Primer and fluorescent probe suitable for dual detection of maize yellow mosaic virus and sugarcane mosaic virus of plants and vector insects and application of primer and fluorescent probe

Abstract

The invention discloses a primer and a fluorescent probe suitable for dual detection of maize yellow mosaic virus and sugarcane mosaic virus of plants and vector insects and application of the primer and the fluorescent probe, and belongs to the technical field of biology. The detection technology established by the invention not only can efficiently detect the target pathogen in the main food crop corn, but also can identify various related weeds and aphids. The characteristic that one method has multiple purposes greatly expands the application scene of the technology, and provides a strong support for comprehensively monitoring the distribution and dynamic state of the pathogen in a farmland ecosystem. Meanwhile, by means of the detection technology system, migration paths of pathogens in different seasons and among different hosts can be tracked, key links of disease circulation of the pathogens are successfully revealed, a solid theoretical foundation is laid for formulating scientific and effective cross-crop and cross-seasonal prevention and control strategies, and important practical guiding significance is achieved.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to primers and fluorescent probes for the dual detection of maize yellow mosaic virus and sugarcane mosaic virus in plants and vector insects, and their applications. Background Technology

[0002] corn( Zea mays Corn (L.) is my country's largest crop, accounting for 23% of global annual production. my country is a major global producer and consumer of corn, and corn has been my country's largest grain crop for many consecutive years. Besides its edible value, corn is also an important raw material for high-quality feed and light industry. Pests and diseases are the main disasters affecting corn production, among which corn viral diseases are one of the major threats to corn production safety. Under natural conditions, various viruses can infect corn and cause yield reduction. Maize yellow mosaic virus (MaYMV), a novel baculovirus, is widely distributed in Asia, America, and Africa. It often mixes with other RNA viruses and can be detected in cereal crops such as corn, as well as weeds. Studies have shown that MayMcV and SCMV (sugarcane mosaic virus) can co-infect corn through the corn aphid. Furthermore, MayMcV isolates cause mosaic symptoms when infecting corn alone, and exhibit more severe symptoms when mixed with SCMV. Infected maize plants typically exhibit symptoms such as yellowing leaves, mosaic patterns, and stunting. In severe cases, it can lead to plant death, affecting maize yield and quality, and seriously jeopardizing the safety of maize agricultural production. Studies have found that MayMV infection can cause yellowing and stunting symptoms in wheat in Henan Province, and also lead to red leaf disease in various weeds in the field.

[0003] After being injected into the phloem of corn by the stylets of aphids, the corn yellow mosaic virus replicates and multiplies extensively within plant cells. The viral genome and proteins systematically move throughout the plant (via the vascular bundles), spreading to the entire plant, including leaves, stems, and even roots, accumulating to very high concentrations. However, for the aphid, the vector, feeding on infected plants involves ingesting virus-containing plant sap. Virus particles must penetrate the aphid's intestinal barrier to enter the hemolymphatic system, then circulate to the salivary glands. When the aphid feeds again, the virus is injected into a new, healthy plant via saliva. This means the virus cannot replicate and multiply within the aphid. The aphid is merely a passive "transporter," and the amount of virus within it depends entirely on the amount it recently ingested from infected plants. Over time, the virus is metabolized or degraded, and its concentration gradually decreases. Therefore, the viral load within aphids remains consistently low.

[0004] MaYMV and SCMV compound infection causes systemic collapse of maize through "physiological plunder + defense disintegration + synergistic proliferation", which is much more harmful than single virus. The core of prevention and control lies in accurate monitoring of double viruses at seedling stage and innovation of disease-resistant germplasm, and the establishment of "one management and double detection" RPA field rapid detection technology is the primary defense line to block the spread of disasters.

[0005] Currently, there is no rapid detection method for maize yellow mosaic virus and sugarcane mosaic virus. In addition, there are few studies on the detection method of maize yellow mosaic virus and sugarcane mosaic virus, so the establishment of the detection method is of great significance for disease prevention and control. Even in the case of limited technology and means in aphid virus detection research, the detection result can be obtained conveniently and quickly, which helps to ensure the yield and quality of maize and ensure the healthy development of maize industry. SUMMARY

[0006] The purpose of the present application is to provide a primer and fluorescent probe for double detection of maize yellow mosaic virus and sugarcane mosaic virus suitable for plants and intermediate insects, and application thereof, in order to solve the problems existing in the prior art.

[0007] To achieve the above-mentioned purpose, the present application provides the following solutions: One of the technical solutions of the present application is a primer and fluorescent probe for detecting maize yellow mosaic virus, which comprises an upstream primer as shown in SEQ ID NO. 1 and a downstream primer as shown in SEQ ID NO. 2; the sequence of the fluorescent probe is shown in SEQ ID NO. 5.

[0008] The second technical solution of the present application is a primer and fluorescent probe for detecting sugarcane mosaic virus, which comprises an upstream primer as shown in SEQ ID NO. 3 and a downstream primer as shown in SEQ ID NO. 4; the sequence of the fluorescent probe is shown in SEQ ID NO. 6.

[0009] The third technical solution of the present application is a primer and fluorescent probe for double detection of maize yellow mosaic virus and sugarcane mosaic virus, which comprises the primer and fluorescent probe for detecting maize yellow mosaic virus and the primer and fluorescent probe for detecting sugarcane mosaic virus.

[0010] The fourth technical solution of the present application is a kit for double detection of maize yellow mosaic virus and sugarcane mosaic virus, which comprises the primer and fluorescent probe for double detection of maize yellow mosaic virus and sugarcane mosaic virus.

[0011] The fifth technical solution of the application is a double detection method of maize yellow mosaic virus and sugarcane mosaic virus for non-disease diagnosis or treatment purposes. The RNA of the sample to be detected is used as a template, and the primer and fluorescent probe for double detection of maize yellow mosaic virus and sugarcane mosaic virus are used for amplification reaction, and whether the sample to be detected contains maize yellow mosaic virus and sugarcane mosaic virus is judged according to the amplification reaction result.

[0012] Based on the above technical solution, the application has the following technical effects: 1. The application is based on RPA-LFD detection technology. This detection method can complete the amplification of MaYMV and SCMV within 15 minutes, and the visual result can be obtained through the test strip within 5 minutes. This detection method can meet the needs and is rapid and instant, which is of great significance for timely discovery and control of virus transmission. This method does not require complex instruments and equipment and high temperature conditions, and is suitable for use in fields, basic research units and on-site detection environments with limited conditions. Moreover, the operation is simple and can be completed by non-professionals, thereby reducing the detection threshold.

[0013] 2. The detection method has high sensitivity and can detect low-concentration virus nucleic acids, which helps to discover infection early and take corresponding prevention and control measures. This method has good specificity and can specifically detect target viruses without cross-reaction, thereby ensuring the accuracy of the detection result.

[0014] 3. The detection technology established by the application can detect maize yellow mosaic virus that was newly discovered in 2016 and first reported in 2021 to cause maize red leaf disease, thereby providing a key tool for timely response to its potential threat. Moreover, the established detection technology breaks through the species limitation and has wide applicability. It can not only efficiently detect target pathogens in maize, a major food crop, but also identify in multiple related weeds and aphids. This "one method for multiple uses" greatly expands the application scenarios of the technology and provides strong support for comprehensive monitoring of the distribution and dynamics of the pathogen in the farmland ecosystem. Meanwhile, through the detection technology system of the application, the migration path of the pathogen between different seasons and different hosts can be tracked, and the key link of the disease cycle is successfully revealed, thereby laying a solid theoretical foundation for formulating scientific and effective cross-crop and cross-season prevention and control strategies, which has important practical guiding significance. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The figure is a display diagram and weblogo diagram of the genomic structure and conserved sequence of MaYMV.

[0016] Figure 2 The figure is a display diagram and weblogo diagram of the genomic structure and conserved sequence of SCMV.

[0017] Figure 3 For screening and optimization of MaYMV probe concentration (A), primer concentration (B), time (C) and temperature (D).

[0018] Figure 4 In the above, A is to detect plasmid DNA containing MaYMV cp gene with different concentrations by PCR instrument, and B is to detect plasmid DNA containing MaYMV cp gene with different concentrations by RPA-LFD.

[0019] Figure 5 For the detection results specific to MaYMV, wherein A is to use the nucleic acids of MCMV and SCMV as templates to perform PCR instrument detection with MaYMV primers and probes, and B is to use the nucleic acids of MCMV and SCMV as templates to perform RPA-LFD detection with MaYMV primers and probes.

[0020] Figure 6 For screening of time (A) and temperature (B) conditions of MaYMV in aphids by extracting RNA from the body of the transmission vector aphid.

[0021] Figure 7 In the above, A is PCR detection of different varieties of corn, B is RPA-LFD detection of different varieties of corn, C is PCR detection of toxic aphids and non-toxic aphids, D is RPA-LFD detection of toxic aphids and non-toxic aphids, E is PCR detection of weeds, and F is RPA-LFD detection of weeds.

[0022] Figure 8 For screening of SCMV probe concentration and primer concentration, four groups of controls and four groups of treatments are set. Among them, A is probe concentration screening, and B is primer concentration screening.

[0023] Figure 9 In the above, A is to detect plasmid DNA containing SCMV cp gene with different concentrations by PCR instrument, and B is to detect plasmid DNA containing SCMV cp gene with different concentrations by RPA-LFD.

[0024] Figure 10 For screening of the conditions of double detection, the probe addition ratio and the primer addition ratio of MaYMV and SCMV are screened respectively. Among them, A is probe addition ratio screening, and B is primer addition ratio screening. DETAILED DESCRIPTION

[0025] The technical solutions described in the present application are all conventional solutions in the art if not specifically stated, and the reagents or raw materials used are purchased from commercial channels or are already disclosed if not specifically stated.

[0026] The embodiment of the present application provides a primer and a fluorescent probe for detecting maize mosaic virus, wherein the primer comprises an upstream primer shown as SEQ ID NO. 1 and a downstream primer shown as SEQ ID NO. 2; and the sequence of the fluorescent probe is shown as SEQ ID NO. 5.

[0027] In some specific embodiments, the 5' end of the fluorescent probe shown as SEQ ID NO. 5 is modified by FAM, the 3' end is modified by Spacer, and the middle is modified by THF.

[0028] The embodiment of the present application also provides a primer and a fluorescent probe for detecting sugarcane mosaic virus, wherein the primer comprises an upstream primer shown as SEQ ID NO. 3 and a downstream primer shown as SEQ ID NO. 4; and the sequence of the fluorescent probe is shown as SEQ ID NO. 6.

[0029] In some specific embodiments, the 5' end of the fluorescent probe shown as SEQ ID NO. 6 is modified by Digoxigenin, the 3' end is modified by Spacer, and the middle is modified by THF.

[0030] The embodiment of the present application also provides a primer and a fluorescent probe for detecting maize mosaic virus and sugarcane mosaic virus, which comprises the primer and the fluorescent probe for detecting maize mosaic virus and the primer and the fluorescent probe for detecting sugarcane mosaic virus.

[0031] The embodiment of the present application also provides a kit for detecting maize mosaic virus and sugarcane mosaic virus, which comprises the primer and the fluorescent probe for detecting maize mosaic virus and sugarcane mosaic virus.

[0032] The embodiment of the present application also provides a method for detecting maize mosaic virus and sugarcane mosaic virus for non-disease diagnosis or treatment purposes, wherein RNA of a sample to be detected is used as a template to perform an amplification reaction by using the primer and the fluorescent probe for detecting maize mosaic virus and sugarcane mosaic virus, and whether the sample to be detected contains maize mosaic virus and sugarcane mosaic virus is determined according to a result of the amplification reaction.

[0033] In some specific embodiments, the system of the amplification reaction is as follows: 29.4 μL of A buffer, 2.5 μmol·L - 1 2 μL of RPA-F, 2.5 μmol·L -1 2 μL of RPA-R, 1 μmol·L -1 0.6 μL of probe, 8.5 μL of ddH2O, 5 μL of RNA and 2.5 μL of B buffer.

[0034] The optimal primer concentration of MaYMV and SCMV is 2.5 μmol·L -1 The optimal probe concentration of MaYMV and SCMV is 1 μmol·L -1 The optimal primer addition amount of MaYMV is 1.5 μL, and the optimal primer addition amount of SCMV is 0.5 μL; the optimal probe addition amount of MaYMV is 0.4 μL, and the optimal probe addition amount of SCMV is 0.2 μL In some specific embodiments, the ratio of the primers of the maize yellow mosaic virus and the sugarcane mosaic virus is 3:1, and the ratio of the probes of the maize yellow mosaic virus and the sugarcane mosaic virus is 2:1.

[0035] In some specific embodiments, the reaction condition of the amplification reaction is 42℃ for 30 min.

[0036] The application provides a single and double RPA-LFD on-demand real-time detection method suitable for corn virus diseases of plants and intermediate insects, and establishes a single and double RPA-LFD system of corn virus diseases MaYMV and SCMV, and simultaneously establishes a detection system of a virus-carrying intermediate insect MaYMV, establishes a more sensitive and convenient virus disease detection technology suitable for plants and intermediate insects, can detect MaYMV and SCMV on demand, and performs field distribution annual dynamic monitoring and early warning, establishes the system, provides strong support for accurate detection of corn, aphids and weed viruses and annual cycle law of corn weeds, deeply explores the disaster mechanism of MaYMV and SCMV, and lays a solid foundation for effectively preventing and controlling the two viruses and guaranteeing the healthy development of the corn industry.

[0037] On the one hand, the application provides construction of a single detection method of a maize yellow mosaic virus (MaYMV), construction of a virus detection method of a virus-carrying intermediate insect aphid, establishment of a single detection method of a sugarcane mosaic virus (SCMV), and establishment of a double RPA-LFD detection method of MaYMV and SCMV.

[0038] Preferably, the establishment of the above-mentioned single detection method of MaYMV and SCMV RPA-LFD is to design primers and probes respectively, to screen single detection reaction conditions, to screen aphid detection reaction conditions, to screen double detection reaction conditions, to perform specificity detection, to perform sensitivity detection, and to perform field sample detection.

[0039] Preferably, the above-mentioned reaction condition screening is to screen primer concentration, probe concentration, reaction time and reaction temperature according to actual needs.

[0040] Preferably, the optimal primer concentration of MaYMV and SCMV in the above-mentioned single detection method is 2.5 μmol·L -1, the optimal probe concentration is 1 μmol·L -1 , the optimal time is 14 min, and the optimal temperature is 41℃. The optimal detection time for aphids is 30 min, the optimal temperature is 42℃, the optimal primer ratio for duplex is 3:1, and the optimal probe ratio for duplex is 2:1.

[0041] Preferably, under the above strictly screened conditions, the specificity and sensitivity of MaYMV and the sensitivity of SCMV are comprehensively detected.

[0042] In the second aspect, the application of the method for constructing the virus infection system, the application is selected from any one or more of the following: (1) Study of early diagnosis and prevention and control of corn virus disease; (2) Study of corn virus disease detection efficiency; (3) Study of virus disease detection on aphids, corn, and weeds; (4) Study of the impact of corn virus disease on corn production safety; (5) Study of the popularization and application of corn virus disease prevention and control technology; (6) Study of the annual transmission mechanism of corn red leaf disease through aphid vectors on corn and weeds.

[0043] Preferably, the above corn virus disease has no obvious symptoms in the early stage of the disease, and the virus is small and difficult to observe with the naked eye, which is easily ignored. The rapid detection method can quickly detect the presence of the virus in the latent period or the early stage of the disease, so that prevention and control measures can be taken in time to prevent the spread and transmission of the virus.

[0044] Preferably, the above detection method based on RPA technology is a high-efficiency detection method that can quickly process a large number of samples and improve detection efficiency.

[0045] Preferably, the above rapid detection method can timely detect virus diseases, reduce yield reduction and economic losses caused by virus diseases, and has important significance for preventing virus invasion, cutting off virus transmission, producing non-toxic seed seedlings, disease monitoring and early warning, and green prevention and control, large-scale monitoring in the field, and timely detection of the epidemic trend of virus diseases.

[0046] Example 1 RPA primer and probe synthesis 1. According to the partial conservative sequence of MaYMV cp (NC_075108) and SCMV cp (AY042184) genes, 2 pairs of specific primers and probes were designed using NCBI.

[0047] MaYMV primer: RPA-F (SEQ ID NO. 1): 5'-GGGAGGTAGAAATGGACGCAGAGCT-3'; RPA-R (SEQ ID NO. 2): 5'-Biotin-GATGGCGCATTGATCTTCTGATGAGT-3' (Biotin).

[0048] SCMV primers: RPA-F (SEQ ID NO. 3): 5'-CCAAGGAGGGGGAAGTGGATCTGGAACAAC-3'; RPA-R (SEQ ID NO. 4): (5'-Biotin-GGCGCATTTTCTTCGACATTGCCTTAAGCTT-3'; MaYMV probe (SEQ ID NO. 5): [5'FAM]-AAGACGACCAAGTGGAAACACTGCAGGAAG[THF]CCTGGAGTCAGACGA-[3'C3spacer]; SCMV probe (SEQ ID NO. 6): [5'digoxigenin]-CAGGTGGATCATCAGGAAACAATGGAGGC[THF]GCCAATCAGGTTCAGA-[3'C3spacer]).

[0049] The 5' end of the RPA amplification primer RPA-R is modified with biotin. The 5' end of the RPA probe is modified with FAM (carboxyfluorescein FAM) and Digoxigenin (digoxin Digoxigenin), the 3' end is modified with Spacer (spacer), and the middle is modified with THF (tetrahydrofuran THF). The primers and probes are entrusted to Beijing Chengke Biotechnology Co., Ltd. for synthesis.

[0050] Example 2 Optimization of MaYMV and SCMV detection system reaction conditions 1. Optimization of MaYMV detection system reaction conditions According to the steps required by the RNA isothermal rapid amplification kit (Anfu Future Changzhou Biotechnology Co., Ltd.), a preliminary RPA-LFD detection system was established.

[0051] The reaction system is: A buffer 29.4 μL, 10 μmol·L -1 RPA-F 2 μL, 10 μmol·L -1 RPA-R 2 μL, probe 0.6 μL, ddH2O 8.5 μL, RNA 5 μL, B buffer 2.5 μL.

[0052] After adding each reaction component into the dry powder tube, mix well by inverting the tube rapidly. After mixing, centrifuge at 800 r·min -1 After rapid centrifugation, quickly place in a 42 ℃ water bath for 15 min.

[0053] After the reaction is completed, dilute 10 μL of the reaction product 20-fold with ddH2O, and then add 80 μL of the reaction product to the sample well of the colloidal gold test strip (Anpu Future Changzhou Biological Technology Co., Ltd.). When a red line appears on the quality control line (C line) of the colloidal gold test strip and no red line appears on the detection line (T line), the detection result is negative; when two red lines (C line and T line) appear on the colloidal gold test strip, the detection result is positive.

[0054] When the probe concentration is 5, 1 and 0.5 μmol·L -1 , the T line of the colloidal gold test strip for detecting MaYMV-infected corn leaf samples appears a red band, and as the probe concentration decreases, the T line has no red band, i.e., MaYMV cannot be detected, and no red band appears on the healthy samples. When the probe concentration is high, the colloidal gold test strip is prone to false positives, and when the concentration is too low, the band is too shallow, so 1 μmol·L -1 is selected as the optimal probe concentration of the RPA-LFD detection system.

[0055] When the RPA primer concentration is 2.5 and 2 μmol·L -1 , the T line of the colloidal gold test strip for detecting MaYMV-infected corn leaf samples appears a red band, and as the primer concentration decreases, the T line has no red band. When the primer concentration is low, RPA amplification fails, and the colloidal gold test strip cannot develop color, so a higher concentration of 2.5 μmol·L -1 is selected as the optimal primer concentration of the RPA-LFD detection system.

[0056] When the RPA reaction temperature is 41, 39 and 37 ℃, respectively, the T line of the colloidal gold test strip for detecting MaYMV-infected corn leaf samples appears a red band, and no red band appears on the healthy samples. When the temperature is 41 ℃, the band color is clearer, so 41 ℃ is selected as the optimal reaction temperature of the RT-RPA-LFD detection system.

[0057] When the RPA reaction time is 13 min, the T line of the colloidal gold test strip for detecting MaYMV-infected corn leaf samples has a weak red band, and as the reaction time increases, the T line red band color deepens, and no red band appears on the healthy samples. When the RPA reaction time is too short, amplification cannot be completed, so while reducing the time cost, it is also necessary to ensure that RPA amplification is completed, so 13 min is selected as the optimal reaction time of the RPA-LFD detection system.

[0058] 2 Specificity of the RPA-LFD detection method for MaYMV As shown in Figure 5 , the RT-PCR detection of MaYMV, SCMV and MCMV was carried out using primers MaYMV-F / R, and the results of agarose gel electrophoresis showed that the expected bands were obtained after amplification of the three plant viruses. Using the established RPA-LFD detection system, only the T line of the colloidal gold test strip for MaYMV appeared a red band, and the T lines of the other two plant viruses did not appear, indicating that the detection method can specifically detect MaYMV.

[0059] 3 Sensitivity of the RPA-LFD detection method for MaYMV The ordinary PCR detection was carried out on plasmid DNA containing MaYMV cp gene with different concentrations, and the results of agarose gel electrophoresis showed that the minimum detection limit was 2.341×10 -7 ng·μL -1 . Using RPA-LFD detection, when the concentration of plasmid DNA was 2.341×10 -11 ng·μL -1 , the red band of the T line of the colloidal gold test strip was weakened, and when the concentration of plasmid DNA was 2.341×10 -14 ng·μL -1 , the T line had no red band, i.e. the minimum limit of RPA-LFD detection of MaYMV was 2.341×10 -14 ng·μL -1 . It was shown that the RPA-LFD detection method was 10 7 times more sensitive than ordinary PCR detection.

[0060] 4 Optimization of the reaction conditions of the aphid detection system As shown in Figure 6 , the RPA reaction time was selected as 35, 30, 25, 20, 15 and 10 min, respectively. When the time was 15 min, the T line of the colloidal gold test strip for detecting aphid-containing samples had a weak red band, and with the increase of reaction time, the color of the T line red band deepened, and no red band appeared in healthy samples. When the RPA reaction time was too short, the amplification could not be completed. Therefore, 30 min was selected as the best reaction time of the RPA-LFD detection system.

[0061] The reaction temperature of RPA was selected as 42, 41, 40, 39, 38 and 37℃, respectively. When the temperature was 38℃, the T line of the colloidal gold test strip for detecting aphid-containing samples appeared a weak red band, and no red band appeared in healthy samples. When the temperature was 42℃, the band color was clearer, and therefore 42℃ was selected as the optimum reaction temperature of the RT-RPA-LFD detection system.

[0062] 5 Optimization of reaction conditions of SCMV detection system As Figure 8 shown, when the probe concentration was 1.5, 1 and 0.5 μmol·L -1 , the colloidal gold test strip T line of the corn leaf sample infected by SCMV appeared a red band, and with the decrease of the probe concentration, the T line had no red band, that is, SCMV could not be detected, and the healthy sample had no red band. When the probe concentration was high, the colloidal gold test strip was prone to false positive, and when the concentration was too low, the strip was too shallow, so 1 μmol·L -1 was selected as the optimal probe concentration of the RPA-LFD detection system.

[0063] When the RPA primer concentration was 5, 2.5 and 0.5 μmol·L -1 , the colloidal gold test strip T line of the corn leaf sample infected by MaYMV appeared a red band, and with the decrease of the primer concentration, the T line had no red band. When the primer concentration was low, RPA amplification failed, and the colloidal gold test strip could not develop color, so a higher concentration of 2.5 μmol·L -1 was selected as the optimal primer concentration of the RPA-LFD detection system.

[0064] 6 Sensitivity of RPA-LFD detection method of SCMV As Figure 9 shown, the plasmid DNA containing different concentrations of SCMV cp gene was detected by ordinary PCR, and the agarose gel electrophoresis result showed that the minimum detection limit was 2.217×10 -1 ng·μL -7 . When the plasmid DNA concentration was 2.217×10 -1 ng·μL - , the colloidal gold test strip T line red band was weakened, and when the plasmid DNA concentration was 2.217×10 9 ng·μL -1 , the T line had no red band, that is, the minimum limit of RPA-LFD detection of MaYMV was 2.217×10 -9 ng·μL -1 . It was shown that the RPA-LFD detection method was 10 8 times more sensitive than the ordinary PCR detection.

[0065] Example 3 Optimization of reaction conditions of MaYMV and SCMV double detection system As Figure 10 shown, when the probe was added in the ratio of 1:1, 2:1, 5:1 μmol·L -1When the colloidal gold test strips for detecting MayMV and SCMV infected maize leaf samples showed red bands on the T line, as the proportion of MayMV probe added decreased, the MayMV T line showed no red band, meaning MayMV could not be detected. By adjusting the proportion to achieve consistent band color, a 2:1 ratio was chosen as the optimal probe addition ratio for the dual RPA-LFD detection system of MayMV and SCMV.

[0066] When the RPA primer concentration is 1:1, 3:1, or 9:1 μmol·L⁻¹ -1 At the time of detection, a red band appeared on the T line of the colloidal gold test strip for maize leaf samples infected with MayMV and SCMV. As the proportion of MayMV primer added decreased, no red band appeared on the T line for MayMV, indicating that MayMV could not be detected. By adjusting the ratio to achieve consistent band color, a 3:1 primer addition ratio was selected as the optimal primer addition ratio for the dual RPA-LFD detection system of MayMV and SCMV.

[0067] Example 4 First, PCR detection was performed on different maize varieties, and gel electrophoresis images were obtained. Then, the established RPA-LFD system was used to detect different maize varieties, and the results were consistent, indicating that it is suitable for detecting maize infected with MayMV in the field. PCR detection was then performed on both poisonous and non-poisonous aphids, and gel electrophoresis images were obtained. The established RPA-LFD system was then used to detect both poisonous and non-poisonous aphids, and the results were consistent, indicating that it is suitable for aphid detection. PCR detection was performed on common weeds in the field, and gel electrophoresis images were obtained. The established RPA-LFD system was then used to detect weeds. PCR could not detect MayMV, but RPA-LFD could, indicating that RPA-LFD has higher sensitivity and is also suitable for weed detection.

[0068] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A primer and fluorescent probe for detecting maize yellow mosaic virus, characterized in that, The primers include an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2; the sequence of the fluorescent probe is shown in SEQ ID NO.

5.

2. The primers and fluorescent probes according to claim 1, characterized in that, The fluorescent probe shown in SEQ ID NO.5 has its 5′ end modified with FAM, its 3′ end modified with Spacer, and its middle end modified with THF.

3. A primer and fluorescent probe for detecting sugarcane mosaic virus, characterized in that, The primers include an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4; the sequence of the fluorescent probe is shown in SEQ ID NO.

6.

4. The primers and fluorescent probes according to claim 3, characterized in that, The fluorescent probe shown in SEQ ID NO.6 has a 5′ end modified with Digoxigenin, a 3′ end modified with Spacer, and a middle section modified with THF.

5. A primer and fluorescent probe for dual detection of maize yellow mosaic virus and sugarcane mosaic virus, characterized in that, It includes the primers and fluorescent probes for detecting maize yellow mosaic virus as described in claim 1, and the primers and fluorescent probes for detecting sugarcane mosaic virus as described in claim 3.

6. A kit for dual detection of maize yellow mosaic virus and sugarcane mosaic virus, characterized in that, Includes the primers and fluorescent probes for dual detection of maize yellow mosaic virus and sugarcane mosaic virus as described in claim 5.

7. A dual detection method for maize yellow mosaic virus and sugarcane mosaic virus for non-disease diagnosis or treatment purposes, characterized in that, Using the RNA of the sample to be tested as a template, an amplification reaction is performed using the primers and fluorescent probes for dual detection of maize yellow mosaic virus and sugarcane mosaic virus as described in claim 5. The amplification reaction results are used to determine whether the sample to be tested contains maize yellow mosaic virus and sugarcane mosaic virus.

8. The dual detection method according to claim 7, characterized in that, The amplification reaction system consisted of: 29.4 μL of buffer A and 2.5 μmol·L⁻¹. -1 RPA-F 2μL, 2.5μmol·L -1 RPA-R 2μL, 1μmol·L -1 0.6 μL probe, 8.5 μL ddH2O, 5 μL RNA, and 2.5 μL B buffer; The probe ratio of MaYMV to SCMV is 2:1; the primer ratio of MaYMV to SCMV is 3:

1.

9. The dual detection method according to claim 8, characterized in that, The primer ratio for maize yellow mosaic virus and sugarcane mosaic virus was 3:1; the probe ratio for maize yellow mosaic virus and sugarcane mosaic virus was 2:

1.

10. The dual detection method according to claim 7, characterized in that, The reaction conditions for the amplification reaction were: 42℃ for 13 min.

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