Method for simultaneous detection of multiple strawberry pathogens and application thereof

Abstract

The application discloses a method for synchronously detecting multiple strawberry pathogens and application, and the method for synchronously detecting multiple strawberry pathogens comprises a primer combination capable of synchronously detecting multiple strawberry pathogens, the primer combination comprises 18 specific primers and 2 universal primers, and can be used for detecting strawberry mottle virus, strawberry light yellow edge virus, strawberry crinkle virus, strawberry vein banding virus, strawberry powdery mildew, strawberry gray mold, strawberry anthracnose, strawberry xanthomonas and strawberry internal reference beta-Actin1, and corresponding detection methods, detection reagents and detection kits are simultaneously provided. The application realizes one-pot reaction for simultaneously detecting nine targets, greatly improves the detection efficiency, effectively avoids false negative results through the internal reference gene monitoring, provides strong technical support for early diagnosis of strawberry diseases, and has great application value and market prospect.

Description

Technical Field

[0001] This invention relates to agricultural biotechnology and plant disease detection, and particularly to a method and application for the simultaneous detection of multiple strawberry pathogens, including a primer set and its application for multiplex PCR combined with capillary electrophoresis detection of strawberry mottle virus (SMoV), strawberry mild yellow edge virus (SMYEV), strawberry wrinkle virus (SCV), strawberry vein virus (SVBV), strawberry powdery mildew (Podosphaera aphanis), strawberry gray mold (Botrytis cinerea), strawberry anthracnose (Colletotrichum spp.), and Xanthomonas fragariae. Background Technology

[0002] Strawberry (Fragaria × ananassa Duch.) is a berry crop with extremely high economic value. Due to its high nutritional and edible value and good taste, it is widely cultivated worldwide. It is cold-hardy and shade-tolerant, but not drought-tolerant, waterlogging-tolerant, heat-intolerant, or cold-hardy. However, during its cultivation, strawberry is highly susceptible to various pathogens, including viruses, fungi, and bacteria, leading to serious economic losses.

[0003] Strawberry Mottle Virus (SMoV), Strawberry Vein Virus (SVBV), Strawberry Shrink Virus (SCV), and Strawberry Mild Yellow Edge Virus (SMYEV) are the main viruses that harm strawberries. These viruses are primarily transmitted through vectors such as aphids. Infection causes symptoms such as stunted growth, leaf mottling, wrinkling, yellowing, and veining, leading to reduced fruit yield and poorer fruit quality. These viruses often cause multiple infections, resulting in complex and subtle symptoms that are difficult to diagnose accurately with the naked eye.

[0004] Strawberry powdery mildew (caused by *Podosphaera aphanis*), strawberry gray mold (caused by *Botrytis cinerea*), and strawberry anthracnose (mainly caused by *Colletotrichum spp.*) are the three major fungal diseases causing strawberry diseases. Strawberry powdery mildew affects leaves, strawberry gray mold affects flowers, and strawberry anthracnose affects fruit. They are highly susceptible to outbreaks and epidemics under suitable temperature and humidity conditions, causing leaf blight, fruit rot and mold, severely affecting the marketability and storage life of fresh fruit. Strawberry angular leaf spot, caused by *Xanthomonas fragariae*, is a seed-borne and seedling-borne bacterial disease that causes angular lesions on leaves; in severe cases, leaves wither and die, weakening plant growth.

[0005] Currently, the main detection methods for the above-mentioned pathogens include: observation of biological symptoms, serological methods, conventional PCR technology and real-time quantitative PCR (qPCR).

[0006] Among them, the observation of biological symptoms relies on experience and is highly subjective. It is difficult to accurately judge latent infections and complex infections, and early diagnosis cannot be achieved.

[0007] Serological methods, such as ELISA, are simple to operate and suitable for screening large numbers of samples, but they have low sensitivity, long antibody preparation cycles, high costs, and may miss viral variants.

[0008] Conventional PCR technology is highly sensitive and specific, but it can usually only detect one pathogen per reaction. When multiple pathogens need to be detected, multiple PCR reactions need to be performed separately, which is cumbersome, time-consuming, labor-intensive, costly, and consumes a large amount of samples.

[0009] Real-time quantitative PCR, or qPCR, combines qualitative and quantitative capabilities with extremely high sensitivity. However, throughput is limited by the number of instrument channels, and simultaneous detection of multiple pathogens requires complex multicolor fluorescent labeling and probe design, which is extremely expensive. Furthermore, it is difficult to achieve simultaneous detection of more than 4-5 targets in a single reaction tube.

[0010] Therefore, existing detection methods are insufficient to meet the high-throughput, multi-target, rapid, accurate, and cost-effective detection requirements of modern agriculture for strawberry seedling health, early disease warning, and integrated field control. Thus, there is an urgent need to develop an integrated detection technology capable of simultaneously detecting multiple key strawberry pathogens. This technology is of paramount importance to the healthy development of the strawberry industry. Summary of the Invention

[0011] In view of the shortcomings of existing detection methods in detecting strawberry pathogens, this invention provides a method and application for simultaneous detection of multiple strawberry pathogens, which can meet the detection needs of high throughput, multi-target, rapid, accurate and cost-controllable detection in strawberry seedling health, early disease warning and field integrated control.

[0012] To achieve the above objectives, the embodiments of the present invention adopt the following technical solutions:

[0013] A method for simultaneously detecting multiple strawberry pathogens, characterized in that the method includes the following steps:

[0014] Take a standard RNA sample, determine its concentration and purity, and then dilute it to the required concentration. The standard RNA sample is an RNA sample obtained by synthesizing a plasmid of the candidate target region sequence for the corresponding pathogen detection and then transcribing it using an in vitro high-efficiency transcription kit.

[0015] The amplification system was prepared using primer combinations, buffer solutions, enzyme mixtures, nuclease-free pure water, and DNA templates.

[0016] RNA samples were added to the PCR reaction tubes, and nucleic acid-free pure water was used as a negative control.

[0017] Perform PCR amplification;

[0018] The amplification products were detected by capillary electrophoresis.

[0019] Data analysis;

[0020] Result determination;

[0021] The primer set includes 18 specific primers and 2 universal primers: specific primers against strawberry mottle virus as shown in SEQ ID NO.1 and SEQ ID NO.2; specific primers against strawberry mild yellow edge virus as shown in SEQ ID NO.3 and SEQ ID NO.4; specific primers against strawberry shrunken virus as shown in SEQ ID NO.5 and SEQ ID NO.6; specific primers against strawberry veining virus as shown in SEQ ID NO.7 and SEQ ID NO.8; specific primers against strawberry powdery mildew fungus as shown in SEQ ID NO.9 and SEQ ID NO.10; specific primers against strawberry gray mold fungus as shown in SEQ ID NO.11 and SEQ ID NO.12; specific primers against strawberry anthracnose fungus as shown in SEQ ID NO.13 and SEQ ID NO.14; specific primers against strawberry xanthomonas as shown in SEQ ID NO.15 and SEQ ID NO.16; and specific primers against the strawberry β-actin gene Actin1 as shown in SEQ ID NO.1. The sequences are shown in NO.17 and SEQ ID NO.18; the universal primer sequences are shown in SEQ ID NO.19 and SEQ ID NO.20.

[0022] According to one aspect of the invention, the 5' end of the universal primer is fluorescently labeled, and the fluorescent label may be FAM or HEX.

[0023] According to one aspect of the present invention, the concentration of the primer combination is 100 μmol / L, wherein the addition amount of each primer pair is as follows: the concentration range of the primer pair specifically targeting strawberry mottle virus is 0.4-0.6 μL; the concentration range of the primer pair specifically targeting strawberry mild yellow edge virus is 0.4-0.6 μL; the concentration range of the primer pair specifically targeting strawberry shrunken virus is 0.4-0.6 μL; the concentration range of the primer pair specifically targeting strawberry vein virus is 0.5-1.0 μL; and the concentration range of the primer pair specifically targeting strawberry mottle virus is 0.5-1.0 μL. The concentration range of the specific primer pairs for powdery mildew is 0.4-0.8 μL; the concentration range of the specific primer pairs for amplifying strawberry gray mold is 0.4-0.8 μL; the concentration range of the specific primer pairs for amplifying strawberry anthracnose is 0.5-1.0 μL; the concentration range of the specific primer pairs for amplifying strawberry Xanthomonas is 0.5-1.0 μL; the concentration range of the specific primer pairs for amplifying strawberry β-actin gene Actin1 is 0.4-0.6 μL; and the concentration range of the universal primers is 8-15 μL.

[0024] According to one aspect of the invention, the multiplex amplification system includes a PCR mixture and a DNA template.

[0025] According to one aspect of the present invention, the PCR mixture is used in a 20 μl multiplex amplification system, the PCR mixture comprising 10 μl one-step RT-PCR Buffer, 2 μl primer combination, 1.5 μl one-step enzyme mixture, and 5.5 μl nuclease-free purified water.

[0026] According to one aspect of the present invention, the DNA template is used in a 20 μl multiplex amplification system, wherein the amount of DNA template used is 1 μl.

[0027] According to one aspect of the invention, the PCR mixture comprises a one-step RT-PCR buffer, a primer set, a one-step enzyme mixture, and nuclease-free purified water.

[0028] According to one aspect of the present invention, the amplification program of the multiplex amplification system is as follows: reverse transcription at 50°C for 30 min; denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s as one cycle, for a total of 35 cycles; then holding at 72°C for 10 min, and then cooling to 4°C after completion.

[0029] A kit for simultaneous detection of multiple strawberry pathogens, the kit comprising the aforementioned multiplex amplification system.

[0030] According to one aspect of the present invention, the application of a multiplex amplification system and kit in the simultaneous detection of multiple strawberry pathogens.

[0031] The advantages of this invention are as follows: Through the above technical solution, eight important pathogens and one internal reference gene can be detected simultaneously, significantly reducing operation time, labor costs, and reagent consumption. It is suitable for rapid screening of large batches of samples and diagnosis of complex infections. Introducing the strawberry Actin1 gene as an internal standard effectively monitors the quality of the entire process from RNA extraction to PCR amplification, avoiding false negatives caused by sample degradation or reaction inhibition, and ensuring the reliability of the test results. The universal primer-mediated amplification strategy effectively overcomes the bottleneck of multiplex PCR, significantly improving amplification efficiency and sensitivity, while specific primers ensure accurate identification. It has strong scalability. To add new detection targets, only specific primers with the same universal adapter need to be designed, without changing the entire detection system and labeling method, resulting in good platform compatibility. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The present invention describes the detection results of amplification of strawberry wrinkle virus (SCV) pathogen samples, strawberry mottle virus (SMoV) pathogen samples, strawberry mild yellow edge virus (SMYEV) pathogen samples, strawberry vein virus (SVBV) pathogen samples, strawberry powdery mildew (P. aphanius) pathogen samples, strawberry gray mold (B. cinerea) pathogen samples, strawberry anthracnose (Colletotrichum spp.) pathogen samples, strawberry Xanthomonas fragariae pathogen samples, and strawberry samples using the specific primer pairs for strawberry wrinkle virus (SCV) described in this invention.

[0034] Figure 2 The sequence result of cloning and sequencing of the specific primer amplification product of the strawberry shrunken virus described in this invention;

[0035] Figure 3 The above are the amplification detection results of strawberry powdery mildew, strawberry xanthomonas, and strawberry gray mold as described in this invention;

[0036] Figure 4 The results of amplification detection of strawberry shriveling virus, strawberry mottle virus, and strawberry mild yellow edge virus described in this invention;

[0037] Figure 5 The results of the detection of strawberry vein virus, strawberry anthracnose bacterium, and virus-free and disease-free strawberry enzyme RNA as described in this invention;

[0038] Figure 6 The image shows the detection results of some actual samples described in this invention. Samples F1 and F2 were found to contain strawberry powdery mildew, while samples G1 and G2 were found to contain both strawberry mottle virus and strawberry powdery mildew. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] This embodiment provides a method for simultaneous detection of multiple strawberry pathogens. The method uses a primer combination to detect strawberry pathogens. The primer combination contains 18 specific primers and 2 universal primers.

[0042] Access the NCBI database and download strawberry pathogen-related sequence information. Search for the target pathogen using its standard name or classification ID, selecting representative and complete gene or genomic sequences. After downloading, use ApE software to identify highly conserved genomic regions of the pathogen as candidate target regions, and use these as templates to design appropriate primer design parameters, such as primer length, annealing temperature, and PCR product length. Combine BLAST analysis to design specific primers. The specificity of primer pairs is further determined using single-step RT-PCR and cross-amplification. Taking the screening of specific primers for strawberry shrunken virus (SCV) as an example, specific primer pairs for SCV were used to amplify and detect the virus using standard RNA samples of other pathogens as templates. The primer pairs that amplified only in SCV samples and did not show significant amplification in samples of other species were selected and tentatively designated as specific primer pairs for SCV. After removing the fluorescent label, the SCV samples were amplified using the above-mentioned specific primer pairs for SCV. The target band was then recovered and purified using a gel extraction kit and cloned for sequencing. The sequencing results were compared and analyzed with the NCBI database to further determine the specificity of the amplification primers for SCV. After identification, specific primers for strawberry mottle virus (SMoV), strawberry mild yellow edge virus (SMYEV), strawberry vein virus (SVBV), strawberry powdery mildew fungus (P. aphanius), strawberry gray mold fungus (B. cinerea), strawberry anthracnose fungus (Colletotrichum spp.), and strawberry xanthomonas (X. fragariae) were obtained using this method. Primers for each site were tested and screened, and the final primer combination sequence of this invention is shown in Table 1.

[0043] Table 1

[0044]

[0045] The underlined part is a pair of identical, pre-designed universal sequences attached to the 5' end of each pair of specific primers, namely the universal sequences shown in SEQ ID NO.19-20. The forward primer of the universal primer is labeled with FAM fluorescent at the 5' end.

[0046] Example 2

[0047] This embodiment provides a method for the simultaneous detection of multiple strawberry pathogens using a multiplex amplification system. The multiplex amplification system includes a one-step RT-PCR buffer, primer set, one-step enzyme mixture, nuclease-free purified water, and template RNA. The Hieff Unicon® V Universal Multiplex One Step RT-qPCR Probe Kit (product number 11213ES60) was used, and the reaction system was prepared according to its instructions. The system included: 10 μL of One-Step RT-PCR Buffer, 2 μL of primer set, 1.5 μL of One-Step Enzyme Mix, 5.5 μL of nuclease-free purified water, and 1 μL of template RNA. The primer concentration was 100 μmol / L. The components and amounts of the primer set are shown in Table 2.

[0048] Table 2

[0049]

[0050] The PCR reaction program was as follows: reverse transcription at 50℃ for 30 min; denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s as one cycle, for a total of 35 cycles; then hold at 72℃ for 10 min, and finally cool to 4℃. The height of the CE detection-specific peak in each result spectrum represents a specific pathogen.

[0051] Example 3

[0052] Eight known standard pathogen RNA samples and one virus-free strawberry RNA sample were amplified and detected. The specific steps are as follows:

[0053] 1. Sample preparation

[0054] Take standard species RNA samples, determine the concentration and purity of RNA using NanoDrop2000 (Thermo), dilute the RNA to the corresponding concentration, and store at 4℃ or -20℃ for later use.

[0055] 2. Prepare the amplification system

[0056] Prepare the PCR amplification system according to the components in Table 3, vortex to mix, and then dispense according to the number of samples.

[0057] Table 3

[0058]

[0059] 3. Add RNA template

[0060] Add 1 µL of each prepared RNA sample to the corresponding PCR reaction tube, and set 1 µL of nucleic acid-free pure water as a negative control.

[0061] 4. PCR amplification

[0062] Place each reaction tube into the reaction chamber of the PCR amplification instrument and set the reaction volume to 20 µL. Perform one-step RT-PCR amplification according to the following procedure: reverse transcription at 50℃ for 30 min; denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s as one cycle, for a total of 35 cycles; then hold at 72℃ for 10 min, and finally cool to 4℃.

[0063] 5. Perform capillary electrophoresis to detect the amplification products.

[0064] Prepare a loading mixture containing the molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × number of samples to be tested. Vortex to mix for 10-15 seconds. Use a pipette to dispense 9 μL of the formamide and internal standard mixture into each well. Add 1 μL of the amplification product to the formamide and internal standard mixture, and cover with the sealing tape. Perform the test according to the instructions in the user manual.

[0065] 6. Data Analysis

[0066] Import the relevant files into the GeneMapper software, input the raw data from the detector, and analyze the data.

[0067] 7. Result Determination

[0068] Graphs showing the results of capillary detection of eight pathogens in strawberry samples. The results demonstrate that this detection system can accurately detect eight pathogens in strawberries.

[0069] Example 4

[0070] Eight known standard pathogen RNA samples and one virus-free strawberry RNA sample were amplified and detected. The specific steps are as follows:

[0071] 1. Sample preparation

[0072] Take standard species RNA samples, determine the concentration and purity of RNA using NanoDrop2000 (Thermo), and dilute the RNA to 1 ng / μL, then store at 4℃ or -20℃ for later use.

[0073] 2. Prepare the amplification system

[0074] Prepare the PCR amplification system according to the components in Table 3, vortex to mix, and then dispense according to the number of samples.

[0075] 3. Add RNA template

[0076] Add 1 µL of each prepared RNA sample to the corresponding PCR reaction tube, and set 1 µL of nucleic acid-free pure water as a negative control.

[0077] 4. PCR amplification

[0078] Place each reaction tube into the reaction chamber of the PCR amplification instrument and set the reaction volume to 20 µL. Perform one-step RT-PCR amplification according to the following procedure: reverse transcription at 50℃ for 30 min; denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s as one cycle, for a total of 35 cycles; then hold at 72℃ for 10 min, and finally cool to 4℃.

[0079] 5. Perform capillary electrophoresis to detect the amplification products.

[0080] Prepare a loading mixture containing the molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × number of samples to be tested. Vortex to mix for 10-15 seconds. Use a pipette to dispense 9 μL of the formamide and internal standard mixture into each well. Add 1 μL of the amplification product to the formamide and internal standard mixture, and cover with the sealing tape. Perform the test according to the instructions in the user manual.

[0081] 6. Data Analysis

[0082] Import the relevant files into the GeneMapper software, input the raw CE values ​​from the detector, and analyze the data.

[0083] Example 5

[0084] Eight known standard pathogen RNA samples and one virus-free strawberry RNA sample were amplified and detected. The specific steps are as follows:

[0085] 1. Sample preparation

[0086] Take standard species RNA samples, determine the concentration and purity of RNA using NanoDrop2000 (Thermo), and dilute the RNA to 0.1 ng / μL. Store at 4℃ or -20℃ for later use.

[0087] 2. Prepare the amplification system

[0088] Prepare the PCR amplification system according to the components in Table 3, vortex to mix, and then dispense according to the number of samples.

[0089] 3. Add RNA template

[0090] Add 1 µL of each prepared RNA sample to the corresponding PCR reaction tube, and set 1 µL of nucleic acid-free pure water as a negative control.

[0091] 4. PCR amplification

[0092] Place each reaction tube into the reaction chamber of the PCR amplification instrument and set the reaction volume to 20 µL. Perform one-step RT-PCR amplification according to the following procedure: reverse transcription at 50℃ for 30 min; denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s as one cycle, for a total of 35 cycles; then hold at 72℃ for 10 min, and finally cool to 4℃.

[0093] 5. Perform capillary electrophoresis to detect the amplification products.

[0094] Prepare a loading mixture containing the molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × number of samples to be tested. Vortex to mix for 10-15 seconds. Use a pipette to dispense 9 μL of the formamide and internal standard mixture into each well. Add 1 μL of the amplification product to the formamide and internal standard mixture, and cover with the sealing tape. Perform the test according to the instructions in the user manual.

[0095] 6. Data Analysis

[0096] Import the relevant files into the GeneMapper software, input the raw CE values ​​from the detector, and analyze the data.

[0097] Example 6

[0098] Eight known standard pathogen RNA samples and one virus-free strawberry RNA sample were amplified and detected. The specific steps are as follows:

[0099] 1. Sample preparation

[0100] Take standard species RNA samples, determine the concentration and purity of RNA using NanoDrop2000 (Thermo), and dilute the RNA to 0.01 ng / μL, then store at 4℃ or -20℃ for later use.

[0101] 2. Prepare the amplification system

[0102] Prepare the PCR amplification system according to the components in Table 3, vortex to mix, and then dispense according to the number of samples.

[0103] 3. Add RNA template

[0104] Add 1 µL of each prepared RNA sample to the corresponding PCR reaction tube, and set 1 µL of nucleic acid-free pure water as a negative control.

[0105] 4. PCR amplification

[0106] Place each reaction tube into the reaction chamber of the PCR amplification instrument and set the reaction volume to 20 µL. Perform one-step RT-PCR amplification according to the following procedure: reverse transcription at 50℃ for 30 min; denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s as one cycle, for a total of 35 cycles; then hold at 72℃ for 10 min, and finally cool to 4℃.

[0107] 5. Perform capillary electrophoresis to detect the amplification products.

[0108] Prepare a loading mixture containing the molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × number of samples to be tested. Vortex to mix for 10-15 seconds. Use a pipette to dispense 9 μL of the formamide and internal standard mixture into each well. Add 1 μL of the amplification product to the formamide and internal standard mixture, and cover with the sealing tape. Perform the test according to the instructions in the user manual.

[0109] 6. Data Analysis

[0110] Import the relevant files into the GeneMapper software, input the raw CE values ​​from the detector, and analyze the data.

[0111] Example 7

[0112] Eight known standard pathogen RNA samples and one virus-free strawberry RNA sample were amplified and detected. The specific steps are as follows:

[0113] 1. Sample preparation

[0114] Take standard species RNA samples, determine the concentration and purity of RNA using NanoDrop2000 (Thermo), and dilute the RNA to 0.001 ng / μL, then store at 4℃ or -20℃ for later use.

[0115] 2. Prepare the amplification system

[0116] Prepare the PCR amplification system according to the components in Table 3, vortex to mix, and then dispense according to the number of samples.

[0117] 3. Add RNA template

[0118] Add 1 µL of each prepared RNA sample to the corresponding PCR reaction tube, and set 1 µL of nucleic acid-free pure water as a negative control.

[0119] 4. PCR amplification

[0120] Place each reaction tube into the reaction chamber of the PCR amplification instrument and set the reaction volume to 20 µL. Perform one-step RT-PCR amplification according to the following procedure: reverse transcription at 50℃ for 30 min; denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s as one cycle, for a total of 35 cycles; then hold at 72℃ for 10 min, and finally cool to 4℃.

[0121] 5. Perform capillary electrophoresis to detect the amplification products.

[0122] Prepare a loading mixture containing the molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × number of samples to be tested. Vortex to mix for 10-15 seconds. Use a pipette to dispense 9 μL of the formamide and internal standard mixture into each well. Add 1 μL of the amplification product to the formamide and internal standard mixture, and cover with the sealing tape. Perform the test according to the instructions in the user manual.

[0123] 6. Data Analysis

[0124] Import the relevant files into the GeneMapper software, input the raw CE values ​​from the detector, and analyze the data.

[0125] Example 8

[0126] Eight known standard pathogen RNA samples and one virus-free strawberry RNA sample were amplified and detected. The specific steps are as follows:

[0127] 1. Sample preparation

[0128] Take standard species RNA samples, determine the concentration and purity of RNA using NanoDrop2000 (Thermo), and dilute the RNA to 0.0001 ng / μL, then store at 4℃ or -20℃ for later use.

[0129] 2. Prepare the amplification system

[0130] Prepare the PCR amplification system according to the components in Table 3, vortex to mix, and then dispense according to the number of samples.

[0131] 3. Add RNA template

[0132] Add 1 µL of each prepared RNA sample to the corresponding PCR reaction tube, and set 1 µL of nucleic acid-free pure water as a negative control.

[0133] 4. PCR amplification

[0134] Place each reaction tube into the reaction chamber of the PCR amplification instrument and set the reaction volume to 20 µL. Perform one-step RT-PCR amplification according to the following procedure: reverse transcription at 50℃ for 30 min; denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s as one cycle, for a total of 35 cycles; then hold at 72℃ for 10 min, and finally cool to 4℃.

[0135] 5. Perform capillary electrophoresis to detect the amplification products.

[0136] Prepare a loading mixture containing the molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × number of samples to be tested. Vortex to mix for 10-15 seconds. Use a pipette to dispense 9 μL of the formamide and internal standard mixture into each well. Add 1 μL of the amplification product to the formamide and internal standard mixture, and cover with the sealing tape. Perform the test according to the instructions in the user manual.

[0137] 6. Data Analysis

[0138] Import the relevant files into the GeneMapper software, input the raw CE values ​​from the detector, and analyze the data.

[0139] The detection results of Examples 4-8 are statistically summarized in Table 4:

[0140] Table 4

[0141]

[0142] The test results showed that all species could be effectively detected when the template dosage was greater than or equal to 0.0001 ng.

[0143] Example 9

[0144] 1. Sample preparation

[0145] Fourteen strawberry leaf samples were collected, and RNA / DNA was extracted using the Kangwei Reagent Pathogenic Microorganism DNA / RNA Extraction Kit (Catalog No.: CW3030S) according to the instructions. The samples were then stored at 4℃ or -20℃ for later use.

[0146] 2. Prepare the amplification system

[0147] Prepare the PCR amplification system according to the components in Table 3, vortex to mix, and then dispense according to the number of samples.

[0148] 3. Add RNA template

[0149] Add 1 µL of each prepared RNA sample to the corresponding PCR reaction tube, and set 1 µL of nucleic acid-free pure water as a negative control.

[0150] 4. PCR amplification

[0151] Place each reaction tube into the reaction chamber of the PCR amplification instrument and set the reaction volume to 20 µL. Perform one-step RT-PCR amplification according to the following procedure: reverse transcription at 50℃ for 30 min; denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 30 s as one cycle, for a total of 35 cycles; then hold at 72℃ for 10 min, and finally cool to 4℃.

[0152] 5. Perform capillary electrophoresis to detect the amplification products.

[0153] Prepare a loading mixture containing the molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × number of samples to be tested. Vortex to mix for 10-15 seconds. Use a pipette to dispense 9 μL of the formamide and internal standard mixture into each well. Add 1 μL of the amplification product to the formamide and internal standard mixture, and cover with the sealing tape. Perform the test according to the instructions in the user manual.

[0154] 6. Data Analysis

[0155] Import the relevant files into the GeneMapper software, input the raw CE values ​​from the detector, and analyze the data.

[0156] The actual sample test results are shown in Table 5:

[0157] Table 5

[0158]

[0159] "-" indicates that nothing was detected; "+" indicates that nothing was detected.

[0160] All 14 samples (A1 to G2) successfully amplified the specific band of the strawberry β-Actin1 gene. This result proves that the nucleic acid extraction quality of all samples was up to standard, and there were no inhibitors in the reaction system. The entire detection process, from nucleic acid extraction to PCR amplification and electrophoresis detection, was effective and reliable. The successful detection of the internal control gene fundamentally eliminated the possibility of "false negative" results, ensuring the accuracy and reliability of subsequent pathogen detection results. Infections with strawberry powdery mildew (P. aphanius), strawberry gray mold (B. cinerea), and strawberry mottle virus (SMoV) were successfully detected in the 14 samples. The detection rate of strawberry powdery mildew was high, which is consistent with the high prevalence of this disease in the field. Sample C1 showed simultaneous detection of strawberry powdery mildew and strawberry gray mold, while samples G1 and G2 showed simultaneous detection of strawberry mottle virus and strawberry powdery mildew, demonstrating the method's ability to detect co-infections.

[0161] The advantages of this invention are as follows: Through the above technical solution, eight important pathogens and one internal reference gene can be detected simultaneously, significantly reducing operation time, labor costs, and reagent consumption. It is suitable for rapid screening of large batches of samples and diagnosis of complex infections. Introducing the strawberry Actin1 gene as an internal standard effectively monitors the quality of the entire process from RNA extraction to PCR amplification, avoiding false negatives caused by sample degradation or reaction inhibition, and ensuring the reliability of the test results. The universal primer-mediated amplification strategy effectively overcomes the bottleneck of multiplex PCR, significantly improving amplification efficiency and sensitivity, while specific primers ensure accurate identification. It has strong scalability. To add new detection targets, only specific primers with the same universal adapter need to be designed, without changing the entire detection system and labeling method, resulting in good platform compatibility.

[0162] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for simultaneous detection of multiple strawberry pathogens, characterized in that, The method for simultaneous detection of multiple strawberry pathogens includes the following steps: Take a standard RNA sample, determine its concentration and purity, and then dilute it to the required concentration. The standard RNA sample is an RNA sample obtained by synthesizing a plasmid of the candidate target region sequence for the corresponding pathogen detection and then transcribing it using an in vitro high-efficiency transcription kit. A multiplex amplification system was prepared using primer combinations, buffer solutions, enzyme mixtures, nuclease-free pure water, and RNA templates. RNA samples were added to the PCR reaction tubes, and nucleic acid-free pure water was used as a negative control. Perform PCR amplification; The amplification products were detected by capillary electrophoresis. Data analysis; Result determination; The primer set includes 18 specific primers and 2 universal primers: specific primers against strawberry mottle virus as shown in SEQ ID NO.1 and SEQ ID NO.2; specific primers against strawberry mild yellow edge virus as shown in SEQ ID NO.3 and SEQ ID NO.4; specific primers against strawberry shrunken virus as shown in SEQ ID NO.5 and SEQ ID NO.6; specific primers against strawberry veining virus as shown in SEQ ID NO.7 and SEQ ID NO.8; specific primers against strawberry powdery mildew fungus as shown in SEQ ID NO.9 and SEQ ID NO.10; specific primers against strawberry gray mold fungus as shown in SEQ ID NO.11 and SEQ ID NO.12; specific primers against strawberry anthracnose fungus as shown in SEQ ID NO.13 and SEQ ID NO.14; specific primers against strawberry Xanthomonas bacillus as shown in SEQ ID NO.15 and SEQ ID NO.16; and specific primers against the strawberry β-actin gene Actin1 as shown in SEQ ID NO.

1. The sequences are shown in NO.17 and SEQ ID NO.18; the universal primer sequences are shown in SEQ ID NO.19 and SEQ ID NO.

20.

2. The method for simultaneous detection of multiple strawberry pathogens according to claim 1, characterized in that, The 5' end of the universal primer is fluorescently labeled, and the fluorescent label is selected from FAM and HEX.

3. The method for simultaneous detection of multiple strawberry pathogens according to claim 1, characterized in that... The primer combination concentration was 100 μmol / L, with the following concentrations of each primer: 0.4-0.6 μL for the specific primer against strawberry mottle virus; 0.4-0.6 μL for the specific primer against strawberry mild yellow edge virus; 0.4-0.6 μL for the specific primer against strawberry shriveling virus; 0.5-1.0 μL for the specific primer against strawberry vein virus; and 0.5-1.0 μL for the specific primer against strawberry powdery mildew. The concentration range of the primers is 0.4-0.8 μL; the concentration range of the specific primers against strawberry gray mold is 0.4-0.8 μL; the concentration range of the specific primers against strawberry anthracnose is 0.5-1.0 μL; the concentration range of the specific primers against strawberry xanthomonas is 0.5-1.0 μL; the concentration range of the specific primers against the strawberry β-actin gene Actin1 is 0.4-0.6 μL; and the concentration range of the universal primers is 8-15 μL.

4. The method for simultaneous detection of multiple strawberry pathogens according to claim 1, characterized in that, The multiplex amplification system includes a PCR mixture and a DNA template.

5. The method for simultaneous detection of multiple strawberry pathogens according to claim 4, characterized in that, The PCR mixture is used in a 20 μl multiplex amplification system. The PCR mixture includes 10 μl of one-step RT-PCR Buffer, 2 μl of primer combination, 1.5 μl of one-step enzyme mixture, and 5.5 μl of nuclease-free pure water.

6. The method for simultaneous detection of multiple strawberry pathogens according to claim 4, characterized in that, The DNA template is used in a 20 μl multiplex amplification system, and the amount of DNA template used is 1 μl.

7. The method for simultaneous detection of multiple strawberry pathogens according to claim 1, characterized in that, The amplification program of the multiplex amplification system is as follows: reverse transcription at 50℃ for 30 min; denaturation at 95℃ for 3 min; denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, and extension at 72℃ for 30 s as one cycle, for a total of 35 cycles; then hold at 72℃ for 10 min, and then cool down to 4℃.

8. The method for simultaneous detection of multiple strawberry pathogens according to claim 1, characterized in that, The steps for capillary electrophoresis detection of the amplified products are as follows: prepare a loading mixture containing molecular weight internal standard and formamide, vortex and mix for 10-15 seconds; use a pipette to dispense 9 μL of the formamide and internal standard mixture into each detection well; add 1 µL of the amplified product to the formamide and internal standard mixture, and cover with the sealing glue cap.

9. A kit for simultaneous detection of multiple strawberry pathogens, characterized in that, The kit for simultaneous detection of multiple strawberry pathogens comprises the multiplex amplification system as described in any one of claims 1-7.

10. The application of the multiplex amplification system according to any one of claims 1-7, or the kit according to claim 9, in the simultaneous detection of multiple strawberry pathogens.

Images