of 2 × Easy Taq Supermix DNA polymerase (formulation: 2 μ L10 × Easy Taqbuffer, 1.60 μ L2.5 mM dNTPs, 0.4 μ L Easy Taq DNA polymerase,6 μ L L H₂O)。

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; then 33 cycles of 94 ℃ for 30s, 58 ℃ for 30s and 72 ℃ for 60 s; finally, extension is carried out for 7min at 72 ℃, and the reaction is finished after heat preservation at 4 ℃.

1.3 sequence alignment

And (3) directly sequencing the PCR amplification product, and splicing the DNA sequencing result by utilizing SeqMan software. Alignment of chloroplast ccsA gene sequences of yellow fruit and other ginseng varieties using ClustalW Omega software is shown in FIG. 1. At the 635 th base of the ccsA gene, the Huangguo ginseng is G at this base, and the other ginseng is A. The mutation site is a non-synonymous mutation, resulting in a change of the corresponding amino acid from glutamine (Q) to arginine (R). The chloroplast ccsA gene sequence of the yellow fruit ginseng is shown as SEQ ID No: 3, the chloroplast ccsA gene sequence of other ginseng varieties is shown as SEQ ID No: 4, respectively.

Secondly, designing a special primer for specifically identifying the yellow fruit ginseng

According to the specific SNP site of the yellow fruit ginseng found in the sequence alignment, a forward primer HgF is designed at the 438 th to 458 th bases of the picture 1, and the sequence is 5'-CTATGCAGCTCTTCTATGTGG-3' (SEQ ID No: 1 in the sequence table). A reverse primer HgR is designed at the 635-654 th base, and the sequence is 5'-ATCCAACTGTTGAATCAGCC-3' (SEQ ID No: 2 in the sequence table). To ensure the absolute specificity of HgR for Huangguo ginseng, the penultimate base A at the 3' end was changed to G, so that HgR has a two-base mismatch for ginseng species other than Huangguo ginseng. The amplification interval of the positive primer and the negative primer is 438 bp-654 bp, so that the specific amplification fragment length of the yellow fruit ginseng is 217 bp. The relative positions of the primers and the length of the fragment expected to be amplified are shown in FIG. 1.

Example 2 routine PCR identification of Panaxanthus gracilis

PCR amplification was performed using the primers HgF and HgR using genomic DNAs of different ginseng species shown in Table 1 as templates.

The total volume of PCR reaction is 20 mul, which comprises 1 mul (10ng) of genome DNA of a sample to be detected, 2 mul (0.5 muM) of primer HgF, 2 mul (0.5 muM) of primer HgR, 5 mul of sterilized ultrapure water and 10 mul 2 × Easy Taq Supermix DNA polymerase. the reaction conditions of PCR are that pre-denaturation is carried out for 4min at 94 ℃, 33 thermal cycle parameters comprise denaturation for 30s at 94 ℃, annealing for 30s at 62 ℃ and extension for 30s at 72 ℃, extension for 5min at 72 ℃ is continued after the cycle is finished, and the reaction is finished after the heat preservation at 4 ℃.

mu.L of the PCR product was electrophoretically detected in a 1% agarose gel containing ethidium bromide, and the results were visualized using a gel imaging system. As shown in FIG. 2 (lanes 1-5 are Huangguo, 6-7 are Dama teeth, 8-9 are Era teeth, 10-11 are edgings, 12-13 are long necks, 14-15 are round winged Roots), only Huangguo ginseng produced specific bands amplified by HgF and HgR, but none of the other ginseng species produced bands. Recovering the specific band of the xanthoceras sorbifolia bunge and sequencing, wherein the sequence length is 217bp, and the sequences (SEQ ID No: 5 in a sequence table) and SEQ ID No: and 3, the corresponding sequence results are completely consistent, and the reliability of the experimental result is verified. The gel electrophoresis detection result shown in FIG. 2 shows neat and bright target sequence bands, and no non-specific amplification band or sheet-shaped dragging occurs, so that the 217bp specific band generated by the yellow fruit ginseng can be used for accurately distinguishing the yellow fruit ginseng from other ginseng varieties.

Example 3 fluorescent quantitative PCR identification of Panaxanthus gracilis

The genomic DNA of different ginseng varieties is used as a template, and the primers HgF and HgR are used for carrying out fluorescent quantitative PCR amplification. Sample materials of different ginseng species are shown in table 1.

The 10. mu.L fluorescent quantitative PCR reaction system comprises 1. mu.L (10ng) of genome DNA of a sample to be detected, 1. mu.L (0.5. mu.M) of primer HgF, 1. mu.L (0.5. mu.M) of primer HgR, 2. mu.L of sterilized ultrapure water and 5. mu.L of 2 × SYBR Green I Mastermix.

The fluorescent quantitative PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; the 40 thermal cycle parameters include denaturation at 95 ℃ for 10s, annealing at 62 ℃ for 15s and extension at 72 ℃ for 20s, and the melting step after the cycle is finished is to increase from 72 ℃ to 95 ℃ and increase by 1 ℃ in each step. After the melting step was completed, the experimental results were analyzed using end point analysis.

The end point analysis results of EndPoint are shown in FIG. 3 (sample 1 is ultrapure water as a negative control, 2-6 are big horse teeth, 7-11 are two horse teeth, 12-16 are edgings, 17-21 are long necks, 22-26 are round winged reed, and 27-50 are yellow fruits). SYBR Green I is a dye with a Green excitation wavelength that binds to all double-stranded DNA double-helix minor groove regions. At the beginning of PCR amplification, dye molecules not bound to the double-stranded DNA emit weak fluorescence, and the fluorescence signal intensity of SYBRGreen I is gradually increased along with the amplification of the double-stranded PCR product. Thus, the presence of a particular allele in a target can be determined by detection of a fluorescent signal. As can be seen from FIG. 3, all the yellow fruit ginseng samples were produced with amplification products, and the fluorescence Signal levels (Signal Level%) thereof were all 80 or more, while the negative control and other ginseng species were unable to perform PCR amplification due to the specificity of the primers for the yellow fruit ginseng species, and only weak fluorescence signals emitted from dye molecules not bound to the double-stranded DNA could be detected. Therefore, the primer and the fluorescent quantitative PCR technology can be used for easily distinguishing and identifying the Huangguo ginseng from other ginseng varieties. Compared with other non-gel electrophoresis analysis such as TaqMan and molecular beacon, the fluorescent quantitative PCR method developed by the research does not need to mark the primer, the result identification time does not exceed two hours, a large number of samples can be analyzed in a short time, and the field large-scale rapid identification and screening of the radix polygonati officinalis can be effectively realized.

Sequence listing

<110> smoke desk university

<120> a special primer and PCR method for specific identification of Chinese Huangguo ginseng

<141>2020-05-08

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ctatgcagct cttctatgtg g 21

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<212>DNA

<213> reverse primer HgR (Panax ginseng) of Huangguo ginseng

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atccaactgt tgaatcagcc 20

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<212>DNA

<213> chloroplast ccsA gene sequence (Panax ginseng) of xanthoceras sorbifolia

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atgatatttt caactttaga gcatatatta actcatattt ccttttcaat cgtttcaatt 60

gtaattacaa ttcatttgat aaccttatta gtcgatgaaa tcataaaact atatgattcg 120

tcagaaaagg gaatgatagc tattttttta tgtataacag gattattagt cactcgttgg 180

atttattcga gacatttccc actaagtgat ttatatgaat cattaatctt tctttcatgg 240

agtttatcag ttattcatat agttccgtat ttcaaaaaaa agaaaaaaaa tttaagcaca 300

ataactgcgt caagtgttat ttttacccag ggctttgcta cttcaggtct tttaactgaa 360

atacatcaat ccgcaatatt agtacccgct ctccaatccg agtggttaat aatgcacgta 420

agtatgatga tattgagcta tgcagctctt ctatgtggat cattattatc agtagcactt 480

ctagtcatta catttcgaaa aaacagaaat attttttgta aaaggaatcc tttattaaat 540

gagttatttt cctttggtga aatccaatac atgaatgaaa gaaacaatgt tttaagaact 600

actttttttt ctgctaagaa ttattacagg tcccggttga ttcaacagtt ggattattgg 660

agttatcgtg tcattagtct agggtttatc tttttaacca taggtattct ttcgggagca 720

gtatgggcta atgaagcatg gggatcatat tggaattggg acccaaagga aacttgggca 780

tttattactt ggatcgtatt tgcgatttat ttacacactc gaaccaatat aaaattacgg 840

ggtgcaaatt ccgcaattgt ggcgtctata ggctttctta taatttggat atgctatttt 900

ggagtcaatc tattaggaat aggactacac agttatggtt catttacgtt aacgtctaat 960

tga 963

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atgatatttt caactttaga gcatatatta actcatattt ccttttcaat cgtttcaatt 60

gtaattacaa ttcatttgat aaccttatta gtcgatgaaa tcataaaact atatgattcg 120

tcagaaaagg gaatgatagc tattttttta tgtataacag gattattagt cactcgttgg 180

atttattcga gacatttccc actaagtgat ttatatgaat cattaatctt tctttcatgg 240

agtttatcag ttattcatat agttccgtat ttcaaaaaaa agaaaaaaaa tttaagcaca 300

ataactgcgt caagtgttat ttttacccag ggctttgcta cttcaggtct tttaactgaa 360

atacatcaat ccgcaatatt agtacccgct ctccaatccg agtggttaat aatgcacgta 420

agtatgatga tattgagcta tgcagctctt ctatgtggat cattattatc agtagcactt 480

ctagtcatta catttcgaaa aaacagaaat attttttgta aaaggaatcc tttattaaat 540

gagttatttt cctttggtga aatccaatac atgaatgaaa gaaacaatgt tttaagaact 600

actttttttt ctgctaagaa ttattacagg tcccagttga ttcaacagtt ggattattgg 660

agttatcgtg tcattagtct agggtttatc tttttaacca taggtattct ttcgggagca 720

gtatgggcta atgaagcatg gggatcatat tggaattggg acccaaagga aacttgggca 780

tttattactt ggatcgtatt tgcgatttat ttacacactc gaaccaatat aaaattacgg 840

ggtgcaaatt ccgcaattgt ggcgtctata ggctttctta taatttggat atgctatttt 900

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ctatgcagct cttctatgtg gatcattatt atcagtagca cttctagtca ttacatttcg 60

aaaaaacaga aatatttttt gtaaaaggaa tcctttatta aatgagttat tttcctttgg 120

tgaaatccaa tacatgaatg aaagaaacaa tgttttaaga actacttttt tttctgctaa 180

gaattattac aggtcccggt tgattcaaca gttggat 217

Claims

1. The PCR special primer for identifying the specific property of the yellow fruit ginseng is based on the short single copy area of the yellow fruit ginseng in chloroplastccsAThe 635 th base yellow fruit from the 5' end is designed to be G.

2. The PCR primer special for the specific identification of the xanthophyllum chinense Pursh as claimed in claim 1, wherein: the nucleotide sequences of the PCR special primers of the yellow fruit ginseng are respectively shown as SEQ ID No: 1 and SEQ ID No: 2, respectively.

3. The PCR primer special for the specific identification of the ginseng Huangguo according to claim 1 or 2, wherein: chloroplast of the yellow fruit and other ginseng speciesccsAThe gene sequences are respectively shown as SEQ ID No: 3 and SEQ ID No: 4, respectively.

4. The method for performing the specific identification of the xanthophylls that is described in claim 1, 2 or 3, characterized in that: firstly, carrying out conventional PCR amplification on a sample to be detected, and then carrying out 1% agarose gel electrophoresis detection on a PCR amplification product, wherein if a single band with the length of 217bp appears, the sample source is the xanthoceras sorbifolia bunge.

5. The identification method according to claim 4, characterized in that the total volume of the PCR reaction is 20 μ L, the genomic DNA comprises 1 μ L (10ng) of a sample to be detected, 2 μ L (0.5 μ M) of the primer HgF, 2 μ L (0.5 μ M) of the primer HgR, 5 μ L of sterilized ultrapure water, and 10 μ L2 × Easy Taq Supermix DNA polymerase, wherein the reaction conditions of the PCR reaction are that pre-denaturation at 94 ℃ is carried out for 4min, pre-denaturation at 94 ℃ is carried out for 30s, annealing at 62 ℃ is carried out for 30s, elongation at 72 ℃ is carried out for 30s, after the circulation is finished, elongation at 72 ℃ is carried out for 5min, and the reaction is finished at 4 ℃.

6. The method for performing the specific identification of the xanthophylls that is described in claim 1, 2 or 3, characterized in that: firstly, carrying out fluorescence quantitative PCR amplification on a sample to be detected, and after the amplification cycle is finished, heating the temperature from 72 ℃ to 95 ℃ to perform a melting step; analyzing an experimental result by using an EndPoint end point analysis method after the melting step is finished; the melting step after the circulation is finished is that the temperature is increased from 72 ℃ to 95 ℃, and the temperature is increased by 1 ℃ in each step; and (3) analyzing by using an EndPoint end point analysis method after the melting step is finished, wherein if the fluorescence Signal Level (Signal Level%) is more than 50, the sample source is the yellow fruit ginseng.

7. The identification method according to claim 6, characterized in that the 10 μ L fluorescent quantitative PCR reaction system is 1 μ L (10ng) of genomic DNA of a sample to be tested, 1 μ L (0.5 μ M) primer HgF, 1 μ L (0.5 μ M) primer HgR, 2 μ L sterilized ultrapure water, 5 μ L2 × SYBR Green I Mastermix;

the fluorescent quantitative PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; the 40 thermal cycle parameters comprise denaturation at 95 ℃ for 10s, annealing at 62 ℃ for 15s and extension at 72 ℃ for 20s, and the melting step after the cycle is increased from 72 ℃ to 95 ℃ and is increased by 1 ℃ in each step; after the melting step was completed, the experimental results were analyzed using end point analysis.