Kit for detecting cucumber green mottle mosaic virus based on enzyme-mediated dual-amplification nucleic acid amplification, detection method and application

Abstract

The invention belongs to the technical field of viral nucleic acid molecular diagnosis, and particularly relates to a kit for detecting cucumber green mottle mosaic virus based on enzyme-mediated dual-amplification nucleic acid amplification, a detection method and application. The detection system comprises a primer pair DNA upstream primer (SEQ ID NO.2) and a DNA downstream primer (SEQ ID NO.3) for amplifying a coat protein gene of the cucumber green mottle mosaic virus, and a probe (RNA primer (SEQ ID NO.1)) for detecting a CP gene, a nucleic acid amplification group enzyme system, a signal amplification group enzyme system, a nucleic acid scavenging enzyme, an activating enzyme NTP, a buffer solution and a freeze-drying protective agent, wherein the system is prepared into pre-subpackaged freeze-dried balls. The kit provided by the invention can realize RNA detection of aM concentration, and can realize early screening of cucumber green mottle mosaic virus due to high detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of viral nucleic acid molecular diagnostic technology, specifically relating to a kit, detection method, and application for detecting cucumber green mottle mosaic virus based on enzyme-mediated double amplification of nucleic acid. Background Technology

[0002] Cucumber green mottle mosaic virus (CGMMV) belongs to the genus Tobamovirus in the family Cladoviridae. Its virus particles are rod-shaped, approximately 300 nm long and 18 nm in diameter, and are non-enveloped, composed of over 2100 capsid protein subunits and genomic RNA. Under natural conditions, CGMMV primarily infects cucurbitaceous crops such as watermelon, melon, cucumber, bottle gourd, calabash, pumpkin, loofah, and bitter melon. Under artificial inoculation conditions, it can also infect plants such as amaranth, quinoa, purslane, datura, common tobacco, tobacco Bunsen, tobacco trifoliate, tobacco sansilica, and petunia. As an important pathogen of cucurbitaceous crops, this virus can cause symptoms such as mosaic leaf formation, plant stunting, and fruit drop, severely impacting the yield and quality of cucurbitaceous crops.

[0003] Currently, quantitative real-time PCR (polymerase chain reaction) is the primary technology for high-sensitivity molecular detection and is widely used in various scenarios. However, this technology requires expensive and inconvenient instruments, and the detection time typically exceeds 40 minutes. These drawbacks significantly limit its application in point-of-care testing (POCT). To address the limitations of quantitative real-time PCR in rapid nucleic acid detection, various techniques for rapid nucleic acid amplification under isothermal conditions have become a focus of research in recent years. These include: transcription-mediated amplification and its derivatives, which, due to their reaction mechanism, have long overall detection times and limited sensitivity, failing to achieve high-sensitivity detection in a short time; loop-mediated isothermal amplification, which is rapid and sensitive, but its weak resistance to contamination leading to false positives limits its application in rapid nucleic acid detection; and recombinase polymerase amplification (RPA) technology and its derivatives, which have been extensively studied, but few products are currently available for clinical use. Its complex operation and weak fluorescence signal make it difficult to meet the demands for ultra-rapid and highly sensitive detection.

[0004] More importantly, during nucleic acid amplification, the large number of target nucleic acid fragments generated are highly susceptible to forming tiny aerosol particles due to operational procedures (such as opening the lid, pipetting, and shaking) or environmental factors (such as airflow). These aerosols containing high concentrations of amplification products are extremely stable and can persist and diffuse in the experimental environment (including instrument surfaces, consumables, and even the air) for extended periods. Once contaminated with subsequent reaction systems, even trace amounts of contaminated nucleic acid can be efficiently identified and amplified by subsequent amplification reactions, leading to severe false-positive results. This contamination is characterized by its insidious nature, cumulative effect, and difficulty in complete removal, posing a significant threat to the accuracy, reliability, and reproducibility of test results. While existing technologies include measures such as strict zoning, ultraviolet irradiation, and the use of UNG-dUTP anti-contamination systems, they all have significant limitations: physical zoning cannot completely prevent aerosol diffusion, ultraviolet irradiation is ineffective in dead zones and may damage reagents, and the UNG system can only prevent specific types of contamination (including dU) and is ineffective against single-stranded DNA. Even in a laboratory environment, it is difficult to completely avoid the risk of contamination. In point-of-care testing (POCT) scenarios with limited resources or rudimentary conditions, the risk of contamination increases exponentially, which greatly limits the reliable application of nucleic acid testing technology, especially the simple-to-operate isothermal amplification technology, at the grassroots level, bedside, or in the field.

[0005] Therefore, inventing a rapid on-site molecular detection technology and applying it to field testing and monitoring is the most effective way to fundamentally cut off the source of cucumber green mottle mosaic virus. Specifically, there is an urgent need to provide a rapid on-site detection kit and method for cucumber green mottle mosaic virus that offers accuracy comparable to professional laboratory tests while also being fast, simple, and portable. Simultaneously, a nucleic acid detection method that can effectively block or significantly reduce the risk of aerosol contamination at its source is of urgent technical demand and significant application value for improving the reliability of test results, ensuring diagnostic accuracy, expanding the applicable scenarios of the technology (especially rapid on-site detection), and reducing the risk of misdiagnosis and mistreatment due to false positives and subsequent costs. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides a kit, detection method, and application for detecting cucumber green mottle mosaic virus based on enzyme-mediated double amplification of nucleic acid, which has high sensitivity and accuracy.

[0007] The technical solution of this invention is as follows: On one hand, the present invention provides a kit for detecting cucumber green mottle mosaic virus based on enzyme-mediated double amplification of nucleic acid, comprising an RNA probe, an upstream DNA primer and a downstream DNA primer; the sequence of the probe is shown in SEQ ID NO.1; the sequence of the upstream DNA primer is shown in SEQ ID NO.2; and the sequence of the downstream DNA primer is shown in SEQ ID NO.3.

[0008] Specifically, the final concentration of the RNA probe can be 300-500 nM, and the final concentration of the upstream and downstream DNA primers can be 150-250 nM.

[0009] Preferably, the final concentration of the RNA probe can be 350-450 nM, and the final concentrations of the upstream and downstream DNA primers are both 180-220 nM.

[0010] Preferably, the final concentration of the RNA probe is 400 nM, and the final concentrations of the upstream and downstream DNA primers are both 200 nM.

[0011] Specifically, the kit further includes a nucleic acid amplification enzyme system and a signal amplification enzyme system; the nucleic acid amplification enzyme system includes recombinase, single-stranded binding protein, polymerase and ATP energy regenerating enzyme; the signal amplification enzyme system includes transcriptase, reverse transcriptase, poly-L-lysine and cleavage enzyme.

[0012] Preferably, the final concentration of the nucleic acid amplification enzyme system can be: 10-30 nM recombinase, 40-60 nM single-stranded binding protein, 15-25 nM polymerase, and 0.5-1.5 μM ATP energy regenerating enzyme.

[0013] Preferably, the final concentration of the nucleic acid amplification enzyme system can be: 15-25 nM recombinase, 45-55 nM single-stranded binding protein, 18-22 nM polymerase, and 0.8-1.2 μM ATP energy regenerating enzyme.

[0014] Preferably, the final concentration of the nucleic acid amplification enzyme system can be: 20 nM recombinase, 50 nM single-stranded binding protein, 20 nM polymerase, and 1 μM ATP energy regenerating enzyme.

[0015] Specifically, the final concentration of the signal amplification enzyme system can be: 0.5-1.5 μM transcriptase, 0.5-1.5 μM reverse transcriptase, 0.5-1.5 μM poly-L-lysine, and 0.5-1.5 μM cleavage enzyme.

[0016] Preferably, the final concentration of the signal amplification enzyme system can be: 0.8-1.2 μM transcriptase, 0.8-1.2 μM reverse transcriptase, 0.8-1.2 μM poly-L-lysine, and 0.8-1.2 μM cleavage enzyme.

[0017] Preferably, the final concentration of the signal amplification enzyme system can be: 1 μM transcriptase, 1 μM reverse transcriptase, 1 μM poly-L-lysine, and 1 μM cleavage enzyme.

[0018] Specifically, the kit also includes a nucleic acid removal enzyme, nucleoside triphosphate, buffer solution, and lyophilization protectant.

[0019] Preferably, the final concentration of the nucleic acid clearing enzyme can be 0.5-1.5 μM; and the final concentration of the nucleoside triphosphate is 50-70 μM.

[0020] Preferably, the final concentration of the nucleic acid clearing enzyme can be 0.8-1.2 μM; and the final concentration of the nucleoside triphosphate is 55-65 μM.

[0021] Preferably, the final concentration of the nucleic acid clearing enzyme can be 1 μM; the final concentration of the nucleoside triphosphate is 60 μM.

[0022] Specifically, the buffer solution comprises: Tris-HAc, potassium acetate, magnesium acetate, mercaptoethanol, and ATP; the lyophilization protectant is polyethylene glycol.

[0023] Preferably, the buffer solution comprises: 10-30 mM Tris-HAc, 20-80 mM potassium acetate, 2-5 mM magnesium acetate, 1-4 mM mercaptoethanol and 0.5-1 mM ATP; the lyophilization protectant is polyethylene glycol 1-3% v / v.

[0024] Preferably, the buffer solution comprises: 20 mM Tris-HAc, 50 mM potassium acetate, 5 mM magnesium acetate, 2 mM mercaptoethanol and 1 mM ATP; the lyophilization protectant is polyethylene glycol 2% v / v.

[0025] In another aspect, the present invention provides a method for rapid on-site detection of cucumber green mottle mosaic virus, comprising the following steps: S1. Sample collection and processing: Collect plant tissue samples and add them to lysis buffer; S2. Heat the lysis buffer containing the sample from step S1 to release the nucleic acid; S3. Add the sample processed in step S2 to the reagents in the aforementioned kit, mix well, and obtain the reaction solution; S4. Place the reaction mixture from step S3 into the testing device, and interpret the results after testing.

[0026] Specifically, the instrument for the heating treatment in step S2 is a handheld metal bath; the detection device in step S4 is a handheld fluorescence detector.

[0027] Specifically, the sample is one or more of the following: watermelon, cantaloupe, cucumber, gourd, bottle gourd, pumpkin, loofah, bitter melon, amaranth, quinoa, purslane, datura, common tobacco, Bunsen tobacco, Sanssa tobacco, Shansi tobacco, or petunia.

[0028] In another aspect, the present invention provides the application of the aforementioned kit or method in the detection of cucumber green mottle mosaic virus.

[0029] The beneficial effects of this invention are as follows: (1) The RNA extraction steps provided by the present invention are simplified. It only requires treating the sample with sample soaking solution, adding it to the lysis tube, and heat treatment for 10 minutes to complete the process.

[0030] (2) The enzyme-mediated dual amplification nucleic acid amplification technology for detecting cucumber green mottle mosaic virus of the present invention, by integrating the nucleic acid amplification enzyme system, can amplify the target nucleic acid to 109 times within 10-30 minutes under constant temperature of 42 degrees Celsius. By integrating the signal amplification enzyme system, one nucleic acid amplification product can generate more than 10,000 fluorescence signals under constant temperature of 42 degrees Celsius, ultimately achieving efficient and rapid detection of samples.

[0031] (3) The present invention performs molecular detection of cucumber green mottle mosaic virus with high specificity and sensitivity as low as 1 copy / μL, which can detect samples infected with cucumber green mottle mosaic virus at an early stage.

[0032] (4) The present invention can effectively block or significantly reduce the risk of aerosol pollution from the source, improve the credibility of the detection results and ensure the accuracy of the diagnosis.

[0033] (5) This invention can be used in conjunction with a portable handheld metal bath and a handheld fluorescence detector, and can be carried to any location such as rural sites to carry out detection, meeting the application scenarios of field detection and monitoring.

[0034] (6) The operation process of this invention is simple, suitable for anyone to use, and has a wide range of application prospects.

[0035] (7) The kit of the present invention can realize RNA detection at the aM level (aM is converted to copy number, which is basically at the single copy level). In addition to rapid qualitative detection of cucumber green mottle mosaic virus, it can also realize early screening of cucumber green mottle mosaic virus. Through sequence analysis and comparison, the false negative rate caused by the method of the present invention is extremely low. Attached Figure Description

[0036] Figure 1 The graphs are for Example 1 and Comparative Examples 1-11 in Example 1.

[0037] Figure 2 The graphs are for the detection of nucleic acid at 13 fg / μL in Example 1, Comparative Examples 1-11 in Example 2.

[0038] Figure 3The graphs are for the detection of nucleic acid at 1.3 fg / μL in Example 1, Comparative Examples 1-11 in Example 2.

[0039] Figure 4 The graphs are for the detection of nucleic acid at 0.13 fg / μL in Example 1 and Comparative Examples 1-11 in Example 2.

[0040] Figure 5 The graphs are for the detection of nucleic acid at 0.013 fg / μL in Example 1, Comparative Examples 1-11 in Example 2.

[0041] Figure 6 The detection curves for cucumber green mottle mosaic virus RNA, positive control, other control viruses, and negative control were obtained according to the protocol of Example 1.

[0042] Figure 7 A gel image illustrating the effect of nucleic acid removal enzymes on nucleic acid removal. Detailed Implementation

[0043] The present invention will be further clearly and completely illustrated below through embodiments. These embodiments are only some examples of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional experiments, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0044] Example 1 This embodiment provides a method for rapid on-site detection of cucumber green mottle mosaic virus based on enzyme-mediated double amplification of nucleic acid.

[0045] 1.1 Reagents (1) Enzyme system of nucleic acid amplification group: recombinase (Lyo-ready T4 UvsY Protein, Thermo Fisher Scientific, A72125; Lyo-ready T4 UvsX Protein, Thermo Fisher Scientific, A72124), single-strand binding protein (Lyo-ready T4 Gene 32 Protein, Thermo Fisher Scientific, A72123), polymerase (Lyo-ready Bst DNA Polymerase, Thermo Fisher Scientific, A56655) and ATP energy regeneration enzyme (Creatine Kinase, Yisheng Biotechnology, 14502ES05).

[0046] (2) Signal amplification enzyme system: transcriptase (MAXIscript™ T7 transcription kit, Thermo Fisher Scientific, AM1314), reverse transcriptase (M-MuLV Reverse Transcriptase, NEB, M0253S), poly-L-lysine (Yisheng Biotechnology, 60716ES08) and cleavage enzyme (RNase H, NEB, M0297L).

[0047] (3) Nucleic acid clearance enzyme (Exonuclease III (E. coli), NEB, M0206S).

[0048] (4) Activating enzyme NTP: nucleoside triphosphate (Ribonucleotide Solution Mix, NEB, NO466S).

[0049] (5) RNA probe (SEQ ID NO.1), DNA upstream primer (SEQ ID NO.2) and DNA downstream primer (SEQ ID NO.3).

[0050] (6) Buffer (10-30mM pH7.2-7.8 Tris-HAc; 20-80mM potassium acetate (KAc); 2-5mM magnesium acetate (Mg(Ac)2); 1-4mM mercaptoethanol; 0.5-1mM ATP), RNase-free water, lyophilization protectant (polyethylene glycol).

[0051] Primer and probe sequences are shown in Table 1: Table 1 Primer and probe sequences

[0052] The above-mentioned actual preparation into pre-packaged freeze-dried pellets is carried out using the following method: First, prepare the buffer solution according to the formula: 20 mM pH7.5 Tris-HAc; 50 mM potassium acetate (KAc); 5 mM magnesium acetate (Mg(Ac)2); 2 mM mercaptoethanol; 1 mM ATP; then prepare the mixture before lyophilization according to Table 2.

[0053] Table 2

[0054] Using a 50 μL pipette, aspirate the above mixture and add 50 μL of the mixture to liquid nitrogen until all liquids are prepared. Remove the pre-frozen tray, pour in enough liquid nitrogen to submerge the monolayer microspheres, add the microspheres to the tray, and then place it in a freeze dryer to freeze dry according to the parameters provided by the manufacturer (freeze dryer (model: 100F) purchased from Ningbo Xinzhi Biotechnology Co., Ltd.).

[0055] 1.2 Detection Method The specific testing steps are as follows: S1. Collect samples (leaf samples from previous years that were diseased, collected by Ningxia Agricultural Technology Extension Station). After treatment with sample soaking solution (10mM Tris (pH 8), 50 mM NaCl, 1 mM EDTA, 0.1% SDS, and 2% PVP-40), add to lysis buffer (10mM Tris (pH 8), 50 mM NaCl, 1 mM EDTA, 0.1% SDS, and 2% PVP-40, 1 mg / μL colorless peptidase (Sigma-Aldrich, A3547)) tubes and incubate at 95℃ for 10 minutes to extract RNA from the samples.

[0056] S2. Using the RNA from step S1 as a template, add 50 μL of the extracted RNA to a pre-packaged lyophilized bulb, mix well with a shaker and centrifuge briefly, then perform an isothermal amplification reaction at 42°C. One cycle is performed per minute, for a total of 30 cycles. Illumination is performed during each cycle, with the fluorescence channel being FAM.

[0057] S3. The reaction fluorescence signal Tt value obtained in step S2 is used as the detection result of cucumber green mottle mosaic virus. If there is no Tt value in the cycle, it means that the sample to be tested is not infected with cucumber green mottle mosaic virus and the detection result is negative. If there is a Tt value, it means that the sample to be tested is infected with cucumber green mottle mosaic virus and the detection result is positive.

[0058] Comparative Example A comparative example was set up with reference to Example 1. The differences between the comparative example and the example are shown in Table 3: Table 3

[0059] Note: SEQ ID NO.10 and SEQ ID NO.11 in Comparative Example 7 are from: "Establishment of Real-time Fluorescent Quantitative PCR Detection System for Cucumber Green Mottle Mosaic Virus", Liu Mei, Wang Yuhong, Peng Bin, Wu Huijie, Han Yage, Gu Qinsheng, Journal of Plant Protection, 2022, 49(4):1063-1070.

[0060] Example 1: Detection Performance RNA was extracted from watermelon seeds using the FastPure Viral DNA / RNA Mini Kit (RC311-C1). (Sample source: melon seeds and seedlings from Yuanzhou District, Guyuan City, Ningxia; sampled variety: watermelon; name: Dabao No. 1; origin: Jiuquan, Gansu; RT-qPCR result was positive). TE was used as a negative control, and RNase-free water was used as a blank control. The detection performance was compared according to the reaction system, time, and temperature of Example 1, Comparative Examples 1-11.

[0061] The results showed that Example 1 had the best detection performance, while the comparative examples had poor detection performance. Comparative examples 7-11 were completely unable to detect anything, and the negative control was undetectable. This indicates that the primer and probe combinations screened in this invention, as well as the various components in the reaction, all have a strong effect on the detection performance and the reaction system. Figure 1 ).

[0062] Example 2: Sensitivity 50 μL of sample nucleic acid (the stock nucleic acid concentration after RNA virus extraction from positive watermelon seeds (origin: Guyuan County, Ningxia; watermelon: Jinyan No. 4) was 1.3 ng / μL, and then serially diluted with RNase-free water to 13 fg / μL, 1.3 fg / μL, 0.13 fg / μL, and 0.013 fg / μL, respectively) was added to the lyophilized bulbs of Example 1 and Comparative Examples 1-11. The samples were then capped, vortexed for 10 s, centrifuged for 10 s, and then analyzed. A real-time PCR instrument was used with a reaction temperature of 42℃, signal acquisition performed once per cycle, for a total of 30 cycles, with a reaction time of 30 minutes. The reporter group was set to FAM.

[0063] The results showed that, based on the experimental findings, the screened primers and probes could detect RNA virus extracted nucleic acid at a minimum dilution of E7 (0.13 fg / μL), exhibiting high sensitivity; while comparative examples 1-6 could only detect up to 13 fg / μL, and comparative examples 7-11 were practically undetectable at a nucleic acid dilution of E5, demonstrating poor sensitivity. Figure 2-5 ).

[0064] Example 3 Specificity Nucleic acid extraction was performed on cucumber green mottle virus positive watermelon seed samples (source: Guyuan County, Ningxia; watermelon: Jinyan No. 4, name: Jinyan No. 4) using the FastPure Viral DNA / RNA Mini Kit (RC311-C1). A commercially synthesized cucumber green mottle mosaic virus plasmid was used as a positive control (Suzhou Genewiz Biotechnology Co., Ltd.). Genomic RNA of cucumber mosaic virus (CMV), melon yellow spot virus (MYSV), and zucchini yellow mosaic virus (ZYMV) (source: nucleic acid extracted from previously virus-positive samples from Ningxia Agricultural Technology Extension Station, stored at -80 degrees Celsius) was tested and evaluated. Healthy watermelon seeds were used as negative controls, and ultrapure water was used as a blank control. Specificity detection was performed according to the reaction system, time, and temperature specified in Example 1.

[0065] The results showed that cucumber green mottle mosaic virus RNA and the positive control were detected, while other viruses and the negative control were not detected, indicating that the primer and probe combination in Example 1 of this invention has high specificity. Figure 6 ).

[0066] Example 4: Accuracy Watermelon seeds or leaves from the laboratory were used as test samples (samples of diseased leaves and seeds collected by Ningxia Agricultural Technology Extension Station in previous years). After soaking and rubbing the samples in sample soaking solution (purchased from Suzhou Jingrui Biotechnology Co., Ltd., Su Su Medical Device Registration No.: 20211362), they were added to lysis tubes (purchased from Suzhou Jingrui Biotechnology Co., Ltd., Su Su Medical Device Registration No.: 20211362) and rapidly extracted RNA at 95℃ for 10 minutes. Cucumber green mottle mosaic virus RNA was used as a positive control, TE as a negative control, and RNase-free water as a blank control. The effectiveness of this method in actual samples was determined according to the sample loading system, time, and temperature of Example 1 and Comparative Examples 1-11. Twenty samples were randomly selected from the laboratory (16 positive samples and 4 negative samples) for testing, and the remaining samples were used for qPCR detection. The results showed that the detection results of Example 1 were consistent with the qPCR detection results, with an accuracy of 100% (as shown in Table 4). The accuracy rates of the detection results of Comparative Examples 1-6 compared with those of qPCR were 60%, 75%, 75%, 60%, 80%, and 45%, respectively.

[0067] Table 4 Comparison of results between the method in Example 1 and the qPCR detection method.

[0068] Note: "-" indicates no TT value or no Ct value, which is negative.

[0069] Table 5 shows the detection results of Comparative Example 1 and Comparative Example 2.

[0070] Note: "-" indicates no TT value or no Ct value, which is negative.

[0071] Table 6 shows the detection results of Comparative Examples 3 and 4.

[0072] Note: "-" indicates no TT value or no Ct value, which is negative.

[0073] Table 7 shows the test results for Comparative Examples 5 and 6.

[0074] Note: "-" indicates no TT value or no Ct value, which is negative.

[0075] Table 8 shows the detection results of Comparative Example 7 and Comparative Example 8.

[0076] Note: "-" indicates no TT value or no Ct value, which is negative.

[0077] Table 9 shows the test results for Comparative Examples 9 and 10.

[0078] Note: "-" indicates no TT value or no Ct value, which is negative.

[0079] Table 10 shows the test results of Comparative Example 11.

[0080] Note: "-" indicates no TT value or no Ct value, which is negative.

[0081] Example 5: Nuclease Degradation Product Test Test system: At time point 0, 50 μL of target nucleic acid (1000 copies / µL, commercially synthesized cucumber green mottle mosaic virus plasmid (Suzhou Genewiz Biotechnology Co., Ltd.)) was added to a test tube containing the lyophilized pellets from Example 1. After capping, the tube was vortexed for 10 seconds, centrifuged for 10 seconds, and then tested. A standard PCR instrument was used at 42°C, with 30 cycles of 1 minute each, for a total reaction time of 30 minutes. After the reaction, the tubes were removed from the instrument and placed at room temperature. The above test procedure was repeated at 6 hours, 8 hours, 10 hours, 11 hours, and 11.5 hours. After each reaction, the test tubes were removed and placed at room temperature. After the last reaction tube had finished reacting and been placed at room temperature for 30 minutes, 10 µL of nucleic acid product was aspirated from each tube for gel electrophoresis.

[0082] Control system: At time point 0, two test tubes were taken, and 50 μL of target nucleic acid was added to each lyophilized bulb (the difference from Example 1 is that it does not contain nuclease). After capping, the tubes were vortexed for 10 seconds, centrifuged for 10 seconds, and then analyzed. The same standard PCR instrument was used for the reaction, at a temperature of 42°C, with each cycle lasting 1 minute, for a total of 30 cycles and a reaction time of 30 minutes. After the reaction, the tubes were removed from the instrument and allowed to stand at room temperature for 12 hours. Then, 10 μL of each tube was taken and combined with the test system for gel electrophoresis. Imaging analysis was then performed.

[0083] From the glue map ( Figure 7 As can be seen, the reaction system with added nucleic acid removal enzyme showed no amplification bands from 0.5h to 12h, while the control group showed obvious bands.

[0084] Application Example 1: Rapid On-site Nucleic Acid Sampling To improve ease of use on-site, in addition to the testing system, the sampling method and sample processing plan are also extremely important. For positive seed samples (1-6) (seed samples from previous years that were diseased and collected by Ningxia Agricultural Technology Extension Station) and negative seed samples (7-12), one spoonful of sample was taken with a 0.02g spoon and added to the lysis buffer (purchased from Suzhou Jingrui Biotechnology Co., Ltd., SuSu Medical Device Registration No.: 20211362), and heated at 95℃ for 10 min using a handheld metal bath. For positive leaf samples (13-18) (leaf samples from previous years that were diseased and collected by Ningxia Agricultural Technology Extension Station) and negative leaf samples (19-24), the operator wore disposable gloves, tore off a piece of leaf about the size of a fingernail, added it to the lysis buffer (purchased from Suzhou Jingrui Biotechnology Co., Ltd., SuSu Medical Device Registration No.: 20211362), and heated at 95℃ for 10 min using a handheld metal bath. Using cucumber green mottle mosaic virus RNA as a positive control and TE as a negative control, the samples were tested using the method described in Example 1, along with the two types of samples (seed samples and leaf samples). The results showed that the rapid sampling method could detect positive samples normally, and no negative samples were detected, with a detection accuracy of 100%.

[0085] Table 11 Tt values ​​of seed and leaf samples obtained through rapid sampling and testing.

[0086] Note: "-" indicates no TT value or no Ct value, which is negative.

[0087] Application Example 2: Sample Processing Scheme Optimization To achieve rapid on-site testing and verification, the shorter the sample processing time, the better. For positive watermelon seeds (1-4), the extraction time was shortened to 5 minutes. Each seed was cut in half and added to two separate tubes of lysis buffer. For leaf samples (5-8), each leaf was divided in half and added to two separate tubes of lysis buffer, then heated at 95°C for 5 minutes and 10 minutes respectively. Cucumber green mottle mosaic virus RNA was used as a positive control, and TE as a negative control. The samples were tested using the method described in Example 1, along with the two types of samples mentioned above. The results showed that shortening the extraction time to 5 minutes did not significantly affect performance, and heating for 5 minutes can be used as a rapid on-site sample processing procedure.

[0088] Table 12 Test Tt values ​​for shortened extraction time

[0089] Note: "-" indicates no TT value or no Ct value, which is negative.

[0090] Application Example 3: Simplified Operation of Freeze-Dried Balls To meet the needs of rapid on-site testing, the shaking and centrifugation operations after adding lyophilized bulbs need to be performed manually, without relying on instruments. The detection method of Example 1 was used to perform shaking, centrifugation, and manual mixing followed by hand-shaking to collect the liquid, and the detection performance was compared. The results showed no significant difference between manual and machine operation, indicating that manual mixing can be directly used on-site. All samples were positive samples from the Ningxia Agricultural Technology Extension Station.

[0091] Table 13 Comparison of Tt values ​​between manual and machine mixing results

[0092] Application Example 4: On-site Rapid Testing Sampling was performed using a spoon and disposable gloves. Sample processing time was 5 minutes, and testing time was 20 minutes. A handheld metal bath and handheld fluorescence detector were used on-site to test seeds and leaves. Cucumber green mottle mosaic virus RNA was used as a positive control, and TE as a negative control. Both types of samples were tested using the freeze-dried bulbs described in Example 1. The specific procedures are as follows: For 3 positive seed samples (samples 1-3 collected by the Ningxia Agricultural Technology Extension Station from previous years' disease-affected seed samples) and 3 negative seed samples (samples 4-6), one spoonful (0.02g) was scooped up and added to the lysis buffer. For 3 positive leaf samples and 3 negative leaf samples, the operator, wearing disposable gloves, manually tore off a piece of leaf about the size of a fingernail and added it to the lysis buffer. The above samples were then heated at 95℃ for 5 minutes using a handheld metal bath. A lyophilized bulb for cucumber green mottle mosaic virus detection was taken out, and 50μL of the above-treated sample was added to it. The bulb was capped, manually shaken to mix for 10 seconds, and the liquid was collected and shaken to the bottom of the tube. The bulb was then placed in the handheld fluorescence detector, started with one button, and the detection program was run automatically. After 20 minutes, the instrument automatically displayed the detection Tt value and the positive / negative result based on its internal algorithm. The entire process took no more than 30 minutes, and strongly positive results could be displayed within 10 minutes. Scenario 1 involves using a power outlet to power the handheld metal bath and the handheld fluorescence detector, while Scenario 2 involves using an 84Wh power bank to power the two portable instruments. The results are shown in Table 14.

[0093] Table 14. Field test TT values ​​of seeds and leaves

[0094] Note: "-" indicates no TT value or no Ct value, which is negative.

[0095] In summary, Example 1 of this invention provides an optimal primer and probe combination with the best specificity, sensitivity, and accuracy, enabling DNA detection at the aM level. The necessary reagents are pre-prepared in lyophilized bulbs for convenient on-site rapid testing, and the process is quick and simple, achieving rapid qualitative detection of cucumber green mottle mosaic virus.

[0096] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A kit for detecting cucumber green mottle mosaic virus based on enzyme-mediated double amplification of nucleic acid, characterized in that, It includes an RNA probe, a DNA upstream primer, and a DNA downstream primer; the sequence of the RNA probe is shown in SEQ ID NO.1; the sequence of the DNA upstream primer is shown in SEQ ID NO.2; and the sequence of the DNA downstream primer is shown in SEQ ID NO.

3.

2. The reagent kit according to claim 1, characterized in that, The final concentration of the RNA probe is 300-500 nM, and the final concentrations of the upstream and downstream DNA primers are both 150-250 nM.

3. The reagent kit according to claim 1, characterized in that, The final concentration of the RNA probe is 400 nM, and the final concentrations of the upstream and downstream DNA primers are both 200 nM.

4. The reagent kit according to claim 1, characterized in that, The kit also includes a nucleic acid amplification enzyme system and a signal amplification enzyme system; the nucleic acid amplification enzyme system includes recombinase, single-stranded binding protein, polymerase and ATP energy regenerating enzyme; the signal amplification enzyme system includes transcriptase, reverse transcriptase, poly-L-lysine and cleavage enzyme.

5. The reagent kit according to claim 4, characterized in that, The final concentrations of the enzyme system in the nucleic acid amplification group are: 10-30 nM recombinase, 40-60 nM single-stranded binding protein, 15-25 nM polymerase, and 0.5-1.5 μM ATP energy regenerating enzyme.

6. The reagent kit according to claim 4, characterized in that, The final concentrations of the nucleic acid amplification enzyme system were: 20 nM recombinase, 50 nM single-stranded binding protein, 20 nM polymerase, and 1 μM ATP energy regenerating enzyme.

7. The reagent kit according to claim 4, characterized in that, The final concentrations of the signal amplification enzyme system are: 0.5-1.5 μM transcriptase, 0.5-1.5 μM reverse transcriptase, 0.5-1.5 μM poly-L-lysine, and 0.5-1.5 μM cleavage enzyme.

8. The reagent kit according to claim 4, characterized in that, The final concentrations of the signal amplification enzyme system were: 1 μM transcriptase, 1 μM reverse transcriptase, 1 μM poly-L-lysine, and 1 μM cleavage enzyme.

9. The reagent kit according to claim 1, characterized in that, The kit also includes a nuclease, nucleoside triphosphate, buffer, and lyophilization protectant.

10. The reagent kit according to claim 9, characterized in that, The final concentration of the nucleic acid clearing enzyme is 0.5-1.5 μM; the final concentration of the nucleoside triphosphate is 50-70 μM.

11. The reagent kit according to claim 9, characterized in that, The final concentration of the nucleic acid clearing enzyme is 1 μM; the final concentration of the nucleoside triphosphate is 60 μM.

12. The reagent kit according to claim 9, characterized in that, The buffer solution comprises: Tris-HAc, potassium acetate, magnesium acetate, mercaptoethanol, and ATP; the lyophilization protectant is polyethylene glycol.

13. The reagent kit according to claim 12, characterized in that, The buffer solution comprises: 10-30 mM Tris-HAc, 20-80 mM potassium acetate, 2-5 mM magnesium acetate, 1-4 mM mercaptoethanol and 0.5-1 mM ATP; the lyophilization protectant is polyethylene glycol 1-3% v / v.

14. A method for rapid on-site detection of cucumber green mottle mosaic virus, characterized in that, Includes the following steps: S1. Sample collection and processing: Collect plant tissue samples and add them to lysis buffer; S2. Heat the lysis buffer containing the sample from step S1 to release the nucleic acid; S3. Add the sample processed in step S2 to the reagents of the kit according to any one of claims 1-13, mix well, and obtain the reaction solution; S4. Place the reaction mixture from step S3 into the testing device, and interpret the results after testing.

15. The method according to claim 14, characterized in that, The instrument for the heat treatment in step S2 is a handheld metal bath; the detection device in step S4 is a handheld fluorescence detector.

16. The method according to claim 14 or 15, characterized in that, The samples are one or more of the following: watermelon, cantaloupe, cucumber, gourd, bottle gourd, pumpkin, loofah, bitter melon, amaranth, quinoa, purslane, datura, common tobacco, Bunsen tobacco, Sanssa tobacco, Shansi tobacco, or petunia.

17. The use of the kit according to any one of claims 1-13 or the method according to any one of claims 14-16 in the detection of cucumber green mottle mosaic virus.

Images