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

Abstract

This invention belongs to the field of biodetection technology, specifically relating to a primer set, kit, and application for detecting *Alternaria solani* brown spot disease. The base sequences of the primer set are shown in SEQ ID NO.1~SEQ ID NO.5. Based on the primer set, this invention utilizes loop-mediated isothermal amplification technology for the rapid detection of *Alternaria solani*, 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 solani* DNA. Using the rapid detection system provided by this invention, *Alternaria solani* can be detected rapidly, conveniently, 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.

Description

A primer set, reagent kit, and application for detecting syphilis lentigines. 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] *Prunus domestica* L., a cultivated species of plum in the genus *Prunus* of the family Rosaceae, originated in West Asia and Europe. It is one of Serbia's main fruit varieties and is considered its national fruit. Epidemiological studies show that *Prunus domestica* has potential medicinal value. 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 domestica* 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, brown spot disease of plums caused by *Alternaria alternata* has frequently broken out in major producing areas such as Gashi County and Yingjisha County, causing increasingly serious damage. This disease mainly affects plum fruits and has a long incubation period in the field. Early symptoms are difficult to detect, and the disease tends to be concentrated and explosive. Typically, small red spots first form on the fruit peel, gradually deepening to dark brown lesions. These lesions expand outwards, forming large spots 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 of plums. Traditional detection methods, such as tissue isolation and culture, are time-consuming, and PCR requires sophisticated thermal cycling equipment. These limitations make these methods difficult to apply quickly in the field. While LAMP technology has the advantage of isothermal amplification, its detection performance is highly dependent on the specificity and sensitivity of the designed primers. However, there is a lack of LAMP detection methods that can effectively distinguish Alternaria alternata from its closely related bacteria and maintain high sensitivity 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:

[0006] 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.

[0007] A second aspect of the present invention provides a kit for detecting syphilis lentigines, the kit comprising the aforementioned primer set.

[0008] A third aspect of the invention provides the application of the primer set or the kit for the detection of brown spot disease and / or Alternaria alternata.

[0009] Preferably, the method for detecting Alternaria alternata using the primer set is as follows:

[0010] Collect infected plants, culture the pathogens from the infected plants, and extract the DNA from the pathogens;

[0011] Prepare mixed primers using the primer set described above;

[0012] 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.

[0013] If a specific band appears, the pathogen to be tested is *Alternaria alternata*; if no specific band appears, the pathogen to be tested is not *Alternaria alternata*.

[0014] Preferably, the molar ratio of each component in the mixed primer is:

[0015] 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.

[0016] Preferably, the reaction system used for amplification is any one of the following:

[0017] 1) 2.5×Bst 4.0 LowSaltMix 10μL, 10×Red pH Dye 2.5μL, 10× Mixed Primers 2.5μL, DNA 3μL, ddH2O 7μL;

[0018] 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.

[0019] Preferably, the amplification conditions are 65℃~66℃ for 45 min.

[0020] Preferably, the inactivation conditions are 85°C for 10 minutes.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] This invention provides a primer set for detecting brown spot disease of *Alternaria alternata*, the base sequences of which are shown in SEQ ID NO.1~SEQ ID NO.5. This invention provides a set of primers for rapid detection of brown spot disease of *Alternaria alternata* by comparing the ITS sequence of *Alternaria alternata* with different pathogens belonging to the genus *Alternaria*. Based on this primer set, this invention uses loop-mediated isothermal amplification technology for rapid detection of *Alternaria alternata*, accurately detecting *Alternaria alternata* from the complex pathogenic environment in diseased plant tissues. The rapid detection system described in this invention has good specificity and high sensitivity, with a detection limit of 0.246 fg / μL for *Alternaria alternata* DNA. Using the rapid detection system provided by this invention, *Alternaria alternata* 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.

[0023] 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

[0024] Figure 1 shows the agarose gel electrophoresis results based on the specificity screening of four primer groups using A. alternata and A. ochraceus.

[0025] Figure 2 shows the agarose gel electrophoresis results of primer set sensitivity screening based on different template concentrations.

[0026] Figure 3 shows the agarose gel electrophoresis results of field adaptability testing with different primer sets.

[0027] Figure 4 shows the 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.

[0028] Figure 5 shows the LAMP sensitivity 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.

[0029] Figure 6 shows the universality detection of AltPD-1 using LAMP. 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.

[0030] Figure 7 shows the validation of LAMP-SYBR Green I real-time fluorescence detection on infected and healthy materials obtained from four different regions. A: Material from Jiashi County; B: Material from Shache County; C: Material from Yingjisha County; D: Material from Maigati County.

[0031] Figure 8 shows the LAMP-Cresol Red visualization verification of infected and healthy materials obtained from four different regions. A: Visualization results in centrifuge tubes; B: Agarose gel electrophoresis results in Figure A.

[0032] Figure 9 shows the optimization of the LAMP-Cresol Red visualization system. A: Optimization of DNA addition amount; B: Optimization of temperature; C: Optimization of reaction time.

[0033] Figure 10 shows the optimization of the LAMP-SYBR Green I real-time fluorescence quantitative detection system. A: Optimization of DNA addition amount; B: Optimization of temperature; C: Optimization of SYBR Green I concentration. Detailed Implementation

[0034] 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.

[0035] The inventive concept of this invention is as follows:

[0036] 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.

[0037] Therefore, by comparing the ITS sequences of Alternaria alternata, Alternaria arborescens, Alternaria citriarbusti, Alternaria gaisen, Alternaria mali and Alternaria toxicogenica, which belong to the genus Alternaria, this invention provides a primer set for detecting new syphilis brown spot disease, the base sequences of which are shown in SEQ ID NO.1~SEQ ID NO.5.

[0038] 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.

[0039] The list of abbreviations for this invention is shown in Table 1.

[0040] Table 1 List of Abbreviations

[0041]

[0042] Example 1

[0043] A primer set for detecting syphilitic brown spot disease is as follows:

[0044] 1. Design primer sequences.

[0045] By comparing the ITS sequences of Alternaria alternata, Alternaria arborescens, Alternaria citriarbusti, Alternaria gaisen, Alternaria mali and Alternaria toxicogenica, which belong to the same genus Alternaria, this invention provides four sets of primers. The specific primer information is shown in Table 2.

[0046] Table 2 Primer Information

[0047]

[0048] 2. Experimental materials and methods.

[0049] 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*.

[0050] Experimental methods:

[0051] 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 mixed primers for the 10×LAMP Primer Mix system based on the primer sets shown in AltPD-1~AltPD4, as detailed in Table 3.

[0053] Table 3 10×LAMP Primer Mix System

[0054]

[0055] 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.

[0056] 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.

[0057] Table 4 LAMP-Cresol Red Visualization System

[0058]

[0059] Note: In Table 4, "-" indicates that this item is not present.

[0060] 3. Primer-specific screening.

[0061] To ensure primer specificity, the four designed primer sets were initially screened. The pathogen A. alternata and the common pathogen of plum A. ochraceus were tested for specificity together. ddH2O was used 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.

[0062] The results are shown in Figure 1. In Figure 1, M represents the 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 template for lanes 1, 4, 7, and 10 was *A. alternata*; the amplification template for lanes 2, 5, 8, and 11 was *A. ochraceus*; and the amplification template for lanes 3, 6, 9, 12, and 13 was ddH2O.

[0063] Since the AltPD-2 primer set did not amplify the target band, no further experimental verification of the AltPD-2 primer set was conducted.

[0064] 4. Primer set sensitivity screening.

[0065] 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. In this experiment, different concentrations of *A. alternata* DNA 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.

[0066] The results are shown in Figure 2. In Figure 2, M: 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.

[0067] 5. Field applicability testing.

[0068] 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.

[0069] The results are shown in Figure 3. In Figure 3, M: 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 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.

[0070] Therefore, primer set AltPD-1 showed the best performance for the detection of Alternaria alternata.

[0071] Example 2

[0072] An application for detecting brown spot disease in new syphilis is as follows:

[0073] 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.

[0074] (1) The LAMP-Cresol Red visualization detection method based on the AltPD-1 primer set has the following steps:

[0075] 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.

[0076] S2. Prepare the mixed primers for the 10×LAMP Primer Mix system: see Table 3 for details.

[0077] 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.

[0078] (2) The LAMP-SYBR Green I real-time fluorescence quantitative detection method based on the AltPD-1 primer set has the following steps:

[0079] 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.

[0080] 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.

[0081] Table 5 LAMP-SYBR Green I Real-Time Quantitative Detection System

[0082]

[0083] Note: In Table 5, "-" indicates that this item is not available.

[0084] Based on the two methods described above, specific detection was performed on common bacterial strains Aspergillus ochraceus, Chaetomium globosum, Neoscytalidium dimidiatum, Diaporthe phaseolorum, Cytosporachrysosperma, Botryosphaeria dothidea, Fusarium oxysporum, Fusarium verticillioides, and Alternaria alternata, the pathogen of new plum brown spot disease.

[0085] 1. Specificity verification.

[0086] The results are shown in Figure 4. The experimental results show that only when the template is *Alternaria alternata* did the LAMP-CresolRed visualization system turn yellow, and the LAMP-SYBR Green I real-time fluorescence quantitative detection system showed specific amplification. When the template is other pathogens or sterile water, the LAMP-CresolRed visualization system remained red, and the LAMP-SYBR Green I real-time fluorescence quantitative detection system did not show specific amplification curves. Both methods were verified by agarose gel electrophoresis, and the results were consistent with the observations.

[0087] In Figure 4B, lanes 1 through 10 are, in order: *Alternaria alternata*, *Aspergillus ochraceus*, *Chaetomium globosum*, *Neoscytalidium dimidiatum*, *Diaporthe phaseolorum*, *Cytospora chrysosperma*, *Botryosphaeria dothidea*, *Fusarium oxysporum*, *Fusarium verticillioides*, and NTC. In Figure 4C, centrifuge tubes 1 through 10 are, in order: *Alternaria alternata*, *Aspergillus ochraceus*, *Chaetomium globosum*, *Neoscytalidium dimidiatum*, *Diaporthe phaseolorum*, *Cytospora chrysosperma*, *Botryosphaeria dothidea*, *Fusarium oxysporum*, *Fusarium verticillioides*, and NTC. In Figure 4D, lanes 1 through 10 are, in order: Alternaria alternata, Aspergillus ochraceus, Chaetomium globosum, Neoscytalidium dimidiatum, Diaporthe phaseolorum, Cytospora chrysosperma, Botryosphaeria dothidea, Fusarium oxysporum, Fusarium verticillioides, and NTC.

[0088] 2. Sensitivity detection.

[0089] The results are shown in Figure 5. Alternaria alternata DNA at a concentration of 246 μg / ml was serially diluted tenfold using ddH2O to obtain DNA 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.

[0090] In Figures 5B and D, lanes 1-9 are, in order: 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. In Figure 5C, centrifuge tubes 1-9 are, in order: 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.

[0091] 3. Universality testing.

[0092] Alternaria alternata pathogens extracted from multiple plants at different locations were subjected to LAMP-Cresol Red visualization and LAMP-SYBR Green I real-time quantitative PCR detection. ddH2O was used as a negative control, while other reaction conditions remained unchanged.

[0093] The results are shown in Figure 6. All eight *Alternaria alternata* strains extracted from different plant varieties showed a yellow positive reaction in the LAMP-Cresol Red visualization system, and ladder-like amplification bands were also observed upon verification 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 upon verification 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 *Alternaria alternata* strains from different plant varieties in different regions. The LAMP-Cresol Red visualization system and the LAMP-SYBR Green I real-time quantitative PCR system developed in this invention have good versatility.

[0094] In Figure 6, lanes 1-9 in B and D are, in order: AltFC-127, AltFC-039, AltFC-067, AltFC-074, AltFC-030, AltPO-047, AltPO-096, AltAJ-084, NTC. In Figure 6, centrifuge tubes 1-9 are, in order: AltFC-127, AltFC-039, AltFC-067, AltFC-074, AltFC-030, AltPO-047, AltPO-096, AltAJ-084, NTC.

[0095] The strain information in Figure 6 is shown in Table 6.

[0096] Table 6. Strain Information

[0097]

[0098] Note: "+" in Table 6 indicates a positive test result.

[0099] 4. Field applicability testing.

[0100] 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.

[0101] The results are shown in Figures 7 and 8. The experimental results showed that crude DNA extracted from infected fruits obtained from four different regions exhibited a yellow positive reaction in the LAMP-Cresol Red visualization system, and also showed ladder-like amplification bands 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 also showed ladder-like amplification bands 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.

[0102] In Figure 8A, centrifuge tubes 1-3 contain, in order: two infected strains and one healthy strain from Gashi County; centrifuge tubes 4-6 contain, in order: two infected strains and one healthy strain from Shache County; centrifuge tubes 7-9 contain, in order: two infected strains and one healthy strain from Yingjisha County; centrifuge tubes 10-12 contain, in order: two infected strains and one healthy strain from Maigati County; centrifuge tube 13 is NTC. In Figure 8B, lanes 1-13 correspond to centrifuge tubes 1-13.

[0103] 5. Optimization of the LAMP-Cresol Red visualization system.

[0104] Based on the AltPD-1 primer set, the temperature, time, and proportion of each component in the LAMP reaction system for detection by Alternaria alternata strain were optimized.

[0105] First, the amount of DNA in the reaction system was optimized using gradients 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 gradients of 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, and 69℃ to determine the optimal reaction temperature. Subsequently, the reaction time was adjusted at the optimal temperature for 30 min, 35 min, 40 min, 45 min, and 50 min to determine the optimal reaction time. The results are shown in Figure 9. The results indicate that the optimal reaction temperature for the LAMP-CresolRed visualization system is 65℃, the optimal reaction time is 45 min, the optimal amount of DNA is 3 μL, and the optimal ratio of inner to outer primers is 8:1.

[0106] Figure 9A shows the optimization of DNA addition volume, with centrifuge tubes 1-8 containing 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, and 8 μL respectively. Figure 9B shows the optimization of temperature, with centrifuge tubes 1-8 containing 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, and 69℃ respectively. Figure 9C shows the optimization of reaction time, with centrifuge tubes 1-5 containing 30 min, 35 min, 40 min, 45 min, and 50 min respectively.

[0107] 6. Optimization of the LAMP-SYBR Green I real-time fluorescence quantitative detection system.

[0108] Based on the AltPD-1 primer set, the temperature, time, and proportion of each component were optimized for LAMP-SYBR Green I real-time fluorescence quantitative detection of Alternaria alternata strain.

[0109] (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.

[0110] (2) Subsequently, the temperature of the reaction system was optimized by gradient optimization at 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃ and 69℃.

[0111] (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.

[0112] The results are shown in Figure 10. The optimal reaction temperature for the LAMP-SYBR Green I real-time quantitative PCR system was 66℃, and the optimal amount of DNA was 3 μL. Based on the amplification data, most of the amplification reactions began with a burst of amplification at around 15 min, reaching their maximum value at approximately 50 min. Regarding the most crucial aspect of this experiment—the control and analysis of 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 hardly 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. 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.

[0113] 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.

[0114] 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 Alternaria alternata, characterized in that, The base sequences of the primer set are shown in SEQ ID NO.1 to SEQ ID NO.

5.

2. A kit for detecting Alternaria alternata, 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 Alternaria alternata and / or new plum brown spot disease caused by Alternaria alternata in plant samples.

4. The application as described in claim 3, characterized in that, The application includes the following steps: collecting susceptible plants, culturing the pathogens from the susceptible plants, and extracting the DNA of the pathogens; preparing mixed primers using the primer set; using the DNA as a template, amplifying the pathogens using the mixed primers at 62℃~69℃ for 45min~50min, inactivating the pathogens, and determining the results by agarose gel electrophoresis; if a specific band appears, the pathogen to be detected is Alternaria alternata; if no specific band appears, the pathogen to be detected 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) 10 μL of 2.5×Bst 4.0 LowSaltMix, 2.5 μL of 10×Red pH Dye, 2.5 μL of 10× mixed primers, 3 μL of DNA, and 7 μL of ddH2O; 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 are 85℃ for 10 minutes.

Images