Detection method and detection kit for wheat leaf blight bacteria based on enzyme-mediated double-index amplification technology

Abstract

The invention discloses a wheat leaf blight bacterium detection method based on an enzyme-mediated double-index amplification technology and a detection kit thereof. The invention belongs to the technical field of biology, and particularly relates to a wheat leaf blight bacterium detection method based on an enzyme-mediated double index amplification technology and a detection kit thereof. The composition for researching and detecting wheat leaf blight bacteria comprises a primer F5, a primer R3 and an RNA probe, the nucleotide sequence of the primer F5 is shown as SEQ ID No: 5, the nucleotide sequence of the primer R3 is shown as SEQ ID No: 9, and the nucleotide sequence of the RNA probe is shown as SEQ ID No: 16. According to the present invention, based on the EmDEA technology, the specific screening design is performed on the DNA primer and the RNA probe, such that the rapid detection method of the wheat leaf blight bacteria is established, and the method has good specificity, can detect the minimum DNA content of the wheat leaf blight bacteria of 100 fg, and has application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a detection method for wheat leaf blight pathogen based on enzyme-mediated double exponential amplification technology and a detection kit thereof. Background Art

[0002] Currently, detection of wheat leaf blight pathogens relies primarily on morphological observation and molecular biology techniques. Traditional morphological methods identify the pathogen by microscopically observing spore morphology, compartmentalization, and cultural characteristics. However, these methods have significant limitations. First, the spore morphology of similar species within the genus Alternaria (such as Alternaria alternata and Alternaria tenuissima) is highly similar, making differentiation difficult. Second, in the early stages of the disease or when seeds carry the pathogen, symptoms are subtle and pure culture is difficult, leading to a high risk of missed detection. Molecular detection techniques such as DNA barcoding, conventional PCR, and real-time fluorescence PCR have improved specificity but still face challenges. For example, conventional PCR requires sophisticated instrumentation and complex procedures and is susceptible to interference from sample inhibitors. DNA barcoding relies on database integrity and has limited resolution for similar species with small genetic differences. Existing industry standards (such as SN / T 4733-2016) carry the risk of false positives due to insufficient conservation of target genes (ITS).

[0003] Real-time fluorescence quantitative RPA (RPA) builds upon the RPA system by introducing a specific fluorescent probe. This technology utilizes a dual-labeled nucleic acid probe with a fluorescent group (Tamra or FAM) at the 5' end and a quencher at the 3' end to achieve quantitative RPA detection. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quencher group. When the target gene is present in the reaction, the probe binds to it, and the exonuclease in the system cleaves the probe, releasing the fluorescent group. The intensity of the generated fluorescent signal is proportional to the number of target genes, and the fluorescence intensity increases with RPA amplification. This method does not require precise temperature control and can be performed at relatively low temperatures (37°C to 42°C). The reaction results are displayed through fluorescence detection, significantly shortening the detection time to within 30 minutes. Enzyme-mediated duplex exponential amplification (EmDEA) is a recently developed real-time fluorescence quantitative RPA technique that performs specific isothermal amplification of the target nucleic acid sequence and directional signal amplification, which is then amplified through fluorescence signal reaction. The reagents used in isothermal fluorescence RPA reactions are mostly freeze-dried and pre-mixed, making them convenient for rapid testing in the field. Samples can be quickly processed based on test results, significantly reducing testing time and costs and enabling rapid on-site testing. This study established a rapid detection method for wheat leaf blight pathogens based on EmDEA technology, hoping to provide a reference for on-site testing at ports of entry and field disease monitoring. Summary of the Invention

[0004] The main problem to be solved by the present invention is how to quickly and accurately detect wheat leaf blight pathogens.

[0005] In order to solve the above problems, the present invention provides a composition for detecting wheat leaf blight pathogen.

[0006] The composition for detecting wheat leaf blight pathogen provided by the present invention includes primer F5, primer R3 and RNA probe, the nucleotide sequence of primer F5 is shown in SEQ ID No: 5, the nucleotide sequence of primer R3 is shown in SEQ ID No: 9, and the nucleotide sequence of RNA probe is shown in SEQ ID No: 16.

[0007] In the above composition, the 5' end of the RNA probe is labeled with a reporter group, and the 3' end is labeled with a quencher group.

[0008] Furthermore, the fluorescent group can be selected from at least one of FAM, VIC, HEX, TRT, CY3, CY5, ROX, JOE, FITC, TET, NED, TAMRA, LC RED640, LC RED705, Quasar705 or Texas Red.

[0009] Furthermore, the quenching group can be selected from at least one of TAMRA, BHQ1, BHQ2, BHQ3, MGB, and Dabcy1.

[0010] In the above composition, the reporter group is FAM, and the quencher group is BHQ1.

[0011] The present invention also provides a kit for detecting wheat leaf blight pathogens, wherein the kit comprises the composition described above.

[0012] Furthermore, the kit also includes detection reagents required for enzyme-mediated dual exponential amplification nucleic acid detection technology.

[0013] Furthermore, the kit also contains a basic dry powder and an activator. The basic dry powder includes the lyophilized product of the enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase, and signal amplifier enzyme, and a lyophilization protectant. The activator can be NTP (nucleoside triphosphate) and a buffer.

[0014] The kit may further include a nucleic acid extraction reagent, which is used to extract nucleic acids from a biological sample (sample to be tested).

[0015] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0016] The present invention also provides a method for detecting wheat leaf blight pathogen, which comprises using the composition or the kit described above to perform an amplification reaction on a sample to be tested, and determining whether the sample to be tested contains wheat leaf blight pathogen or is wheat leaf blight pathogen based on the amplification result, wherein the amplification reaction is performed based on enzyme-mediated double exponential amplification nucleic acid detection technology.

[0017] Furthermore, the method comprises the following steps:

[0018] A1) extracting DNA from the sample to be tested;

[0019] A2) using the test sample DNA as a template, performing enzyme-mediated dual exponential amplification nucleic acid detection using the composition described above to generate an amplification curve;

[0020] A3) Determine the test result based on the Ct value.

[0021] Furthermore, the smaller the Ct value and the larger the peak value, the better the detection effect or the higher the DNA concentration.

[0022] The result determination method is as follows:

[0023] 1) If the Ct value is less than or equal to 30, the sample to be tested contains wheat leaf blight pathogen or is wheat leaf blight pathogen;

[0024] 2) If the Ct value is greater than 30, the sample to be tested does not contain wheat leaf blight pathogen or is not wheat leaf blight pathogen.

[0025] In a specific embodiment, the reaction system for the enzyme-mediated dual exponential amplification of nucleic acid is: 1 μL (10 μmol / L) of upstream primer F5, 1 μL (10 μmol / L) of downstream primer R3, 1 μL (1 μmol / L) of RNA probe N4, 7 μL of DNA of the sample to be tested, 10 μL of activation solution, and one tube of basic dry powder.

[0026] The basic dry powder may include enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase, and freeze-dried products of signal amplification enzyme, and freeze-dried protective agents.

[0027] The activator may be NTP (nucleoside triphosphate) and a buffer.

[0028] The reaction conditions of the enzyme-mediated dual exponential amplification of nucleic acid are: constant temperature at 42° C., collecting a signal once per minute, and collecting a total of 30 signals.

[0029] The present invention also provides the use of the above-mentioned composition in detecting wheat leaf blight pathogens.

[0030] The present invention also provides the use of the above-mentioned composition in preparing a product for detecting wheat leaf blight pathogens.

[0031] The present invention also provides the use of the above-mentioned kit in detecting wheat leaf blight pathogens.

[0032] Based on EmDEA technology, this study established a rapid detection method for wheat leaf blight pathogen by specifically screening and designing DNA primers and RNA probes. This method has good specificity and can detect a minimum plasmid DNA content of 100 fg of wheat leaf blight pathogen, which is practical. The detection time of the EmDEA method is greatly shortened to less than 0.5 h, which is time-saving and can meet the daily detection and monitoring work of ports. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 These are the results of RNA probe screening for wheat leaf blight pathogen.

[0034] Figure 2 These are the results of screening for downstream primers of wheat leaf blight pathogen.

[0035] Figure 3 These are the results of upstream primer screening for wheat leaf blight pathogen.

[0036] Figure 4 This is the specific detection result of wheat leaf blight pathogen.

[0037] Figure 5 These are the sensitivity test results for wheat leaf blight pathogen.

[0038] Figure 6 To simulate the actual sample detection test. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0040] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0041] Unless otherwise specified, the quantitative tests in the following examples were performed three times, and the results were averaged.

[0042] The wheat leaf blight pathogen CBS121461 in the following examples has been described in: Xu Ran, Mou Guiping, Gong Jingru, et al. Establishment of a real-time fluorescence PCR detection method for wheat leaf blight pathogen [J]. Plant Quarantine, 2025, 39(01): 26-30. The public may obtain the biological material from the applicant for use only in repeating the experiments of the present invention and may not be used for other purposes.

[0043] The ATCC 36205 in the following examples is from Zhang Yan, Zhang Xianglin, Wang Chong, et al. Detection of wheat leaf blight pathogen using PCR [J]. Journal of Biosafety, 2015, 24(01): 72-77. The public may obtain this biological material from the applicant for use only in repeating the experiments of the present invention and may not be used for other purposes.

[0044] The CGMCC 3.9868 in the following examples was purchased from the China General Microbiological Culture Collection Center. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0045] The strains listed in Table 1 of the following examples are described by Lü Youhong, Zhu Xiaoqiong, Liu Fang, and Duan Weijun. [A rapid detection method for wheat leaf blight pathogens based on TaqMan-MGB probes is available in the Journal of Biosafety.] The public may obtain these biological materials from the applicant for use solely in replicating the experiments described herein and not for any other purpose. Detailed information on the experimental strains is shown in Table 1.

[0046] Table 1. Experimental strains used in the present invention

[0047]

[0048]

[0049] The culture medium used in the following examples was potato dextrose agar (PDA). The nucleic acid extraction kit was the MAGPlant DNA Extraction Kit (Plant) from Zhuhai Lifei Technology Service Co., Ltd., the ultraRADAR fluorescence constant temperature amplification detection kit (RR032) was purchased from Suzhou Jingrui Biotechnology Co., Ltd., and the high-throughput tissue disruptor (Retsch, MM400) was purchased from Retsch, Germany.

[0050] The real-time fluorescence PCR instrument was the qTOWER high-speed fluorescence quantitative PCR instrument from Jena, the ultramicro-spectrophotometer was the NanoDrop 2000C from ThermoFisher, and the biochemical incubator was the Friocell 222 from MMM.

[0051] Example 1: Establishment of a detection method for wheat leaf blight pathogen based on enzyme-mediated dual exponential amplification technology

[0052] 1. Design and synthesis of primers and probes

[0053] DNA primers and RNA probe sequences were designed based on the wheat leaf blight pathogen (Alternaria triticina) EF sequence determined in our laboratory and published in GenBank (Genbank number: MZ073960.1, updated on November 7, 2021). The primer and probe sequence information is shown in Table 2. DNA primers were synthesized by BGI, and RNA probes were synthesized by Suzhou Jingrui Biotechnology Co., Ltd. The RNA probe reporter group was FAM, and the quencher group was BHQ1.

[0054] Table 2. Primer and probe sequence information

[0055]

[0056]

[0057] 2. DNA Extraction

[0058] Culture the wheat leaf blight pathogen ATCC 36205 on PDA medium at 25°C for 10 days. Scrape the mycelium from the plate and extract DNA using a nucleic acid extraction kit. Concentration of the extracted DNA was determined using a NanoDrop assay and stored at -80°C until further use.

[0059] 3. RNA probe screening

[0060] The enzyme-mediated dual exponential amplification (EmDEA) reaction system (20 μL) used in this study was as follows: 1 μL of DNA upstream primer (10 μmol·L -1 ), DNA downstream primer 1 μL (10 μmol·L -1 ), RNA probe 1 μL (1 μmol·μL -1 ), 7 μL DNA, 10 μL activation solution, and one tube of dry powder. The dry powder and activation solution were from Suzhou Jingrui Co., Ltd.'s basic fluorescence constant-temperature amplification detection kit (RR032). The reaction temperature was constant at 42°C, and signals were collected once per minute for a total of 30 times.

[0061] RNA probe screening was performed using ATCC 36205 as a DNA template. First, the middle primers F2, R2, F2, and R3 were cross-combined to obtain four primer pairs: F2R2, F2R3, F3R2, and F3R3. These four primer pairs were cross-combined with the six RNA probes N1, N2, N3, N4, N5, and N6, forming a total of 24 primer-probe combinations. Each of these 24 combinations was tested on the instrument. After the amplification test results were obtained, the six probes from the same primer set were first compared, and the RNA probe with the lowest Ct value and the highest endpoint fluorescence value was selected. Three replicates and one negative control were set for each primer-probe combination. The negative control used Nuclease-free Water as the template, and all other conditions remained unchanged. The selected combination required the negative control to have no fluorescent signal.

[0062] Depend on Figure 1 All probes in the four primer combinations produced amplification signals, but N4 exhibited an earlier peak than the other probes, so N4 was selected as the target probe. The F2R2 group had a lower Ct value of 8.89 and a higher fluorescence value. The corresponding negative control showed no signal, so N4F2R2 was selected for subsequent experiments.

[0063] 4. Initial round of DNA downstream primer screening

[0064] Downstream primer screening was performed using ATCC 36205 as a DNA template, immobilized RNA probe N4, and DNA upstream primer F2. Six DNA downstream primers (R1, R2, R3, R4, R5, and R6) were tested in combination with the immobilized RNA probe and DNA upstream primer. The DNA downstream primer with the lowest Ct value and the highest endpoint fluorescence value was selected. Three replicates and a negative control were set for each primer-probe combination. The negative control used nuclease-free water as the template. All other conditions remained unchanged. The selected combination required the negative control to show no fluorescence signal.

[0065] Depend on Figure 2 It can be seen that the Ct value of primer R3 is smaller, which is 7.82, and the fluorescence value is the highest. In addition, the corresponding negative control has no signal. Therefore, primer R3 is selected as the downstream primer.

[0066] 5. Initial round of DNA upstream primer screening

[0067] Upstream primer screening was performed using ATCC 36205 as a DNA template, immobilized RNA probe N4, and DNA downstream primer R3. Six DNA upstream primers (F1, F2, F3, F4, F5, and F6) were tested in combination with the immobilized RNA probe and DNA downstream primer. The DNA downstream primer with the lowest Ct value and the highest endpoint fluorescence value was selected. Three replicates and a negative control were set for each primer-probe combination. The negative control used water as the template, while other conditions remained unchanged. The selected combination required the negative control to show no fluorescence.

[0068] Depend on Figure 3 As can be seen, F5 has a smaller Ct value of 7.95, the highest fluorescence value, and no signal in the corresponding negative control. Therefore, primer F5 is the upstream primer screened for this experiment. Based on the above three screening experiments, N4F5R3 was ultimately selected as the primer-probe combination for this experiment.

[0069] In summary, the reaction system of enzyme-mediated dual exponential amplification detection technology (EmDEA) for wheat leaf blight pathogen is as follows: DNA upstream primer F5 1 μL (10 μmol·L -1 ), DNA downstream primer R3 1 μL (10 μmol·L -1 ), RNA probe N4 1 μL (1 μmol·μL -1 ), 7 μL of test sample DNA, 10 μL of activation solution, and one tube of dry powder.

[0070] The reaction conditions for enzyme-mediated double exponential amplification to detect wheat leaf blight pathogens are: constant temperature at 42°C, collecting signals once per minute, and collecting signals 30 times in total.

[0071] After the reaction, the test results are judged according to the Ct value. The judgment criteria are as follows:

[0072] 1) If the Ct value is less than or equal to 30, the sample to be tested contains wheat leaf blight pathogen or is wheat leaf blight pathogen;

[0073] 2) If the Ct value is greater than 30, the sample to be tested does not contain wheat leaf blight pathogen or is not wheat leaf blight pathogen.

[0074] Example 2: Study on the specificity and sensitivity of the enzyme-mediated dual exponential amplification method for detecting wheat leaf blight pathogens

[0075] 1. Specificity experiment

[0076] The N4F5R3 primer-probe combination was used as the experimental primer-probe combination. The three strains of wheat leaf blight pathogen and 23 similar strains of Alternaria species listed in Table 1 were used as test samples. DNA from the test samples was extracted and used as templates. The enzyme-mediated dual exponential amplification method optimized in Example 1 was used to perform a specific experiment for wheat leaf blight pathogen detection.

[0077] The results are as follows Figure 4 As shown in Table 1, three strains of wheat leaf blight pathogen CBS121461, CGMCC 3.9868, and ATCC36205 were all effectively detected, while 23 similar species of Alternaria could not be detected, indicating that the N4F5R3 primer-probe combination has good specificity for enzyme-mediated dual exponential amplification detection.

[0078] 2. Sensitivity test

[0079] A sensitivity test was conducted using ATCC 36205 as the DNA template and N4F5R3 as the primer-probe combination required for the experiment, using the enzyme-mediated dual exponential amplification method optimized in Example 1. The ATCC 36205 plasmid DNA concentration was determined to be 100 ng·μL⁻¹. The DNA was diluted 10-fold with water to form six concentration gradients, with three replicates for each concentration gradient.

[0080] The results show that ( Figure 5 ), the lowest dilution gradient that the primer probe can detect is 10 -6 , that is, the sensitivity of this combination is 100 fg of total DNA content in 20 μL reaction system.

[0081] Example 3: Application of enzyme-mediated dual exponential amplification in detecting wheat leaf blight pathogens

[0082] 1. Treatment of simulated disease samples

[0083] 30g of wheat from Australia was weighed and ground into powder using a homogenizer. 1g of wheat flour was then added to 100mg, 200mg, and 300mg of PDA plates, respectively, to collect mycelium of the wheat leaf blight pathogen. The wheat flour and mycelium mixture was then placed in a 50mL grinding jar, added with a sterile steel ball, and frozen overnight at -80°C. The mixture was then thoroughly ground in a high-throughput tissue disruptor (Retsch, MM400). DNA was extracted using an automated nucleic acid extraction instrument according to the instructions for the MAG Plant DNA Extraction Kit. The extracted DNA was then tested for wheat leaf blight using an optimized real-time fluorescence PCR method.

[0084] 2. Use enzyme-mediated double exponential amplification method to detect samples

[0085] The DNA of the sample to be tested was used to detect wheat leaf blight pathogen using the enzyme-mediated double exponential amplification method optimized in Example 1.

[0086] The results are as follows: DNA was extracted from the samples and tested. Using ATCC 36205 as the positive control, all three simulated DNA samples tested positive, while the negative control, Nuclease-free water, tested negative. This demonstrates that the N4F5R3 combination performs well and can be used in real-world sample testing.

[0087] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A composition for detecting wheat leaf blight pathogen, characterized in that: The composition includes primer F5, primer R3 and an RNA probe, the nucleotide sequence of the primer F5 is shown as SEQ ID No: 5, the nucleotide sequence of the primer R3 is shown as SEQ ID No: 9, and the nucleotide sequence of the RNA probe is shown as SEQ ID No:

16.

2. The composition according to claim 1, characterized in that The 5' end of the RNA probe is labeled with a reporter group, and the 3' end is labeled with a quencher group.

3. The composition according to claim 2, characterized in that The reporter group is FAM, and the quencher group is BHQ1.

4. A kit for detecting wheat leaf blight pathogen, characterized in that: The kit comprises the composition according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also contains basic dry powder and an activator; the basic dry powder includes enzymes required for detection: DNA recombinase, shrimp alkaline phosphatase, T7 RNA polymerase, reverse transcriptase and freeze-dried products of signal amplifier enzyme and freeze-dried protective agent; the activator is nucleoside triphosphate and buffer.

6. A method for detecting wheat leaf blight pathogen, characterized in that: The method comprises performing an amplification reaction on a sample to be tested using the composition according to any one of claims 1 to 3 or the kit according to claim 4 or 5, and determining whether the sample to be tested contains wheat leaf blight pathogen or is wheat leaf blight pathogen based on the amplification result, wherein the amplification reaction is performed based on enzyme-mediated dual exponential amplification nucleic acid detection technology.

7. The method according to claim 6, characterized in that The method comprises the following steps: A1) extracting DNA from the sample to be tested; A2) using the test sample DNA as a template, performing enzyme-mediated dual exponential amplification nucleic acid detection using the composition of any one of claims 1 to 3 to form an amplification curve; A3) Determine the test results based on the Ct value.

8. Use of the composition according to any one of claims 1 to 3 in detecting wheat leaf blight pathogens.

9. Use of the composition according to any one of claims 1 to 3 in the preparation of a product for detecting wheat leaf blight pathogens.

10. Use of the kit according to claim 4 or 5 in detecting wheat leaf blight pathogens.

Images