RPA (recombinase polymerase amplification)-based rapid molecular detection method for puccinia hordei

Abstract

The invention relates to the technical field of molecular detection of agricultural biological diseases, in particular to an RPA (recombinase polymerase amplification)-based rapid molecular detection method for puccinia hordei, which comprises the following steps: S1, collecting, separating and purifying puccinia hordei for test and a reference strain to respectively obtain a puccinia hordei purified strain and a reference strain purified strain; s2, preparing a barley rust and reference bacterial system DNA template; s3, respectively carrying out PCR (Polymerase Chain Reaction) amplification and purification by using the barley rust DNA template and the reference bacterial system DNA template obtained in the step S2; s4, connection and transformation of the vector and a target fragment; s5, designing and screening primers; and S6, establishment of a barley rust rust RPA detection system and primer specificity verification. According to the invention, specific primers of barley rust rust are designed through genome sequence differences, and an efficient and high-sensitivity rapid molecular detection system of barley rust rust is established through system optimization.

Description

Technical Field

[0001] This invention relates to the field of molecular detection technology for agricultural biological diseases, and in particular to a rapid molecular detection method for barley crown rust based on RPA. Background Technology

[0002] Barley is the fourth largest cereal crop after corn, rice, and wheat. It has a long history of cultivation and is widely planted in temperate and warm-temperate alpine regions of both hemispheres. my country is one of the earliest countries to cultivate barley, which is mainly grown on the Qinghai-Tibet Plateau and in the northwest and southwest regions. Barley has a short growing season, is early-maturing and high-yielding, highly adaptable, and has wide-ranging uses, giving it a unique and important agricultural position. Rust diseases (stripe rust, leaf rust, stem rust, crown rust) are devastating fungal diseases affecting barley and other cereal crops worldwide, often causing severe yield losses. Crown rust, in particular, is a significant disease affecting cereal crops and other weeds. Barley crown rust was first discovered in the United States in 1991 and first reported in my country in 2016. Barley crown rust is a biotrophic, obligate parasitic fungus that can spread over long distances across regions via high-altitude air currents, posing a serious potential threat to barley production in my country. Therefore, timely and effective control of barley crown rust is an urgent need to be addressed in ensuring food security.

[0003] Currently, research on crown rust mainly focuses on oats and grassy weeds, while studies on barley crown rust are limited to pathogenicity, resistance, and host range. The pathogen requires a nearly ten-day incubation period to develop symptoms after invading the host; therefore, once it occurs, it spreads rapidly, often posing a potential epidemic risk. More importantly, the symptoms of barley crown rust and barley stripe rust are extremely similar, often leading to misdiagnosis and affecting effective control. However, molecular detection methods for barley crown rust fungus are still lacking. Therefore, this invention establishes a rapid molecular detection technology for barley crown rust fungus, enabling accurate detection and providing technical support for rapid and efficient diagnosis, early monitoring and warning, and timely and effective control of barley crown rust. This has significant practical implications for reducing disease damage and ensuring barley production.

[0004] Traditional biological methods for identifying pathogenic fungi generally involve steps such as symptom description, pathogen isolation, purification culture, and microscopic observation. In fungal classification, the emergence of new pathogenic fungi necessitates descriptions of their morphology, physiological and biochemical characteristics, and the symptoms they cause. However, relying solely on morphology for fungal identification is unreliable and impractical for real-world applications. With the rapid development of molecular biology techniques, DNA data has greatly facilitated fungal classification and identification. A series of molecular biological identification technologies have completely overcome the shortcomings of traditional biological methods, simplifying the detection and identification of pathogenic fungi and providing more accurate results. Recombinase polymerase amplification (RPA) is a novel multi-enzyme isothermal nucleic acid amplification technology. Compared to conventional PCR amplification, RPA reactions can be performed at a constant temperature of 37 to 42°C, typically achieving specific amplification of nucleic acid sequences within 5-30 minutes.

[0005] Barley crown rust is a new barley disease discovered in Qinghai Province in recent years. Its symptoms are extremely similar to those of barley stripe rust, making misdiagnosis easy. Accurate identification of the pathogen is the primary step and scientific foundation for effective disease control. Therefore, establishing a rapid molecular detection method for barley crown rust based on RPA (Rapid Prognostic Acid Detection) is more efficient and reliable for the detection and diagnosis of barley crown rust pathogens. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a rapid molecular detection method for *Rhizoctonia solani* based on RPA. Specific primers for *Rhizoctonia solani* are designed based on genomic sequence differences, and after system optimization, a high-efficiency and high-sensitivity rapid molecular detection system for *Rhizoctonia solani* is established.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A rapid molecular detection method for *Crassula capitella* based on RPA includes the following steps:

[0009] S1, Collection, isolation and purification of the tested barley crown rust fungus and the reference strain, respectively obtaining purified barley crown rust strain and purified reference strain;

[0010] S2, Preparation of DNA templates for *Begonia solani* and reference strains: DNA was extracted from the purified strains of *Begonia solani* and reference strains obtained in S1 to prepare DNA templates for *Begonia solani* and reference strains.

[0011] S3, PCR amplification and purification were performed using the *Begonia rust* DNA template obtained in step S2 and the reference strain DNA template, respectively.

[0012] The primer sequences are as follows:

[0013] EF1-983F:5´-GCYCCYGGHCAYCGTGAYTTYAT-3´

[0014] EF1-1567R: 5´-ACHGTCCRATACCACCSATCTT-3´;

[0015] S4, Ligation and transformation of vector and target fragment: The ligation vector was formed using pMD™ 18-T Vector Cloning Kit (TaKaRa), and transformed using DH5α competent cells from Weidi Biotechnology. Positive clones were screened and then sequenced.

[0016] S5, Primer Design and Screening: Sequence alignment, screening for specific fragments of *Bacillus crown rust* and other reference strains, and designing PCR primers and RPA primers using Primer Premier 5.0 software;

[0017] The PCR primer sequences are:

[0018] F: 5´-GTTCTGTCCCTACTGACATTCTGGG-3´;

[0019] R: 5´-GATGAGGGAAATGGAGCAAGTCTG-3´;

[0020] The RPA primer sequences are:

[0021] F: 5´-TTCCCCTAGTTCTGTCCCTACTGACATTCTGGG-3´;

[0022] R: 5´-GAATTGATGAGGGAAATGGAGCAAGTCTGAACC-3´;

[0023] S6. Establishment of RPA detection system for barley crown rust and verification of primer specificity.

[0024] Preferably, the specific method for obtaining the purified bacterial strain in step S1 includes the following steps:

[0025] S11, the barley variety Guoluo and the wheat varieties required for the reference strain, are cultivated in a seedling room with 13℃ cyclic light, i.e., 16 h of light and 8 h of darkness.

[0026] S12, when the seedling grows to the point where one leaf is fully unfolded, use an eyelash pen to pick up a single urediniospore and inoculate it on the front of the leaf. After inoculation, spray an appropriate amount of water mist, cover with an isolation cover and cover with a lid to keep it moist. Place it in a moist room at 10°C and 70%~80% relative humidity in the dark for 36 hours.

[0027] S13, after the moisturizing period, was transferred to the incubation greenhouse for further cultivation. After the sporulation and urediniospores ruptured, the strain was collected in sterile test tubes and temporarily stored in a 4°C refrigerator to obtain purified strains of *Begonia solani* and purified strains of the reference strain.

[0028] Preferably, the reference strain in step S1 is selected from pathogens of common diseases of wheat crops, including wheat stripe rust, wheat stem rust, wheat leaf rust, barley stripe rust, barley leaf rust, oat crown rust, oat stem rust, barley sheath rot pathogen, and wheat powdery mildew pathogen.

[0029] Preferably, the specific method for preparing the DNA templates of *Crassula ovata* and the reference strain in step S2 is as follows: the collected bacterial strain is placed in a 2 ml centrifuge tube, two 3 mm steel balls are added, the strain is broken up with a tissue homogenizer, and the *Crassula ovata* DNA template and the reference strain DNA template are prepared separately using the Bevo Medical Technology Fungal Genomic DNA Extraction Kit according to the operating instructions.

[0030] Preferably, the specific operation of PCR amplification in step S3 is as follows: using the *Begonia rotundifolia* DNA template and the reference strain DNA template obtained in step S2, select amplification primers, and the total PCR amplification system is 50 μL; wherein, the total amplification system includes forward primers, reverse primers, *Begonia rotundifolia* DNA template or reference strain DNA template, 2 × Es Taq MasterMix (Dye) PCR mixture and ddH2O.

[0031] The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 35 cycles of amplification, each cycle including 94℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, and final extension at 72℃ for 5 min to ensure that all amplification products complete the extension, and finally enter the 16℃ hold state.

[0032] Preferably, the primers selected for PCR amplification are EF1-983F / EF1-1567R, synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd.; the 2 × Es Taq MasterMix (Dye) PCR mixture is from Kangwei Century Biotechnology Co., Ltd.

[0033] Preferably, the method for establishing the barley crown rust fungus RPA detection system in step S6 is as follows: using the genomic DNA of barley crown rust fungus and other reference strains prepared in step S2 as templates, and using the primers designed in S5, conventional PCR and RPA system amplification reactions are performed; ddH2O is used as a negative control, and the experiment is repeated more than 3 times.

[0034] Preferably, the specific steps for establishing the barley crown rust RPA detection system in step S6 are as follows:

[0035] PCR amplification reaction: The total amplification system includes forward primers, reverse primers, *Bacillus thuringiensis* DNA template or reference strain DNA template, 2×Es Taq MasterMix (Dye) PCR mixture, and ddH2O;

[0036] The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 33 cycles of amplification, each cycle including 94℃ denaturation for 10 s, 63℃ annealing for 30 s, 72℃ extension for 20 s, and a final extension at 72℃ for 5 min to ensure that all amplification products have completed the extension, and finally enter the 16℃ holding state.

[0037] RPA reaction: Refer to the RPA reaction kit instructions. The total reaction system includes buffer A, forward primer, reverse primer, *Crassula ovata* DNA template or reference strain DNA template, ddH2O, and buffer B. Place the reaction tube in a metal bath and incubate at 39°C for 20 min until the reaction is complete.

[0038] Preferably, the specific procedures for verifying the primer specificity of the barley crown rust RPA detection system are as follows: PCR amplification products are detected by agarose gel electrophoresis, and the amplification product bands are observed by ultraviolet gel imaging.

[0039] After the RPA reaction, the reaction product was added to DNA extraction buffer containing Tris-saturated phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:1. After mixing, the mixture was centrifuged at 12,000 rpm for 5 min. The supernatant was then mixed with 6× Loading buffer and detected by agarose gel electrophoresis. After electrophoresis, the amplified product bands were observed using a UV gel imaging system.

[0040] A rapid molecular detection method for barley crown rust fungus based on RPA, as described in any of the above, is used for the rapid detection of barley crown rust fungus and is suitable for field monitoring of barley crown rust fungus.

[0041] The beneficial effects of this invention are:

[0042] This invention focuses on the pathogen of barley crown rust. Specific primers for barley crown rust were designed based on genomic sequence differences. After system optimization, a rapid molecular detection system for barley crown rust that is efficient, sensitive, and easy to diagnose was established. This system is used for the early diagnosis and accurate identification of the disease, and also provides a means of monitoring the spread of the disease.

[0043] This invention, based on RPA technology, enables rapid molecular detection of barley crown rust. Monitoring and early warning of plant pathogens are the cornerstone of effective disease control and ensuring agricultural production safety. Although conventional PCR technology is widely used in this field, its operation usually depends on laboratory conditions, making it difficult to meet the needs of rapid field detection. RPA technology, due to its simple operation, high speed, high specificity, and high sensitivity, shows significant advantages in the field of nucleic acid amplification. This technology, through isothermal amplification, overcomes the dependence of traditional PCR on thermal cyclers, greatly promoting detection applications under resource-scarce field conditions.

[0044] The RPA detection system established in this invention yields results consistent with conventional PCR amplification, both specifically detecting barley crown rust. Its detection limit is 1 pg / μL, demonstrating high sensitivity.

[0045] The RPA detection system established in this invention can detect barley crown rust fungus in the genomic DNA of barley seedlings that have not yet shown symptoms after inoculation. It can provide technical support for the rapid detection of barley crown rust in the early stage in the field and is of great significance for the early warning and control of barley crown rust in my country. Attached Figure Description

[0046] Figure 1 To verify the PCR primer specificity of this invention using 2% agarose gel electrophoresis, the following primers were used: M. 600 DNA Marker I; 1 and 2 were barley crown rust fungi; 3 was wheat stripe rust fungi; 4 was wheat stem rust fungi; 5 was wheat leaf rust fungi; 6 was barley stripe rust fungi; 7 was barley leaf rust fungi; 8 was oat crown rust fungi; 9 was oat stem rust fungi; 10 was barley sheath rot fungus; 11 was wheat powdery mildew fungus; 12 was a healthy barley leaf; and 13 was a negative control ddH2O.

[0047] Figure 2 To verify the primer specificity of the RPA system using 2% agarose gel electrophoresis, the following primers were used: M. 600 DNA Marker I; 1 and 2 were *Balticia pinnatifida*; 3 was *Bacillus stripe rust*; 4 was *Bacillus stem rust*; 5 was *Bacillus leaf rust*; 6 was *Bacillus stripe rust*; 7 was *Bacillus leaf rust*; 8 was *Bacillus oatus*; 9 was *Bacillus oatus*; 10 was *Bacillus sheath rot*; 11 was *Bacillus thuringiensis*; 12 was a healthy *Golgotha* leaf; and 13 was a negative control (ddH2O).

[0048] Figure 3To verify the sensitivity of the RPA system using 2% agarose gel electrophoresis, the following parameters were used: M. 600 DNA Marker I; 1-8 were 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL, respectively; 9 was the negative control ddH2O.

[0049] Figure 4 This invention relates to the 2% agarose gel electrophoresis detection of the RPA system for different time periods of infection with *Berberis crown rust*. M. 600 DNA Marker I; lanes 1-9 represent days 1-9 after *Berberis crown rust* inoculation with the barley variety *Guoluo*, respectively; lane 10 is the negative control ddH2O. Detailed Implementation

[0050] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to embodiments and accompanying drawings. The content mentioned in the embodiments is not intended to limit the present invention.

[0051] Example 1

[0052] Establishment of RPA detection system for barley crown rust and primer specificity verification experiment:

[0053] 1. Collection, isolation, and purification of the tested barley crown rust fungus and reference strains.

[0054] The barley crown rust standard was collected from Qilian County, Qinghai Province.

[0055] The reference strains were selected from pathogens of common diseases of wheat crops: wheat stripe rust, wheat stem rust, wheat leaf rust, barley stripe rust, barley leaf rust, oat crown rust, oat stem rust, barley sheath rot, and wheat powdery mildew. The tested pathogens were provided by the State Key Laboratory of Dryland Crop Stress at Northwest A&F University.

[0056] The barley variety Guoluo and the wheat varieties required for the aforementioned reference strain were cultivated in a seedling room under a 13°C, cyclic light (16 h light / 8 h dark) system. When the seedlings reached the stage where one leaf was fully expanded, a single urediniospore was picked up with an eyelash pen and inoculated onto the upper surface of the leaf. After inoculation, a suitable amount of water mist was sprayed, and the seedlings were covered with an isolation cover and then sealed to maintain humidity. They were placed in a humidified room at 10°C and 70%–80% relative humidity for 36 h in the dark. After the humidification period, the seedlings were transferred to a cultivation greenhouse for further cultivation. After conidia and urediniospore rupture, the inoculum was collected using sterile test tubes and temporarily stored in a 4°C refrigerator to obtain purified strains of *Berberis crown rust* and purified strains of the reference strain.

[0057] 2. Preparation of DNA templates from *Balticia pinnatifida* and reference strains.

[0058] The bacterial strains collected in step 1 were placed in a 2 ml centrifuge tube, two 3 mm steel balls were added, and the mixture was broken up using a tissue homogenizer. The DNA templates of *Crassula ovata* and reference strains were prepared using the Bevo Medical Technology Fungal Genomic DNA Extraction Kit according to the instructions.

[0059] 3. PCR amplification and purification

[0060] Using the genomic DNA extracted in section 2 above as a template (Barley rust DNA template and reference strain DNA template), the universal primers selected for PCR amplification were EF1-983F / EF1-1567R, which were synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd.

[0061] The total PCR amplification system was 50 μL: 2 μL each of forward and reverse primers (10 μM), 2 μL of template DNA (50 ng / μL), 25 μL of 2×Es Taq MasterMix (Dye) PCR mixture (Kangwei Century Biotechnology Co., Ltd.), and 19 μL of ddH2O.

[0062] The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 35 cycles of amplification, each cycle including 94℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, and final extension at 72℃ for 5 min to ensure that all amplification products complete the extension, and finally enter the 16℃ hold state.

[0063] The primer sequences are:

[0064] EF1-983F:5´-GCYCCYGGHCAYCGTGAYTTYAT-3´

[0065] EF1-1567R:5´-ACHGTRCCRATACCACCSATCTT-3´

[0066] After amplification, the PCR products were detected by 1% agarose gel electrophoresis.

[0067] After the assay was completed, the product was purified using an omega gel recovery kit.

[0068] 4. Ligation and transformation of the vector and the target fragment

[0069] The ligation vector was obtained using the pMD™ 18-T Vector Cloning Kit (TaKaRa), and the cells were transformed with DH5α competent cells from Weidi Biotechnology. After screening for positive clones, the cells were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0070] 5. Primer design and screening

[0071] Sequence alignment was performed to screen for specific fragments from *Bacillus crown rust* and other reference strains. Primer Premier 5.0 software was used to design conventional PCR primers and RPA primers.

[0072] The PCR primer sequences are:

[0073] F: 5´-GTTCTGTCCCTACTGACATTCTGGG-3´

[0074] R: 5´-GATGAGGGAAATGGAGCAAGTCTG-3´

[0075] The RPA primer sequences are:

[0076] F: 5´-TTCCCCTAGTTCTGTCCCTACTGACATTCTGGG-3´

[0077] R: 5´-GAATTGATGAGGGAAATGGAGCAAGTCTGAACC-3´

[0078] 6. Establishment of RPA detection system for barley crown rust and verification of primer specificity

[0079] Genomic DNA from *Balticia pinnatifida* and other common pathogens affecting cereal crops was used as templates (*Balticia pinnatifida* DNA template or reference strain DNA template) for conventional PCR and RPA amplification reactions. ddH2O was used as a negative control, and the experiment was repeated at least three times.

[0080] The total PCR amplification system was 50 μL: 2 μL each of forward and reverse primers (10 μM), 2 μL template DNA (50 ng / μL), 25 μL 2×Es Taq MasterMix (Dye) PCR mixture (Kangwei Century Biotechnology Co., Ltd.), and 19 μL ddH2O. The PCR amplification reaction program was as follows: 94℃ pre-denaturation for 5 min; 33 cycles of amplification, each cycle including 94℃ denaturation for 10 s, 63℃ annealing for 30 s, 72℃ extension for 20 s, and a final extension at 72℃ for 5 min, ensuring all amplification products completed the extension, and finally holding at 16℃. PCR amplification products were detected by 2% (m / v) agarose gel electrophoresis, and the amplification product bands were observed using a UV gel imaging system.

[0081] Following the instructions of the RPA reaction kit (Anpu Future Biotechnology Co., Ltd.), the total reaction volume was 50 μL: 29.4 μL Abuffer, 2 μL each of forward and reverse primers (10 μM), 2 μL template (10 ng / μL), and ddH2O added to a final volume of 47.5 μL. After thorough mixing, 2.5 μL of B buffer was added. The reaction tube was incubated at 39°C for 20 min in a metal bath. After the reaction, 50 μL of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1 v / v) DNA extraction buffer was added to the reaction product. After mixing, the mixture was centrifuged at 12000 rpm for 5 min. 5 μL of the supernatant was mixed with 1 μL of 6× Loading buffer and analyzed by 2% agarose gel electrophoresis. After electrophoresis, the amplified product bands were observed using a UV gel imaging system.

[0082] The results are as follows Figure 1 As shown in (conventional PCR) and (RPA), except for the specific target bands appearing in lanes 1 and 2, i.e., the amplification system of *Begonia rotundifolia* genome, no bands were observed in the other lanes, which is the same as the negative control. This indicates that the reaction has high specificity.

[0083] Example 2

[0084] Sensitivity validation test of the rapid molecular detection system for barley crown rust based on RPA.

[0085] 1. Propagate barley crown rust fungus strain for later use.

[0086] The barley variety Guoluo was planted, and the cultivation conditions were the same as in Example 1. When the barley leaves were fully expanded, fresh *Crassula ovata* monouretic spore strain was placed in a 0.5 mL centrifuge tube, and an appropriate amount of electronic fluoride solution was added to prepare a spore suspension. The spore suspension was then pipetted and evenly applied to the upper surface of the barley leaves. After inoculation, the mixture was kept in darkness and moist (as in Example 1), and then transferred to a cultivation greenhouse for further cultivation and collection of the inoculum (as in Example 1) to obtain a large number of propagated inoculum.

[0087] 2. Preparation and serial dilution of DNA template from *Begonia solani*.

[0088] Genomic DNA template of *Bolfosca crown rust* was extracted (same as in Example 1). The extracted *Bolfosca crown rust* genomic DNA was serially diluted with an initial concentration of 10 ng / μL. The concentration gradients were: 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL.

[0089] 3. RPA Sensitivity Verification

[0090] Using different concentrations of *Bacillus crown rust* genomic DNA as templates, RPA amplification reactions were performed according to the RPA kit (Anpu Future Biotechnology Co., Ltd.) instructions (same as in Example 1). After the reaction, the RPA was detected by 2% agarose gel electrophoresis to determine the sensitivity of the RPA detection. ddH2O was used as a negative control, and the experiment was repeated at least three times.

[0091] The results are as follows Figure 3 As shown, agarose gel electrophoresis revealed that the RPA detection bands gradually faded as the template DNA concentration decreased. Specific amplification bands appeared at DNA concentrations of 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, and 1 pg / μL, amplifying a target band of 234 bp. However, no specific amplification bands were detected at DNA concentrations below 1 pg / μL, indicating that the detection limit of the established RPA detection system is 1 pg / μL.

[0092] Example 3

[0093] Experiments simulating early diagnosis and incubation period detection of disease caused by field infection.

[0094] 1. Indoor inoculation with *Crassula ovata* to simulate extremely low spore infection in the field.

[0095] When barley seedlings of the Guoluo variety have grown to the point where one leaf is fully expanded, mark a 5 cm section on the front of one leaf. Sprinkle a small amount of urediniospores on a glass slide, and under a stereomicroscope, use an eyelash pen to pick up a single urediniospore and inoculate it into the marked area on the leaf. After inoculation, spray an appropriate amount of water mist, cover the slide with an isolation cover and cover it with a lid to keep it moist. Place it in a humidified room at 10℃ and 70%~80% relative humidity, away from light, for 24 hours.

[0096] 2. Sample Collection

[0097] After the dark and moisturizing period, the samples were transferred to a cultivation greenhouse (16℃ for 16 hours during the day / 13℃ for 8 hours at night) for further cultivation. Samples were taken every 24 hours after inoculation, and the areas marked on the leaves were collected. According to observations in this experiment, chlorosis began to appear at the inoculation site on day 7 post-inoculation, and spores gradually formed. A total of 9 days' worth of leaves were collected for this experiment as samples.

[0098] 3. Sample DNA extraction

[0099] Leaves collected at different infection times after inoculation were placed in 2 ml centrifuge tubes, two 3 mm steel balls were added, and the tissues were broken up using a tissue homogenizer. Genomic DNA templates were extracted using a modified CTAB method for later use.

[0100] 4. RPA Detection

[0101] Using the extracted genomic DNA as a template, an RPA amplification reaction was performed (same as in Example 1). After the reaction, the results were detected by 2% agarose gel electrophoresis. ddH2O was used as a negative control, and the experiment was repeated at least three times.

[0102] The results are as follows Figure 4 As shown, distinct specific amplification bands appeared from day 6 onwards. This indicates that healthy barley seedlings infected with barley crown rust can be detected by the RPA system established in this invention during the early sporulation and disease development stage.

[0103] In summary, this invention designed RPA primers targeting the genome sequence of *Bombyx mori* (barley crown rust fungus) and conducted tests on the specificity, sensitivity, and applicability of the detection system, establishing an RPA detection method for *Bombyx mori*. This method exhibits high specificity and sensitivity, effectively detecting even trace amounts of DNA. Therefore, the RPA-based molecular detection method for *Bombyx mori* proposed in this invention enables rapid detection of *Bombyx mori*, providing technical support for rapid and efficient diagnosis of this fungus and showing promising application prospects in field monitoring of barley crown rust pathogens.

[0104] All technical features in this embodiment can be modified in appearance according to actual needs.

[0105] The above embodiments are preferred implementations of the present invention. In addition, the present invention can be implemented in other ways. Any obvious substitutions without departing from the concept of the present technical solution are within the protection scope of the present invention.

Claims

1. A rapid molecular detection method for *Crassula ovata* based on RPA, characterized in that: Includes the following steps: S1, Collection, isolation and purification of the tested barley crown rust fungus and the reference strain, respectively obtaining purified barley crown rust strain and purified reference strain; S2, Preparation of DNA templates for *Begonia solani* and reference strains: DNA was extracted from the purified strains of *Begonia solani* and reference strains obtained in S1 to prepare DNA templates for *Begonia solani* and reference strains. S3, PCR amplification and purification were performed using the *Begonia rust* DNA template obtained in step S2 and the reference strain DNA template, respectively. The primer sequences are as follows: EF1-983F:5´-GCYCCYGGHCAYCGTGAYTTYAT-3´ EF1-1567R: 5´-ACHGTCCRATACCACCSATCTT-3´; S4, Ligation and transformation of vector and target fragment: The ligation vector was ligated using the pMD™ 18-T Vector Cloning Kit (TaKaRa), and transformed using DH5α competent cells from Weidi Biotechnology. Positive clones were screened and then sequenced. S5, Primer Design and Screening: Sequence alignment, screening for specific fragments of *Bacillus crown rust* and other reference strains, and designing PCR primers and RPA primers using Primer Premier 5.0 software; The PCR primer sequences are: F: 5´-GTTCTGTCCCTACTGACATTCTGGG-3´; R: 5´-GATGAGGGAAATGGAGCAAGTCTG-3´; The RPA primer sequences are: F: 5´-TTCCCCTAGTTCTGTCCCTACTGACATTCTGGG-3´; R: 5´-GAATTGATGAGGGAAATGGAGCAAGTCTGAACC-3´; S6. Establishment of RPA detection system for barley crown rust and verification of primer specificity.

2. The rapid molecular detection method for *Crassula ovata* based on RPA according to claim 1, characterized in that: The specific method for obtaining the purified bacterial strain in step S1 includes the following steps: S11, the barley variety Guoluo and the wheat varieties required for the reference strain, are cultivated in a seedling room with 13℃ cyclic light, i.e., 16 h of light and 8 h of darkness. S12, when the seedling grows to the point where one leaf is fully unfolded, use an eyelash pen to pick up a single urediniospore and inoculate it on the front of the leaf. After inoculation, spray an appropriate amount of water mist, cover with an isolation cover and cover with a lid to keep it moist. Place it in a moist room at 10°C and 70%~80% relative humidity in the dark for 36 hours. S13, after the moisturizing period, was transferred to the incubation greenhouse for further cultivation. After the sporulation and urediniospores ruptured, the strain was collected in sterile test tubes and temporarily stored in a 4°C refrigerator to obtain purified strains of *Begonia solani* and purified strains of the reference strain.

3. The rapid molecular detection method for *Crassula ovata* based on RPA according to claim 1, characterized in that: The reference strains in step S1 are pathogens of common diseases of wheat crops, including wheat stripe rust, wheat stem rust, wheat leaf rust, barley stripe rust, barley leaf rust, oat crown rust, oat stem rust, barley sheath rot pathogen, and wheat powdery mildew pathogen.

4. The rapid molecular detection method for *Crassula ovata* based on RPA according to claim 1, characterized in that: The specific method for preparing the DNA templates of *Begonia rotundifolia* and the reference strain in step S2 is as follows: the collected bacterial strains are placed in a 2 ml centrifuge tube, two 3 mm steel balls are added, and the mixture is broken up with a tissue homogenizer. The DNA templates of *Begonia rotundifolia* and the reference strain are prepared separately using the Bevo Medical Technology Fungal Genomic DNA Extraction Kit according to the instructions.

5. The rapid molecular detection method for *Crassula ovata* based on RPA according to claim 1, characterized in that: The specific operation of PCR amplification in step S3 is as follows: using the *Begonia rotundifolia* DNA template and the reference strain DNA template obtained in step S2, select amplification primers, and the total PCR amplification system is 50 μL; wherein, the total amplification system includes forward primers, reverse primers, *Begonia rotundifolia* DNA template or reference strain DNA template, 2 × Es Taq MasterMix (Dye) PCR mixture and ddH2O. The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 35 cycles of amplification, each cycle including 94℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, and final extension at 72℃ for 5 min to ensure that all amplification products complete the extension, and finally enter the 16℃ hold state.

6. The rapid molecular detection method for *Crassula ovata* based on RPA according to claim 5, characterized in that: The primers used for PCR amplification were EF1-983F / EF1-1567R, synthesized and provided by Sangon Biotech (Shanghai) Co., Ltd.; the 2 × Es Taq MasterMix (Dye) PCR mixture was from Kangwei Century Biotechnology Co., Ltd.

7. The rapid molecular detection method for *Crassula ovata* based on RPA according to claim 1, characterized in that: The method for establishing the barley crown rust RPA detection system in step S6 is as follows: using the genomic DNA of barley crown rust fungus and other reference strains prepared in step S2 as templates, and using the primers designed in S5, conventional PCR and RPA system amplification reactions are performed; ddH2O is used as a negative control, and the experiment is repeated more than 3 times.

8. The rapid molecular detection method for *Crassula ovata* based on RPA according to claim 7, characterized in that: The specific steps for establishing the RPA detection system for barley crown rust in step S6 are as follows: PCR amplification reaction: The total amplification system includes forward primers, reverse primers, *Bacillus thuringiensis* DNA template or reference strain DNA template, 2×Es Taq MasterMix (Dye) PCR mixture, and ddH2O; The PCR amplification reaction program is as follows: 94℃ pre-denaturation for 5 min; 33 cycles of amplification, each cycle including 94℃ denaturation for 10 s, 63℃ annealing for 30 s, 72℃ extension for 20 s, and a final extension at 72℃ for 5 min to ensure that all amplification products have completed the extension, and finally enter the 16℃ holding state. RPA reaction: Refer to the RPA reaction kit instructions. The total reaction system includes buffer A, forward primer, reverse primer, *Crassula ovata* DNA template or reference strain DNA template, ddH2O, and buffer B. Place the reaction tube in a metal bath and incubate at 39°C for 20 min until the reaction is complete.

9. The rapid molecular detection method for *Crassula ovata* based on RPA according to claim 8, characterized in that: The specific procedures for verifying the primer specificity of the barley crown rust RPA detection system are as follows: PCR amplification products are detected by agarose gel electrophoresis, and the amplification product bands are observed by ultraviolet gel imaging. After the RPA reaction, the reaction product was added to DNA extraction buffer containing Tris-saturated phenol, chloroform and isoamyl alcohol in a volume ratio of 25:24:

1. After mixing, the mixture was centrifuged at 12,000 rpm for 5 min. The supernatant was then mixed with 6× Loading buffer and detected by agarose gel electrophoresis. After electrophoresis, the amplified product bands were observed using a UV gel imaging system.

10. A rapid molecular detection method for *Crassula ovata* based on RPA according to any one of claims 1-9, characterized in that: This method is used for the rapid detection of barley crown rust and is suitable for field monitoring and early warning of barley crown rust.

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