CN116042892A - Multiple primer group, kit and detection method for simultaneously identifying 3 types of highly toxic mushrooms - Google Patents
Multiple primer group, kit and detection method for simultaneously identifying 3 types of highly toxic mushrooms Download PDFInfo
- Publication number
- CN116042892A CN116042892A CN202211409430.XA CN202211409430A CN116042892A CN 116042892 A CN116042892 A CN 116042892A CN 202211409430 A CN202211409430 A CN 202211409430A CN 116042892 A CN116042892 A CN 116042892A
- Authority
- CN
- China
- Prior art keywords
- primer
- amanita
- highly toxic
- kit
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- 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
- C12Q1/6876—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
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
-
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- Mycology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of food safety detection, and particularly relates to a multiplex primer group, a kit and a detection method for simultaneously identifying 3 types of highly toxic mushrooms. The invention provides a group of multiplex primer groups for simultaneously identifying 3 types of highly toxic mushrooms, the sequences of which are shown in SEQ ID NO. 1-SEQ ID NO.9, and a tube of multiplex primer groups are combined to prepare a kit for simultaneously identifying 3 types of highly toxic mushrooms. The multiplex primer group or the kit can simultaneously carry out fluorescent PCR amplification in the same reaction tube, thereby realizing simultaneous detection and identification of 3 major 12 highly toxic mushrooms, greatly improving the detection efficiency and reducing the detection cost. The multiplex real-time fluorescence PCR kit provided by the invention is simple and quick to operate, has the advantages of strong detection specificity, high sensitivity and the like, is suitable for samples such as mushrooms and processed products, and can provide technical support for emergency treatment of food-borne mushroom poisoning.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to a multiplex primer group, a kit and a detection method for simultaneously identifying 3 types of highly toxic mushrooms.
Background
The food safety problem in China is highly concerned by the public, wherein food-borne toxic mushroom poisoning is frequent in recent years, and serious threat is caused to the life health of people. The toxic and lethal event caused by eating the wild mushrooms by mistake occurs every year, so that the method is very important for detecting whether the wild mushrooms have the toxic and lethal effect. Currently known, main species causing poisoning and mortality of food-borne mushrooms in China are highly toxic amanita, pholiota nameko and russula vinosa.
There are 900-1000 species of amanita worldwide, of which about 50 species of highly toxic amanita. It is reported that >90% of the global food-borne mushroom poisoning lethal events are caused by such toxic mushrooms. Currently, there are 12 types of highly toxic amanita found and identified in our country, which are, respectively, amanita cinerea (Amanitin exitialis), amanita cinerea (a. Fujinenia Hongo), amanita pseudoamanita cinerea (a. Fujineniae), amanita molluscum (a. Mollinusa), amanita pallida (a. Pallida), amanita parvula (a. Parviexifolia), amanita meretrica (a. Rimosa), amanita sub-gray (a. Fujiginea), huang Gaie amanita (a. Fujiquiria), amanita pseudoamanita pallida (a. Fujillidoroseea), amanita glaucophyllum (a. Griseuorea) and amanita leptospora (a. Virosa), etc. In addition, in recent years, the pholiota nameko and the russula vinosa have caused more mushroom poisoning and killing events in China, and bring about high public attention. Analysis of toxic mushroom poisoning situation issued by China center for prevention and control of diseases 2019-2021 shows that: these two highly toxic mushrooms have become the main species that are lethal to food-borne mushrooms in China. Only 2020-2021, the pholiota nameko resulted in 30 mushroom poisoning events, resulting in 74 people poisoning and 8 deaths; russula vinosa caused 26 poisoning events, resulting in 76 poisoning and 10 deaths.
The detection and identification methods for the toxic mushrooms mainly comprise a morphological identification method and a molecular identification method. The morphological identification method is characterized by the characteristic morphology of mushrooms, needs enough experience and expertise, is difficult to accurately identify for common people, and is difficult to identify by the morphology after processing the mushrooms. Molecular biological identification techniques represented by PCR have been reported to be applied to the identification of highly toxic mushrooms. For example, a conventional PCR method is adopted to carry out single species identification, and loop-mediated isothermal amplification (LAMP) technology is adopted to carry out detection and identification of the virulent amanita, the pholiota nameko and the russula vinosa. The technical means can only identify single-class highly toxic mushrooms, and once a suspected mushroom poisoning event occurs, different reaction systems and reaction programs are needed to carry out detection and identification for multiple times, so that the detection cost is increased, the identification efficiency is reduced, and the emergency treatment of the mushroom poisoning event is not facilitated. Therefore, development of a detection and identification method for simultaneously identifying multiple types of highly toxic mushrooms is needed to solve the technical problem of emergent treatment of existing mushroom poisoning.
Disclosure of Invention
The invention aims to provide a group of multiplex primer groups, a kit and a detection method for simultaneously identifying 3 types of highly toxic mushrooms, which are simple and convenient to operate, overcome the tedious steps of the conventional single-molecule detection technology, provide technical support for emergency treatment and prevention of food-borne toxic mushrooms, and have high detection flux and efficiency, good specificity and good sensitivity.
The invention provides a group of multiple primer groups for simultaneously identifying 3 types of highly toxic mushrooms, wherein the types of the highly toxic mushrooms comprise highly toxic amanita, pholiota nameko and russula vinosa;
the primer set designed for the highly toxic amanita comprises an upstream Universal primer Universal A.spp-F, a downstream Universal primer Universal A.spp-R and a probe Universal A.spp-P, wherein the nucleotide sequence of the upstream Universal primer Universal A.spp-F is shown as SEQ ID NO.1, the nucleotide sequence of the downstream Universal primer Universal A.spp-R is shown as SEQ ID NO.2, and the nucleotide sequence of the probe Universal A.spp-P is shown as SEQ ID NO. 3;
the primer set designed for the Phlebopus portentosus comprises an upstream primer L.bru-F, a downstream primer L.bru-R and a probe L.bru-P, wherein the nucleotide sequence of the upstream primer L.bru-F is shown as SEQ ID NO.4, the nucleotide sequence of the downstream primer L.bru-R is shown as SEQ ID NO.5, and the nucleotide sequence of the probe L.bru-P is shown as SEQ ID NO. 6;
the primer group designed for russula vinosa comprises an upstream primer R.sub-F, a downstream primer R.sub-R and a probe R.sub-P, wherein the nucleotide sequence of the upstream primer R.sub-F is shown as SEQ ID NO.7, the nucleotide sequence of the downstream primer R.sub-R is shown as SEQ ID NO.8, and the nucleotide sequence of the probe R.sub-P is shown as SEQ ID NO. 9.
Preferably, the highly toxic amanita includes amanita cinerea (Amanitin exitialis), amanita cinerea (a. Fulginena Hongo), amanita pseudoamanita cinerea (a. Fulginenides), amanita pallida (a. Pallidurosisea), amanita schizophyllum (a. Rimosa), huang Gaie amanita (a. Subjunillea), amanita soft-touch (a. Molluscula), amanita gratifolia (a. Subfurginenia), amanita pseudoamanita pallida (a. Subbpalidomorosea) and amanita leptospiricola (a. Virosa).
Preferably, each probe is labeled with a different fluorescent group at the 5 'end and a quenching group at the 3' end.
Preferably, the fluorescent group is selected from any one of FAM, VIC, ROX and CY5, and the quenching group is BHQ1 or BHQ2.
The invention also provides a group of kit for simultaneously identifying 3 types of highly toxic mushrooms, which comprises the mixed solution of the multiple primer groups.
Preferably, in the mixed solution, the concentration ratio of the upstream primer, the downstream primer and the probe is 2:2:1,
and the concentration ratio of the multiple primer groups designed for the amanita fuliginea, the pholiota nameko and the russula vinosa is 1:1:1.
preferably, the kit further comprises a reaction solution, a positive quality control product, a negative quality control product and water without nuclease.
Preferably, the working concentration of each primer in the kit is 0.16-0.72 mu M, and the working concentration of each probe is 0.08-0.36 mu M.
The invention also provides a multiplex real-time fluorescence PCR method for simultaneously identifying 3 types of highly toxic mushrooms, which comprises the following steps of: and (3) preparing a reaction system by using DNA of the sample to be detected as a template and a mixed solution of the multiple primer groups or a reagent in the kit, and performing multiple real-time fluorescent PCR (polymerase chain reaction), wherein an amplification curve of the sample to be detected is an S-shaped curve, ct is less than or equal to 35, and the sample to be detected is judged to be positive.
Preferably, the reaction program of the multiplex real-time fluorescent PCR reaction comprises: pre-denaturation at 94 ℃ for 180s; denaturation at 94℃for 5s, annealing at 53℃for 15s, elongation at 72℃for 30s,40-50 cycles.
The beneficial effects are that: the invention provides a group of multiplex primer groups for simultaneously identifying 3 types of highly toxic mushrooms, which is used for designing primers and probes aiming at specific common conserved gene sequences of highly toxic amanita as target genes, and simultaneously designing primers and probes aiming at specific sequences of russula sarcochrous and russula vinosa as target genes respectively, combining the primers and the probes as a multiplex primer group, and can be used for preparing a kit for simultaneously identifying 3 types of highly toxic mushrooms. The multiplex primer group or the kit can simultaneously carry out fluorescent PCR amplification in the same reaction tube, thereby realizing simultaneous detection and identification of 3 major 12 highly toxic mushrooms, greatly improving the detection efficiency and reducing the detection cost. The multiplex real-time fluorescence PCR kit provided by the invention is simple and quick to operate, has the advantages of strong detection specificity, high sensitivity (detection limit of 0.01 pg/mu L DNA) and the like, is suitable for samples of mushrooms, processed products (mushroom residues, soup and the like) and the like, and can provide technical support for emergency treatment of food-borne mushroom poisoning.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart and a result chart of a specific primer probe combination for screening highly toxic mushrooms by agarose gel electrophoresis; wherein (a) - (c) are PCR amplified gel maps of the primer probe combinations 1-3 of the highly toxic amanita; (d) - (f) PCR amplified gel map of the Phlebopus portentosus primer probe combinations 4, 6 and 7; (g) - (i) PCR amplified gel map of russula vinosa primer probe combinations 9, 11, 13;
FIG. 2 is a graph showing the result of optimizing the primer probe concentration of a real-time fluorescence PCR detection system for highly toxic mushrooms, wherein the probe concentration is respectively 80nM, 120nM, 160nM, 200nM, 240nM, 280nM, 320nM and 360nM, and the ratio of the primer/probe concentration is 2/1; wherein A is a real-time fluorescence PCR amplification curve of russula vinosa; b is a real-time fluorescence PCR amplification curve of the Phlebopus portentosus; c is a real-time fluorescence PCR amplification curve of the highly toxic amanita; A. the dark curves indicated by the arrows in B and C are PCR amplification curves at optimal concentrations (primer 240nM, probe 120 nM);
FIG. 3 is a diagram showing the result of the specificity verification of the real-time fluorescence PCR detection method of highly toxic mushrooms; wherein the blue streak is a fluorescence PCR amplification curve of CY5 channel highly toxic amanita, the green dotted line is an amplification curve of HEX channel russula sarcochrous, the red solid line is a fluorescence PCR amplification curve of FAM channel russula vinosa, and the black line represents an amplification curve of other mushroom samples;
FIG. 4 is a diagram of the sensitivity verification result of the real-time fluorescence PCR detection kit for highly toxic mushrooms; in the figure, three types of highly toxic mushroom mixed DNA templates are diluted in a gradient manner from left to right, wherein the concentrations are as follows: 10 ng/. Mu.L, 1.0 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 0.1 pg/. Mu.L, 0.01 pg/. Mu.L and ddH 2 O; the blue streak is the PCR amplification curve of CY5 channel highly toxic amanita, the red solid line is the PCR amplification curve of FAM channel russula vinosa, and the green dotted line is the PCR amplification curve of HEX channel russula vinosa.
Detailed Description
The invention provides a group of multiple primer groups for simultaneously identifying 3 types of highly toxic mushrooms, wherein the types of the highly toxic mushrooms comprise highly toxic amanita, pholiota nameko and russula vinosa; the multiple primer sets designed for different types of highly toxic mushrooms are shown in SEQ ID NO. 1-9 in Table 1.
TABLE 1 multiplex primers and probes designed for different classes of highly toxic mushrooms according to the present invention
The highly toxic amanita of the present invention preferably includes amanita cinerea (Amanitin exitialis), amanita cinerea (a. Fuliginea Hongo), amanita pseudoamanita cinerea (a. Fuligineoides), amanita pallida (a. Pallidorosea), amanita schizophyllum (a. Rimosa), huang Gaie amanita (a. Subjunillea), amanita soft-touch (a. Molluscula), amanita subformis (a. Subfuruginea), amanita pseudoamanita pallida (a. Subbpalidorosea) and amanita leptospiricola (a. Virosa).
In the invention, preferably, different fluorescent groups are respectively marked at the 5 'end of each probe, and quenching groups are marked at the 3' end of each probe; and the fluorescent group is preferably selected from any one of FAM, VIC, ROX and CY5, and the quenching group is preferably BHQ1 or BHQ2.
The invention also provides a group of kit for simultaneously identifying 3 types of highly toxic mushrooms, which comprises the mixed solution of the multiple primer groups.
In the mixed solution, the concentration ratio of the upstream primer to the downstream primer to the probe is preferably 2:2:1, and the concentration ratio of the multiple primer sets designed for the amanita fulgidus, the pholiota nameko and the russula vinosa is preferably 1:1:1. as in the present example, all primer probes were diluted to 10 μm separately, and each set of primer probes was used as the upstream primer: a downstream primer: probe = 2:2:1 volume ratio, and the three groups of primer probe compositions are mixed according to the volume ratio of 1:1:1 to obtain a final primer and probe mixture.
The kit of the invention preferably further comprises a reaction solution, a positive quality control product, a negative quality control product and nuclease-free water, wherein the reaction solution preferably comprises Taq DNA polymerase, dNTP and MgSO 4 Solutions, etc.; the positive quality control product is preferably a DNA template containing the highly toxic amanita, the armillaria luteo-virens and the russula vinosa, and the negative quality control product is preferably a Lentinus edodes DNA template.
The kit according to the present invention is preferably a 50-time format kit, and the stock of each reagent is shown in Table 2.
Table 2 three kinds of real-time fluorescence PCR detection kit component tables for highly toxic mushrooms
The invention also provides a multiplex real-time fluorescence PCR method for simultaneously identifying 3 types of highly toxic mushrooms, which comprises the following steps of: and (3) preparing a reaction system by using DNA of the sample to be detected as a template and a mixed solution of the multiple primer groups or a reagent in the kit, and performing multiple real-time fluorescent PCR (polymerase chain reaction), wherein an amplification curve of the sample to be detected is an S-shaped curve, ct is less than or equal to 35, and the sample to be detected is judged to be positive.
When the kit is used for detection, the method preferably comprises the steps of preparing a reaction system with the concentration of 25 mu L, and respectively performing real-time fluorescence PCR amplification by using a positive quality control product and a negative quality control product, wherein the reaction system for real-time fluorescence PCR amplification preferably comprises the following components: 4.5. Mu.L of DNA, 12.5. Mu.L of reaction solution, 4.5. Mu.L of primer mixture and dd H 2 O. In the reaction system, the DNA concentration is preferably 0.1 to 30 ng/. Mu.L; in the reaction system, the concentration of each primer is 0.6-1.2 mu M; the concentration of each probe is 0.3-0.6 mu M. The PCR amplification procedure of the present invention preferably comprises: pre-denaturation at 94 ℃ for 180s; denaturation at 94℃for 5s, annealing at 53℃for 15s, elongation at 72℃for 30s,40-50 cycles.
In the invention, different types of highly toxic mushrooms are marked with different fluorescent groups on the probe, so that the light emission is different, and the type of highly toxic mushrooms can be distinguished by the same light fluorescence, for example, in the embodiment, blue streaks are fluorescent PCR amplification curves of CY5 channel highly toxic amanita, green dotted lines are amplification curves of HEX channel sarcophachus cinus, and red solid lines are FAM channels.
After the multiplex real-time fluorescent PCR reaction is finished, the invention can judge whether positive amplification occurs or not by observing fluorescent signals and amplification curves, wherein the amplification curve of the sample to be detected is an S-shaped curve, ct is less than or equal to 35, and the sample to be detected is judged as positive; the sample to be tested has no amplification curve, ct is more than 40, and the sample to be tested is judged as negative; the amplification curve of the sample to be detected has a logarithmic growth phase, but Ct is more than 35 and less than or equal to 40, and the re-detection is carried out after the sample is re-extracted.
The type of the sample to be tested is not particularly limited, and preferably includes dried fruiting body of mushroom, fresh fruiting body of mushroom, processed mushroom residue or soup.
For further explanation of the present invention, a set of multiplex primer sets, kits and detection methods for simultaneously identifying 3 kinds of highly toxic mushrooms, which are provided by the present invention, will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Design of highly toxic mushroom primer probe combination
And (5) designing a general primer probe for the highly toxic amanita fungus. The ITS gene sequences of 10 highly toxic amanits, including Huang Gaie amanita (A.subjuniperea), amanita pallidum (A.pallidum), amanita griseus (A.fulgineahongo), amanita fatal (A.exilianis), amanita pseudogriseus (A.fulginenides), amanita meracilis (A.rimosa), amanita bisporus (A.bisporigenera), amanita pseudoamanita pallidum (A.subbpalirobiosea), amanita graminearum (A.subfurgifera), and amanita glauca (A.phagos), were downloaded from NCBI GeneBank database. Meanwhile, a part of amanita species of the same genus as the highly toxic amanita, which is toxic or edible, is downloaded, and 9 species including amanita pantherina (a. Pantherina), amanita parviflora (a. Pantherina), globus-base amanita (a. Subglobosa), amanita javanica (a. Javanica), amanita cinerea (a. Griseof ola), amanita sinica (a. Caojizong), amanita horn (a. Spissacea), amanita europaea (a. Obowlanelana), amanita glabra (a. Frigillia) and the like are included. 3-5 ITS sequences were downloaded per species. The common conserved gene sequence among the highly toxic amanita species is determined through gene sequence comparison analysis, and a proper TaqMan probe position is searched between the upstream Primer and the downstream Primer, the Primer and the probe are designed by combining biological software such as Primer Express 3.0, oligo 6.0 and the like, and the specificity of each pair of Primer probes is verified through BLAST comparison. And 3 groups of highly toxic amanita universal primer probe combinations are designed, each pair of combinations comprises 1 upstream primer, 1 downstream primer and 1 probe, CY5 fluorescent groups are marked at the 5 'end of the probe sequence, BHQ2 quenching groups are marked at the 3' end, and the sequences are shown in table 1.
Design of primer probes for Phlebopus portentosus and Phlebopus rarius. Downloading ITS gene sequences of russula vinosa and russula vinosa from NCBI GeneBank database, designing primers and probes by combining biological software such as Primer Express 3.0 and Oligo 6.0, and performing BLAST comparison test on the designed Primer probes on NCBI to ensure the specificity of the primers and probes. Aiming at the Phlebopus portentosus, 4 groups of primer probe combinations are designed, HEX fluorescent groups are marked at the 5 'end of a probe sequence, and BHQ2 quenching groups are marked at the 3' end of the probe sequence; for russula vinosa, 6 groups of primer probe combinations are designed, FAM fluorescent groups are marked at the 5 'end of the probe sequence, BHQ1 quenching groups are marked at the 3' end of the probe sequence, and the sequences are shown in Table 1.
2. Screening of highly toxic mushroom primer probe combinations
DNA templates of positive and negative quality control were detected from the combination of a plurality of groups of highly toxic mushroom primer probes in Table 1. And screening out the optimal primer probe combination with good specificity and repeatability by combining gel electrophoresis with a fluorescence quantitative PCR instrument. As shown in figure 1, the results show that the primer probe combinations 2, 6 and 13 are respectively specific primer probes of the virulent amanita, the pholiota nameko and the russula vinosa, and have no amplified bands to other mushroom species.
3. Optimization of real-time fluorescent PCR reaction system
(1) Optimization of primer probe concentration
Under the condition that other components in the reaction system are unchanged, three groups of screened specific primer probe combinations are added into the same reaction system, different primer concentrations (160 nM-720nM of primers and 80nM increment) and probe (80 nM-360nM of each probe and 40nM increment) concentrations are set, and the optimal primer probe concentration of the multiplex PCR reaction is explored. As shown in Table 3 and FIG. 2, when the total primer concentration is 240nM and the probe concentration is 120nM, the real-time fluorescence PCR amplification reaction shows lower Ct value and stronger fluorescence intensity for 3 types of highly toxic mushrooms. Thus, primer 240nM and probe 120nM are selected as the optimal primer probe concentrations.
TABLE 3 influence of primer probe concentration on fluorescence Ct value of class 3 highly toxic mushrooms
(2) Optimization of annealing temperature
Taking 12 kinds of 3 kinds of extremely toxic mushrooms as positive templates, setting different annealing temperatures (50-60 ℃), and examining the optimal conditions of multiple real-time fluorescent PCR reactions. The RT-PCR reaction conditions are as follows: a pre-denaturation step, wherein the condition is 94 ℃ pre-denaturation for 180s; a cycling step of denaturation at 94℃for 5s, annealing for 15s and elongation at 72℃for 30s,35-45 cycles. As shown in Table 4, the annealing temperature between 51 and 60 ℃ has no significant effect on Ct value of the Phlebopus portentosus (p.gtoreq.0.05) but has significant effect on the highly toxic amanita species and the russia acutifolia (p < 0.05) through Duncan test analysis. In addition, the fluorescence signal intensity increases with the increase of the annealing temperature, and the optimum annealing temperature is finally selected to be 53 ℃ by combining the influence of the Ct value and the fluorescence intensity.
TABLE 4 Effect of annealing temperature on fluorescence Ct value of class 3 highly toxic mushrooms
Note that: the same column of different letters represents a significant difference (P < 0.05, n=3) by analysis using the Duncan's multiple range test method.
3. Real-time fluorescence PCR detection method specificity verification
(1) Collecting and preserving mushroom samples
The collected mushroom samples comprise 34 types of highly toxic amanita, pholiota nameko, russula vinosa and other russula vinosa, toxic or edible amanita and the like, and specifically comprise: huang Gaie Amanita (A.sub-jungulea Imai), propanus (A.panllidorosea), amanita grisea (A.fuliginea Hongo), amanita fatal (A.exitalis), amanita pseudoash (A.fuliginea honiae), amanita schizophyllata (A.rimosa), amanita rubra (A.siamensis), amanita horn Amanita (A.spisasa), amanita pseudo Huang Gaie (A.pseudo-glama), amanita grisea (A.griseoffa), amanita glabra (A.fritiglia), amanita pseudoleopata (A.pseudo-glabra), amanita globosa (A.sub-glabra), amanita gallica (A.caujizoensis), amanita javanica (A.java), and Amanita (A.leopata) and Amanita (A.pantopata). Amanita aweto (a.virgineoides), amanita pestilence (a.sinocirina), amanita parviflora (a.parvipantherina), amanita europaea (a.oberwinklelana), amanita odorifera (a.kotohiraes), a.curtis, pholiota nameko (l.brunneyanata), l.andeavensis, l.sordida, clitocybe coronaria (l.cristata), lentinus edodes (l.edodes), russula vin (r.subnigrica), russula vinosa (r.nigricans), russula japonica (r.japonica hong), russet russula (r.emerica), russet russula vinosa (r.emollica), russia (r.rugosa), and shiveri (c.d.) of the eye. And after the mushroom sample is collected and dried, species identification is carried out through ITS sequencing, and the mushroom sample is preserved at-20 ℃ for later use.
(2) Extraction of mushroom DNA
The DNA of the mushroom sample collected in the above (1) or a commercially available plant DNA extraction kit is extracted by CTAB method.
(3) Real-time fluorescent PCR amplification
The real-time fluorescent PCR reaction system is calculated by 25 mu L and comprises: 4.5. Mu.L of DNA, 12.5. Mu.L of reaction solution, 4.5. Mu.L of primer probe mixture and nuclease-free water. Each reaction reagent is added into a 0.2mLPCR tube, fully vortex and mix evenly, and put into a fluorescent PCR instrument, and the set procedure is as follows: pre-denaturation at 94 ℃ for 180s; denaturation at 94℃for 5s, annealing at 53℃for 15s, elongation at 72℃for 30s,40-50 cycles. After the reaction is finished, whether positive amplification occurs or not is judged by observing a fluorescent signal and an amplification curve. The amplification curve of the sample to be detected is an S-shaped curve, ct is less than or equal to 35, and the sample to be detected is judged to be positive; the sample to be tested has no amplification curve, ct is more than 40, and the sample to be tested is judged as negative; the amplification curve of the sample to be detected has a logarithmic growth phase, but Ct is more than 35 and less than or equal to 40, and the re-detection is carried out after the sample is re-extracted. As shown in FIG. 3, only the highly toxic mushrooms such as the highly toxic amanita, the armillaria sarcochromene and the russian mushroom show an S amplification curve, and no amplification curve appears in other 26 mushroom species, so that the detection method has good specificity.
Example 2
1. Real-time fluorescence PCR detection kit for highly toxic mushrooms and instruction for use
(1) Real-time fluorescence PCR kit for highly toxic mushrooms
A real-time fluorescence PCR detection kit for simultaneously identifying 3 kinds of 12 kinds of highly toxic mushrooms. The kit consists of the following components: primer probe mixed solution (a), reaction solution (B), positive quality control (C), negative quality control (D) and nuclease-free water (E) shown in table 2.
Wherein the primer probe mixed liquid (A) comprises primer probe combinations of 3 types of highly toxic mushrooms. All primer probes were diluted to 10 μm, each set of primer probes was used as the upstream primer: a downstream primer: probe = 2:2:1 volume ratio, and the three groups of primer probe compositions are mixed according to the volume ratio of 1:1:1 to obtain a final mixed solution of the primer and the probe; the reaction solution comprises Taq DNA polymerase, dNTP and MgSO 4 Solutions, etc.; the positive quality control product is a DNA template containing highly toxic amanita, pholiota nameko and russula vinosa; the negative quality control material is Lentinus Edodes DNA template.
In addition, the kit also comprises a PCR tube and a part of instruction for use.
(2) Method for using kit
Multiplex real-time fluorescent PCR total reaction system: extracting DNA in mushroom sample by CTAB method or commercial plant DNA extraction kit, mixing according to table 2, lightly covering PCR eight-connecting tube, centrifuging, mixing, and placing in fluorescent quantitative PCR instrument for amplification detection. The procedure was set up on the machine and the reaction conditions were as follows: pre-denaturation at 94 ℃ for 180s; denaturation at 94℃for 5s, annealing at 53℃for 15s, elongation at 72℃for 30s,40-50 cycles. Meanwhile, positive and negative quality control tests are performed.
And (3) result judgment: positive result judgment: the amplification curve of the sample to be detected is an S-shaped curve, ct is less than or equal to 35, and the sample to be detected is judged to be positive; negative result judgment: the sample to be tested has no amplification curve, ct is more than 40, and the sample to be tested is judged as negative; the amplification curve of the sample to be detected has a logarithmic growth phase, but Ct is more than 35 and less than or equal to 40, and the re-detection is carried out after the sample is re-extracted.
(3) Sensitivity test of kit
The extracted DNA of the Huang Gaie paste, the Phlebopus portentosus and the Phlebopus portentosus was diluted in a 10-fold gradient, specifically 10 ng/. Mu.L, 1.0 ng/. Mu.L, 100 pg/. Mu.L, 10 pg/. Mu.L, 1 pg/. Mu.L, 0.1 pg/. Mu.L, 0.01 pg/. Mu.L and ddH 2 O is marked as numbers 1 to 8 in turn. The reaction solutions were mixed in the proportions described in step (2) of example 2, and then amplified by qPCR. As shown in FIG. 4, all three fluorescent channels were amplified, and were detected even at a DNA template concentration of 0.01 pg/. Mu.L, indicating that the detection sensitivity of the kit was high.
(4) Stability test of kit
The DNA mixture of the drastic amanita, the pholiota nameko and the russula vinosa is used as a template, and templates with gradient concentrations of 10 ng/. Mu.L, 1 ng/. Mu.L and 0.1 ng/. Mu.L are prepared. Detecting by using the method established by the invention, repeating each concentration for 3 times, and carrying out in-group repeatability analysis; 3 different times are selected, DNA mixtures with different concentrations are used as templates, the repetitive analysis among groups is carried out, the variation coefficient is calculated, and the repeatability of the method is analyzed. Negative controls were also established. The coefficients of variation (%) between and within the groups were calculated, respectively, and the reproducibility of the method was examined based on the coefficients of variation. The results are shown in Table 6 when the template concentration is 10-10 -1 When the corresponding Ct value is about 19-29, the intra-group variation coefficient is 0.05% -5.61%; the inter-group variation coefficient is 1.10% -8.82%, which indicates that the established triple fluorescence assayThe quantitative PCR detection method has good stability and repeatability.
Table 5 stability test of three types of highly toxic mushrooms real-time fluorescence PCR detection kit
Example 3
Application of real-time fluorescence PCR detection kit for highly toxic mushrooms
In order to verify the applicability of the invention in practical samples, the situation that toxic bacteria are mixed in edible mushrooms is simulated, and the embodiment simulates the influence of mushroom mixture and processing process on the accuracy of detection results. The specific method comprises the following steps: mixing the mass of the drastic amanita, the dried samples of the pholiota nameko and the russula vinosa, etc. to prepare a premix, wherein the mass of the mixture is 150mg, and then mixing the premix and the mushroom samples according to different mass ratios, and the mass ratio of the premix to the mushroom is 1/0,1/1,1/3,1/9,1/99 and 1/999 respectively. 50mg of the mixed mushroom sample was boiled with 1.5mL of pure water for 15min, simulating the cooking process of mushrooms. Taking mushroom residual fruiting body and soup respectively, extracting DNA by adopting the CTAB method in the step 3 of the embodiment 1, and then measuring actual samples according to the real-time fluorescence PCR detection kit and the method in the embodiment 2. As shown in Table 6, when a sample containing 1% of extremely toxic mushrooms was examined, 3 kinds of extremely toxic mushrooms could be detected; when 0.1% of the highly toxic mushrooms are contained, russula vinosa and russula vinosa can be detected. The results show that once mushroom poisoning occurs, mushroom fruiting body residues or soup can be collected as samples for detection.
TABLE 6 analysis of test results of highly toxic mushrooms in simulated mushroom processing samples
Note that: nd is undetected.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A set of multiplex primer sets for simultaneously identifying 3 types of highly toxic mushrooms, wherein the types of highly toxic mushrooms comprise highly toxic amanita, pholiota nameko and russula vinosa;
the primer set designed for the highly toxic amanita comprises an upstream Universal primer Universal A.spp-F, a downstream Universal primer Universal A.spp-R and a probe Universal A.spp-P, wherein the nucleotide sequence of the upstream Universal primer Universal A.spp-F is shown as SEQ ID NO.1, the nucleotide sequence of the downstream Universal primer Universal A.spp-R is shown as SEQ ID NO.2, and the nucleotide sequence of the probe Universal A.spp-P is shown as SEQ ID NO. 3;
the primer set designed for the Phlebopus portentosus comprises an upstream primer L.bru-F, a downstream primer L.bru-R and a probe L.bru-P, wherein the nucleotide sequence of the upstream primer L.bru-F is shown as SEQ ID NO.4, the nucleotide sequence of the downstream primer L.bru-R is shown as SEQ ID NO.5, and the nucleotide sequence of the probe L.bru-P is shown as SEQ ID NO. 6;
the primer group designed for russula vinosa comprises an upstream primer R.sub-F, a downstream primer R.sub-R and a probe R.sub-P, wherein the nucleotide sequence of the upstream primer R.sub-F is shown as SEQ ID NO.7, the nucleotide sequence of the downstream primer R.sub-R is shown as SEQ ID NO.8, and the nucleotide sequence of the probe R.sub-P is shown as SEQ ID NO. 9.
2. The multiplex primer set according to claim 1, wherein the highly toxic amanita comprises a deadly amanita (Amanitin exitialis), an ash amanita (a. Fuliginea Hongo), an amanita pseudoash (a. Fuligineas), a light red amanita (a. Palidorosea), a split skin amanita (a. Rimosaa), a Huang Gaie amanita (a. Subjunquillea), a soft-touch amanita (a. Moliuscula), an ash-inferior amanita (a. Subfuliginea), a false red amanita (a. Subpalalidorosea) and a light-handle amanita (a. Virusa).
3. The multiplex primer set according to claim 1, wherein each probe has a different fluorescent group at the 5 'end and a quenching group at the 3' end.
4. The multiplex primer set according to claim 3, wherein the fluorescent group is selected from any one of FAM, VIC, ROX and CY5, and the quenching group is BHQ1 or BHQ2.
5. A kit for simultaneously identifying 3 types of highly toxic mushrooms, comprising the mixed solution of the multiplex primer set according to any one of claims 1 to 4.
6. The kit of claim 5, wherein the concentration ratio of the upstream primer, the downstream primer and the probe in the mixed solution is 2:2:1,
and the concentration ratio of the multiple primer groups designed for the amanita fuliginea, the pholiota nameko and the russula vinosa is 1:1:1.
7. the kit according to claim 5, wherein the kit further comprises a reaction solution, a positive quality control product, a negative quality control product and water without a nuclease.
8. The kit according to claim 5, wherein the working concentration of each primer in the kit is 0.16 to 0.72. Mu.M, and the working concentration of each probe is 0.08 to 0.36. Mu.M.
9. A multiplex real-time fluorescent PCR method for simultaneously identifying class 3 highly toxic mushrooms, comprising the steps of: preparing a reaction system by using DNA of a sample to be detected as a template and the mixed solution of the multiplex primer group of any one of claims 1-4 or the reagent in the kit of any one of claims 5-8, and performing multiplex real-time fluorescent PCR reaction, wherein if the amplification curve of the sample to be detected is an S-shaped curve and Ct is less than or equal to 35, judging as positive.
10. The multiplex real-time fluorescent PCR method as set forth in claim 9, wherein the reaction procedure of the multiplex real-time fluorescent PCR reaction includes: pre-denaturation at 94 ℃ for 180s; denaturation at 94℃for 5s, annealing at 53℃for 15s, elongation at 72℃for 30s,40-50 cycles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211409430.XA CN116042892B (en) | 2022-11-11 | 2022-11-11 | Multiple primer group, kit and detection method for simultaneously identifying 3 types of highly toxic mushrooms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211409430.XA CN116042892B (en) | 2022-11-11 | 2022-11-11 | Multiple primer group, kit and detection method for simultaneously identifying 3 types of highly toxic mushrooms |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116042892A true CN116042892A (en) | 2023-05-02 |
| CN116042892B CN116042892B (en) | 2023-08-25 |
Family
ID=86126239
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211409430.XA Active CN116042892B (en) | 2022-11-11 | 2022-11-11 | Multiple primer group, kit and detection method for simultaneously identifying 3 types of highly toxic mushrooms |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116042892B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100267019A1 (en) * | 2007-11-09 | 2010-10-21 | Michigan State University | Identification And Use Of Genes Encoding Amatoxin And Phallotoxin |
| CN102719544A (en) * | 2012-06-27 | 2012-10-10 | 广东省微生物研究所 | Characterized nucleotide sequence, nucleic acid molecular probes and method for identifying Amanita exitialis |
| CN106568769A (en) * | 2016-11-10 | 2017-04-19 | 中国科学院昆明植物研究所 | Extremely toxic mushroom rapid detection method |
| CN111575397A (en) * | 2020-06-01 | 2020-08-25 | 中国检验检疫科学研究院 | Toxic amanita species identification method based on DNA micro-barcode technology and application |
| CN114058726A (en) * | 2021-10-29 | 2022-02-18 | 中国检验检疫科学研究院 | Primer group, kit and method for identifying poisonous mushroom ringscale amantake |
| CN114875174A (en) * | 2022-06-28 | 2022-08-09 | 上海市农业科学院 | LAMP primer group, kit and detection method for rapidly identifying russula vinosa |
-
2022
- 2022-11-11 CN CN202211409430.XA patent/CN116042892B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100267019A1 (en) * | 2007-11-09 | 2010-10-21 | Michigan State University | Identification And Use Of Genes Encoding Amatoxin And Phallotoxin |
| CN102719544A (en) * | 2012-06-27 | 2012-10-10 | 广东省微生物研究所 | Characterized nucleotide sequence, nucleic acid molecular probes and method for identifying Amanita exitialis |
| CN106568769A (en) * | 2016-11-10 | 2017-04-19 | 中国科学院昆明植物研究所 | Extremely toxic mushroom rapid detection method |
| CN111575397A (en) * | 2020-06-01 | 2020-08-25 | 中国检验检疫科学研究院 | Toxic amanita species identification method based on DNA micro-barcode technology and application |
| CN114058726A (en) * | 2021-10-29 | 2022-02-18 | 中国检验检疫科学研究院 | Primer group, kit and method for identifying poisonous mushroom ringscale amantake |
| CN114875174A (en) * | 2022-06-28 | 2022-08-09 | 上海市农业科学院 | LAMP primer group, kit and detection method for rapidly identifying russula vinosa |
Non-Patent Citations (2)
| Title |
|---|
| SARA EPIS等: "Molecular detection of poisonous mushrooms in different matrices", MYCOLOGUA, vol. 102, no. 3, pages 747 - 754 * |
| 章轶哲等: "基于Taqman-MGB 探针的亚稀褶红菇实时荧光定量PCR 检测 方法", 菌物学报, vol. 39, no. 5, pages 937 - 943 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116042892B (en) | 2023-08-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111235232B (en) | Visual rapid nucleic acid detection method based on CRISPR-Cas12a system and application | |
| CN106811513B (en) | Eucalyptus component real-time fluorescence PCR detection method and kit thereof | |
| CN114875174B (en) | LAMP primer group, kit and detection method for rapidly identifying russula vinosa | |
| US20240158873A1 (en) | Dna barcode for screening floccularia luteovirens with high antioxidant activity | |
| US20240158783A1 (en) | Dna barcode for screening total soluble protein content index of floccularia luteovirens | |
| CN109777861A (en) | The loop-mediated isothermal amplification method of mispairing tolerance and application | |
| CN116042892B (en) | Multiple primer group, kit and detection method for simultaneously identifying 3 types of highly toxic mushrooms | |
| CN111690759B (en) | Specific primer, kit and method for detecting RPA of citrus canker pathogen | |
| CN103710463B (en) | Rapid detection kit and method of strawberry mild yellow edge virus | |
| CN112852995A (en) | Primer probe composition, kit and detection method for detecting fusarium graminearum | |
| CN116179752B (en) | Primer group for identifying toxic mushroom species and PCR detection method | |
| CN116516036A (en) | Primer and probe combination for detecting Pantoea ananatis by LAMP method and application | |
| CN103667526A (en) | Rapid detection kit and method for strawberry mottle virus | |
| WO2023087787A1 (en) | Dna barcode for screening floccularia luteovirens using content of total soluble amino acid as index | |
| WO2023087791A1 (en) | Dna barcode for identifying origin of floccularia luteovirens, primer group, and application | |
| WO2023087784A1 (en) | Dna barcode for screening floccularia luteovirens using content of total polyphenol as index | |
| CN114231603B (en) | Primer, reagent, identification method and kit for identifying paeonia boita | |
| CN112048573B (en) | RPA primer and kit for detecting cotton leaf curl virus, and detection method and application thereof | |
| CN110923331B (en) | Primer pair and application thereof in identification of limnodrilus | |
| CN107435064B (en) | qPCR (quantitative polymerase chain reaction) method for rapidly and quantitatively detecting harmful golden algae in chlorella culture | |
| CN117535392B (en) | RPA primer and kit for identifying sex of swan and application | |
| CN116875724A (en) | Composition and kit for detecting russula vinosa and application of composition and kit | |
| CN114182039B (en) | Primer group and kit for identifying ocher deer fungus and application of primer group and kit | |
| CN112481393B (en) | PCR detection kit for identifying Hirudo japonica | |
| CN112680441B (en) | Complete set of reagents and method for detecting 4 alfalfa RNA viruses |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |