Specific primer for purple gyrophora and application of specific primer
By designing specific primers based on the characteristic nucleotide sequence of Gyrotrichum purpurogenum, the problem of rapid and accurate detection of Gyrotrichum purpurogenum in the wild environment was solved, and effective tracking and monitoring of bionic cultivation and conservation and propagation promotion was achieved.
Patent Information
- Application Number
- CN202510754850.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-06
- Publication Date
- 2025-09-16
AI Technical Summary
Existing technologies are unable to quickly and accurately identify Gyrotrichum purpurogenum in the wild environment, resulting in the inability to timely evaluate the preliminary work of bionic cultivation and conservation and propagation promotion, affecting research and application promotion.
Specific primers were designed based on the characteristic nucleotide sequence of Gyrotrichum purpurogenum for PCR detection, and the presence of Gyrotrichum purpurogenum in the sample was determined by the characteristic bands of 223 bp and 194 bp.
It has achieved rapid and accurate detection of Gyrotrichum purpurogenum in complex wild environments, provided a means of early tracking and monitoring for bionic cultivation and conservation and propagation promotion, and supported early dynamic tracking and mushroom fruiting prediction.
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Figure CN120648831A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biotechnology gene detection, and in particular relates to a Gyrotrichum purpurogenum-specific primer and application thereof. Background Art
[0002] In recent years, researchers have carried out habitat surveys, strain isolation and cultivation, and preliminary exploration of the ecological niche of Purple Gyrobacterium. However, the biological characteristics of Purple Gyrobacterium remain inconclusive, making it impossible to artificially breed it. Wild bionic cultivation and conservation and propagation promotion techniques have gradually become the focus of researchers.
[0003] Existing liquid fermentation technology has basically met the demand for strains for biomimetic cultivation, conservation and propagation promotion of Purple Gyroli. However, the development of mycelium cannot be evaluated in time after the strain is moved to the wild. The success of biomimetic cultivation can only be judged by the production of fruiting bodies or by morphological characteristics and ecological habits. However, this judgment is often delayed and requires a long time to wait for the strain to grow before it can be used. Therefore, this method is not conducive to timely evaluation. Traditional fungal identification is generally carried out using ITS sequences, and the amplification primers are also universal primers. Usually, the fruiting bodies of Purple Gyroli are identified. If it is necessary to determine in advance that it is Purple Gyroli, the soil around the growth of Purple Gyroli can only be directly tested. However, this will lead to excessive detection errors. This is because the complex growth environment of Purple Gyroli inevitably contains ectomycorrhizal fungi in the sample. Generally, the poor specificity of ITS universal primers cannot effectively distinguish the target fungi from other fungi, resulting in the inability to effectively evaluate the preliminary work of biomimetic cultivation, conservation and propagation promotion of Purple Gyroli, which greatly affects the research and application promotion of related technologies.
[0004] Based on the above background, it is very important to find a suitable and accurate identification method for the field maturity of Purple Gyrotrichum, which is of great significance to the research and promotion of bionic cultivation, conservation and propagation technology of Purple Gyrotrichum. Summary of the Invention
[0005] In view of this, the purpose of the present invention is to provide a nucleotide sequence, specific primers and applications for identifying Purple Gyrobacterium, which can quickly and accurately identify Purple Gyrobacterium from field environments and field soils.
[0006] To achieve the above-mentioned purpose of the invention, the present invention discloses a characteristic nucleotide for identifying Gyrotrichum purpurogenum, and the nucleotide sequence is shown in SEQ ID NO.1 and SEQ ID NO.2.
[0007] The purple gyroscope bacteria is deposited in the General Microbiology Center of the China Culture Collection Administration Committee. The deposit date is May 23, 2022. The deposit number is CGMCC No. 40193. The deposit unit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is Purple gyroscope bacteria Gomphus purpuraceus.
[0008] Furthermore, the nucleotide is used in identifying or detecting Gyrotrichum purpurogenum.
[0009] According to the above characteristic nucleotide sequences SEQ ID NO.1 and SEQ ID NO.2, primers specific for Gyrotrichum purpurogenum were designed respectively. The primers specific for Gyrotrichum purpurogenum include:
[0010] Specific primer 1:
[0011] Forward primer: 5′-cacagccgatctacctacacttacatg-3′;
[0012] Reverse primer: 5′-gggatgccactatcaacactcttctg-3′;
[0013] Specific primer 2:
[0014] Forward primer: 5′-tccagtgctcaaagaataccctaatgc-3′;
[0015] Reverse primer: 5′-ttcctgatgcgatactgccacataac-3′.
[0016] Furthermore, the specific primers are used in the detection or identification of Gyrotrichum purpurogenum.
[0017] Furthermore, the specific primers are used in detecting or identifying whether a product contains Gyrotrichum purpurogenum.
[0018] The specific primers are used to identify or detect whether it is Gyrotrichum purpurogenum, or to identify whether Gyrotrichum purpurogenum is contained.
[0019] Furthermore, the specific primers can also be used in the preparation of reagents or kits for the detection or identification of Gyrotrichum purpurogenum.
[0020] Furthermore, the specific primers are used in the preparation of reagents or kits for detecting or identifying whether a substance contains Gyrotrichum purpurogenum.
[0021] Furthermore, the sample to be detected or identified is one or more of soil, mycelium, and fruiting body.
[0022] The specific primers of the present invention or the reagents or kits prepared based on the specific primers have high specificity. In addition to being able to identify conventional fruiting body samples, mycelium samples can also be tested to identify whether they contain Purple Gyrotrichum purpurogenum. In addition, the specific primers of the present invention can be particularly used for testing in complex soil environments to detect whether Purple Gyrotrichum purpurogenum is contained in the soil, which is of great significance for the research and promotion of bionic cultivation, conservation, and propagation promotion technologies.
[0023] Furthermore, a method for detecting or identifying Gyrotrichum purpurogenum is provided, wherein the detection method comprises:
[0024] (1) Extract DNA from the sample using a DNA extraction kit;
[0025] (2) Using the DNA extracted in step 1 as a template, PCR amplification was performed using specific primers 1 and 2 respectively; the PCR reaction system was 50 μL, including 25 μL of 2× SanTaq PCR Mix (containing blue dye), 3 μL of DNA template, 2 μL of upstream primer (10 μmol / L), 2 μL of downstream primer (10 μmol / L), and Sterilized ddH2O was added to 50 μL;
[0026] (3) PCR amplification was performed using the reaction system in step 2. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 50-60°C for 30 s, and extension at 72°C for 45 s, for a total of 35 cycles; final extension at 72°C for 10 min; and storage at 4°C.
[0027] (4) Analyze the PCR products by agarose gel electrophoresis with an agarose concentration of 1.5% and a voltage of 120 V. The results are determined based on the characteristic bands.
[0028] Judgment standard: If the PCR products are detected by lipose gel electrophoresis and characteristic bands of 223 bp and 194 bp appear, it indicates that the sample contains Gyrotrichum purpurogenum.
[0029] Beneficial effects:
[0030] The present invention is based on the characteristic nucleotides of Gomphus purpuraceus, and on this basis, specific primers are constructed to establish its PCR detection system, which can quickly and accurately detect Gomphus purpuraceus from complex growth environments in the wild. The detection reagent or kit operation method constructed according to this method is mature and stable, easy to operate, has good specificity, and high sensitivity. In addition, the primers provided by the present invention can be used for the early tracking and monitoring of the bionic cultivation and conservation and promotion of Gomphus purpuraceus, providing a fast, accurate, simple and effective method for the early tracking and monitoring of Gomphus purpuraceus related breeding technology, and providing important means and technical support for the growth and development dynamics of Gomphus purpuraceus in the wild and the prediction of mushroom fruiting. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] Figure 1 : is a gel imaging image of DNA extracted from the fruiting bodies of Gyrotrichum purpurogenum and soil samples in Example 1;
[0032] Figure 2 : is a gel imaging image of the PCR amplification product of the soil sample in Example 2;
[0033] Figure 3 : is a gel imaging image of the PCR amplification product of the soil sample in Example 3;
[0034] Figure 4 : Gel imaging of PCR amplification products of all samples in Example 4. DETAILED DESCRIPTION
[0035] The present invention will be described in detail below with reference to specific embodiments and accompanying drawings:
[0036] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.
[0037] Gomphus purpuraceus is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms. The deposit date is May 23, 2022. The deposit number is CGMCC No. 40193. The depository address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The classification name is Gomphus purpuraceus.
[0038] A characteristic nucleotide sequence for identifying Gyrotrichum purpurogenum, the nucleotide sequence of which is shown in SEQ ID NO.1 and SEQ ID NO.2, is used to design Gyrotrichum purpurogenum-specific primers based on the two characteristic nucleotide sequences. The Gyrotrichum purpurogenum-specific primers include:
[0039] Specific primers 1 designed based on SEQ ID NO. 1: forward primer: 5'-cacagccgatctacctacacttacatg-3'; reverse primer: 5'-gggatgccactatcaacactcttctg-3';
[0040] Specific primer 2 designed according to SEQ ID NO. 2: forward primer: 5'-tccagtgctcaaagaataccctaatgc-3'; reverse primer: 5'-ttcctgatgcgatactgccacataac-3'.
[0041] Sample collection sites: Anlong County, Guizhou Province, and Longli County, Guizhou Province;
[0042] sample:
[0043] 1. Soil sample C1 was collected in Anlong County and soil sample C2 was collected in Longli County. These soil samples did not contain Gyrotrichum purpurogenum.
[0044] 2. The fruiting body samples FB of Gyrotrichum purpurogenum were collected in Longli County;
[0045] 3. Soil sample S1 was collected in Anlong County and soil sample S2 was collected in Longli County. The above soil samples were collected from the soil containing purple gyroscope hyphae just below the fruiting bodies of purple gyroscope.
[0046] The following experiments were performed based on the above specific primers and collected samples:
[0047] Example 1: Gel test of DNA samples extracted from purple gyroscope and field soil samples
[0048] (1) Template DNA was extracted from the samples. The Rapid Fungi Genomic DNA Isolation Kit (Sangon Biotech) was used for the fruiting body samples of Gyrifica gyrifera; the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech) was used for the soil samples. The concentrations of the DNA samples extracted by different methods were determined using the Qsep100 fully automatic nucleic acid and protein analysis system.
[0049] (2) Take 5 μL of the extracted DNA sample and mix it with 1 μL of 6× DNA Loading Buffer. After mixing, use a pipette to slowly add the marker (OM5000) and the sample mixture to the submerged gel loading wells in sequence. Turn on the power supply and set the voltage to 120V to run the gel. When the indicator moves to 3 / 4 of the gel plate, stop the electrophoresis. Use a gel imager to observe the electrophoresis bands and their positions. Use Tanon Gel Image System software to compare with the marker and analyze the sample molecular size.
[0050] The results obtained are as follows Figure 1 As shown, the analysis results show that:
[0051] Among them, the numbers in the figure are M: OM5000 marker;
[0052] line F: fruiting body sample FB;
[0053] Line 1: soil sample C1;
[0054] Line 2: soil sample C2;
[0055] line 3: soil sample S1;
[0056] line 4: soil sample S2;
[0057] Depend on Figure 1 It can be seen that the DNA in the sample can be extracted well according to this embodiment, and the bands in the gel electrophoresis are clear and bright, and the product is approximately 6652 bp in size.
[0058] Example 2: Specific PCR detection of field soil samples C1 and C2
[0059] (1) The soil sample DNA was extracted using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech), and the concentration was determined using the Qsep100 fully automatic nucleic acid and protein analysis system.
[0060] (2) Prepare 50 μL of PCR reaction system, including 25 μL of 2× SanTaq PCR Mix (containing blue dye), 3 μL of DNA template, 2 μL of the forward sequence of specific primer 1 (10 μmol / L), 2 μL of the reverse sequence of specific primer 1 (10 μmol / L), and add sterilized ddH2O to 50 μL.
[0061] 2 μL each of the forward primer (10 μmol / L) of specific primer 2 and the reverse sequence (10 μmol / L) of specific primer 2 were also mixed according to the above PCR reaction system.
[0062] (3) Two PCR reaction systems prepared according to the above steps were used for amplification using a T100 PCR instrument (Bio-Rad). The PCR reaction conditions were as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 50-60°C for 30 s, and extension at 72°C for 45 s; final extension at 72°C for 10 min; and storage at 4°C.
[0063] (4) Mix 5 μL of the amplified DNA sample with 1 μL of 6× DNA Loading Buffer. After mixing, use a pipette to slowly add the marker (OM5000) and the sample mixture into the submerged gel loading wells. Turn on the power supply, set the voltage to 120V, and run the gel. When the indicator moves to 3 / 4 of the gel plate, stop the electrophoresis. Use a gel imager to observe the electrophoresis bands and their positions. Use Tanon Gel Image System software to compare with the marker and analyze the sample molecular size. The results are as follows: Figure 2 As shown, the analysis results show that:
[0064] Figure 2 The markers are M: OM5000 marker;
[0065] Line 1: DNA product amplified from soil sample C1 using specific primer 1;
[0066] Line 2: DNA product amplified from soil sample C1 using specific primer 2;
[0067] Line 3: DNA product amplified from soil sample C2 using specific primer 1;
[0068] Line 4: DNA product amplified from soil sample C2 using specific primer 2;
[0069] The specific primer sequences designed by screening, specific primer 1 and specific primer 2, could not amplify effective DNA fragments in soil that did not contain Gyrotrichum purpurogenum.
[0070] Example 3: Specific PCR detection of field soil samples S1 and S2
[0071] (1) The soil sample DNA was extracted using the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech), and the concentration was determined using the Qsep100 fully automatic nucleic acid and protein analysis system.
[0072] (2) Prepare 50 μL of PCR reaction system, including 25 μL of 2× SanTaq PCR Mix (containing blue dye), 3 μL of DNA template, 2 μL of the forward sequence of specific primer 1 (10 μmol / L), 2 μL of the reverse sequence of specific primer 1 (10 μmol / L), and add sterilized ddH2O to 50 μL.
[0073] 2 μL each of the forward primer (10 μmol / L) of specific primer 2 and the reverse sequence (10 μmol / L) of specific primer 2 were also mixed according to the above PCR reaction system.
[0074] (3) Two PCR reaction systems prepared according to the above steps were used for amplification using a T100 PCR instrument (Bio-Rad). The PCR reaction conditions were as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 50-60°C for 30 s, and extension at 72°C for 45 s; final extension at 72°C for 10 min; and storage at 4°C.
[0075] (4) Mix 5 μL of the amplified DNA sample with 1 μL of 6× DNA Loading Buffer. After mixing, use a pipette to slowly add the marker (OM5000) and the sample mixture into the submerged gel loading wells. Turn on the power supply, set the voltage to 120V, and run the gel. When the indicator moves to 3 / 4 of the gel plate, stop the electrophoresis. Use a gel imager to observe the electrophoresis bands and their positions. Use Tanon Gel Image System software to compare with the marker and analyze the sample molecular size. The results are as follows: Figure 3 As shown, the analysis results show that:
[0076] Figure 3 The markers are M: OM5000 marker;
[0077] Line 1: DNA product amplified by soil sample S1-specific primer 1;
[0078] Line 2: DNA product amplified by soil sample S1-specific primer 2;
[0079] Line 3: DNA product amplified from soil sample S2 using specific primer 1;
[0080] Line 4: DNA product amplified from soil sample S2 using specific primer 2;
[0081] The specific primers 1 and 2 designed by screening can amplify effective DNA fragments for soil containing purple gyroscope bacteria, and the fragment sizes are 226bp and 196bp, respectively, indicating that the specific primers 1 and 2 disclosed in the present invention can successfully detect or identify whether the soil sample contains purple gyroscope bacteria, and the bands can be cleaned.
[0082] Example 4: Specific PCR detection of Gyrotrichum purpurogenum and field soil samples
[0083] (1) The fruiting body samples of Gyricularia purpurogena and the DNA samples extracted in Example 1 were subjected to PCR amplification. The PCR reaction system was 50 μL, including 25 μL of 2×SanTaq PCR Mix (containing blue dye), 3 μL of DNA template, 2 μL of the forward sequence of specific primer 1 (10 μmol / L), 2 μL of the reverse sequence of specific primer 1 (10 μmol / L), and Sterilized ddH2O was added to 50 μL.
[0084] 2 μL each of the forward sequence of specific primer 2 (10 μmol / L) and the reverse sequence of specific primer 2 (10 μmol / L) were also mixed according to the above PCR reaction system.
[0085] (3) Two PCR reaction systems prepared according to the above steps were used for amplification using a T100 PCR instrument (Bio-Rad). The PCR reaction conditions were as follows: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 50-60°C for 30 s, and extension at 72°C for 45 s; final extension at 72°C for 10 min; and storage at 4°C.
[0086] (4) Mix 5 μL of the amplified DNA sample with 1 μL of 6× DNA Loading Buffer. After mixing, use a pipette to slowly add the marker (OM5000) and the sample mixture into the submerged gel loading wells. Turn on the power supply, set the voltage to 120V, and run the gel. When the indicator moves to 3 / 4 of the gel plate, stop the electrophoresis. Use a gel imager to observe the electrophoresis bands and their positions. Use Tanon Gel Image System software to compare with the marker and analyze the sample molecular size. The results are as follows: Figure 4 As shown, the analysis results show that:
[0087] Figure 4 The markers are M: OM5000 marker;
[0088] Line 1: DNA product amplified from fruiting body sample FB using specific primer 1;
[0089] Line 2: DNA product amplified from fruiting body sample FB using specific primer 2;
[0090] Line 3: DNA product amplified from soil sample C1 using specific primer 1;
[0091] Line 4: DNA product amplified from soil sample C1 using specific primer 2;
[0092] Line 5: DNA product amplified from soil sample C2 using specific primer 1;
[0093] Line 6: DNA product amplified from soil sample C2 using specific primer 2;
[0094] Line 7: DNA product amplified from soil sample S1 using specific primer 1;
[0095] Line 8: DNA product amplified from soil sample S1 using specific primer 2;
[0096] Line 9: DNA product amplified from soil sample S2 using specific primer 1;
[0097] Line 10: DNA product amplified from soil sample S2 using specific primer 2.
[0098] The specific primers 1 and 2 designed by screening could amplify effective DNA fragments in the soil and fruiting bodies containing Gyrotrichum purpurogenum, with the fragment sizes being 222 bp and 193 bp respectively.
[0099] In summary, the primer sequences provided by the present invention are highly specific and can be used to detect complex soil samples or fruiting body samples in the field. They can effectively identify the purple gyroscope in the samples, providing effective technical support for the rapid and accurate identification of the purple gyroscope in the field environment.
[0100] The above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art will appreciate that modifications or improvements may be made based on the present invention. Therefore, such modifications or improvements, without departing from the spirit of the present invention, are intended to fall within the scope of protection claimed in the present invention.
Claims
1. A characteristic nucleotide for identifying Gyrotrichum purpurogenum, characterized in that: The nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.
2.
2. The use of the characteristic nucleotide according to claim 1, characterized in that: The application of the nucleotide in identifying or detecting Gyricularia purpurogena.
3. A primer specific for Gyrotrichum purpurogenum, characterized in that: The specific primers of the purple gyroscope bacteria include: Specific primer 1: Forward primer: 5′-cacagccgatctacctacacttacatg-3′; Reverse primer: 5′-gggatgccactatcaacactcttctg-3′; Specific primer 2: Forward primer: 5′-tccagtgctcaaagaataccctaatgc-3′; Reverse primer: 5′-ttcctgatgcgatactgccacataac-3′.
4. An application of a purple gyroscope-specific primer, characterized in that: Use of the specific primers described in claim 3 in the detection or identification of Gyrophila purpurogena.
5. The use of a purple gyroscope-specific primer according to claim 4, characterized in that: The specific primer is used for detecting or identifying whether a product contains purple gyroscope.
6. An application of a purple gyroscope-specific primer, characterized in that: Use of the specific primer according to claim 3 in the preparation of a reagent or kit for the detection or identification of Gyrophila purpurogena.
7. The use of a purple gyroscope-specific primer according to claim 6, characterized in that: The specific primer is used in preparing a reagent or a kit for detecting or identifying whether the bacteria contain Gyrotrichum purpurogenum.
8. The use according to any one of claims 3 to 6, characterized in that: The sample to be tested or identified is one or more of soil, mycelium, and fruiting body.
9. A method for detecting or identifying Gyrococcus purpurogenus, characterized in that: The detection method comprises: (1) Extract DNA from the sample using a DNA extraction kit; (2) Using the DNA extracted in step 1 as a template, PCR amplification reaction was performed using the specific primer 1 and specific primer 2 described in claim 3 respectively; the PCR reaction system was 50 μL, including 25 μL of 2×SanTaq PCR Mix, 3 μL of DNA template, 2 μL of upstream primer (10 μmol / L), 2 μL of downstream primer (10 μmol / L), and sterilized ddH2O was added to 50 μL; (3) PCR amplification was performed using the reaction system in step 2. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 50-60°C for 30 s, and extension at 72°C for 45 s, for a total of 35 cycles; final extension at 72°C for 10 min; and storage at 4°C. (4) Analyze the PCR products by agarose gel electrophoresis with an agarose concentration of 1.5% and a voltage of 120 V. The results are determined based on the characteristic bands.
10. The method according to claim 9, characterized in that The PCR products are detected by lipose gel electrophoresis. If characteristic bands appear on all of them, it indicates that the sample contains Gyrotrichum purpurogenum.