Primer group and kit for detecting new plum brown spot and application of primer group and kit

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a primer group and a kit for detecting new plum brown spot and application of the primer group and the kit, and base sequences of the primer group are shown as SEQ ID NO.1-SEQ ID NO.5. Based on the primer group, the loop-mediated isothermal amplification technology is used for rapid detection of the alternaria nepalensis, and the alternaria nepalensis can be accurately detected from a complex pathogenic bacterium environment in diseased plant tissues. The rapid detection system disclosed by the invention is good in specificity and high in sensitivity, and the detection limit of the DNA of the alternaria nepalensis is 0.246 fg / mu L. By utilizing the rapid detection system provided by the invention, the alternaria nepalensis can be rapidly, simply, accurately and sensitively detected from a field sample within 45 minutes, and compared with a traditional symptom recognition method and a common molecular detection method, the rapid detection system is simpler and more efficient.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a primer set, reagent kit, and application for detecting syphilis brown spot disease. Background Technology

[0002] Xinmei ( Prunus domestica *Prunus spp.*, a cultivated species of the genus *Prunus* in the family Rosaceae, originated in West Asia and Europe. It is one of Serbia's main fruit varieties and is considered the national fruit of Serbia. Epidemiological studies show that *Prunus spp.* has potential medicinal value; and due to its sweet and sour taste and extremely high nutritional value, it has been widely introduced and cultivated in many parts of China, mainly distributed in Xinjiang, Shaanxi, Hebei, and Henan provinces. *Prunus spp.* is a rich source of major antioxidants and phenolic compounds, such as caffeic acid, chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid. These antioxidants and bioactive compounds can effectively treat and prevent gastrointestinal diseases, bone health, and cardiovascular diseases, and maintain blood sugar levels.

[0003] However, in recent years, Alternaria neonata ( Alternaria alternata Brown spot disease, caused by [unspecified pathogen], has frequently broken out in major plum-producing areas such as Jiashi County and Yingjisha County, with increasingly serious damage. This disease primarily affects new plum fruits and has a long incubation period in the field. Early stages are difficult to detect, and the disease is relatively concentrated and prone to outbreaks. Typically, small red spots first form on the fruit peel, gradually deepening to dark brown lesions. These lesions expand outwards, forming large lesions with a sunken center. Finally, the affected area turns reddish-black, and the diseased fruit is often deformed and falls off prematurely. Therefore, early detection of the pathogen is crucial for controlling brown spot disease in new plums. Traditional detection methods, such as tissue isolation and culture, are time-consuming, and PCR requires sophisticated thermal cycling equipment, making rapid application in the field difficult. While LAMP technology offers the advantage of isothermal amplification, its detection performance is highly dependent on the specificity and sensitivity of the designed primers; and current technologies lack effective methods to distinguish [unspecified pathogens]. Alternaria alternata A LAMP detection protocol that is closely related to bacteria and maintains high sensitivity even in complex field sample backgrounds. Summary of the Invention

[0004] The purpose of this invention is to provide a primer set for detecting syphilis lentigines, which solves the problems existing in the prior art.

[0005] The technical solution adopted in this invention is: This invention provides a primer set for detecting syphilis lentigines, the base sequences of which are shown in SEQ ID NO.1~SEQ ID NO.5.

[0006] A second aspect of the present invention provides a kit for detecting syphilis lentigines, the kit comprising the primer set as described in claim 1.

[0007] A third aspect of the invention provides the application of the primer set or the kit, the application being for the detection of brown spot disease and / or Alternaria alternata .

[0008] Preferably, the primer set is used for detection. Alternaria alternata The method is as follows: Collect infected plants, culture the pathogens from the infected plants, and extract the DNA from the pathogens; Prepare mixed primers using the primer set described above; Using DNA as a template, amplification was performed at 62℃~69℃ for 45min~50min using mixed primers, followed by inactivation, and the results were determined by agarose gel electrophoresis. If a specific band appears, the pathogen to be detected is... Alternaria alternata If no specific band is found, then the pathogen being tested is not... Alternaria alternata .

[0009] Preferably, the molar ratio of each component in the mixed primer is: SEQ ID NO.1: SEQ ID NO.2: SEQ ID NO.3: SEQ ID NO.4: SEQ ID NO.5=1:1:8:8:2.

[0010] Preferably, the reaction system used for amplification is any one of the following: 1) 2.5×Bst 4.0 LowSaltMix 10μL, 10×Red pH Dye 2.5μL, 10× Mixed Primers 2.5μL, DNA 3μL, ddH2O 7μL; 2) 10 μL of 2.5×Bst 4.0 LowSaltMix, 2.5 μL of 10×SYBR Green I, 2.5 μL of 10× mixed primers, 3 μL of DNA, and 7 μL of ddH2O.

[0011] Preferably, the amplification conditions are 65℃~66℃ for 45 min.

[0012] Preferably, the inactivation conditions are 85°C for 10 minutes.

[0013] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a primer set for detecting syphilitic brown spot disease, the base sequences of which are shown in SEQ ID NO.1~SEQ ID NO.5. This invention utilizes... Alternaria alternataThe ITS sequence of *Alternaria* was compared with that of different pathogens belonging to the genus *Alternaria*, providing a set of primers for rapid detection of *Alternaria pruinata* brown spot disease. Based on this primer set, this invention utilizes loop-mediated isothermal amplification (LAMP) technology for rapid detection of *Alternaria pruinata*, accurately detecting the fungus from the complex pathogenic environment in diseased plant tissues. The rapid detection system described in this invention exhibits high specificity and sensitivity, with a detection limit of 0.246 fg / μL for *Alternaria pruinata* DNA. Using the rapid detection system provided by this invention, *Alternaria pruinata* can be detected rapidly, simply, accurately, and sensitively from field samples within 45 minutes, which is simpler and more efficient than traditional symptom identification methods and ordinary molecular detection methods.

[0014] The method described in this invention has higher specificity and sensitivity than conventional PCR methods, and can detect various morphologies of Alternaria alternata, such as hyphae and spores. It is of great significance for early warning of Alternaria alternata outbreaks and pathogen monitoring in epidemic areas. At the same time, it can eliminate the need for expensive equipment investment and is easy to promote and use at the grassroots level. Attached Figure Description

[0015] Figure 1 For based on A. alternata and A.ochraceus Agarose gel electrophoresis results for the four primer-specific screenings.

[0016] Figure 2 The results of agarose gel electrophoresis show the sensitivity screening of primer sets based on different template concentrations.

[0017] Figure 3 Agarose gel electrophoresis results for field adaptability testing of different primer sets.

[0018] Figure 4 For LAMP-specific detection of AltPD-1. A: LAMP-SYBR Green I real-time fluorescence quantitative detection curve; B: Agarose gel electrophoresis results in Figure A; C: LAMP-Cresol Red visualization results; D: Agarose gel electrophoresis results in Figure C.

[0019] Figure 5 The LAMP sensitivity detection of AltPD-1 is shown in Figure A. A: LAMP-SYBR Green I real-time quantitative PCR curve; B: Agarose gel electrophoresis results (Figure A); C: LAMP-Cresol Red visualization results; D: Agarose gel electrophoresis results (Figure C).

[0020] Figure 6This is a general LAMP assay for AltPD-1. A: LAMP-SYBR Green I real-time quantitative PCR curve; B: Agarose gel electrophoresis results (Figure A); C: LAMP-Cresol Red visualization results; D: Agarose gel electrophoresis results (Figure C).

[0021] Figure 7 To validate the use of LAMP-SYBR Green I real-time fluorescence detection on infected and healthy materials obtained from four different regions: A: Material from Gashi County; B: Material from Shache County; C: Material from Yingjisha County; D: Material from Maigati County.

[0022] Figure 8 To validate the LAMP-Cresol Red visualization detection of infected and healthy materials obtained from four different regions. A: Visualization results in centrifuge tubes; B: Agarose gel electrophoresis results from Figure A.

[0023] Figure 9 Optimization of the LAMP-Cresol Red visualization system. A: Optimization of DNA addition amount; B: Optimization of temperature; C: Optimization of reaction time.

[0024] Figure 10 Optimization of the LAMP-SYBR Green I real-time quantitative PCR system. A: Optimization of DNA addition amount; B: Optimization of temperature; C: Optimization of SYBR Green I concentration. Detailed Implementation

[0025] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0026] The inventive concept of this invention is as follows: Molecular diagnostics based on nucleic acid amplification technology is playing an increasingly important role in pathogen detection, with polymerase chain reaction (PCR) having been established as the authoritative standard. Although PCR technology is relatively mature and stable, it still has significant limitations. The most significant challenge lies in its reliance on expensive equipment and skilled operators, a drawback that greatly restricts its application in actual field or complex environments. In 2000, Japanese scholars Notomi Tsugunori et al. first proposed loop-mediated isothermal DNA amplification technology internationally. This technology only requires temperature-controlled equipment and is well-suited for complex field environments.

[0027] Therefore, this invention compares species belonging to the same genus *Alternaria*. Alternaria alternata , Alternaria arborescent , Alternaria citriarbustum , Alternaria gaisen , Alternaria blight and Alternaria toxicogenica The ITS sequence provides a primer set for detecting new syphilis lentigines, the base sequences of which are shown in SEQ ID NO.1~SEQ ID NO.5.

[0028] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0029] The list of abbreviations for this invention is shown in Table 1.

[0030] Table 1 List of Abbreviations Example 1 A primer set for detecting syphilitic brown spot disease is as follows: 1. Design primer sequences.

[0031] By comparing with other species belonging to the genus Alternaria Alternaria alternata , Alternaria arborescens , Alternaria citrus shrub , Alternaria gaisen , Alternaria blight and Alternaria toxicogenic For the ITS sequence, this invention provides four sets of primers, and the specific primer information is shown in Table 2.

[0032] Table 2 Primer Information 2. Experimental materials and methods.

[0033] Experimental materials: The *Alternaria neonata* strain used in this invention is published in GenBank, accession number PX122072.1. The strain used in this invention was extracted from *Prunus mume*, *Ficus carica*, *Albizia julibrissin*, and *Platanus orientalis*.

[0034] Experimental methods: S1. Extraction of pathogen DNA: The pathogen was cultured at 25°C for 7 days in a constant temperature incubator. Surface hyphae were scraped off, and then thoroughly ground using an automated rapid sample grinder. DNA was extracted using a DNA kit, and the concentration and quality of the DNA were detected using a UV spectrophotometer. The qualified DNA was immediately stored at -20°C for later use.

[0035] S2. Prepare mixed primers for the 10×LAMP Primer Mix system based on the primer sets shown in AltPD-1~AltPD4, as detailed in Table 3.

[0036] Table 3 10×LAMP Primer Mix System Note: Since AltPD-2 does not have LB primers, the missing part is made up with water. In Table 3, "-" indicates that this item is not available.

[0037] S3. LAMP-Cresol Red Visualization Detection: First, prepare the LAMP-Cresol Red visualization system. Incubate the LAMP-Cresol Red visualization system in a 65℃ constant temperature apparatus for 45 min, then inactivate it at 85℃ for 10 min. After the reaction, observe for the appearance of specific bands using wt 1.2% agarose gel electrophoresis. The presence of specific bands indicates a positive result; otherwise, it indicates a negative result. The preparation method for the LAMP-Cresol Red visualization system is shown in Table 4.

[0038] Table 4 LAMP-Cresol Red Visualization System Note: In Table 4, "-" indicates that this item is not present.

[0039] 3. Primer-specific screening.

[0040] To ensure primer specificity, the four sets of primers were initially screened to identify pathogens. A. alternata Common pathogens in plum trees A.ochraceus Specific detection was performed together, with ddH2O as a negative control NTC. The LAMP reaction was carried out at 65℃ until it was fully reacted. After the reaction, the results were analyzed by agarose gel electrophoresis.

[0041] See results Figure 1 . Figure 1 In the diagram, M stands for DNA 2K plus Marker. Lanes 1-3 represent the amplification results of primer set AltPD-1; lanes 4-6 represent the amplification results of primer set AltPD-2; lanes 7-9 represent the amplification results of primer set AltPD-3; and lanes 10-12 represent the amplification results of primer set AltPD-4. The amplification templates for lanes 1, 4, 7, and 10 are... A. alternata The amplification templates for lanes 2, 5, 8, and 11 are: A.ochraceus The amplification template for lanes 3, 6, 9, 12 and 13 was ddH2O.

[0042] Since the AltPD-2 primer set did not amplify the target band, no further experimental verification of the AltPD-2 primer set was conducted.

[0043] 4. Primer set sensitivity screening.

[0044] To ensure the sensitivity of the primer set, several designed primer sets were initially screened. In the specificity screening, primer set AltPD-2 did not amplify any bands; therefore, sensitivity screening was only performed on the remaining three sets: AltPD-1, AltPD-3, and AltPD-4. Different concentrations of [missing information - likely a specific primer set] were used in this experiment. A. alternata Using DNA as a template and ddH2O as a negative control, the reaction was carried out at 65℃. After the reaction, the results were analyzed by agarose gel electrophoresis.

[0045] See results Figure 2 . Figure 2 In the diagram, M stands for DNA 2K plus Marker. Lanes 1 and 2 represent the amplification results of primer set AltPD-1; lanes 3 and 4 represent the amplification results of primer set AltPD-3; lanes 5 and 6 represent the amplification results of primer set AltPD-4; lane 7 is the negative control. The DNA template concentration in lanes 1, 3, and 5 is 0.246 pg / μL; the DNA template concentration in lanes 2, 4, and 6 is 24.6 fg / μL.

[0046] 5. Field applicability testing.

[0047] DNA was extracted from healthy and diseased plum fruits using 10×TE lysis buffer. These samples were used as templates, with ddH2O as a negative control. The reaction was carried out at 65℃, and the results were analyzed by agarose gel electrophoresis after the reaction was completed.

[0048] See results Figure 3 . Figure 3 In the diagram, M stands for DNA 2K plus Marker. Lanes 1 and 2 represent amplification results using primer set AltPD-1; lanes 3 and 4 represent amplification results using primer set AltPD-3; lanes 5 and 6 represent amplification results using primer set AltPD-4; lane 7 is the negative control. The amplification templates in lanes 1, 3, and 5 are DNA from diseased new plum fruits; the amplification templates in lanes 2, 4, and 6 are DNA from healthy new plum fruits.

[0049] Therefore, it can be seen that, for Alternaria neonatorum Alternaria alternataThe detection results showed that the primer set AltPD-1 performed best.

[0050] Example 2 An application for detecting brown spot disease in new syphilis is as follows: Based on the AltPD-1 primer set in Example 1, this example provides two rapid detection methods for Alternaria neonatorum: one is the LAMP-Cresol Red visualization detection method based on the AltPD-1 primer set, and the other is the LAMP-SYBR Green I real-time fluorescence quantitative detection method based on the AltPD-1 primer set.

[0051] (1) The LAMP-Cresol Red visualization detection method based on the AltPD-1 primer set has the following steps: S1. Extraction of pathogen DNA: The pathogen was cultured at 25°C for 7 days in a constant temperature incubator. Surface hyphae were scraped off, and then thoroughly ground using an automated rapid sample grinder. DNA was extracted using a DNA kit, and the concentration and quality of the DNA were detected using a UV spectrophotometer. The qualified DNA was immediately stored at -20°C for later use.

[0052] S2. Prepare the mixed primers for the 10×LAMP Primer Mix system: see Table 3 for details.

[0053] S3. LAMP-Cresol Red Visualization Detection: First, prepare the LAMP-Cresol Red visualization system. Incubate the LAMP-Cresol Red visualization system in a 65℃ constant temperature device for 45 min, then inactivate it at 85℃ for 10 min. After the reaction, observe the color change of cresol red; red indicates a negative result, and yellow indicates a positive result. Alternatively, observe whether a specific band appears by wt 1.2% agarose gel electrophoresis. The presence of a specific band indicates a positive result, and the rest are negative. The preparation method of the LAMP-Cresol Red visualization system is shown in Table 4.

[0054] (2) The LAMP-SYBR Green I real-time fluorescence quantitative detection method based on the AltPD-1 primer set has the following steps: The extraction of pathogen DNA and the preparation of the 10×LAMP Primer Mix system were carried out in the same manner as described above.

[0055] LAMP-SYBR Green I Real-Time Quantitative Detection: First, prepare the LAMP-SYBR Green I real-time quantitative detection system. Place the system in a 66℃ constant temperature apparatus for 45 min, then inactivate it at 85℃ for 10 min. After the reaction, observe the amplification curve. An S-shaped amplification curve indicates a positive result, while others indicate a negative result. Alternatively, observe the presence of specific bands using wt 1.2% agarose gel electrophoresis. The presence of specific bands indicates a positive result, while others indicate a negative result. The preparation method for the LAMP-SYBR Green I real-time quantitative detection system is shown in Table 5.

[0056] Table 5 LAMP-SYBR Green I Real-Time Quantitative Detection System Note: In Table 5, "-" indicates that this item is not available.

[0057] Based on the two methods mentioned above, the following section discusses common strains. Aspergillus ochraceus、Chaetomium globosum, Neoscytalidium dimidiatum, Diaporthe phaseolorum, Cytospora chrysosperma、Botryosphaeria dothidea、Fusarium oxysporum、Fusarium whorl-shaped The pathogen of new plum brown spot disease Alternaria alternata They were tested together for specificity.

[0058] 1. Specificity verification.

[0059] See results Figure 4 Experimental results show that: only when the template is Alternaria alternata hour ， The LAMP-CresolRed visualization system turned yellow, while the LAMP-SYBR Green I real-time quantitative PCR system showed specific amplification. However, when the template was other pathogens or sterile water, the LAMP-CresolRed visualization system remained red, and the LAMP-SYBR Green I real-time quantitative PCR system did not show any specific amplification curve. Both methods were verified by agarose gel electrophoresis, and the results were consistent with the observations.

[0060] Figure 4 In Chinese B, lanes 1 through 10 are as follows: Alternaria alternata、Aspergillus ochraceus, Chaetomium globosum, Neoscytalidium dimidiatum, Diaporthe phaseolorum、Cytospora chrysosperma、Botryosphaeria dothidea、Fusarium oxysporum、Fusarium verticillioides、 NTC. Figure 4 In centrifuge tube C, tubes 1 through 10 are as follows: Alternaria alternata, Aspergillus ochraceus, Chaetomium globosum, Neoscytalidium dimidiatum, Diaporthe phaseolorum, Cytospora chrysosperma, Botryosphaeria dothidea, Fusarium oxysporum, Fusarium verticillioides, NTC. Figure 4 In Chinese D, lanes 1 through 10 are as follows: Alternaria alternata, Aspergillus ochraceus, Chaetomium globosum, Neoscytalidium dimidiatum, Diaporthe phaseolorum, Cytospora chrysosperma, Botryosphaeria dothidea, Fusarium oxysporum, Fusarium verticillioides, NTC.

[0061] 2. Sensitivity detection.

[0062] See results Figure 5 A concentration of 246 μg / ml was used. Alternaria alternata DNA was serially diluted tenfold using ddH2O to obtain concentration gradients of 24.6 ng / μL, 2.46 ng / μL, 0.246 ng / μL, 24.6 pg / μL, 2.46 pg / μL, 0.246 pg / μL, 24.6 fg / μL, 2.46 fg / μL, and 0.246 fg / μL, which were then added to the system. When detected using the LAMP-Cresol Red visualization system, a yellow positive change was still observed when detecting DNA at a concentration of 0.246 fg / μL. Verification by 1.2% agarose gel electrophoresis also showed a ladder-like band pattern, indicating that the LAMP-Cresol Red visualization system can detect pathogens at the fg level. When the LAMP-SYBR Green I real-time quantitative PCR system detected DNA at a concentration of 0.246 fg / μL, the specific amplification curve was normal, and ladder-like bands were also amplified normally by 1.2% agarose gel electrophoresis. These results indicate that the LAMP-SYBR Green I real-time quantitative PCR system can also detect DNA at the fg level. Therefore, both the LAMP-Cresol Red visualization system and the LAMP-SYBR Green I real-time quantitative PCR system developed in this invention have good sensitivity.

[0063] Figure 5 In lanes B and D, lanes 1-9 are as follows: 2.46 ng / μL, 0.246 ng / μL, 24.6 pg / μL, 2.46 pg / μL, 0.246 pg / μL, 24.6 fg / μL, 2.46 fg / μL, 0.246 fg / μL, NTC. Figure 5 In C, centrifuge tubes 1-9 contain the following concentrations in sequence: 2.46 ng / μL, 0.246 ng / μL, 24.6 pg / μL, 2.46 pg / μL, 0.246 pg / μL, 24.6 fg / μL, 2.46 fg / μL, 0.246 fg / μL, NTC.

[0064] 3. Universality testing.

[0065] Extracted from multiple plants from different locations Alternaria alternataThe pathogens were detected by LAMP-Cresol Red visualization and LAMP-SYBR Green I real-time quantitative PCR, respectively. ddH2O was used as a negative control, and other reaction conditions remained unchanged.

[0066] See results Figure 6 Extracts were taken from 8 different plant species. Alternaria alternata All strains showed a yellow positive reaction in the LAMP-Cresol Red visualization system, and ladder-like amplification bands were also observed when verified by 1.2% agarose gel electrophoresis. Specific amplification curves were also observed in the LAMP-SYBR Green I real-time quantitative PCR system, and ladder-like amplification bands were also observed when verified by 1.2% agarose gel electrophoresis. The negative control, ddH2O, did not react. The experimental results indicate that both the LAMP-Cresol Red visualization system and the LAMP-SYBR Green I real-time quantitative PCR system can accurately and rapidly identify strains from different plant varieties in different regions. Alternaria alternata The strains, the LAMP-Cresol Red visualization system and the LAMP-SYBR Green I real-time fluorescence quantitative detection system developed in this invention have good versatility.

[0067] Figure 6 Lanes B and D, and lanes 1-9 are as follows: AltFC-127, AltFC-039, AltFC-067, AltFC-074, AltFC-030, AltPO-047, AltPO-096, AltAJ-084, NTC. Figure 6 In C, centrifuge tubes 1 to 9 are, in order: AltFC-127, AltFC-039, AltFC-067, AltFC-074, AltFC-030, AltPO-047, AltPO-096, AltAJ-084, and NTC.

[0068] Figure 6 The strain information is shown in Table 6.

[0069] Table 6. Strain Information Note: "+" in Table 6 indicates a positive test result.

[0070] 4. Field applicability testing.

[0071] New plum fruits collected from four different regions were sorted. Two diseased fruits and one healthy fruit were selected from each region. Approximately 0.5 cm of the cut was made at the boundary between the diseased and healthy fruit using a scalpel. 2Fruit peels were placed in 2ml centrifuge tubes as the experimental group, and healthy new plum fruit peels from the corresponding locations were used as the control group. After adding grinding beads, the samples were thoroughly ground in an automated rapid sample grinder, and then 100μL of 10×TE buffer was added to the centrifuge tubes. The tubes were then boiled in 95℃ hot water for 2 minutes, followed by boiling in 85℃ hot water for 1 minute. After removal, the tubes were centrifuged at 10000rpm for 30 seconds in a high-speed refrigerated centrifuge. This step yielded crude DNA extracts of the new plum brown spot pathogen from different regions. Field detection experiments were conducted using the LAMP-Cresol Red visualization detection method and the LAMP-SYBR Green I real-time fluorescence quantitative detection method.

[0072] See results Figure 7 and Figure 8 The experimental results showed that crude DNA extracted from infected fruits obtained from four different regions showed a yellow positive reaction in the LAMP-Cresol Red visualization system, and ladder-like amplification bands were also observed on 1.2% agarose gel electrophoresis. However, crude DNA extracted from healthy fruits obtained from the four regions did not show any positive changes. In the LAMP-SYBR Green I real-time quantitative PCR system, crude DNA extracted from infected fruits obtained from the four different regions all showed specific amplification curves, and ladder-like amplification bands were also observed on 1.2% agarose gel electrophoresis. Crude DNA extracted from healthy fruits obtained from the four regions also did not show any positive changes. The experimental results indicate that both the LAMP-Cresol Red visualization system and the LAMP-SYBR Green I real-time quantitative PCR system can accurately and rapidly identify crude DNA extracted from infected plants. Both the LAMP-Cresol Red visualization system and the LAMP-SYBR Green I real-time quantitative PCR system developed in this invention have good field applicability.

[0073] Figure 8 In centrifuge tube A, tubes 1-3 contain, in order: two infected materials and one healthy material from Gashi County; tubes 4-6 contain, in order: two infected materials and one healthy material from Shache County; tubes 7-9 contain, in order: two infected materials and one healthy material from Yingjisha County; tubes 10-12 contain, in order: two infected materials and one healthy material from Maigati County; and tube 13 contains NTC. Figure 8 In section B, lanes 1-13 and centrifuge tubes 1-13 correspond to each other in sequence.

[0074] 5. Optimization of the LAMP-Cresol Red visualization system.

[0075] Based on the AltPD-1 primer set, targeting Alternaria alternataThe temperature, time, and proportions of each component in the LAMP reaction system for strain detection were optimized.

[0076] First, the amount of DNA in the reaction system was optimized using a gradient of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, and 8 μL. Then, the reaction temperature was optimized using a gradient of 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, and 69℃ to determine the optimal reaction temperature. Subsequently, at the optimal temperature, the reaction time was determined to be 30 min, 35 min, 40 min, 45 min, and 50 min. The reaction results are shown below. Figure 9 The results showed that the optimal reaction temperature for the LAMP-CresolRed visualization system was 65℃, the optimal reaction time was 45 min, the optimal amount of DNA was 3 μL, and the ratio of inner primer to outer primer was 8:1.

[0077] Figure 9 A represents the optimization of the amount of DNA added. The values ​​for centrifuge tubes 1-8 are 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, and 8 μL, respectively. Figure 9 B represents temperature optimization. Centrifuge tubes 1-8 are set at the following temperatures: 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, and 69℃, respectively. Figure 9 C represents the optimized reaction time. Centrifuge tubes 1-5 were set to 30 min, 35 min, 40 min, 45 min, and 50 min, respectively.

[0078] 6. Optimization of the LAMP-SYBR Green I real-time fluorescence quantitative detection system.

[0079] Based on the AltPD-1 primer set, targeting Alternaria alternata The temperature, time, and proportion of each component in LAMP-SYBR Green I real-time fluorescence quantitative PCR for strain detection were optimized.

[0080] (1) First, optimize the amount of DNA in the reaction system by gradient optimization at 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL and 8 μL.

[0081] (2) Subsequently, the temperature of the reaction system was optimized by gradient optimization at 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃ and 69℃.

[0082] (3) The concentration of SYBR Green I added to the LAMP-SYBR Green I system was adjusted and analyzed in gradients of 0.2×, 0.4×, 0.6×, 0.8×, 1.0×, 1.2×, 1.4×, 1.6×, 1.8×, and 2.0×. After completion, the amplification curve of the real-time fluorescence quantitative detection system was observed for judgment.

[0083] See results Figure 10 The optimal reaction temperature for the LAMP-SYBR Green I real-time quantitative PCR system is 66℃, and the optimal DNA volume is 3 μL. Based on the amplification data, most of the amplification reactions began with a burst of amplification at around 15 minutes, reaching their maximum value at approximately 50 minutes. Regarding the crucial aspect of this experiment—controlling and analyzing the concentration of SYBR Green I added to the system—the figure shows that when the concentration of SYBR Green I added to the system was ≤0.6×, the real-time quantitative PCR instrument could barely detect the fluorescence intensity. However, when the concentration of SYBR Green I added to the system was ≥1.2×, the real-time quantitative PCR instrument initially detected extremely high non-specific fluorescence intensity, but subsequently, due to the inhibitory effect of SYBR Green I on DNA chimerism, the reaction could not proceed, causing a sharp drop in fluorescence intensity. The reaction time for 0.8× and 1.0× SYBR Green I was not significantly different, but the fluorescence intensity of 1.0× SYBR Green I was higher. Therefore, the optimal amount of 10× SYBR Green I was 2.5 μL.

[0084] The technical features of the above embodiments can be combined in any way. For the sake of brevity, 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, they should be considered to be within the scope of this specification.

[0085] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A primer set for detecting brown spot disease in new syrup, characterized in that, The base sequences of the primer set are shown in SEQ ID NO.1 to SEQ ID NO.

5.

2. A reagent kit for detecting syphilis brown spot disease, characterized in that, The kit includes the primer set as described in claim 1.

3. The application of the primer set as described in claim 1 or the kit as described in claim 2, characterized in that, The application refers to the detection of brown spot disease and / or Alternaria alternata .

4. The application as described in claim 3, characterized in that, Detection using the primer set Alternaria alternata The method is as follows: Collect infected plants, culture the pathogens from the infected plants, and extract the DNA from the pathogens; Prepare mixed primers using the primer set described above; Using DNA as a template, amplification was performed at 62℃~69℃ for 45min~50min using mixed primers, followed by inactivation, and the results were determined by agarose gel electrophoresis. If a specific band appears, the pathogen to be detected is... Alternaria alternata If no specific band is found, then the pathogen being tested is not... Alternaria alternata .

5. The application as described in claim 4, characterized in that, The molar ratio of each component in the mixed primer is: SEQ ID NO.1: SEQ ID NO.2: SEQ ID NO.3: SEQ ID NO.4: SEQ ID NO.5=1:1:8:8:

2.

6. The application as described in claim 4, characterized in that, The reaction system used for amplification can be any one of the following: 1) 2.5×Bst 4.0 LowSaltMix 10μL, 10×Red pH Dye 2.5μL, 10× Mixed Primers 2.5μL, DNA 3μL, ddH2O 7μL; 2) 10 μL of 2.5×Bst 4.0 LowSaltMix, 2.5 μL of 10×SYBR Green I, 2.5 μL of 10× mixed primers, 3 μL of DNA, and 7 μL of ddH2O.

7. The application as described in claim 4, characterized in that, The amplification conditions were 65℃~66℃ for 45 min.

8. The application as described in claim 4, characterized in that, The inactivation conditions were 85℃ for 10 minutes.

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