A detection kit for Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight

Abstract

The present invention discloses a detection kit for Pseudomonas amylovora, Pseudomonas syringae, and / or mulberry blight. Based on the gene encoding a hypothetical protein of the mulberry blight pathogens Pseudomonas amylovora and / or Pseudomonas syringae, as shown in SEQ ID NO: 1, a primer set for detecting the mulberry blight pathogens Pseudomonas amylovora, Pseudomonas syringae, and / or mulberry blight is designed. The primer set is used for PCR and LAMP detection. The nucleotide sequences of the primer set for PCR detection are shown in SEQ ID NOs: 8-9, and the nucleotide sequences of the primer set for LAMP detection are shown in SEQ ID NOs: 32-35. The primer set has high specificity and sensitivity, and the reaction results can be identified by the naked eye. The PCR and LAMP detection kit containing the primer set is used for rapid detection of the infectious mulberry blight pathogens Pseudomonas amylovora, Pseudomonas syringae, and / or mulberry blight in mulberry trees and mulberry seedlings in mulberry gardens.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection of plant pathogens, and in particular to a detection kit for Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight. Background Art

[0002] The sericulture industry is plagued by bacterial, fungal, and viral diseases of mulberry trees. Mulberry blight, along with bacterial wilt and mulberry wilt, are considered the three major infectious bacterial diseases of mulberry trees, severely impacting the industry's development. Early identification of the pathogen responsible for mulberry blight is crucial for preventing and controlling mulberry wilt. Mulberry blight, commonly known as "top rot," has typical characteristics and affects the entire plant. The top leaves curl, rot at the veins, and fall off, while the top buds and leaves turn black and rot, forming a "top rot" phenomenon.

[0003] The disease has occurred in mulberry gardens in most of the silkworm-producing areas in China, including Jiangsu, Zhejiang, Anhui, Hubei, Hunan, Sichuan, Shaanxi, Yunnan, Jiangxi, and Guangxi. In particular, the susceptible mulberry varieties of the "Tongxiangqing", "Huanglutou", "Husang No. 7" and "Qiangsang" series have a morbidity rate of up to 70% to 80%, causing serious economic losses to sericulture production (Dai Hefang, Zhou Shuiliang, Du Heming, et al. Study on bacterial black blight of mulberry trees - leaf shrinkage disease [J]. Sericulture Science, 1980(03):150-154.; Bao Qi, Cao Mengqi, Zhou Yu, et al. Detection of Pseudomonas syringae based on real-time fluorescence quantitative PCR technology [J]. Sericulture Science, 2016, 42(02):210-218.). In summer and autumn, when there is more rain, especially in a hot, humid and windy environment, the pathogen can easily multiply and spread in large numbers, leading to widespread infection and increased damage. The pathogen of mulberry blight can survive in the soil and water for several months, and can also survive with diseased branches until the summer and autumn of the following year. In mulberry orchards where the disease has occurred, comprehensive disinfection of the soil, water and other environments should be carried out, and diseased branches should be handled in a timely and proper manner to prevent the disease from breaking out again next year.

[0004] Mulberry blight is a bacterial disease of mulberry trees caused by infection with Pseudomonas syringae pv. Mori (a heteromorphic synonym of Pseudomonas amygdali pv. mori). It is highly pathogenic and contagious (Kuai Yuanzhang. Research Progress on Mulberry Pathogenic Prokaryotes and Their Diseases (I) [J]. Sericulture Science, 2012, 38(01): 152-164.). Recent studies suggest that the mulberry-derived Pseudomonas syringae should be Pseudomonas amygdal i (Yamamoto S, Kasai H, Arnold DL, et al. Phylogeny of the genus Pseudomonas: Intrageneric structure reconstructed from the nucleotide sequences of gyrB and rpoD gen es[J]. Microbiology(Reading), 2000, 146(10): 2385-2394; Baltrus, DA, Nishimura, MT, Romanchuk, A., et al.,. Dynamic evolution of pathogenicity revealed by sequencing and comparative genomics of 19 Pseudomonas syringae isolates. PLoS pathogens, 2011, 7(7), e1002 132. https: / / doi.org / 10.1371 / journal.ppat.1002132). Mulberry blight, caused by the pathogen infecting mulberry buds and leaves, primarily harms the leaves at the top of branches and tender shoots, and in severe cases can easily lead to necrosis and dieback of mulberry trees. Symptoms of mulberry blight primarily include blackened and withered leaves and leaf shrinkage. Diseased plants exhibit blackened and withered leaves at the top of the mulberry tree, with brownish-brown linear lesions of varying thickness forming on the branches, severely impacting mulberry leaf yield and quality. The pathogen can survive on diseased branches until May of the following year under different treatments (sterilized soil, natural soil surface, and 10 cm within natural soil). In sterilized soil and natural soil surface, it can survive until June of the following year.

[0005] At present, there is no method to completely cure mulberry blight disease. Therefore, there is an urgent need for a method for early detection of mulberry blight pathogens in production. It is urgent to establish a method for detecting and identifying the pathogens of mulberry blight disease in order to facilitate early warning and prevention and control. Summary of the Invention

[0006] The object of the present invention is to overcome the above-mentioned deficiencies in the prior art and to provide a detection kit for Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight.

[0007] The first object of the present invention is to provide a gene and / or detection reagent for use in detecting Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight.

[0008] The second object of the present invention is to provide a detection reagent for detecting Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight.

[0009] The third object of the present invention is to provide the use of the detection reagent in preparing a detection kit for Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight.

[0010] The fourth object of the present invention is to provide a detection kit for Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight.

[0011] A fifth object of the present invention is to provide a method for detecting Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight for non-therapeutic and / or diagnostic purposes.

[0012] In order to achieve the above object, the present invention is implemented through the following scheme:

[0013] A use of a gene and / or a detection reagent in detecting Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight, characterized in that the nucleotide sequence of the gene is as shown in SEQ ID NO: 1, and the detection reagent is used to detect the nucleotide sequence as shown in SEQ ID NO: 1 or a fragment thereof.

[0014] A reagent for detecting Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight, wherein the reagent is used for detecting the nucleotide sequence shown in SEQ ID NO: 1 or a fragment thereof.

[0015] Preferably, the reagent contains a primer set having nucleotide sequences as shown in SEQ ID NOs: 8 to 9 or a primer set having nucleotide sequences as shown in SEQ ID NOs: 32 to 35.

[0016] More preferably, the primer set whose nucleotide sequences are shown as SEQ ID NOs: 8 to 9 is a PCR detection primer set, and the primer set whose nucleotide sequences are shown as SEQ ID NOs: 32 to 35 is a LAMP detection primer set.

[0017] Most preferably, the nucleotide sequences of the outer primers F3 and B3 in the LAMP detection primer set are shown as SEQ ID NOs: 32-33, respectively, and the nucleotide sequences of the inner primers FIP and BIP are shown as SEQ ID NOs: 34-35, respectively.

[0018] The reagent is used in preparing a detection kit for Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight, or in detecting Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight.

[0019] A detection kit for Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight, comprising the reagent.

[0020] Preferably, the detection kit is a PCR detection kit, which contains a primer set having nucleotide sequences as shown in SEQ ID NOs: 8-9.

[0021] More preferably, the PCR detection kit further contains 2× reaction buffer, DNA template and ddH2O.

[0022] Preferably, the detection kit is a LAMP detection kit, which contains a primer set having nucleotide sequences as shown in SEQ ID NOs: 32 to 35.

[0023] More preferably, in the LAMP detection kit, the nucleotide sequences of the outer primers F3 and B3 in the primer set are shown as SEQ ID NOs: 32-33, respectively, and the nucleotide sequences of the inner primers FIP and BIP are shown as SEQ ID NOs: 34-35, respectively.

[0024] More preferably, the kit further contains 2×LAMP reaction buffer, Bst DNA polymerase, 1 μL of DNA template, fluorescent dye STYO 9 and ddH2O.

[0025] A method for detecting Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight for non-therapeutic and / or diagnostic purposes, comprising designing a primer set for the gene with the nucleotide sequence shown in SEQ ID NO: 1 to detect Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight.

[0026] Preferably, the primer set contains primers with nucleotide sequences as shown in SEQ ID NOs: 8 to 9, and a PCR amplification reaction is performed.

[0027] More preferably, the PCR amplification reaction system is: 12-13 μL of 2× reaction buffer, 0.45-0.55 μL of a primer containing 9.8-10.2 μM nucleotide sequence such as SEQ ID NO: 8, 0.45-0.55 μL of a primer containing 9.8-10.2 μM nucleotide sequence such as SEQ ID NO: 9, 1.8-2.2 μL of DNA template, and ddH2O to 20-25 μL.

[0028] More preferably, the PCR amplification reaction system is: 12.5 μL of 2× reaction buffer, 0.5 μL of a primer with a 10 μM nucleotide sequence such as SEQ ID NO: 8, 0.5 μL of a primer with a 10 μM nucleotide sequence such as SEQ ID NO: 9, 2 μL of DNA template, and ddH2O to 25 μL.

[0029] More preferably, the PCR amplification reaction procedure is: pre-denaturation at 93-97°C for 2.8-3.2 min; denaturation at 76-100°C for 28-32 s, annealing for 28-32 s, annealing temperature at 58-62°C, extension at 70-74°C for 0.8-1.2 min, 35 cycles; extension at 70-74°C for 4.8-5.2 min.

[0030] More preferably, the PCR amplification reaction program is: pre-denaturation at 95°C for 3 min; denaturation at 98°C for 30 s, annealing for 30 s, annealing temperature at 60°C, extension at 72°C for 1 min, 35 cycles; extension at 72°C for 5 min.

[0031] Preferably, the primer set contains a primer set with nucleotide sequences as shown in SEQ ID NOs: 32 to 35, and performs a LAMP amplification reaction.

[0032] More preferably, the nucleotide sequences of the outer primers F3 and B3 in the primer set are shown as SEQ ID NOs: 32-33, respectively, and the nucleotide sequences of the inner primers FIP and BIP are shown as SEQ ID NOs: 34-35, respectively, and a LAMP amplification reaction is performed.

[0033] More preferably, the final concentration ratio of the outer primers to the inner primers in the primer set is 1:(4-10), the final concentrations of the outer primers F3 and B3 are the same, and the final concentrations of the inner primers FIP and BIP are the same.

[0034] More preferably, the final concentration ratio of the outer primers to the inner primers in the primer set is 1:8, the final concentrations of the outer primers F3 and B3 are the same, and the final concentrations of the inner primers FIP and BIP are the same.

[0035] More preferably, the LAMP reaction system comprises: 12-13 μL of 2×LAMP reaction buffer, 0.45-0.55 μL of Bst DNA polymerase, final concentrations of outer primers F3 and B3 whose nucleotide sequences are represented by SEQ ID NOs: 32-33, respectively, at 0.08-0.12 μM each, final concentrations of inner primers FIP and BIP whose nucleotide sequences are represented by SEQ ID NOs: 34-35, respectively, at 0.6-1 μM each, 0.8-1.2 μL of DNA template, 0.45-0.55 μL of fluorescent dye STYO 9, and ddH2O to 20-25 μL; the amplification reaction procedure comprises: constant temperature reaction at 62-64° C. for 30-60 min.

[0036] More preferably, the LAMP reaction system is: 12.5 μL of reaction buffer, 0.5 μL of Bst DNA polymerase, the final concentrations of the outer primers F3 and B3 whose nucleotide sequences are shown in SEQ ID NOs: 32 to 33 are each 0.1 μM, the final concentrations of the inner primers FIP and BIP whose nucleotide sequences are shown in SEQ ID NOs: 34 to 35 are each 0.8 μM, 1 μL of DNA template, 0.5 μL of fluorescent dye STYO 9, and ddH2O made up to 25 μL.

[0037] More preferably, the determination method of the LAMP reaction is a real-time fluorescence curve method, a colorimetric method, and / or a gel electrophoresis method; the real-time fluorescence curve method determines that a fluorescence curve is detected as positive, and no fluorescence curve is detected as negative; the colorimetric method adds SYBR Green I to the reaction solution, and the reaction solution is green, which is determined to be positive, and brown, which is negative; the LAMP amplification product is detected by gel electrophoresis, and the presence of multiple bands is determined to be positive, and the absence of multiple bands is determined to be negative.

[0038] Preferably, the Pseudomonas amygdalus is the pathogen of mulberry blight disease Pseudomonas amygdalus and / or the pathogen of mulberry blight disease Pseudomonas syringae.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention discloses a detection kit for Pseudomonas amygdali, Pseudomonas syringae, and / or mulberry blight. Based on a gene encoding a hypothetical protein (hp) of the mulberry blight pathogen Pseudomonas amygdali and / or Pseudomonas syringae, as shown in SEQ ID NO: 1, a primer set for detecting the mulberry blight pathogens Pseudomonas amygdali, Pseudomonas syringae, and / or mulberry blight is designed. The primer set can be used for PCR and LAMP detection. The nucleotide sequences of the primer set for PCR detection are shown in SEQ ID NOs: 8-9, and the nucleotide sequences of the primer set for LAMP detection are shown in SEQ ID NOs: 32-35. The primer set has high specificity and sensitivity, and the reaction results can be identified by the naked eye. The PCR and LAMP detection kits containing the above primer sets can be used for the rapid detection of infectious mulberry blight pathogens Pseudomonas amygdali, Pseudomonas syringae and / or mulberry blight in mulberry trees and mulberry seedlings in mulberry gardens. They have great application value in the quarantine and prevention of mulberry bacterial diseases and are worthy of promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the colony morphology of Pseudomonas amygdali, the pathogen of mulberry blight, on LB nutrient agar medium.

[0042] Figure 2 Figure 1 shows the PCR primer screening for the pathogen of mulberry blight; lanes 1 to 3 are primers SYB1, lanes 4 to 6 are primers SYB2, lanes 7 to 9 are primers SYB3, lanes 10 to 12 are primers SYB4, lanes 13 to 15 are primers SYB5, lanes 16 to 18 are primers SYB6, and lanes 19 to 21 are primers SYB7.

[0043] Figure 3 The results of PCR sensitivity test for mulberry blight pathogen are shown in the figure; M is a 2000 bp marker; lanes 1 to 8 are 1 ng / μL, 1×10 -1 ng / μL, 1×10 -2 ng / μL, 1×10 -3 ng / μL, 1×10 -4 ng / μL, 1×10 -5 ng / μL, 1×10 - 6 ng / μL, 1×10 -7ng / μL of Pseudomonas amygdali DNA, the pathogen of mulberry blight; lane 9 is sterile water.

[0044] Figure 4 Figure 1 is the specific detection result of PCR for the pathogen of mulberry blight; where M is a 2000 bp marker; lane 1 is the pathogen of mulberry blight, Pseudomonas amygdali; lane 2 is the pathogen of mulberry blight, Pseudomonas amygdali; lane 3 is Pseudomonas syringae pv. phaseolicola; lane 4 is Pseudomonas fulva; lane 5 is Enterobacter cloacae; lane 6 is Ralstonia solanacearum; lane 7 is Klebsiella pneumoniae; lane 8 is Klebsiella oxytoca; lane 9 is Pseudomonas aeruginosa. aeruginosa), lane 10 is Bacillus subtilis, and lane 11 is sterile water (negative control).

[0045] Figure 5 The figure shows the PCR test results of field samples with mulberry blight; M is a 2000bp marker; lanes 1 to 3 are total DNA from field samples with mulberry blight in Zhejiang; lanes 4 to 6 are total DNA from field samples with mulberry blight in Anhui; lanes 7 to 9 are total DNA from field samples with mulberry blight in Jiangsu; lanes 10 to 11 are total DNA from field samples with mulberry blight in Shaanxi; lane 13 is a healthy mulberry tree sample (negative control); lane 14 is sterile water (blank control).

[0046] Figure 6 This is a real-time fluorescence curve graph of the LAMP primer set screening; among them, labels 1 to 5 are primer sets DX-1, DX-2, DX-3, DX-4 and DX-5, respectively.

[0047] Figure 7 Real-time fluorescence curves of LAMP reaction at different temperatures.

[0048] Figure 8 The figure is a real-time fluorescence curve of different final primer concentration ratios in the LAMP reaction; among them, the final concentration ratios of outer primers:inner primers numbered 1 to 5 are 1:2, 1:4, 1:8, and 1:10, respectively.

[0049] Figure 9The graph is a real-time fluorescence curve of the primer specificity experiment of the LAMP reaction; wherein, label 1 is the pathogen of mulberry blight, Pseudomonas amygdali, label 2 is Pseudomonas syringae pv.phaseolicola, label 3 is Pseudomonasfluorescens, label 4 is Pseudomonas fulva, label 5 is Pseudomonas aeruginosa, label 6 is Enterobacter cloacae, label 7 is Klebsiella Pneumoniae, label 8 is Klebsiella oxytoca, label 9 is Pantoea pineapple ananatis, label 10 is Bacillus subtilis, label 11 is Stenotrophomonas maltophilia, label 12 is Enterobacter asburiae, label 13 is Azomonas sp., and label 14 is sterile water (negative control).

[0050] Figure 10The figure shows the color display results of LAMP specific detection of different mulberry blight disease samples, among which, label 1 is the pathogen of mulberry blight disease, Pseudomonas amygdali, label 2 is Pseudomonas syringae pv.phaseolicola, label 3 is Pseudomonasfluorescens, label 4 is Pseudomonas fulva, label 5 is Pseudomonas aeruginosa, label 6 is Enterobacter cloacae, label 7 is Klebsiella pneumoniae, label 8 is Klebsiella oxytoca, label 9 is Pantoea pineapple ananatis, label 10 is Bacillus subtilis, label 11 is Stenotrophomonas maltophilia, label 12 is Enterobacter asburiae, label 13 is Azomonas sp., and label 14 is sterile water (negative control).

[0051] Figure 11The electrophoresis diagrams of LAMP detection results of different mulberry blight disease samples are shown in FIG1 , wherein, number 1 is the pathogen of mulberry blight disease, Pseudomonas amygdali, number 2 is Pseudomonas syringae pv.phaseolicola, number 3 is Pseudomonasfluorescens, number 4 is Pseudomonas fulva, number 5 is Pseudomonas aeruginosa, number 6 is Enterobacter cloacae, number 7 is Klebsiella Pneumoniae, number 8 is Klebsiella oxytoca, number 9 is Pantoea pineappleensis, and number 10 is Pseudomonas fluorescens. ananatis, label 10 is Bacillus subtilis, label 11 is Stenotrophomonas maltophilia, label 12 is Enterobacter asburiae, label 13 is Azomonas sp., and label 14 is sterile water (negative control).

[0052] Figure 12 The figure shows the real-time fluorescence curves of LAMP detection of different mulberry blight disease samples; among them, label 1 is the DNA of mulberry blight pathogen Pseudomonas amygdalus (positive control), labels 2 to 7 are the total DNA of mulberry blight field samples, the samples were collected in Haining City, Zhejiang Province, and the mulberry variety was Qiangsang No. 1; labels 8 to 13 are the total DNA of the diseased samples after Koch's postulate inoculation, and the mulberry variety was Qiangsang No. 1, and label 14 is sterile water (blank control).

[0053] Figure 13 The figure shows the color development results of LAMP detection of different mulberry blight disease samples; among them, label 1 is the DNA of mulberry blight pathogen Pseudomonas amygdalus (positive control), labels 2 to 7 are the total DNA of mulberry blight field samples, the samples were collected in Haining City, Zhejiang Province, and the mulberry variety was Qiangsang No. 1; labels 8 to 13 are the total DNA of diseased samples after Koch's postulate recovery, the mulberry variety is Qiangsang No. 1, and label 14 is sterile water (blank control).

[0054] Figure 14The electrophoresis diagram of the LAMP test results of different mulberry blight samples; among them, label 1 is the DNA of the mulberry blight pathogen Pseudomonas amygdalus (positive control), labels 2 to 7 are the total DNA of mulberry blight field samples, the samples were collected in Haining City, Zhejiang Province, and the mulberry variety was Qiangsang No. 1; labels 8 to 13 are the total DNA of the diseased samples after Koch's postulate recovery, the mulberry variety is Qiangsang No. 1, and label 14 is sterile water (blank control). DETAILED DESCRIPTION

[0055] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0056] Example 1 Determination of Specific Target Gene Sequences of Pseudomonas amygdali, the Pathogen of Mulberry Blight

[0057] 1. Experimental Methods

[0058] 1. Bacterial Whole Genome Sequencing

[0059] (1) Sample confirmation

[0060] The morphology of Pseudomonas amygdali, the pathogen of mulberry blight, on LB nutrient agar medium is as follows: Figure 1 As shown; the DNA of Pseudomonas amygdali, the pathogen of mulberry blight disease, was extracted using the Ezup column bacterial genomic DNA extraction kit. The extraction method is described in the kit instructions, and the quality of the DNA sample of Pseudomonas amygdali, the pathogen of mulberry blight disease, was tested.

[0061] (2) Whole genome sequencing

[0062] The extracted DNA of Pseudomonas amygdali, the pathogen of mulberry blight, was quality tested. For qualified DNA samples, a double-end sequencing library with an insert size of 500 bp was constructed according to the Illumian DNA library construction standard process.

[0063] After the library passed the test, it entered the high-throughput sequencing process. Sequencing was performed using the Illumina HiSeq high-throughput sequencing platform, sequencing mode: PE150 (Pair-end 150).

[0064] 2. Target gene sequence screening

[0065] The obtained whole genome sequence was compared with the sequences of Pseudomonas amygdali reported in the database, and the specific regions were screened and verified using the online software BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0066] 2. Experimental Results

[0067] The full-length cDNA of the hp gene, a specific hypothetical protein (hp) of the mulberry blight pathogen Pseudomonas amygdali, can be used as a target gene sequence for detecting Pseudomonas amygdali. The nucleotide sequence thereof is shown in SEQ ID NO: 1.

[0068] Example 2 PCR detection primer design

[0069] 1. Experimental Methods

[0070] 1. Extraction of Pseudomonas amygdali genomic DNA

[0071] The Ezup column-based bacterial genomic DNA extraction kit was used to extract Pseudomonas amygdali DNA. The extraction method was described in the kit instructions.

[0072] 2. Primer design, reaction system and procedure

[0073] Specific PCR primers were designed for the target gene sequence of Pseudomonas amygdali. The nucleotide sequence of the target gene sequence is shown in SEQ ID NO: 1. A specific gene segment encoding the hypothetical gene shown in SEQ ID NO: 1 in Example 1 was selected and primers were designed using the online software PrimerExplorer V4 (http: / / primerexplorer.jp / elamp4.0.0 / index.html). A total of seven primer sets (SEQ ID NOs: 2-15) were obtained, as shown in Table 1.

[0074] Table 1 Nucleotide sequences of PCR primers

[0075]

[0076] Example 3 Determination of PCR detection primers

[0077] 1. Experimental Methods

[0078] Using Pseudomonas amygdali genomic DNA as a template, PCR amplification reactions were performed using the seven sets of primers designed in Example 2. The PCR amplification reaction system is shown in Table 2.

[0079] Table 2 PCR amplification reaction system

[0080]

[0081] The PCR amplification reaction program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 98°C for 30 s, annealing for 30 s, annealing temperature at 60°C, extension at 72°C for 1 min, 35 cycles; and extension at 72°C for 5 min.

[0082] 2. Experimental Results

[0083] The PCR amplification products were detected by 10g / L agarose gel electrophoresis. Figure 2 As shown in the figure, among the seven primer pairs, SYB-1 and SYB-7 only amplified two samples, indicating that the primers were unstable. However, SYB-4 amplified a single, clearer band at approximately 506 bp, with no nonspecific amplification bands such as primer dimers. Therefore, SYB-4 (SEQ ID NOs: 8-9) was selected as the optimal primer.

[0084] Example 4 A PCR Detection Kit for Pseudomonas amygdali, the Pathogen of Mulberry Blight

[0085] 1. Composition

[0086] In Example 2, primers with nucleotide sequences as shown in SEQ ID NOs: 8 to 9, 2×Taq Master Mix, ddH 2 O, and a DNA template of the mulberry blight pathogen Pseudomonas amygdali were used as positive controls, and sterile water was used as a negative control.

[0087] 2. Usage

[0088] 1. DNA extraction from plant samples

[0089] The collected branch tissue from the diseased mulberry plant was rinsed with sterile water, air-dried, and cut into 3-4 cm segments. The segments were placed in a funnel with a switch (with a filter at the outlet). 75% alcohol was added to the funnel on a clean bench, and the funnel was soaked for 15 seconds before the alcohol was drained. The segments were then treated with mercuric chloride for 5 minutes using the same method, and rinsed 3-4 times with sterile water. The segments were removed and air-dried on the bench. The cortex of the segments was torn off with sterile tweezers to expose the wood. The longitudinal strips of the wood were then scraped off with a sterile scalpel, placed in a sterile mortar, and thoroughly ground with 5 mL of sterile water. DNA was extracted using a plant genomic DNA extraction kit; refer to the kit instructions for specific steps.

[0090] 2. PCR testing

[0091] The DNA sample to be tested was used as a template and PCR amplification was performed using the primers with nucleotide sequences as shown in SEQ ID NOs: 8 to 9 in Example 2. The PCR amplification was performed using the reaction system and procedure in Example 3.

[0092] 3. Result judgment

[0093] The PCR amplification product was detected by 10 g / L agarose gel electrophoresis. If an amplified band was found at about 506 bp, it indicated that the sample to be tested contained the pathogen of mulberry blight, Pseudomonas amygdali; otherwise, it did not contain the pathogen of mulberry blight, Pseudomonas amygdali.

[0094] Example 5 PCR detection sensitivity determination

[0095] 1. Experimental Methods

[0096] The DNA of Pseudomonas amygdali, the pathogen of mulberry blight, was diluted tenfold, respectively, to 1 ng / μL, 1×10 -1 ng / μL, 1×10 -2 ng / μL, 1×10 -3 ng / μL, 1×10 -4 ng / μL, 1×10 -5 ng / μL, 1×10 -6 ng / μL, 1×10 -7 ng / μL and sterile water, using the diluted DNA of each concentration as a template, PCR detection was performed using the detection kit of Example 4 to determine the sensitivity of the primers whose nucleotide sequences in Example 2 are shown in SEQ ID NOs: 8-9.

[0097] 2. Experimental Results

[0098] The results are as follows Figure 3 As shown in Example 2, the primers with nucleotide sequences as shown in SEQ ID NOs: 8-9 have good sensitivity and can only amplify 1×10 -2 ng / μL DNA of Pseudomonas amygdaloids, the pathogen of mulberry blight, the target band size is about 506bp (see lanes 1 to 3).

[0099] Example 6 PCR Detection Specificity Detection

[0100] 1. Experimental Methods

[0101] PCR detection was performed using the detection kit of Example 4, using the genomes of Pseudomonas amygdali pv. phaseolicola, Pseudomonas fulva, Enterobacter cloacae, Ralstonia solanacearum, Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, and Bacillus subtilis as templates, the genome of Pseudomonas amygdali, the pathogen of mulberry blight, as a positive control, and sterile water as a negative control.

[0102] 2. Experimental Results

[0103] The results are as follows Figure 4 As shown, the primers of Example 2, whose nucleotide sequences are shown in SEQ ID NOs: 8-9, have good specificity and can only amplify the pathogen of mulberry blight, Pseudomonas amygdali, with a target band size of about 506 bp (see lanes 1-2), and cannot amplify other strains.

[0104] Example 7 PCR detection of mulberry blight field samples

[0105] 1. Experimental Methods

[0106] For rapid detection of mulberry blight, longitudinal DNA of samples of mulberry blight diseased plants was extracted as templates (disease samples were collected in Haining City, Zhejiang Province, Yingshang County, Fuyang City, Anhui Province, Sheyang County, Yancheng City, Jiangsu Province, and Hanyin City, Shaanxi Province, with 3 samples of mulberry blight in each province), healthy mulberry tree samples (variety Qiangsang No. 1) and sterile water were used as negative controls, and the mulberry blight pathogen Pseudomonas amygdalus was used as a positive control, and detection was performed using the PCR detection kit of Example 4.

[0107] 2. Experimental Results

[0108] The results are as follows Figure 5 As shown, DNA from mulberry blight disease samples from four provinces can amplify a band of about 506 bp (lanes 1 to 12), which means that the mulberry blight pathogen Pseudomonas amygdali can be detected in all of them, while no band was detected in healthy samples and sterile water as negative control samples.

[0109] Example 8 LAMP detection primer design

[0110] A specific gene segment encoding the hypothetical gene shown in SEQ ID NO: 1 in Example 1 was selected, and primers were designed using the online software PrimerExplorer V4 (http: / / primerexplorer.jp / elamp4.0.0 / index.html). A total of 5 primer sets were obtained, as shown in Table 3.

[0111] Table 3 Nucleotide sequences of 5 primer sets

[0112]

[0113]

[0114] LAMP amplification reactions were performed using genomic DNA of Pseudomonas amygdali, the pathogen of mulberry blight, as a template using each of the five primer sets described above. The LAMP reaction system consisted of 12.5 μL of reaction buffer, 0.5 μL of BstDNA polymerase, 0.1 μM final concentrations of outer primers F3 and B3 (nucleotide sequences shown in SEQ ID NOs: 32-33), and 0.8 μM final concentrations of inner primers FIP and BIP (nucleotide sequences shown in SEQ ID NOs: 34-35). The DNA template was 1 μL, and the fluorescent dye STYO 9 was added to a volume of 25 μL in ddH2O. The reaction was incubated at 63°C for 30 min. Results were determined by fluorescence curve analysis.

[0115] 2. Experimental Results

[0116] The results are as follows Figure 6 As shown, among the five primer sets, primer sets 1, 2, 4, and 5 can all amplify effective curves. Among them, primer set 5 (i.e., the DX-5 primer set in Table 3) has the shortest amplification time, so this primer set was selected.

[0117] Example 9 Determination of the Optimal Temperature for LAMP Primer Set Amplification Reaction

[0118] 1. Experimental Methods

[0119] Different LAMP amplification reaction temperatures of 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, and 65°C were designed. The primer set DX-5 and the amplification reaction system of Example 8 were used to perform the LAMP reaction, and the results were determined by the fluorescence curve method.

[0120] 2. Experimental Results

[0121] The results are as follows Figure 7 As shown in the figure, curves can be amplified at the five set temperatures, and as the temperature increases, the fluorescence threshold of the curve gradually increases. Therefore, 63°C is selected as the optimal temperature for the LAMP reaction.

[0122] Example 10 Determination of the final concentration ratio of primers in the LAMP primer set amplification reaction

[0123] 1. Experimental Methods

[0124] The final concentration ratios of different inner and outer primers in the LAMP reaction system were determined, and the following different final primer concentration ratios were designed: (1) outer primer: inner primer = 1:2; (2) outer primer: inner primer = 1:4; (3) outer primer: inner primer = 1:8; (4) outer primer: inner primer = 1:10. LAMP reactions were performed using the primer set DX-5 of Example 8 and the amplification reaction system, and the results were determined by fluorescence curve analysis.

[0125] 2. Experimental Results

[0126] Results After testing and screening, the results were as follows Figure 8 As shown in the figure, four primers with different ratios can all amplify curves. Curves 2, 3, and 4 appear faster, but curve 3 appears the fastest and has the highest threshold. Therefore, the final concentration ratio of inner primers to outer primers in the final LAMP amplification reaction system is selected to be 1:8.

[0127] Example 11 A LAMP Detection Kit for Pseudomonas amygdali, the Pathogen of Mulberry Blight

[0128] 1. Composition

[0129] A LAMP detection kit for Pseudomonas amygdali, the pathogen of mulberry blight, comprises the primer set DX-5 of Example 8, i.e., the primer set having nucleotide sequences as shown in SEQ ID NOs: 32 to 35.

[0130] The kit also contains reaction buffer, Bst DNA polymerase, Pseudomonas amygdali, a pathogen of mulberry blight, as a positive control DNA template, sterile water as a negative control, fluorescent dye STYO 9, and ddH2O.

[0131] 2. Usage

[0132] The LAMP reaction was performed using the test sample DNA as a template using a kit. The amplification reaction system was as follows: 12.5 μL of reaction buffer, 0.5 μL of Bst DNA polymerase, 0.5 μL of STYO 9 fluorescent dye, the final concentrations of the inner primers FIP and BIP were 0.8 μM each, the final concentrations of the outer primers F3 and B3 were 0.1 μM each, 1 μL of DNA template, and ddH2O was added to 25 μL. The amplification reaction procedure was as follows: constant temperature reaction at 63°C for 30 min.

[0133] After the reaction is completed, the amplified product is determined to be infected with Pseudomonas amygdali by real-time fluorescence curve method, colorimetric method or gel electrophoresis.

[0134] The judgment method is as follows: the fluorescence curve is detected by real-time fluorescence curve method and judged as positive, and no fluorescence curve is detected and judged as negative; or by colorimetric observation, after the reaction is completed, SYBR Green I is added to the reaction solution, and the reaction solution is green and judged as positive, and brown and judged as negative; or the amplified product is taken for gel electrophoresis detection, and the appearance of multiple bands is judged as positive, and no appearance is judged as negative.

[0135] Example 12 Specificity detection of LAMP primer set

[0136] 1. Experimental Methods

[0137] The pathogen of mulberry blight, Pseudomonas amygdali, was used as a positive control. Pseudomonas syringae pv.phaseolicola, Pseudomonasfluorescens, Pseudomonas fulva, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae, Klebsiella oxytoca, Pantoea ananatis, Bacillus subtilis, Stenotrophomonas maltophilia, Enterobacter asburiae, Azomonas sp.) as a template and sterile water as a negative control, and LAMP detection was performed using the detection kit and method of Example 11.

[0138] 2. Experimental Results

[0139] The results of real-time fluorescence curve method are as follows Figure 9 As shown, except for the genomic DNA of the positive control bacteria Pseudomonas amygdali, the pathogen of mulberry blight, which showed an amplification curve, the genomic DNA of other bacteria did not show an amplification curve.

[0140] The results of the colorimetric method are as follows Figure 10 As shown, the positive control bacteria, the pathogen of mulberry blight, Pseudomonas amygdali (label 1), the remaining bacterial genomic DNA (labels 2 to 13) and sterile water (label 14) showed negative (brown), and the results were consistent with the fluorescence curve method.

[0141] Gel electrophoresis results Figure 11 As shown, the positive control bacteria, Pseudomonas amygdali (label 1), the pathogen of mulberry blight, and the other bacterial genomic DNA (labels 2 to 13) and sterile water (label 14) showed no bands. The results were consistent with those of the fluorescence curve method and the colorimetric method.

[0142] This indicates that the primer set DX-5 of Example 8 has good specificity and can be used to specifically detect the pathogen of mulberry blight, Pseudomonas amygdali.

[0143] Example 13 Detection of Different Mulberry Blight Diseased Plant Samples

[0144] 1. Experimental Methods

[0145] To rapidly detect mulberry blight caused by the pathogen Pseudomonas amygdali, total DNA was extracted from diseased mulberry blight samples collected from various locations. Sterile water served as a negative control (labeled 14). LAMP assays were performed using the detection kit and method described in Example 11. After completion of the reaction, results were evaluated using real-time fluorescence curve analysis, colorimetry, and gel electrophoresis.

[0146] Among them, 1 is the DNA of Pseudomonas amygdali, the pathogen of mulberry blight disease (positive control), numbers 2 to 7 are the total DNA of field samples of mulberry blight disease, the samples were collected in Haining City, Zhejiang Province, the mulberry variety is Qiangsang No. 1, and the experiment was repeated 6 times; numbers 8 to 13 are the total DNA of disease samples after Koch's postulate recovery, the variety is Qiangsang No. 1, and the experiment was repeated 6 times; number 14 is sterile water (blank control).

[0147] 2. Experimental Results

[0148] The results of real-time fluorescence curve analysis are as follows Figure 12 As shown, fluorescence curves can be detected for the positive control (label 1), field samples of mulberry blight (labels 2 to 7) and samples after recovery (labels 8 to 13), while no fluorescence curve can be detected for sterile water (label 14).

[0149] The results of the colorimetric method are as follows Figure 13 As shown, the color development results of the positive control (label 1), field samples of mulberry blight (labels 2 to 7) and samples after relapse (labels 8 to 13) showed positive (green), while sterile water (label 14) showed negative (brown), and the results were consistent with the fluorescence curve method.

[0150] Gel electrophoresis results Figure 14 As shown, the positive control (label 1), field samples of mulberry blight (labels 2 to 7) and samples after re-infection (labels 8 to 13) showed multiple bands in electrophoresis, while sterile water (label 14) showed no bands.

[0151] The LAMP primer set DX-5 of Example 8 and the detection kit of Example 11 of the present invention can be used to detect mulberry blight diseased plants infected with the mulberry blight pathogen Pseudomonas amygdali, which is of great significance for the rapid detection of mulberry blight in mulberry orchards. It has great application value in disease quarantine and prevention and is worthy of promotion.

[0152] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention. Sequence Listing <110> South China Agricultural University <120> A detection kit for Pseudomonas amygdalus, Pseudomonas syringae and / or mulberry blight <160> 35 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1185 <212> DNA <213> Artificial Sequence <400> 1 ctccatagtc tcatgccagt taccttcagc tcgcctcata acgccccttc ttaactgtta 60 tcgtccatgt atgattcctt gaggttaccc tgcagacaag cggcagagaa tgctagcttt 120 ccgatagtga ccaccaccgc tcaaataggg aaaccactac agaagatcaa aacttaaata 180 ccctaggccc ccttggcgtg ctctaaattg gtaaccaagt tatccattgt cgatctcatc 240 acccgcacct gctcaagagc ctcccggaat gatctttacct cagtccctgc cagaaccccc 300 acaacaccgg ggatggccat tgtctggtta tagcggcgac cttcgacatc tctaaatgaa 360 tcagcagctt ccataatcaa attcatggtc gcagtctgga cagactcgcc tgcggtagac 420 atcaaggacg caacctgcga agtgatgaag tcgtagacca actccccata ctggatcgcc 480 tcttcggtcg aaggaatata ccccttatga acgacgctgt ttcgaaaggc tttccaatcc 540 tttgttttgc cagtcgcccc tgcgggcttc accttctcag gcttcggcga tacggacgtc 600 ccataatgaa gaaggtgaag catgaggtag gcgccatatt gccgctctga tgaattaact 660 aagtcctgcc atgtctgctt catcgcagcc tgtgcaatct ggttcctctg ggcgactact 720 cgaataaaaa actcaaaaaa acgctcctgt gaggaagcga aacgagtaat tgcctcgccg 780 ggatagccat ctaataatgc ccgagcgcca agctcgaaca ggatctcgaa cttatgttgc 840 tgtactacag acaccgtctt atggcctctc gggcactccg tcagataggc tcctcggtca 900 ttgacctcgt gccaagctgt gtggcaattc aacggatgac cgtcctcctg aaaacatccc 960 gcacacatga ccaagacctt cattgacagc tctccctgta gtgttaattc atcttggctg 1020 ctggatagag gccaatcaac cacaaaaaaa gcactgcagg atttggcagg atttcgcgaa 1080 tcgttcagct tccctgctgc tgagcctaat cgtaaaactt tggcccacgg cggtagcgtg 1140 aaacagaaag gtttggatgc aaggacttaa gccgatagca tatgc 1185 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 ttatagcggc gaccttcgac 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 gagcctatct gacggagtgc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 accaccaccg ctcaaatagg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 gtcgaaggtc gccgctataa 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 ccccttatga acgacgctgt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <400> 7 ttggcgctcg ggcattatta 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <400> 8 tgtctggtta tagcggcgac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <400> 9 agatcctgtt cgagcttggc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 tggttatagc ggcgaccttc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 ttggcgctcg ggcattatta 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 gatagtgacc accaccgctc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <400> 13 cagcgtcgtt cataaggggt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <400> 14 gttaccttca gctcgcctca 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 gtcgccgcta taaccagaca 20 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <400> 16 tgccatgtct gcttcatcg 19 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <400> 17 tatctgacgg agtgcccg 18 <210> 18 <211> 41 <212> DNA <213> Artificial Sequence <400> 18 cgcttcctca caggagcgtt ttagcctgtg caatctggtt c 41 <210> 19 <211> 39 <212> DNA <213> Artificial Sequence <400> 19 cgagcgccaa gctcgaacag aggccataag acggtgtct 39 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <400> 20 cggtcattga cctcgtgc 18 <210> twenty one <211> 18 <212> DNA <213> Artificial Sequence <400> twenty one agcagggaag ctgaacga 18 <210> twenty two <211> 42 <212> DNA <213> Artificial Sequence <400> twenty two gtcttggtca tgtgtgcggg ataagctgtg tggcaattca ac 42 <210> twenty three <211> 41 <212> DNA <213> Artificial Sequence <400> twenty three atcttggctg ctggatagag gctcgcgaaa tcctgccaaa t 41 <210> twenty four <211> 18 <212> DNA <213> Artificial Sequence <400> twenty four gcctcttcgg tcgaagga 18 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <400> 25 agcagacatg gcaggactt 19 <210> 26 <211> 41 <212> DNA <213> Artificial Sequence <400> 26 caggggcgac tggcaaaaca ataccccctta tgaacgacgc t 41 <210> 27 <211> 40 <212> DNA <213> Artificial Sequence <400> 27 cttctcaggc ttcggcgata cgggcaatat ggcgcctacc 40 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <400> 28 attgtcgatc tcatcacccg 20 <210> 29 <211> 18 <212> DNA <213> Artificial Sequence <400> 29 atgtctaccg caggcgag 18 <210> 30 <211> 39 <212> DNA <213> Artificial Sequence <400> 30 aatggccatc cccggtgttg ctcaagagcc tcccggaat 39 <210> 31 <211> 41 <212> DNA <213> Artificial Sequence <400> 31 ggttatagcg gcgaccttcg actccagact gcgaccatga a 41 <210> 32 <211> 18 <212> DNA <213> Artificial Sequence <400> 32 tcgcctgcgg tagacatc 18 <210> 33 <211> 19 <212> DNA <213> Artificial Sequence <400> 33 gcctacctca tgcttcacc 19 <210> 34 <211> 40 <212> DNA <213> Artificial Sequence <400> 34 cgaccgaaga ggcgatccag tagacgcaac ctgcgaagtg 40 <210> 35 <211> 40 <212> DNA <213> Artificial Sequence <400> 35 tcctttgttt tgccagtcgc ccgacgtccg tatcgccgaa 40

Claims

1. A test kit for detecting Pseudomonas amygdaloids for non-therapeutic and / or diagnostic purposes Pseudomonas amygdali ) and / or mulberry blight, characterized in that, The detection reagent contains a primer set with nucleotide sequences as shown in SEQ ID NOs: 8-9.

2. A test kit for detecting Pseudomonas amygdaloids for non-therapeutic and / or diagnostic purposes Pseudomonas amygdali ) and / or mulberry blight, characterized in that, The detection reagent contains a primer set with nucleotide sequences as shown in SEQ ID NOs: 32-35.

3. A method for detecting Pseudomonas amygdaloids ( Pseudomonas amygdali ) and / or mulberry blight, characterized in that, The reagent contains a primer set having nucleotide sequences as shown in SEQ ID NOs: 8 to 9 or a primer set having nucleotide sequences as shown in SEQ ID NOs: 32 to 35.

4. The reagent according to claim 3 is used in the preparation of Pseudomonas amygdaloids ( Pseudomonas amygdali ) and / or mulberry blight disease detection kit.

5. A Pseudomonas amygdalae ( Pseudomonas amygdali ) and / or a detection kit for mulberry blight, characterized in that, The detection kit contains the detection reagent according to claim 3.

6. A method for detecting Pseudomonas amygdaloids for non-therapeutic and / or diagnostic purposes Pseudomonas amygdali ) and / or mulberry blight, characterized in that, PCR amplification reaction was performed using the primer set with the nucleotide sequence shown in SEQ ID NOs: 8-9.

7. A method for detecting Pseudomonas amygdaloids for non-therapeutic and / or diagnostic purposes Pseudomonas amygdali ) and / or mulberry blight, characterized in that, The primer set with the nucleotide sequences shown in SEQ ID NOs: 32 to 35 was used for LAMP amplification reaction.

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