Primer, kit and method for detecting crop root rot fusarium solani and application

Abstract

The invention relates to the technical field of phytopathogen detection, and particularly discloses a primer, a kit and a method for detecting crop root rot fusarium solani and application, the primer comprises a specific RPA primer RPA-F and a specific RPA primer RPA-R; the specific RPA primer RPA-F is as shown in SEQ ID NO. 1, and the specific RPA primer RPA-R is as shown in SEQ ID NO. 2; the kit further comprises a guide RNA primer crRNA, and the guide RNA primer crRNA is as shown in SEQ ID NO. 3. The kit comprises a specific RPA primer RPA-F, a specific RPA primer RPA-R, a Cas12 protein, a T7 transcriptase, a crRNA DNA template, a DNA polymerase and a Cas12 / 13 nucleic acid detection test strip. The kit has the characteristics of good sensitivity, relatively good specificity and enough reliability.

Description

Technical Field

[0001] This invention relates to the field of plant pathogen detection technology, and in particular to primers, kits, methods, and applications for detecting Fusarium solani, a root rot pathogen in crops. Background Technology

[0002] Fusarium solani is a global soil-borne pathogenic fungus that infects various crops, including eggplant, soybean, potato, tomato, and apple, primarily through soil transmission, seedling spread, wound infection, and airborne spore transmission. It causes root rot, stem rot, and fruit rot, resulting in significant economic losses. Current detection methods mainly include: morphological identification (time-consuming, 7-10 days, requires pure culture, and is highly dependent on experience); conventional PCR / real-time fluorescent PCR (high sensitivity, but relies on expensive instruments and specialized laboratories, making field application difficult); and LAMP technology (while achieving isothermal amplification, primer design is complex, prone to false positives, and results require turbidity or fluorescence readings, limiting visualization). Therefore, existing technologies cannot simultaneously meet the four requirements of "high sensitivity, high specificity, instrument-free operation, and rapid on-site detection." Patent document CN116622900A discloses a primer composition and detection method for detecting the pathogen of Atractylodes lancea root rot based on loop-mediated isothermal amplification (LAMP). It mainly designs LAMP-specific primer sets for Fusarium oxysporum, Fusarium solani, and Fusarium arvense. LAMP isothermal amplification is used, and after the amplification reaction, a colorimetric reaction is performed, allowing direct visual observation of green fluorescence. The LAMP-mediated isothermal amplification technology established for the pathogen of Atractylodes lancea root rot can achieve rapid detection of the pathogen in diseased tissues, with advantages such as high specificity, high sensitivity, short reaction time, visualized detection results, and low cost, providing a reliable technical basis for the prevention and control of Atractylodes lancea root rot. However, this method has drawbacks, including complex primer design, susceptibility to false positives, and the need for turbidity or fluorescence readings, resulting in limited visualization. Patent document CN118834994A discloses a multiplex PCR detection method for the pathogen of ginger root rot. The method uses a primer combination consisting of primer pairs E1, E2, and E3 to identify or assist in the identification of the pathogen. The pathogens causing ginger root rot are identified as *Pythium spp.*, *Fusarium oxysporum*, and / or *Fusarium solani*. Multiplex PCR is performed using these three primer pairs, and the size of the PCR products determines the pathogen species. This method can rapidly and accurately identify the three pathogens and has promising applications. However, this method relies on expensive instruments and specialized laboratories, making it difficult to apply in the field. Therefore, existing methods for detecting *Fusarium solani* in crops suffer from insufficient sensitivity, specificity, and reliability. Summary of the Invention

[0003] In order to solve the above-mentioned technical problems existing in the detection of Fusarium solani in crops, this invention provides primers, reagent kits, methods and applications for detecting Fusarium solani, a root rot fungus in crops. It has the characteristics of good sensitivity, good specificity and sufficient reliability.

[0004] The first technical solution of the present invention: primers for detecting Fusarium solani, a crop root rot pathogen, wherein the primers include specific RPA primer RPA-F and specific RPA primer RPA-R; the specific RPA primer RPA-F is shown in SEQ ID NO.1, and the specific RPA primer RPA-R is shown in SEQ ID NO.2. The specific RPA primers RPA-F and RPA-R of this invention act like "precision-guided radar," rapidly and specifically identifying and replicating large quantities of gene fragments unique to *Fusarium solani* in complex sample environments. This enables highly sensitive detection of *Fusarium solani*, providing a crucial tool for the early diagnosis and control of crop root rot. Based on the complementary base pairing principle (AT, CG), the forward primer RPA-F accurately binds to one strand of the target DNA of *Fusarium solani*, while the reverse primer RPA-R binds to the other strand. The binding site is a sequence unique to *Fusarium solani*, which is not present in the genes of other microorganisms (such as other fungi, bacteria, or the crop's own DNA). This combination is tailor-made for recombinase polymerase amplification (RPA) technology. In the RPA reaction system, these primers bind to the recombinase and single-stranded DNA... Multiple enzymes, including recombinase and DNA polymerase, work synergistically to achieve exponential amplification. The recombinase binds to the primers to form a complex that scans the entire genomic DNA and precisely locates the target DNA site that is perfectly complementary to the primer sequence. This complex then penetrates and opens the DNA double helix, allowing the primer to bind to the template. Starting from the 3' end of the primer, the DNA polymerase rapidly synthesizes a new DNA strand using the target DNA as a template. This newly synthesized DNA strand then serves as the template for the next round of reactions. The entire process is carried out continuously at a constant temperature, amplifying minute amounts of target DNA fragments billions of times within 15–20 minutes, producing sufficient nucleic acid for detection. Using this pair of specific RPA primers for detection offers advantages over traditional methods such as culture identification and conventional PCR, with good sensitivity, high specificity, and sufficient reliability. This is particularly significant due to the primer sequences SEQ ID NO. 1 and SEQ ID NO. 2.2. Specific gene regions targeting *Fusarium solani* can effectively distinguish *Fusarium solani* from other closely related *Fusarium* species, minimizing false positives and exhibiting high specificity. RPA technology itself is extremely sensitive, capable of detecting single-digit pathogen genomes. This means that even in the early stages of infection, when the pathogen's numbers are very low, it can be successfully detected, enabling early warning and demonstrating high sensitivity. The entire testing process, from sample processing to result output, can be shortened to within 30 minutes, making it rapid and efficient, while traditional PCR takes several hours. This is crucial for agricultural production and plant quarantine scenarios requiring rapid on-site decision-making. Yes; the RPA reaction is carried out at a constant temperature, making it simple to operate and eliminating the need for expensive PCR instruments such as thermal cyclers. Simple temperature control, such as holding the device in hand, keeping it close to the body for warmth, or using a simple thermostat, is sufficient for amplification, greatly lowering the equipment barrier and making testing possible in the field and in on-site laboratories. The test results are clear and easy to interpret, reducing human error and ensuring high reliability. Researchers, plant protection personnel, or agricultural experts using this invention can quickly confirm whether Fusarium solani is the culprit causing crop root rot, thus guiding farmers to use pesticides precisely, selecting effective fungicides against this fungus, avoiding indiscriminate use, and reducing economic losses and environmental pesticide residues.

[0005] Preferably, SEQ ID NO.1 is 5'-CTTGACAGGAGGCGCCGCCAACCGCGAACGCTC-3'; and SEQ ID NO.2 is 5'-TCCCGTGAGTGTTGCGTTGCGTCCCCGTGCCGT-3'.

[0006] Preferably, the specific RPA primers RPA-F and RPA-R are obtained by the following steps: (A01) searching for the whole genome sequence of the eggplant root rot pathogen in the NCBI database; (A02) screening the whole genome sequence in step (A01) in the nr database and performing experimental verification to obtain the target gene; (A03) using Primer6 software to detect the conserved region of the target gene in step (A02), designing and obtaining specific RPA primers RPA-F, as shown in SEQ ID NO.1; and designing and obtaining specific RPA primers RPA-R, as shown in SEQ ID NO.2. Using the NCBI database, the complete genome sequences of multiple strains of *Fusarium solani*, a fungus causing eggplant root rot, were located and downloaded. This provided a complete genetic background information for *Fusarium solani*, essentially creating a complete archive and genealogy of the fungus. Having all the genomic information is crucial for identifying the unique "identity characteristics" of this species, preventing primers designed based on only partial information from accidentally targeting other microorganisms or missing strain variations. A non-redundant protein NR database was used to compare and analyze the complete genome sequences, screening the most suitable target genes from the massive amount of genomic information. This identified unique target genes that must be unique to *Fusarium solani*, exhibiting species specificity and significant differences from the gene sequences of other closely related species such as *Fusarium oxysporum*, the crop itself, or common microorganisms in the environment. The gene must be conserved, highly stable across all different strains of *Fusarium solani*, and resistant to mutation. This ensures that the designed primers can detect all variants and will not fail due to minor strain variations. The gene should be single-copy or low-copy, typically... The target gene appears only once in the genome, facilitating precise quantification and avoiding signal interference. Through bioinformatics analysis and experimental verification, a target gene perfectly meeting all the above conditions was obtained. Obtaining the target gene ensures the accuracy and broad applicability of subsequent detection methods. Using the professional primer design software Primer6, primers were designed within conserved regions of the target gene. These conserved regions are the most stable and least prone to change within the gene, serving as a "safe zone" for primer design, ensuring foolproof primer binding. The software comprehensively considers multiple indicators: appropriate primer length to ensure primer specificity; annealing temperature (Tm) values ​​to ensure that RPA-F and RPA-R have similar Tm values, enabling efficient operation under the same isothermal conditions; moderate GC content to avoid secondary structure formation; self- and dimer checks to prevent primer self-binding or mutual binding, ensuring amplification efficiency; and crucial 3' end base pairing, directly affecting amplification initiation efficiency, necessitates ensuring end stability. Through rigorous calculation and screening, the optimal primer pair was finally obtained: the specific RPA primer RPA-F (SEQ ID NO).1) Specific RPA primers RPA-R (SEQ ID NO. 2) are used to ensure high binding efficiency, enabling rapid and accurate binding to the target DNA; zero false positive rate, extremely high specificity, and no binding to non-target sequences; efficient amplification capability, guiding DNA polymerase to efficiently synthesize new strands, achieving exponential signal amplification; the primers obtained through defined steps demonstrate sound scientific rigor, their design not based on empiricism or random guesswork, but following the rigorous and reproducible research paradigm of modern molecular biology, giving the entire detection method a solid theoretical basis and high reliability; it is highly advanced, utilizing the latest genomic database NCBI, bioinformatics tool NR alignment, Primer6, and isothermal amplification technology RPA; it has good reliability, employing a technical path of whole-genome screening → target gene verification → software optimization design to maximize the quality of the final product, meaning this primer pair can stably, accurately, and efficiently complete the detection of Fusarium solani, with extremely low false positive and false negative rates.

[0007] Preferably, the target gene for detection in step (A02) is the XM_046274036.2 gene. The XM_046274036.2 gene was ultimately selected. This number is a unique accession number pointing to a specific predicted protein gene, meaning that after bioinformatics analysis and experimental verification, it perfectly meets all the above conditions and is the "golden target" for detection.

[0008] Preferably, the system also includes a guide RNA primer crRNA, as shown in SEQ ID NO.3. The guide RNA primer crRNA precisely guides the CRISPR-Cas12a system to the target DNA fragment amplified by RPA and activates the "collateral damage" activity of the Cas12a enzyme, indiscriminately cleaving surrounding reporter molecules, thereby generating a strong fluorescent detection signal. The addition of the guide RNA primer crRNA achieves functional separation between "specific recognition" and "signal amplification and output," significantly improving the sensitivity, specificity, and convenience of detection. A portion of the 5' end sequence of the crRNA binds to the Cas12a protein, while another portion binds to the RPA amplification product, XM_046274036.The complete complementarity of specific regions of the two genes allows the Cas12a protein to be precisely delivered to the target DNA. The Cas12a protein is only activated, transitioning from a "standby" state to an "active" state, after the crRNA successfully pairs and binds to the target DNA. The crRNA pre-binds to the Cas12a protein, forming a Cas12a-crRNA complex, which remains "silent" in solution. During the RPA reaction, a large amount of target DNA double-stranded DNA diffuses through the reaction system. The Cas12a-crRNA complex scans this DNA, with the spacer sequence in the crRNA attempting to pair with the complementary sequence on the target DNA. Once a complete pairing is found... The target DNA, a perfectly matched fragment amplified by RPA-F and RPA-R, forms a complex with crRNA. This binding triggers a conformational change in the Cas12a protein, activating it. The activated Cas12a protein exhibits two cleavage activities: cis-cleavage—cutting the double-stranded target DNA, and trans-cleavage—like "runaway scissors," indiscriminately and aggressively cutting any single-stranded DNA in the reaction system, including pre-added reporter molecules such as ssDNA probes with fluorescent and quenching groups. After the reporter molecule is cleaved, the fluorescent and quenching groups separate, resulting in fluorescence. This fluorescence can be detected by RPA-CRISPR-Cas12. A nucleic acid test strip allows for visual reading; the addition of crRNA significantly improves the overall detection system's performance, providing double protection with extremely high specificity. The first layer involves specific amplification using the RPA primer pair, and the second is the specific recognition of the amplification products by crRNA. A signal is only generated when both conditions are met simultaneously, greatly reducing the false positive rate. Even if RPA produces some non-specific amplification products, as long as they cannot be accurately recognized by crRNA, a signal will not be triggered. This mechanism is known as "strict dependence on trans-cleavage." The signal cascade amplification provides extremely high sensitivity. The exponential nucleic acid amplification by RPA performs the first amplification, and an activated Cas12a enzyme can... By cleaving thousands of reporter molecules, Cas12a's trans-cleavage activity is amplified a second time. This dual amplification effect enables detection sensitivity to reach the aM level, theoretically even allowing the detection of single molecules. The results interpretation is revolutionary, offering convenient testing. Results no longer require complex gel electrophoresis and UV light observation; they can be visualized using RPA-CRISPR-Cas12a nucleic acid test strips, similar to COVID-19 antigen test strips, where "one line indicates negative, two lines indicate positive." This makes the technology ideal for rapid on-site testing in fields or grassroots laboratories. Visual interpretation avoids errors caused by manual electrophoresis or analysis software, resulting in more intuitive and reliable results.

[0009] Preferably, the guide RNA primer crRNA is 5'-UAAUUUCUACUAAGUGUAGAUCGGAUAUCGGCUUCUGGACG-3'.

[0010] Preferably, the acquisition of the guide RNA primer crRNA includes the following steps: (B01) screening target sites based on the specific RPA primer RPA-F, specific RPA primer RPA-R, and the PAM pattern of the CRISPR-Cas12a guide RNA; (B02) designing and obtaining the guide RNA primer crRNA based on the target sites in step (B01), as shown in SEQ ID NO.3. The guide RNA primer crRNA obtained according to the defined steps can be correctly loaded by the Cas12a enzyme, can efficiently and specifically recognize the target DNA, and can successfully activate the trans-cleavage activity of Cas12a, ultimately ensuring the reliability, sensitivity, and success rate of the entire detection system; the software or designer uses the amplification products of RPA-F and RPA-R as the search range to find the PAM sequence adjacent to the original spacer sequence, where PAM is a very short specific sequence located at the 3' end downstream of the target DNA sequence. For Cas12a, this pattern is TTTN, where N can be any of A, T, C, G. One type; PAM is the "identification ID" of the Cas12a enzyme. When the Cas12a-crRNA complex scans DNA, it first looks for the PAM sequence. Only when a PAM is found will it stop to check whether the adjacent sequences are complementary to its own crRNA. Without a PAM, even if the sequence is a perfect match, Cas12a will completely ignore it. The TTTA PAM requirement is an inherent characteristic of the Cas12a enzyme and a hard threshold for designing crRNA. This screening step ensures that the selected target site can be effectively recognized by the Cas12a machine. After finding the PAM sequence, such as TTTA, the adjacent... The 20 nucleotides (nt) region at the 5' direction on the PAM is the candidate target site. This 20 nt sequence will be the spacer sequence in the future designed crRNA used to recognize the target DNA. Its length determines specificity and binding efficiency. If it is too short, such as <18 nt, the specificity will decrease, and it may misidentify similar sequences; if it is too long, such as >24 nt, it may reduce binding efficiency and reaction kinetics, and increase synthesis costs. 20 nt is a gold standard length that has been validated by numerous experiments, achieving the best balance between high specificity and high efficiency. Through the above screening, one or more candidate target sites were obtained, each site... Each crRNA consists of: a 20-nt specific sequence followed by a TTTN pattern PAM sequence; the selected 20-nt DNA target sequence is used directly as a template to synthesize its corresponding RNA sequence, which is the spacer sequence of the crRNA. It is responsible for achieving precise localization through complementary base pairing with the target DNA; in addition to this 20-nt variable spacer sequence, the crRNA also needs an inherent scaffold sequence. The structure of this scaffold sequence is common to all crRNAs targeting Cas12a. Its function is like a "handle" to bind firmly to the Cas12a protein.A 20-nt specific spacer sequence was linked to an intrinsic scaffold sequence to chemically synthesize the final, complete guide RNA primer, crRNA. This crRNA is a fully functional "guide," with the scaffold portion ensuring correct loading by the Cas12a protein, and the spacer portion derived from the 20-nt DNA sequence ensuring precise delivery of the Cas12a protein to a specific location on the RPA amplification product. The species-specific XM_046274036.2 gene was selected through bioinformatics screening, ensuring species specificity for the detection, and target selection was performed. Primers RPA-F and primers were designed in the conserved region of this gene. The RPA-R→8 primer ensures efficient amplification of target fragments from all relevant strains, providing ample "ammunition" for subsequent detection and primer design. Within the RPA amplicon range, a 20nt target site is identified according to the PAM=TTTN rule, ensuring the activation of the CRISPR system for crRNA target screening. The selected DNA target is converted into crRNA in RNA form, creating a core "guide" compatible with both Cas12a protein and target DNA for crRNA synthesis. Through this interconnected and logically rigorous design process, the resulting RPA primers (SEQ ID NO.1 and SEQ ID NO.2) and crRNA (SEQ ID NO.3) form a perfectly synergistic "combination weapon." The RPA primers are responsible for large-scale target amplification, while the crRNA accurately identifies targets from thousands of sources. Cas12a is activated under the guidance of crRNA and releases a strong signal. This design ensures that the developed detection method is not only theoretically feasible but also highly efficient and reliable in practice, with an extremely low false positive rate and extremely high sensitivity, perfectly meeting all the requirements for rapid on-site detection.

[0011] Preferably, the PAM mode of the CRISPR-Cas12a guide RNA in step (B01) is TTTN.

[0012] Preferably, the target site in step (B01) is 20 nt.

[0013] The second technical solution of the present invention is a kit for detecting Fusarium solani, a crop root rot pathogen, comprising specific RPA primer RPA-F, specific RPA primer RPA-R, Cas12 protein, T7 transcriptase, crRNA DNA template, DNA polymerase, and RPA-CRISPR-Cas12a nucleic acid detection test strip.This invention's kit is an integrated rapid detection platform combining RPA isothermal amplification and CRISPR-Cas12 fluorescence detection technologies. It allows users to perform accurate, rapid, and visual detection of *Fusarium solani* in fields and grassroots laboratories without complex instruments or professional training. The entire process, from sample to result, is completed within 30-60 minutes. The results are visible to the naked eye through the test strip bands, much like a professional "rapid pathogen detection test strip." Specific RPA primers RPA-F and RPA- act as "scouts" and "copier initiators," specifically recognizing the target gene of *Fusarium solani* and initiating exponential amplification. Under isothermal conditions, the amplification occurs on complex samples... The system searches for and binds to pathogen DNA, guiding DNA polymerase to synthesize billions of target fragments—RPA amplicones—providing sufficient "targets" for subsequent CRISPR detection. The Cas12 protein, acting as both "molecular scissors" and "signal amplifier," is the core actuator of the CRISPR system. When it binds to crRNA and is activated by the target DNA, it performs two functions: cis-cutting, cleaving the target DNA double strand, and trans-cutting, indiscriminately cleaving all surrounding single-stranded DNA, including reporter molecules such as reporter probes on test strips. This amplifies the chemical signal millions of times. Its presence transforms nucleic acid recognition events into readable detections. The key to the signal; T7 transcriptase is used for in vitro transcription of crRNA, acting as a "spot synthesis factory" for crRNA. T7 transcriptase can efficiently synthesize large quantities of functional crRNA on-site using the crRNA DNA as a template; it reduces costs, as chemically synthesized crRNA is expensive and difficult to preserve. Providing crRNA DNA templates and T7 transcriptase allows users to transcribe it themselves, which is more economical and stable; it better ensures activity, as freshly transcribed crRNA has higher activity, avoiding degradation problems that may occur during long-term storage; DNA polymerase, as the "copying engine" in the RPA reaction, plays a crucial role in recombinase and single-stranded binding proteins. With the assistance of RPA-CRISPR-Cas12a, starting with primers and using target DNA as a template, new DNA chains are rapidly synthesized. It is not an ordinary Taq polymerase, but an engineered polymerase specifically designed for isothermal amplification, responsible for achieving rapid nucleic acid amplification at a constant temperature of 37–40°C, eliminating dependence on expensive PCR instruments. The RPA-CRISPR-Cas12a nucleic acid test strip acts as a "display" for the test results, enabling visual interpretation. The interpretation rules are typically: C line color development + T line no color development = negative; C line color development + T line color development = positive. No instruments are required; results can be quickly interpreted by visually observing the bands, greatly improving the convenience and on-site applicability of the technology. The RPA-CRISPR-Cas12a nucleic acid test strip is a dedicated nucleic acid test strip for Cas12 / 13.

[0014] Preferably, the concentration of the specific RPA primer RPA-F is 8 μM to 12 μM; the concentration of the specific RPA primer RPA-F is 9 μM to 11 μM; and the concentration of the specific RPA primer RPA-F is 10 μM. If the concentration is too low (<8 μM), there will be insufficient primers, resulting in low binding efficiency with the template, leading to low amplification efficiency, decreased sensitivity, and potential failure to detect low concentrations of pathogens, causing false negatives. If the concentration is too high (>12 μM), non-specific binding is likely to occur, amplifying erroneous fragments, leading to increased background signal and a higher risk of false positives. It may also promote primer dimer formation, consuming reaction components and inhibiting effective amplification. The 10 μM concentration is the optimal working concentration determined through extensive optimization experiments, achieving a perfect balance between high specificity and high sensitivity, ensuring both high efficiency and accuracy in the amplification reaction.

[0015] Preferably, the concentration of the specific RPA primer RPA-R is 8 μM to 12 μM; the concentration of the specific RPA primer RPA-R is 9 μM to 11 μM; or the concentration of the specific RPA primer RPA-R is 10 μM.

[0016] Preferably, the crRNA DNA template includes a T7 promoter and a guide RNA primer crRNA.

[0017] Preferably, the kit also includes a reaction buffer, a positive control (PC), a negative control (NC), a reporter molecule (ssDNAReporter), a sample DNA extraction reagent, and / or an instruction manual. The reaction buffer provides optimal pH, ionic strength (e.g., Mg²⁺), energy source ATP, and a stable environment for RPA and CRISPR reactions. The positive control (PC) contains a plasmid or synthetic DNA fragment with the target DNA sequence, used to verify the correctness and effectiveness of the entire kit procedure. The negative control (NC) is a solution without target DNA, such as nucleic acid-free water, used to confirm the specificity of the test results and exclude false positives. The reporter molecule (ssDNAReporter) is a short-chain ssDNA with fluorescent groups such as FAM and quencher groups / biotin, serving as the substrate for Cas12 trans-cutting and the source of signal generation. The sample DNA extraction reagent is a simple lysis buffer or column extraction unit for rapid extraction of genomic DNA from plant root tissues, greatly improving convenience and achieving a complete "sample-in, result-out" solution. The instruction manual provides detailed, illustrated operating steps, result interpretation guidelines, precautions, and answers to frequently asked questions, ensuring stable results for different users. Reliable results; The overall workflow of this invention's kit is as follows: DNA is rapidly extracted crudely from crop root samples using the extraction solution provided in the kit; the extracted DNA is mixed with RPA primers, polymerase, buffer, etc., and reacted at a single temperature, such as 39°C, for 15–20 min to amplify the target fragment; the RPA product, Cas12 protein, T7 transcriptase, crRNA DNA template, reporter molecule, etc., are mixed and reacted at the same temperature for another 10–15 min; if the target DNA is present, Cas12 is activated and cleaves the reporter molecule; the above reaction solution is added to the test strip, and the bands are observed after 2–5 min; if only the C line is colored and the T line is not colored, it is negative; if both the C and T lines are colored, it is positive; if the C line is not colored, it is invalid. This invention's kit transforms a complex molecular biology experiment into a simple operation similar to "blood glucose testing" or "pregnancy testing," truly realizing the "democratization" of high technology and having revolutionary significance for the early diagnosis and control of crop diseases.

[0018] The third technical solution of the present invention: a method for detecting Fusarium solani, a root rot pathogen of crops, comprising the following steps: (S01) extracting genomic DNA of the root rot pathogen of the crop to be tested; (S02) placing an appropriate amount of the DNA extract from step (S01) into an RPA reaction system for reaction; (S03) placing an appropriate amount of the RPA reaction product from step (S02) into an RPA-CRISPR-Cas12a test strip detection system for reaction; (S04) adding RNase-free water to the reaction system after the reaction in step (S03) is completed and bringing the volume to the mark; (S05) inserting an RPA-CRISPR-Cas12a nucleic acid detection test strip into the reaction container after the volume is brought to the mark in step (S04), and observing whether there are control lines and detection lines on the test strip, as well as color changes. This invention integrates a streamlined process of rapid sample processing → RPA isothermal amplification → CRISPR-Cas12a detection → lateral flow chromatography test strip visualization reading. It transforms the presence or absence of invisible trace pathogen DNA into visible test strip band signals, achieving on-site, rapid, accurate, and low-cost pathogen detection. Extracting the genomic DNA of the root rot pathogen of the target crop is the starting point and foundation of the entire detection process. Using physical grinding or chemical lysis methods, the cell walls and cell membranes of crop root tissue cells and *Fusarium solani* are broken, releasing the internal genomic DNA. Purification steps remove impurities such as proteins, RNA, and carbohydrates that inhibit subsequent reactions, providing template DNA as "raw materials" for the subsequent RPA amplification reaction. Plant tissues contain large amounts of polysaccharides and polyphenols, which severely inhibit enzyme activity; removing these ensures the high efficiency and reliability of subsequent reactions and avoids false negatives. The method simplifies the sample, transforming complex biological samples into purified DNA solutions suitable for molecular detection. Within the RPA reaction system, the signal is specifically amplified to create the detection target—this is the first exponential amplification of the signal. The extracted DNA template is mixed with specific RPA primers RPA-F, heterologous RPA primers RPA-R, DNA polymerase, recombinase, single-strand binding proteins, and dNTPs. The mixture is reacted at a constant temperature of 37–40°C for 15–20 minutes. The primers specifically bind to the target sequence, guiding the enzyme system to rapidly synthesize billions of target DNA fragments—RPA amplicones. Isothermal amplification eliminates the need for a thermal cycler; a simple thermostat, or even a hand warmer or a water bath, is sufficient, greatly lowering the equipment barrier. Ultra-high sensitivity ensures effective amplification even with only 1–10 pathogens in the sample, providing ample target molecules for detection. The specificity of the RPA primers is the first layer of specificity guarantee for the entire method.Within the RPA-CRISPR-Cas12a test strip detection system, the reaction accurately identifies and triggers signal amplification, representing a second signal amplification and specificity verification. The RPA reaction product is mixed with Cas12a protein, crRNA (or a template for crRNA synthesis and T7 transcriptase), and a reporter molecule (fluorescently labeled ssDNA), and the reaction continues for 10–15 minutes under the same isothermal conditions. If the target sequence is present in the RPA product, the crRNA guides Cas12a to bind to it, activating Cas12a's trans-cleavage activity. The activated Cas12a cleaves the reporter molecule, causing a structural change. This provides a second layer of specificity; Cas12a activation strictly depends on a perfect match between the crRNA and the target sequence, providing double protection. Even with extremely weak non-specific amplification of the RPA, Cas12a cannot be activated, greatly reducing the false positive rate. Finally, the signal is converted from a nucleic acid recognition event into a chemical event of reporter molecule cleavage. Add RNase-free water to adjust the volume and concentration of the detection solution, preparing it for test strip testing. The reaction system is relatively concentrated, and direct use on test strips may result in chromatography speeds that are too slow, too fast, or fail due to non-standard ionic strength, viscosity, or volume. Adding RNase-free water dilutes and adjusts the volume to avoid degrading RNA components. Standardization ensures that the sample volume and concentration are consistent for each test, making the results stable and reproducible. Optimize the chromatography by adjusting the physicochemical properties of the solution to allow it to chromatographically advance onto the test strip at the optimal speed, ensuring that the control line (C line) and test line (T line) develop normally and clearly. This step directly affects the success or failure of the test strip test and the accuracy of the interpretation. The test strip method provides visual results, marking the end of the entire process and transforming the chemical reaction into a straightforward yes / no signal. The prepared reaction solution is added to the sample application area of ​​the test strip, and the solution is chromatographically deposited along the strip via capillary action. The reporter molecule is typically designed with a FAM fluorescent group and a biotin tag. The test line (T line) contains an anti-FAM antibody; the control line (C line) contains streptavidin (which strongly binds to biotin) or other control antibodies. A negative result (no target bacteria) is indicated by the presence of the C line and the absence of the T line; a positive result (target bacteria) is indicated by the presence of both the C and T lines; an invalid result is indicated by no color development on the C line, regardless of the T line, indicating an operational error or a faulty test strip, making the result unreliable and requiring a retest. The process is extremely simple, requiring no expensive instruments, and results are available within 5 minutes with visual interpretation. The results are clear, with highly intuitive band displays that are unlikely to cause ambiguity. Suitable for field testing, it perfectly meets the rapid on-site testing needs of rural areas and grassroots laboratories.This invention transforms complex molecular detection techniques into a simple process of sample input and result output. It is rapid, completing the process from sample to result within 30-60 minutes, far exceeding traditional culture (several days) or even PCR (several hours) methods. It is accurate, with the dual specificity of RPA and CRISPR ensuring extremely reliable results and very low false positive and false negative rates. It is convenient, with isothermal reaction and test strip readings requiring minimal equipment and personnel skills. It is applicable, truly enabling early diagnosis, on-site guidance, and precise control of crop diseases, helping farmers and plant protection personnel make timely decisions and reduce economic losses.

[0019] Preferably, the extraction of genomic DNA of the root rot pathogen of the crop to be tested in step (S01) includes the following steps: (S01-1) Take an appropriate amount of powder of the mycelium of the root rot pathogen of the crop to be tested after grinding with liquid nitrogen and place it in a centrifuge container; (S01-2) Add an appropriate amount of CTAB buffer to the centrifuge container in step (S01-1) and heat it; (S01-3) Add an appropriate amount of chloroform-isoamyl alcohol mixture to the centrifuge container in step (S01-2), stir and centrifuge, and take the supernatant; (S01-4) Add an appropriate amount of pre-cooled anhydrous ethanol to the supernatant in step (S01-3), stir and centrifuge, and take the precipitate; (S01-5) Add an appropriate amount of enzyme-free water to the precipitate in step (S01-4) and mix to obtain the genomic DNA extract of the root rot pathogen of the crop to be tested. This DNA extraction method—CTAB—is a liquid-phase separation-based chemical extraction process. Its core function is to efficiently separate and purify high-quality genomic DNA from complex plant-fungus mixed samples through a series of chemical and physical operations, while maximally removing inhibitors such as proteins, polysaccharides, and polyphenols. This results in a pure, intact DNA template suitable for downstream high-sensitivity amplification detection of RPA-CRISPR. The process begins with sampling to obtain a representative sample. A small section of tissue is scraped or cut from the lesion boundary of the root of a diseased crop, where the pathogen—Fusarium solani—is most likely to be enriched. Ensuring the sample contains the target pathogen and avoiding false negatives due to sampling errors, the sample is placed in a centrifuge tube for subsequent operations. Cells are lysed using CTAB buffer and heating to release DNA and initially suppress impurities. CTAB (hexadecyltrimethylammonium bromide) is a cationic detergent that lyses cells, effectively disrupting plant cell walls, cell membranes, and the tough cell walls of fungi, releasing genomic DNA into the solution. It can bind to impurities, forming complexes with polysaccharides and polyphenols in the sample. These impurities are strong inhibitors of molecular biological reactions, especially abundant in plant tissues. Heating temperatures, typically 55–65°C, accelerate cell lysis, allowing CTAB to function more effectively, while also helping to denature and inactivate proteins. The result is a complex mixture containing DNA, proteins, polysaccharide-CTAB complexes, and cell debris, with the DNA now free in the solution.Chloroform-isoamyl alcohol extraction is used to remove proteins, polysaccharide-CTAB complexes, and cell debris. Chloroform is an organic solvent that denatures proteins and dissolves them in the organic phase. Isoamyl alcohol acts as an antifoaming agent to prevent excessive foaming during vigorous mixing, which could lead to unclear layering and sample loss. Isoamyl alcohol is typically mixed with chloroform in a 24:1 ratio. After centrifugation, the solution separates into three layers: the lower organic phase contains dissolved denatured proteins, lipids, polysaccharide-CTAB complexes, and other hydrophobic impurities; the middle interface consists of a white protein precipitate, which is an aggregate of cell debris and denatured proteins; and the upper aqueous phase contains the desired clear genomic DNA, RNA, and soluble carbohydrates. Taking the supernatant involves carefully aspirating the DNA-rich upper aqueous phase, thus achieving efficient separation of DNA from most impurities. Ethanol is used to precipitate DNA, concentrating and precipitating the DNA, and further removing soluble impurities such as salts. Pre-cooled anhydrous ethanol is used because DNA is insoluble in ethanol. After adding ethanol, it neutralizes the negative charge on the surface of DNA molecules, destroys their hydration layer, and thus causes the DNA to precipitate from the solution. Gently invert and stir to mix the ethanol and aqueous phase thoroughly, inducing the DNA to form a white filamentous precipitate. Centrifuge the flocculent DNA precipitate at high speed to the bottom of the tube, forming a small visible white precipitate. Collecting the precipitate means discarding the supernatant containing remaining soluble impurities. The DNA is highly concentrated and purified, and the solvent system is changed from salt solution to ethanol. The DNA was dissolved in enzyme-free water, and the purified DNA precipitate was redissolved in a buffer suitable for downstream reactions. Enzyme-free water (RNase-free water) was added to the precipitate to ensure that the water was free of RNase (ribonuclease) to avoid degradation of RNA components (such as crRNA or in vitro transcription template) that may be used in subsequent CRISPR reactions. It was also free of DNase to protect the extracted DNA. A pure genomic DNA solution was obtained that can be used for downstream RPA amplification. At this point, the DNA extract was highly pure, free of obvious PCR / RPA inhibitors, and at a suitable concentration, making it an ideal starting material for highly sensitive molecular detection.This DNA extraction method boasts high purity, effectively removing the three main inhibitors—polysaccharides, polyphenols, and proteins—through CTAB combined with impurity and chloroform extraction, ensuring the high efficiency of subsequent enzymatic reactions (RPA and CRISPR). It also exhibits high integrity; the gentle CTAB method extracts high-molecular-weight genomic DNA with superior integrity compared to many aggressive rapid lysis methods, enabling long-fragment amplification. Furthermore, it is highly applicable, particularly suitable for difficult-to-process sample types like plant tissues, making it one of the gold standard methods in plant molecular biology research. Finally, it demonstrates reliability, with highly stable and reliable purification results, laying a solid foundation for the accuracy and repeatability of the entire pathogen detection process. The quality of the extracted DNA is the first and most crucial guarantee against false negatives. This CTAB extraction method serves as the "frontline outpost" of the entire detection process, ensuring that the most authentic and pure pathogen DNA is delivered to the subsequent RPA-CRISPR system; its success directly determines the success or failure of the entire mission.

[0020] Preferably, the amount of the root rot pathogen to be tested in step (S01-1) is 1 mL to 3 mL; the amount of the root rot pathogen to be tested in step (S01-1) is 1.5 mL to 2.5 mL; the amount of the root rot pathogen to be tested in step (S01-1) is 2 mL. These limited amounts of the root rot pathogen to be tested ensure sufficient starting material while matching the reagent volume in downstream steps. Too little material may result in an insufficient concentration of pathogen DNA, which is insufficient to trigger detection after extraction, leading to false negatives; too much material will result in incomplete lysis and excessively high levels of polysaccharide and polyphenol impurities, inhibiting subsequent reactions.

[0021] Preferably, the centrifuge container in step (S01-1) is a centrifuge tube. Centrifuge tubes are specifically designed for centrifugation, are resistant to chemical corrosion, CTAB and chloroform, and have a conical bottom that facilitates the accumulation of precipitates and the aspiration of supernatant. The cap provides a seal to prevent the leakage of harmful vapors and avoid cross-contamination of samples.

[0022] Preferably, the amount of CTAB buffer added in step (S01-2) is 600-800 μL; the amount of CTAB buffer added in step (S01-2) is 650-750 μL; the amount of CTAB buffer added in step (S01-2) is 700 μL. Limiting the amount of CTAB buffer added enables efficient lysis and protection, lysing cells and forming complex precipitates with polysaccharides and polyphenols.

[0023] Preferably, the pH of the CTAB buffer in step (S01-2) is 7–7.5; the pH of the CTAB buffer in step (S01-2) is 7.2–7.4. Limiting the pH of the CTAB buffer provides a neutral-to-alkaline stable environment, which is the pH range in which DNA is most stable, effectively preventing depurination and degradation of DNA under acidic conditions.

[0024] Preferably, the CTAB buffer in step (S01-2) comprises Tris-HCl, EDTA, NaCl, and SDS. Tris-HCl, as the main component of the buffer system, maintains pH stability during the reaction and resists pH changes caused by acidic substances released from cell lysis. EDTA, as a metal ion chelating agent, can strongly bind divalent cations such as Mg²⁺, which are essential cofactors for DNase activity. By "starving" DNase, EDTA effectively protects the released DNA from degradation. NaCl provides a high ionic strength environment, which helps to release proteins such as histones from DNA, allowing DNA to be fully released into the solution, and promotes the formation of insoluble complexes between CTAB and polysaccharides and polyphenols.

[0025] SDS is a strong anionic detergent that is primarily responsible for emulsifying lipids and dissolving membrane proteins. It is a key component in disrupting cell membranes and organelle membranes, and works synergistically with CTAB to completely lyse cells.

[0026] Preferably, the concentration of Tris-HCl used in the CTAB buffer in step (S01-2) is 0.05M to 0.15M; the concentration of Tris-HCl used in the CTAB buffer in step (S01-2) is 0.1M. Limiting the concentration of Tris-HCl can better maintain pH stability during the reaction.

[0027] Preferably, the concentration of EDTA used in the CTAB buffer in step (S01-2) is 40mM to 60mM; the concentration of EDTA used in the CTAB buffer in step (S01-2) is 45mM to 55mM; the concentration of EDTA used in the CTAB buffer in step (S01-2) is 50mM. Limiting the concentration of EDTA allows for better binding of divalent cations such as Mg²⁺.

[0028] Preferably, the concentration of NaCl used in the CTAB buffer in step (S01-2) is 0.4M to 0.6M; the concentration of NaCl used in the CTAB buffer in step (S01-2) is 0.45M to 0.55M; and the concentration of NaCl used in the CTAB buffer in step (S01-2) is 0.5M. Limiting the concentration of NaCl provides a better high ionic strength environment.

[0029] Preferably, the concentration of SDS used in the CTAB buffer in step (S01-2) is 4 mM to 6 mM; the concentration of SDS used in the CTAB buffer in step (S01-2) is 4.5 mM to 5.5 mM; the concentration of SDS used in the CTAB buffer in step (S01-2) is 5 mM. Limiting the concentration of SDS allows for better emulsification of lipids and dissolution of membrane proteins.

[0030] Preferably, the heating method in step (S01-2) is constant temperature water bath heating. Constant temperature water bath heating has the characteristics of uniform and gentle heating, avoiding local overheating that could lead to DNA breakage, and is more ideal than metal baths or dry baths.

[0031] Preferably, the heating temperature in step (S01-2) is 50℃~70℃; the heating temperature in step (S01-2) is 55℃~65℃; the heating temperature in step (S01-2) is 60℃. The defined heating temperature is high enough to accelerate cell lysis, denature proteins, and promote the binding of CTAB to impurities, but not high enough to cause severe DNA breakage.

[0032] Preferably, the heating time in step (S01-2) is 20 min to 45 min; the heating time in step (S01-2) is 25 min to 40 min; and the heating time in step (S01-2) is 30 min. These defined heating times ensure that the sample, especially the tough fungal cell walls, is fully lysed.

[0033] Preferably, the amount of chloroform-isoamyl alcohol mixed solution added in step (S01-3) is equal to the volume of CTAB buffer added in step (S01-2). Adding the same volume of chloroform-isoamyl alcohol mixed solution as the CTAB buffer most effectively achieves phase separation and protein partitioning, ensuring extraction efficiency; organic extraction precisely removes proteins and impurities.

[0034] Preferably, in step (S01-3), the mass ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol mixed solution is 20-28:1; in step (S01-3), the mass ratio of chloroform to isoamyl alcohol is 21-27:1; in step (S01-3), the mass ratio of chloroform to isoamyl alcohol is 22-26:1; in step (S01-3), the mass ratio of chloroform to isoamyl alcohol is 23-25:1; and in step (S01-3), the mass ratio of chloroform to isoamyl alcohol is 24:1. The specific mass ratio of chloroform to isoamyl alcohol ensures that chloroform fully denatures and dissolves the protein; and that isoamyl alcohol effectively reduces foaming during mixing, resulting in a clear interface between the aqueous and organic phases, facilitating the absorption of the supernatant, and preventing cross-contamination.

[0035] Preferably, the centrifugation speed in step (S01-3) is 10000 rpm to 14000 rpm; the centrifugation speed in step (S01-3) is 11000 rpm to 13000 rpm; and the centrifugation speed in step (S01-3) is 12000 rpm. These defined centrifugation speeds ensure that proteins, cell debris, and polysaccharide-CTAB complexes are completely precipitated onto the organic phase and interface, thereby obtaining a clear supernatant.

[0036] Preferably, the stirring temperature in step (S01-3) is 2℃~6℃; the stirring temperature in step (S01-3) is 3℃~5℃; the stirring temperature in step (S01-3) is 4℃. These defined stirring temperatures reduce the risk of DNA degradation at high temperatures.

[0037] Preferably, the centrifugation time in step (S01-3) is 5 min to 15 min; the centrifugation time in step (S01-3) is 7 min to 13 min; the centrifugation time in step (S01-3) is 9 min to 11 min; and the centrifugation time in step (S01-3) is 10 min. The specified centrifugation time ensures complete separation.

[0038] Preferably, in step (S01-4), the amount of pre-cooled anhydrous ethanol added is 1.5 to 2.5 times the volume of the supernatant; in step (S01-4), the amount of pre-cooled anhydrous ethanol added is twice the volume of the supernatant. Limiting the amount of pre-cooled anhydrous ethanol added most effectively neutralizes the DNA charge, allowing it to precipitate completely. Pre-cooling improves the DNA precipitation efficiency and yields a denser precipitate.

[0039] Preferably, the centrifugation speed in steps (S01-4) is 10000 rpm to 14000 rpm; the centrifugation speed in steps (S01-4) is 11000 rpm to 13000 rpm; and the centrifugation speed in steps (S01-4) is 12000 rpm. The defined centrifugation speed, temperature, and time constitute high-speed, low-temperature centrifugation, ensuring that the precipitated DNA settles tightly at the bottom of the tube, forming a visible white flocculent precipitate, facilitating the complete removal of the supernatant, which contains salts and soluble impurities.

[0040] Preferably, the stirring temperature in step (S01-4) is 2℃~6℃; the stirring temperature in step (S01-4) is 3℃~5℃; the stirring temperature in step (S01-4) is 4℃.

[0041] Preferably, the centrifugation time in step (S01-4) is 5 min to 15 min; the centrifugation time in step (S01-4) is 7 min to 13 min; the centrifugation time in step (S01-4) is 9 min to 11 min; the centrifugation time in step (S01-4) is 10 min.

[0042] Preferably, the amount of enzyme-free water added in step (S01-5) is 80 μL to 120 μL; the amount of enzyme-free water added in step (S01-5) is 90 μL to 110 μL; the amount of enzyme-free water added in step (S01-5) is 100 μL. Enzyme-free water prevents RNase contamination, protecting crRNA in subsequent CRISPR reactions, and prevents DNase contamination, which degrades freshly extracted DNA. The limited amount of enzyme-free water added is suitable for the universal and safe final volume of most downstream reactions, concentrating the DNA to a concentration suitable for use as a PCR / RPA template. Too little water may result in an excessively high DNA concentration, inhibiting the reaction; too much water will result in an excessively low concentration. These optimized parameters work together to maximize cell lysis and DNA release through optimized buffer composition and heating conditions; maximize impurity removal through precisely controlled organic extraction and precipitation conditions; maximize DNA yield and purity, ensuring the success of downstream high-sensitivity RPA-CRISPR detection; and ensure consistent results for different operators at different times, exhibiting standardization and reproducibility. High yield: thorough lysis and precipitation ensure the maximum amount of DNA obtained from limited samples; high purity: optimized CTAB formulation, precise organic extraction and precipitation washing thoroughly remove all known potent PCR / RPA inhibitors such as polysaccharides, polyphenols, proteins, and salt ions; integrity: mild lysis and precipitation conditions protect DNA integrity, ensuring the integrity of the target amplification region while not requiring ultra-long fragments; compatibility: the DNA ultimately dissolved in enzyme-free water has a salt concentration, pH, and purity fully compatible with the RPA reaction system and will not inhibit the activity of any enzymes.

[0043] Preferably, the reaction temperature in step (S02) is 35℃~40℃; the reaction temperature in step (S02) is 36℃~39℃; and the reaction temperature in step (S02) is 37℃~38℃. Within this defined reaction temperature range, the core enzymes of the RPA reaction—recombinase, polymerase, and single-strand binding protein—exhibit the highest activity. Recombinase requires this temperature to function effectively, opening the DNA double helix, and polymerase synthesizes DNA with optimal efficiency at this temperature. If the temperature is too low, enzyme activity will be insufficient, leading to a slow reaction or even failure; if the temperature is too high, the recombinase will be inactivated, resulting in a completely ineffective reaction. The defined reaction temperature of 37℃~38℃ is similar to human body temperature, meaning the reaction can even be completed by hand, with close-fitting insulation, or in a simple constant-temperature water bath, greatly reducing the equipment barrier.

[0044] Preferably, the reaction time in step (S02) is 20 min to 50 min; the reaction time in step (S02) is 22 min to 48 min; the reaction time in step (S02) is 30 min to 40 min; and the reaction time in step (S02) is 45 min. With these limited reaction times, the RPA reaction is very rapid, and the limited reaction time range is sufficient and allows for a margin of error. This ensures that even with extremely low pathogen concentrations, there is enough time to complete amplification, guaranteeing high detection sensitivity. Furthermore, this time is much shorter than traditional PCR, typically 1.5 h to 2 h, demonstrating the rapidity of RPA.

[0045] Preferably, the amount of DNA extract used in step (S02) is 0.5 μL to 2 μL; or 1 μL to 1.5 μL. This limited amount of DNA extract is sufficient to contain several copies of the target sequence even if the concentration of pathogen DNA in the sample is very low; the sufficient amount ensures amplification; and it avoids inhibition, as trace amounts of inhibitors may remain in the DNA extract. Using an excessively large volume may introduce these inhibitors into the RPA system, inhibiting enzyme activity and leading to false negatives. The limited amount provides a template while maximally diluting potential inhibitors; thus, it minimizes the risk of false negatives while ensuring high detection sensitivity.

[0046] Preferably, the RPA reaction system in step (S02) includes PA buffer, specific RPA primers RPA-F and RPA-R, enzyme-free water, and a starter. The reaction system components form a precisely proportioned "chemical reactor," a carefully balanced mixture.

[0047] Preferably, the PA buffer concentration in the RPA reaction system of step (S02) is 20 μL to 30 μL; the PA buffer concentration in the RPA reaction system of step (S02) is 25 μL. The PA buffer is the master mixture, which contains all the core enzymes required for the RPA reaction (recombinase, strand displacement polymerase, single-strand binding protein), dNTPs (raw materials for DNA synthesis), energy substances (such as ATP), and an optimized buffer system (providing suitable pH and ionic strength). Users only need to add primers, template, and water, which simplifies the operation and improves reproducibility; the limited concentration balances reagent cost and detection sensitivity.

[0048] Preferably, the concentration of the specific RPA primer RPA-F in the RPA reaction system in step (S02) is 5 μM to 15 μM; the concentration of the specific RPA primer RPA-F in the RPA reaction system in step (S02) is 10 μM.

[0049] Preferably, the content of specific RPA primer RPA-F in the RPA reaction system of step (S02) is 1 μL to 3 μL; the content of specific RPA primer RPA-F in the RPA reaction system of step (S02) is 1.5 μL to 2.5 μL; the content of specific RPA primer RPA-F in the RPA reaction system of step (S02) is 2 μL. The specified concentrations and contents of RPA-F are validated optimal working concentrations. Sufficient concentration ensures that enough primer molecules bind to the template, driving the reaction rapidly; it avoids nonspecificity, as excessively high concentrations increase the risk of primer dimerization and nonspecific amplification, while excessively low concentrations result in low reaction efficiency; it provides high specificity and high-efficiency amplification.

[0050] Preferably, the concentration of the specific RPA primer RPA-R in the RPA reaction system in step (S02) is 5 μM to 15 μM; the concentration of the specific RPA primer RPA-R in the RPA reaction system in step (S02) is 10 μM.

[0051] Preferably, the content of specific RPA primer RPA-R in the RPA reaction system of step (S02) is 1 μL to 3 μL; the content of specific RPA primer RPA-R in the RPA reaction system of step (S02) is 1.5 μL to 2.5 μL; the content of specific RPA primer RPA-R in the RPA reaction system of step (S02) is 2 μL. Limiting the concentration and content of RPA-R is a validated optimal working concentration. Sufficient concentration ensures that enough primer molecules bind to the template, driving the reaction rapidly; it avoids nonspecificity, as excessively high concentrations increase the risk of primer dimerization and nonspecific amplification, while excessively low concentrations result in low reaction efficiency; it provides high specificity and high efficiency amplification.

[0052] Preferably, the enzyme-free water content in the RPA reaction system of step (S02) is 15 μL to 20 μL; the enzyme-free water content in the RPA reaction system of step (S02) is 16 μL to 19 μL; the enzyme-free water content in the RPA reaction system of step (S02) is 17 μL to 18 μL. The defined enzyme-free water content can make up the volume, ensuring the entire reaction system reaches the standard final volume, guaranteeing the accurate concentration of all reaction components, and preventing RNase / DNase contamination, thus protecting the reaction components.

[0053] Preferably, the initiator content in the RPA reaction system of step (S02) is 2 μL to 4 μL; the initiator content in the RPA reaction system of step (S02) is 2.5 μL to 3.5 μL; the initiator content in the RPA reaction system of step (S02) is 3 μL. The initiator is a magnesium acetate solution. Mg²⁺ is an essential cofactor for DNA polymerase activity and is added at the beginning of the reaction to initiate the entire amplification process. The limited initiator content can effectively achieve "one-click start". Adding the initiator after mixing all components can prevent the reaction from starting prematurely during mixing, improving the consistency and controllability of the reaction. These parameters together optimize the RPA recombinase polymerase amplification reaction, enabling it to efficiently and specifically amplify trace amounts of target DNA fragments exponentially under isothermal and rapid conditions, providing sufficient "targets" for subsequent CRISPR detection. The effect is to achieve rapid nucleic acid amplification without the need for a thermal cycler. Rapid amplification: At a constant temperature of 37°C, the target DNA fragment can be amplified billions of times in just 25 minutes, far exceeding the speed of PCR; Ultra-high sensitivity: The optimized system can detect single-digit pathogen genome copies, enabling early diagnosis; Excellent specificity: Carefully designed primers and their optimal concentrations ensure the specificity of amplification, laying a solid foundation for subsequent CRISPR detection; Extremely simple operation: Simply mix the template, primers, water, and master mixture, and finally add the initiator. No complex instruments are required; a temperature block is sufficient, making it perfect for on-site testing; Stable results: Precise component concentrations and volumes ensure intra- and inter-batch repeatability, making the test results reliable and trustworthy.

[0054] Preferably, the reaction temperature in step (S03) is 35℃~40℃; the reaction temperature in step (S03) is 36℃~39℃; the reaction temperature in step (S03) is 37℃~38℃. The defined reaction temperature is completely consistent with the upstream RPA reaction temperature, allowing the RPA reaction tube to be directly transferred to the CRISPR reaction without changing the temperature, achieving seamless integration and simplifying operation. The SrCas12a protease exhibits optimal activity at this temperature, efficiently completing target recognition and trans-cleavage, maintaining the advantage of isothermal detection, eliminating the need for complex instruments, and ensuring the reaction efficiency of the Cas12a enzyme.

[0055] Preferably, the reaction time in step (S03) is 30 min to 55 min; the reaction time in step (S03) is 33 min to 44 min; the reaction time in step (S03) is 36 min to 40 min; and the reaction time in step (S03) is 38 min. This defined reaction time provides a sufficient and efficient reaction window. The first few minutes complete the binding and activation of the Cas12a-crRNA complex with the target DNA. In the remaining minutes, the activated Cas12a enzyme continuously and aggressively cleaves the reporter probe—the FAM Biotin probe—achieving a cascade amplification of the signal. This ensures that even with a low RPA product concentration, there is sufficient time to accumulate and generate a sufficiently strong detection signal.

[0056] Preferably, the amount of RPA reaction product used in step (S03) is 1 μL to 3 μL; the amount of RPA reaction product used in step (S03) is 1.5 μL to 2.5 μL; the amount of RPA reaction product used in step (S03) is 2 μL. This limited amount of RPA reaction product provides sufficient target, balancing target quantity with the risk of inhibition, and contains a large amount of target DNA fragments, sufficient to ensure rapid and efficient activation of the Cas12a system. It also avoids system inhibition; certain components in the RPA reaction system, such as high concentrations of salt, proteins, and dNTPs, may inhibit the Cas12a reaction. Using this limited amount can maximize the dilution of these potential inhibitors, avoiding their impact on the efficiency of the CRISPR reaction. While providing sufficient "trigger," it ensures the "purity" and high efficiency of the CRISPR reaction system.

[0057] Preferably, the RPA-CRISPR-Cas12a test strip detection system in step (S03) includes SrCas12a protein, 10×Buffer, guide RNA, FAM-Biotin probe, and enzyme-free water. The components of the reaction system form a precisely assembled "molecular reconnaissance machine," a precise molecular machine.

[0058] Preferably, the concentration of SrCas12a protein used in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 20 nM to 30 nM; the concentration of SrCas12a protein used in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 22 nM to 28 nM; and the concentration of SrCas12a protein used in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 25 nM.

[0059] Preferably, the SrCas12a protein content in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 0.3 μL to 0.7 μL; the SrCas12a protein content in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 0.4 μL to 0.6 μL; the SrCas12a protein content in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 0.5 μL. As the core actuator of the system, i.e., the "molecular scissors," the concentration of SrCas12a protein is crucial. Too low a concentration results in insufficient signal amplification, leading to decreased sensitivity; too high a concentration may increase non-specific cutting. Specific concentrations and contents of SrCas12a protein are used to achieve a high signal-to-noise ratio.

[0060] Preferably, the content of 10×Buffer in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 1 μL to 3 μL; the content of 10×Buffer in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 1.5 μL to 2.5 μL; and the content of 10×Buffer in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 2 μL. The defined 10×Buffer content provides the optimal biochemical environment for the Cas12a enzyme, including suitable pH, ionic strength (especially Mg²⁺, which is an essential cofactor for Cas12a cleavage activity), and stabilizers, ensuring that the Cas12a enzyme maintains the correct conformation and highest activity.

[0061] Preferably, in step (S03) of the RPA-CRISPR-Cas12a test strip detection system, the concentration of the guiding RNA primer crRNA is 20 nM to 30 nM; in step (S03) of the RPA-CRISPR-Cas12a test strip detection system, the concentration of the guiding RNA primer crRNA is 22 nM to 28 nM; in step (S03) of the RPA-CRISPR-Cas12a test strip detection system, the concentration of the guiding RNA primer crRNA is 25 nM.

[0062] Preferably, the content of the guide RNA primer crRNA in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 0.5 μL to 1.5 μL; the content of the guide RNA primer crRNA in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 0.8 μL to 1.2 μL; the content of the guide RNA primer crRNA in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 1 μL. The guide RNA acts as the system's "navigator" and "activation switch," and its sequence (SEQ ID NO.3) determines the specificity of recognition. Limiting the concentration and content of the guide RNA ensures that a large number of guide molecules can quickly bind to the Cas12a protein to form a complex and rapidly find the target, thus accelerating the reaction speed.

[0063] Preferably, the concentration of the FAM-Biotin probe used in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 700 nM to 800 nM; the concentration of the FAM-Biotin probe used in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 720 nM to 780 nM; and the concentration of the FAM-Biotin probe used in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 750 nM.

[0064] Preferably, the concentration of FAM-Biotin probe in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 1 μL to 3 μL; the concentration of FAM-Biotin probe in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 1.5 μL to 2.5 μL; and the concentration of FAM-Biotin probe in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 2 μL. The FAM-Biotin probe, acting as the system's "signal flare," is typically a short-chain ssDNA with FAM (fluorescent reporter group) and Biotin / TAMRA (quencher or capture group) labeled at both ends. It is the substrate for Cas12a's trans-cleavage activity. When uncleaved, fluorescence is quenched; once cleaved, the reporter group separates, emitting fluorescence. Limiting the concentration and content of the FAM-Biotin probe ensures sufficient substrate for generating a strong signal without waste or excessive background.

[0065] Preferably, the enzyme-free water content in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 10 μL to 15 μL; the enzyme-free water content in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 11 μL to 14 μL; the enzyme-free water content in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 12 μL to 13 μL; and the enzyme-free water content in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 12.5 μL. Limiting the enzyme-free water content ensures the system volume is made up to the standard final volume, guaranteeing accurate final concentrations of all reaction components, preventing RNase contamination, and protecting the integrity of the crRNA. These parameters collectively optimize the CRISPR-Cas12a reaction, enabling it to accurately identify RPA amplification products and, once successful, efficiently trigger a strong signal amplification effect. This results in ultra-high specificity for secondary verification of RPA products and ultra-high sensitivity for converting nucleic acid signals into readable optical / visual signals. Dual Specificity Validation - Ultra-high specificity: Signal generation strictly depends on the simultaneous occurrence of two independent events: (a) specific amplification of the RPA primer pair; (b) specific recognition of the amplification product by crRNA. Any non-specific amplification cannot activate Cas12a, thus reducing the false positive rate to an extremely low level. Secondary Signal Amplification - Ultra-high Sensitivity: RPA performs the first amplification - nucleic acid exponential amplification, and Cas12a's trans-cleavage performs the second amplification - one activated Cas enzyme cleaves thousands of reporter molecules. This cascade amplification effect maximizes detection sensitivity. Isothermal and Rapid: At 37℃, the entire process from recognition to signal amplification can be completed within 25 minutes, perfectly compatible with upstream RPA, maintaining the advantage of isothermal amplification and detection throughout the process. Strong Signal Output Compatibility: The generated FAM fluorescence signal can be used for quantitative analysis with a fluorescence reader or directly for visual detection with lateral flow test strips, offering exceptional flexibility.

[0066] Preferably, in step (S04), the volume is adjusted to 50 μL. Adjusting the volume to 50 μL standardizes the CRISPR reaction product, creating optimal conditions for test strip detection; optimizes ionic strength and viscosity, as the original CRISPR reaction system has a high ionic concentration and is quite viscous. Direct addition may result in slow or uneven chromatography speeds on the test strip, or even blockage of the cellulose membrane, leading to false negatives or invalid results. Diluting with RNase-free water reduces ionic strength and viscosity; standardizes the volume, ensuring that the physicochemical properties of the sample added to the test strip are consistent for each test, thus stabilizing the chromatography speed and time and guaranteeing reproducibility; provides sufficient sample volume, as 50 μL is sufficient to completely immerse the sample pad of the test strip, ensuring that the solution can fully and completely chromatize the entire strip through capillary action; and avoids detection failures due to system incompatibility.

[0067] Preferably, in step (S05), if both the control line and the test line appear simultaneously, it is a positive reaction, indicating the presence of *Fusarium solani*; if neither the control line nor the test line appears simultaneously, it is a negative reaction, indicating the absence of *Fusarium solani*. The result interpretation criteria provide a clear, intuitive, and unambiguous set of rules for determining results; eliminating subjective ambiguity by providing absolute "yes / no" answers, allowing different operators to reach consistent conclusions and avoiding misjudgments caused by differing personal interpretations; regarding reliability, the appearance of the control line (C line) proves that the test strip itself is effective, increasing the credibility of the results.

[0068] Preferably, the reaction time of the RPA-CRISPR-Cas12a nucleic acid test strip in step (S05) within the reaction container after volume adjustment is 3 to 6 minutes; the reaction time of the RPA-CRISPR-Cas12a nucleic acid test strip in step (S05) within the reaction container after volume adjustment is 4 to 5 minutes. The defined reaction time is the optimal observation time window for the test strip's chromatography and color development. If the time is too short, the solution may not be fully chromatographically developed, and the C and T lines may not be fully developed, making it easy to misinterpret weak positives or negatives as false negatives. If the time is too long, the test strip may become excessively dry, or the background signal may deepen, affecting the interpretation of the band clarity. Within the defined reaction time range, chromatography is fully completed, the band color contrast is strongest, the background is clean, and the results are clearest and most stable. This ensures accuracy by interpreting the results at the optimal time point, maximizing the distinction between true positives and true negatives and reducing misinterpretations. It also ensures speed by specifying that the final result can be read after only 5 minutes, meeting the needs of rapid on-site testing. The combined effect of these parameters achieves standardized testing conditions. Volume adjustment unifies the physicochemical properties of the solution, ensuring stable test strip results. It also enables clear result interpretation, defining precise and unambiguous criteria for positive and negative results and optimizing interpretation time, allowing anyone to easily and accurately read the results. Volume adjustment ensures the test strip, acting as a "display," receives a clear and interference-free "input source." The interpretation criteria provide a clear and unambiguous "user manual," instructing users on how to interpret the information displayed on the screen. The reaction time specifies the optimal time to read the "screen," preventing both over-reading and distorted results. Any user, even without a professional background, can obtain a clear and reliable "yes" or "no" answer regarding whether a crop is infected with Fusarium solani within 30-40 minutes after sample processing through a simple "strip reading" step, truly democratizing the application of high-tech molecular detection technology.

[0069] The fourth technical solution of this invention: the application of the RPA-CRISPR-Cas12a test strip detection system in detecting actual pathogen-carrying samples of crops. The ultimate purpose and application scenario of the solution are clearly defined, indicating that this invention is intended to directly serve agricultural production practices, used to diagnose whether crop roots (such as eggplant) carry the pathogen *Fusarium solani*. This sets the application tone for all verification experiments and subsequent promotion of the solution.

[0070] Preferably, the crop is eggplant, tomato, tobacco, soybean, potato, or apple.

[0071] As a preferred method, the RPA-CRISPR-Cas12a specificity of the crop root rot pathogen was tested. Specificity testing verifies the ability of this detection method to "recognize only the target pathogen and not other pathogens," ensuring no false positives. The biggest risk with detection methods is "false positives," misidentifying beneficial or unrelated bacteria as the target pathogen; specificity testing aims to eliminate this risk.

[0072] Preferably, the method for testing the specificity of the RPA-CRISPR-Cas12a test strip for crop root rot pathogens is as follows: the specificity of the RPA-CRISPR-Cas12a test strip for Fusarium solani, Fusarium proliferatum, Fusarium fuijkuroi, Fusarium oxysporum, Fusarium incarnatum, Fusarium commune, Botrytis cinerea, Colletotrichum fructicola, Colletotrichum fructicola, Alternaria alternata, and Fusarium solani is tested sequentially. The test list is highly representative, consisting of closely related species such as *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium oxysporum*, *Fusarium oxysporum*, and *Fusarium oxysporum*. These fungi are closely related to *Fusarium oxysporum* and have high gene sequence similarity, making them the most likely to cause cross-reactions and false positives. The ability to distinguish them demonstrates the extremely high specificity of the primer and crRNA design. Common pathogens, such as *Botrytis cinerea*, *Anthracnose* (*Fructus*, *Colletotrichum*), and *Alternaria*, are also common pathogens in the field, demonstrating that the method is not affected by other common diseases. This demonstrates the high reliability of the detection method.

[0073] As a preferred method, the sensitivity of the RPA-CRISPR-Cas12a test strip for crop root rot pathogens was tested. Sensitivity testing determines the minimum number of pathogens that the method can detect, i.e., its "detection limit." Sensitivity determines whether the method can be used for early diagnosis; only when pathogens are detected at a very low level can timely warnings and control be implemented.

[0074] Preferably, the sensitivity test of the RPA-CRISPR-Cas12a test strip against crop root rot pathogens includes the following steps: (C01) taking an array of genomic DNA extracts of the crop root rot pathogens to be tested and diluting them sequentially to 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; (C02) taking appropriate amounts of the diluted genomic DNA extracts of the crop root rot pathogens to be tested from step (C01) and reacting them with the RPA-CRISPR-Cas12a test strip. A gradient dilution sample is prepared, with a dilution gradient from 10 ng / μL to 1 fg / μL, representing a large dilution series with a total of 8 concentrations, differing by 10 ng / μL. 7 (Times); Unit explanation: fg (femtogram) is one-thousandth of pg (picogram). 1 fg of DNA is roughly equivalent to the weight of several fungal genomes. Diluting DNA to a concentration of 100 fg / μL means that there may only be a few percent of DNA copies on average in 1 μL of sample, which challenges the limits of detection. By preparing samples of known concentrations, it is possible to accurately determine at what concentration the method becomes undetectable, thereby determining its detection limit.

[0075] Preferably, the amount of the diluted genomic DNA extract of the root rot pathogen of the test crop used in step (CO2) is 1 μL to 3 μL; the amount of the diluted genomic DNA extract of the root rot pathogen of the test crop used in step (CO2) is 1.5 μL to 2.5 μL; the amount of the diluted genomic DNA extract of the root rot pathogen of the test crop used in step (CO2) is 2 μL. Each dilution concentration was tested, with a dosage of 2 μL, representing the amount of DNA added to the reaction system at 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg, respectively, demonstrating the system's performance at ultra-low copy numbers. The results show that this method can still provide clear positive signals at levels of 1 pg and even 100 fg, proving its extremely high sensitivity and ability to detect trace amounts of pathogen DNA. In field applications, this means that even a very small amount of Fusarium rot infecting crop roots can be detected promptly, enabling true early diagnosis and saving valuable time for prevention and control. These validations collectively constitute the "performance evaluation certificate" for this RPA-CRISPR-Cas12a test strip detection method. Rigorous experiments have demonstrated that this method possesses the two most critical properties required for practical application: high specificity and high sensitivity, providing solid scientific data support for its reliability and demonstrating its practical application in complex field conditions. Two major validation experiments, specificity and sensitivity, are the "gold standard" for evaluating a diagnostic method. The specificity test answers the question "How accurate is it?", proving that the method will not harm innocent pathogens and that the results are reliable. The sensitivity test answers the question "How effective is it?", proving that the method is highly accurate and can detect early and hidden infections. Through these systematic validations, the RPA-CRISPR-Cas12a test strip detection method has transformed from a theoretically feasible technology into a reliable tool with clearly defined performance parameters and proven in practice. This allows researchers, plant protection experts, and farmers to confidently use this method for the diagnosis and monitoring of crop diseases, as all its performance characteristics have been pre-assessed and proven.

[0076] The present invention has the following beneficial effects: (1) The specific RPA primers RPA-F and RPA-R are like "precision-guided radar", which can quickly and specifically identify and replicate the unique gene fragments of Fusarium solani in complex sample environments, thereby achieving highly sensitive detection of Fusarium solani and providing a key tool for the early diagnosis and control of crop root rot; According to the base complementary pairing principle (AT,CG), the forward primer RPA-F can accurately bind to one strand of the target DNA of Fusarium solani, while the reverse primer RPA-R can bind to the other strand. The binding site is a unique sequence of Fusarium solani. The genes of other microorganisms (such as other fungi, bacteria or crop DNA) do not contain this sequence, which is tailor-made for the recombinase polymerase amplification RPA technology; (2) In the RPA reaction system, the pair of primers interact with the recombinase Multiple enzymes, such as single-stranded DNA binding protein and DNA polymerase, work together to complete exponential amplification; the recombinase binds to the primer to form a complex, which can scan the entire genomic DNA and accurately locate the target DNA site that is completely complementary to the primer sequence, and then invade and open the DNA double strand, so that the primer binds to the template; the DNA polymerase starts from the 3' end of the primer, uses the target DNA as a template, and rapidly synthesizes a new DNA chain, which then serves as the template for the next round of reaction. The whole process is carried out continuously at a constant temperature, and within 15 to 20 minutes, a tiny amount of target DNA fragment can be amplified billions of times to produce enough nucleic acid to be detected; (3) Using this pair of specific RPA primers for detection, compared with the traditional methods of culture identification and ordinary PCR, it has the characteristics of good sensitivity, good specificity and sufficient reliability, among which the primer sequence SEQ ID NO.1 and SEQ ID NO.2 are specific gene regions for Fusarium solani. They can effectively distinguish Fusarium solani from other closely related Fusarium species, minimizing false positive results and exhibiting high specificity. RPA technology itself is extremely sensitive and can detect single-digit pathogen genomes. This means that in the early stages of infection, when the number of pathogens is still very small, they can be successfully detected, enabling early warning and exhibiting high sensitivity. (4) The entire detection process from sample processing to result output can be shortened to within 30 minutes, which is fast and efficient, while traditional PCR takes several hours. This is crucial for agricultural production and plant quarantine scenarios that require rapid on-site decision-making. RPA reaction is carried out at a constant temperature, making it easy to operate. It does not require expensive PCR instruments such as thermal cyclers. Simple temperature control, such as holding it in your hand, keeping it warm, or using a simple thermostat, can complete the amplification, greatly reducing the equipment threshold and making it possible to conduct detection in the field and on-site laboratories. The detection results are clear and easy to interpret, reducing human error and ensuring high reliability.Researchers, plant protection personnel, or agricultural experts can use this invention to quickly confirm whether the culprit causing crop root rot is Fusarium solani, thereby guiding farmers to use pesticides accurately, select effective fungicides against the fungus, avoid blind use of pesticides, and reduce economic losses and environmental pesticide residues; (5) For the first time, RPA-lateral flow chromatography technology was used to establish a rapid detection method for Fusarium solani, and through specificity and sensitivity evaluation, it can be used for the detection of actual samples, providing a sensitive and reliable new method for the field detection of Fusarium solani; The target gene primers selected were obtained through a large number of experimental screenings, with good specificity and no cross-reaction with other pathogens; The primer probes used have good amplification effect and strong band specificity, and can form a high concentration of primer-probe heterodimers in the detection area, thus making the test strip show a strong positive reaction and increasing the detection sensitivity; The established detection method can detect 100 fg of Fusarium solani The genome of Fusarium tumefaciens; (6) The method of detecting Fusarium tumefaciens by combining RPA technology with lateral flow chromatography has the advantages of high sensitivity and high throughput of molecular biological detection, as well as good specificity and simple operation of immunological detection. It does not require complicated instruments and is especially suitable for rapid screening and detection of Fusarium tumefaciens in grassroots laboratories and on-site. The detection speed is fast. Compared with conventional PCR, it does not need to go through the three steps of denaturation, annealing and extension. The optimal temperature of RPA reaction is between 37℃ and 40℃. No denaturation is required. The reaction can be completed in about 20 minutes at room temperature. It does not require complicated instruments and equipment and is suitable for on-site detection. It realizes isothermal amplification. Unlike PCR, which requires thermal cycling, it gets rid of the dependence on thermal cycling instruments. As long as there is a stable heat source, RPA reaction can occur, which greatly expands the scope of RPA use and can truly realize portable on-site rapid nucleic acid detection. (7) It can be used for the rapid detection of Fusarium solani in the tissues of infected crop plants. The detection process can be completed in about 1 hour, making it an effective means of detecting Fusarium solani. The detection method is used for early detection of Fusarium solani and for disease prediction and forecasting, which is of great significance for determining the appropriate control period and effectively controlling the disease. Attached Figure Description

[0077] Figure 1 This is a diagram showing the specificity test results of the RPA-CRISPR-Cas12a test strip for eggplant root rot pathogen in Example 15 of the present invention; Figure 2 This is a graph showing the sensitivity test results of the RPA-CRISPR-Cas12a test strip for eggplant root rot pathogen in Example 16 of the present invention. Figure 3 This is a diagram of the RPA-CRISPR-Cas12a test strip detection system in Example 17 of the present invention detecting actual bacterial-carrying eggplant samples. Detailed Implementation

[0078] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0079] Example 1: Primers for detecting Fusarium solani, a root rot pathogen in crops. The primers include specific RPA primers RPA-F and RPA-R. Specific RPA primer RPA-F is shown in SEQ ID NO.1, and specific RPA primer RPA-R is shown in SEQ ID NO.2. SEQ ID NO.1 is 5'-CTTGACAGGAGGCGCCGCCAACCGCGAACGCTC-3'; SEQ ID NO.2 is 5'-TCCCGTGAGTGTTGCGTTGCGTCCCCGTGCCGT-3'.

[0080] Obtaining specific RPA primers RPA-F and RPA-R involves the following steps: (A01) searching for the whole genome sequence of the eggplant root rot pathogen in the NCBI database; (A02) screening the whole genome sequence in step (A01) based on the nr database and performing experimental verification to obtain the detection target gene; the detection target gene in step (A02) is the XM_046274036.2 gene;

[0081] (A03) Using Primer6 software, the conserved regions of the target gene were detected in step (A02), and specific RPA primers RPA-F were designed and obtained, as shown in SEQ ID NO.1; specific RPA primers RPA-R were designed and obtained, as shown in SEQ ID NO.2.

[0082] Example 2: This example is basically the same as Example 1, except that it also includes a guide RNA primer crRNA, as shown in SEQ ID NO.3. The guide RNA primer crRNA is 5'-UAAUUUCUACUAAGUGUAGAUCGGAUAUCGGCUUCUGGACG-3'.

[0083] Obtaining the guide RNA primer crRNA includes the following steps: (B01) Screening target sites based on the specific RPA primer RPA-F, the specific RPA primer RPA-R, and the PAM pattern of the CRISPR-Cas12a guide RNA; in step (B01), the PAM pattern of the CRISPR-Cas12a guide RNA is TTTN; the target site in step (B01) is 20 nt; (B02) Designing and obtaining the guide RNA primer crRNA based on the target site in step (B01), as shown in SEQ ID NO.3.

[0084] Example 3: A kit for detecting Fusarium solani, a root rot pathogen in crops, includes specific RPA primers RPA-F and RPA-R, Cas12 protein, T7 transcriptase, crRNA DNA template, DNA polymerase, and an RPA-CRISPR-Cas12a nucleic acid detection strip. The concentration of specific RPA primer RPA-F is 8 μM. The concentration of specific RPA primer RPA-R is 8 μM. The crRNA DNA template includes the T7 promoter and the guide RNA primer crRNA. The kit also includes reaction buffer, a positive control (PC), a negative control (NC), a reporter molecule (ssDNAReporter), sample DNA extraction reagents, and / or instructions for use.

[0085] Example 4: This example is basically the same as Example 3, except that the concentration of the specific RPA primer RPA-F is 12 μM. The concentration of the specific RPA primer RPA-R is 12 μM.

[0086] Example 5: This example is basically the same as Example 3, except that the concentration of the specific RPA primer RPA-F is 9 μM. The concentration of the specific RPA primer RPA-R is 9 μM.

[0087] Example 6: This example is basically the same as Example 3, except that the concentration of the specific RPA primer RPA-F is 11 μM. The concentration of the specific RPA primer RPA-R is 11 μM.

[0088] Example 7: This example is basically the same as Example 3, except that the concentration of the specific RPA primer RPA-F is 10 μM. The concentration of the specific RPA primer RPA-R is 10 μM.

[0089] Example 8: A method for detecting Fusarium solani, a root rot pathogen of crops, comprising the following steps: (S01) Extracting genomic DNA of the root rot pathogen to be tested; the extraction of genomic DNA of the root rot pathogen to be tested in step (S01) includes the following steps: (S01-1) Taking an appropriate amount of powder obtained by grinding the mycelium of the root rot pathogen to be tested in liquid nitrogen and placing it in a centrifuge container; the amount of root rot pathogen to be tested used in step (S01-1) is 1 mL; the centrifuge container in step (S01-1) is a centrifuge tube; (S01-2) In step (S01-1), add an appropriate amount of preheated CTAB buffer to the centrifuge container; in step (S01-2), the volume of CTAB buffer added is 600 μL; the pH of the CTAB buffer in step (S01-2) is 7; the CTAB buffer in step (S01-2) contains Tris-HCl, EDTA, NaCl, and SDS; the concentration of Tris-HCl used in the CTAB buffer in step (S01-2) is 0.05 M; in step (S01-2), CTA... The concentration of EDTA used in buffer B is 40 mM; the concentration of NaCl used in CTAB buffer in step (S01-2) is 0.4 M; the concentration of SDS used in CTAB buffer in step (S01-2) is 4 mM; the heating method in step (S01-2) is constant temperature water bath heating; the heating temperature in step (S01-2) is 50℃; the heating time in step (S01-2) is 45 min; (S01-3) add an appropriate amount of... After mixing the chloroform and isoamyl alcohol solution, stir and centrifuge, and collect the supernatant; the volume of the chloroform-isoamyl alcohol solution added in step (S01-3) is equal to the volume of CTAB buffer added in step (S01-2); the mass ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol solution in step (S01-3) is 20:1; the centrifugation speed in step (S01-3) is 10000 rpm; the stirring temperature in step (S01-3) is 2℃; the centrifugation time in step (S01-3) is 15 min;

[0090] (S01-4) Add an appropriate amount of pre-cooled anhydrous ethanol to the supernatant of step (S01-3), stir and centrifuge, and take the precipitate; the amount of pre-cooled anhydrous ethanol added in step (S01-4) is 1.5 times the volume of the supernatant; the centrifugation speed in step (S01-4) is 10000 rpm; the stirring temperature in step (S01-4) is 2℃; the centrifugation time in step (S01-4) is 15 min; (S01-5) Add an appropriate amount of enzyme-free water to the precipitate of step (S01-4) and mix to obtain the genomic DNA extract of the root rot pathogen of the crop to be tested; the amount of enzyme-free water added in step (S01-5) is 80 μL; (S02) Take an appropriate amount The DNA extract in step (S01) is placed in the RPA reaction system for reaction; the reaction temperature in step (S02) is 35℃; the reaction time in step (S02) is 20 min; the amount of DNA extract used in step (S02) is 0.5 μL; the RPA reaction system in step (S02) includes PA buffer, specific RPA primers RPA-F and RPA-R, enzyme-free water, and initiator; the PA buffer content in the RPA reaction system in step (S02) is 20 μL; the concentration of specific RPA primer RPA-F used in the RPA reaction system in step (S02) is 5 μM; step (S02) The specific RPA primer RPA-F in the RPA reaction system is 3 μL; the specific RPA primer RPA-R in step (S02) is 5 μM; the specific RPA primer RPA-R in step (S02) is 3 μL; the enzyme-free water in step (S02) is 15 μL; the initiator in step (S02) is 2 μL; (S03) take an appropriate amount of the RPA reaction product from step (S02) and place it in the RPA-CRISPR-Cas12a test strip detection system for reaction; the reaction temperature in step (S03) is 3... 5℃; the reaction time in step (S03) is 30 min; the amount of RPA reaction product used in step (S03) is 1 μL; the RPA-CRISPR-Cas12a test strip detection system in step (S03) includes SrCas12a protein, 10×Buffer, guide RNA, FAM-Biotin probe, and enzyme-free water; the concentration of SrCas12a protein used in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 20 nM; the content of SrCas12a protein in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 0.7 μL; In step (S03), the 10×Buffer content in the RPA-CRISPR-Cas12a test strip detection system is 1 μL; In step (S03), the concentration of the guide RNA in the RPA-CRISPR-Cas12a test strip detection system is 20 nM; In step (S03), the guide RNA content in the RPA-CRISPR-Cas12a test strip detection system is 1.5 μL; In step (S03), the concentration of the FAM-Biotin probe in the RPA-CRISPR-Cas12a test strip detection system is 700 nM; In step (S03), the FAM-Biotin probe content in the RPA-CRISPR-Cas12a test strip detection system is 3 μL; In step (S03), the RPA-CRISPR-Cas12a test strip detection system... The enzyme-free water content in the 2a test strip detection system is 10 μL; (S04) After the reaction in step (S03) is completed, RNase-free water is added to the reaction system to bring the volume to the mark; in step (S04), the volume is brought to 50 μL; (S05) The RPA-CRISPR-Cas12a nucleic acid test strip is inserted into the reaction container after volume adjustment in step (S04), and the presence of control lines and test lines, as well as color changes, are observed; in step (S05), if both control lines and test lines appear simultaneously, it is a positive reaction, indicating the presence of Fusarium solani; if neither control lines nor test lines appear simultaneously, it is a negative reaction, indicating the absence of Fusarium solani; the reaction time of the RPA-CRISPR-Cas12a nucleic acid test strip in the reaction container after volume adjustment in step (S05) is 3 minutes.

[0091] Example 9: This example is basically the same as Example 8, except that the amount of the root rot pathogen to be tested in step (S01-1) is 3 mL; the amount of CTAB buffer added in step (S01-2) is 800 μL; the pH of the CTAB buffer in step (S01-2) is 7.5; the concentration of Tris-HCl in the CTAB buffer in step (S01-2) is 0.15 M; the concentration of EDTA in the CTAB buffer in step (S01-2) is 60 mM; the concentration of NaCl in the CTAB buffer in step (S01-2) is 0.6 M; and the concentration of SD in the CTAB buffer in step (S01-2) is... The concentration of S used is 6 mM; the heating temperature in step (S01-2) is 70℃; the heating time in step (S01-2) is 20 min; the mass ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol mixed solution in step (S01-3) is 28:1; the centrifugation speed in step (S01-3) is 14000 rpm; the stirring temperature in step (S01-3) is 6℃; the centrifugation time in step (S01-3) is 5 min; the amount of pre-cooled anhydrous ethanol added in step (S01-4) is 2.5 times the volume of the supernatant; the centrifugation speed in step (S01-4) is 14000 rpm; the stirring temperature in step (S01-4) is... The reaction temperature in step (S01-4) is 6℃; the centrifugation time in step (S01-4) is 5 min; the amount of enzyme-free water added in step (S01-5) is 120 μL; the reaction temperature in step (S02) is 40℃; the reaction time in step (S02) is 50 min; the amount of DNA extract used in step (S02) is 2 μL; the content of PA buffer in the RPA reaction system in step (S02) is 30 μL; the concentration of specific RPA primer RPA-F used in the RPA reaction system in step (S02) is 15 μM; the content of specific RPA primer RPA-F in the RPA reaction system in step (S02) is 1 μL; the RPA reaction in step (S02) The concentration of the specific RPA primer RPA-R in the system was 15 μM; the content of the specific RPA primer RPA-R in the RPA reaction system in step (S02) was 1 μL; the content of enzyme-free water in the RPA reaction system in step (S02) was 20 μL; the content of the initiator in the RPA reaction system in step (S02) was 4 μL; the reaction temperature in step (S03) was 40℃; the reaction time in step (S03) was 20 min; the amount of RPA reaction product used in step (S03) was 3 μL; the concentration of SrCas12a protein in the RPA-CRISPR-Cas12a test strip detection system in step (S03) was 30 nM; the content of SrCas12a protein in the RPA-CRISPR-Cas12a test strip detection system in step (S03) was 0.3 μL; the content of 10×Buffer in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 3 μL; the concentration of guide RNA used in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 30 nM. Step (S03): The guide RNA concentration in the RPA-CRISPR-Cas12a test strip detection system is 0.5 μL; the concentration of the FAM-Biotin probe in the RPA-CRISPR-Cas12a test strip detection system is 800 nM; the concentration of the FAM-Biotin probe in the RPA-CRISPR-Cas12a test strip detection system is 1 μL; the concentration of enzyme-free water in the RPA-CRISPR-Cas12a test strip detection system is 15 μL; the reaction time of the RPA-CRISPR-Cas12a nucleic acid detection test strip in the reaction container after volume adjustment is 6 min.

[0092] Example 10: This example is basically the same as Example 8, except that the amount of the root rot pathogen to be tested in step (S01-1) is 2 mL; the amount of CTAB buffer added in step (S01-2) is 700 μL; the pH of the CTAB buffer in step (S01-2) is 7.2; the concentration of Tris-HCl in the CTAB buffer in step (S01-2) is 0.1 M; the concentration of EDTA in the CTAB buffer in step (S01-2) is 50 mM; the concentration of NaCl in the CTAB buffer in step (S01-2) is 0.5 M; and the concentration of SD in the CTAB buffer in step (S01-2) is... The concentration of S used is 5 mM; the heating temperature in step (S01-2) is 60℃; the heating time in step (S01-2) is 30 min; the mass ratio of chloroform to isoamyl alcohol in the chloroform-isoamyl alcohol mixed solution in step (S01-3) is 24:1; the centrifugation speed in step (S01-3) is 12000 rpm; the stirring temperature in step (S01-3) is 4℃; the centrifugation time in step (S01-3) is 10 min; the amount of pre-cooled anhydrous ethanol added in step (S01-4) is twice the volume of the supernatant; the centrifugation speed in step (S01-4) is 12000 rpm; the stirring temperature in step (S01-4) is... 4℃; the centrifugation time in step (S01-4) is 10 min; the amount of enzyme-free water added in step (S01-5) is 100 μL; the reaction temperature in step (S02) is 37℃; the reaction time in step (S02) is 45 min; the amount of DNA extract used in step (S02) is 1 μL; the content of PA buffer in the RPA reaction system in step (S02) is 25 μL; the concentration of specific RPA primer RPA-F used in the RPA reaction system in step (S02) is 10 μM; the content of specific RPA primer RPA-F in the RPA reaction system in step (S02) is 2 μL; the RPA reaction in step (S02) The concentration of the specific RPA primer RPA-R used in the system was 10 μM; the content of the specific RPA primer RPA-R in the RPA reaction system in step (S02) was 2 μL; the content of enzyme-free water in the RPA reaction system in step (S02) was 17 μL; the content of the initiator in the RPA reaction system in step (S02) was 3 μL; the reaction temperature in step (S03) was 37℃; the reaction time in step (S03) was 38 min; the amount of RPA reaction product used in step (S03) was 2 μL; the concentration of SrCas12a protein used in the RPA-CRISPR-Cas12a test strip detection system in step (S03) was 25 nM; the content of SrCas12a protein in the RPA-CRISPR-Cas12a test strip detection system in step (S03) was 0.5 μL; the content of 10×Buffer in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 2 μL; the concentration of guide RNA used in the RPA-CRISPR-Cas12a test strip detection system in step (S03) is 25 μL. nM; Step (S03) The content of guide RNA in the RPA-CRISPR-Cas12a test strip detection system is 1 μL; Step (S03) The concentration of FAM-Biotin probe used in the RPA-CRISPR-Cas12a test strip detection system is 750 nM; Step (S03) The content of FAM-Biotin probe in the RPA-CRISPR-Cas12a test strip detection system is 2 μL; Step (S03) The content of enzyme-free water in the RPA-CRISPR-Cas12a test strip detection system is 12.5 μL; Step (S05) The reaction time of the RPA-CRISPR-Cas12a nucleic acid detection test strip in the reaction container after volume adjustment is 5 min. After the reaction is complete, add 30 μL of Nase-free water to the reaction system to bring the total volume to 50 μL. Then insert the Cas12 / 13 nucleic acid test strip into the reaction tube. After 4–5 minutes, observe the appearance of the control line and test line on the test strip, as well as their color changes. If both the control line (C line) and test line (T line) appear simultaneously, it is a positive reaction, proving the presence of *Fusarium solani*. This invention has high specificity, high sensitivity, accurate results, and high practicality.

[0093] Example 11: Application of the RPA-CRISPR-Cas12a system in detecting actual crop samples carrying pathogens. The RPA-CRISPR-Cas12a specificity of crop root rot pathogens was tested. The method for testing the RPA-CRISPR-Cas12a specificity of crop root rot pathogens was as follows: the RPA-CRISPR-Cas12a test strips for *Fusarium solani*, *Fusarium proliferatum*, *Fusarium fuijkuroi*, *Fusarium oxysporum*, *Fusarium incarnatum*, *Fusarium commune*, *Botrytiscinerea*, *Colletotrichum fructicola*, *Colletotrichum fructicola*, *Alternaria alternata*, and *Fusarium solani* were tested sequentially.

[0094] Example 12: Application of the RPA-CRISPR-Cas12a test strip detection system in detecting actual crop samples carrying pathogens. The sensitivity of the RPA-CRISPR-Cas12a test strip for detecting crop root rot pathogens was tested. The test included the following steps: (C01) Take several samples of genomic DNA extract of the target crop root rot pathogen and dilute them sequentially to 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; (C02) Take an appropriate amount of the diluted genomic DNA extract of the target crop root rot pathogen from step (C01) and react it with the RPA-CRISPR-Cas12a test strip; the amount of the diluted genomic DNA extract of the target crop root rot pathogen used in step (C02) is 1 μL.

[0095] Example 13: This example is basically the same as Example 12, except that the amount of the genomic DNA extract of the root rot pathogen of the crop to be tested after dilution in step (CO2) is 3 μL.

[0096] Example 14: This example is basically the same as Example 12, except that the amount of the genomic DNA extract of the root rot pathogen of the crop to be tested after dilution in step (CO2) is 2 μL.

[0097] Example 15: Application of the RPA-CRISPR-Cas12a system in detecting actual pathogen-carrying samples of crops. The specificity of the RPA-CRISPR-Cas12a test strip for eggplant root rot pathogens was tested. Following the method in Example 10, ten common eggplant pathogens were detected sequentially: *Fusarium solani*, *Fusarium proliferatum*, *Fusarium fuijkuroi*, *Fusarium oxysporum*, *Fusarium incarnatum*, *Fusarium commune*, *Botrytis cinerea*, *Colletotrichum fructicola*, *Colletotrichum fructicola*, and *Alternaria alternata*. Water was used as a negative control. The test results are as follows: Figure 1As shown, only the sample of *Fusarium solani*, the causal agent of eggplant root rot, showed a positive reaction, while other common eggplant pathogens and the water control showed negative reactions. This result indicates that the primers and methods provided in this invention have high specificity for *Fusarium solani*, the causal agent of eggplant root rot, and can effectively distinguish it from other eggplant pathogens.

[0098] Example 16: Application of the RPA-CRISPR-Cas12a test strip detection system in detecting actual crop samples carrying pathogens. The sensitivity of the RPA-CRISPR-Cas12a test strip against *Fusarium solani*, the causal agent of eggplant root rot, was tested. Following the method in Example 10, the genome copy numbers of the target *Fusarium solani* were sequentially diluted to 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg per μL. Each diluted sample was used as a template for the RPA-CRISPR-Cas12a reaction, with 2 μL of DNA template added to each reaction. The test results are as follows: Figure 2 As shown, the reaction results for samples ranging from 10 ng to 100 fg were all positive, indicating that the detection limit of the genome of eggplant root rot pathogens in this invention is as low as 100 fg / μL, demonstrating extremely high sensitivity.

[0099] Example 17: Application of the RPA-CRISPR-Cas12a test strip detection system in detecting actual infected crop samples. Six eggplant root systems infected with eggplant root rot fungus were used as test samples, with healthy eggplant root systems as controls; the test results are as follows. Figure 3 As shown, all six samples tested showed positive reactions, with color changes observed in the test strip bands, while the healthy eggplant sample showed a negative reaction. This result indicates that the present invention has a good detection effect on eggplant root rot pathogens, and can quickly and accurately detect pathogens in complex samples, showing good application prospects.

Claims

1. Primers for detecting Fusarium solani, the root rot pathogen of crops, are characterized by: The primers include specific RPA primer RPA-F and specific RPA primer RPA-R; the specific RPA primer RPA-F is shown in SEQ ID NO.1, and the specific RPA primer RPA-R is shown in SEQ ID NO.

2.

2. The primers for detecting Fusarium solani, a root rot pathogen of crops, according to claim 1, are characterized in that: The specific RPA primers RPA-F and RPA-R are obtained by the following steps: (A01) Locate the complete genome sequence of the fungus causing eggplant root rot in the NCBI database; (A02) Screen the whole genome sequences in step (A01) based on the nr database and perform experimental verification to obtain the target genes for detection; (A03) Use Primer6 software to detect the conserved regions of the target gene in step (A02). The specific RPA primer RPA-F was designed and obtained, as shown in SEQ ID NO.1; The specific RPA primer RPA-R was designed and obtained, as shown in SEQ ID NO.

2.

3. The primers for detecting Fusarium solani, a root rot pathogen of crops, as described in claim 1, are characterized in that: It also includes a guide RNA primer crRNA, as shown in SEQ ID NO.

3.

4. The primers for detecting Fusarium solani, a root rot pathogen of crops, according to claim 3, are characterized in that: Obtaining the guide RNA primer crRNA includes the following steps: (B01) Target sites were screened based on the PAM pattern of RNA guided by specific RPA primers RPA-F and RPA-R and CRISPR-Cas12a. (B02) Design and obtain the guide RNA primer crRNA according to the target site in step (B01), as shown in SEQ ID NO.

3.

5. A kit for detecting Fusarium solani, a root rot pathogen in crops, characterized by: The test includes the specific RPA primer RPA-F, the specific RPA primer RPA-R, the Cas12 protein, the T7 transcriptase, the crRNA DNA template, the DNA polymerase, and the RPA-CRISPR-Cas12a nucleic acid detection strip as described in claim 1.

6. A method for detecting Fusarium solani, a root rot pathogen in crops, characterized by: Including the following step, (S01) Extract genomic DNA from the root rot pathogen of the crop to be tested; (S02) Take an appropriate amount of the DNA extract from step (S01) and place it in the RPA reaction system for reaction; (S03) Take an appropriate amount of the RPA reaction product from step (S02) and place it in the RPA-CRISPR-Cas12a test strip detection system for reaction; (S04) After the reaction in step (S03) is completed, add RNase-free water to the reaction system and bring the volume to the mark; (S05) Insert the RPA-CRISPR-Cas12a nucleic acid test strip into the reaction container after the volume was adjusted in step (S04), and observe whether there are control lines and test lines on the test strip, as well as the color change.

7. The method for detecting Fusarium solani, a root rot pathogen of crops, according to claim 6, is characterized in that: The extraction of genomic DNA from the root rot pathogen of the crop to be tested in step (S01) includes the following steps: (S01-1) Take an appropriate amount of powder after grinding the mycelium of the root rot fungus of the crop to be tested with liquid nitrogen and place it in a centrifuge container. (S01-2) After adding an appropriate amount of CTAB buffer to the centrifuge container from step (S01-1), heat it. (S01-3) After adding an appropriate amount of chloroform-isoamyl alcohol mixed solution to the centrifuge container in step (S01-2), stir and centrifuge, and take the supernatant; (S01-4) After adding an appropriate amount of pre-cooled anhydrous ethanol to the supernatant of step (S01-3), stir and centrifuge to collect the precipitate; (S01-5) Add an appropriate amount of enzyme-free water to the precipitate from step (S01-4) and mix to obtain the genomic DNA extract of the root rot pathogen of the crop to be tested.

8. The method for detecting Fusarium solani, a root rot pathogen of crops, according to claim 6, is characterized in that: The RPA reaction system in step (S02) includes PA buffer, specific RPA primer RPA-F, specific RPA primer RPA-R, enzyme-free water, and initiator.

9. The method for detecting Fusarium solani, a root rot pathogen of crops, according to claim 6, is characterized in that: The RPA-CRISPR-Cas12a test strip detection system in step (S03) includes SrCas12a protein, 10×Buffer, guide RNA primer crRNA, FAM-Biotin probe, and enzyme-free water.

10. Application of the RPA-CRISPR-Cas12a test strip detection system in detecting actual bacterial-carrying crop samples.

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