RT-RPA-LFD method capable of simultaneously, rapidly and visually detecting three pathogens of sugarcane mosaic disease and application of RT-RPA-LFD method

Abstract

The invention belongs to the technical field of plant virus detection, and particularly relates to an RT-RPA-LFD method capable of rapidly and visually detecting three pathogens of sugarcane mosaic disease at the same time and application of the RT-RPA-LFD method. The invention relates to an RT-RPA-LFD primer group capable of simultaneously, rapidly and visually detecting three pathogens of sugarcane mosaic disease. The RT-RPA-LFD primer group comprises a primer group I, a primer group II and a primer group III, wherein the primer group I comprises an SrMV primer pair and an SrMV probe; the primer group II comprises an SCSMV primer pair and an SCSMV probe; and the primer group III comprises an SCMV primer pair and an SCMV probe. According to the multiple RT-RPA-LFD detection method disclosed by the invention, complicated RNA extraction, cDNA reverse transcription, gel electrophoresis and the like are not needed in the whole process, and multiple detection results can be observed by naked eyes within 5 minutes through a lateral flow test strip only by incubating at the constant temperature of 37 DEG C for 10 minutes. The detection system has the characteristics of rapidness, high efficiency, high sensitivity, strong specificity and no dependence on laboratory equipment, is suitable for on-site large-scale detection of field samples and sugarcane introduction materials, and greatly improves the detection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of plant virus detection technology, specifically relating to an RT-RPA-LFD method and its application that can simultaneously and rapidly visualize the detection of three pathogens of sugarcane mosaic virus. Background Technology

[0002] Sugarcane mosaic disease (SMD) is one of the major viral diseases affecting sugarcane production. The main symptoms are irregular yellow-green stripes, streaks, or mottling on the leaves. In severe cases, varying degrees of reddening and necrosis may occur. The primary damage is reduced germination rate of sugarcane seedlings, shortened internodes, and slowed growth, leading to a significant decrease in sugarcane yield and quality. In severe cases, yield losses can exceed 30%, seriously hindering the stable and sustainable development of my country's sugarcane industry. Sugarcane mosaic disease is mainly caused by three pathogens: sugarcane mosaic virus (SCMV), sugarcane streak mosaic virus (SCSMV), and sorghum mosaic virus (SrMV). SrMV and SCMV are both from the Potato Virus Y family (…). Potyviridae Potato virus Y ( ) Potyvirus SCSMV belongs to the genus SCMAV of the family Povidoneviruses (Potatovirus Y family, Poaceae family). Poacevirus (Members). Because these three viruses often cause similar symptoms and co-infections are common, it is impossible to accurately distinguish between these three pathogens based on symptoms alone.

[0003] Because sugarcane is primarily propagated asexually via sugarcane stalks, the virus spreads rapidly throughout sugarcane-growing regions in China and even abroad as the seed stalks are transported. Furthermore, the symptoms caused by sugarcane virus infection are complex and can be latent due to environmental factors, nutritional status, and growth stage, placing new and higher demands on the sensitivity of virus detection. Currently, the key measure for controlling sugarcane virus diseases is early detection and warning of sugarcane viruses. Numerous methods for detecting and identifying sugarcane viruses have been reported, including electron microscopy, serological testing, and molecular biology techniques such as RT-PCR, multiplex RT-PCR, IC-RT-PCR, and antibody-based immunoserological methods. However, these methods differ in applicability and sensitivity, are time-consuming, and require specialized equipment, thus limiting their large-scale application in production. Recombinase polymerase amplification (RPA) is a detection technique that initiates exponential DNA amplification at a constant temperature (22–45°C), rapidly completing nucleic acid amplification within 20 minutes. RPA combined with lateral flow dipstick (LFD) is a rapid, simple, and visual detection method for amplified products. This technology mainly uses a double-antibody sandwich method, where the control line and the test line are covered by colloidal gold particles and biotin ligands, respectively. After the RPA product flows laterally on the test strip, the result is displayed as a color signal on the control line and the test line within 5 to 10 minutes, which can achieve semi-quantitative analysis that is directly visible to the naked eye.

[0004] There are currently no research reports on triple RT-RPA-LFD detection methods for simultaneously detecting SrMV, SSCSMV, and SCMV. This invention, based on conserved segments of the full-length genome sequences of SrMV, SSCSMV, and SCMV published on NCBI, designs primers and probes for multiplex RT-RPA-LFD detection. Through optimization of the reaction system, a triple RT-RPA-LFD detection method for the simultaneous and rapid detection of SrMV, SSCSMV, and SCMV is designed. This method can be used for the quarantine of virus-free healthy sugarcane seedlings and germplasm materials, as well as for the monitoring of sugarcane mosaic virus outbreaks, providing a guarantee for the healthy development of the sugarcane industry.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide an RT-RPA-LFD method and its application for the simultaneous, rapid, and visual detection of three pathogens of sugarcane mosaic disease.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A primer set for the simultaneous, rapid, and visual detection of three pathogens of sugarcane mosaic virus is disclosed. The RT-RPA-LFD primer set includes primer set I, primer set II, and primer set III. Primer set I includes SrMV primer pairs and SrMV probes, and their nucleotide sequences are shown below: SrMV-F: 5'-GTTACGTCGATCTCTTAAACCAAGCATGGGCAG-3'; SrMV-R: 5'Biotin-CATAAACTGTGGTGGAGTTTGGTTGAAATAGAG-3'; SrMV probe: 5'FAM-TTATATTCAATCTGGCGTGTGTACAAAGTC-dSpacer-AGAAATATTACAAGCCGTGC-C3Spacer-3' Primer set II includes SSCMV primer pairs and SSCMV probes, the nucleotide sequences of which are shown below: SCSMV-F: 5'-TTTGGCGAATTGGCGTACAARTGGGTTCAG-3'; SCSMV-R: 5'Biotin-CATCATCTTCYCTACGCAGGTCCGTGTCCTC-3'; SCSMV probe: 5'TAMRA-TCCTCTCACCAGCAGAAATAGACGTGCGTA-dSpacer-CCAGGTGATTAACGCACGCAC-C3 Spacer-3'; Primer set III includes SCMV primer pairs and SCMV probes, the nucleotide sequences of which are shown below: SCMV-F: 5'-GGACTTCAGCGAAATCTCACCGACTATAGCTTAGC-3'; SCMV-R: 5'Biotin-TCTGTATTCTCCTGAGTCTCGCCGACATTTCCGTC-3'; SCMV probe: 5'DIG-CTATGAAATGACTTCACGCACACCAGCTAG-dSpacer-GCTAAGGAAGCCCACATGCAG-C3 Spacer-3'.

[0008] Furthermore, the final concentrations of SrMV-F, SrMV-R, SrMV probe, SSCMV-F, SSCMV-R, SSCMV probe, SCMV-F, SCMV-R and SCMV probe are, in sequence, 40 nM, 40 nM, 12 nM, 40 nM, 40 nM, 12 nM, 40 nM, 40 nM and 12 nM.

[0009] This invention also provides a multiplex RT-RPA-LFD kit for the simultaneous, rapid, and visual detection of three pathogens of sugarcane mosaic virus. The multiplex RT-RPA-LFD kit includes the RT-RPA-LFD primer set as described in claim 1 and Whatman No. 1 cellulose filter paper strips. The cellulose filter paper strips are 22 mm × 2 mm in size, with the upper 20 mm area encapsulated in paraffin and the lower 2 mm area used for nucleic acid binding. Rapid crude nucleic acid extraction is achieved through the following four steps: Pathological tissue is lysed by grinding in buffer [20 mM Tris (pH 8.0), 25 mM NaCl, 2.5 mM EDTA, 0.05% SDS] for 15 s; the cellulose filter paper strips are immersed in a homogenized mixture for 3 s to adsorb nucleic acids; washing is performed in buffer [10 mM Tris (pH 8.0), 0.1% Tween-20] for 3 s; and finally, elution is performed in the RPA reaction mixture for 4 s. The entire process can be completed within 30 s, generating a crude template suitable for rapid on-site detection.

[0010] This invention also provides a triple-target colloidal gold test strip containing anti-DIG, TAMRA, and FAM monoclonal antibodies for detecting multiplex RT-RPA results. The working principle of the multiplex RT-RPA-LFD test strip relies on the specific binding of nucleic acid amplification products labeled with unique fluorophores to the corresponding antibodies coated on the LFD membrane. Specific RPA amplicones double-labeled with different reporter molecules can be captured by different detection antibodies, named anti-DIG monoclonal antibody, anti-TAMRA monoclonal antibody, and anti-FAM monoclonal antibody, corresponding to the T1, T2, and T3 lines on the test strip, respectively. DIG, TAMRA, and FAM are labeled at the 5' end of the probe, and biotin is labeled at the 5' end of the optimal reverse primer. The RPA amplification products are deposited on the three detection lines of the LFD test strip through immunocapture for the LFD detection of SCMV, SSCMV, and SrMV. The T1 line immobilizes DIG antibody for detecting DIG-labeled SCMV-RPA amplicons; the T2 line immobilizes TAMRA antibody for detecting TAMRA-labeled SrMV-RPA amplicons; and the T3 line immobilizes FAM antibody for detecting FAM-labeled SrMV-RPA amplicons. The detection results are interpreted as follows: When the T1 line and control line of the test strip show color, the sample is positive for sugarcane mosaic virus; if there is no T1 line, the sample is negative for sugarcane mosaic virus. When the T2 line and control line of the test strip are visible, the sample is positive for sugarcane mosaic virus; if there is no T2 line, the sample is negative for sugarcane mosaic virus. When the T3 line and control line of the test strip show color, the sample is positive for sorghum mosaic virus; if there is no T3 line, the sample is negative for sorghum mosaic virus.

[0011] The optimized multiplex RT-RPA reaction system (50 μL) of this invention is as follows: 29.4 μL of Abuffer, 2 μL (1 μM) each of the upstream primers of SrMV / SCSMV / SCMV, 2 μL (1 μM) each of the downstream primers of SrMV / SCSMV / SCMV, 0.6 μL (1 μM) each of the SrMV / SCSMV / SCMV probes, and a total volume of template and ddH2O of 4.3 μL. Finally, 2.5 μL of B buffer is added to the reaction tube.

[0012] The RT-RPA amplification conditions used in this invention are: constant temperature incubation at 37℃ for 10 min, RPA amplification product dropped onto colloidal gold test strip, and results observed after 5 min.

[0013] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses Whatman No. 1 cellulose filter paper strips to rapidly extract nucleic acids, which shortens the nucleic acid preparation time from the traditional 2 h to 25 s, significantly improving the detection efficiency.

[0014] (2) The present invention can complete reverse transcription and RPA amplification through a one-step triple RT-RPA-LFD reaction, and can also detect specific visual bands of three pathogens that cause sugarcane mosaic disease at the same time. It is highly specific, fast, accurate, simple to operate and highly sensitive.

[0015] (3) This invention designs and synthesizes three pairs of specific primers and probes for the conserved sequences of three pathogens: sorghum mosaic virus, sugarcane stripe mosaic virus and sugarcane mosaic virus. By optimizing the concentration, reaction temperature and reaction time of these three pairs of specific primers and probes, reverse transcription and RPA amplification can be completed in one step with only one RT-RPA reaction using the crude nucleic acid extracted from the sample as a template. This overcomes the shortcomings of conventional RT-RPA for pathogen detection, which requires two or three RT-RPA reactions, cumbersome operation steps and multiple types of reagents.

[0016] (4) This invention enables the detection of three pathogenic viruses, SrMV, SSCMV, and SCMV, on the test strip even at a concentration of 10 fg / μL, demonstrating very high detection sensitivity. It is 100,000 times more sensitive than multiplex RT-PCR and 1,000 times more sensitive than multiplex RT-LAMP, showing significant advantages in detecting low viral titers, asymptomatic cases, and early stages of viral infection.

[0017] (5) The multiplex RT-RPA-LFD detection method established in this invention has strong specificity and no cross-reaction with other viral pathogens of sugarcane, such as sugarcane yellow leaf virus (SCYLV), sugarcane baculovirus (SCBV), potato virus Y (PVY), and wheat mosaic virus (TriMV). It can be used for the accurate detection of mixed-infected samples.

[0018] (6) This invention successfully developed a large-scale sugarcane virus detection system suitable for field use through multiplex RT-RPA-LFD experiments. The entire process does not require complicated RNA extraction, cDNA reverse transcription, gel electrophoresis, etc. It only requires incubation at 37°C for 10 min, and the multiplex detection results can be observed visually within 5 min using lateral flow test strips. This detection system is characterized by its speed, efficiency, high sensitivity, strong specificity, and independence from laboratory equipment. It is suitable for large-scale on-site detection of field samples and sugarcane introduced materials, greatly improving detection efficiency. Attached Figure Description

[0019] Figure 1The image shows the verification results of rapid nucleic acid extraction using Whatman No.1 cellulose filter paper strips; Figure 2 The diagram shows the optimization results of the triple RT-RPA-LFD system of this invention; Figure 3 This invention provides a sensitivity verification and methodological comparison for triple RT-RPA-LFD detection. Figure 4 This is a graph showing the specificity analysis results of the triple RT-RPA-LFD detection of the present invention; Figure 5 This is a diagram showing the results of field sample testing of sugarcane according to the present invention.

[0020] Explanation of key figure labels: Figure 1 In the diagram, A: Schematic diagram of cellulose filter paper strip structure; B: Schematic diagram of the workflow for rapid nucleic acid extraction using cellulose filter paper strip; C: Comparison of agarose gel electrophoresis of crude nucleic acid extracted from sugarcane with RNA extracted by conventional TRIzol method and DNA extracted by CATB method (NA: nucleic acid; M: DNA molecular weight standard; numbers 1, 2, and 3 represent three independent replicate experiments); D: Using cDNA from crude NA or TRIzol-extracted RNA from sugarcane leaves infected with five viruses (SrMV, SCMV, SSCMV, SCYLV, and SCBV) as templates, the products were reverse amplified by RT-PCR, and the results were detected by agarose gel electrophoresis. ddH2O served as a negative control; E: Using crude NA or TRIzol-extracted RNA as templates, the RT-RPA amplification products of the above five sugarcane viruses were subjected to agarose gel electrophoresis. Figure 2 In the diagram, A: Validation of RT-RPA-LFD primers and probes; Lane 1: Multiplex RT-RPA-LFD detection using primers / probes / templates for sorghum mosaic virus, sugarcane stripe mosaic virus, and sugarcane mosaic virus; Lane 2: Template-free control (NTC) for multiplex RT-RPA-LFD; Lane 3: SrMV-specific RT-RPA-LFD detection; Lane 4: SSCMV-specific RT-RPA-LFD detection; Lane 5: SSCMV-specific RT-RPA-LFD detection; B: Optimization of primer and probe concentrations in the multiplex RT-RPA-LFD detection method; C: Optimization results of RT-RPA-LFD amplification temperature; D: Optimization results of multiplex RT-RPA-LFD amplification time; T1-T3 are detection lines 1-3, and C is the control line.

[0021] Figure 3In the diagram, A: Sensitivity validation of multiplex RT-RPA-LFD: T1-T3 are detection lines 1-3, C is the control line, and NTC is the template-free control; B: Sensitivity validation of multiplex RT-PCR (agarose gel electrophoresis analysis); C: Agarose gel electrophoresis analysis of multiplex RT-LAMP products; D: Visual fluorescence detection of multiplex RT-LAMP products. Figure 4 In the diagram, A represents the detection results of PVY, SCYLV, SCBV, and TriMV viral templates in the multiplex RT-RPA-LFD system; PC represents the positive control; NTC represents the control without a template; B represents the detection performance of multiplex RT-RPA-LFD on single or multiple viral templates; "+" indicates the addition of the corresponding viral template, and "-" indicates the absence of the corresponding viral template. Figure 5 In the table, A: randomly collected sugarcane samples showing suspected mosaic symptoms; B: multiplex RT-RPA-LFD test results of 20 field samples; C: singlex RT-PCR test results; D: multiplex RT-PCR test results; E: comparative analysis of the results of the three detection methods: "+" indicates the presence of the corresponding virus, and "-" indicates the absence of the corresponding virus. Detailed Implementation

[0022] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Example

[0023] 149 sugarcane virus samples were collected from major sugarcane-growing areas in Guangxi Zhuang Autonomous Region, including Nanning, Baise, Liuzhou, Guigang, Fangchenggang, and Chongzuo, as well as virus samples from other sugarcane-growing areas in China. Total RNA was extracted from leaf samples using the RNA isolater Total RNA Extraction Reagent (Catalog No.: R401-01, Nanjing Novizan Biotechnology Co., Ltd.) according to the manufacturer's instructions. Subsequently, cDNA was synthesized using RNA as a template using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Catalog No.: R323-01, Nanjing Novizan Biotechnology Co., Ltd.) to prepare SrMV, SSCMV, and SCMV RNA standards to establish an RT-RPA-LFD experimental analysis system.

[0024] To rapidly extract nucleic acids from sugarcane for the detection of mosaic virus, this study used Whatman No. 1 cellulose filter paper (catalog number: 1001-824, GE Healthcare) pre-cut into 22 mm × 2 mm strips. The top 20 mm of each strip was embedded in paraffin wax, leaving only the bottom 2 mm exposed for nucleic acid adsorption. Lysis buffer [20 mM Tris (pH 8.0), 25 mM NaCl, 2.5 mM EDTA, 0.05% SDS] was pre-added to the grinding bag to release the nucleic acids. 0.5–1 g of sugarcane leaf tissue was placed in the grinding bag and rapidly ground for 15 seconds. The cellulose filter paper strip was then immersed in the lysis buffer for 3 seconds to adsorb the nucleic acids. Subsequently, the filter paper strip was transferred to washing buffer [10 mM Tris (pH 8.0), 0.1% Tween-20] and washed for 3 s to remove amplification inhibitors. Then, it was immersed in the reaction solution for 4 s to release nucleic acids. The resulting crude nucleic acid template can be directly used for recombinase polymerase amplification (RPA) and polymerase chain reaction (PCR).

[0025] Based on the complete genome sequences in GenBank, conserved genomic regions of SrMV, SSCMV, and SCMV were sequence aligned using DNAMAN software, and target regions were selected for recombinase polymerase amplification (RPA) primer design. Virus-specific RPA detection primer pairs (SrMV-F / -R, SSCMV-F / -R, and SCMV-F / -R) were designed using SnapGene 6.0 software, with the forward primer (5' end) unlabeled and the reverse primer (5' end) biotin-labeled. All RPA primers were 30-35 bp in length, with GC content controlled between 35-60%, and amplified products ranging in length from 180-280 bp. Simultaneously, virus-specific hybridization probes of 46-52 nt in length were designed and synthesized, with the 5' ends of the SrMV, SSCMV, and SCMV probes labeled with 6-carboxyfluorescein (FAM), 5-carboxytetramethylrhodamine (TAMRA), and digoxigenin (DIG), respectively. All probes have a C3 spacer blocker at the 3' end and a dSpacer (tetrahydrofuran, THF) is introduced at base 31 as the nfo enzyme recognition site. All primers and probes were synthesized by Nanning Genesys Pharma Co., Ltd. (Nanning, China).

[0026] The recombinase polymerase amplification reaction was performed using the RNA Isothermal Rapid Amplification Kit (Colloidal Gold Test Strip Type II) (Catalog No.: WLN8203KIT, Anpu Future (Changzhou) Biotechnology Co., Ltd.), and the operation procedure was strictly followed according to the instructions. The liquid components in the kit were pre-alicylated and thoroughly mixed by vortexing after thawing at room temperature. Since this kit already contains reverse transcriptase, there is no need to additionally reverse transcribe the RNA template into cDNA.

[0027] The one-step RT-RPA assay was performed in a single tube. The multiplex RT-RPA reaction system (total volume 50 μL) consisted of: 29.4 μL of buffer A in the lyophilized reaction tube; 2 μL each of the upstream primers (1 μM each) and downstream primers (1 μM each) for SrMV / SCSMV / SCMV; 0.6 μL each of the specific probes for SrMV / SCSMV / SCMV (1 μM each); 4.3 μL of template and ultrapure water; and finally, 2.5 μL of buffer B was added. The tube was immediately incubated at 37°C for 10 min. After the reaction, the product was diluted 50-fold with the dilution buffer or ultrapure water provided with the test strip. After thorough mixing, the product was immersed in a three-target nucleic acid detection test strip (catalog number: AJP13-25K, Nanjing Zhongding Biotechnology Co., Ltd.) for color development. The results were determined within 5 min by observing the control line (C line) and the detection line (T line). The result interpretation criteria are as follows: Positive (valid): Both the test line (T line) and the control line (C line) show color; Negative (no target virus): Only the control line (C line) shows color, the test line does not show color; Invalid (retest required): The test line shows color but the control line does not, or neither shows color.

[0028] RPA probes labeled with different fluorescent groups were designed for each target virus, and corresponding monoclonal antibodies targeting these fluorescent groups were immobilized on the binding pad to prepare multiplex lateral flow test strips (LFD). The multiplex RT-RPA-LFD detection method was optimized by simultaneously testing the concentrations and ratios of primers, probes, and templates for SrMV, SSCMV, and SCMV in the same test tube. Specific optimized concentrations are shown in Table 1.

[0029] Table 1. Optimized final concentrations of primers and probes for triple RT-RPA-LFD

[0030] Eight reaction time gradients (3, 5, 7.5, 10, 15, 20, 25 and 30 min) were set to determine the shortest and optimal reaction time; eight reaction temperature gradients (28, 31, 34, 37, 39, 41, 43 and 45℃) were set to screen for the best temperature conditions.

[0031] Sugarcane virus RNA was serially diluted 10-fold to obtain seven concentration gradients: 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and 1 fg / μL. Sensitivity assays were performed using these RNA templates in an optimized reaction system, with a negative control (NTC, template-free control) included. The detection limit of multiplex RT-RPA-LFD was determined by evaluating the clarity and intensity of the detection bands. Sensitivity comparisons were also performed with multiplex reverse transcription polymerase chain reaction (mRT-PCR) and multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) assays.

[0032] To verify the specificity of the multiplex RT-RPA-LFD detection method of the present invention, viral RNA templates of PVY, SCYLV, and SCBV, along with recombinant plasmid DNA containing the TriMV genome sequence (GenBank: FJ263671), were detected together with a positive control (a mixture of SrMV, SCSMV, and SCMV RNA templates) and a negative control (ddH2O). The reaction was incubated at 37°C for 10 min, followed by LFD analysis to assess cross-reactivity with non-target pathogens.

[0033] To evaluate the applicability of the multiplex RT-RPA-LFD platform of this invention in field sample detection, 20 sugarcane samples suspected of being infected with mosaic virus were randomly collected from the field. Crude nucleic acids were extracted from the leaf samples using a rapid nucleic acid extraction method based on cellulose test strips, and these extracted nucleic acids were used as templates for the multiplex RT-RPA-LFD to evaluate its performance in field diagnosis.

[0034] Results and Analysis 1. Nucleic acid extraction process and template quality verification like Figure 1 As shown in Figure A, a rapid nucleic acid adsorption carrier was constructed using Whatman No. 1 cellulose filter paper strips: the upper 20 mm of the strips were sealed and fixed with paraffin wax, while the lower 2 mm of the strips were exposed to form a nucleic acid adsorption area.

[0035] like Figure 1 As shown in B, the lysis system was constructed as follows: A lysis buffer [20 mM Tris (pH 8.0), 25 mM NaCl, 2.5 mM EDTA, 0.05% SDS] was pre-added to the grinding bag. 0.5–1 g of diseased sugarcane leaf tissue was added and rapidly ground for 15 seconds. The tissue was then immersed in filter paper for 3 seconds to complete nucleic acid adsorption. Subsequently, the tissue was washed sequentially with washing buffer [10 mM Tris (pH 8.0), 0.1% Tween-20] for 3 seconds and eluted with reaction solution for 4 seconds. The resulting crude nucleic acid extract could be directly used for RPA / PCR reactions.

[0036] like Figure 1 As shown in Figure C, agarose gel electrophoresis analysis revealed that nucleic acid templates extracted by the cellulose filter paper method (NA), TRIzol method (RNA), and CTAB method (DNA) all exhibited clear bands, proving that the filter paper method can obtain high-quality templates.

[0037] Further PCR amplification was performed using the crude nucleic acid extract as a template, such as... Figure 1 As shown in Figure D, SrMV (1332 bp), SSCMV (319 bp), SCMV (497 bp), SCYLV (604 bp), and SCBV (831 bp) all exhibited single bands of the expected size; the RPA amplification product ( Figure 1 E) The product size was consistent with that extracted RNA by TRIzol, although the band intensity was weaker (due to lower template content), demonstrating that the crude template extract can be effectively used for RPA reactions. This method reduces nucleic acid preparation time from the traditional 2 h to 25 s, significantly improving detection efficiency.

[0038] 2. Optimization of the Multiple RT-RPA-LFD Detection System 2.1 Validation of multiplex RT-RPA-LFD primers and probes like Figure 2 As shown in Figure A, the triple RT-RPA-LFD reaction (lane 1) showed positive red bands at T1, T2, and T3 lines, while the single reaction (lanes 3-5) showed virus-specific bands, confirming that the method is accurate and feasible.

[0039] 2.2 Optimization of multiplex RT-RPA-LFD primer and probe concentrations Six primer / probe concentration combinations were tested (120 nM / 36 nM, 40 nM / 12 nM, 20 nM / 6 nM, 120 nM / 12 nM, 40 nM / 6 nM, 20 nM / 3 nM), such as Figure 2 As shown in Figure B, the three bands showed the most uniform intensity with 40 nM primer / 12 nM probe, and this was selected as the optimal concentration.

[0040] 2.3 Multiple RT-RPA-LFD Temperature Optimization The reaction temperature was tested within the range of 28-45℃. Figure 2 As shown in Figure C, the three bands exhibit the strongest and most uniform intensity at 37°C (close to physiological temperature, suitable for on-site testing), which is determined to be the optimal reaction temperature.

[0041] 2.4 Multiple RT-RPA-LFD Time Optimization Set a gradient reaction time of 3-30 min, such as Figure 2As shown in Figure D, the brightness of the three bands is uniform after 10 minutes, which meets the requirements for rapid detection and is selected as the shortest reaction time.

[0042] 3. Sensitivity Verification and Methodological Comparison 3.1 Multiple RT-RPA-LFD Sensitivity When the mixed RNA of SrMV, SSCSMV, and SCMV was diluted to 10 fg / μL, the LFD band was still clearly visible; at 1 fg / μL, no color development was observed (consistent with the negative control), and the detection limit reached 10 fg / μL. Figure 3 A).

[0043] 3.2 Comparison with mRT-PCR The limit for mRT-PCR detection of cDNA is 1 ng / μL ( Figure 3 B), the sensitivity of the multiple RT-RPA-LFD is about 100,000 times higher than that of the other two.

[0044] 3.3 Comparison with mRT-LAMP The detection limit of mRT-LAMP for cDNA is 100 pg / μL. Figure 3 (C and 3D), the sensitivity of the multiple RT-RPA-LFD is about 1000 times higher than that of the previous one.

[0045] 4. Specificity verification of multiple RT-RPA-LFD like Figure 4 As shown in Figure A, non-target pathogens (PVY, SCYLV, SCBV, TriMV) and negative controls only show the control line (C line), with no detection line (T line) appearing; positive controls (SrMV / SCSMV / SCMV) show three clear red bands. Figure 4 As shown in B, in the detection of samples with one to three infections, the T line only showed color when the target virus was present, and there was no cross-reaction with non-target viruses, proving that the method has excellent specificity.

[0046] 5. Field sample testing and verification Collect 20 sugarcane plants suspected of having mosaic disease ( Figure 5 A), using multiplex RT-RPA-LFD, 6 cases of SrMV, 13 cases of SSCMV, and 1 case of SCMV were detected as positive. Figure 5 B), the results were consistent with those of singlet RT-PCR ( Figure 5 C); among them, SSCMV samples 14 and 16 showed very faint bands in singlet RT-PCR electrophoresis, which may have been missed, highlighting the advantage of the multiplex RT-RPA-LFD visualization detection method. In the five-fold RT-PCR detection, the multiplex RT-RPA-LFD showed a higher detection rate for SSCMV (samples 8, 9, 13, 14, 16, and 20), indicating that its sensitivity is superior to traditional methods. Figure 5 D). The results of on-site sample testing using the three methods were summarized, such as... Figure 5 As shown in E.

[0047] In summary, the multi-RT-RPA-LFD detection system developed in this invention is characterized by its speed, efficiency, and large-scale operation, making it suitable for on-site testing.

[0048] The foregoing description of specific exemplary embodiments of the present invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and many changes and variations are possible. The exemplary embodiments were chosen and described to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. An RT-RPA-LFD primer set capable of simultaneously, rapidly, and visually detecting three pathogens of sugarcane mosaic virus, characterized in that... The RT-RPA-LFD primer set includes primer set I, primer set II, and primer set III; wherein, Primer set I includes SrMV primer pairs and SrMV probes, and their nucleotide sequences are shown below: SrMV-F: 5'-GTTACGTCGATCTCTTAAACCAAGCATGGGCAG-3'; SrMV-R: 5'Biotin-CATAAACTGTGGTGGAGTTTGGTTGAAATAGAG-3'; SrMV probe: 5'FAM-TTATATTCAATCTGGCGTGTGTACAAAGTC-dSpacer-AGAAATATTACAAGCCGTGC-C3Spacer-3' Primer set II includes SSCMV primer pairs and SSCMV probes, the nucleotide sequences of which are shown below: SCSMV-F: 5'-TTTGGCGAATTGGCGTACAARTGGGTTCAG-3'; SCSMV-R: 5'Biotin-CATCATCTTCYCTACGCAGGTCCGTGTCCTC-3'; SCSMV probe: 5'TAMRA-TCCTCTCACCAGCAGAAATAGACGTGCGTA-dSpacer-CCAGGTGATTAACGCACGCAC-C3Spacer-3'; Primer set III includes SCMV primer pairs and SCMV probes, the nucleotide sequences of which are shown below: SCMV-F: 5'-GGACTTCAGCGAAATCTCACCGACTATAGCTTAGC-3'; SCMV-R: 5'Biotin-TCTGTATTCTCCTGAGTCTCGCCGACATTTCCGTC-3'; SCMV probe: 5'DIG-CTATGAAATGACTTCACGCACACCAGCTAG-dSpacer-GCTAAGGAAGCCCACATGCAG-C3Spacer-3'.

2. The RT-RPA-LFD primer set according to claim 1, characterized in that, The final concentrations of SrMV-F, SrMV-R, SrMV probe, SSCMV-F, SSCMV-R, SSCMV probe, SCMV-F, SCMV-R and SCMV probe are, respectively, 40 nM, 40 nM, 12 nM, 40 nM, 40 nM, 12 nM, 40 nM, 40 nM and 12 nM.

3. A multiplex RT-RPA-LFD kit for simultaneous, rapid, and visual detection of three pathogens of sugarcane mosaic virus, characterized in that, Includes the RT-RPA-LFD primer set as described in claim 1, and Whatman No. 1 cellulose filter paper strip.

4. The application of the RT-RPA-LFD primer set according to claim 1 or the multiplex RT-RPA-LFD kit according to claim 3 in the simultaneous rapid detection of three pathogens of sugarcane mosaic virus.

5. The application according to claim 4, characterized in that, The three pathogens of sugarcane mosaic disease are sorghum mosaic virus (SrMV), sugarcane stripe mosaic virus (SCSMV), and sugarcane mosaic virus (SCMV).

6. The application of the RT-RPA-LFD primer set according to claim 1 or the multiplex RT-RPA-LFD kit according to claim 3 in the simultaneous and rapid detection of sorghum mosaic virus SrMV, sugarcane stripe mosaic virus SSCSMV and sugarcane mosaic virus SCMV.

7. A multiplex RT-RPA-LFD detection method for the simultaneous and rapid detection of three pathogens of sugarcane mosaic virus, characterized in that, Includes the following steps: S1. Collect sugarcane samples and complete crude nucleic acid extraction within 25 seconds using Whatman No. 1 cellulose filter paper strips; S2. Using crude extracted nucleic acid as a template, perform an RT-RPA reaction using the RT-RPA-LFD primer set described in claim 1; S3. Take the RT-RPA reaction solution, dilute it 50 times with sterile double-distilled water, and then use the LFD test strip to detect and judge the result.

8. The multiple RT-RPA-LFD detection method according to claim 7, characterized in that, In step S2, the RT-RPA reaction conditions are: reaction at 37°C for 10 min, followed by LFD incubation for 5 min.

9. The multiple RT-RPA-LFD detection method according to claim 7, characterized in that, In step S3, the interpretation of the LFD test strip is as follows: Positive: One red band appears on the control line of the test strip, and one to three red bands appear on the test line; Negative: One red band appears at the control line of the test strip, but no band appears at the test line; Invalid: No bands appeared on the control line of the test strip.

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