CN110904263B - Characteristic nucleotide sequence, nucleic acid molecular primer, kit and method for quantitatively detecting dictyophora rubrovolvata - Google Patents
Characteristic nucleotide sequence, nucleic acid molecular primer, kit and method for quantitatively detecting dictyophora rubrovolvata Download PDFInfo
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- 125000003729 nucleotide group Chemical group 0.000 title abstract description 16
- 241001313734 Dictyophora Species 0.000 claims abstract description 29
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 238000003753 real-time PCR Methods 0.000 claims description 15
- 230000003321 amplification Effects 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 238000007400 DNA extraction Methods 0.000 claims description 2
- 241001313710 Dictyophora indusiata Species 0.000 abstract description 12
- 238000012408 PCR amplification Methods 0.000 abstract description 4
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- 241000233866 Fungi Species 0.000 description 3
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a characteristic nucleotide sequence, a nucleic acid molecular primer, a kit and a method for quantitatively detecting dictyophora rubrovolvata. The characteristic nucleotide sequence of the dictyophora rubrovolvata for quantitative detection is shown as SEQ ID No. 2. The nucleic acid molecular primer for quantitatively detecting the dictyophora rubrovolvata comprises PRSF 5'-TTTTAACGGTCGGGGT-3', PRSR: 5'-TTAAAGGGTCCCAAC-3'. The nucleic acid molecular primers PRSF and PRSR are utilized to carry out fluorescence PCR amplification, only Dictyophora rubrovalvata can excite a fluorescence signal, and other Dictyophora rubrovalvata such as Dictyophora echinata, Dictyophora indusiata and Dictyophora indusiata have no Ct value when the cycle number is less than 30, so that the nucleic acid molecular primers PRSF and PRSR have high specificity, can be used for rapidly detecting authenticity and content of Dictyophora rubrovalvata sporocarp and related products thereof, and can finish detection within 2 h.
Description
Technical Field
The invention belongs to the technical field of rapid detection of authenticity of edible and medicinal fungi by using a molecular technical means, and particularly relates to a characteristic nucleotide sequence, a nucleic acid molecular primer, a kit and a method for quantitatively detecting dictyophora rubrovolvata.
Background
Dictyophora rubrovolvata m.zang, d.g.ji & x.liu, is a new species first published and named by zanu et al in 1976. Taxonomically, Dictyophora rubrovolvata (M.Zang, D.G.Ji & X.X.Liu) was incorporated by Kreisel in 1996 under Phallus, now known as Phallus rubrovolvatus (M.Zang, D.G.Ji & X.X.Liu) Kreisel. For convenience of description, the invention continues to use folk name, Dictyophora rubrovalvata.
Dictyophora rubrovolvata found in Yunnan is an edible fungus which is widely planted in Yunnan, Sichuan, Guizhou, Fujian and the like and has high economic value, wherein Dictyophora rubrovolvata (Dictyophora rubrovolvata) in the Jinzhou region of Guizhou is identified as a national geographic sign product and is highly famous. Dictyophora rubrovalvata has rich nutrition, high water content of 89.86%, soluble protein content of 181.05mg/100g, total amount of free amino acids of 308.78mg/100g FW, glutamic acid of 133.576mg/100g FW, flavonoid content of 22.5mg/100g, Vc content of 106.77mg/100g, and total sugar content of 285.59mg/100 g.
The dictyophora indusiata on the market mainly comprises dictyophora rubrovolvata, dictyophora echinovolvatus (M.Zang, D.R.Zheng & Z.X.Hu) Kreisel, dictyophora indusiatus Vent, dictyophora indusiata Phallus sp, and the like, and the price of the dictyophora indusiata is different greatly, for example, 600-800 yuan/kg of dictyophora rubrovolvata dry product, 200 yuan/kg of dictyophora echinovolvata dry product and 200 yuan/kg of dictyophora indusiata dry product are subjected to spore body cleaning and fungus tray removing processes before the products are on the market, and then are dried and packaged. It is difficult to distinguish the categories from each other in appearance, which also results in the consumer being difficult to correctly identify and suffer from losses. Therefore, the research and development of technologies for rapidly and quantitatively detecting the authenticity and content of dictyophora rubrovolvata and related products thereof have important significance for the healthy development of the dictyophora rubrovolvata industry and have substantial application value for the commercial production and product inspection of the dictyophora rubrovolvata.
Disclosure of Invention
The first purpose of the invention is to provide a characteristic nucleotide sequence which can be used for rapidly detecting the authenticity of dictyophora rubrovolvata fruiting bodies and related products and detecting the content of dictyophora rubrovolvata.
The characteristic nucleotide sequence of dictyophora rubrovolvata is derived from the partial sequence of the LSU gene of dictyophora rubrovolvata (the nucleotide sequence of the LSU gene is shown as SEQ ID NO.1 and totally contains 895 basic groups). The characteristic nucleotide sequence comprises partial basic groups of dictyophora rubrovolvata 28S rDNA, the nucleotide sequence is shown as SEQ ID NO.2, and the characteristic nucleotide sequence totally comprises 161 basic groups.
The second purpose of the invention is to provide a nucleic acid molecule primer for amplifying the characteristic nucleotide sequence of dictyophora rubrovolvata for quantitative and rapid detection, wherein the nucleic acid molecule primer comprises:
PRSF:5′-TTTTAACGGTCGGGGT-3′；
PRSR:5′-TTAAAGGGTCCCAAC-3′。
the annealing temperatures of the nucleic acid molecule primer sequences PRSF and PRSR are similar. The nucleic acid molecular primer does not react with Dictyophora rubrovolvata, Dictyophora indusiata, yellow Dictyophora phalloidea Phallus luteus (Liou & L.Hwang) T.Kasuya, Dictyophora rubrovolvata and the like, and only has extremely high binding specificity with Dictyophora rubrovolvata. The content of Dictyophora rubrovalvata DNA in the substrate is detected according to the strength of a fluorescent signal in the fluorescent quantitative PCR process, and the sensitivity is high. Therefore, the nucleic acid molecular primer can be used for rapidly detecting the authenticity of dictyophora rubrovolvata fruiting bodies and related products thereof and measuring the content of the dictyophora rubrovolvata fruiting bodies and the related products thereof through fluorescent quantitative PCR amplification.
The third purpose of the invention is to provide a kit for quantitatively and rapidly detecting dictyophora rubrovolvata, which comprises the nucleic acid molecular primers PRSF and PRSR, and a conventional DNA extraction reagent and a fluorescent quantitative PCR reaction reagent.
The fourth purpose of the invention is to provide a method for quantitatively and rapidly detecting dictyophora rubrovolvata, which adopts the nucleic acid molecular primers PRSF and PRSR as amplification primers, and uses dictyophora rubrovolvata DNA genome as a template, and utilizes a fluorescent quantitative PCR method to rapidly detect dictyophora rubrovolvata.
The invention designs specific nucleic acid molecular primers PRSF and PRSR based on the LSU gene sequence (SEQ ID NO.1) of dictyophora rubrovolvata, takes genome DNA of dictyophora rubrovolvata as a template, and carries out PCR (see embodiment 2 for details) according to a conventional fluorescent quantitative PCR method, only dictyophora rubrovolvata can excite a fluorescent signal, and other dictyophora such as dictyophora echinata, dictyophora indusiata, yellow dictyophora indusiata and winter dictyophora all have no Ct value (as shown in figure 1) when the cycle number is less than 30. The quantitative detection of dictyophora rubrovolvata is realized according to a standard curve (shown in figure 2) of dictyophora rubrovolvata LSU genes, the correlation coefficient is 0.999, the linear range of the Ct value is 17.70-31.79, the regression equation is-3.5628 x +26.565, and the relative content of the detected sample of the dictyophora rubrovolvata can be measured according to the regression equation. The invention adopts the fluorescent quantitative PCR technology for detection, the limit of detection is as low as 0.03 ng/mu L, the method is simple, the specificity is good, the time consumption is short, and the detection can be completed within 2 hours.
Drawings
Fig. 1 is an amplification curve of real-time fluorescent quantitative PCR performed on different dictyophora samples by using nucleic acid molecules PRSF and PRSR as primers according to a fluorescent quantitative PCR method, as shown in fig. 1, point a is a Ct value of dictyophora rubrovolvata of 25.43 ± 0.49, and when only the dictyophora rubrovolvata has a Ct value of about 25 (25.43 ± 0.49), the fluorescent value reaches a threshold value, while other dictyophora rubrovolvata does not reach the threshold value in the whole PCR cycle process (within 30 cycles).
FIG. 2 shows a standard curve of Dictyophora rubrovalvata LSU gene obtained by amplification according to fluorescent quantitative PCR method using PRSF and PRSR as primers according to DNA of Dictyophora rubrovalvata samples with different concentrations as templates, wherein the correlation coefficient is 0.999, the linear range of Ct value is 17.70-31.79, and the regression equation is-3.5628 x + 26.565. The relative content of the dictyophora rubrovolvata detected samples can be measured according to the regression equation.
Detailed Description
The present invention will be better understood by those skilled in the art from the following examples. The examples are described only to illustrate the invention and should not be construed as limiting the invention as detailed in the claims.
Example 1: extraction of Dictyophora rubrovalvata genome DNA and obtaining of LSU gene sequence
Weighing 0.2g Dictyophora rubrovalvata sample, placing in a sterile mortar, pouring liquid nitrogen, rapidly grinding, and selectingDictyophora rubrovolvata genomic DNA was extracted using the HiPure Fungal DNAMini Kit (Meyer Biotech, Guangzhou, No. D3171-03) from magenta. Nucleic acid molecular primers LR5 and LR0R (LR5: 5'-TCCTGAGGGAAACTTCG-3' and LR0R: 5'-ACCCGCTGAACTTAAGC-3' are synthesized by Huada Gene Co.), and PCR amplification is carried out by taking Dictyophora rubrovolvata genome DNA as a template to obtain the Dictyophora rubrovolvata LSU gene sequence (SEQ ID NO. 1). The method comprises the following specific steps: using Premix TaqTM(Ex TaqTMVersion 2.0plus dye) kit (Takara, accession number: RR902A) were subjected to PCR reaction. The reaction system is 25 mu L of total volume, wherein Premix Taq TM10 μ L, LR5(10 μmol/L)0.5 μ L, LR0R (10 μmol/L)0.5 μ L, template DNA0.5 μ L, complement ddH2O to 25. mu.L. The PCR reaction program is pre-denaturation at 94 ℃ for 5min, 30s at 94 ℃, 30s at 55 ℃, 80s at 72 ℃, 35 cycles and 8min at 72 ℃. The PCR product was subjected to agarose gel electrophoresis, and purified using a SanPrep column DNA gel recovery kit (Shanghai Biotech engineering Co., Ltd., No. B518131-0100). The purified PCR product was sequenced by the Bwayasu major Gene (BGI). The nucleotide sequence of the obtained Dictyophora rubrovalvata LSU gene fragment is shown in SEQ ID NO.1 and comprises 895 basic groups.
Example 2: fluorescent PCR amplification of Dictyophora rubrovalvata specific primer
According to Dictyophora rubrovalvata LSU gene (SEQ ID NO.1), a pair of sequences consisting of 15-16 nucleotides, namely PRSF: 5'-TTTTAACGGTCGGGGT-3' and PRSR: 5'-TTAAAGG GTCCCAAC-3', is designed by using primer design software primer5.0, and the primers are synthesized by Huada Gene corporation (BGI). The sequence obtained by primer amplification is shown as SEQ ID NO.2, and the total number of 161 bases. SYBR Premix EX TaqTM II kit (Takara, number: RR820A) is used for carrying out fluorescent quantitative PCR reaction, and genome DNA of dictyophora rubrovolvata, dictyophora echinata, dictyophora indusiata, yellow dictyophora indusiata and dictyophora phalloidea are respectively used as templates, and primers PRSF and PRSR are used for carrying out PCR amplification. The reaction system was 25 μ L total volume: SYBR Premix EX TaqTM II 10.0. mu.L, ROX 0.4. mu.L, PRSF 0.5. mu.L (10. mu. mol/L), PRSR 0.5. mu.L (10. mu. mol/L), template DNA 0.5. mu.L, plus ddH2O to 25. mu.L. The reaction procedure is pre-denaturation at 95 deg.C for 2min, at 95 deg.C for 15s, at 50 deg.C for 15s, at 72 deg.C20s, 30 cycles. Using a fluorescent quantitative PCR instrument (Applied Biosystems QuantStaudio 6)&7Real-Time PCR System, Thermo Fisher Co.). The results are shown in FIG. 1. The fluorescence value of Dictyophora rubrovalvata reaches a threshold value when the Ct average value is 25.4 (namely 25.4 PCR cycles); and other dictyophora indusiata do not reach the threshold value in the whole PCR circulation process. Can realize the specificity detection of Dictyophora rubrovalvata, and the characteristic nucleotide sequence of Dictyophora rubrovalvata is shown in SEQ ID NO.2, and has 161 basic groups.
Example 3: quantitative detection of Dictyophora rubrovalvata DNA content
According to the known dictyophora rubrovolvata genome DNA samples with different concentrations as templates, PRSF: 5'-TTTTAACGGTCGGGGT-3' and PRSR: 5'-TTAAAGGGTCCCAAC-3' are used as primers to amplify according to the fluorescent quantitative PCR method of the example 2, and the Ct value of the obtained dictyophora rubrovolvata rDNALSU gene fluorescent PCR (shown in table 1) is plotted to obtain a standard curve (shown in figure 2). The correlation coefficient is 0.999, the Ct value linear range is 17.70-31.79, and the regression equation is-3.5628 x + 26.565. The relative content of the dictyophora rubrovolvata detected sample can be measured according to the regression equation. The minimal detection assay was performed according to the above-described fluorescent quantitative PCR method, and the results showed that the detection limit was as low as 0.03 ng/. mu.L. Can realize the quantitative detection of Dictyophora rubrovalvata.
TABLE 1 Ct values of Dictyophora rubrovalvata DNA samples of different concentrations
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
<120> characteristic nucleotide sequence, nucleic acid molecular primer, kit and method for quantitatively detecting dictyophora rubrovolvata
<160> 2
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ttcggccgtc cgagttgtaa tctggagaag cgttttcagt gccggcccgc gtacaagtcc 120
cctggaacgg ggcgtcgcag agggtgagaa tcccgtctct gacgcggtca tgccggcgcg 180
ctgcgatgcg ctctcgaaga gtcgagttgt ttgggaatgc agctcaaaac gggtggtaaa 240
ttccatctaa agctaaatac tggcgaaaga ccgatagcga acaagtaccg tgagggaaag 300
atgaaaagca ctttggaaag agagtcaaac agtacgtgaa attgttgaaa gggaaacgct 360
tgaagtcagt cgcgtctccc gggacttcag tcgcgctctc aaaggcgcgg cgtacttccc 420
gggtctggac gggccagcgt cgatttcgac cgtcgtaaaa aggtacgagg aacgtggcac 480
ctccgggtgt gttatagcct cgtgttccgc atgcgacggt ggggatcgag ggacgcagcg 540
cgccttttaa cggtcggggt tcgcccacgt aacgcgcttg ggatgctggc ttaatggctt 600
taagcgaccc gtcttgaaac acggaccaag gagtctaaca tgctcgcgag tgttcgggtg 660
gaaaacccgt gcgcgtaatg aaagtgaaag gttgggaccc tttaagaggg ggcaccgacg 720
cccggacttg agctgctgcg acggttccga ggcagagcgc gtatgttggg acccgaaaga 780
tggtgaacta tgcctgagta gggcgaagcc agaggaaact ctggtggagg ctcgtagcga 840
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acccgtgcgc gtaatgaaag tgaaaggttg ggacccttta a 161
Claims (3)
1. A nucleic acid molecule primer for rapid quantitative detection of Dictyophora rubrovalvata is characterized by comprising the following primer pairs:
PRSF: 5′-TTTTAACGGTCGGGGT-3′；
PRSR: 5′-TTAAAGGGTCCCAAC-3′。
2. a kit for rapidly and quantitatively detecting Dictyophora rubrovalvata, which is characterized by comprising the nucleic acid molecular primers PRSF and PRSR as claimed in claim 1, a DNA extraction reagent and a fluorescent quantitative PCR reaction reagent.
3. A method for rapidly and quantitatively detecting dictyophora rubrovolvata is characterized in that the nucleic acid molecular primers PRSF and PRSR in claim 1 are used as amplification primers, a dictyophora rubrovolvata DNA genome is used as a template, and a fluorescent quantitative PCR method is used for detecting the dictyophora rubrovolvata.
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| Nutritional characteristics of proteins from the volva and pileus in cultivated mushroom Dictyophora rubrovolvata;Yongliang Zhuang 等;《Int J Food Sci Nutr》;20110201;第62卷(第4期);第392-396页 * |
| 基于ITS中华散尾鬼笔的分类及抑菌活性研究;王瑞琦等;《黑龙江畜牧兽医》;20171210(第23期);第178-181页 * |
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