Specific primer and kit for identifying microspecies O and microspecies T of bipolaris maydis and application of specific primer and kit
By designing specific primers and PCR detection methods, the accuracy and sensitivity of identification of small species O and small species T of corn spot bacteria were solved, and efficient and rapid disease monitoring and variety breeding were achieved.
Patent Information
- Application Number
- CN202510418446.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-07-08
AI Technical Summary
The prior art lacks accuracy and sensitivity in the identification of small species O and small species T of corn spot bacteria, making it difficult to meet the fast and specific detection needs.
Specific primers were designed and PCR detection methods were established. The genome-specific segments of O and T species were used to identify O and T species through PCR amplification, and the observation results were combined with agarose gel electrophoresis.
It has achieved high specificity, high sensitivity and rapid identification of small species O and small species T of corn spot bacteria, and is suitable for disease monitoring and breeding.
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Figure CN120272631A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of molecular detection of pathogenic bacteria, and more specifically, to a specific primer, a kit and their applications for identifying race O and race T of Bipolaris maydis. Background Art
[0002] Southern leaf blight of maize is one of the important fungal diseases that occur on maize and is prevalent in major maize-producing regions around the world. The occurrence of southern leaf blight of maize reduces the yield and quality of maize, seriously affecting the development of the maize industry. Bipolaris maydis belongs to Cochliobolus heterostrophus in the phylum Ascomycota of the fungal kingdom. Bipolaris maydis has obvious physiological differentiation. According to the cytoplasmic types of maize varieties, maize can be divided into four types: T type, C type, S type, and normal N type. According to the cytoplasmic specialization of different differential hosts, Bipolaris maydis is divided into four physiological races, namely T, C, S, and O. Among them, race O and race T are the dominant races and highly virulent races.
[0003] In the control of southern leaf blight of maize, the accurate identification of the physiological races of Bipolaris maydis plays a key role in determining which cytoplasmic type of maize variety to plant and its control. Currently, the commonly used techniques for detecting the physiological races of Bipolaris maydis are the plate method using Escherichia coli transfected with T-toxin receptor and the differential host technique for identification, and their accuracy and sensitivity need to be further improved. Therefore, there is an urgent need to establish a method for identifying race O and race T of Bipolaris maydis with strong specificity, high sensitivity, high accuracy and short identification period, so as to lay a foundation for the control of southern leaf blight of maize. Summary of the Invention
[0004] For the specific identification of race O and race T of Bipolaris maydis, the inventors designed corresponding specific primers based on the specific regions and gene sequences in the genomes of race O and race T of Bipolaris maydis, and established a PCR detection method. Using the genomic DNA of the sample as a template, it can efficiently and specifically identify race O and race T of Bipolaris maydis, which is of great significance for the control of diseases caused by this pathogen.
[0005] To achieve the above object, the technical solution of the present invention is as follows:
[0006] A specific primer for identifying race O and race T of Bipolaris maydis includes the upstream primer and downstream primer of race O, and the upstream primer and downstream primer of race T; among them, the nucleic acid sequence of the upstream primer of race O is shown in SEQ ID NO.1, and the nucleic acid sequence of the downstream primer of race O is shown in SEQ ID NO.2; the nucleic acid sequence of the upstream primer of race T is shown in SEQ ID NO.3, and the nucleic acid sequence of the downstream primer of race T is shown in SEQ ID NO.4.
[0007] Specifically, the sequences of the primers are as follows:
[0008] Table 1 Information of specific primers for race O and race T of Bipolaris maydis
[0009]
[0010] The second object of the present invention is to provide a kit for identifying race O and race T of Bipolaris maydis, and the kit includes the above-mentioned specific primers.
[0011] It can be understood that the kit may further include one or a combination of several of PCR premix, ddH2O, template DNA to be detected, negative control, and positive control.
[0012] The present invention also aims to provide the application of the above-mentioned specific primers or the above-mentioned kit in identifying race O and race T of Bipolaris maydis.
[0013] The present invention also aims to provide a method for identifying race O and race T of Bipolaris maydis, including the following steps: performing PCR amplification on the DNA of the sample to be identified using the above-mentioned primers or the kit.
[0014] Furthermore, the reaction system for the PCR amplification includes: 2.0 μL of DNA template, 1.0 μL of upstream and downstream primers with a concentration of 10 μM, 10 μL of 2×Easy Taq PCR Super Mix, and ddH2O is added to make up to 20 μL; the upstream primer includes the sequences shown in SEQ ID NO.1 and / or SEQ ID NO.3, and the downstream primer includes the sequences shown in SEQ ID NO.2 and / or SEQ ID NO.4.
[0015] Furthermore, the reaction conditions for the PCR amplification include: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, 35 cycles, annealing at 58°C or 60°C for 30 s, 35 cycles, extension at 72°C for 30 s, 35 cycles, and continued extension at 72°C for 10 min, 35 cycles.
[0016] Further, after the PCR amplification product is electrophoresed on a 1% agarose gel, it is photographed and observed using a gel imager.
[0017] Further, the conditions for gel electrophoresis are 100V for 30 minutes.
[0018] Further, the upstream and downstream primers of race O specifically amplify a 941bp band, and the upstream and downstream primers of race T specifically amplify an 837bp band.
[0019] Compared with the prior art, the present invention has the following advantages:
[0020] The specific primer pair provided by the present invention for detecting race O and race T has the advantages of strong specificity, high sensitivity, high accuracy, rapid and simple operation, etc., and can effectively identify Bipolaris maydis race O and race T.
[0021] Moreover, the identification method is simple and widely applicable, and has good application prospects in disease monitoring and early warning, variety breeding, etc. Description of the Drawings
[0022] Figure 1 It is the electrophoresis pattern results of Example 1 and Example 2 of the present invention; wherein, A is the specific identification electrophoresis pattern of Bipolaris maydis race O of the present invention, where: M: DL2000 marker; 1 - 5: Bipolaris maydis race O standard strain C5, Bipolaris maydis race T standard strain C4, Curvularia lunata, Bipolaris sorokiniana, Bipolaris setariae; 6: ddH2O blank control; B is the specific verification electrophoresis pattern of Bipolaris maydis race T of the present invention, where: M: DL2000 marker; 1 - 5: Bipolaris maydis race T standard strain C4, Bipolaris maydis race O standard strain C5, Curvularia lunata, Bipolaris sorokiniana, Bipolaris setariae; 6: ddH2O blank control.
[0023] Figure 2 It is the electrophoresis pattern result of Example 3 of the present invention; wherein, A is the sensitivity verification electrophoresis pattern of the primer pair of race O, M: Marker; 1: 10ng / μL; 2: 1ng / μL; 3: 100pg / μL; 4: 10pg / μL; 5: 1pg / μL; 6: 100fg / μL; 7: ddH2O blank control; B is the sensitivity verification electrophoresis pattern of the primer pair of race T, M: Marker; 1: 10ng / μL; 2: 1ng / μL; 3: 100pg / μL; 4: 10pg / μL; 5: 1pg / μL; 6: 100fg / μL; 7: ddH2O blank control.
[0024] Figure 3Electrophoresis pattern results in Example 4 of the present invention; wherein, A is the electrophoresis pattern for specific identification of race O, M: Marker; 1: Standard strain C5 (Cochliobolus heterostrophus) of Bipolaris maydis; 2: Standard strain C4 of race T of Bipolaris maydis (Cochliobolus heterostrophus); 3: Setosphaeria turcica; 4: Cercospora maydis-zeae; 5: Cercospora zeina; 6: Setophoma zeae-maydis; 7: Epicoccum latusicollum; 8: Fusarium verticillioides; 9: F. graminearum; 10: Puccinia polysora; 11: P. sorghi; 12: ddH2O blank control; B is the electrophoresis pattern for specific identification of race T, M: Marker; 1: Standard strain C4 (Cochliobolus heterostrophus) of race T of Bipolaris maydis; 2: Standard strain C5 of race O of Bipolaris maydis (Cochliobolus heterostrophus); 3: Setosphaeriaturcica; 4: Cercospora maydis-zeae; 5: Cercosporazeina; 6: Setophoma zeae-maydis; 7: Epicoccumlatusicollum; 8: Fusarium verticillioides; 9: F. graminearum; 10: Puccinia polysora; 11: P. sorghi; 12: ddH2O blank control.
[0025] Figure 4Electrophoresis pattern results in Example 5 of the present invention; wherein, A is the identification result using the primer of race O, M: Marker; 1: Cochliobolus heterostrophus C5 standard strain (race O); 2: Cochliobolus heterostrophus C4 standard strain (race T); 3: Cochliobolus heterostrophus BMSY-1; 4: Cochliobolus heterostrophus BMSY-2; 5: Cochliobolus heterostrophus BMSY-3; 6: Cochliobolus heterostrophus BMSY-4; 7: Cochliobolus heterostrophus BMSY-5; 8: Cochliobolus heterostrophus BMSY-6; 9: Cochliobolus heterostrophus BMSY-7; 10: Cochliobolus heterostrophus BMSY-8; 11: Cochliobolus heterostrophus BMLY-1; 12: Cochliobolus heterostrophus BMLY-2; 13: Cochliobolus heterostrophus BMLY-3; 14: Cochliobolus heterostrophus BMLY-4; 15: Cochliobolus heterostrophus BMLY-5; 16: Cochliobolus heterostrophus BMLY-6; 17: Cochliobolus heterostrophus BMLY-7; 18: Cochliobolus heterostrophus BMLY-8; 19: Cochliobolus heterostrophus BMDY-1; 20: Cochliobolus heterostrophus BMDY-2; 21: Cochliobolus heterostrophus BMDY-3; 22: Cochliobolus heterostrophus BMDY-4; 23: Cochliobolus heterostrophus BMDY-5; 24: ddH2O blank control; B is the identification result using the primer of race T, M: Marker; 1: (Cochliobolus heterostrophus)C4 standard strain (race T); 2: (Cochliobolus heterostrophus)C5 standard strain (race O); 3: Cochliobolus heterostrophus BMSY-1; 4: Cochliobolus heterostrophus BMSY-2; 5: Cochliobolus heterostrophus BMSY-3; 6: Cochliobolus heterostrophus BMSY-4; 7: Cochliobolus heterostrophus BMSY-5; 8: Cochliobolus heterostrophus BMSY-6; 9: Cochliobolus heterostrophus BMSY-7; 10: Cochliobolus heterostrophus BMSY-8; 11: Cochliobolus heterostrophus BMLY-1; 12: Cochliobolus heterostrophus BMLY-2; 13: Cochliobolus heterostrophus BMLY-3; 14: Cochliobolus heterostrophus BMLY-4; 15: Cochliobolus heterostrophus BMLY-5; 16: Cochliobolus heterostrophus BMLY-6; 17: Cochliobolus heterostrophus BMLY-7; 18: Cochliobolus heterostrophus BMLY-8; 19: Cochliobolus heterostrophus BMDY-1; 20: Cochliobolus heterostrophus BMDY-2; 21: Cochliobolus heterostrophus BMDY-3; 22: Cochliobolus heterostrophus BMDY-4; 23: Cochliobolus heterostrophus BMDY-5; 24: ddH2O blank control. Detailed implementation manners
[0026] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the spirit of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.
[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. The terms used in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention.
[0028] Unless otherwise specified, the materials and reagents used in the present invention can be obtained from commercial products in this field.
[0029] Example 1 Specificity Verification of Primers for Cochliobolus heterostrophus Race O
[0030] Using the genomic DNA of Cochliobolus heterostrophus race O standard strain C5, Cochliobolus heterostrophus race T standard strain C4, Curvularia lunata, Cochliobolus sativus ) ) and Cochliobolus setariae as templates, and ddH2O as a blank control for PCR amplification. The PCR reaction system was 20 μL: 2.0 μL of DNA template, 1.0 μL of 10 μM upstream (SEQ ID NO.1) and downstream primers (SEQ ID NO.2), 10 μL of 2×Easy Taq PCR Super Mix, and made up to 20 μL with ddH2O. ddH2O was used to replace the DNA template as a negative control. The PCR reaction program: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, 35 cycles, annealing at 60 °C for 30 s, 35 cycles, extension at 72 °C for 30 s, 35 cycles, continued extension at 72 °C for 10 min, 35 cycles; stored at 4 °C. The PCR amplification products were electrophoresed on a 1% agarose gel at 100 V for 30 min, and then photographed and observed with a gel imager.
[0031] As Figure 1 shown in the gel electrophoresis results of Figure A, a band of about 941 bp was amplified from the genomic DNA of Cochliobolus heterostrophus race O standard strain C5, while no bands were amplified from the genomic DNAs of Cochliobolus heterostrophus race T standard strain C4, Curvularia lunata, Cochliobolus sativus, Cochliobolus setariae and other corn pathogens belonging to the same genus and the blank control, indicating that the primers are specific primers for Cochliobolus heterostrophus race O.
[0032] Example 2 Specificity Verification of Primers for Race T of Bipolaris maydis
[0033] Using the genomic DNA of the standard strain C4 of race T of Bipolaris maydis, the standard strain C5 of race O of Bipolaris maydis, Curvularia lunata, Bipolaris sativus, and Bipolaris setariae as templates, and ddH2O as a blank control for PCR amplification. The PCR reaction system was 20 μL: 2.0 μL of DNA template, 1.0 μL of 10 μM upstream (SEQ ID NO.3) and downstream primers (SEQ ID NO.4), 10 μL of 2×Easy Taq PCR Super Mix, and supplemented with ddH2O to 20 μL. ddH2O was used to replace the DNA template as a negative control. The PCR reaction procedure: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, 35 cycles, annealing at 58 °C for 30 s, 35 cycles, extension at 72 °C for 30 s, 35 cycles, continued extension at 72 °C for 10 min, 35 cycles; stored at 4 °C. After the PCR amplification products were electrophoresed on a 1% agarose gel at 100 V for 30 min, they were photographed and observed with a gel imager.
[0034] As Figure 1 shown in the gel electrophoresis diagram of B, a band of about 837 bp was amplified from the genomic DNA of the standard strain C4 of race T of Bipolaris maydis, while no band was amplified in the standard strain C5 of race O of Bipolaris maydis, Curvularia lunata, Bipolaris sativus, Bipolaris setariae and other maize pathogenic bacteria of the same genus and the blank control, indicating that the primer is a specific primer for race T of Bipolaris maydis.
[0035] Example 3 Sensitivity Verification Experiment
[0036] Sensitivity verification of primers for race O: Prepare genomic DNA of race O at different concentrations (10 ng / μL; 1 ng / μL; 100 pg / μL; 10 pg / μL; 1 pg / μL; 100 fg / μL), and perform PCR amplification reactions according to the system and procedure in Example 1 respectively.
[0037] The results are as Figure 2 shown in A, and it can be seen that the detection limit of race O is 100 fg / μL.
[0038] Verification of the sensitivity of the primer pair for Race T: Genomic DNA of Race T with different concentrations (10 ng / μL; 1 ng / μL; 100 pg / μL; 10 pg / μL; 1 pg / μL; 100 fg / μL) was prepared, and PCR amplification reactions were carried out respectively according to the system and procedure in Example 2.
[0039] The results are as Figure 2 shown in B. It can be seen that the detection limit of Race T is 100 pg / μL.
[0040] Example 4
[0041] Using the genomic DNA of the standard strain C5 (Cochliobolus heterostrophus) of Race O of Bipolaris maydis, the standard strain C4 of Race T of Bipolaris maydis, Setosphaeria turcica, Cercospora maydis-zeae, Cercospora zeina, Setophoma zeae-maydis, Epicoccum latusicollum, Fusarium verticillioides, F. graminearum, Puccinia polysora, and Puccinia sorghi of maize as templates, PCR amplification was carried out with ddH2O as a blank control. The amplification system and procedure were the same as in Example 1.
[0042] The results of gel electrophoresis are as Figure 3 shown in A. The primer for Race O amplified a band only in the standard strain C5 of Race O and no bands were amplified in the pathogenic fungi of the main leaf diseases of maize, further indicating that this primer is a specific primer for Bipolaris maydis Race O.
[0043] Using the genomic DNA of the standard strain C4 (Cochliobolus heterostrophus) of race T of Bipolaris maydis, the standard strain C5 of race O of Bipolaris maydis, Setosphaeria turcica, Cercospora maydis-zeae, Cercospora zeina, Setophoma zeae-maydis, Epicoccum latusicollum, Fusarium verticillioides, F. graminearum, Puccinia polysora, and Puccinia sorghi as templates, PCR amplification was carried out with ddH2O as the blank control. The amplification system and procedure were the same as in Example 2.
[0044] The results of gel electrophoresis were as Figure 3 shown in B. The primer specific to race T amplified a band only in the standard strain C4 of race T and no bands were amplified in the pathogenic fungi of major maize leaf diseases, further indicating that this primer is a specific primer for race T of Bipolaris maydis.
[0045] Example 5
[0046] Using the genomic DNA of the standard strain C5 (race O) of Bipolaris maydis, the standard strain C4 (race T) of Bipolaris maydis, Bipolaris maydis BMSY-1, Bipolaris maydis BMSY-2, Bipolaris maydis BMSY-3, Bipolaris maydis BMSY-4, Bipolaris maydis BMSY-5, Bipolaris maydis BMSY-6, Bipolaris maydis BMSY-7, Bipolaris maydis BMSY-8, Bipolaris maydis BMLY-1, Bipolaris maydis BMLY-2, Bipolaris maydis BMLY-3, Bipolaris maydis BMLY-4, Bipolaris maydis BMLY-5, Bipolaris maydis BMLY-6, Bipolaris maydis BMLY-7, Bipolaris maydis BMLY-8, Bipolaris maydis BMDY-1, Bipolaris maydis BMDY-2, Bipolaris maydis BMDY-3, Bipolaris maydis BMDY-4, and Bipolaris maydis BMDY-5 as templates, PCR amplification was carried out with ddH2O as the blank control. The amplification system and procedure were the same as in Example 1.
[0047] The results of gel electrophoresis were asFigure 4 As shown in Figure A, with the standard strain C5 of race O as the positive control, specific bands were amplified in all 21 Bipolaris maydis strains, indicating that all 22 strains were Bipolaris maydis race O.
[0048] Using the genomic DNA of the standard strain C4 (race T) of Bipolaris maydis, the standard strain C5 (race O) of Bipolaris maydis, Bipolaris maydis BMSY-1, Bipolaris maydis BMSY-2, Bipolaris maydis BMSY-3, Bipolaris maydis BMSY-4, Bipolaris maydis BMSY-5, Bipolaris maydis BMSY-6, Bipolaris maydis BMSY-7, Bipolaris maydis BMSY-8, Bipolaris maydis BMLY-1, Bipolaris maydis BMLY-2, Bipolaris maydis BMLY-3, Bipolaris maydis BMLY-4, Bipolaris maydis BMLY-5, Bipolaris maydis BMLY-6, Bipolaris maydis BMLY-7, Bipolaris maydis BMLY-8, Bipolaris maydis BMDY-1, Bipolaris maydis BMDY-2, Bipolaris maydis BMDY-3, Bipolaris maydis BMDY-4, and Bipolaris maydis BMDY-5 as templates, and ddH2O as the blank control, PCR amplification was carried out. The amplification system and procedure were the same as in Example 2.
[0049] The results of gel electrophoresis are as Figure 4 As shown in Figure B, with the standard strain C4 of race T as the positive control, specific bands were not amplified in all 22 Bipolaris maydis strains, indicating that all 22 strains were not Bipolaris maydis race T.
[0050] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.
[0051] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.
Claims
1. A specific primer for identifying race O and race T of Bipolaris maydis, characterized in that, It includes the upstream primer and downstream primer of race O, as well as the upstream primer and downstream primer of race T; among them, the nucleic acid sequence of the upstream primer of race O is shown in SEQ ID NO.1, and the nucleic acid sequence of the downstream primer of race O is shown in SEQ ID NO.2; the nucleic acid sequence of the upstream primer of race T is shown in SEQ ID NO.3, and the nucleic acid sequence of the downstream primer of race T is shown in SEQ ID NO.
4.
2. A kit for identifying race O and race T of Bipolaris maydis, characterized in that, It includes the specific primer described in claim 1.
3. Application of the specific primer described in claim 1 or the kit described in claim 2 in identifying race O and race T of Bipolaris maydis.
4. A method for identifying race O and race T of Bipolaris maydis, characterized in that, It includes the following steps: performing PCR amplification on the DNA of the sample to be identified by using the primer described in claim 1 or the kit described in claim 2.
5. The method according to claim 4, characterized in that, The reaction system for the PCR amplification includes: 2.0 μL of DNA template, 1.0 μL of upstream and downstream primers with a concentration of 10 μM, 10 μL of 2×Easy Taq PCR Super Mix, and ddH2O is added to make up to 20 μL; the upstream primer includes the sequence shown in SEQ ID NO.1 and / or SEQ ID NO.3, and the downstream primer includes the sequence shown in SEQ ID NO.2 and / or SEQ ID NO.
4.
6. The method according to claim 4 or 5, characterized in that, The reaction conditions for the PCR amplification include: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, 35 cycles, annealing at 58 °C or 60 °C for 30 s, 35 cycles, extension at 72 °C for 30 s, 35 cycles, and continued extension at 72 °C for 10 min, 35 cycles.
7. The method according to claim 4, characterized in that, After the PCR amplification product is electrophoresed on 1% agarose gel, it is photographed and observed with a gel imager.
8. The method according to claim 7, characterized in that, The conditions for the gel electrophoresis are electrophoresis at 100 V for 30 min.
9. The method according to claim 4, wherein The upstream primer and downstream primer of race O specifically amplify a 941 bp band, and the upstream primer and downstream primer of race T specifically amplify an 837 bp band.