A rt-lamp freeze-dried powder reagent and detection method for on-site detection of sweet potato virus

Abstract

The application belongs to the technical field of viral reagents, and particularly relates to a RT-LAMP reagent for detecting sweet potato virus on site and a method for detecting sweet potato virus on site. The RT-LAMP freeze-dried powder reagent buffer, non-enzyme component, enzyme component, pH indicator, freeze-dried protection reagent, direct spread type additive and specific LAMP primer are used for identifying and amplifying sweet potato virus in a sample to be detected, the specific LAMP primer comprises 0.2 muM outer primer F3 / B3, 0.5 muM loop primer LF / LB and 1.3 muM inner primer FIP / BIP, and the specific LAMP primer is at least one of SPFMV primer group, SPCSV primer group or SPLCV primer group; the SPFMV primer group is composed of 6 primers with the sequence shown in SEQ ID NO:1; the SPCSV primer group is composed of 6 primers with the sequence shown in SEQ ID NO:2; and the SPLCV primer group is composed of 6 primers with the sequence shown in SEQ ID NO:3. The reagent and method can quickly and accurately diagnose sweet potato virus, and have the advantages of high efficiency, easy operation and low cost.

Description

Technical Field

[0001] This invention belongs to the field of sweet potato virus reagent technology, specifically relating to an RT-LAMP freeze-dried powder reagent and detection method for on-site detection of sweet potato virus. Background Technology

[0002] Sweet potato is an important food, feed, and industrial raw material crop in my country. Viral diseases are one of the main limiting factors affecting sweet potato yield and quality, among which sweet potato feather mottling virus (SPFMV), sweet potato chlorosis dwarf virus (SPCSV), and sweet potato leafroll virus (SPLCV) are the three most widespread viruses. SPFMV is the most widely distributed sweet potato virus globally, and can cause feather mottling and stunted growth when it infects alone. SPCSV often co-infects with SPFMV, causing devastating sweet potato virus disease (SPVD), resulting in significant losses in tuber yield. SPLCV can cause leaf curling, yellowing, and stunted growth, severely affecting photosynthesis. In my country's main sweet potato producing areas, the detection rate and co-infection rate of these viruses are generally high.

[0003] Currently, the detection of sweet potato viral diseases mainly relies on serological methods (such as ELISA) and molecular biological methods (such as RT-PCR and real-time quantitative RT-PCR). However, these methods have significant shortcomings: they are difficult to meet the needs of rapid on-site detection. Serological methods (ELISA) are cumbersome and time-consuming (usually 2-3 hours), have limited sensitivity, low detection rate for low-concentration infected samples, and antibodies are easily affected by cross-reactions, making it difficult to distinguish virus strains. Conventional RT-PCR requires expensive precision thermal cycling equipment, and agarose gel electrophoresis analysis is required after amplification, which is complex, time-consuming, and carries the risk of contamination upon opening the container, making it unsuitable for on-site use. Although real-time quantitative RT-PCR (qPCR) has high sensitivity and specificity, it heavily relies on expensive instruments and professional skills, and requires complex nucleic acid extraction, making it difficult to promote in grassroots units and fields.

[0004] Although loop-mediated isothermal amplification (LAMP) technology has been applied to sweet potato virus detection, it still has significant limitations. On the one hand, there are significant differences in sequence specificity among different sweet potato viruses, and existing primer designs fail to adequately account for the variation characteristics of the viral genome, resulting in insufficient detection specificity. On the other hand, when multiple viruses are present in a sample, such as SPFMV, SPCSV, and SPLCV, the specific reactions of each virus interfere with each other, preventing simultaneous multiplex detection by the LAMP method. Specific identification of each virus individually is still required, making the process cumbersome and inefficient.

[0005] In agricultural production practice, the control measures for sweet potato viral diseases are generally not differentiated based on the type of virus—once a viral infection is confirmed, a uniform treatment strategy can be adopted without the need for precise identification of the specific virus type. However, the existing testing procedures still adhere to a step-by-step identification model, which not only increases time and cost but also fails to meet the actual needs of rapid screening.

[0006] Therefore, there is an urgent need to establish a universal detection scheme that can simultaneously detect multiple sweet potato viruses without the need for typing, in order to simplify operation, improve efficiency, and better serve rapid field diagnosis and disease management. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides an RT-LAMP reagent and a field detection method for sweet potato viruses. This reagent and method can detect not only single sweet potato viruses but also multiple viruses simultaneously, especially covering the three major viruses SPFMV, SPCSV, and SPLCV. It is a rapid on-site qualitative detection technology with high sensitivity, high specificity, simple operation, intuitive results, and no need for complex instruments, which can support early warning, precise control, and healthy seedling production of sweet potato diseases.

[0008] To solve the above-mentioned technical problems, the following technical solutions are provided: A RT-LAMP lyophilized powder reagent for field detection of sweet potato viruses is provided, the RT-LAMP lyophilized powder reagent comprising: A buffer, wherein the buffer is composed of 1-3 mM Tris-HCl, 1-3 mM borax and 1-3 mM glycine, and has a pH of 8-9; The non-enzymatic component is composed of 10-40 mM KCl, 5-10 mM MgSO4, 8-12 mM (NH4)2SO4 and 1-3 mM dNTPs. The enzyme component consists of 200-300 U reverse transcriptase, 50-70 U RNase inhibitor, and 20-30 U BstDNA polymerase. A pH indicator comprising 0.05–0.2% (w / v) phenol red and 0.01–0.04% (w / v) cresol red; The lyophilization protectant is composed of 1-3% (w / v) mannitol, 2-4% (w / v) PEG8000, 8-12% (w / v) trehalose, 1-3% (w / v) glycine and 0.02-0.05% (w / v) Tween 20; A direct-expansion additive, wherein the direct-expansion additive comprises 0.5-1.5M betaine, 8-12mM DTT, 0.5-1.5% PVP, 10-20mM acetylcysteine, and 0.2-0.8 mg / mL BSA; and Specific LAMP primers for identifying and amplifying sweet potato virus in a test sample, wherein the specific LAMP primers comprise 0.2 μM outer primer F3 / B3, 0.5 μM loop primer LF / LB, and 1.3 μM inner primer FIP / BIP, and wherein the specific LAMP primers are at least one of the SPFMV primer set, SPCSV primer set, or SPLCV primer set; The SPFMV primer set consists of 6 primers with sequences as shown in SEQ ID NO:1; The SPCSV primer set consists of 6 primers with sequences as shown in SEQ ID NO:2; The SPLCV primer set consists of 6 primers with sequences as shown in SEQ ID NO:3.

[0009] It also provides a method for on-site detection of sweet potato viruses, including the following steps: Step 1: Cut the sweet potato leaves to be tested into small pieces, add the nucleic acid release agent and mix well. Then heat-treat at 65℃~95℃ for 5min~15min, centrifuge and take the supernatant as a template. Step 2: Add the RT-LAMP lyophilized powder reagent according to claim 1 to the first reactor, and simultaneously add the supernatant, mix evenly to reconstitute the RT-LAMP lyophilized powder reagent; Step 3: Place the first reactor in a constant temperature device at 60~65℃ for 30min~60min and observe the color of the first reaction solution: When performing single-target identification and detection, the specific LAMP primers in the RT-LAMP lyophilized powder reagent are selected from SPFMV primers, SPCSV primers, or SPLCV primers. If the solution changes from purple to yellow, it indicates that the sample contains the target sweet potato virus, indicating that the specific sweet potato virus is positive; otherwise, the target sweet potato virus is not present. When performing a combined infection test, the specific LAMP primers in the RT-LAMP lyophilized powder reagent are a mixture of SPFMV primers, SPLCV primers, and SPCSV primers. If the solution changes from purple to yellow, it indicates the presence of sweet potato virus in the sample, indicating a positive result for sweet potato virus; otherwise, sweet potato virus is not present.

[0010] Furthermore, a positive false virus detection group was set up, which included: The RT-LAMP reagent was added to the second reactor, along with a fake sweet potato virus standard template. The mixture was stirred until homogeneous. The second reactor was placed in a constant temperature device at 60-65°C for 30-60 minutes to obtain the second reaction solution. If a sweet potato sample shows a positive result, and both the first and second reaction solutions are bright yellow, then the positive result is accurate.

[0011] Furthermore, a negative control group was also set up, which included, The RT-LAMP reagent is added to the third reactor and mixed evenly. The third reactor is placed in a constant temperature device at 60-65°C and reacted for 30-60 minutes to obtain the third reaction solution. If the sweet potato sample shows a positive result and the third reaction solution is purple-red, it indicates that the on-site reagents are normal.

[0012] Furthermore, the first reactor, the second reactor, and the third reactor are each unit tubes in a PCR conjugate.

[0013] Furthermore, the constant temperature device is a water bath, a metal bath, or a portable constant temperature amplification instrument.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention employs a ternary buffer system (pH 8-9) composed of Tris-HCl, borax, and glycine. This system not only effectively maintains a suitable pH environment for the amplification reaction but also significantly buffers the acidification effect caused by CO2 dissolution in the air, preventing nonspecific discoloration of the reaction solution due to pH drift. Even after being exposed to air for several hours after opening the container, the negative reaction solution maintains a stable purplish-red color, greatly enhancing the reliability and operational tolerance of the reagent in open field environments.

[0015] This invention designs three specific LAMP primer sets (each containing an outer primer F3 / B3, an inner primer FIP / BIP, and a loop primer LF / LB) based on the highly conserved CP target gene regions in the genomes of three sweet potato viruses: SPFMV, SPCSV, and SPLCV. Experimental verification shows that each primer set exhibits no cross-reactivity with non-target sweet potato viruses, achieving 100% specificity. Therefore, this invention can perform precise typing detection against a single virus, or combine the three primer sets for simultaneous screening and multiplex detection in a single reaction, effectively overcoming the technical bottleneck of traditional LAMP's inability to perform multiplex detection, and significantly reducing detection costs and time.

[0016] This invention introduces a direct amplification additive composed of betaine, DTT, PVP, acetylcysteine, and BSA into the reaction system. This composition effectively neutralizes PCR inhibitors (such as polyphenols and polysaccharides) released from the lysis of sweet potato leaves, allowing the crude extract after simple heat treatment with a nucleic acid releasing agent to be directly used as a template for loading without any RNA purification steps. This direct amplification strategy shortens the operation to three steps: chopping, heating, and loading, greatly simplifying the process and lowering the experimental threshold.

[0017] This invention, using the aforementioned direct amplification system and lyophilized reagent, only requires mixing the sample to be tested with the nucleic acid releasing agent, heating, reconstituted the lyophilized reagent with the supernatant, and placing it in a 60-65℃ constant temperature device (water bath, metal bath, or household thermos) for 30-60 minutes. The result can then be interpreted by visually observing the color. The entire process can be completed by personnel without professional experimental background within 20-50 minutes. The positive and negative concordance rate between the test results and RT-qPCR reaches 100%, truly achieving rapid on-site diagnosis with "sample in, result out".

[0018] This invention uses a pseudovirus standard as a positive control. The pseudovirus particles encapsulate the target RNA fragment and can fully participate in the entire nucleic acid release and amplification process. When the test sample is positive, if the positive control reaction tube also turns bright yellow simultaneously, it confirms that the amplification system and operating procedures are normal, effectively eliminating the risk of false negatives and improving the reliability of on-site testing results.

[0019] The multiple detection mode of this invention is not only applicable to the joint detection of RNA viruses (SPFMV, SPCSV), but also to the simultaneous detection of DNA viruses (SPLCV), realizing the co-detection of RNA / DNA complex pathogens in the same tube. This versatile design allows for flexible configuration according to actual field needs, taking into account the detection requirements of different disease scenarios, and has good prospects for widespread application. Attached Figure Description

[0020] Figure 1 This is a diagram of the LAMP primer set structure for Example 1.

[0021] Figure 2 The graph shows the sensitivity detection results of SPFMV RT-LAMP in Experiment Example 3.

[0022] Figure 3 The graph shows the sensitivity detection results of SPCSV RT-LAMP in Experiment Example 3.

[0023] Figure 4 The figure shows the sensitivity detection results of SPLCV RT-LAMP in Experiment Example 3.

[0024] Figure 5The figure shows the sensitivity detection results of the triple RT-LAMP detection system in Experiment Example 4.

[0025] Figure 6 The results show the sensitivity detection of the triple RT-LAMP detection system in Experiment 5. The left side shows the RT-LAMP reagent from Experiment 2, and the right side shows the purchased RT-LAMP reagent.

[0026] Figure 7 The result of the SPFMV RT-LAMP reaction in Experiment Example 6 is shown in the figure.

[0027] Figure 8 The image shows the reaction results of SPCSV RT-LAMP in Experiment Example 6.

[0028] Figure 9 The image shows the reaction results of SPLCV RT-LAMP in Experiment Example 6.

[0029] Figure 10 This is a diagram showing the results of multiple RT-LAMP reactions of random field samples in Experiment Example 7.

[0030] Figure 11 The image shows the results of the multiplex RT-LAMP reaction of field samples in Experiment 8.

[0031] Figure 12 The image shows the results of the multiple RT-LAMP reaction in a constant temperature metal bath for field samples in Experiment Example 8.

[0032] Figure 13 This is a diagram showing the results of the multiple RT-LAMP reaction of field samples from a household thermos cup in Experiment Example 8.

[0033] Figure 14 The results of RT-LAMP detection in different colorimetric systems in Experiment Example 9 are shown, where the upper layer is phenol red-cresol red indicator and the lower layer is calcein indicator. Detailed Implementation

[0034] To make the technical problems, technical solutions, and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0035] Example 1

[0036] To illustrate the primer acquisition process in this case, this embodiment discloses the following primer acquisition process: The genome sequences of all sweet potato SPLMV, SPCSV, and SPLCV virus strains (including globally representative genome sequences) were downloaded from public databases. Multiple sequence alignment was performed to screen for highly conserved target gene regions. The SPFMV detection primer set was designed based on a highly conserved CP gene segment and optimized to cover major strains worldwide. The SPCSV detection primer set was designed for the CP genes in conserved RNA1 or RNA2 regions of the genome. The SPLCV detection primer set was designed for gene replication-related protein genes or CP genes. Using PrimerExplorer V5 software, a set of specific RT-LAMP primers was designed for this conserved region, including one pair of outer primers (F3 / B3), one pair of inner primers (FIP / BIP), and circular primers (LF, LB). The primers were synthesized as follows... Figure 1 As shown, based on the results of multiple sequence alignment, the 3' ends of F2 and B2 are designed on the target virus-specific sequences, and base matching cannot be completed with other closely related species, thus achieving the purpose of specificity.

[0037] Primer-BLAST was used to verify the specificity of its matching with the target viral sequence, ensuring that it had no significant homology with non-target pathogens (such as SPVG, SPV2, SPMMV, etc.) and avoiding cross-reactivity.

[0038] The MFEprimer 3.1 multiple primer analysis software was used to perform multiple alignment analysis on all primer sequences of the three viruses to ensure that no obvious dimers or hairpin structures were formed between the primers.

[0039] Send the appropriate primer set to a primer synthesis company for synthesis.

[0040] Preparation of Standards

[0041] Using the primer sequence as the core, sequences were extended at both ends to a total length of approximately 700 bp. These sequences were sent to a biosynthesis service company for RNA pseudovirus or DNA plasmid preparation, and copy number quantification was performed using ddPCR. The primer sequences for SPFMV are shown in SEQ ID NO:1; SPCSV primers are shown in SEQ ID NO:2; and SPLCV primers are shown in SEQ ID NO:3. The sequences of each viral primer set are shown in Table 1. The LAMP primer set structure diagram is shown below. Figure 1 .

[0042] Table 1 Primer information for SPCSV, SPFMV, and SPLCV viruses used in LAMP

[0043] Example 2

[0044] This embodiment discloses an RT-LAMP lyophilized powder reagent for field detection of sweet potato viruses, the components of which are as follows: Buffer: Composed of 2.5 mM Tris-HCl, 1 mM borax and 1 mM glycine, pH 8.0; Non-enzymatic components: composed of 10 mM KCl, 5 mM MgSO4, 8 mM (NH4)2SO4 and 1 mM dNTPs; Enzyme components: Composed of 200 U reverse transcriptase, 50 U RNase inhibitor, and 20 U Bst DNA polymerase; pH indicator: Contains 0.05% (w / v) phenol red and 0.01% (w / v) cresol red; Lyophilization protection reagent: composed of 1% (w / v) mannitol, 2% (w / v) PEG8000, 8% (w / v) trehalose, 1% (w / v) glycine and 0.02% (w / v) Tween 20; Direct-expansion additive: composed of 0.5 M betaine, 8 mM DTT, 0.5% PVP, 10 mM acetylcysteine, and 0.2 mg / mL BSA; and Specific LAMP primers are used to identify and amplify sweet potato virus in test samples. The specific LAMP primers contain 0.2 μM outer primer F3 / B3, 0.5 μM loop primer LF / LB, and 1.3 μM inner primer FIP / BIP. The specific LAMP primers are SPFMV primers, and their primer sequence set is shown in SEQ ID NO:1. They are used to detect single sweet potato virus sPFMV.

[0045] Example 2

[0046] An RT-LAMP lyophilized powder reagent for field detection of sweet potato viruses has the following components: Buffer: Composed of 2 mM Tris-HCl, 2 mM borax and 2 mM glycine, pH 8.5; Non-enzymatic components: composed of 25 mM KCl, 7.5 mM MgSO4, 10 mM (NH4)2SO4 and 2 mM dNTPs; Enzyme components: Composed of 250 U reverse transcriptase, 60 U RNase inhibitor and 25 U Bst DNA polymerase; pH indicator: Contains 0.12% (w / v) phenol red and 0.025% (w / v) cresol red; Lyophilization protection reagent: composed of 2% mannitol, 3% PEG8000, 10% trehalose, 2% glycine and 0.035% Tween 20; Direct-expansion additive: composed of 1.0 M betaine, 10 mM DTT, 1.0% PVP, 15 mM acetylcysteine, and 0.5 mg / mL BSA; and Specific LAMP primers are used to identify and amplify sweet potato virus in test samples. The specific LAMP primers contain 0.2 μM outer primers F3 / B3, 0.5 μM loop primers LF / LB, and 1.3 μM inner primers FIP / BIP. The specific LAMP primers are SPCSV primers, and their primer sequence set is shown in SEQ ID NO:2. They are used to detect single sweet potato virus SPCSV.

[0047] Example 3

[0048] An RT-LAMP lyophilized powder reagent for field detection of sweet potato viruses has the following components: Buffer: Composed of 3 mM Tris-HCl, 3 mM borax and 3 mM glycine, pH 9.0; Non-enzymatic components: composed of 40 mM KCl, 10 mM MgSO4, 12 mM (NH4)2SO4 and 3 mM dNTPs; Enzyme components: Composed of 300 U reverse transcriptase, 70 U RNase inhibitor, and 30 U Bst DNA polymerase; pH indicator: Contains 0.2% (w / v) phenol red and 0.04% (w / v) cresol red; Lyophilization protection reagent: composed of 3% mannitol, 4% PEG8000, 12% trehalose, 3% glycine and 0.05% Tween 20; Direct-expansion additive: composed of 1.5 M betaine, 12 mM DTT, 1.5% PVP, 20 mM acetylcysteine, and 0.8 mg / mL BSA; and Specific LAMP primers are used to identify and amplify sweet potato viruses in test samples. The specific LAMP primers contain 0.2 μM outer primer F3 / B3, 0.5 μM loop primer LF / LB, and 1.3 μM inner primer FIP / BIP. The specific LAMP primers are SPCSV, SPFMV, and SPLCV, and their primer sequence sets are shown in SEQ ID NO:2, SEQ ID NO:1, and SEQ ID NO:3. They are used to detect single sweet potato viruses SPCSV, SPFMV, and SPLCV.

[0049] To further illustrate the effects of the present invention, the following experiments were conducted: Experimental Example 1 The RT-LAMP lyophilized powder reagent for detecting sweet potato viruses was prepared with the following composition: Buffer system: 2mM Tris-HCl, 2mM borax, 2mM glycine, pH 8.5.

[0050] Non-enzymatic components: 30 mM KCl, 8 mM MgSO4, 10 mM (NH4)2SO4 and 1.5 mM dNTPs.

[0051] Enzyme components: Contains 250U of reverse transcriptase, 60U of RNAase inhibitor, and 24U of Bst DNA polymerase.

[0052] Lyophilization protectant: 2% mannitol, 3% PEG8000, 10% trehalose, 2% glycine, 0.1% Tween 20.

[0053] Direct-diffusion additives: 1.0M betaine, 10mM DTT, 1.0% PVP, 15mM acetylcysteine, and 0.5mg / mL BSA

[0054] Colorimetric indicator: 0.1% (w / v) phenol red, 0.03% (w / v) cresol red.

[0055] Primer combinations: F3 / B3 0.2uM; LF / LB 0.5uM; FIP / BIP 1.3uM.

[0056] Experiment Example 2

[0057] Using the RT-LAMP lyophilized powder reagent from Experiment 1, after reconstitution with 25 μL of water or reconstitution solvent, gradient cross-validation experiments were conducted with corresponding pseudoviruses or DNA plasmids to determine the primer concentration ratios for SPFMV, SPCSV, and SPLCV viruses. The primer concentration ratios with the best specificity (obtained by no color change in the negative water control) and the highest amplification efficiency (color change in the shortest time) were selected. After multiple adjustments and validations, an optimal set of primer concentration ratios was obtained. The optimal concentration values ​​for each viral primer set are shown in Table 2.

[0058] Table 2. Optimal primer concentration combinations for each virus in single RT-LAMP reactions.

[0059] Based on the primer concentration of the single-virus detection system, the concentration system of the multiplex primer for simultaneous detection of three viruses was adjusted and verified. The detailed results are shown in Table 3.

[0060] Table 3 Optimal primer concentration combinations for each virus in triple RT-LAMP reaction

[0061] Experimental Example 3

[0062] SPFMV, SPCSV pseudoviral RNA standards, and SPLCV DNA plasmids were serially diluted to concentrations of 1000 / 100 / 10 / 1 copy / µL. The lyophilized LAMP reagent from Example 1 was used, with the optimal primer set concentration from Example 2 added. 5µL of each standard was used as template, and the reaction was carried out at 65°C for 45 minutes. The results are as follows. Figures 2-4 .

[0063] This shows that the detection limit for all three viruses is 5 copies / reaction (1 copy / uL is used as 5uL as template), indicating that the sensitivity for the three SPFMV, SPCSV and SPLCV viruses can reach at least 5 copies.

[0064] Experiment Example 4

[0065] SPFMV, SPCSV, and SPCV virus standards at a concentration of 100 copies / µL were mixed in a 1:1:1 ratio to form a mixed positive standard, which was then diluted with nuclease-free water to achieve a concentration of 5 copies / µL for each virus in the mixed standard. A triplet RT-LAMP reaction system was prepared using the lyophilized LAMP reagent from Example 2 and the primer concentrations from the multiplex detection in Example 3. This mixed system was then used to detect the mixed standard, pure 5 copy / µL SPFMV virus, SPCSV virus, and SPCV virus standards, respectively. 2µL of template (total 10 copies of standard) was added to each reaction. After incubation at 65°C for 45 minutes, color changes were observed. Results Figure 5 .

[0066] This demonstrates that the multiplex detection system is effective not only for detecting multiple infected samples (mixed standards) but also for detecting individual viral targets. The three targets do not interfere with each other, thus not affecting the detection accuracy of any single virus. This greatly meets the needs of low-cost and safe grain production, eliminating the need for separate detection of individual sweet potato viruses. The sensitivity of the multiplex detection reaches at least 10 copies / reaction.

[0067] Experimental Example 5

[0068] A high-performance general-purpose RT-LAMP reagent was purchased from publicly available sources and its detection performance was compared with that of the lyophilized RT-LAMP reagent in Experiment Example 1 of this invention. Triple RT-LAMP reaction systems were prepared using the purchased reagent and the reagent of this invention, according to the primer concentrations for multiplex detection in Experiment Example 2. Then, SPFMV, SPCSV, and SPLCV mixed positive nucleic acids; SPFMV positive nucleic acids; SPCSV positive nucleic acids; and SPLCV positive nucleic acids were detected in Experiment Example 4, with 5 copies added to each reaction and incubated at 65°C for 35 min. The results are as follows: Figure 6.

[0069] Therefore, it can be seen that both the RT-LAMP reagent of Experiment 1 of the present invention and the purchased reagent can effectively detect triple and single sweet potato viruses. Specifically, the reagent of Experiment 1 of the present invention shows obvious color change in both multiplex and singlex detection, indicating good amplification efficiency and high sensitivity. In contrast, the purchased reagent shows less change in SPCSV and SPLCV during singlex target detection, indicating that multiple targets interfere with each other during multiplex detection with the purchased reagent, causing a decrease in the LAMP amplification efficiency of one or two targets and a decrease in detection sensitivity. This further demonstrates that the reagent and primers of the present invention can perform multiplex detection.

[0070] Experimental Example 6

[0071] The RT-LAMP lyophilized powder reagent from Example 2 was used, with the optimal primer set concentration from Example 2 added. SPFMV, SPCSV, SPLCV, and SPVG standards were tested in each viral reaction system, and the results are as follows. Figure 7 , 8 9.

[0072] The results show that only the SPFMV standard changes color in the SPFMV system, only the SPCSV standard changes color in the SPCSV system, and only the SPLCV standard changes color in the SPLCV system. There is no significant color change for non-target standards, demonstrating good cross-specificity.

[0073] Experimental Example 7

[0074] This experiment included field sample adaptability testing, direct amplification testing of field samples, and positive / negative concordance rate testing.

[0075] Ten sweet potato leaf samples exhibiting varying degrees of SPFMV infection symptoms were collected from the field, along with five healthy leaf samples. Each sample was divided into two portions. Total RNA was extracted from one portion using a plant total RNA rapid extraction kit (purchased mature extraction kit). The sample was then processed with a nucleic acid release agent, the components of which are shown in Table 4. Table 4

[0076] Total RNA extraction was performed according to the instructions of the finished kit; the sample nucleic acid release process is as follows: Using sterile scissors, cut 4 to 6 strips of approximately 2mm x 8mm from each sample and transfer these strips to a 1.5mL centrifuge tube.

[0077] Add 500uL of nucleic acid release agent, ensuring the sample is completely submerged.

[0078] Place the centrifuge tubes on a constant temperature metal bath and heat at 92~95℃ for 10-15 minutes. Then remove the centrifuge tubes and place them at room temperature, or put them in a refrigerator for 3-5 minutes to allow the sample temperature to drop to room temperature.

[0079] Take 25 μL of sample supernatant treated with nucleic acid release agent and add it directly to the multiplex 3-lyophilized RT-LAMP reagent in Experiment Example 4. Vortex mix for 2-5 seconds to completely dissolve the lyophilized bulbs or powder. Then place the PCR tube in a constant temperature metal bath and react at 65°C for 30-50 minutes. Finally, observe the color change to determine whether the sample is infected with SPLMV.

[0080] Total RNA was extracted and detected by RT-qPCR using a primer-probe kit containing SPLMV, following the kit's instructions. The amplification volume was 25 μL, with forward and reverse primer concentrations of 150 nM. The amplification program was as follows: 50℃ for 15 min, one cycle; 95℃ for 3 min, one cycle; 95℃ for 15 s, 60℃ for 30 s, fluorescence collection, 45 cycles.

[0081] Therefore, it can be seen that the following Figure 10 As shown, all leaf samples with varying degrees of SPFMV infection symptoms (01, 02, 03, 04, 06, 07, 09, 11, 12, 14) changed from their original red color to yellow, indicating SPFMV positivity; 05, 08, 10, 13, and 15 were healthy leaves, maintaining their original color and indicating SPFMV negativity. RT-qPCR results are shown in Table 5.

[0082] Table 5. Results of RT-qPCR detection in random field samples

[0083] Therefore, it can be seen that the freeze-dryable RT-LAMP reagent of the present invention has good adaptability and detection capability for real field samples.

[0084] The freeze-dryable RT-LAMP reagent of this invention, when combined with another nucleic acid release agent invented by our company, enables a rapid and simple direct amplification process without nucleic acid extraction, greatly facilitating end-user use and reducing technical barriers and equipment requirements.

[0085] The detection results of the freeze-dryable RT-LAMP reagent of the present invention are highly consistent with the detection results of RT-qPCR. According to the random sampling detection results, the positive and negative concordance rate reaches 100%.

[0086] Experimental Example 8

[0087] In a sweet potato planting base in Guangdong, an agricultural technician with no molecular experimental experience was randomly invited to use the kit of this invention. Following the kit instructions, three field samples showing obvious symptoms of infection with three viruses, including SPFMV, were analyzed using a multiplex assay. Parallel comparisons were conducted using a PCR instrument, a constant-temperature metal bath, and a household thermos. The results are as follows. Figure 11 , 12 13.

[0088] The results show that samples from all different reaction devices yielded the corresponding results, i.e., positive samples all changed color, while NTC did not. Furthermore, all experimental operations were independently performed by agricultural technicians without molecular experimental experience, achieving results consistent with those obtained by professional biotechnologists in the laboratory. This demonstrates that the product of this invention has excellent field applicability and tolerance to simple reaction devices, as well as great user-friendliness. It is simple to operate, provides clear results, is fast, and is very suitable for field use.

[0089] Experimental Example 9

[0090] This experimental example demonstrates testing with different colorimetric systems. The system of this invention is also applicable to RT-LAMP detection using ionic indicator methods; this experimental example is conducted to illustrate this.

[0091] Four samples were selected from Example 7, two positive and two negative. Samples were processed using the direct amplification method described in Example 7. RT-LAMP detection was performed using the pH indicator method, following the procedure in Example 7. A universal LAMP reagent of our invention was used, in which the phenol red and cresol red indicator combination was replaced with calcein. 0.1-0.2 mM calcein and 0.4-1.0 mM MnCl2 ions were added to each reaction. Then, the calcein-indicated RT-LAMP reaction was performed using the same LAMP primer concentration and enzyme mix amount as in Example 7, with the reaction temperature and time consistent with the parameters of Example 7.

[0092] The reaction results are shown below. Figure 14 As shown in the figure, both indicator schemes yielded the same positive and negative results, indicating that the system of this invention can choose between the pH indicator method or the ion indicator method according to actual needs. The ion indicator method can use calcein, HNB, or Eriochrome Black T, etc.

[0093] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An RT-LAMP lyophilized powder reagent for on-site detection of sweet potato virus, characterized in that, The RT-LAMP lyophilized powder reagent includes: A buffer, wherein the buffer is composed of 1-3 mM Tris-HCl, 1-3 mM borax and 1-3 mM glycine, and has a pH of 8-9; The non-enzymatic component is composed of 10-40 mM KCl, 5-10 mM MgSO4, 8-12 mM (NH4)2SO4 and 1-3 mM dNTPs; The enzyme component consists of 200-300 U reverse transcriptase, 50-70 U RNase inhibitor, and 20-30 U Bst DNA polymerase. A pH indicator comprising 0.05–0.2% (w / v) phenol red and 0.01–0.04% (w / v) cresol red; The lyophilization protectant is composed of 1-3% (w / v) mannitol, 2-4% (w / v) PEG8000, 8-12% (w / v) trehalose, 1-3% (w / v) glycine and 0.02-0.05% (w / v) Tween 20; A direct-expansion additive, wherein the direct-expansion additive comprises 0.5-1.5M betaine, 8-12mM DTT, 0.5-1.5% PVP, 10-20mM acetylcysteine, and 0.2-0.8 mg / mL BSA; and, Specific LAMP primers for identifying and amplifying sweet potato virus in a test sample, wherein the specific LAMP primers comprise 0.2 μM outer primer F3 / B3, 0.5 μM loop primer LF / LB, and 1.3 μM inner primer FIP / BIP, and wherein the specific LAMP primers are at least one of the SPFMV primer set, SPCSV primer set, or SPLCV primer set; The SPFMV primer set consists of 6 primers with sequences as shown in SEQ ID NO:1; The SPCSV primer set consists of 6 primers with sequences as shown in SEQ ID NO:2; The SPLCV primer set consists of 6 primers with sequences as shown in SEQ ID NO:

3.

2. A method for on-site detection of sweet potato virus, characterized in that, Includes the following steps: Step 1: Cut the sweet potato leaves to be tested into small pieces, add the nucleic acid release agent and mix well. Then heat-treat at 65℃~95℃ for 5 min~15 min, centrifuge, and take the supernatant as a template. Step 2: Add the RT-LAMP lyophilized powder reagent according to claim 1 to the first reactor, and simultaneously add the supernatant, mix evenly to reconstitute the RT-LAMP lyophilized powder reagent; Step 3: Place the first reactor in a constant temperature device at 60~65℃ for 30min~60min and observe the color of the first reaction solution: When performing single-target identification and detection, the specific LAMP primers in the RT-LAMP lyophilized powder reagent are selected from SPFMV primers, SPCSV primers, or SPLCV primers. If the solution changes from purple to yellow, it indicates that the sample contains the target sweet potato virus, indicating that the specific sweet potato virus is positive; otherwise, the target sweet potato virus is not present. When performing a combined infection test, the specific LAMP primers in the RT-LAMP lyophilized powder reagent are a mixture of SPFMV primers, SPLCV primers, and SPCSV primers. If the solution changes from purple to yellow, it indicates the presence of sweet potato virus in the sample, indicating a positive result for sweet potato virus; otherwise, sweet potato virus is not present.

3. The method for on-site detection of sweet potato virus according to claim 2, characterized in that, A positive false virus detection group was also set up, which included: The RT-LAMP reagent was added to the second reactor, along with a fake sweet potato virus standard template. The mixture was stirred until homogeneous. The second reactor was placed in a constant temperature device at 60-65°C for 30-60 minutes to obtain the second reaction solution. If a sweet potato sample shows a positive result, and both the first and second reaction solutions are bright yellow, then the positive result is accurate.

4. The method for on-site detection of sweet potato virus according to claim 3, characterized in that, A negative control group was also set up, which included, The RT-LAMP reagent is added to the third reactor and mixed evenly. The third reactor is placed in a constant temperature device at 60-65°C and reacted for 30-60 minutes to obtain the third reaction solution. If the sweet potato sample shows a positive result and the third reaction solution is purple-red, it indicates that the on-site reagents are normal.

5. The method for on-site detection of sweet potato virus according to claim 4, characterized in that, The first reactor, the second reactor, and the third reactor are each unit tubes in a PCR conjugate.

6. The method for on-site detection of sweet potato virus according to any one of claims 2 to 5, characterized in that, The constant temperature device is a water bath, a metal bath, or a portable constant temperature amplification instrument.

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