Dual-mode detection method for sugarcane smut fungi

Abstract

The invention belongs to the technical field of sugarcane germ detection, and particularly relates to a dual-mode detection method for sugarcane smut fungi. According to the dual-mode detection method for the sugarcane smut fungi, a fluorescence-naked eye reading dual-mode lateral chromatography platform for detecting the sugarcane smut fungi genes is developed by utilizing a composite material RuPd (at) vCDs and based on a rolling circle amplification and Cas12a active RCA-Cas platform. The method depends on a target chain as a primer, triggers an RCA process to generate a DNA product, and then activates the trans-cleavage activity of Cas12a, so that DNAC is catalytically cleaved. CDs is used as a luminescent probe for detecting a fluorescence signal, and RuPd bimetallic can catalytically decompose hydrogen peroxide by using own catalase-like activity, so that an alginate-tyramine conjugate forms gel for measuring a flow distance. The dual-mode detection realizes ultra-sensitive and rapid detection of the smut smut of sugarcane, and shows a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of sugarcane pathogen detection, and in particular to a dual-mode detection method for sugarcane smut pathogen. Background Art

[0002] Sugarcane smut, caused by a sporozoite, is the most common and serious disease in sugarcane production. Infection causes premature budding and the formation of black whips at the growing points. This disease not only reduces sugarcane stalk yield, but also leads to a decrease in sucrose content and quality. Therefore, early prediction and diagnosis of sugarcane smut are crucial for improving the economic benefits of the sugarcane industry.

[0003] There are several main methods for detecting sugarcane smut. Optical microscopy-based detection primarily involves observing the presence of dark spots on sugarcane leaves and stem nodes, as well as the presence of black mycelium within these spots. This method is simple to use and low-cost, making it an important method for on-site diagnosis of sugarcane smut. ELISA-based detection utilizes the binding interaction between smut fungus antigens and antibodies to produce a color reaction on an ELISA plate, thereby detecting the fungus. This method offers advantages such as simplicity, rapidity, and ease of use. Jiang Fuqiang et al. developed an ELISA-based detection method using a monoclonal antibody specific for the core antigen of the fungus. This method boasts high sensitivity, simplicity, and portability, making it suitable for rapid diagnosis of sugarcane smut. However, the long-term storage and complex cross-linking process of enzyme-labeled antibodies in ELISAs can lead to enzyme inactivation, further reducing the sensitivity of the ELISA. PCR-based detection methods amplify specific gene fragments of the sugarcane smut fungus for detection. Liu Chunyan et al. used PCR to design specific primers for the detection and identification of sugarcane smut. These methods offer high sensitivity and accuracy, but the equipment is expensive and generally requires specialized personnel. However, because these techniques rely on expensive equipment or complex processing by specialized personnel, they are generally unsuitable for field analysis. Therefore, a simple, rapid, and low-cost method for detecting sugarcane smut is needed.

[0004] Lateral flow assays (LFAs) are constructed using low-cost, environmentally friendly paper materials. These materials are porous matrices that facilitate fluid movement and flexibly immobilize the matrix, enabling interactions between biomolecules. Fluorescent LFAs offer numerous advantages over colorimetric LFAs, including high sensitivity, quantitative analysis, and robustness to sample matrix interference. Fluorescent LFAs require UV illumination for visualization, followed by fluorescence readings from a portable fluorescence dry-well analyzer. Building on this, we propose using changes in hydrogel permeability to directly measure distance for quantitative analysis. In distance-based analysis, a critical step is converting the signal into a result that correlates with visual distance. Conventional LFAs suffer from limited sensitivity, and their single-mode readout is susceptible to environmental influences, resulting in insufficient precision. Therefore, it is crucial to construct promising hydrogel-based LFAs with rapid response, controllable sensitivity, and excellent scalability. Summary of the Invention

[0005] In view of the above problems, the present invention provides a dual-mode detection method for sugarcane smut pathogen, which realizes rapid and ultrasensitive detection of sugarcane smut pathogen genes and shows broad application prospects.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: A dual-mode detection method for sugarcane smut pathogen comprises the following steps: (1) Signal amplification based on rolling circle probe: 2-5 μL padlock probe, 2-5 μL target of specific concentration or prepared sugarcane sample solution and 5-8 μL T4 ligase buffer were annealed at 90-100 °C and then slowly cooled to room temperature; 1-3 μL 350 U T4 DNA ligase was added and the resulting solution was incubated at room temperature for 2-2.5 h to generate a circular rolling circle probe; then a mixture containing 10-15 μL of the above circular rolling circle probe, 2-5 μL dNTPs, 0.3-0.6 μL 4 U phi29 polymerase, 0.5-1 μL 5 U Nt.AlWI, 0.5-1 μL 100× BSA, 2-5 μL 10× phi29 buffer and 4.5-5 μL ddH2O was incubated at 35-40 °C for 2.5-3 h for target detection amplification reaction. h to obtain a reaction solution; finally, the reaction solution was incubated at 75-80 °C for 20 min to inactivate the enzyme; (2) Determination on test strips: The reaction solution after the above enzyme inactivation was incubated with Cas12a-crRNA at a certain volume ratio at room temperature for 10-15 min to form a ternary complex system; then 10-15 μL Bio-DNA1 was added and incubated at 35-40 °C for 2-2.5 h; then 50-60 μL RuPd@vCDs-DNA2 was added and incubated at 35-40 °C for 1-1.5 h to obtain the product; finally, the product was introduced into the sample pad of the lateral flow test strip platform for detecting sugarcane smut fungus, which includes a sample pad, a nitrocellulose membrane and an absorption pad; after the product migrated on the lateral flow test strip by capillary force for 10 -15min later, add 100-110μL of Tween-20 and PVP mixed solution to the sample pad and observe the capture effect of the test line / control line; ① Fluorescence quantitative analysis is performed on the test area and control area using PFD; ② Cut off the absorbent pad of the test paper and replace the original absorbent pad with a filter paper strip, drop 5-10μL of a mixed solution of alginate-tyramine conjugate and 4 mM H2O2 on the test line, and wait for 30-50s; in the presence of RuPd and H2O2, the conjugate is catalyzed to form a gel that adheres to the T line, and 70-80μL DPBS is introduced into the sample pad and flows on the NC membrane by capillary action. Gel formation will hinder the flow of DPBS solution. The amount of RuPd on the T line can be correlated with the amount of gel formation. Therefore, the flow distance of DPBS penetrating the hydrogel on the filter paper can be directly measured with a ruler to determine the capture amount of RuPd on the T line, thereby obtaining the content of the target object.

[0007] Preferably, the preparation process of the alginate-tyramine conjugate is as follows: 0.1-0.3 g of alginate is dissolved in 9-10 g of 0.05 M MES buffer and stirred at 25-30 ° C for 4-5 h; 5-8 mL of 45 mM EDC and 5-8 mL of 45 mM NHS are added in sequence and stirred at room temperature for 1-1.5 h; then, 1-2 g of tyramine is added to the above solution and stirred at 20-25 ° C overnight; then, ethanol is added to the above solution to obtain a precipitate, which is the alginate-tyramine conjugate; washing is performed until the absorbance peak of tyramine at 275 nm is no longer detectable in the washing solution; finally, the alginate-tyramine conjugate is freeze-dried.

[0008] Preferably, the nitrocellulose membrane is treated as follows before use: the capture probe Bio-DNA labeled with biotin is T Incubate with streptavidin at 4-8 °C for 1 h to obtain SA-Bio-DNA T Probe solution; SA-Bio-DNA TThe probe solution and streptavidin (SA) solution were sprayed onto the nitrocellulose membrane respectively to form SA-Bio-DNA T The solution fixed area is used as the test line, and the SA solution fixed area is used as the control line. The distance between the test line and the control line is 5 mm, and the treated nitrocellulose membrane is obtained. The treated nitrocellulose membrane is dried completely and stored in a dry container.

[0009] Preferably, the volume ratio of the reaction solution to Cas12a-crRNA is 1:1.

[0010] Preferably, the preparation process of the lateral flow test paper platform for detecting sugarcane smut pathogen is as follows: a sample pad, a nitrocellulose membrane, and an absorption pad are sequentially attached and assembled on a polyvinyl chloride plate from left to right, with an overlap of 2 mm between the nitrocellulose membrane, the sample pad, and the absorption pad; after assembly, the lateral flow test paper is cut into single strips of 4 mm; the sample pad is pretreated by immersing it in a buffer solution, and is fully saturated after 5 minutes; and then, the sample pad is dried at 65°C for 24 hours.

[0011] Preferably, the buffer solution is a mixed solution comprising 0.1 M Tris-hydrochloric acid, 1% sodium chloride, 0.5% Tween 20, 1% BSA, 2% sucrose and 1% Triton X-100, and the pH of the buffer solution is 8.0.

[0012] Preferably, the sugarcane sample solution is prepared by taking 2-3 mg of leaves, whips, and stems of different sugarcane varieties, both normal and infected with smut fungi, respectively; completely chopping the samples and continuously grinding them with liquid nitrogen until the leaves, whips, and stems of each variety become powdered; then adding 5-8 mL of PBS to each powdered sample and soaking them at 3-6°C overnight; filtering the mixture with filter paper to obtain an extract; then filtering the extract with a 0.22 μm filter membrane; and after the above pretreatment, storing the sample solution at -20°C for future use.

[0013] Preferably, the preparation method of the RuPd@vCDs-DNA2 (i.e., a composite material based on RuPd bimetallic nanozyme) comprises the following steps: 1) Dissolve 2-2-dithiosalicylic acid and melamine in acetic acid at a mass ratio of 2-3:1 and ultrasonically treat. Then, add the mixed solution to a reactor and react at 160-180°C for 10-12 hours. After the reaction, cool to room temperature, wash the product in boiling water, filter with suction, collect the solid, and dry to obtain CDs powder. 2) Dissolving the dried CDs powder in dimethyl sulfoxide, adding 2,3-epoxypropyl methacrylate to the solution, and then allowing the mixture to react at room temperature for 22-24 hours. After the reaction, the product is washed, filtered, and dried to obtain vCDs powder. The vCDs powder is lyophilized and stored at low temperature until use. 3) Mix Na2PdCl4 solution and RuCl3 solution in a 1:1 volume ratio and incubate in the dark for 10-15 minutes. Then, add sodium borohydride solution and stir until the solution becomes colorless. Centrifuge, wash, and dry the sample to obtain the RuPd complex. Finally, suspend the RuPd complex in ultrapure water and store at low temperature until ready to use. 4) Using K2S2O8 as a crosslinker, mix it with vCDs powder, RuPd, and PVP aqueous solution, and react at 70-80°C for 1-2 hours to obtain RuPd@vCDs. 5) Incubate 40-45 μL of RuPd@vCDs with 10-15 μL of DNA2 (10 μM) at 30-40 °C for 1-1.5 h to obtain RuPd@vCDs-DNA2.

[0014] Preferably, the usage ratio of acetic acid to melamine is 1 mL:5-6 mg; the usage ratio of CDs powder, dimethyl sulfoxide and 2,3-epoxypropyl methacrylate is 1 g:5 mL:10 mL.

[0015] Preferably, the volumes of the Na2PdCl4 solution and the RuCl3 solution are both 5 mL, and the concentrations are both 20 mM; the amount of sodium borohydride solution added is 50 mL, and the concentration is 20 mM; the mass ratio of the K2S2O8, vCDs powder material and RuPd is 10:1:1, and the usage ratio of the PVP aqueous solution to RuPd is 1 mL:2-2.5 mg.

[0016] The carbon dots (CDs) described in this invention are a novel carbon-based nanomaterial with excellent biocompatibility, exceptional optical properties, low cost, and high stability. The RuPd bimetallic nanozyme is simple to synthesize, requiring only a single reducing agent for a one-step synthesis. The RuPd bimetallic nanozyme is uniform in size and exhibits high catalytic activity and stability. The peroxidase activity of RuPd is not only higher than that of conventional HRP, but also higher than that of individual Ru and Pd nanoparticles, indicating a synergistic effect between RuPd and Pd. This synergistic RuPd nanozyme can efficiently catalyze hydrogen peroxide, enabling the formation of a hydrogel from alginate-tyramine conjugates.

[0017] The CRISPR / Cas12a (Cpf1) enzyme in this invention is a type of endonuclease guided by a single crRNA. Under the guidance of the crRNA, it specifically recognizes and cleaves double-stranded DNA (dsDNA) targets bearing a PAM (5'-TTTN-3' or 5'-TTN-3'), creating double-strand breaks and sticky ends. Subsequently, the trans-cleavage activity of Cas12a is activated, catalyzing the cleavage of nearby ssDNA. Through its specific target recognition and trans-cleavage activity, CRISPR / Cas12a-based biosensors offer improved accuracy, adaptability, portability, timeliness, and effectiveness compared to traditional methods. Therefore, CRISPR / Cas12a biosensor systems can meet diverse requirements and enable application-specific detection in various fields, including disease diagnosis, food safety inspection, and environmental analysis. Rolling circle amplification (RCA) is a simple and effective isothermal enzymatic amplification technology that uses a short circular oligonucleotide as a template and dNTPs / NTPs as raw materials to amplify a long repetitive single-stranded DNA / RNA under the action of DNA / RNA polymerase.

[0018] The principle of lateral flow chromatography based on lateral flow test paper for detecting sugarcane smut pathogens in the present invention is as follows: This method relies on the target as a primer to trigger the RCA process, generating long single-stranded DNA (ssDNA) products and activating the trans-cleavage activity of Cas12a for fluorescence detection. Simply put, when the target strand (target) is added and hybridized with the prepared rolling circle probe, the phi29 DNA polymerase rapidly initiates the RCA reaction, generating a large amount of ssDNA. Finally, hybridization between the Cas12a / crRNA complex and the amplified ssDNA product activates the trans-cleavage activity of the Cas12a protein and initiates the catalytic cleavage of Bio-DNA1. This results in the control line (line C) being unable to capture the fluorescent probe and generating no fluorescent signal response. However, a fluorescent signal response is generated on the detection line (line T). Conversely, in the absence of the target, the RCA reaction cannot be triggered, thus preventing downstream reactions and inhibiting subsequent trans-cleavage by the Cas12a protein. The intact Bio-DNA1 binds to the fluorescent probe and is immobilized on the C line, generating a strong fluorescent signal. Furthermore, the cross-linking degree of the alginate-tyramide conjugate is significantly correlated with the concentration of the RuPd bimetallic nanozyme. Because the amount of RuPd bimetallic nanozyme immobilized on the T-line varies, the degree of hydrogel formation by the catalytic conjugate and the permeability of the hydrogel also vary. Therefore, the concentration of the RuPd bimetallic nanozyme can be used to determine the amount of capture on the detection line, and then the concentration of the detected substance can be quantitatively analyzed.

[0019] By adopting the above technical solution, the beneficial effects of the present invention are: This study combines optically strong carbon dots with a RuPd bimetallic catalyst with catalase-like activity. Using the composite RuPd@vCDs-DNA2, and leveraging the rolling circle amplification (RCA) and Cas12a activity in the RCA-Cas platform, a dual-mode lateral flow platform with fluorescence and visual readout has been developed for rapid, ultrasensitive, and specific detection of sugarcane smut pathogen genes. This method relies on the target as a primer to trigger the RCA process to generate DNA products, which then activates the trans-cleavage activity of Cas12a, catalyzing the cleavage of DNA1. The CDs serve as luminescent probes for fluorescence signal detection, while the RuPd bimetallic catalyst utilizes its catalase-like activity to catalyze the decomposition of hydrogen peroxide, resulting in the formation of an alginate-tyramine conjugate hydrogel for visual readout. During the enzyme-catalyzed hydrogel formation process, the permeability of the hydrogel is related to the cross-linking degree of the alginate-tyramine conjugate. Furthermore, the cross-linking degree of the alginate-amine conjugate is significantly correlated with the concentration of the RuPd bimetallic nanozyme. Therefore, the concentration of the nanozyme can be determined by the permeability of the hydrogel, and the capture amount on the detection line can be determined by the concentration of the nanozyme, and then the concentration of the detection object can be quantitatively analyzed.

[0020] Therefore, the present invention successfully constructed an RCA-Cas platform based on rolling circle amplification and Cas12a activity for rapid, ultrasensitive and specific detection of sugarcane smut pathogen genes. This method combines high RCA reaction efficiency and powerful Cas12a anti-cleavage activity, greatly improving the detection sensitivity and improving the selectivity, reproducibility and stability of the LFA platform. At the same time, a quantitative lateral flow analysis method based on the naked eye readout distance of the RuPd bimetallic nanozyme-catalyzed hydrogel permeability change provides a new detection method. The use of dual-mode detection further improves the reliability of the detection method. The present invention provides a new method for the detection and prevention of sugarcane smut pathogens, which has good practical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a detection principle diagram of a dual-mode detection method for sugarcane smut pathogens according to the present invention.

[0022] Figure 2 (A) TEM image of vCDs, (B) SEM image of RuPd, (C) SEM image of RuPd@vCDs, (D) TEM image of RuPd@vCDs, and (E) energy spectrum of RuPd@vCDs in the present invention.

[0023] Figure 3 (A) Fluorescence emission spectra and (B) FT-IR spectra of RuPd@vCDs and vCDs in the present invention.

[0024] Figure 4For different concentrations (10 -15 ~ 10 -8 M) (A) Fluorescence phenomenon of the LFA platform, (B) flow distance photograph, (C) fluorescence value linear curve, (D) flow distance linear curve.

[0025] Figure 5 This is the specificity of the LFA platform in the present invention.

[0026] Figure 6 Long-term stability of (A) fluorescence and (B) flow distance of the LFA platform of the present invention; (C) fluorescence and (D) reproducibility of flow distance. DETAILED DESCRIPTION

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. Example

[0028] The detection principle diagram of the sugarcane smut pathogen detection method of the present invention is as follows: Figure 1 As shown, the specific steps are as follows: (1) Synthesis of alginate-tyramine conjugates First, 0.1 g of alginate (Alg, 1.0 wt %) was dissolved in 9.9 g of 0.05 M MES buffer (pH 6.0) and stirred at 25°C for 4 h. 5 mL of 45 mM EDC and 5 mL of 45 mM NHS were added sequentially, and the mixture was stirred at room temperature for 1 h. Next, 1 g of tyramine (Tyr, 5.0 wt %) was added to the above solution and stirred at 25°C overnight. The alginate-tyramine (Alg-Tyr) conjugate was then precipitated with 90% ethanol and washed until the tyramine absorbance peak at 275 nm was no longer detectable in the wash solution. Finally, the UV-cured Alg-Tyr conjugate was lyophilized.

[0029] (2) Hydrogel formation The RuPd bimetallic nanozyme-catalyzed Alg-Tyr conjugate formed a hydrogel by dissolving the Alg-Tyr conjugate in 0.5 wt% DPBS to obtain a conjugate solution. A mixture of freshly prepared RuPd bimetallic nanozyme (50 μL) and hydrogen peroxide (50 μL) was then added to 3 mL of the conjugate solution (RuPd concentration was 0.24 mg / mL; H₂O₂ concentration was 4 mM). The absence of flow within 1 minute after inverting the bottle was considered to indicate a hydrogel state.

[0030] (3) Treatment of nitrocellulose membrane Biotin-labeled capture probe Bio-DNA T Incubate with streptavidin at 4 °C for 1 h to obtain SA-Bio-DNA T probe solution; then, the SA-Bio-DNA was transferred to the T The probe solution and streptavidin (SA) solution were sprayed onto nitrocellulose membrane (NC membrane) to form SA-Bio-DNA T The solution-fixed area is used as the test line (T line), and the SA solution-fixed area is used as the control line (C line). The distance between the test line and the control line is 5 mm, and the treated nitrocellulose membrane is obtained. The treated nitrocellulose membrane is dried completely and stored in a dry container.

[0031] (4) Preparation of lateral flow assay platform (LFA) The LFA used to detect sugarcane smut fungus consists of several components: a sample pad, a NC membrane, and an absorbent pad. These components are attached to a polyvinyl chloride plate from left to right, with a 2 mm overlap between the NC membrane and the other components to ensure smooth flow of the reaction fluid through the strip. After assembly, the material is cut into 4 mm strips to prepare a complete test strip. To enhance the release of RuPd@vCDs-DNA2 and facilitate the flow of sample solution from the sample pad to the absorbent pad, the sample pad is pretreated by immersing it in a buffer solution (0.1 M Tris-HCl, 1% NaCl, 0.5% Tween 20, 1% BSA, 2% sucrose, and 1% Triton X-100, pH 8.0) for 5 minutes to achieve full saturation. The sample pad is then dried at 65°C for 24 hours.

[0032] (5) Detection of target objects 1) Signal amplification based on rolling circle probe: 2 μL padlock probe, 2 μL target of specific concentration (Target, 10 -15 ~ 10 -8M) or the prepared sugarcane sample solution was annealed with 5 μL of T4 ligase buffer at 90°C for 1 minute, then slowly cooled to room temperature. 350 U of T4 DNA ligase (1 μL) was added, and the resulting solution was incubated at room temperature for 2 hours to generate a circular rolling circle probe (padlock probe).

[0033] The target detection amplification reaction was performed in a 20 μL mixture at 37°C for 2.5 hours. The mixture consisted of 10 μL of the ligation product, 2 μL of 10 mM dNTPs, 4 U of phi29 polymerase (10 U / mL, 0.3 μL), 5 U of t.AlWI (10 U / mL, 0.5 μL), 0.5 μL of 100× BSA, 2 μL of 10× phi29 buffer, and 4.7 μL of ddH2O. Finally, the reaction solution was incubated at 75°C for 20 minutes to inactivate the enzyme.

[0034] On-strip assay: 20 μL of the above enzyme-inactivated reaction solution was incubated with 20 μL of Cas12a-crRNA at room temperature for 10 min to form a ternary complex system; then 10 μL of Bio-DNA1 (1 μM) was added and incubated at 37 °C for 2 h. Next, 50 μL of RuPd@vCDs-DNA2 was added to 50 μL of the above solution, and the product was obtained after incubation at 37 °C for 1 h. Finally, the product was introduced into the sample pad of the lateral flow test paper platform for detecting sugarcane smut fungus, which includes a sample pad, a nitrocellulose membrane and an absorption pad. After the product migrated on the lateral flow test paper by capillary force for 10 minutes, 100 μL of a mixed solution of Tween-20 (5%) and PVP (5%) was added to the sample pad to observe the capture effect of the test line / control line. ① Fluorescence quantitative analysis was performed on the test area and the control area using PFD. ② The absorbent pad of the test paper was cut off and replaced with a filter paper strip. A mixed solution of 5 μL of alginate-tyramine conjugate and 4 mM H2O2 was dropped onto the test line and waited for 40 seconds. In the presence of RuPd and H2O2, the conjugate was catalyzed to form a gel that adhered to the T line. 70 μL DPBS is introduced into the sample pad and flows on the NC membrane by capillary action. Gel formation will hinder the flow of DPBS solution. The amount of RuPd on the T line can be correlated with the amount of gel formation. Therefore, using a ruler to directly measure the flow distance of DPBS penetrating the hydrogel on the filter paper can determine the capture amount of RuPd on the T line, thereby deriving the content of the target substance.

[0035] Figure 1Activated means that the amplified chain (ssDNA) binds to RNA, and unactivated means that there is no amplified chain if there is no target.

[0036] The preparation method of RuPd@vCDs-DNA2 composite material based on RuPd bimetallic nanozyme comprises the following steps: (1) Synthesis of vCDs First, 544.0 mg of 2-2-dithiosalicylic acid (DTSA) and 201.6 mg of melamine (MA) were dissolved in 40 mL of acetic acid and sonicated. The mixture was then added to an 80 mL polytetrafluoroethylene reactor and reacted at 180°C for 10 hours. After the reaction was completed, the mixture was cooled to room temperature and washed in 1 L of boiling water. The solid was then collected by suction filtration and dried at 25°C to obtain CDs powder.

[0037] vCDs were prepared by reacting GMA with surface amines or hydroxyl groups on CDs. 1 g of dried vCDs powder was dissolved in 5 mL of dimethyl sulfoxide (DMSO), and 10 mL of 2,3-epoxypropyl methacrylate (GMA) was added to the solution. The mixture was then allowed to react at 25°C for 24 h. The product was then washed with 1 L of water, filtered by suction, and the solid collected. The vCDs powder was then dried at 25°C. The vCDs were further lyophilized and stored at 4°C.

[0038] (2) Synthesis of RuPd Mix Na2PdCl4 (5 mL, 20 mM) and RuCl3 (5 mL, 20 mM). Incubate in the dark for 10 minutes. To stimulate metal ion reduction, add freshly prepared sodium borohydride solution (50 mL, 20 mM) while stirring. Bubbles will form during this process, followed by a black solid floating on the surface of the reaction solution. Continue stirring until the solution becomes colorless.

[0039] The sample was centrifuged (10,000 rpm, 10 min), washed several times with water, and then vacuum dried overnight. Finally, the RuPd complex was suspended in 30 mL of ultrapure water and stored at 4 °C for further use.

[0040] (3) Synthesis of RuPd@vCDs and preparation of RuPd@vCDs-DNA2 240 mg of K2S2O8 was used as a crosslinker, mixed with 24 mg of vCDs, 24 mg of RuPd, and 10 ml of a 5% PVP aqueous solution. The mixture was reacted at 80 °C for 1 h to produce RuPd@vCDs (2.4 mg / mL). Then, 40 μL of RuPd@vCDs was incubated with 10 μL of DNA2 (10 μM) at 37 °C for 1 h to produce RuPd@vCDs-DNA2.

[0041] The sugarcane sample solution was prepared by collecting approximately 2 mg of leaves, rhizomes, and stems from different sugarcane varieties (Guitang 44 and 05136) infected with the smut fungus and normal sugarcane. Surface impurities were cleaned with alcohol. The samples were then completely chopped and continuously ground with liquid nitrogen to form a powder. 5 mL of PBS was then added to each powdered sample and the mixture was soaked overnight at 4°C. The mixture was filtered through filter paper to obtain an extract, which was then filtered through a 0.22 μm filter membrane. After the above pretreatment, the sample solution was stored at -20°C until use.

[0042] To verify the actual detection performance of the LFA platform, sample solutions were introduced into the RCA-Cas platform and the LFA platform, and a portable fluorescence dry-well analyzer was used to detect whether there was a fluorescent signal response. The results are shown in Table 1. It can be seen that fluorescent signal responses were observed in the leaves, stems, and whips of sugarcane samples infected with sugarcane smut fungus.

[0043]

[0044] Next, DPBS was introduced, and the flow distance was visually read to determine the amount of RuPd on the T-line, allowing for quantitative analysis of the target compound. When a normal sample was introduced, the T-line on the LFA platform failed to detect a fluorescent signal, and the DPBS flow distance visually read was greater than 30 cm. When a diseased sample was introduced, the T-line on the LFA platform detected a fluorescent signal, and the DPBS flow distance visually read was less than 30 cm. These results demonstrate the feasibility of this LFA platform for analyzing real samples infected with sugarcane smut pathogens.

[0045] The oligonucleotide sequence information used in the present invention is shown in Table 2 below.

[0046]

[0047] Note: The nucleic acid sequences used in this experiment were provided by Shanghai Bioengineering.

[0048] To demonstrate the successful synthesis of RuPd@vCDs composites, the applicant characterized RuPd, vCDs, and RuPd@vCDs.

[0049] like Figure 2 , SEM and TEM images show the morphology and size of vCDs. Figure 2 As shown in A, the particle size of vCDs is 5-6 nm, and the lattice spacing is about 0.2063 nm ( Figure 2 A, upper right inset). SEM image of RuPd ( Figure 2 B) shows that RuPd is composed of interconnected porous structures. SEM image of RuPd@vCDs ( Figure 2 C) RuPd and vCDs combined together showing the porous structure. Figure 2 D is the TEM image of RuPd@vCDs. By analyzing the lattice spacing of RuPd and vCDs in RuPd@vCDs, it is found that the lattice spacing of RuPd is 0.2353 nm and the lattice spacing of vCDs is 0.2065 nm. It is concluded that the lattice spacing of the composite RuPd@vCDs is almost the same as that of the individual vCDs. Energy dispersive X-ray spectroscopy (EDS) mapping of PdRu ( Figure 2 E) Shows the ordered distribution of vCDs and PdRu elements throughout the nanostructure. This demonstrates the successful synthesis of RuPd@vCDs composites.

[0050] like Figure 3 The fluorescence emission spectra of RuPd@vCDs and vCDs showed that under the excitation of 365 nm, their emission wavelengths were both 615 nm ( Figure 3 A). FT-IR spectrum shows that the spectrum at 1610 cm is due to the stretching vibration of the C=O double bond in vCDs. -1 A strong absorption peak appears at 1220 cm -1 The strong absorption peak at 1220 cm is due to the stretching vibration of CO. -1 A strong absorption peak will appear at 1220 cm -1 and 1610 cm -1 There are similar characteristic peaks, which can prove the successful synthesis of RuPd@vCDs composite materials ( Figure 3 B).

[0051] In addition, to verify the effectiveness of the present invention, the applicant also analyzed the performance (including sensitivity, specificity, stability and reproducibility, etc.) of the LFA platform of the present application. The analysis results are as follows: Under 365 nm ultraviolet light, a preliminary judgment can be made by observing the fluorescence intensity on the T line. Subsequently, a quantitative analysis is performed by reading the fluorescence intensity of the test line using PFD. For example, when the target concentration is low, the ssDNA product generated by the RCA process is less, the activated Cas12a decreases, the ability to cut Bio-DNA1 decreases, and a large amount of Bio-DNA1 is fixed on the C line, which reduces the fluorescence intensity on the T line. Figure 4 As shown in A, when the concentration of the target substance gradually increases, the color of the strip on the test line deepens as the T line fluorescence signal increases, and the detection limit of the naked eye reading is as low as 10 fM. Figure 4 As shown in C, at 10 -15 ~ 10 -8 Within the M range, the logarithm of the target concentration and the T-line fluorescence intensity value show a good linear relationship, and the linear equation is y=2750.1logC-922.8, R 2 =0.994, with a detection limit of 28.05 aM (S / N = 3).

[0052] like Figure 4 As shown in B, the flow distance response of DPBS under different concentrations of target is recorded. When the concentration of target is low, the number of fluorescent probes bound to the T line decreases, that is, the number of bimetallic nanozymes RuPd decreases, and the ability to catalyze gel formation becomes weaker. Figure 4 As shown in D, with the increase of target concentration, the gel formation speed is faster and the flow distance is gradually shortened due to the increase in the number of bimetallic nanozymes RuPd fixed on the T line. -15 ~ 10 -8 Within the range of M, the logarithm of the target concentration and the flow distance value show a good linear relationship, and the linear equation is y=-1.243logC+28.375, R 2 =0.993, with a limit of detection of 35.17 aM (S / N = 3). This LFA-PFD platform is simple to construct, cost-effective, and exhibits good accuracy. Its low limit of detection and wide linear range enable more sensitive detection of sugarcane smut pathogen genes.

[0053] In order to study the specificity of the prepared LFA to the sugarcane smut pathogen gene, the present invention selected single base mismatch of the sugarcane smut pathogen gene, double base mismatch of the sugarcane smut pathogen gene, sugarcane tip rot pathogen gene, mixture of sugarcane tip rot pathogen gene and sugarcane smut pathogen gene and blank sample for testing. Figure 5 As shown, Target is the target of the present invention, smHei, tmHei, NC, ITS1 are the sequences that have been screened out, and Mix is ​​a mixed solution of the target sequence and smHei, tmHei, NC, ITS1. Figure 5 It can be seen that when sugarcane smut pathogen is present, the T-line fluorescence signal intensity on the LFA platform is the highest and the corresponding flow distance value is the lowest. This result shows that the LFA platform can specifically detect the sugarcane smut pathogen gene.

[0054] Stability is a key indicator for evaluating LFA performance. It ensures the stability and reliability of test results and directly affects its service life and maintenance costs. To evaluate the stability of the LFA platform, the stability of test strips ( Figure 6 A and 6B). Over time, we observed that the fluorescence response and mobility distance remained relatively stable over 28 days, with a loss of approximately 10.71% in fluorescence and 5.94% in mobility distance. These results demonstrate the robustness of this LFA platform.

[0055] Reproducibility refers to the consistency of LFA performance when prepared with the same raw materials at different times and locations under the same experimental conditions. To evaluate the reproducibility of LFA, 10 different batches of test paper, such as Figure 6 As shown in Figures C and 6D, the fluorescence response results of the test strips from different batches were similar, with a relative standard deviation (RSD) of 1.8%. The flow distance values ​​were also similar, with an RSD of 0.98%. This demonstrates the good reproducibility of the LFA platform.

[0056] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A dual-mode detection method for sugarcane smut pathogen, characterized in that: The steps include: (1) Signal amplification based on rolling circle probe: 2-5 μL padlock probe, 2-5 μL target of specific concentration or prepared sugarcane sample solution and 5-8 μL T4 ligase buffer were annealed at 90-100 °C and then slowly cooled to room temperature; 1-3 μL 350 U T4 DNA ligase was added and the resulting solution was incubated at room temperature for 2-2.5 h to generate a circular rolling circle probe; then a mixture containing 10-15 μL of the above circular rolling circle probe, 2-5 μL dNTPs, 0.3-0.6 μL 4 U phi29 polymerase, 0.5-1 μL 5 U Nt.AlWI, 0.5-1 μL 100× BSA, 2-5 μL 10× phi29 buffer and 4.5-5 μL ddH2O was incubated at 35-40 °C for 2.5-3 h for target detection amplification reaction. h to obtain a reaction solution; finally, the reaction solution was incubated at 75-80 °C for 20 min to inactivate the enzyme; (2) Determination on test strips: The reaction solution after the above enzyme inactivation was incubated with Cas12a-crRNA at a certain volume ratio at room temperature for 10-15 min to form a ternary complex system; then 10-15 μL Bio-DNA1 was added and incubated at 35-40 °C for 2-2.5 h; then 50-60 μL RuPd@vCDs-DNA2 was added and incubated at 35-40 °C for 1-1.5 h to obtain the product; finally, the product was introduced into the sample pad of the lateral flow test strip platform for detecting sugarcane smut fungus, which includes a sample pad, a nitrocellulose membrane and an absorption pad; after the product migrated on the lateral flow test strip by capillary force for 10 -15min later, add 100-110μL of a mixed solution of Tween-20 and PVP to the sample pad and observe the capture effect of the test line / control line; ① Perform fluorescence quantitative analysis on the test area and control area; ② Cut off the absorbent pad of the test paper and replace the original absorbent pad with a filter paper strip, drop 5-10μL of a mixed solution of alginate-tyramine conjugate and 4 mM H2O2 on the test line, and wait for 30-50s; in the presence of RuPd and H2O2, the conjugate is catalyzed to form a gel that adheres to the T line, and 70-80μL of DPBS is introduced into the sample pad and flows on the NC membrane by capillary action. Gel formation will hinder the flow of DPBS solution. The amount of RuPd on the T line can be correlated with the amount of gel formation. Therefore, using a ruler to directly measure the flow distance of DPBS penetrating the hydrogel on the filter paper can determine the capture amount of RuPd on the T line, thereby deriving the content of the target object.

2. The dual-mode detection method for sugarcane smut pathogen according to claim 1, characterized in that: The preparation process of the alginate-tyramine conjugate is as follows: 0.1-0.3 g of alginate is dissolved in 9-10 g of 0.05 M MES buffer and stirred at 25-30° C. for 4-5 hours; 5-8 mL of 45 mM EDC and 5-8 mL of 45 mM NHS are added in sequence and stirred at room temperature for 1-1.5 hours; then, 1-2 g of tyramine is added to the above solution and stirred at 20-25° C. overnight; then, ethanol is added to the above solution to obtain a precipitate, which is the alginate-tyramine conjugate; washing is performed until the absorbance peak of tyramine at 275 nm is no longer detectable in the washing solution; and finally, the alginate-tyramine conjugate is freeze-dried.

3. The dual-mode detection method for sugarcane smut pathogen according to claim 1, characterized in that: The nitrocellulose membrane was treated as follows before use: the capture probe Bio-DNA labeled with biotin was T Incubate with streptavidin at 4-8 °C for 1 h to obtain SA-Bio-DNA T Probe solution; SA-Bio-DNA T The probe solution and streptavidin SA solution were sprayed onto the nitrocellulose membrane respectively to form SA-Bio-DNA T The solution fixed area is used as the test line, and the SA solution fixed area is used as the control line. The distance between the test line and the control line is 5 mm, and the treated nitrocellulose membrane is obtained. The treated nitrocellulose membrane is dried completely and stored in a dry container.

4. The dual-mode detection method for sugarcane smut pathogen according to claim 1, wherein: The volume ratio of the reaction solution to Cas12a-crRNA is 1:

1.

5. The dual-mode detection method for sugarcane smut pathogen according to claim 1, wherein: The preparation process of the lateral flow test paper platform for detecting sugarcane smut pathogens is as follows: a sample pad, a nitrocellulose membrane, and an absorption pad are sequentially attached and assembled on a polyvinyl chloride plate from left to right, with a 2 mm overlap between the nitrocellulose membrane, the sample pad, and the absorption pad; after assembly, the lateral flow test paper is cut into individual 4 mm strips; the sample pad is pretreated by immersing it in a buffer solution, which is fully saturated after 5 minutes; and the sample pad is then dried at 65°C for 24 hours.

6. The dual-mode detection method for sugarcane smut pathogen according to claim 5, characterized in that: The buffer solution is a mixed solution containing 0.1 M Tris-hydrochloric acid, 1% sodium chloride, 0.5% Tween 20, 1% BSA, 2% sucrose and 1% Triton X-100, and the pH of the buffer solution is 8.

0.

7. The dual-mode detection method for sugarcane smut pathogen according to claim 1, wherein: The sugarcane sample solution is prepared by taking 2-3 mg of leaves, whips, and stems of different sugarcane varieties, both normal and infected with smut fungi, respectively; completely chopping the samples; and continuously grinding the leaves, whips, and stems of each variety with liquid nitrogen until the leaves, whips, and stems are powdered. 5-8 mL of PBS is then added to each powdered sample, and the mixture is soaked overnight at 3-6°C. The mixture is filtered through filter paper to obtain an extract, and the extract is then filtered through a 0.22 μm filter membrane. After the above pretreatment, the sample solution is stored at -20°C for future use.

8. The dual-mode detection method for sugarcane smut pathogen according to claim 1, wherein: The preparation method of RuPd@vCDs-DNA2 comprises the following steps: 1) Dissolve 2-2-dithiosalicylic acid and melamine in acetic acid at a mass ratio of 2-3:1 and ultrasonically treat. Then, add the mixed solution to a reactor and react at 160-180°C for 10-12 hours. After the reaction, cool to room temperature, wash the product in boiling water, filter with suction, collect the solid, and dry to obtain CDs powder. 2) Dissolving the dried CDs powder in dimethyl sulfoxide, adding 2,3-epoxypropyl methacrylate to the solution, and then allowing the mixture to react at room temperature for 22-24 hours. After the reaction, the product is washed, filtered, and dried to obtain vCDs powder. The vCDs powder is lyophilized and stored at low temperature until use. 3) Mix Na2PdCl4 solution and RuCl3 solution in a 1:1 volume ratio and incubate in the dark for 10-15 minutes. Then, add sodium borohydride solution and stir until the solution becomes colorless. Centrifuge, wash, and dry the sample to obtain the RuPd complex. Finally, suspend the RuPd complex in ultrapure water and store at low temperature until ready to use. 4) Using K2S2O8 as a crosslinker, mix it with vCDs powder, RuPd, and PVP aqueous solution, and react at 70-80°C for 1-2 hours to obtain RuPd@vCDs. 5) Incubate 40-45 μL of RuPd@vCDs with 10-15 μL of DNA2 (10 μM) at 30-40 °C for 1-1.5 h to obtain RuPd@vCDs-DNA2.

9. The dual-mode detection method for sugarcane smut pathogen according to claim 8, wherein: The usage ratio of the acetic acid to the melamine is 1 mL:5-6 mg; the usage ratio of the CDs powder, dimethyl sulfoxide and 2,3-epoxypropyl methacrylate is 1 g:5 mL:10 mL.

10. The dual-mode detection method for sugarcane smut pathogen according to claim 8, wherein: The volumes of the Na2PdCl4 solution and the RuCl3 solution are both 5 mL, and the concentrations are both 20 mM; the amount of sodium borohydride solution added is 50 mL, and the concentration is 20 mM; the mass ratio of the K2S2O8, vCDs powder material and RuPd is 10:1:1, and the usage ratio of the PVP aqueous solution to RuPd is 1 mL:2-2.5 mg.

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