A reagent, kit, method and application of rpa-cr ispr / cas12a-lfd for detecting fusarium oxysporum f. sp. lycopersici
By combining RPA-CRISPR/Cas12a with lateral flow chromatography technology, the need for high sensitivity, high specificity, and ease of detection of Fusarium oxysporum in eggplant has been addressed. This enables rapid and accurate detection in both primary laboratories and fields, making it suitable for rapid detection and screening of Fusarium oxysporum in primary agricultural fields.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
- Filing Date
- 2026-05-06
- Publication Date
- 2026-06-02
AI Technical Summary
Existing technologies cannot simultaneously meet the requirements of high sensitivity, high specificity, instrument-free operation, and rapid on-site detection of Fusarium oxysporum in eggplant. They also suffer from time-consuming processes, reliance on complex equipment, and a tendency to produce false positives.
The RPA-CRISPR/Cas12a combined lateral flow chromatography technique, using specific RPA primers and Cas12 protein, combined with lateral flow chromatography test strips, enables rapid detection of Fusarium oxysporum in eggplant.
It achieves highly sensitive and specific on-site detection, and can complete the test within 20 minutes. It is suitable for rapid screening in grassroots laboratories and fields, reducing reliance on complex instruments and minimizing the risk of false positives.
Smart Images
Figure CN122128467A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant pathogen detection technology, specifically relating to a reagent, kit, method, and application for detecting RPA-CRISPR / Cas12a-LFD of Fusarium oxysporum var. solanum. Background Technology
[0002] Fusarium oxysporum (eggplant) Fusarium oxysporum f. sp. melongenae Fusarium wilt (FU) is a globally prevalent soil-borne vascular pathogen. This fungus primarily infects eggplants through soil, seedlings, water, and agricultural operations, causing severe wilt disease. After invading the vascular bundles, the fungus blocks the vessels and secretes toxins, leading to plant wilting and death, resulting in significant economic losses. Early, rapid, and accurate detection of this pathogen is crucial for disease control. Currently, detection methods for this pathogen mainly include the following: (1) Morphological identification: It takes a long time (usually 7–10 days), requires a pure culture process, and is highly dependent on the experience of the operator.
[0003] (2) Conventional PCR or real-time fluorescence PCR technology: Although the detection sensitivity is high, it must rely on expensive temperature-controlled amplification instruments and professional molecular biology laboratories, making it difficult to apply rapidly in the field or at grassroots agricultural stations.
[0004] (3) LAMP (loop-mediated isothermal amplification) technology: Although it can achieve isothermal amplification, its primer design is very complex (usually requiring 4-6 primers), which is prone to non-specific amplification leading to false positives. Moreover, the results usually need to be read by turbidity or fluorescence, which limits the degree of visualization.
[0005] Therefore, existing technologies cannot simultaneously meet the four requirements of "high sensitivity, high specificity, no instrument required, and rapid on-site operation". Summary of the Invention
[0006] To address the problems of existing technologies in detecting Fusarium oxysporum in eggplant, such as long processing time, reliance on complex and expensive instruments, high skill requirements for operators, and the susceptibility to false positives or low visualization in some isothermal amplification techniques, the present invention aims to provide a detection primer, kit, and method based on RPA-CRISPR / Cas12a combined with lateral flow chromatography test strip technology, so as to achieve accurate detection of Fusarium oxysporum with high sensitivity and specificity, without the need for complex instruments, and with rapid on-site result reading.
[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The first aspect of the present invention provides a reagent for detecting RPA-CRISPR / Cas12a-LFD of Fusarium oxysporum, the reagent comprising a specific RPA primer pair and a guide RNA primer, the specific RPA primer pair comprising FomRPA-F and FomRPA-R, the guide RNA primer comprising Fom-crRNA, the nucleotide sequence of FomRPA-F being shown in SEQ ID NO.1, the nucleotide sequence of FomRPA-R being shown in SEQ ID NO.2, and the nucleotide sequence of Fom-crRNA being shown in SEQ ID NO.3.
[0008] A second aspect of the present invention provides a kit containing the above-described RPA-CRISPR / Cas12a-LFD reagent for detecting Fusarium oxysporum.
[0009] Furthermore, the kit also contains Cas12 protein, T7 transcriptase, DNA polymerase, FAM probe, and Cas12 / 13 specific nucleic acid detection strips.
[0010] Furthermore, the Cas12 protein is SrCas12a.
[0011] Furthermore, the Cas12 / 13 dedicated nucleic acid test strip is a side-flow chromatography test strip.
[0012] A third aspect of the present invention provides the use of the above-described reagents and / or kits in any of the following: (1) Early diagnosis, field screening and disease prediction of Fusarium wilt in solanaceous crops; (2) Prepare products for detecting Fusarium oxysporum in eggplant.
[0013] The fourth aspect of the present invention provides a method for detecting Fusarium oxysporum solani based on RPA-CRISPR / Cas12a-LFD, including the steps of using the above-described kit.
[0014] Furthermore, the method includes the following steps: S.1 Extracting DNA from the sample to be tested; S.2 The DNA extracted in step S.1 was subjected to RPA amplification using the RPA amplification system to obtain RPA amplification products; S.3 The RPA amplification product obtained in step S.2 was incubated using the RPA-CRISPR / Cas12a-LFD reaction system; S.4 After adding water to make up the volume, add the diluted incubation product to the sample application end of the Cas12 / 13 special nucleic acid test strip; observe whether there are control lines and test lines on the nucleic acid test strip, and their color changes.
[0015] Furthermore, the RPA amplification system in S.2 includes PA buffer, FomRPA-F, FomRPA-R, template DNA, and enzyme-free water; the amplification conditions are 35-40℃ for 10-30 min.
[0016] Furthermore, the RPA-CRISPR / Cas12a-LFD reaction system in S.3 includes SrCas12a, Fom-crRNA, 10×Buffer, FAM probe, RPA clean-recovered reaction product, and enzyme-free water; the reaction conditions are 35-40℃ for 10-30 min.
[0017] The present invention has the following beneficial effects: 1) This invention is the first to establish a rapid detection method for *Fusarium oxysporum* sp. *spinosa* using RPA-CRISPR / Cas12a combined with lateral flow chromatography. Through specificity and sensitivity evaluation, it has been shown to be applicable to the detection of actual samples, providing a sensitive and reliable new method for the on-site detection of *Fusarium oxysporum* sp. *spinosa*. The target gene primers selected in this invention were obtained through extensive experimental screening, exhibiting good specificity and no cross-reactivity with other pathogens. The primers and probes used in this invention demonstrate excellent amplification effects and strong band specificity, forming a high concentration of primer-probe heterodimers in the detection region, thus resulting in a strong positive reaction on the test strip and increasing detection sensitivity. The detection method established in this invention can detect 10 fg of *Fusarium oxysporum* genome.
[0018] 2) The method for detecting Fusarium oxysporum in eggplant using the RPA technology combined with lateral flow chromatography of the present invention 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 oxysporum in primary laboratories and on-site.
[0019] 3) Fast detection speed: 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 42℃. No denaturation is required. The reaction can be completed in about 20 minutes at room temperature.
[0020] 4) No complex instruments or equipment are required, making it suitable for on-site testing. It achieves isothermal amplification, unlike PCR which requires thermal cycling. This eliminates the dependence on thermal cycling equipment; as long as a stable pyrogen source is available, the RPA reaction can occur, greatly expanding the scope of RPA applications and enabling truly portable, rapid on-site nucleic acid testing.
[0021] 5) This invention can be used for the rapid detection of Fusarium oxysporum in the tissues of infected solanaceous plants. The detection process can be completed in about 1 hour, which is an effective means of detecting Fusarium oxysporum.
[0022] 6) The detection method of this invention is used for early detection and prediction of Fusarium wilt in solanaceous plants, which is of great significance for determining the appropriate time for prevention and control and effectively controlling the disease. Attached Figure Description
[0023] Figure 1 Process flow diagram; Figure 2 Sensitivity detection results of RPA-CRISPR / Cas12a-LFD for Fusarium oxysporum in eggplant; CK is the sterile water blank control group; Figure 3 Specificity detection results of RPA-CRISPR / Cas12a-LFD for Fusarium oxysporum var. solanum; CK is the sterile water blank control group. Figure 4 Application of RPA-CRISPR / Cas12a-LFD technology in detecting actual bacterial contamination in eggplant samples; CK is a negative control of eggplant inoculated with sterile water. Detailed Implementation
[0024] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions or according to the manufacturer's recommended experimental conditions. Unless otherwise specified, the materials and reagents used are commercially available.
[0025] Example 1: Detection method for Fusarium oxysporum in eggplant The process flow diagram for the detection method of Fusarium oxysporum in eggplant is as follows: Figure 1 As shown.
[0026] 1. Primer Design and Synthesis: The complete genome sequences of *Fusarium oxysporum* (GCA_000149955.2) and *Fusarium oxysporum* (GCA_001888865.1) were retrieved from the NCBI database. Based on screening in the NR database and specificity experiments, a pair of specific RPA primers were designed using Primer6 software in their relatively conserved regions. The primers are as follows: FomRPA-F: 5'-TATACCACGCCCCTCCCGACGGGATATT-3' (SEQ ID NO. 1); FomRPA-R: 5'-ACAGCAGCTGCATCGAGAGCTTCCTTTC-3' (SEQ ID NO. 2).
[0027] 2. Guided RNA synthesis: Based on the specific region amplified by RPA in step 1 and the PAM mode (TTTN) of CRISPR / Cas12a guided RNA, target sites of ~20 nt were screened, and guide RNA primers were designed as follows: Fom-crRNA: 5'-UAAUUUCUACUAAGUGUAGAUACGGAUAUGUACGGUAUACC-3' (SEQ ID NO. 3).
[0028] 3. RPA-CRISPR / Cas12a detection of Fusarium wilt in eggplant 1) Extraction of genomic DNA from the fungus causing eggplant wilt After grinding an appropriate amount of the sample to be tested into powder, place it in a 2 mL centrifuge tube and add 700 μL of CTAB (preheated to 60℃) buffer (0.1M Tris-HCl, 50mM EDTA, 0.5M NaCl, 5mM SDS, pH 7.0). After incubating in a 65℃ water bath for 30-60 min, add an equal volume (700 μL) of chloroform:isoamyl alcohol mixture (24:1), invert and mix well, let stand for 3-5 min, centrifuge at 12000 rpm and 4℃ for 10 min, take the supernatant and add 2 volumes of pre-cooled anhydrous ethanol to precipitate, centrifuge at 12000 rpm and 4℃ for 10 min, remove the supernatant from the bottom of the DNA precipitate tube, blow off excess anhydrous ethanol in a clean bench, add 100 μL of enzyme-free water and measure the concentration to obtain the genomic DNA extract.
[0029] 2) The RPA reaction system is as follows: A buffe: 29.4 μL; 10 μM fomRPA-F: 2 μL; 10 μM fomRPA-R: 2 μL; Sample gDNA to be tested: 5 μL; Enzyme-free water: 12.5 μL; B bufffer: 2.5 μL.
[0030] The optimal reaction conditions for the above system are: 37℃ for 10-30 min.
[0031] 3) The RPA-CRISPR / Cas12a-LFD reaction system is as follows: 25 nM SrCas12a: 0.5 μL; 10×Buffer: 2 μL; 25 nM guide RNA: 1 μL; 750 nM FAM probe: 2 μL; RPA clean recovery of reactants: 2 μL; Enzyme-free water: 12.5 μL.
[0032] The optimal reaction conditions for the above system are: 37℃ for 10-30 min.
[0033] 4) Analysis and interpretation of test results After the reaction is complete, add 30 μL of RNase-free water to the reaction system to bring the total volume to 50 μL. Then insert the test strip into the reaction tube and observe the test strip for the appearance of the control line and the test line, as well as their color changes, after 4-5 minutes. If both the control line (C line) and the test line (T line) appear simultaneously, it is a positive reaction, indicating the presence of Fusarium oxysporum.
[0034] Example 2: Specificity test of RPA-CRISPR / Cas12a-LFD for Fusarium oxysporum esculentum. Following the RPA-CRISPR / Cas12a-LFD detection method described above, nine common eggplant pathogens, *Fusarium oxysporum*, were sequentially tested. F. oxysporum f. sp. melongenae Fusarium moniliforme ( ) F. proliferatum ), Fusarium oxysporum ( F. fuijkuroi Fusarium solani () F. solani Fusarium rosenbergii ( ), F. incarnatum Fusarium moniliforme () F. commune ), gray mold ( Botrytis cinerea ), fruit anthracnose ( Colletotrichum fructicola Alternaria ( Alternaria alternata The test was conducted, with water as a negative control.
[0035] Test results: such as Figure 2 As shown, only the sample containing *Fusarium oxysporum*, the causal agent of eggplant wilt, showed a positive reaction, while other common eggplant pathogens and the water control showed negative reactions. This result demonstrates that the primers and method provided in this invention have high specificity for *Fusarium oxysporum*, the causal agent of eggplant wilt, and can effectively distinguish it from other eggplant pathogens.
[0036] Example 3: Sensitivity test of RPA-CRISPR / Cas12a-LFD for Fusarium oxysporum var. eggplantis Following the RPA-CRISPR / Cas12a-LFD detection method described above, the genome copy number of the *Fusarium oxysporum* strain to be tested was sequentially diluted to 10 ng / uL, 1 ng / uL, 100 pg / uL, 10 pg / uL, 1 pg / uL, 100 fg / uL, 10 fg / uL, and 1 fg / uL. Each of these was then used as a template for the RPA-CRISPR / Cas12a-LFD reaction, with 5 μL of DNA template added to each reaction.
[0037] Test results: such as Figure 3 As shown, the reaction results for samples ranging from 10 ng / uL to 10 fg / uL were all positive, indicating that the detection limit of the genome of Fusarium wilt in eggplant is as low as 10 fg / uL, demonstrating extremely high sensitivity.
[0038] Example 4: Application of RPA-CRISPR / Cas12a-LFD technology in detecting actual bacterial-carrying samples Select eggplant seedlings with 2-3 true leaves. After gently washing the root substrate with sterile water, cut off 0.5-1 cm of the main root tip with sterile scissors. Immerse the roots in a solution with a concentration of 1×10⁻⁶. 6 ~1×10 7 Eggplant seedlings were treated with a suspension of *Fusarium oxysporum* spores / mL for 30 min. The control group roots were immersed in sterile water. After inoculation, seedlings were transplanted into a sterile substrate (nutrient soil: vermiculite = 3:1) and cultured at 28℃ under 16 h light / 8 h darkness. Eggplant root tissue samples were collected at 0, 3, 5, 7, and 9 days post-inoculation, and DNA was extracted using the CTAB method. The obtained DNA was used as a template for RPA amplification. The amplification product was added to a CRISPR / Cas12a-LFD detection system (containing Cas12a protein, crRNA, FAM probe, T7 transcriptase, etc.), and the results were read using nucleic acid detection test strips. Test results: as shown. Figure 4 As shown, the tests on the 3d, 5d, 7d, and 9d samples all showed positive reactions, with color changes observed at the test bands on the test strip. The 1d samples and the healthy eggplant samples showed negative reactions. These results demonstrate that the present invention has a good detection effect on eggplant wilt disease, and can rapidly and accurately detect the pathogen in complex samples, showing promising application prospects.
Claims
1. A reagent for detecting RPA-CRISPR / Cas12a-LFD of Fusarium oxysporum, characterized in that, The reagent includes a specific RPA primer pair and a guide RNA primer. The specific RPA primer pair includes FomRPA-F and FomRPA-R, and the guide RNA primer includes Fom-crRNA. The nucleotide sequence of FomRPA-F is shown in SEQ ID NO.1, the nucleotide sequence of FomRPA-R is shown in SEQ ID NO.2, and the nucleotide sequence of Fom-crRNA is shown in SEQ ID NO.
3.
2. A kit containing the RPA-CRISPR / Cas12a-LFD reagent for detecting Fusarium oxysporum as described in claim 1.
3. The kit according to claim 2, characterized in that, It also contains Cas12 protein, T7 transcriptase, DNA polymerase, FAM probe, and Cas12 / 13 dedicated nucleic acid test strips.
4. The kit according to claim 3, characterized in that, The Cas12 protein is SrCas12a.
5. The reagent kit as described in claim 3, characterized in that, The Cas12 / 13 dedicated nucleic acid test strip is a side-flow chromatography test strip.
6. The use of the reagent as described in claim 1 and / or the kit as described in any one of claims 2-5 in any of the following: (1) Early diagnosis, field screening and disease prediction of Fusarium wilt in solanaceous crops; (2) Prepare products for detecting Fusarium oxysporum in eggplant.
7. A method for detecting *Fusarium oxysporum* sp. *spinosa* based on RPA-CRISPR / Cas12a-LFD, characterized in that... The steps include using the kit described in any one of claims 2-5.
8. The detection method as described in claim 7, characterized in that, The method includes the following steps: S.1 Extracting DNA from the sample to be tested; S.2 The DNA extracted in step S.1 was subjected to RPA amplification using the RPA amplification system to obtain RPA amplification products; S.3 The RPA amplification product obtained in step S.2 was incubated using the RPA-CRISPR / Cas12a-LFD reaction system; S.4 After adding water to make up the volume, add the diluted incubation product to the sample application end of the Cas12 / 13 special nucleic acid test strip; observe whether there are control lines and test lines on the nucleic acid test strip, and their color changes.
9. The detection method as described in claim 8, characterized in that, The RPA amplification system in S.2 includes PA buffer, FomRPA-F, FomRPA-R, template DNA, and enzyme-free water; the amplification conditions are 35-40℃ for 10-30 min.
10. The detection method as described in claim 8, characterized in that, The RPA-CRISPR / Cas12a-LFD reaction system in S.3 includes SrCas12a, Fom-crRNA, 10×Buffer, FAM probe, RPA clean-recovered reaction product, and enzyme-free water; the reaction conditions are 35-40℃ for 10-30 min.