RPA detection primer for colletotrichum gloeosporioides of green vegetables and application of RPA detection primer

Abstract

The invention provides RPA (recombinase polymerase amplification) detection primers for colletotrichum gloeosporioides of green vegetables and application of the RPA detection primers and belongs to the technical field of agriculture. In order to solve the problems of long time consumption (2-3 hours), low sensitivity (5 ng / mu L) and dependence on precise equipment in the existing PCR detection, the invention provides RPA primers 56F1 (5 '-GAAAAGCGGCAT-3') and 56R1 (5 '-CGTTGGAAAATACA-3') of targeted pathogenic bacteria specific genes NC030956, as well as an RPA primer 62F1 / F1 and an RPA primer 62F2 / F2, and establishes a 39 DEG C constant-temperature 20-minute amplification system. The method realizes three major breakthroughs: 1, the detection sensitivity reaches 0.05 ng / mu L and is improved by 100 times compared with PCR (Polymerase Chain Reaction); 2, seven related pathogens such as colletotrichum nicotianae are strictly distinguished, zero cross reaction is realized, and the sample accuracy rate is 100%; 3, the whole process only needs 1.5 hours, the DNA crude extraction time of diseased leaves is 30 minutes, the RPA amplification time is 20 minutes, the electrophoresis detection time is 35 minutes, the cost is low, and a thermal cycler is not needed. The invention provides an on-site solution with the characteristics of rapidness, accuracy and saving for prevention and control of the cruciferous anthracnose, and solves the problem that the traditional technology cannot give consideration to both speed and sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and particularly relates to an RPA detection primer for anthracnose of green vegetables and an application thereof. Background Art

[0002] Anthracnose of green vegetables is a major vegetable disease caused by the fungus Colletotrichum higginsianum. It causes dark brown lesions on leaves and stems, leading to severe yield losses. Currently, methods for detecting anthracnose of green vegetables primarily include traditional pathogen isolation and culture and conventional PCR. Traditional methods are time-consuming and susceptible to interference from environmental bacteria. While conventional PCR offers a certain degree of specificity, its sensitivity is limited, making it difficult to meet the demand for rapid early disease diagnosis. Furthermore, many fungi within the genus Colletotrichum share similar morphology and high genome sequence similarity, such as C. nicotianae, C. truncatum, C. gloeosporioide, C. siamense, C. fruticola, and C. orbiculare. Their ITS sequences also share high similarity with closely related species, making it difficult for traditional detection and ITS sequencing methods to accurately distinguish them, leading to delayed disease prevention and control measures. Therefore, developing a rapid, specific and sensitive method for detecting anthracnose of green vegetables is of great significance for early warning and scientific prevention and control of the disease. Summary of the Invention

[0003] The present invention provides an RPA detection primer for Colletotrichum oleraceus and application thereof, aiming to solve the technical problem of low detection sensitivity in the prior art.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0005] In a first aspect, the present invention provides an RPA detection primer for anthracnose of green vegetables, wherein the primer comprises at least one of the following three pairs of primers:

[0006] Primer 62F1: 5′-GCTCCAGATCAAGAGCCGTTGGCTATCGTAT-3′;

[0007] Primer 62R1: 5′-CCGTATTGGTGAGTATCCAGCCCATTGAGGG-3′;

[0008] Primer 62F2: 5′-CTGTTGTTCCTGCGATAGACTAACCTACTTGC-3′;

[0009] Primer 62R2: 5′-TGTTTTCCTTCAACCCATAACCTAGCTTGG-3′;

[0010] Primer 56F1: 5′-GAAAGCGGCAATCTTCAAGACATCACTGTT-3′;

[0011] Primer 56R1: 5′-CGTTGGGAATAGCCAGGGAGACAATACA-3′.

[0012] Preferably, the RPA detection primers include:

[0013] Primer 56F1: 5′-GAAAGCGGCAATCTTCAAGACATCACTGTT-3′;

[0014] Primer 56R1: 5′-CGTTGGGAATAGCCAGGGAGACAATACA-3′.

[0015] Based on the above scheme, experiments have shown that 56F1 / 56R1 is the best choice as detection primer based on comprehensive amplification efficiency and product specificity.

[0016] In a preferred embodiment, the amplification product of the primer pair 56F1 and the primer pair 56R1 has a size of 181 bp.

[0017] In a second aspect, the present invention provides use of the RPA detection primer for Colletotrichum oleraceus described in the first aspect in detecting Colletotrichum oleraceus.

[0018] In a third aspect, the present invention provides a kit for detecting anthracnose pathogens of green vegetables, comprising the RPA detection primers described in the first aspect.

[0019] The preferred solution includes RPA amplification reagents.

[0020] In a preferred embodiment, the RPA amplification reagent includes 2×RPA reaction buffer and 280 mM magnesium acetate solution.

[0021] Based on the above scheme, since the recombinase UvsX is most suitable for a weakly acidic environment, the buffer maintains the pH of the reaction system at 6.0-6.5, maintains the enzyme activity environment, and prevents nuclease inactivation. In addition, the 2×RPA reaction buffer contains KCl / NaCl to provide ion concentration, promotes the binding of the recombinase-primer complex to the DNA template, and also has the function of stabilizing the reaction components and providing amplification raw materials. The magnesium ions (Mg 2+ ) is an essential cofactor of DNA polymerase, which can catalyze the formation of phosphodiester bonds by binding to the active center of the enzyme, and Mg 2+Neutralizes the negative charge of the phosphate group on the DNA backbone, promotes stable primer-template hybridization, and thus maintains the stability of the nucleic acid structure.

[0022] In a fourth aspect, the present invention provides a method for detecting anthracnose pathogens of green vegetables, comprising the following steps:

[0023] S1. Extract DNA from the sample to be tested;

[0024] S2. Using DNA as a template, perform RPA amplification using at least one pair of primers selected from 62F1 / 62R1, 62F2 / 62R2, or 56F1 / 56R1;

[0025] S3. Detect the amplified product. When using the primer pair 62F1 / 62R1, if a specific fragment of 210 bp is present, the sample is determined to carry the green vegetable anthracnose pathogen; when using the primer pair 62F2 / 62R2, if a specific fragment of 228 bp is present, the sample is determined to carry the green vegetable anthracnose pathogen; when using the primer pair 56F1 / 56R1, if a specific fragment of 181 bp is present, the sample is determined to carry the green vegetable anthracnose pathogen.

[0026] Preferably, the RPA reaction system includes: 29.5 μL of 2×RPA reaction buffer, RPA detection primers, 1.0 μL of template DNA, sterile deionized water to 47.5 μL, and 2.5 μL of 280 mM magnesium acetate.

[0027] Based on the above scheme, 2×RPA buffer provides an enzyme protection environment, dNTP raw materials and anti-inhibitor components; the primer pair specifically targets the pathogen gene and defines the amplification region, the template DNA provides the target sequence for amplification, and the final volume is adjusted to 47.5μL with water to ensure stable osmotic pressure of the reaction solution; magnesium acetate is used as a key activator, with a final concentration of 14mM to accurately activate DNA polymerase and initiate chain extension reactions. The entire system achieves specific amplification at room temperature (39°C) and rapidly (20min).

[0028] In a preferred embodiment, the RPA amplification is carried out at a constant temperature of 39° C. for 20 minutes, and the amplified product is detected by 1.5% agarose gel electrophoresis.

[0029] Based on the above scheme, the results are specifically interpreted by fragment size (181bp), excluding the interference of primer dimers (<100bp), with an accuracy rate of >95%, realizing portable, rapid, and visual end-point detection, which is suitable for rapid field detection.

[0030] The beneficial effects of the present invention are:

[0031] This paper designs RPA primers for the first time based on the C. higginsianum genes NC_030962 and NC_030956. Through systematic screening of three pairs of candidate primers (62F1 / R1, 62F2 / R2, and 56F1 / R1), combined with a "buffer-magnesium ion" synergistic system, it achieves fast speed, high sensitivity, and strong specificity, providing a rapid detection solution that can be deployed in the field for the prevention and control of crucifer anthracnose.

[0032] 1. Double improvement in detection efficiency and sensitivity: Existing PCR technology takes 2-3 hours and has a detection limit of only 5ng / μL, while the present invention can complete amplification in just 20 minutes and achieve single-copy detection at 0.05ng / μL (sensitivity is 100 times higher than PCR), solving the problem of traditional methods missing trace pathogens in the latent period.

[0033] 2. Strict species specificity: Zero cross-reaction against seven congener / closely related pathogens such as tobacco anthrax (C.nicotianae) and flat-headed anthrax (C.truncatum) and common plant fungi (such as Alternaria solani), only accurately amplifying the 181bp fragment of the target pathogen, and the electrophoresis interpretation accuracy rate is 100%.

[0034] 3. Strong field adaptability: In the detection of simulated disease samples, the detection accuracy of 15 artificially inoculated diseased leaves and isolated strains reached 100%, and there were zero false positives in healthy plant samples, proving that it can tolerate interference from complex plant matrices. It is low-cost and does not require professional instruments. The operation can be started at body temperature, and it only takes 1.5 hours from sampling to result output. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 This is a diagram showing the amplification effects of three pairs of RPA primers on the DNA of Colletotrichum pekinensis.

[0037] Figure 2 This is the specificity detection diagram of primers 56F1 / 56R1.

[0038] Figure 3 This is the sensitivity detection chart of primers 56F1 / 56R1.

[0039] Figure 4 This is the RPA detection image of the actual disease sample. DETAILED DESCRIPTION

[0040] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0041] Example 1:

[0042] This embodiment provides an RPA detection primer for Colletotrichum oleraceus, wherein the primer comprises at least one of the following three pairs of primers:

[0043] Primer 62F1: 5′-GCTCCAGATCAAGAGCCGTTGGCTATCGTAT-3′;

[0044] Primer 62R1: 5′-CCGTATTGGTGAGTATCCAGCCCATTGAGGG-3′;

[0045] Primer 62F2: 5′-CTGTTGTTCCTGCGATAGACTAACCTACTTGC-3′;

[0046] Primer 62R2: 5′-TGTTTTCCTTCAACCCATAACCTAGCTTGG-3′;

[0047] Primer 56F1: 5′-GAAAGCGGCAATCTTCAAGACATCACTGTT-3′;

[0048] Primer 56R1: 5′-CGTTGGGAATAGCCAGGGAGACAATACA-3′.

[0049] Among them, a better solution: the RPA detection primers include:

[0050] Primer 56F1: 5′-GAAAGCGGCAATCTTCAAGACATCACTGTT-3′;

[0051] Primer 56R1: 5′-CGTTGGGAATAGCCAGGGAGACAATACA-3′.

[0052] The amplification product of the primers 56F1 and 56R1 is 181 bp in size.

[0053] Example 2:

[0054] This embodiment provides an application of the RPA detection primer for Colletotrichum oleraceus described in Example 1 in detecting Colletotrichum oleraceus.

[0055] Example 3:

[0056] This embodiment provides a kit for detecting anthracnose pathogens of green vegetables, comprising the RPA detection primers described in Example 1.

[0057] Wherein, the kit includes RPA amplification reagent.

[0058] The RPA amplification reagent includes 2×RPA reaction buffer and 280 mM magnesium acetate solution.

[0059] Example 4:

[0060] This embodiment provides a method for detecting anthrax of green vegetables, comprising the following steps:

[0061] S1. Extract DNA from the sample to be tested;

[0062] S2. Using DNA as a template, perform RPA amplification using at least one pair of primers selected from 62F1 / 62R1, 62F2 / 62R2, or 56F1 / 56R1;

[0063] S3. Detect the amplified product. When using the primer pair 62F1 / 62R1, if a specific fragment of 210 bp is present, the sample is determined to carry the green vegetable anthracnose pathogen; when using the primer pair 62F2 / 62R2, if a specific fragment of 228 bp is present, the sample is determined to carry the green vegetable anthracnose pathogen; when using the primer pair 56F1 / 56R1, if a specific fragment of 181 bp is present, the sample is determined to carry the green vegetable anthracnose pathogen.

[0064] The RPA reaction system includes: 29.5 μL of 2×RPA reaction buffer, the RPA detection primer pair described in Example 1, 1.0 μL of template DNA, sterile deionized water to 47.5 μL, and 2.5 μL of 280 mM magnesium acetate.

[0065] Specifically, the RPA detection primer pair includes an upstream primer and a downstream primer, wherein, in the RPA reaction system, 2.4 μL of 10 μM upstream primer and 10 μM downstream primer are each taken, and the RPA detection primer pair can be 62F1 / R1, 62F2 / R2 or 56F1 / R1. In a preferred solution, the RPA detection primer pair is 56F1 / R1.

[0066] The RPA amplification was carried out at a constant temperature of 39° C. for 20 min, and the amplified product was detected by 1.5% agarose gel electrophoresis.

[0067] The present invention will be further explained below in conjunction with experiments:

[0068] 1. Screening of RPA primers based on specific genes of Colletotrichum oleraceus

[0069] A1. Gene Sequence Acquisition: The genome sequence of C. higginsianum (IMI 349063) was downloaded from NCBI. Specific genes were screened by comparing it with seven closely related anthrax species (C. nicotianae, C. truncatum, etc.). After verification with the GenBank NR / nt database, NC_030962 and NC_030956 were selected.

[0070] A2. Primer design: Use Primer Premier 5.0 to design three pairs of RPA primers in the conserved region of the gene:

[0071] 62F1 / R1 (targeting NC_030962), 62F2 / R2 (targeting NC_030962), and 56F1 / R1 (targeting NC_030956), the sequences are as follows:

[0072] 62F1: 5′-GCTCCAGATCAAGAGCCGTTGGCTATCGTAT-3′;

[0073] 62R1: 5′-CCGTATTGGTGAGTATCCAGCCCATTGAGGG-3′;

[0074] 62F2: 5′-CTGTTGTTCCTGCGATAGACTAACCTACTTGC-3′;

[0075] 62R2: 5′-TGTTTTCCTTCAACCCATAACCTAGCTTGG-3′;

[0076] 56F1: 5′-GAAAGCGGCAATCTTCAAGACATCACTGTT-3′;

[0077] 56R1: 5′-CGTTGGGAATAGCCAGGGAGACAATACA-3′.

[0078] A3. DNA extraction: The CTAB method was used to extract genomic DNA of pure cultured C. higginsianum strains (concentration > 50 ng / μL).

[0079] A4. RPA reaction system: 29.5 μL of 2×RPA buffer, 2.4 μL of each 10 μM primer, 1.0 μL of template DNA, and ddH2O to 47.5 μL. Mix well, add 2.5 μL of 280 mM magnesium acetate (final concentration 14 mM), and incubate at 39°C for 20 min.

[0080] A5. Product detection: Take 2 μL of amplified product, run electrophoresis on 1.5% agarose gel (120 V, 20 min), and stain with EB for imaging.

[0081] 1.1 Test results

[0082] Primer screening (using C. higginsianum DNA as template)

[0083] Table 1 RPA primer screening results

[0084] Primer pairs Product size Amplification efficiency Band clarity Screening conclusion 62F1 / R1 210bp +++ ++ Available 62F2 / R2 228bp ++ + Available 56F1 / R1 181bp ++++ +++ Better

[0085] See Figure 1 , M is DL2000DNAMarker, 1, 3, and 5 use primers 62F1 / 62R1, 62F2 / 62R2, and 56F1 / 56R1 to amplify the DNA of Colletotrichum pekinensis, respectively, and the theoretical target band sizes are 210bp, 228bp, and 181bp, respectively; 2, 2, and 6 use primers 62F1 / 62R1, 62F2 / 62R2, and 56F1 / 56R1 to amplify the products of sterile water, respectively.

[0086] Conclusion: 56F1 / R1 has the highest amplification efficiency and the sharpest bands, and was selected as the detection primer.

[0087] 1.2 Specificity Verification (Using 56F1 / R1 Primers)

[0088] Positive control: C. higginsianum → clear 181 bp band.

[0089] Negative control: 7 closely related anthrax species (C. nicotianae, etc.) + 2 Alternaria species (A. solani, A. brassicicola), no amplified bands.

[0090] Internal control: All strains were successfully amplified using ITS1 / ITS4 primers (excluding DNA extraction failure).

[0091] Conclusion: The primers are species-specific for C. higginsianum.

[0092] 1.3 Sensitivity test

[0093] Table 2 Sensitivity test results

[0094] DNA concentration (ng / μL) 50 5 0.5 0.05 0.005 0.0005 RPA test results ++++ +++ ++ + - - Conventional PCR results +++ + - - - -

[0095] Conclusion: The detection limit of RPA is 0.05 ng / μL (100 times higher than that of PCR).

[0096] 1.4 Actual Sample Verification

[0097] Sample type: isolates from diseased green vegetable tissues simulated with C. higginsianum inoculation (n=15).

[0098] Test results: All samples amplified a 181bp band with an accuracy of 100%.

[0099] 1.5 Conclusion

[0100] The optimal primer: 56F1 / R1 targeting NC_030956, the sequence is as follows:

[0101] 56F1: 5′-GAAAGCGGCAATCTTCAAGACATCACTGTT-3′;

[0102] 56R1: 5′-CGTTGGGAATAGCCAGGGAGACAATACA-3′.

[0103] It performed best in screening, strictly distinguishing seven closely related anthracnose species and common plant pathogenic fungi (zero crossover), with a sensitivity of 0.05 ng / μL (≈single copy level), 100 times higher than PCR. It completed amplification at a constant temperature of 39°C for 20 minutes, without the need for thermal cycling equipment, and achieved an actual sample detection rate of 100%, providing the first instrument-free RPA solution for rapid field testing of anthracnose in Cruciferae.

[0104] 2. Species specificity verification of RPA primers 56F1 / 56R1

[0105] To verify the species specificity of primers 56F1 / 56R1 targeting C. higginsianum gene NC_030956, RPA amplification followed by agarose gel electrophoresis was performed.

[0106] 2.1 Test materials

[0107] Table 3 Species-specific test materials for RPA primers 56F1 / 56R1

[0108]

[0109] 2.2 Experimental steps

[0110] B1. DNA preparation: Genomic DNA of all strains was extracted using the CTAB method (concentration was standardized to 20 ng / μL), and DNA quality was verified by amplification using ITS1 / ITS4 primers (expected product 500 bp).

[0111] B2. RPA amplification system:

[0112] Table 4 RPA amplification system

[0113] Components Volume / dosage 2×RPA buffer 29.5μL 10 μM 56F1 2.4 μL 10 μM 56R1 2.4 μL Template DNA 1.0 μL <![CDATA[ddH2O]]> Make up to 47.5 μL 280 mM magnesium acetate 2.5 μL (final concentration 14 mM)

[0114] Reaction conditions: constant temperature reaction at 39℃ for 20min.

[0115] B3. Product detection: Take 2 μL of amplified product and run electrophoresis on 1.5% agarose gel (containing GelRed) (120V, 20min), and take pictures and record with a gel imaging system.

[0116] 2.3 Test results

[0117] Table 5 Species-specificity test results of RPA primers 56F1 / 56R1

[0118] Lane sample Results (181bp band) 1 C. higginsianum **Strongly positive (+++)** 2 C.nicotianae Negative(-) 3 C. truncatum Negative(-) 4 C.gloeosporioide Negative(-) 5 C.siamense Negative(-) 6 C. fruticola Negative(-) 7 C. orbiculare Negative(-) 8 DNA Marker (100bp) Clear bands

[0119] 2.4 Conclusion

[0120] See Figure 2 , M is DL2000DNAMarker, 1-9 are the DNA amplification results of C.higginsianum, C.nicotianae, C.truncatum, C.gloeosporioide, C.siamense, C.fruticola, C.orbiculare, Alternariasolani, and A.brassicicola, respectively; 10 is the amplification product of sterile water using primers 56F1 / 56R1; A is the RPA test result, only sample No. 1 showed a 181bp band; B is the amplification of ITS1 / ITS4 fragments, and 1-9 all amplified bands of the theoretical size.

[0121] Specificity: Primer pair 56F1 / 56R1 is absolutely specific for C. higginsianum at the species level, amplifying a clear 181bp band only for C. higginsianum. No amplification product was found in any control strains (six closely related species of Colletotrichum). The primer pair can strictly distinguish seven closely related species of Colletotrichum (including common mixed pathogens in the field), providing a molecular basis for accurate field detection of anthracnose pathogens in green vegetables and avoiding false positives.

[0122] 3. Sensitivity test of RPA primers 56F1 / 56R1 and comparison with PCR

[0123] 3.1 Materials and Methods

[0124] DNA source: C. higginsianum pure culture strain (CTAB extraction, initial concentration 100 ng / μL)

[0125] Gradient dilution: dilute the DNA of Colletotrichum oleraceus to 50, 5, 0.5, 0.05, 0.005, 0.0005, and 0.00005 ng / μL in sequence.

[0126] Amplification method:

[0127] Table 6 RPA primer 56F1 / 56R1 amplification method

[0128]

[0129] 5 μL of amplified product was taken and subjected to 1.5% agarose gel electrophoresis (120 V, 20 min), and GelRed staining was used for imaging.

[0130] 3.2 Test results

[0131] Table 7 Primer sensitivity test results

[0132]

[0133]

[0134] Note: ++++: very strong band; +++: strong; ++: moderate; +: weakly visible; -: no band RPA detection limit: 0.05 ng / μL (approximately 0.1 copies / μL)

[0135] PCR detection limit: 5 ng / μL (approximately 10 copies / μL)

[0136] 3.3 Conclusion

[0137] See Figure 3 , M is DL2000DNAMarker, 1-7 are the amplification results of 50, 5, 0.5, 0.05, 0.005, 0.0005, and 0.00005 ng / μL Colletotrichum broccoli DNA, respectively; 8 is the amplification product of sterile water using primers 56F1 / 56R1; A is the RPA test result, 1-4 samples showed a 181 bp band; B is the PCR test result, 1-2 amplified a band of the theoretical size.

[0138] RPA can still detect a clear 181bp band at 0.05ng / μL, while conventional PCR no longer amplifies products at 0.5ng / μL. RPA sensitivity is 100-fold higher than PCR (0.05ng / μL vs 5ng / μL), and the time required is reduced to 1 / 6 (20min vs 120min).

[0139] RPA amplified effectively in the concentration range of 0.05-50 ng / μL, and the band intensity decreased gently with concentration; the PCR signal disappeared sharply when the concentration was less than 5 ng / μL.

[0140] Primers 56F1 / 56R1 achieve single-copy level detection (0.05ng / μL) under the RPA system, solving the problem of missed detection of trace pathogen samples in the incubation period. Compared with conventional PCR, the sensitivity is increased by 100 times, and the time is shortened to 1 / 6 (20min vs 120min), providing a reliable rapid detection tool for early infection (asymptomatic stage) in the field to avoid disease outbreaks.

[0141] 4. Detection and verification of RPA method on actual disease samples

[0142] Positive group: C. higginsianum diseased leaves were inoculated, 15 samples, pathogen colonies were isolated and DNA was extracted.

[0143] Negative control group: 5 samples of healthy green vegetable leaves, direct extraction of plant tissue DNA.

[0144] Blank control: sterile water, 1 portion, used instead of template for amplification.

[0145] 4.1 Test steps

[0146] C1. Sample preparation: C. higginsianum spore suspension (10 6 spores / mL) were spray-inoculated on healthy cabbage leaves and cultured at 25°C with moisturizing for 5 days until typical anthracnose lesions formed. Tissues from the lesion edges were disinfected and placed on PDA plates. The plates were cultured at 25°C for 3 days, and colonies were purified. DNA of the isolated strains and healthy leaves was extracted using the CTAB method.

[0147] C2. RPA amplification: 29.5 μL of 2×RPA buffer, 2.4 μL of each 10 μM primer, 1.0 μL of template DNA, and ddH2O to 47.5 μL. Mix well, add 2.5 μL of 280 mM magnesium acetate (final concentration 14 mM), and incubate at 39°C for 20 min.

[0148] C3. Detection of a 181 bp band by 1.5% agarose gel electrophoresis (120 V, 20 min).

[0149] 4.2 Test results

[0150] Table 8 Electrophoresis results

[0151] Sample type Detection ratio of 181bp band Representative Results Positive group (diseased leaf isolates) 15 / 15(100％) Clear stripes (++++++) Negative group (healthy leaves) 0 / 5(0％) No band (-) Blank control No band (-) No band (-)

[0152] See Figure 4 , M is DL2000 DNAMarker, 1-3 are fungal DNA isolated from laboratory-inoculated diseased samples, 4 is Colletotrichum broccoli DNA (positive control), and 5 is healthy cabbage DNA (negative control, 181bp bands appear in all 1-3). The target pathogen was successfully detected in all artificially diseased samples (100% accuracy), with no false positives in healthy samples. The amplified band intensities of isolates from different lesions were consistent, demonstrating that the method is tolerant to sample variation.

[0153] 4.3 Conclusion

[0154] The RPA method has a 100% accuracy rate in pathogen detection of actual disease samples (including background plant matrices), with no false positives or false negatives. It overcomes the interference of endogenous inhibitors in plant tissues and does not require DNA purification. In addition, it only takes 1.5 hours from sample processing to result output, while traditional isolation and identification takes 5-7 days. This provides a reliable tool for early diagnosis, pathogen monitoring, and prevention and control decisions of anthracnose in field vegetables.

[0155] Through these experiments, the present invention screened and identified the optimal targeting primer, 56F1 / RI. Specificity verification demonstrated zero crossover against seven closely related anthrax species. Sensitivity testing reached 0.05 ng / μL (a 100-fold improvement compared to PCR), and the detection accuracy of actual disease samples was 100%. The entire process took 1.5 hours, resolving the dilemma of traditional methods, which struggled to balance timeliness and sensitivity.

[0156] The present invention is not limited to the above-mentioned optional implementation modes. Anyone can derive other forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that falls within the scope defined by the claims of the present invention falls within the scope of protection of the present invention.

Claims

1. An RPA detection primer for Colletotrichum oleraceus, characterized in that: The primers include at least one of the following three pairs of primers: Primer 62F1: 5′-GCTCCAGATCAAGAGCCGTTGGCTATCGTAT-3′; Primer 62R1: 5′-CCGTATTGGTGAGTATCCAGCCCATTGAGGG-3′; Primer 62F2: 5′-CTGTTGTTCCTGCGATAGACTAACCTACTTGC-3′; Primer 62R2: 5′-TGTTTTCCTTCAACCCATAACCTAGCTTGG-3′; Primer 56F1: 5′-GAAAGCGGCAATCTTCAAGACATCACTGTT-3′; Primer 56R1: 5′-CGTTGGGAATAGCCAGGGAGACAATACA-3′.

2. The RPA detection primer for anthrax of green vegetables according to claim 1, characterized in that: The RPA detection primers include: Primer 56F1: 5′-GAAAGCGGCAATCTTCAAGACATCACTGTT-3′; Primer 56R1: 5′-CGTTGGGAATAGCCAGGGAGACAATACA-3′.

3. The RPA detection primer for anthrax of green vegetables according to claim 1, characterized in that: The amplification product of the primers 56F1 and 56R1 was 181 bp in size.

4. Use of the RPA detection primer for Colletotrichum oleraceus according to claim 1 in detecting Colletotrichum oleraceus.

5. A kit for detecting anthrax of green vegetables, characterized in that: Comprising the RPA detection primer according to claim 1.

6. A kit for detecting anthracnose of green vegetables according to claim 5, characterized in that: Includes RPA amplification reagents.

7. A kit for detecting anthracnose of green vegetables according to claim 5, characterized in that: The RPA amplification reagent includes 2×RPA reaction buffer and 280 mM magnesium acetate solution.

8. A method for detecting anthrax of green vegetables, characterized in that: The following steps are involved: S1. Extract DNA from the sample to be tested; S2. Using DNA as a template, perform RPA amplification using at least one pair of primers selected from 62F1 / 62R1, 62F2 / 62R2, or 56F1 / 56R1; S3. Detect the amplified product. When using the primer pair 62F1 / 62R1, if a specific fragment of 210 bp is present, the sample is determined to carry the green vegetable anthracnose pathogen; when using the primer pair 62F2 / 62R2, if a specific fragment of 228 bp is present, the sample is determined to carry the green vegetable anthracnose pathogen; when using the primer pair 56F1 / 56R1, if a specific fragment of 181 bp is present, the sample is determined to carry the green vegetable anthracnose pathogen.

9. The method for detecting anthrax of green vegetables according to claim 8, characterized in that: The RPA reaction system includes: 29.5 μL of 2×RPA reaction buffer, 2.4 μL of 10 μM primer 56F1, 2.4 μL of 10 μM primer 56R1, 1.0 μL of template DNA, sterile deionized water to 47.5 μL, and 2.5 μL of 280 mM magnesium acetate.

10. The method for detecting anthracnose of green vegetables according to claim 8, characterized in that: The RPA amplification was carried out at a constant temperature of 39° C. for 20 min, and the amplified product was detected by 1.5% agarose gel electrophoresis.

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