LAMP detection primer set and detection method for Pseudomonas aeruginosa

Abstract

The present invention discloses a LAMP detection primer set and a detection method for Stagonosporopsis chrysanthemi, which is a chrysanthemum stem and leaf blight disease, and belongs to the technical field of plant pathogen molecular detection. The LAMP detection primer set includes an outer primer pair, an inner primer pair and a loop primer pair, whose nucleotide sequence is shown in SEQ ID NO: 1-6. The present invention also discloses the application of the LAMP detection primer set in detecting chrysanthemum stem and leaf blight disease. The present invention can not only quickly detect the pathogenic bacteria purified culture Stagonosporopsis.chrysanthemi, but also directly perform rapid diagnosis on chrysanthemum plants in the field, effectively avoiding the dependence on a variety of complex instruments in the detection, and the LAMP detection method provided by the present invention has the advantages of high detection specificity, high sensitivity, short detection time, direct observation by naked eyes, etc., and has a wide range of practical application value.

Description

Technical Field

[0001] The invention relates to the technical field of plant pathogen molecular detection, in particular to a LAMP detection primer set and a detection method for Pseudomonas aeruginosa causing chrysanthemum stem and leaf blight. Background Art

[0002] Chrysanthemum morifolium cv. Fubaiju is a perennial herb of the Asteraceae family with high ornamental value and health benefits. It can be processed into beverages and used as medicine. Chrysanthemum extract has many functions such as improving cardiovascular health, inhibiting inflammation and delaying aging, and therefore has important economic value.

[0003] At present, there are many types of chrysanthemum fungal diseases. In 1970, G.M. A. M. R. Strong et al. first reported chrysanthemum wilt caused by Fusarium oxysporum. In 1983, Gao Qichao et al. conducted a survey on medicinal chrysanthemum diseases and discovered chrysanthemum downy mildew in Huangshan City, Anhui Province. The disease harmed tribute chrysanthemum and caused a large reduction in its production. In 2011, a disease suspected to be caused by Sclerotinia sclerotiorum was found on chrysanthemums, which was determined to be chrysanthemum sclerotinia after research. In 2015, Zhao Xiting et al. isolated and identified the pathogen of a major disease that caused blackening and death of leaves on Huaihuang chrysanthemum, and preliminarily determined that the disease was Huaihuang chrysanthemum black spot caused by Alternaria of the genus Alternaria. In 2024, Lv Ruiling et al. of our research group discovered a new fungal disease on Fubai chrysanthemum in Macheng, Hubei, which can cause ulcer necrotic spots on the leaves, stems and flowers of Fubai chrysanthemum. The pathogen was identified as Stagonosporopsis chrysanthemi by microscopic observation and multi-gene molecular identification, which is a quarantine disease on chrysanthemum in my country. The disease occurs at a frequency of about 40%, which seriously affects the yield and quality of Fubaiju. Stagonosporopsis sp. fungi can cause devastating diseases of many plants, causing serious economic losses, and have been reported all over the world. In 2021, Zhou Jiawei and others isolated the pathogen from the diseased Pinellia tissue and determined that it was caused by this genus of fungi. In September 2022, Wassim Habib and others first discovered that this genus of fungi can also cause Italian cauliflower leaf spot disease, which attracted widespread attention.

[0004] S. chrysanthemi can infect chrysanthemum varieties such as Fubaiju and Hangbaiju, causing serious damage to Asteraceae plants. The symptoms of different fungal diseases in the field are relatively similar, which causes trouble for the diagnosis of chrysanthemum diseases. Conventional disease diagnosis techniques mainly include isolation of pathogens, obtaining pure cultures, determining pathogens by Koch's postulates, and then identifying species through microscopic morphological observation, molecular identification, and sequencing of pathogens. The whole process takes at least one month and as long as half a year. In addition, the whole process requires the use of certain experimental instruments to assist, which is not conducive to the rapid identification of pathogens, especially for some explosive, destructive, and fast-spreading quarantine diseases, and it is easy to miss the best prevention and control opportunities.

[0005] In recent years, molecular biology technology has developed rapidly, and loop-mediated isothermal amplification (LAMP) technology is one of them. This technology has the advantages of not relying on large-scale instruments and equipment, simple operation, strong specificity, high sensitivity and easy results, and is widely used in the rapid detection of plant pathogens. At present, there is no report on the LAMP rapid detection technology for chrysanthemum stem and leaf blight S. chrysanthemi. Therefore, the purpose of the present invention is to provide a LAMP primer set and detection method for rapid detection of chrysanthemum stem and leaf blight Pseudomonas chrysanthemi. Summary of the invention

[0006] The purpose of the present invention is to provide a LAMP detection primer set and a detection method for Pseudomonas aeruginosa, a chrysanthemum stem and leaf blight disease, so as to solve the problems existing in the above-mentioned prior art. The detection method provided by the present invention can quickly diagnose the chrysanthemum stem and leaf blight disease of chrysanthemum plants in the field, has the advantages of high detection specificity, high sensitivity, short detection time, direct observation by the naked eye, etc., provides technical support for grassroots plant protection agencies in disease diagnosis and quarantine departments in import and export quarantine inspection, and has broad practical application value.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a LAMP detection primer set for Stagonosporopsis chrysanthemi, which comprises an outer primer pair, an inner primer pair and a loop primer pair;

[0009] The outer primer pair consists of a forward outer primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse outer primer with a nucleotide sequence as shown in SEQ ID NO: 2; the inner primer pair consists of a forward inner primer with a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse inner primer with a nucleotide sequence as shown in SEQ ID NO: 4; the loop primer pair consists of a forward loop primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a reverse loop primer with a nucleotide sequence as shown in SEQ ID NO: 6.

[0010] The invention also provides a LAMP detection kit for identifying Stagonosporopsis chrysanthemi, which causes chrysanthemum stem and leaf blight, comprising the LAMP detection primer set.

[0011] The present invention also provides application of the LAMP detection primer set in preparing a product for identifying Stagonosporopsis chrysanthemi, which causes chrysanthemum stem and leaf blight.

[0012] The present invention also provides a LAMP detection method for Stagonosporopsis chrysanthemi, which comprises the following steps:

[0013] The DNA of the chrysanthemum sample to be tested is used as a template, and the LAMP detection primer set is used to perform LAMP amplification. According to the electrophoresis detection result of the LAMP amplification product and / or the fluorescent dye color development result, it is determined whether the chrysanthemum sample to be tested contains Stagonosporopsis chrysanthemi, a chrysanthemum stem and leaf blight fungus.

[0014] Furthermore, if the electrophoresis test results show waterfall-like trapezoidal characteristic bands and / or the fluorescent dye color development result is fluorescent green, it is judged that Stagonosporopsis chrysanthemi, the chrysanthemum stem and leaf blight disease, is present in the chrysanthemum sample to be tested; if the electrophoresis test results do not have waterfall-like trapezoidal characteristic bands and / or the fluorescent dye color development result is orange-yellow, it is judged that Stagonosporopsis chrysanthemi, the chrysanthemum stem and leaf blight disease, is not present in the chrysanthemum sample to be tested.

[0015] Furthermore, the reaction condition of the LAMP amplification is incubation at 65° C. for 50 min.

[0016] Furthermore, the detection system of the LAMP amplification is: 10×Isothermal Amplification Buffer 2.5 μL, dNTP Mix 2.5 μL, MgSO4 1 μL, forward and reverse inner primers 4 μL each, forward and reverse loop primers 1 μL each, forward and reverse outer primers 0.5 μL each, Bst 2.0 Warm Start DNA Polymerase 1 μL, DNA template 1 μL, ddH2O is supplemented to 25 μL.

[0017] The present invention also provides the use of the LAMP detection primer set or the LAMP detection kit in detecting chrysanthemum stem and leaf blight.

[0018] The present invention discloses the following technical effects:

[0019] (1) High specificity: The LAMP detection method uses multiple specific primers designed for multiple regions of the target DNA sequence, ensuring that only target sequences that fully match the primers can be effectively amplified. This design principle makes the LAMP test extremely accurate in identifying the pathogen of chrysanthemum stem and leaf blight. The experiment can effectively distinguish between Pseudomonas aeruginosa and other non-target microorganisms, reducing the possibility of false detection and missed detection.

[0020] (2) High sensitivity: The LAMP test for chrysanthemum leaf blight does have a high sensitivity, which plays a key role in pathogen detection and early warning. Even if the pathogen content is very low, as long as there is enough target DNA sequence, the LAMP reaction can still effectively amplify and accurately detect Pseudomonas aeruginosa, the pathogen of chrysanthemum leaf blight.

[0021] (3) Fast and easy: LAMP testing is performed at a relatively stable temperature (usually 60-65°C) for 40-60 minutes, and the test results can be obtained by visual observation. It does not require temperature cycling and complex operations like PCR programs, and does not require expensive experimental instruments and equipment. This allows the test to be performed anytime and anywhere in quarantine inspection departments, farm fields, and other places without the need for technical support from large laboratories.

[0022] (4) Simple sample processing: In the LAMP test for chrysanthemum stem and leaf blight, the collected leaf samples only need to be properly crushed and extracted to serve as a template for LAMP testing. This simple sample processing method reduces pre-processing time and improves detection efficiency.

[0023] The present invention can not only quickly detect the purified culture of pathogenic bacteria S. chrysanthemi, but also directly quickly diagnose chrysanthemum plants in the field, effectively avoiding the dependence on a variety of complex instruments in the detection. In addition, the LAMP detection method provided by the present invention has the advantages of high detection specificity, high sensitivity, short detection time, direct observation by the naked eye, etc., providing technical support for grassroots plant protection agencies in disease diagnosis and quarantine departments in import and export quarantine inspection, and has wide practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is the result of phylogenetic analysis of Pseudomonas aeruginosa (strains HGNU2021-5 and HGNU2021-18) based on the Tub2 gene; Note: The NJ method was used with 1000 repetitions;

[0026] Figure 2 Electrophoresis detection diagram of the amplification effect of the LAMP primer set designed for RPB2, LSU and Tub2 genes as target genes; Note: From left to right: 1: Colletotrichum sojae; 2: Rhizoctonia solani; 3: Alternaria alternata; 4: Ephemerococcus nigromaculata; 5: Fusarium graminearum; 6: Micronodule; 7: Chaetomium globosum; 8: Botrytis cinerea; 9: Nigrospora oryzae; 10: Arthrospora phragmites; 11: Pseudomonas pseudoseptoria;

[0027] Figure 3 Electrophoresis detection diagram of the amplification effect of 5 groups of LAMP primers designed for Tub2 gene as target gene; Note: From left to right: 1: Rhizoctonia solani; 2: Alternaria alternata; 3: Micronodularia solani; 4: Ephemerococcus nigromaculata; 5: Fusarium graminearum; 6: Pseudomonas pseudospora; 7: Chaetomium globosum; 8: Botrytis cinerea; 9: Trichosanthis tricolor; 10: Cucurbita pekinensis;

[0028] Figure 4 The results of the optimal reaction temperature of LAMP detection of Pseudomonas aeruginosa for chrysanthemum stem and leaf blight; A in the figure is visual observation, and B is electrophoresis detection; the temperature range is 51℃-73℃, with a gradient of 2℃;

[0029] Figure 5The results of the optimal reaction time of LAMP detection of Pseudomonas aeruginosa for chrysanthemum stem and leaf blight; A in the figure is visual observation, and B is electrophoresis detection; 8 reaction times are set from left to right, namely 10min, 20min, 30min, 40min, 50min, 60min, 70min and 80min;

[0030] Figure 6 The results of LAMP specificity detection of Stagonosporopsis chrysanthemi, the cause of chrysanthemum stem and leaf blight; A in the figure is visual observation, and B is electrophoresis detection; from left to right: 1: Stagonosporopsis chrysanthemi, 2: Colletotrichum sp., 3: Alternaria alternata, 4: Fusarium graminearum, 5: Rhizoctonia solani, 6: Epicoccumnigrum, 7: Microdochium nivale, 8: Chaetomium globosum, 9: Apiospora arrundinis, CK: negative control;

[0031] Figure 7 The results of LAMP sensitivity test for Pseudomonas aeruginosa, a chrysanthemum stem and leaf blight disease; A in the figure is visual observation, and B is electrophoresis detection; No. 1 is DNA stock solution (concentration 4484.0 ng / μL), and No. 2-7 are DNA stock solutions after 10 3 , 10 6 , 10 9 , 10 12 , 10 15 , 10 18 Results after dilution; CK is the negative control;

[0032] Figure 8 The results of LAMP practicality test for Pseudomonas aeruginosa stem and leaf blight of chrysanthemum; A in the figure is naked eye observation, and B is electrophoresis detection; from left to right are genomic DNA amplification reaction, DNA extraction reaction from 5 diseased chrysanthemum leaves, and DNA extraction reaction from 3 healthy chrysanthemum leaves, and CK is the negative control. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Preliminary studies of the present invention have identified that chrysanthemum stem and leaf blight is caused by infection with Stagonosporopsis chrysanthemi, but there has been no report on the LAMP rapid detection technology for the rapid detection of Stagonosporopsis chrysanthemi, the pathogen of chrysanthemum stem and leaf blight. It is well known in the art that the pathogen of watermelon, melon and trichosanthes vine blight is Stagonosporpsis. citrulli; the pathogen of cucumber and zucchini vine blight is Stagonosporpsis. cucurbita; in the present invention, the pathogen of chrysanthemum stem and leaf blight is Stagonosporopsis chrysanthemi, and these three are all different species of the genus Stagonosporopsis. There are obvious differences in classification between different species, such as the size and morphology of conidia, and there are also obvious differences at the genomic DNA level. Taking the Tub2 gene as an example, there are many differences in the gene sequences of the three. The phylogenetic tree was constructed using the Tub2 gene sequence ( Figure 1 ) to reflect the differences among the three. Usually, the relatives of the same branch are closer, and the relatives of different branches or the farther the genetic distance is, the farther the relationship is. Figure 1 It can be reflected that the chrysanthemum stem and leaf blight pathogen strains HGNU2021-5 and HGNU2021-18 used in the present invention (strains HGNU2021-5 and HGNU2021-18 are both recorded in the document "First Report of Stem and Foliage Blight of Chrysanthemum morifolium cv. Fubaiju Caused by Stagonosporopsis chrysanthemi in China") are clustered in the same branch with S. chrysanthemi, with a close relationship, and a distant relationship with S. citrulli and S. cucurbita, while S. citrulli and S. cucurbita are closely related. It is confirmed that the Stagonosporopsis chrysanthemi species targeted by the present invention is distantly related to other species reported in the prior art, such as S. citrulli and S. cucurbita, and there are many differences in the target gene sequences, so the detection technology of S. citrulli and S. cucurbita cannot be directly borrowed.

[0039] In order to improve the detection efficiency of chrysanthemum stem and leaf blight, the present invention has carried out the following research:

[0040] The test strains used in the following examples are shown in Table 1, all of which were provided by the Microbiology Laboratory of the College of Biology and Agricultural Resources of Huanggang Normal University.

[0041] Table 1 Test strains used for LAMP specificity analysis

[0042]

[0043]

[0044] Example 1

[0045] 1 Screening of specific target sequences for Stagonosporopsis chrysanthemi, the pathogen causing chrysanthemum stem and leaf blight

[0046] The RPB2, LSU and Tub2 genes of Stagonosporopsis chrysanthemi HGNU2021-18 strain were used as target genes, and primers were designed using the online LAMP primer design software Primer Explorer V5 (http: / / primerexplorer.jp / e) to obtain three sets of Stagonosporopsis chrysanthemi LAMP primer sets. Each set of primers consisted of three pairs of outer primers, inner primers and loop primers. The sequences of each primer are shown in Table 2. Detected by LAMP program, LAMP detection system (25μL): 10×Isothermal Amplification Buffer 2.5μL, dNTP Mix (10mM) 2.5μL, MgSO4 (100mM) 1μL, forward and reverse inner primers (concentration 10μM) 4μL each, forward and reverse loop primers (concentration 10μM) 1μL each, forward and reverse outer primers (concentration 10μM) 0.5μL each, Bst 2.0Warm Start DNA Polymerase (8000U / mL) 1μL, DNA template 1μL, ddH2O to make up the total volume 25μL. The reaction conditions are incubation at 65℃ for 50min.

[0047] The amplified LAMP amplification product was detected by agarose gel electrophoresis and visually observed with fluorescent dyes to determine the test results. Agarose gel electrophoresis detection: 2 μL of LAMP amplification product was mixed with 1 μL of 10× Loading buffer, and then subjected to 1% agarose gel electrophoresis. Positive amplification results showed waterfall-like trapezoidal characteristic bands, and negative amplification results showed no bands. Fluorescent dye visual observation: After the LAMP reaction was completed, 0.2 μL of SYBR-Green I dye was added to the product, and the reaction results were determined based on the color change of the dye. The reaction results that showed fluorescent green were positive, and the reaction results that remained orange-yellow were negative.

[0048] Agarose gel electrophoresis showed that the primer set designed with RPB2 as the target gene could not amplify bands for Pseudomonas aeruginosa, and the primer set designed with LSU as the target gene could not amplify bands for all tested strains. The primer set designed with Tub2 as the target gene could amplify two non-specific target bands for Botrytis cinerea, Nigrospora oryzae, Arthrocosporium phragmites and Pseudomonas aeruginosa, while other strains (Colletotrichum sojae, Rhizoctonia solani, Alternaria alternata, Echinococcus nigrostriatus, Fusarium graminearum, Micronoduleus and Chaetomium globosum) could not amplify any bands ( Figure 2 ). Therefore, Tub2 gene was selected as the target gene to further design LAMP primers.

[0049] Table 2 Design of LAMP primer sets with RPB2, LSU and Tub2 genes as target genes

[0050]

[0051] 2 Design of LAMP specific primers for Pseudomonas aeruginosa blight on chrysanthemum stem and leaf

[0052] Through the above determination of beta-Tubulin (Tub2) gene fragments of Pseudomonas aeruginosa and other plant pathogens, primers were designed using the online LAMP primer design software Primer Explorer V5 (http: / / primerexplorer.jp / e), and a total of 5 sets of Pseudomonas aeruginosa LAMP primer sets were obtained. Each set of primers consisted of 3 pairs of outer primers, inner primers and loop primers. The sequences of each primer are shown in Table 3. The products detected by LAMP program were detected by agarose gel electrophoresis. It was found that primer set 1 and primer set 5 had no specificity for all strains and could amplify bands. Primer set 2 and primer set 3 could not amplify any bands for all strains. Primer set 4 could amplify bands for Pseudomonas aeruginosa, a pathogenic blight on chrysanthemum stems and leaves, while other strains (Rhizoctonia solani, Alternaria alternata, Ephemerococcus nigromaculata, Fusarium graminearum, Micronodule bacteria, Chaetomium globosum, Botrytis cinerea, Trichoderma spp. and Pseudomonas pekinensis) could not amplify bands ( Figure 3 ). Therefore, primer set 4 of Tub2 gene was selected as the LAMP primer in this study.

[0053] Table 3 Design of LAMP primer sets with Tub2 gene as target gene

[0054]

[0055]

[0056] 3 Establishment and optimization of the initial LAMP system for Pseudomonas aeruginosa against chrysanthemum stem and leaf blight

[0057] 3.1 LAMP initial detection system (25 μL): 10×IsothermalAmplification Buffer 2.5 μL, dNTP Mix (10 mM) 2.5 μL, MgSO4 (100 mM) 1 μL, primer 4FIP / 46BIP (concentration 10 μM) 4 μL each, primer 4LF / 4LB (concentration 10 μM) 1 μL each, primer 4F3 / 4B3 (concentration 10 μM) 0.5 μL each, Bst 2.0Warm Start DNA Polymerase (8000 U / mL) 1 μL, DNA template 1 μL, ddH2O to make up the total volume to 25 μL. The reaction conditions were incubation at 65°C for 50 min.

[0058] 3.2 Reaction temperature optimization

[0059] The above reaction system was used to screen the optimal reaction temperature. The experimental results are as follows: Figure 4 As shown. When the reaction temperature is 55-69℃, the LAMP reaction is more complete and the color is darker. When the temperature is 53℃, the color of positive amplification is lighter, and the LAMP amplification is incomplete. In addition, if the temperature is too low or too high, LAMP cannot be amplified. In summary, 65℃ is selected as the optimal reaction temperature for sufficient amplification of the LAMP reaction.

[0060] 3.3 Response time optimization

[0061] The optimal reaction time was screened using the above reaction system and optimal reaction temperature. The experimental results are as follows: Figure 5 As shown. At 10 minutes of reaction, LAMP has not started to amplify. At 20 minutes of reaction, a ladder-shaped band appears on the electrophoresis graph but is not obvious. After staining with SYBR-Green I, it becomes slightly fluorescent green, and the LAMP reaction begins to amplify. In order to ensure sufficient amplification of LAMP and improve detection efficiency, 50 minutes is selected as the optimal reaction time for LAMP detection.

[0062] 4 LAMP specific detection of Pseudomonas aeruginosa causing chrysanthemum stem and leaf blight

[0063] The genomic DNA of the samples was extracted by CTAB method, and LAMP specificity detection was performed according to the above optimized detection system. The detection results ( Figure 6 ) It can be seen that the LAMP test is only positive for Pseudomonas aeruginosa, which is the cause of chrysanthemum stem and leaf blight. When other plant pathogenic fungi (Soybean Anthracnose, Alternaria alternata, Fusarium graminearum, Rhizoctonia solani, Black Ephedra, Micronodule Pseudomonas, Chaetomium globosum, and Arthritis phragmites) are used as templates, no positive reaction is observed. Whether the evaluation is based on agarose gel electrophoresis detection or fluorescent dye visual observation, the experimental results are consistent. Therefore, it is shown that the LAMP detection method established by the present invention is specific to Pseudomonas aeruginosa, which is the cause of chrysanthemum stem and leaf blight, and can be used to distinguish Pseudomonas aeruginosa from other fungal species.

[0064] 5. Determination of LAMP sensitivity of Pseudomonas aeruginosa against chrysanthemum stem and leaf blight

[0065] LAMP sensitivity detection is a standard that reflects the detection limit of the reaction system and is also an important indicator for achieving field detection.

[0066] The extracted bacterial genomic DNA was tested for DNA concentration using an ultra-micro spectrophotometer, and the concentration was 4484.0 ng / μL. 3 The sample was diluted in a series of gradients, and 7 concentrations were obtained by continuous dilution. 1 μL of each concentration was taken as a template for amplification. The results showed that ( Figure 7 ), after 10 18After dilution, obvious bands can still be seen in agarose gel electrophoresis, and green fluorescence is shown after staining with SYBR-Green I. Therefore, the sensitivity of LAMP reaction is extremely high.

[0067] 6 Practical detection of Pseudomonas aeruginosa causing chrysanthemum stem and leaf blight using LAMP

[0068] In order to evaluate the field effect of LAMP method in detecting chrysanthemum stem and leaf blight, eight chrysanthemum leaf samples (including five diseased samples and three healthy samples) were collected and subjected to LAMP detection after rapid DNA extraction.

[0069] result( Figure 8 ) showed that the five diseased leaf samples showed green fluorescence after LAMP amplification and SYBR-Green I staining, while the healthy chrysanthemum leaves did not show green fluorescence. The agarose gel electrophoresis results were consistent with the results after SYBR-Green I staining. At the same time, Pseudomonas aeruginosa, the chrysanthemum stem and leaf blight pathogen, was successfully isolated from the diseased and healthy junction of the diseased chrysanthemum leaves by tissue separation, indicating that the LAMP detection method established in this study has a high accuracy rate and can be used for the detection of chrysanthemum stem and leaf blight in the field.

[0070] The invention is innovative and practical, which not only enriches the technical means of plant disease detection, but also provides a reference for field diagnosis of chrysanthemum stem and leaf blight and the formulation of effective prevention and control measures. By applying for a patent, it aims to protect the technological innovation results of the invention, promote the popularization and application of the technology, and contribute to the sustainable development of the chrysanthemum planting industry. The inventor team hopes that through the authorization of this patent, it can further promote the research and application of the LAMP method in the field of plant disease detection and provide solid technical support for the healthy development of agricultural production.

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A LAMP detection primer set for Stagonosporopsis chrysanthemi, which is characterized by: It includes outer primer pairs, inner primer pairs and loop primer pairs; The outer primer pair is composed of a forward outer primer having a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse outer primer having a nucleotide sequence as shown in SEQ ID NO: 2; the inner primer pair is composed of a forward inner primer having a nucleotide sequence as shown in SEQ ID NO: 3 and a reverse inner primer having a nucleotide sequence as shown in SEQ ID NO: 4; the loop primer pair is composed of a forward loop primer having a nucleotide sequence as shown in SEQ ID NO: 5 and a reverse loop primer having a nucleotide sequence as shown in SEQ ID NO: 6; The LAMP detection primer set is designed by taking the Tub2 gene of Stagonosporopsis chrysanthemi, the pathogen causing chrysanthemum stem and leaf blight, as a target gene.

2. A LAMP detection kit for identifying Stagonosporopsis chrysanthemi, which is characterized by: The method comprises the LAMP detection primer set according to claim 1.

3. Use of the LAMP detection primer set according to claim 1 in the preparation of a product for identifying Stagonosporopsis chrysanthemi, the pathogen causing chrysanthemum stem and leaf blight.

4. A LAMP detection method for Stagonosporopsis chrysanthemi, which is characterized by: The following steps are involved: Using the DNA of the chrysanthemum sample to be tested as a template, LAMP amplification is performed using the LAMP detection primer set described in claim 1, and judging whether Stagonosporopsis chrysanthemi, a chrysanthemum stem and leaf blight pathogen, is present in the chrysanthemum sample to be tested based on the electrophoresis detection result of the LAMP amplification product and / or the fluorescent dye color development result.

5. The LAMP detection method according to claim 4, characterized in that If the electrophoresis test result shows a waterfall-shaped trapezoidal characteristic band and / or the fluorescent dye color development result is fluorescent green, it is judged that Stagonosporopsis chrysanthemi, the chrysanthemum stem and leaf blight disease, is present in the chrysanthemum sample to be tested; if the electrophoresis test result shows no waterfall-shaped trapezoidal characteristic band and / or the fluorescent dye color development result is orange-yellow, it is judged that Stagonosporopsis chrysanthemi, the chrysanthemum stem and leaf blight disease, is not present in the chrysanthemum sample to be tested.

6. The LAMP detection method according to claim 4, characterized in that The reaction condition of the LAMP amplification was incubation at 65° C. for 50 min.

7. The LAMP detection method according to claim 4, characterized in that The detection system of the LAMP amplification is: 10×Isothermal Amplification Buffer 2.5μL, dNTP Mix 2.5μL, MgSO4 1μL, forward and reverse inner primers 4μL each, forward and reverse loop primers 1μL each, forward and reverse outer primers 0.5μL each, Bst 2.0Warm Start DNA Polymerase 1μL, DNA template 1μL, ddH2O is added to 25μL.

8. Use of the LAMP detection primer set according to claim 1 or the LAMP detection kit according to claim 2 in detecting chrysanthemum stem and leaf blight.

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