CN114854898A - Primer and probe for simultaneously detecting authenticity of Mongolian white mushroom and agaricus bisporus - Google Patents
Primer and probe for simultaneously detecting authenticity of Mongolian white mushroom and agaricus bisporus Download PDFInfo
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- CN114854898A CN114854898A CN202210613347.8A CN202210613347A CN114854898A CN 114854898 A CN114854898 A CN 114854898A CN 202210613347 A CN202210613347 A CN 202210613347A CN 114854898 A CN114854898 A CN 114854898A
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Abstract
The invention discloses a primer and a probe for simultaneously detecting authenticity of Mongolian white mushroom and agaricus bisporus, wherein the sequences of the primer and the probe are as follows: the sequence of the forward primer is shown as SEQ ID No. 1; the reverse primer sequence is shown as SEQ ID No. 2; the Mongolian white-mushroom probe sequence is shown in SEQ ID No. 3; the agaricus bisporus probe sequence is shown as SEQ ID No. 4; the quality control probe sequence is shown in SEQ ID No. 5. The primer and the probe have good specificity and high sensitivity, can realize quality control same-tube detection of Mongolian white-mushroom, agaricus bisporus and can carry out quantitative detection of Mongolian white-mushroom and 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
The Mongolian white mushroom has mild property and sweet taste, and has effects of dispersing intestine, invigorating qi, clearing heat, and relieving exterior syndrome. The fruit body is large, the mushroom meat is compact and thick, the texture is fresh and tender, and the mushroom is unique in fragrance and is a famous wild edible mushroom. The Mongolian baili mushroom is famous and precious in having fragrant and fresh taste, contains a large amount of balsam volatile substances, and delicate flavor substances and flavor enhancing substances such as sodium glutamate, baili mushroom ammonia acid, 5' -bird glycol and the like, has endless fragrant and fresh taste, and can be smelled far away. The Mongolian baili mushroom not only has high nutritive value, but also is rich in polysaccharide, polypeptide, polyunsaturated fatty acid, 8 kinds of amino acids essential to human body, and various vitamins and minerals such as iron, calcium, phosphorus, selenium, etc., has medical health care effects of resisting oxidation, aging, tumor, lowering blood pressure, blood lipid, blood sugar, etc., and can treat symptoms such as dryness-heat restlessness, etc. The amino acid components of the health food can be balanced with amino acid components in animal food, has good absorption effect in human body, can regulate human body function, and can enhance immunity, and is suitable for dyspepsia, abdominal pain, and stomach pain. It also has effects in lowering blood pressure, and can be used for treating chondropathy. Has antibacterial effect, and can be used for treating trauma and removing toxic substance by folk method. It is a low calorie food, and is suitable for people with weight reducing and body building effects. Due to the influence of human factors and climatic factors, the population quantity of the Mongolian baili mushroom is reduced, and the quality and the yield of the Mongolian baili mushroom are gradually reduced, so that the requirements of commercialization and marketization are difficult to meet, and the price of the Mongolian baili mushroom is increased. Due to the high price and the unique rare value, the phenomenon of confusing fake and inferior products and other wild bacteria often appears in the market. And merchants often sell Mongolian baili mushrooms as a wide source, readily available mushroom. For example, commercial edible mushrooms such as Pleurotus eryngii, Pleurotus geesteranus and Agaricus bisporus are used to make the mushroom paste of "Baili" mushroom.
The research on the edible fungus authenticity identification technology is started late in China, and the research in the field is still in a preliminary stage. Therefore, the realization of a safe and efficient detection technology with the independent property rights of China is a research subject which is full of opportunities and extremely challenging at the same time in the current research field of the edible fungus authenticity identification technology. The Silibinoguole union as the producing area of Mongolian Baili mushroom urgently needs to develop relevant mushroom authenticity identification technology and detection standard of Mongolian Baili mushroom and agaricus bisporus with independent intellectual property rights so as to protect local characteristic mushrooms and maintain the legal rights and interests of consumers. At present, the technical research, the detection standard, the invention patent and the commercial kit related to the mushroom authenticity identification mainly focus on the technology of common PCR ITS, and the real-time fluorescent PCR direct detection and the multi-channel quality control detection with the same tube have fewer reports. False negative is also a bottleneck which troubles the wide application of PCR technology in mushroom authenticity identification. The same-control quality control is an effective means for removing false negative.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: how to provide a primer and a probe for high-efficiency and strong-specificity quality control same-tube detection of Mongolian baili mushroom and agaricus bisporus in mushrooms, and solve the problem of qualitative and quantitative detection of the source components of the Mongolian baili mushroom and the agaricus bisporus in the mushrooms.
The technical scheme of the invention is as follows: the primer and probe combination for the quality control same-tube detection of the Mongolian white mushroom and the agaricus bisporus comprises the following sequences:
the sequence of the forward primer is shown as SEQ ID No. 1;
the reverse primer sequence is shown as SEQ ID No. 2;
the Mongolian white-mushroom probe sequence is shown in SEQ ID No. 3;
the agaricus bisporus probe sequence is shown as SEQ ID No. 4;
the quality control probe sequence is shown in SEQ ID No. 5.
Furthermore, the Mongolian white mushroom probe, the Agaricus bisporus probe and the quality control probe sequence are modified with a reporter group at the 5 'end and a quenching group at the 3' end, wherein the reporter group is any one of FAM, HEX, ROX or CY5, and the quenching group is any one of TAMRA, MGB, BHQ1 or BHQ 2.
The primer and probe combination is applied to the detection of Mongolian white mushroom and agaricus bisporus.
A kit comprises the primer and probe combination.
The method for detecting Mongolian white mushroom source, agaricus bisporus source and quality control in mushrooms in the same tube comprises the following steps:
(1) extracting DNA of mushroom sample;
(2) taking the DNA in the step (1) as a template, performing 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 white-mushroom and agaricus bisporus as positive control, sterilized deionized water as negative control, and taking blank control of DNA extraction as a control group of the extraction method; (3) setting Threshold as automatic after the Real-time PCR reaction is finished, and reading Ct values of the Mongolian white-mushroom, the agaricus bisporus and the quality control corresponding probes and Ct values of positive control, negative control and blank control; the judgment of the corresponding probe source result can be carried out only when the Ct of the quality control is less than or equal to 40, the Ct of the positive control is less than or equal to 40, and the Ct of the negative control and the Ct of the blank control are 0; when the Ct of the corresponding probe is less than or equal to 40, the result is judged to have corresponding source, and the Ct of a plurality of probes is less than or equal to 40, and the result is judged to have corresponding two mushrooms;
(4) making a DNA quantitative standard curve by using Mongolian white mushroom and agaricus bisporus positive standard substances;
(5) and (3) obtaining a quantitative detection result of the corresponding mushroom source in the mushrooms by utilizing 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 is 94 ℃, 30s, the denaturation temperature is 94 ℃, 5s, the annealing extension temperature is 60 ℃, 31s and 40 cycles.
Further, the Real-time PCR reaction system is as follows: 10 mu L of Probe qPCR premix solution and 1 mu L of forward primer shown in SEQ ID No.1, wherein the concentration is 10 mu mol/L; 1 mu L of reverse primer shown in SEQ ID No.2, and the concentration is 10 mu mol/L; 0.5 mu L of Mongolian white-mushroom probe shown in SEQ ID No.3, and the concentration is 10 mu mol/L; 0.5 mu L of agaricus bisporus probe shown in SEQ ID No.4 with the concentration of 10 mu mol/L and 0.5 mu L of quality control probe shown in SEQ ID No.5 with the concentration of 10 mu mol/L; DNA template 2. mu.L, sterile deionized water 4.5. mu.L, total volume 20. mu.L.
According to the invention, the ITS genome sequences of 17 edible fungi such as Mongolian white mushrooms, agaricus bisporus, fragrant pleurotus eryngii, pleurotus geesteranus, lentinus edodes, pleurotus eryngii, flammulina velutipes, hypsizygus marmoreus, pleurotus cornucopiae, pleurotus eryngii, cordyceps, morel, matsutake, morel, agrocybe cylindracea, agaricus blazei and the like are compared, and 10 varieties or strains of ITS genome sequences are selected for each edible fungus. And comparing the 170 sequences by using biological information software, screening out conserved and specific sequences of the Mongolian white mushroom and the agaricus bisporus, and designing primers and probes by using primer design software. The innovativeness of the design is that two ends of conserved and middle specific sequences need to be screened out on a sequence of 100-150bp, primers are designed at the positions conserved at the two ends, and probes are designed at the positions specific in the middle. The conservative primers and the specific probes can effectively reduce the mismatch between the primers and the competition of a plurality of PCR reactions on reaction resources, and can ensure the progress of multiple real-time fluorescent quantitative PCR reactions. The multiplex real-time fluorescent quantitative PCR reaction is the basis of the detection of multiple source components. The annealing temperatures of the primers and probes are controlled at 55-60 ℃ and 65-70 ℃, and secondary structures affecting the annealing efficiency are not generated, and the design ensures that the primers and probes can be used for subsequent qualitative and quantitative detection to ensure that the primers and probes have high specificity on ITS genes.
The invention researches and develops Mongolian white mushroom, agaricus bisporus, quality control and other 3-channel detection primers and probes, and optimizes the primer and probe combination of multi-channel multi-mushroom detection and quality control detection of the same tube. In the process, the problems of influence among various primers and probes in the same PCR reaction system and competition between the template and PCR reaction resources are solved, and the effect that the PCR reaction system can simultaneously carry out multiple real-time fluorescent 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 the Mongolian white-mushroom and the agaricus bisporus in the mushrooms, can simultaneously detect the Mongolian white-mushroom, the agaricus bisporus and the quality control, saves working procedures and reduces cost.
Drawings
FIG. 1 shows the real-time fluorescence quantitative PCR detection of 15 kinds of mushrooms such as Pleurotus eryngii, Pleurotus geesteranus, Lentinus edodes, Pleurotus eryngii, Flammulina velutipes, Hypsizygus marmoreus, Pleurotus pulmonarius, Pleurotus citrinopileatus, Cordyceps militaris, Morchella esculenta, Tricholoma matsutake, Morchella esculenta, Agrocybe aegerita and Agaricus blazei by using HEX and TAMRA modified probe-labeled Pleurotus mongolicus, FAM and MGB modified probe-labeled Agaricus bisporus and ROX and BHQ2 modified probe-labeled quality control.
FIG. 2 shows real-time fluorescence quantitative PCR detection of 15 kinds of mushrooms (other mushrooms) such as Pleurotus eryngii, Pleurotus geesteranus, Lentinus edodes, Pleurotus eryngii, enoki mushroom, Hypsizygus marmoreus, Pleurotus nebrodensis, Pleurotus cornucopiae, Pleurotus citrinopileatus, Cordyceps, Mycoplasma comatus, Morchella esculenta, Agrocybe aegerita and Agaricus blazei, with HEX and TAMRA modified probes labeled Pleurotus nebrodensis, FAM and MGB modified probes labeled Agaricus bisporus, and ROX and BHQ2 modified probes labeled quality control.
FIG. 3 shows that the probe for white mushroom can detect 1pg of white mushroom DNA by HEX and TAMRA modified probes in a detection sensitivity amplification experiment on white mushroom DNA (100ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng, 0.0005ng, 0.00025ng, 0.0001ng and 0.00001 ng). The above results indicate that the Mongolian white mushroom probe has a higher sensitivity in mushroom detection.
FIG. 4 shows that Agaricus bisporus DNA (100ng, 10ng, 1ng, 0.1ng, 0.01ng, 0.001ng, 0.0005ng, 0.00025ng, 0.0001ng and 0.00001ng) is subjected to a detection sensitivity amplification experiment by marking Agaricus bisporus with FAM and MGB modified probes, and Agaricus bisporus DNA of 0.25pg can be detected by the Agaricus bisporus probes. The above results indicate that the agaricus bisporus probe has high sensitivity in mushroom detection.
Fig. 5 a baily mushroom mongolica test standard curve: the method is used for quantitative detection of Mongolian white mushroom in mushrooms.
FIG. 6 Agaricus bisporus detection Standard Curve: the method is used for quantitative detection of agaricus bisporus in mushrooms.
Detailed Description
1. The detection method comprises the following steps:
(1) extracting DNA of mushroom sample, and establishing extraction blank control (control group for subsequent extraction method).
(2) The concentration and quality of the DNA were determined and the concentration was diluted to 100-200 ng/. mu.L.
(3) The diluted DNA is subjected to amplification detection by using multiple fluorescent quantitative PCR primers and probes, Mongolian white mushroom and agaricus bisporus positive standard substances are used as positive controls, sterilized deionized water is used as a negative control, a blank control for DNA extraction is used as a 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 (I) | Volume (microliter) |
| Probe |
10 |
| Forward primer | 1 |
| Reverse directionPrimer and method for producing the same | 1 |
| Mongolian white mushroom probe | 0.5 |
| Agaricus bisporus probe | 0.5 |
| Quality control probe | 0.5 |
| |
2 |
| Sterilized deionized water | 4.5 |
| |
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 (I) | Volume (microliter) |
| Probe |
10 |
| Forward primer | 1 |
| Reverse primer | 1 |
| Agaricus bisporus probe | 0.5 |
| Quality control probe | 0.5 |
| |
2 |
| Sterilized deionized water | 5 |
| |
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 Mongolian white-mushroom, the agaricus bisporus and the quality control corresponding probes and Ct values of positive control, negative control and blank control; the judgment of the corresponding mushroom probe result can be carried out only when the Ct of the quality control is less than or equal to 40, the Ct of the positive control is less than or equal to 40, and the Ct of the negative control and the Ct of the blank control are 0; 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.
2. Design of primer and Probe sequences
Since the difference of the DNA of the fungus on ITS is small, the ITS genome of the fungus DNA is selected to design Mongolian white mushroom, agaricus bisporus and quality control detection primers and probes. The synthesis method of the primer and the probe comprises the following steps: beijing Rui Boxing Biol.A.was entrusted with the synthesis and purification according to the invented sequence.
A forward primer: 5'CTTGCGCTCCTTGGTATTC 3' (SEQ ID No.1),
reverse primer: 5'GCTAATGYWTTTMAGAGGAGC 3' (SEQ ID No.2),
mongolian white mushroom probe: 5'CTTTTCAGCTTTTGCGAGTTGGATTG 3' (SEQ ID No.3),
agaricus bisporus probe: 5'TATTCTCAACTCTCCAATACTTTGTT 3' (SEQ ID No.4),
quality control probe: 5'AGGAGCATGCCTGTTTGAGTGTCAT 3' (SEQ ID No. 5);
the Mongolian white mushroom, the agaricus bisporus and the quality control probe sequence are modified with a reporter group at the 5 'end and a quenching group at the 3' end, wherein the reporter group is any one of FAM, HEX, ROX or CY5, and the quenching group is any one of TAMRA, MGB, BHQ1 or BHQ 2.
3. Specific detection of primers and probes
The Real-time PCR reaction system for single mushroom source detection is shown in the following table
| Composition (I) | Volume (microliter) |
| Probe |
10 |
| Forward primer | 1 |
| Reverse primer | 1 |
| Corresponding probe derived from mushroom | 0.5 |
| Quality control probe | 0.5 |
| |
2 |
| Sterilized deionized water | 4.5 |
| |
20 |
The QPCR detection is carried out on Mongolian white-mushroom, agaricus bisporus, fragrant pleurotus eryngii, pleurotus geesteranus, shiitake mushroom, pleurotus eryngii, flammulina velutipes, hypsizygus marmoreus, pleurotus cornucopiae, russula marmoreus, cordyceps sinensis, morchella esculenta, matsutake, morchella esculenta, agrocybe aegerita and agaricus blazei by utilizing HEX and MGB modified probes to label Mongolian white-mushroom, FAM and MGB modified probes and ROX and BHQ2 modified probe label quality control contrast.
The detection results are as follows:
ct value: mean (three groups of data) ± standard deviation;
the results show that: ct less than 40 (not 0) indicates the corresponding mushroom origin in the sample. The detection result accords with the mushroom source of the sample. Mongolian baili mushroom origin was detected in Mongolian baili mushroom, agaricus bisporus origin was detected in agaricus bisporus, and Mongolian baili mushroom origin and agaricus bisporus origin were not detected in its mushrooms.
4. Detection limit experiment of corresponding primers and probes for mushroom source detection
Sterilizing ddH from genomic DNA of Mongolian white mushroom and Agaricus bisporus 2 O dilution was performed so that the concentrations thereof were 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, and the amplification test for the detection limit of the primer and the probe was performed by diluting 10 times in total. As is clear from the results below, the Mongolian white mushroom probe detected 1pg of Mongolian white mushroom DNA in the sample, and the Agaricus bisporus probe detected 0.25pg of Agaricus bisporus DNA in the sample. The results show that the detection limit of the autonomously developed Mongolian white mushroom and mushroom primer and probe reaches the pg level, and the detection sensitivity is higher.
The detection results are as follows:
ct value: mean (three groups of data) ± standard deviation; N/A not suitable for detection
5. Marking Mongolian white mushroom and FAM by using HEX and MGB modified probes and marking agaricus bisporus by using the same to respectively establish standard curves of the Mongolian white mushroom and the agaricus bisporus.
The detection results are as follows:
the amplification efficiency is close to 90% -110%, and the linear fitting R 2 The result is more than or equal to 0.98, and the results show that the primer and the probe for the independently developed Mongolian baili mushroom and agaricus bisporus have good standard curves and can carry out quantitative detection.
Sequence listing
<110> Sino academy of profession, Sino Kuo Yuan Biotechnology Ltd
<120> primer and probe for simultaneously detecting authenticity of Mongolian white mushroom 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 (7)
1. The primer and probe combination for the quality control same-tube detection of the Mongolian white mushroom and the agaricus bisporus is characterized in that the sequences of the primer and the probe are as follows:
the sequence of the forward primer is shown as SEQ ID No.1,
the reverse primer sequence is shown as SEQ ID No.2,
the Mongolian white-mushroom probe sequence is shown in SEQ ID No.3,
the agaricus bisporus probe sequence is shown in SEQ ID No.4,
the quality control probe sequence is shown in SEQ ID No. 5.
2. The Mongolian white-mushroom probe, agaricus bisporus and quality control concatemer detection primer and probe according to claim 1, wherein the Mongolian white-mushroom 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, the reporter group is any one of FAM, HEX, ROX or CY5, and the quencher group is any one of TAMRA, MGB, BHQ1 or BHQ 2.
3. Use of the primer and probe combination of claim 1 or 2 for the detection of white mushroom Mongolian and Agaricus bisporus.
4. A kit comprising the primer and probe combination of claim 1 or 2.
5. The method for detecting Mongolian white mushroom, agaricus bisporus and quality control in the mushrooms is characterized by comprising the following steps:
(1) extracting DNA of mushroom sample;
(2) taking the DNA in the step (1) as a template, performing 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 white-mushroom and agaricus bisporus as positive control, sterilized deionized water as negative control, and taking blank control of DNA extraction as a control group of the extraction method;
(3) setting Threshold as automatic after the Real-time PCR reaction is finished, and reading Ct values of the Mongolian white-mushroom, the agaricus bisporus and the quality control corresponding probes and Ct values of positive control, negative control and blank control; the judgment of the corresponding mushroom probe result can be carried out only when the Ct of the quality control is less than or equal to 40, the Ct of the positive control is less than or equal to 40, and the Ct of the negative control and the Ct of the blank control are 0; when the Ct of the corresponding probe is less than or equal to 40, the result is judged to have corresponding mushroom origin, and the Ct of the probes is less than or equal to 40, and the result is judged to have corresponding two mushroom origins;
(4) making a DNA quantitative standard curve by using Mongolian white mushroom and agaricus bisporus positive standard substances;
(5) and (3) obtaining a quantitative detection result of the corresponding mushroom source in the mushrooms by utilizing the Ct value of the corresponding mushroom source in the mushrooms and a formula in a standard curve.
6. The method of claim 5, wherein Real-time PCR amplification parameters are as follows: the pre-denaturation temperature is 94 ℃, 30s, the denaturation temperature is 94 ℃, 5s, the annealing extension temperature is 60 ℃, 31s and 40 cycles.
7. The method for quality control of mushroom of claim 5, wherein the Real-time PCR reaction system is as follows: 10 mu L of Probe qPCR premix solution and 1 mu L of forward primer shown in SEQ ID No.1, wherein the concentration is 10 mu mol/L; 1 mu L of reverse primer shown in SEQ ID No.2, and the concentration is 10 mu mol/L; 0.5 mu L of Mongolian white-mushroom probe shown in SEQ ID No.3, and the concentration is 10 mu mol/L; 0.5 mu L of agaricus bisporus probe shown in SEQ ID No.4 with the concentration of 10 mu mol/L and 0.5 mu L of quality control probe shown in SEQ ID No.5 with the concentration of 10 mu mol/L; DNA template 2. mu.L, sterile deionized water 4.5. mu.L, total volume 20. mu.L.
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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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