CN114854898B - Primer and probe for simultaneously detecting authenticity of Mongolian Bai Limo and agaricus bisporus - Google Patents
Primer and probe for simultaneously detecting authenticity of Mongolian Bai Limo and agaricus bisporus Download PDFInfo
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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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- 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
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- 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
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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 primer and a probe for simultaneously detecting the authenticity of Mongolian Bai Limo and agaricus bisporus, wherein the sequences of the primer and the probe are as follows: the forward primer sequence is shown as SEQ ID No. 1; the reverse primer sequence is shown as SEQ ID No. 2; the sequence of the Mongolian Bai Limo probe is shown as SEQ ID No. 3; the agaricus bisporus probe sequence is shown in SEQ ID No. 4; the quality control probe sequence is shown as SEQ ID No. 5. The primer and the probe have good specificity and high sensitivity, can realize the detection of the Mongolian white mushroom, the agaricus bisporus and the quality control same tube, and can perform the quantitative detection of the Mongolian Bai Limo and the agaricus bisporus.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to the field of mushroom detection in edible mushrooms.
Background
Mongolian Bai Limo has flat property, sweet taste and effects of diffusing intestine, tonifying qi, dissipating heat and relieving exterior syndrome. Because of the large fruiting body, the fungus meat is compact and fat, has fresh and tender texture and unique fragrance, and is a wild edible fungus which is far and near known. Mongolia Bai Limo is rare in that the fragrant and delicious taste is possessed, and the fragrant and delicious taste contains a large amount of balsam volatile substances, sodium glutamate, bai Limo amino acid, 5' -guan glycol and other delicious substances and flavor enhancing substances, and the fragrant and delicious aftertaste is endless, and can be smelled at a long distance. The white mushroom in Mongolian is high in nutritive value, is rich in polysaccharide, polypeptide, polyunsaturated fatty acid, 8 amino acids necessary for human body, various vitamins and minerals such as iron, calcium, phosphorus and selenium, has the medical health care effects of resisting oxidation, resisting aging, resisting tumor, reducing blood pressure, blood fat, blood sugar and the like, and can treat symptoms such as dryness-heat disorder. The amino acid component can be balanced with amino acid component in animal food, has better absorption effect in human body, can regulate human body function, and can enhance immunity, and is suitable for dyspepsia, abdominal distending pain and stomach ache. Can also reduce blood pressure, treat cartilage disease, and has anticancer effect. Has bactericidal effect, and can be used for treating traumatic injury and toxic materials by recording Bai Limo of Mongolian. It belongs to low calorie food, and is very suitable for people with weight loss and body building. Due to the influence of human factors and climate factors, the population quantity of the Mongolian white mushrooms is reduced, the quality and the yield of the Mongolian white mushrooms are lower, the requirements of commercialization and marketization are difficult to meet, and the price of the Mongolian white mushrooms is increased. Because of the high price and the high specific and rare value, the phenomenon of confusing counterfeit and inferior products and other kinds of wild bacteria often occurs in the market. Whereas merchants often sell as Mongolian Gu Baili mushrooms, which are widely available and readily available. For example, commercial edible mushrooms such as pleurotus eryngii, pleurotus geesteranus, agaricus bisporus and the like are often used to make Bai Limo mushroom sauce.
The research in the field of edible fungi identification technology in China starts late, and the research in the field is still in a preliminary stage. Therefore, the current research field in the edible fungus authenticity identification technology aspect realizes the safe and efficient detection technology with independent property rights in China, and is a research subject which is full of opportunities and has challenges at the same time. Tin Lin Guole used as a producing area of the white mushroom in Mongolia is urgently required to develop a related mushroom authenticity identification technology and detection standard of the white mushroom Bai Limo and the agaricus bisporus with independent intellectual property rights so as to protect local special mushrooms and maintain legal rights and interests of consumers. At present, the technical researches, detection standards, invention patents and commercial kits related to mushroom authenticity identification are mainly concentrated on the technology of common PCR ITS, and the reports of real-time fluorescence PCR direct detection and multichannel homogeneous control quality control detection are less. False negatives are also a bottleneck which plagues the wide application of PCR technology in the identification of mushrooms. The same quality control is an effective means of removing false negatives.
Disclosure of Invention
The technical problems to be solved by the invention are as follows: how to provide a primer and a probe for detecting Mongolian white mushroom, agaricus bisporus and quality control same tube in high-efficiency and high-specificity mushrooms, and solve the problems of qualitative and quantitative detection of Mongolian Bai Limo and agaricus bisporus source components in mushrooms.
The technical scheme of the invention is as follows: primer and probe combinations for detecting the white mushroom, the agaricus bisporus and the quality control same tube, and the sequences of the primer and the probe are as follows:
the forward primer sequence is shown as SEQ ID No. 1;
the reverse primer sequence is shown as SEQ ID No. 2;
the sequence of the Mongolian Bai Limo probe is shown as SEQ ID No. 3;
the agaricus bisporus probe sequence is shown in SEQ ID No. 4;
the quality control probe sequence is shown as SEQ ID No. 5.
Further, the 5 'end of the Mongolian Bai Limo probe, the agaricus bisporus probe and the quality control probe sequence is modified with a reporter group, the 3' end is modified with a quenching group, the reporter group is any one of FAM, HEX, ROX or CY5, and the quenching group is any one of TAMRA, MGB, BHQ or BHQ 2.
The primer and probe combination is applied to detection of the white mushroom and the agaricus bisporus.
A kit comprising the primer and probe combination described above.
The method for detecting the mongolian Bai Limo source, the agaricus bisporus source and the quality control common tube in the mushrooms comprises the following steps:
(1) Extracting DNA of mushroom samples;
(2) Taking the DNA of the step (1) as a template, carrying out multiplex fluorescence quantitative PCR amplification by using primers and probes of SEQ ID No. 1-SEQ ID No.5, taking positive standard substances of Mongolian Bai Limo and agaricus bisporus as positive control, taking sterilized deionized water as negative control, and taking blank control of DNA extraction as a control group of an extraction method; (3) Setting Threshold as automatic after the Real-time PCR reaction is finished, and reading Ct values of the corresponding probes of the white mushroom, the agaricus bisporus and the quality control and Ct values of the positive control, the negative control and the blank control; judging the source result of the corresponding probe only when the quality control Ct is less than or equal to 40 and the positive control Ct is less than or equal to 40 and the negative control Ct and the blank control Ct are 0; when the Ct of the corresponding probe is less than or equal to 40, the result is judged to have the corresponding source, and the Ct of the plurality of probes is less than or equal to 40, and the result is judged to have the corresponding mushrooms;
(4) Using Mongolian Bai Limo and agaricus bisporus positive standard as a standard curve of DNA quantification;
(5) And obtaining a quantitative detection result of the corresponding mushroom source in the mushrooms by using the Ct value of the corresponding mushroom source in the mushrooms and a formula in a standard curve.
Further, real-time PCR amplification parameters were: the pre-denaturation temperature was 94 ℃,30s, denaturation temperature 94 ℃,5s, annealing extension temperature 60 ℃,31s,40 cycles.
Further, the Real-time PCR reaction system is as follows: 10 mu L of Probe qPCR premix, 1 mu L of forward primer shown in SEQ ID No.1 and 10 mu mol/L concentration; 1. Mu.L of the reverse primer shown in SEQ ID No.2 at a concentration of 10. Mu. Mol/L; mongolian Bai Limo probe shown in SEQ ID No.3 is 0.5 mu L with the concentration of 10 mu mol/L; the agaricus bisporus probe shown in SEQ ID No.4 is 0.5 mu L, the concentration is 10 mu mol/L, and the quality control probe shown in SEQ ID No.5 is 0.5 mu L, and the concentration is 10 mu mol/L; DNA template 2. Mu.L, sterilized deionized water 4.5. Mu.L, total volume 20. Mu.L.
17 edible fungi ITS genomes of white mushroom, agaricus bisporus, apricot mushroom, pleurotus geesteranus, lentinus edodes, pleurotus eryngii, flammulina velutipes, hypsizigus marmoreus, white beech mushroom, stropharia rugoso-annulata, tricholoma matsutake, morchella, agrocybe cylindracea, agaricus blazei and the like are compared, and ITS genome sequences of 10 varieties or strains are selected for each edible fungus. The 170 sequences are compared through bioinformatics software, the conserved and specific sequences of Mongolian Bai Limo and agaricus bisporus are screened, and primer and probe are designed through primer design software. The innovation of the design is that the sequences with the conservation at both ends and the specificity in the middle are needed to be screened out on the sequences with the conservation at both ends, the primers are designed at the conservation positions at both ends, and the probes are designed at the specificity in the middle. The conserved primer and the specific probe can effectively reduce the mismatch between the primers and the competition of a plurality of PCR reactions to reaction resources, and can ensure the performance of multiple real-time fluorescent quantitative PCR reactions. Multiplex real-time fluorescent quantitative PCR reactions are the basis for detection of multisource components. The annealing temperature of the primer and the probe is controlled between 55 ℃ and 60 ℃ and 65 ℃ and 70 ℃, the secondary structure which affects the annealing efficiency is avoided, the primer and the probe are ensured to have high specificity on ITS genes, and the design ensures that the primer and the probe can be used for subsequent qualitative and quantitative detection.
The invention develops 3-channel detection primers and probes for Mongolian white mushroom, agaricus bisporus, quality control and the like, and optimizes the primer and probe combination for multi-channel multi-mushroom detection and same-control quality control detection. In the process, the problems of influence between various primers and probes in the same PCR reaction system and competition between a template and PCR reaction resources are solved, and the effect that the PCR reaction system can simultaneously perform multiple real-time fluorescence PCR is achieved.
Compared with the prior art, the invention has the following beneficial effects:
the primer and the probe have strong specificity and high sensitivity, can realize qualitative and quantitative detection of Mongolian Bai Limo and agaricus bisporus in mushrooms, can detect Mongolian white mushroom, agaricus bisporus and quality control simultaneously, saves working procedures and reduces cost.
Drawings
In FIG. 1, the quality control controls of the agaricus bisporus marked by HEX and TAMRA modified probes, the agaricus bisporus marked by FAM and MGB modified probes and the agaricus bisporus marked by ROX and BHQ2 modified probes were used for detecting real-time fluorescence quantitative PCR of 15 mushrooms (other mushrooms) such as apricot, pleurotus geesteranus, lentinus edodes, pleurotus eryngii, flammulina velutipes, hypsizigus marmoreus, money mushroom, stropharia rugoso-annulata, tricholoma matsutake, morchella, tea tree mushroom and agaricus blazei, and the amplification curves appear in the agaricus bisporus, and the amplification curves do not appear in the other mushrooms, which indicates that the agaricus bisporus primers and probes have specificity.
FIG. 2 shows the detection of real-time fluorescence quantitative PCR of 15 mushrooms (other mushrooms) such as Pleurotus eryngii, pleurotus geesteranus, lentinus edodes, pleurotus eryngii, flammulina velutipes, hypsizygus marmoreus, pleurotus geesteranus, stropharia rugoso-annulata, morchella, agrocybe cylindracea, agrocybe aegerita, agaricus blazei, etc., using HEX and TAMRA modified probes to label Mongolian Bai Limo, FAM and MGB modified probes to label agaricus bisporus, and ROX and BHQ2 modified probes to label quality control controls, showing that no amplification curves appear in agaricus bisporus, indicating that the agaricus primer and probe have specificity.
FIG. 3 detection sensitivity amplification experiments were performed on Mongolian Bai Limo DNA (100 ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng, 0.0005ng, 0.00025ng, 0.0001ng and 0.00001 ng) using HEX and TAMRA modified probes to label Mongolian Bai Limo, and Mongolian Bai Limo DNA of 1pg could be detected by Mongolian white mushroom probes. The results above demonstrate that the mongolian Bai Limo probe has higher sensitivity in mushroom detection.
FIG. 4 detection sensitivity amplification experiments on agaricus bisporus DNA (100 ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng, 0.0005ng, 0.00025ng, 0.0001ng and 0.00001 ng) using FAM and MGB modified probe-labeled agaricus bisporus, the agaricus bisporus probe can detect 0.25pg of agaricus bisporus DNA. The above results indicate that the agaricus bisporus probe has higher sensitivity in the aspect of mushroom detection.
Fig. 5 mongolian white mushroom detection standard curve: the method is used for quantitative detection of Mongolia Bai Limo in mushrooms.
FIG. 6 agaricus bisporus detection standard curve: the method is used for quantitatively detecting agaricus bisporus in the mushrooms.
Detailed Description
1. The detection method comprises the following steps:
(1) DNA of the mushroom sample was extracted, and an extraction blank (control group for the subsequent extraction method) was established.
(2) The concentration and quality of DNA were measured, and the concentration was diluted to 100-200 ng/. Mu.L.
(3) The diluted DNA is amplified and detected by using multiplex fluorescent quantitative PCR primers and probes, mongolian Bai Limo and agaricus bisporus positive standard substances are used as positive control, sterilized deionized water is used as negative control, blank control of DNA extraction is used as control group of the extraction method, a Real-time PCR reaction system is shown in table 1, and Real-time PCR amplification parameters are shown in table 4.
TABLE 1 Real-time PCR reaction System (Simultaneous detection of Mongolian white mushroom, agaricus bisporus and quality control)
| Composition of the components | Volume (microliter) |
| Probe qPCR premix | 10 |
| Forward primer | 1 |
| Reverse primer | 1 |
| Mongolian Bai Limo probe | 0.5 |
| Agaricus bisporus probe | 0.5 |
| Quality control probe | 0.5 |
| DNA | 2 |
| Sterilized deionized water | 4.5 |
| Total volume of | 20 |
TABLE 2 Real-time PCR reaction system (Mongolian white mushroom and quality control simultaneous detection)
TABLE 3 Real-time PCR reaction System (Agaricus bisporus and quality control Simultaneous detection)
| Composition of the components | Volume (microliter) |
| Probe qPCR premix | 10 |
| Forward primer | 1 |
| Reverse primer | 1 |
| Agaricus bisporus probe | 0.5 |
| Quality control probe | 0.5 |
| DNA | 2 |
| Sterilized deionized water | 5 |
| Total volume of | 20 |
TABLE 4 Real-time PCR amplification parameters
(4) Setting Threshold as automatic after the Real-time PCR reaction is finished, and reading Ct values of the corresponding probes of the white mushroom, the agaricus bisporus and the quality control and Ct values of the positive control, the negative control and the blank control; only when the quality control Ct is less than or equal to 40 and the positive control Ct is less than or equal to 40, and the negative control and the blank control Ct are 0, judging the corresponding mushroom probe result; when Ct of the corresponding probe is less than or equal to 40, the result is judged to have the corresponding mushroom source, while Ct of the plurality of probes is less than or equal to 40, and the result is judged to have the corresponding two mushroom sources.
2. Design of primer and Probe sequences
Since the difference in ITS of fungal DNA is small, fungal DNA ITS genome was selected to design the mongolica, agaricus bisporus and quality control detection primers and probes. The synthesis method of the primer and the probe comprises the following steps: the Beijing Rui Boxing family biological company is entrusted to synthesize and purify according to the invented sequence.
Forward primer: 5'CTTGCGCTCCTTGGTATTC 3' (SEQ ID No. 1),
reverse primer: 5'GCTAATGYWTTTMAGAGGAGC 3' (SEQ ID No. 2),
mongolian Bai Limo probe: 5'CTTTTCAGCTTTTGCGAGTTGGATTG 3' (SEQ ID No. 3),
agaricus bisporus probe: 5'TATTCTCAACTCTCCAATACTTTGTT 3' (SEQ ID No. 4),
and (3) a quality control probe: 5'AGGAGCATGCCTGTTTGAGTGTCAT 3' (SEQ ID No. 5);
the 5 'end of the sequence of the Mortierella mongolica, the agaricus bisporus and the quality control probe is modified with a reporter group, the 3' end of the sequence of the Mortierella mongolica, the agaricus bisporus and the quality control probe is modified with a quenching group, wherein the reporter group is any one of FAM, HEX, ROX or CY5, and the quenching group is any one of TAMRA, MGB, BHQ or BHQ 2.
3. Specific detection of primers and probes
Real-time PCR reaction system for single mushroom origin detection is shown in the following table
| Composition of the components | Volume (microliter) |
| Probe qPCR premix | 10 |
| Forward primer | 1 |
| Reverse primer | 1 |
| Corresponding mushroom-derived probes | 0.5 |
| Quality control probe | 0.5 |
| DNA | 2 |
| Sterilized deionized water | 4.5 |
| Total volume of | 20 |
And (3) performing qPCR detection on Mongolian white mushroom, agaricus bisporus, apricot shiitake mushroom, pleurotus geesteranus, mushroom, pleurotus eryngii, flammulina velutipes, seafood mushrooms, white beech mushrooms, white mushroom, money mushrooms, worm mushrooms, beef tripe mushrooms, pine mushrooms, morchella, tea mushrooms and agaricus blazei by utilizing HEX and MGB modified probes to mark Mongolian Bai Limo, FAM and MGB modified probes to mark agaricus bisporus and ROX and BHQ2 modified probes to mark quality control contrast.
The detection results are as follows:
ct value: mean (three sets of data) ± standard deviation;
the results illustrate: ct less than 40 (not 0) indicates a corresponding mushroom origin in the sample. The detection result accords with the mushroom source of the sample. Mongolian Bai Limo source was detected in the Mongolian white mushroom, agaricus bisporus source was detected in the agaricus bisporus, and Mongolian Bai Limo source and agaricus bisporus source were not detected in the agaricus bisporus.
4. Detection limit experiment of primer and probe for detecting mushroom origin
Sterilizing genomic DNA of Mongolian Bai Limo and Agaricus bisporus with ddH 2 The amplification experiments were performed at detection limits of the primers and probes by diluting with O to concentrations of 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 0.1 ng/. Mu.L, 0.01 ng/. Mu.L, 0.001 ng/. Mu.L, 0.0005 ng/. Mu.L, 0.00025 ng/. Mu.L, 0.0001 ng/. Mu.L and 0.00001 ng/. Mu.L for 10 times. From the following results, it was found that the Mongolian probe could detect 1pg of Mongolian Bai Limo DNA in the sample, and the agaricus bisporus probe could detect 0.25pg of agaricus bisporus DNA in the sample. The above results demonstrate that the detection limits of the independently developed mongolian Bai Limo and mushroom primers and probes reach the pg level, and the detection sensitivity is higher.
The detection results are as follows:
ct value: mean (three sets of data) ± standard deviation; N/A is not suitable for detection
5. And (3) marking Mongolian Bai Limo by using HEX and MGB modified probes and marking agaricus bisporus by using FAM and MGB modified probes, and respectively establishing standard curves of Mongolian Bai Limo and agaricus bisporus.
The detection results are as follows:
the amplification efficiency is close to 90% -110%, and R is linearly fitted 2 The results show that the standard curves of the independently developed Mongolian Bai Limo and agaricus bisporus primers and probes are good and can be quantitatively detected.
Sequence listing
<110> tin Lin Guole professional college tin Lin Guole allied Kun Yuan Biotechnology Co., ltd
<120> a primer and probe for simultaneously detecting the authenticity of Mongolian Bai Limo and agaricus bisporus
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
cttgcgctcc ttggtattc 19
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gctaatgywt ttmagaggag c 21
<210> 3
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
cttttcagct tttgcgagtt ggattg 26
<210> 4
<211> 26
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
tattctcaac tctccaatac tttgtt 26
<210> 5
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
aggagcatgc ctgtttgagt gtcat 25
Claims (1)
1. The method for detecting the Mongolian white mushroom, the agaricus bisporus and the quality control common tube in the mushrooms is characterized by comprising the following steps:
(1) Extracting DNA of mushroom samples, and setting up an extracted blank control;
(2) Taking the DNA of the step (1) as a template, carrying out multiplex fluorescence quantitative PCR amplification by using primers and probes of SEQ ID No. 1-SEQ ID No.5, taking positive standard substances of Mongolian Bai Limo and agaricus bisporus as positive control, taking sterilized deionized water as negative control, and taking blank control of DNA extraction as control group of an extraction method;
(3) Setting Threshold as automatic after the Real-time PCR reaction is finished, and reading Ct values of the corresponding probes of the white mushroom, the agaricus bisporus and the quality control and Ct values of the positive control, the negative control and the blank control; only when the quality control Ct is less than or equal to 40 and the positive control Ct is less than or equal to 40, and the negative control and the blank control Ct are 0, judging the corresponding mushroom probe result; when the Ct of the corresponding probe is less than or equal to 40, the result is judged to have the corresponding mushroom source, and meanwhile, the Ct of the plurality of probes is less than or equal to 40, and the result is judged to have the corresponding two mushroom sources;
(4) Using Mongolian Bai Limo and agaricus bisporus positive standard as a standard curve of DNA quantification;
(5) The quantitative detection result of the corresponding mushroom source in the mushrooms can be obtained by utilizing the Ct value of the corresponding mushroom source in the mushrooms and a formula in a standard curve;
the Real-time PCR amplification parameters were: pre-denaturation temperature 94 ℃,30s, denaturation temperature 94 ℃,5s, annealing extension temperature 60 ℃,31s,40 cycles;
the Real-time PCR reaction system is as follows: 10 mu L of Probe qPCR premix; 1 mu L of forward primer shown in SEQ ID No.1 with concentration of 10 mu mol/L; 1. Mu.L of the reverse primer shown in SEQ ID No.2 at a concentration of 10. Mu. Mol/L; mongolian Bai Limo probe shown in SEQ ID No.3 is 0.5 mu L with the concentration of 10 mu mol/L; the agaricus bisporus probe shown in SEQ ID No.4 is 0.5 mu L and the concentration is 10 mu mol/L; the quality control probe shown in SEQ ID No.5 is 0.5 mu L, and the concentration is 10 mu mol/L; 2. Mu.L of DNA template; and 4.5. Mu.L of sterilized deionized water in a total volume of 20. Mu.L.
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| JP2014155486A (en) * | 2013-01-16 | 2014-08-28 | Japan Health Sciences Foundation | Mushroom identification method and identification kit |
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| CN112662570A (en) * | 2021-01-21 | 2021-04-16 | 拉萨市高原生物研究所 | Brown agaricus bisporus XZ2 strain and application thereof |
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|---|---|---|---|---|
| CN103298953A (en) * | 2010-10-28 | 2013-09-11 | 株式会社雪国舞茸 | Method for selecting fungus body and kit |
| JP2014155486A (en) * | 2013-01-16 | 2014-08-28 | Japan Health Sciences Foundation | Mushroom identification method and identification kit |
| CN109706085A (en) * | 2018-12-25 | 2019-05-03 | 内蒙古自治区农牧业科学院 | No. 7 bacterial strains of one plant of Tricholoma mongolicum prairie Tricholomataceae and its acclimation method |
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