CN114854899B - Primer probe combination for detecting amygdalina and agaricus bisporus and detection method - Google Patents
Primer probe combination for detecting amygdalina and agaricus bisporus and detection method Download PDFInfo
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- 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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- 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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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The invention discloses a primer probe combination for detecting amygdalina and agaricus bisporus, which comprises forward and reverse primers, an amygdalina probe, an agaricus bisporus probe and a quality control probe; the sequences of the forward and reverse primers are shown as SEQ ID No.1 and SEQ ID No. 2; the sequences of the Pleurotus eryngii probe, the Agaricus bisporus probe and the quality control probe are sequentially shown as SEQ ID No.3-SEQ ID No. 5. The invention also discloses a method for detecting the amygdalina and agaricus bisporus by using the primer probe combination. The primer probe combination in the invention has strong specificity and high sensitivity, realizes qualitative and quantitative detection of the amygdalina and the agaricus bisporus, and obviously improves the detection accuracy of the amygdalina and the agaricus bisporus; the same-tube detection can be carried out on the amygdalina, the agaricus bisporus and the quality control, so that the detection procedures and time are effectively saved, and the detection cost is obviously reduced.
Description
Technical Field
The invention relates to the technical field of food detection, in particular to a primer probe combination for detecting amygdalina and agaricus bisporus and a detection method.
Background
Agaricus bisporus is also known as white mushroom, tricholoma matsutake, mushroom etc. The agaricus bisporus fruiting body is medium and large, the fungus cover is 5-12 cm wide, the fungus cover is semi-spherical, flat, white and smooth after being flattened, slightly dry and gradually changed into yellow, and the edge is rolled inwards in the initial stage. The fungus meat is white and thick, and turns light red after injury, and has the special smell of mushrooms. The fungus pleat is pink, and turns brown to black brown, is dense, narrow, free of growth, unequal in length, 4.5-9 cm long in length, 1.5-3.5 cm thick, white, smooth, mercerized, nearly cylindrical, soft or medium in interior, single-layer in fungus ring, white, membranous and easy to fall off in the middle of the raw fungus handle. The agaricus bisporus has delicious taste and is an edible fungus with large cultivation scale and wide cultivation range. The protein content is up to 42% (dry weight), the amino acid variety is rich, and the nucleotide and vitamin are also rich. Agaricus bisporus is also pharmaceutically acceptable. The composition contains a large amount of tyrosinase, and has effect in lowering blood pressure. Can also be made into auxiliary therapeutic agent for pneumonia. Some countries have also found broad spectrum antibiotics containing anticancer substances and antibacterial agents. As the research of submerged culture is successful, people can also use mushroom mycelia to produce protein, oxalic acid, mycose and other substances.
The armillaria mellea is also called tiger skin armeniaca, wampee, tricholoma matsutake and armeniaca. The fruiting bodies are generally moderately large. The fungus cover is hemispherical to be flat, smooth, not sticky, white or light earthy yellow to light earthy red, and is internally curled at the edge. The fungus meat is white and fat. The fungus folds are white or slightly yellowish, dense, narrow, curved and growing, and of unequal length. The stipe is white or slightly yellowish, with streaks and internal solids. The mushrooms are clustered or formed on grasslands in summer and autumn. The mushroom has rich meat, good flavor and delicious taste, and has the trade names of apricot, apricot slices, etc. The Xiangmu mushroom has rich nutritive value, contains more protein, carbohydrate, vitamins, trace elements, minerals and the like, and can enhance the immunity of human body by eating more. The Pleurotus eryngii contains abundant monosaccharides, disaccharides and polysaccharides.
However, due to the influence of human factors and climate factors, the population quantity of the amygdalina is reduced, the quality and the yield of the amygdalina are lower, the requirements of commercialization and marketization are difficult to meet, and the price of the amygdalina 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. And merchants often sell as xiang apricot shiitake mushrooms, which are widely available and easily available.
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, more accurate detection means are required for detecting the Pleurotus eryngii and agaricus bisporus.
Disclosure of Invention
The invention aims to solve the problem that the authenticity of the amygdalina and the agaricus bisporus is difficult to identify in the prior art, and provides a primer probe combination and a detection method for detecting the amygdalina and the agaricus bisporus, which can detect the amygdalina and the agaricus bisporus simultaneously and accurately, qualitatively and quantitatively.
In order to solve the technical problems, the invention adopts the following technical scheme: the primer probe combination for detecting the amygdalina and the agaricus bisporus comprises forward and reverse primers, an amygdalina probe, an agaricus bisporus probe and a quality control probe; the sequences of the forward and reverse primers are shown as SEQ ID No.1 and SEQ ID No. 2; the sequences of the Pleurotus eryngii probe, the Agaricus bisporus probe and the quality control probe are sequentially shown as SEQ ID No.3-SEQ ID No. 5.
Preferably, the 5 'end of the Pleurotus eryngii probe, the Agaricus bisporus probe and the quality control probe is modified with a reporter group, and the 3' end is modified with a quenching group.
Preferably, the reporter group is one of FAM, HEX, ROX or CY 5; the quenching group is one of TAMRA, MGB, BHQ1 or BHQ 2.
The invention also provides a method for detecting the amygdalina and the agaricus bisporus, which uses the DNA of the mushroom sample to be detected as a template, uses the primer probe combination to carry out Real-timePCR amplification, and carries out qualitative and quantitative detection on the DNA content of the amygdalina and the agaricus bisporus in the mushroom sample to be detected according to an amplification curve.
Preferably, the Real-time PCR amplification parameters are: the pre-denaturation temperature was 94 ℃,30s, denaturation temperature 94 ℃,5s, annealing extension temperature 60 ℃,31s,40 cycles.
Preferably, the reaction system for Real-time PCR amplification is as follows: 10 mu L of Probe qPCR premix, 2 mu L of mushroom sample DNA template to be detected and 4.5 mu L of sterilized deionized water; 1 mu L of each forward and reverse primer, and the concentration is 10 mu mol/L; the concentration of each 0.5 mu L of the Pleurotus eryngii probe, the Agaricus bisporus probe and the quality control probe is 10 mu mol/L; the total volume of the system was 20. Mu.L.
The invention has the beneficial effects that:
firstly, the primer probe combination in the invention has strong specificity and high sensitivity, realizes qualitative and quantitative detection of the amygdalina and the agaricus bisporus, and obviously improves the detection accuracy of the amygdalina and the agaricus bisporus;
secondly), the primer probe combination in the invention overcomes the influence between a plurality of primers and probes in the same PCR reaction system and the competition between a template and PCR reaction resources in the Real-timePCR amplification process, achieves the effect that one PCR reaction system can simultaneously perform multiple Real-time fluorescence PCR, can perform the same-tube detection on the Pleurotus eryngii, the agaricus bisporus and the quality control, effectively saves detection procedures and time, and obviously reduces detection cost.
Drawings
FIG. 1 is a standard curve of Pleurotus eryngii in example 2;
FIG. 2 is a standard curve of agaricus bisporus in example 2;
FIG. 3 is a detection curve of real-time fluorescent quantitative PCR performed on 17 mushrooms in example 3;
FIG. 4 is a detection curve of real-time fluorescent quantitative PCR performed on 17 mushrooms in example 3;
FIG. 5 is a test result of the DNA detection sensitivity of Pleurotus citrinopileatus in example 4;
FIG. 6 is a test result of the sensitivity of the agaricus bisporus probe to agaricus bisporus DNA detection in example 4.
Detailed Description
The invention will be further described with reference to the drawings and the specific examples.
Example 1 sequence design of primers and probes
17 edible fungus ITS genomes of armeniaca, agaricus bisporus, mongolian Bai Limo, pleurotus geesteranus, lentinus edodes, pleurotus eryngii, flammulina velutipes, hypsizigus marmoreus, white beech mushroom, stropharia rugoso-annulata, tricholoma matsutake, morchella esculenta, agrocybe cylindracea, agaricus blazei and the like are compared, and each edible fungus selects ITS genome sequences of 10 varieties or strains. The 170 sequences are compared through bioinformatics software, the conserved and specific sequences of the amygdalina and agaricus bisporus are screened, and the primer and the probe are designed by using primer design software. The sequences of the forward and reverse primers are shown as SEQ ID No.1 and SEQ ID No. 2; the sequences of the Pleurotus eryngii probe, the Agaricus bisporus probe and the quality control probe are sequentially shown as SEQ ID No.3-SEQ ID No. 5.
And (3) marking the Pleurotus citrinopileatus by using HEX and MGB modified probes, marking the agaricus bisporus by using FAM and MGB modified probes, and marking quality control by using ROX and BHQ2 modified probes.
Example 2 establishment of qualitative and quantitative detection methods for Pleurotus eryngii and Agaricus bisporus
(1) And extracting DNA of the mushroom sample to be tested.
(2) The concentration and quality of the sample DNA were measured, and the concentration was diluted to 100-200 ng/. Mu.L.
(3) The primers and probes prepared in example 1 were used for amplification detection of diluted DNA, positive standards of Pleurotus eryngii and Agaricus bisporus were used as positive controls, sterilized deionized water was used as negative controls, blank controls of DNA extraction were used as controls of the extraction method, the Real-time PCR reaction system is shown in Table 1, and the Real-time PCR amplification parameters are shown in Table 2.
TABLE 1Real-timePCR reaction System (Simultaneous detection of Pleurotus eryngii, agaricus bisporus and quality control)
TABLE 2 Real-timeEPCR 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 Pleurotus eryngii, 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.
(5) And respectively establishing standard curves of the amygdalus comatus and the agaricus bisporus by using HEX and MGB modified probes to mark the amygdalus comatus and FAM and MGB modified probes to mark the agaricus bisporus. The detection results are shown in fig. 1, fig. 2 and table 3:
TABLE 3 Standard Curve data for Pleurotus eryngii and agaricus bisporus
Table 3 shows that the amplification efficiency is close to 90% -110%, the linear fitting R2 is more than or equal to 0.98, and the results show that the standard curves of the Pleurotus eryngii, the agaricus bisporus primers and the probes are good, and the quantitative detection can be carried out.
Example 3 specificity identification test of 17 mushrooms with Pleurotus eryngii and agaricus bisporus
qPCR was performed on 17 kinds of mushrooms (agaricus bisporus, pleurotus eryngii, mongolian Bai Limo, pleurotus geesteranus, lentinus edodes, pleurotus eryngii, flammulina velutipes, hypsizygus marmoreus, white beech mushroom, money mushroom, cordyceps, bonus edodes, morchella, agrocybe cylindracea and Agaricus blazei) using the primers and probes in example 1 according to the detection method of example 2. The results of the measurements are shown in FIG. 3, FIG. 4 and Table 4. The amplification curves in the Pleurotus eryngii of FIG. 3 were shown, and none of the other mushrooms were shown, indicating that Pleurotus eryngii primers and probes were specific. In FIG. 4, the amplification curves were shown in agaricus bisporus, but not in other agaricus bisporus, indicating that the agaricus bisporus primers and probes were specific.
TABLE 4 results of specificity identification test of 17 mushrooms with Pleurotus eryngii and agaricus bisporus
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. The method has the advantages that the amygdalina source is detected in the amygdalina, the agaricus bisporus source is detected in the agaricus bisporus, and the amygdalina source and the agaricus bisporus source are not detected in other mushrooms, so that the primers and the probes have very high accuracy in detecting the amygdalina and the agaricus bisporus, and can be used for identifying the amygdalina and the agaricus bisporus in the mushrooms.
Example 4 detection Limit experiment of primers and probes for corresponding Mushroom Source detection
Genomic DNA of Pleurotus eryngii and Agaricus bisporus was diluted with sterilized ddH2O to a concentration 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 total dilutions, respectively, as templates. The detection limit amplification test of the primers and probes was performed on the DNA template in this example according to the detection method of example 2 using the primers and probes in example 1. As a result, as shown in FIG. 5, FIG. 6 and Table 5, the Pleurotus eryngii probe could detect 0.25pg of Pleurotus eryngii 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 Pleurotus citrinopileatus, the mushroom primers and the probes reach pg level, and the detection sensitivity is high.
TABLE 5 detection limits of primers and probes for corresponding mushroom origin detection
Ct value: mean (three sets of data) ± standard deviation; N/A is not suitable for detection
In conclusion, the probe of the invention can detect 0.25pg of Pleurotus eryngii DNA in the sample, and the probe of the invention can detect 0.25pg of Agaricus bisporus DNA in the sample, which shows that the primer and the probe of the invention have very high accuracy and sensitivity for detecting Pleurotus eryngii and Agaricus bisporus; the primers and the probes can detect the amygdalus comosus, the agaricus bisporus and the quality control in the same tube, effectively remove the false negative problem existing in the identification of the true and false of the mushrooms by using the PCR technology, effectively save detection procedures and time, obviously reduce detection cost, and can be widely applied to the detection and identification of the mushrooms.
The specification and figures are to be regarded in an illustrative rather than a restrictive sense, and one skilled in the art, in light of the teachings of this invention, may make various substitutions and alterations to some of its features without the need for inventive faculty, all being within the scope of this invention.
Sequence listing
<110> tin Lin Guole professional college tin Lin Guole allied Kun Yuan Biotechnology Co., ltd
<120> primer probe combinations for detecting Pleurotus eryngii and agaricus bisporus and detection method
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Claims (3)
1. The primer probe combination for detecting the amygdalina and the agaricus bisporus is characterized by comprising forward and reverse primers, an amygdalina probe, an agaricus bisporus probe and a quality control probe; the sequences of the forward and reverse primers are shown as SEQ ID No.1 and SEQ ID No. 2; the sequences of the Pleurotus citrinopileatus probe, the agaricus bisporus probe and the quality control probe are sequentially shown as SEQ ID No.3-SEQ ID No. 5;
performing Real-time PCR amplification by using the primer probe combination, wherein the Real-time PCR amplification parameters are as follows: pre-denaturation temperature 94 ℃,5s, annealing extension temperature 60 ℃,31s,40 cycles;
the reaction system of Real-time PCR amplification is as follows: 10 mu L of Probe qPCR premix, 2 mu L of mushroom sample DNA template to be detected and 4.5 mu L of sterilized deionized water; 1 mu L of each forward and reverse primer, and the concentration is 10 mu mol/L; the concentration of each 0.5 mu L of the Pleurotus eryngii probe, the Agaricus bisporus probe and the quality control probe is 10 mu mol/L; the total volume of the system is 20 mu L;
and (3) carrying out qualitative and quantitative detection on the DNA content of the amygdalina and the agaricus bisporus in the mushroom sample to be detected according to the PCR amplification curve.
2. The primer probe combination according to claim 1, wherein the armeniaca probe, the agaricus bisporus probe and the quality control probe are modified with a reporter group at the 5 'end and a quencher group at the 3' end.
3. The primer probe combination of claim 2, wherein the reporter group is one of FAM, HEX, ROX or CY 5; the quenching group is one of TAMRA, MGB, BHQ1 or BHQ 2.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200928363A (en) * | 2007-12-20 | 2009-07-01 | Of Food And Drug Analysis Dept Of Health Bureau | A rapid detection method for identification of poisonous mushroom |
| 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 |
| CN104498368A (en) * | 2014-12-02 | 2015-04-08 | 忻州市沐野食用菌研究所 | Agaricus bisporus strain and cultivation method of fruiting body of agaricus bisporus strain |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200928363A (en) * | 2007-12-20 | 2009-07-01 | Of Food And Drug Analysis Dept Of Health Bureau | A rapid detection method for identification of poisonous mushroom |
| 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 |
| CN104498368A (en) * | 2014-12-02 | 2015-04-08 | 忻州市沐野食用菌研究所 | Agaricus bisporus strain and cultivation method of fruiting body of agaricus bisporus strain |
Non-Patent Citations (1)
| Title |
|---|
| 用rDNA-ITS方法鉴别内蒙古多种野生食用菌;刘晓婷等;《食药用菌》;第23卷(第05期);摘要，第1-2节 * |
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