A kit for detecting cucumber green mottle mosaic virus based on enzyme-mediated double amplification of nucleic acid amplification, a detection method and application

Abstract

The application belongs to the technical field of virus nucleic acid molecule diagnosis, and particularly relates to a kit for detecting cucumber green mottle mosaic virus based on enzyme-mediated double amplification of nucleic acid, a detection method and application. The detection system of the application comprises a primer pair for amplifying the coat protein gene of the cucumber green mottle mosaic virus, i.e. a DNA upstream primer (SEQ ID NO. 2) and a DNA downstream primer (SEQ ID NO. 3), a probe for detecting the CP gene (an RNA primer (SEQ ID NO. 1)), a nucleic acid amplification enzyme system, a signal amplification enzyme system, a nucleic acid clearing enzyme, an activating enzyme NTP, a buffer, and a freeze-drying protective agent, which are prepared into a pre-dispensed freeze-dried ball. The kit can realize RNA detection at aM concentration, and early screening of the cucumber green mottle mosaic virus can be realized due to high detection sensitivity.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of virus nucleic acid molecule diagnosis, and particularly relates to a kit for detecting cucumber green mottle mosaic virus based on enzyme-mediated double amplification of nucleic acid, a detection method and application. BACKGROUND

[0002] Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus of the Tobamovirus family, and the virus particle is rod-shaped, about 300 nm long, and 18 nm in diameter without envelope, which is composed of more than 2100 coat protein subunits and genomic RNA. Under natural conditions, CGMMV mainly infects watermelon, melon, cucumber, gourd, bottle gourd, pumpkin, luffa, bitter gourd and other cucurbitaceae crops. Under artificial inoculation conditions, it can also infect amaranth, quinoa, pigweed, datura, common tobacco, benson tobacco, three tobacco, shansi tobacco and petunia. As an important pathogen of cucurbitaceae crops, the virus can cause leaf mottle, plant dwarfing and fruit reverse cupping, etc., which seriously affects the yield and quality of cucurbitaceae crops.

[0003] At present, the high-sensitivity molecular detection technology is mainly fluorescence quantitative PCR (polymerase chain reaction), which is widely used in various scenes of molecular detection. However, this technology needs to configure expensive and inconvenient to carry instruments to realize detection, and the detection time is usually more than 40 minutes, which greatly limits the application of this technology in the field of POCT (point-of-care test). In order to solve the deficiency of fluorescence quantitative PCR in the field of rapid nucleic acid detection, a variety of rapid nucleic acid amplification technologies under isothermal conditions have become the focus of research in recent years, including the following main technologies: transcription-mediated amplification and its derivative technologies, due to the restriction of reaction mechanism, the overall detection time is long, and the sensitivity is also limited, which cannot achieve short-time high-sensitivity detection; loop-mediated isothermal amplification has the characteristics of rapidness and sensitivity, but the weak anti-pollution ability leads to false positive, which limits its application in the field of nucleic acid rapid detection; there are more researches on recombinase polymerase amplification technology (RPA technology) or its derivative technology, but there are few products applied in clinic, and the operation is complex, the fluorescence signal is weak, which makes it difficult to meet the demand of ultra-fast high-sensitivity detection.

[0004] More importantly, during the nucleic acid amplification process, a large number of target nucleic acid fragments generated are prone to form tiny aerosol particles due to operation (such as opening, pipetting, and oscillation) or environmental factors (such as air flow). These aerosols containing high concentrations of amplification products have strong stability and can remain and spread in the experimental environment (including instrument surfaces, consumables, and even air) for a long time. Once contaminated, even a trace amount of contaminated nucleic acid will be efficiently recognized and amplified by the subsequent amplification reaction, resulting in serious false positive results. This contamination is hidden, cumulative, and difficult to completely eliminate, posing a major threat to the accuracy, reliability, and repeatability of the detection results. In the prior art, although measures such as strict partitioning operation, ultraviolet irradiation, and the use of UNG-dUTP anti-pollution systems exist, they all have significant limitations: physical partitioning cannot completely prevent aerosol diffusion, ultraviolet irradiation is ineffective for dead angles and may damage reagents, and the UNG system can only prevent contamination of specific types (containing dU) and is ineffective for single-stranded DNA. In the laboratory environment, it is still difficult to completely avoid the risk of contamination, and in the point-of-care testing (POCT) scene where resources are limited or conditions are poor, the risk of contamination is multiplied, greatly limiting the application of nucleic acid detection technology, especially the isothermal amplification technology that relies on simple operation, in the grassroots, bedside, or field.

[0005] Therefore, developing a field molecular rapid detection technology and applying it to field detection and monitoring scenarios 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 cucumber green mottle mosaic virus rapid detection kit and detection method that is as accurate as a professional laboratory and has the advantages of rapidness, simplicity, portability, etc. At the same time, a nucleic acid detection method that can effectively block or significantly reduce the risk of aerosol contamination from the source has an urgent technical need and great application value for improving the reliability of detection results, ensuring the accuracy of diagnosis, expanding the application scenarios of the technology (especially field rapid detection), reducing the risk of misdiagnosis and mistreatment due to false positives, and subsequent costs. SUMMARY

[0006] To overcome the above-mentioned deficiencies, the present application provides a kit and method for detecting cucumber green mottle mosaic virus based on enzyme-mediated double-amplification nucleic acid amplification, which has high sensitivity and accuracy.

[0007] The technical solution of the present application is as follows:

[0008] On the one hand, the present application provides a kit for detecting cucumber green mottle mosaic virus based on enzyme-mediated double-amplification nucleic acid amplification, which comprises an RNA probe, a DNA upstream primer, and a DNA downstream primer. The sequence of the probe is shown in SEQ ID NO. 1. The sequence of the DNA upstream primer is shown in SEQ ID NO. 2. The sequence of the DNA downstream primer is shown in SEQ ID NO. 3.

[0009] Specifically, the final concentration of the RNA probe can be 300-500 nM, and the final concentration of the DNA upstream primer and the DNA downstream primer can be 150-250 nM.

[0010] Preferably, the final concentration of the RNA probe can be 350-450 nM, and the final concentration of the DNA upstream primer and the DNA downstream primer can be 180-220 nM.

[0011] Preferably, the final concentration of the RNA probe is 400 nM, and the final concentration of the DNA upstream primer and the DNA downstream primer is 200 nM.

[0012] Specifically, the kit further comprises a nucleic acid amplification group enzyme system and a signal amplification group enzyme system; the nucleic acid amplification group enzyme system comprises a recombinase, a single-strand binding protein, a polymerase and an ATP energy regeneration enzyme; and the signal amplification group enzyme system comprises a transcription enzyme, a reverse transcription enzyme, a poly-L-lysine and a cleavage enzyme.

[0013] Preferably, the final concentration of the nucleic acid amplification group enzyme system can be: 10-30 nM recombinase, 40-60 nM single-strand binding protein, 15-25 nM polymerase and 0.5-1.5 μM ATP energy regeneration enzyme.

[0014] Preferably, the final concentration of the nucleic acid amplification group enzyme system can be: 15-25 nM recombinase, 45-55 nM single-strand binding protein, 18-22 nM polymerase and 0.8-1.2 μM ATP energy regeneration enzyme.

[0015] Preferably, the final concentration of the nucleic acid amplification group enzyme system can be: 20 nM recombinase, 50 nM single-strand binding protein, 20 nM polymerase and 1 μM ATP energy regeneration enzyme.

[0016] Specifically, the final concentration of the signal amplification group enzyme system can be: 0.5-1.5 μM transcription enzyme, 0.5-1.5 μM reverse transcription enzyme, 0.5-1.5 μM poly-L-lysine and 0.5-1.5 μM cleavage enzyme.

[0017] Preferably, the final concentration of the signal amplification group enzyme system can be: 0.8-1.2 μM transcription enzyme, 0.8-1.2 μM reverse transcription enzyme, 0.8-1.2 μM poly-L-lysine and 0.8-1.2 μM cleavage enzyme.

[0018] Preferably, the final concentration of the signal amplification group enzyme system can be: 1 μM transcription enzyme, 1 μM reverse transcription enzyme, 1 μM poly-L-lysine and 1 μM cleavage enzyme.

[0019] Specifically, the kit further comprises a nucleic acid scavenger, a nucleoside triphosphate, a buffer and a freeze-drying protective agent.

[0020] Preferably, the final concentration of the nucleic acid scavenger can be 0.5-1.5 μM; the final concentration of the nucleoside triphosphate is 50-70 μM.

[0021] Preferably, the final concentration of the nucleic acid scavenger can be 0.8-1.2 μM; the final concentration of the nucleoside triphosphate is 55-65 μM.

[0022] Preferably, the final concentration of the nucleic acid scavenger can be 1 μM; the final concentration of the nucleoside triphosphate is 60 μM.

[0023] Specifically, the buffer comprises Tris-HAc, potassium acetate, magnesium acetate, mercaptoethanol and ATP; and the freeze-drying protective agent is polyethylene glycol.

[0024] Preferably, the buffer 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; and the freeze-drying protective agent is polyethylene glycol 1-3% v / v.

[0025] Preferably, the buffer comprises 20 mM Tris-HAc, 50 mM potassium acetate, 5 mM magnesium acetate, 2 mM mercaptoethanol and 1 mM ATP; and the freeze-drying protective agent is polyethylene glycol 2% v / v.

[0026] In another aspect, the application provides a method for rapid detection of cucumber green mottle mosaic virus in situ, comprising the following steps:

[0027] S1, sample collection and processing: collecting plant tissue samples and adding the samples into a lysis solution;

[0028] S2, heating the lysis solution containing the samples in step S1 to release nucleic acids;

[0029] S3, adding the sample after step S2 into the reagents of the kit and mixing to obtain a reaction solution;

[0030] S4, placing the reaction mixture in step S3 into a detection device and reading the results after detection.

[0031] Specifically, the heating instrument in step S2 is a palm metal bath; and the detection device in step S4 is a palm fluorescence detector.

[0032] Specifically, the sample is one or more of watermelon, muskmelon, cucumber, gourd, bottle gourd, pumpkin, loofah, bitter gourd, amaranth, quinoa, pigweed, jimsonweed, common tobacco, bengal tobacco, three-leafed tobacco, shandong tobacco, or petunia.

[0033] In another aspect, the present application provides the use of the foregoing kit or method in the detection of cucumber green mottle mosaic virus.

[0034] The present application has the following beneficial effects:

[0035] (1) The RNA extraction step provided by the present application is simplified, only needs to use a sample soaking solution to treat the sample, then add it into a lysis tube, and heat treat for 10 minutes to complete.

[0036] (2) The enzyme-mediated double amplification nucleic acid amplification technology for detecting cucumber green mottle mosaic virus in the present application can amplify the target nucleic acid to 109 times within 10-30 minutes under the condition of constant temperature at 42 degrees through the integration of nucleic acid amplification group enzymes. Through the integration of signal amplification group enzymes, more than 10,000 fluorescent signals can be generated from one nucleic acid amplification product under the condition of constant temperature at 42 degrees, and finally the sample detection is high-efficiency and rapid.

[0037] (3) The present application detects cucumber green mottle mosaic virus by molecular detection, has strong specificity, and the sensitivity can be as low as 1 copy / μL, so that the sample infected with cucumber green mottle mosaic virus can be found early.

[0038] (4) The present application can effectively block or significantly reduce the risk of aerosol pollution from the source, improve the reliability of the detection result, and ensure the accuracy of the diagnosis.

[0039] (5) The present application can be used with a portable palm metal bath and a palm fluorescent detector, and can be carried to any place such as a rural site for detection, meeting various field detection and monitoring application scenarios.

[0040] (6) The operation process of the present application is simple and suitable for anyone to use, and has wide application prospects.

[0041] (7) The kit of the present application can realize aM level RNA detection (aM is converted into copy number, basically in single copy level), in addition to rapid qualitative detection of cucumber green mottle mosaic virus, early screening of cucumber green mottle mosaic virus can also be realized, and through sequence analysis and comparison, the false negative rate caused by the method of the present application is extremely low. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Detection curve graphs of example 1, example 2, comparative example 1, comparative example 2, comparative example 3, comparative example 4, comparative example 5, comparative example 6, comparative example 7, comparative example 8, comparative example 9, comparative example 10, and comparative example 11 in effect example 1.

[0043] Figure 2Detection curve graph for Example 1, Comparative Examples 1-11 in Effect Example 2 for 13 fg / μL of nucleic acid.

[0044] Figure 3 Detection curve graph for Example 1, Comparative Examples 1-11 in Effect Example 2 for 1.3 fg / μL of nucleic acid.

[0045] Figure 4 Detection curve graph for Example 1, Comparative Examples 1-11 in Effect Example 2 for 0.13 fg / μL of nucleic acid.

[0046] Figure 5 Detection curve graph for Example 1, Comparative Examples 1-11 in Effect Example 2 for 0.013 fg / μL of nucleic acid.

[0047] Figure 6 Detection curve graph for detection of Cucumber Green Mottle Mosaic Virus RNA, positive control, other control viruses and negative control according to the protocol of Example 1.

[0048] Figure 7 Effect gel for nucleic acid removal by nucleic acid removal enzyme. DETAILED DESCRIPTION

[0049] The application will be further clarified by the following examples which are intended to be exemplary of the application. The following examples are not intended to limit the application but merely to illustrate it. The experimental methods used in the following examples are routine experiments unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0050] Example 1

[0051] This example provides a method for on-site rapid detection of Cucumber Green Mottle Mosaic Virus based on enzyme-mediated double amplification of nucleic acid amplification.

[0052] 1.1 Reagents

[0053] (1) Nucleic acid amplification enzyme system: recombinant enzyme (Lyo-ready T4 UvsY Protein, Thermo, A72125; Lyo-ready T4 UvsX Protein, Thermo, A72124), single-strand binding protein (Lyo-ready T4 Gene 32 Protein, Thermo, A72123), polymerase (Lyo-ready Bst DNA Polymerase, Thermo, A56655), and ATP energy regeneration enzyme (Creatine Kinase, Yixing Biological, 14502ES05).

[0054] (2) Signal amplification enzyme group: Transcription enzyme (MAXIscript™ T7 Transcription Kit, Thermo, AM1314), reverse transcriptase (M-MuLV Reverse Transcriptase, NEB, M0253S), poly-L-lysine (Poly-L-lysine, Yikesheng Biology, 60716ES08) and cutting enzyme (RNase H, NEB, M0297L).

[0055] (3) Nucleic acid removal enzyme (Exonuclease III (E. coli), NEB, M0206S).

[0056] (4) Activating enzyme NTP: Ribonucleotide Solution Mix (NEB, N0466S).

[0057] (5) RNA probe (SEQ ID NO. 1), DNA upstream primer (SEQ ID NO. 2) and DNA downstream primer (SEQ ID NO. 3).

[0058] (6) Buffer (10-30 mM pH 7.2-7.8 Tris-HAc; 20-80 mM potassium acetate (KAc); 2-5 mM magnesium acetate (Mg(Ac)2); 1-4 mM mercaptoethanol; 0.5-1 mM ATP), RNase-free Water, freeze-drying protectant (polyethylene glycol).

[0059] The primer and probe sequences are shown in Table 1:

[0060] Table 1 Primer and probe sequences

[0061]

[0062] The above actual preparation is made into pre-packaged freeze-dried balls, and the specific method is as follows:

[0063] First, prepare the buffer according to the formula: 20 mM pH 7.5 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 freeze-drying according to Table 2.

[0064] Table 2

[0065]

[0066] Using a 50 μL pipette to suck the above mixture, 50 μL of the above mixture was injected into liquid nitrogen until all the liquid was prepared. Take out the pre-frozen tray, pour the liquid nitrogen that can cover the single layer of microspheres, pour the microspheres into the tray, and then put it into the freeze dryer according to the parameters provided by the manufacturer (freeze dryer (model: 100F) purchased from Ningbo Xinzhi Biological Technology Co., Ltd.).

[0067] 1.2 Detection method

[0068] The specific detection steps are as follows:

[0069] S1, collect samples (last year's diseased leaf samples collected by Ningxia Agricultural Technology Promotion Center), treat with sample soaking solution (10 mM Tris (pH 8), 50 mM NaCl, 1 mM EDTA, 0.1% SDS, and 2% PVP-40), add lysis solution (10 mM Tris (pH 8), 50 mM NaCl, 1 mM EDTA, 0.1% SDS, and 2% PVP-40, 1 mg / μL achromopeptidase (Sigma-Aldrich, A3547)) tube, 95℃ 10 minutes, extract sample RNA.

[0070] S2, using the RNA of step S1 as a template, adding 50 μL of extracted RNA to the pre-packaged freeze-dried ball, mixing and centrifuging with a shaker, then performing isothermal amplification reaction, the reaction temperature is 42℃, one cycle per minute, a total of 30 cycles, each cycle is lighted, and the fluorescence channel is FAM.

[0071] S3, the reaction fluorescence signal Tt value obtained in step S2 is taken 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 Tt value, it means that the sample to be tested is infected with cucumber green mottle mosaic virus, and the detection result is positive.

[0072] Comparative example

[0073] The comparative example is set according to the reference example 1, and the difference between the comparative example and the example is shown in table 3:

[0074] Table 3

[0075]

[0076] Note: SEQ ID NO. 10 and SEQ ID NO. 11 in Comparative Example 7 are derived 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, Plant Protection, 2022, 49(4): 1063-1070.

[0077] Effect Example 1 Detection Performance

[0078] The FastPure Viral DNA / RNA Mini Kit viral DNA / RNA extraction kit (RC311-C1) was used to extract watermelon seed RNA (sample source: melon seeds and seedlings in Yuanzhou District, Guyuan City, Ningxia, sample variety: watermelon, name: Dabaoyihao, origin: Jiuquan, Gansu, RT-qPCR detection result is positive), TE was used as a negative control, and RNase-free water was used as a blank control. The reaction system, time, and temperature of Example 1, Comparative Examples 1-11 were compared for detection performance.

[0079] The results show that the detection performance of Example 1 is the best, and the detection performance of Comparative Examples is poor, among which Comparative Examples 7-11 cannot be normally detected, and the negative control is not detected, indicating that the primers, probe combinations screened by the application, and each component in the reaction have a strong effect on the detection performance and the reaction system. Figure 1 ).

[0080] Effect Example 2 Sensitivity

[0081] 50 μL of sample nucleic acid (positive watermelon seed (source: Guyuan County, Ningxia, watermelon: Jinyan No. 4, name: Jinyan No. 4) RNA virus extract nucleic acid stock solution concentration is 1.3 ng / μL) was added to the lyophilized balls of Example 1, Comparative Examples 1-11, and the viral nucleic acid was gradient diluted with RNase-free water to 13 fg / μL, 1.3 fg / μL, 0.13 fg / μL, and 0.013 fg / μL, respectively. The above concentrations of nucleic acids were used for detection, the lid was covered, and it was shaken and mixed for 10 s, then centrifuged for 10 s, and then detected on the machine. The fluorescence quantitative PCR instrument reaction temperature was 42℃, signal acquisition was performed once per cycle, a total of 30 cycles, the reaction time was 30 minutes, and the reporter group was set to FAM.

[0082] The results show that according to the experimental results, the selected primers and probes can detect RNA virus extract nucleic acid dilution / E7 (0.13 fg / μL) with high sensitivity; while Comparative Examples 1-6 can only detect 13 fg / μL, and Comparative Examples 7-11 cannot detect nucleic acid dilution / E5, and have poor sensitivity.Figures 2-5 ).

[0083] Effect Example 3 Specificity

[0084] For the cucumber green mottle virus positive watermelon seed sample (source: Guyuan County, Ningxia, watermelon: Jinyan No. 4, name: Jinyan No. 4), nucleic acid extraction was performed using the FastPure Viral DNA / RNA Mini Kit viral DNA / RNA extraction kit (RC311-C1), and the commercially synthesized cucumber mosaic virus plasmid was used as a positive control (Suzhou Jinyuzhi Biological Technology Co., Ltd.), and the cucumber mosaic virus (CMV), melon yellow spot virus (MYSV), and zucchini yellow mosaic virus (ZYMV) genomic RNA (source: nucleic acid extracted from previous virus positive samples from the Ningxia Agricultural Technology Promotion Center, stored at -80°C) were detected and evaluated. Healthy watermelon seeds were used as a negative control, and ultrapure water was used as a blank control. The reaction system, time, and temperature of Example 1 were used for specific detection.

[0085] The results showed that the cucumber green mottle virus RNA and the positive control were detected, and the other viruses, the negative control were not detected, indicating that the primer and probe combination of Example 1 of the present application had high specificity. Figure 6 ).

[0086] Effect Example 4 Accuracy

[0087] Laboratory watermelon seeds or leaves were used as test samples (disease leaves and seed samples collected by the Ningxia Agricultural Technology Promotion Center in previous years), and the samples were soaked and kneaded with sample soaking solution (purchased from Suzhou Jingrui Biological Technology Co., Ltd., Suzhou Hardware Record: 20211362) and then added to a lysis tube (purchased from Suzhou Jingrui Biological Technology Co., Ltd., Suzhou Hardware Record: 20211362). The sample RNA was quickly extracted at 95°C for 10 minutes. Cucumber green mottle virus RNA was used as a positive control, TE was used as a negative control, and RNase-free water was used as a blank control. The sample addition system, time, and temperature of Example 1 and Comparative Examples 1-11 were used to determine the effectiveness of the method in actual samples. Twenty samples were randomly selected from the laboratory (16 positive samples and 4 negative samples) for detection, 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% (Table 4). The detection results of Comparative Examples 1-6 and the qPCR results had an accuracy of 60%, 75%, 75%, 60%, 80%, and 45%, respectively.

[0088] Table 4 Comparison of Example 1 method and qPCR detection method results

[0089]

[0090] Note: "-" means no TT value or no Ct value, which is negative.

[0091] Table 5 Test results of Comparative Example 1 and Comparative Example 2

[0092]

[0093] Note: "-" means no TT value or no Ct value, which is negative.

[0094] Table 6 Test results of Comparative Example 3 and Comparative Example 4

[0095]

[0096] Note: "-" means no TT value or no Ct value, which is negative.

[0097] Table 7 Test results of Comparative Example 5 and Comparative Example 6

[0098]

[0099] Note: "-" means no TT value or no Ct value, which is negative.

[0100] Table 8 Test results of Comparative Example 7 and Comparative Example 8

[0101]

[0102] Note: "-" means no TT value or no Ct value, which is negative.

[0103] Table 9 Test results of Comparative Example 9 and Comparative Example 10

[0104]

[0105] Note: "-" means no TT value or no Ct value, which is negative.

[0106] Table 10 Test results of Comparative Example 11

[0107]

[0108] Note: "-" means no TT value or no Ct value, which is negative.

[0109] Example 5 Nuclease degradation product test

[0110] Test system: At time point 0, 50 μL of target nucleic acid (1000 copies / μL, commercial synthetic cucumber green mottled mosaic virus plasmid (Suzhou Jinyuzhi Biotechnology Co., Ltd.)) was added to the detection tube containing the lyophilized ball of Example 1, and after covering the cap, it was shaken and mixed for 10 s, and after centrifugation for 10 s, it was detected on the instrument. The reaction was carried out using a general PCR instrument, the reaction temperature was 42°C, each cycle was 1 min, a total of 30 cycles, and the reaction time was 30 min. After the reaction was completed, it was taken out of the instrument and placed at room temperature. After 6 h, 8 h, 10 h, 11 h, and 11.5 h, respectively, the above detection operation was repeated, and after the reaction was completed, the detection tube was taken out and placed at room temperature. After the last reaction tube was completed and placed at room temperature for 30 min, 10 μL of nucleic acid product was taken from each tube for gel electrophoresis experiment.

[0111] Control system: At time point 0, two detection tubes were taken, and 50 μL of target nucleic acid was added to the lyophilized ball (the difference from Example 1 is that it does not contain nucleic acid scavenger), and after covering the cap, it was shaken and mixed for 10 s, and after centrifugation for 10 s, it was detected on the instrument. The same general PCR instrument was used for reaction, the reaction temperature was 42°C, each cycle was 1 min, a total of 30 cycles, and the reaction time was 30 min. After the reaction was completed, it was taken out of the instrument and placed at room temperature for 12 h, and then 10 μL was taken and subjected to gel electrophoresis experiment together with the test system, and after completion, imaging analysis was performed.

[0112] From the gel map ( Figure 7 ), it can be seen that the reaction system with added nucleic acid scavenger had no amplification band from 0.5 h to 12 h, while the control group had obvious bands.

[0113] Application Example 1: On-site rapid sampling of nucleic acid

[0114] To improve the convenience of field use, in addition to the detection system, the sampling method and sample processing scheme are also very important. For positive seed samples (1-6) (collected by Ningxia Agricultural Technology Promotion Station in previous years) and negative seed samples (7-12), a small spoon of 0.02 g was scooped into a spoon, and a lysis solution (purchased from Suzhou Jingrui Biotechnology Co., Ltd., Suzhou Machinery Record: 20211362) was added, and a palm metal bath was used for heating treatment at 95°C for 10 min. For positive leaf samples (13-18) (collected by Ningxia Agricultural Technology Promotion Station in previous years) and negative leaf samples (19-24), the operator wore disposable gloves, tore off a fingernail-sized leaf, and added it to the lysis solution (purchased from Suzhou Jingrui Biotechnology Co., Ltd., Suzhou Machinery Record: 20211362), and used a palm metal bath for heating treatment at 95°C for 10 min. Cucumber green mottle mosaic virus RNA was used as a positive control, and TE was used as a negative control. The above two types of samples (seed samples and leaf samples) were detected using the method described in Example 1. The results showed that the rapid sampling method could normally detect positive samples, and no negative samples were detected, with a detection accuracy of 100%.

[0115] Table 11 Tt values of rapid sampling detection of seed samples and leaf samples

[0116]

[0117] Note: “-” represents no TT value or no Ct value, which is negative.

[0118] Application Example 2 Sample processing scheme optimization

[0119] In order to realize on-site rapid testing and verification, the shorter the sample processing time is, the better. The extraction time was shortened to 5 min to test positive watermelon seeds (1-4), and the single seed was cut in half and added to two tubes of lysis solution; for leaf samples (5-8), a piece of leaf was divided into two, and two tubes of lysis solution were added, and heated at 95°C for 5 min / 10 min. Cucumber green mottle mosaic virus RNA was used as a positive control, and TE was used as a negative control. The above two types of samples were detected using the method described in Example 1. The results showed that there was no obvious difference in performance when the extraction time was shortened to 5 min, and heating for 5 min could be used as a rapid sample processing procedure in the field.

[0120] Table 12 Tt values of extraction time reduction test

[0121]

[0122] Note: “-” represents no TT value or no Ct value, which is negative.

[0123] Application Example 3 Simplification of freeze-dried ball operation

[0124] To adapt to the on-site rapid detection needs, the shaking mixing and centrifugation operation after the addition of the freeze-dried ball sample needs to be separated from the instrument dependence and manually operated. The detection methods of Example 1 were used to perform shaking mixer mixing, centrifuge, and manual mixing, and the liquid was collected by hand, and the detection performance was compared. The results showed that there was no significant difference between manual operation and machine operation, and manual mixing operation could be directly used on site. The samples were all positive samples from the Ningxia Agricultural Technology Promotion Station.

[0125] Table 13 Comparison of detection results of Tt value of manual mixing and machine mixing

[0126]

[0127] Application Example 4 On-site Rapid Detection

[0128] The sample was taken using a spoon and disposable gloves, the sample processing time was 5 minutes, the detection time was 20 minutes, and the on-site palm metal bath and palm fluorescent detector were used in combination to test the seeds and leaves. The cucumber green mottled mosaic virus RNA was used as a positive control, and TE was used as a negative control, and the freeze-dried balls described in Example 1 were used for detection with the above two types of samples. The specific operation is as follows: for 3 positive seed samples (seed samples 1-3 collected by the Ningxia Agricultural Technology Promotion Station in previous years) and 3 negative seed samples (sample 4- sample 6), a small spoon of 0.02 g was used to scoop a spoonful and add lysis solution; for 3 positive leaf samples and 3 negative leaf samples, the operator wore disposable gloves and tore the leaf to the size of the nail cover, and added lysis solution; the above samples were heated at 95°C for 5 min using the palm metal bath; the cucumber green mottled mosaic virus detection freeze-dried ball was taken, 50 μL of the above treated sample was added to the freeze-dried ball, the lid was covered, and the liquid was shaken and mixed manually for 10 s, and then the liquid was collected and shaken to the bottom of the tube, and then placed into the palm fluorescent detector, and one key was started to automatically run the detection program. After 20 minutes, the instrument automatically displays the detection Tt value and the detection of positive and negative according to the internal algorithm. The whole process does not exceed 30 minutes, and the strong positive result can be displayed within 10 minutes. Scene 1 uses a socket to power the palm metal bath and palm fluorescent detector, and scene 2 uses an 84 Wh mobile power supply to power the above two portable instruments, and the results are shown in Table 14.

[0129] Table 14 On-site test TT value of seeds and leaves

[0130]

[0131] Note: “-” represents no TT value or no Ct value, which is negative.

[0132] In summary, the primer and probe combination of the embodiment 1 of the present application has the best specificity, sensitivity and accuracy, and can realize aM level DNA detection; the reagents necessary for the test are pre-prepared in the freeze-dried balls, which is convenient for on-site rapid detection, and the process is fast, simple and convenient, and the rapid qualitative detection of the cucumber green mottle mosaic virus is realized.

[0133] The above detailed description is a specific description of one of the feasible embodiments of the present application, which is not used to limit the patent scope of the present application. It should be pointed out that any equivalent implementation or change made without departing from the present application should be included in the scope of the technical solutions of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A kit for the detection of Cucumber Green Mottle Mosaic Virus based on enzyme mediated double-amplification nucleic acid amplification, characterized in that, consists of: an RNA probe, a DNA upstream primer, a DNA downstream primer, a nucleic acid amplification enzyme group, a signal amplification enzyme group, a nucleic acid scavenger, nucleoside triphosphates, a buffer and a freeze-drying protective agent; 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. The nucleic acid amplification enzyme group comprises a recombinase, a single-strand binding protein, a polymerase and an ATP energy regeneration enzyme. The signal amplification enzyme group comprises a transcription enzyme, a reverse transcription enzyme, poly-L-lysine and a cleavage enzyme.

2. The kit of claim 1, wherein The final concentration of the RNA probe is 300-500 nM, and the final concentration of the DNA upstream primer and the DNA downstream primer is 150-250 nM.

3. The kit of claim 1, wherein The final concentration of the RNA probe is 400 nM, and the final concentration of the DNA upstream primer and the DNA downstream primer is 200 nM.

4. The kit of claim 1, wherein The final concentration of the nucleic acid amplification enzyme group is: 10-30 nM recombinase, 40-60 nM single-strand binding protein, 15-25 nM polymerase and 0.5-1.5 μM ATP energy regeneration enzyme.

5. The kit of claim 1, wherein The final concentration of the nucleic acid amplification enzyme group is: 20 nM recombinase, 50 nM single-strand binding protein, 20 nM polymerase and 1 μM ATP energy regeneration enzyme.

6. The kit of claim 1, wherein The final concentration of the signal amplification enzyme group is: 0.5-1.5 μM transcription enzyme, 0.5-1.5 μM reverse transcription enzyme, 0.5-1.5 μM poly-L-lysine and 0.5-1.5 μM cleavage enzyme.

7. The kit of claim 1, wherein The final concentration of the signal amplification enzyme group is: 1 μM transcription enzyme, 1 μM reverse transcription enzyme, 1 μM poly-L-lysine and 1 μM cleavage enzyme.

8. The kit of claim 1, wherein The final concentration of the nucleic acid scavenger is 0.5-1.5 μM, and the final concentration of the nucleoside triphosphates is 50-70 μM.

9. The kit of claim 1, wherein The final concentration of the nucleic acid scavenger is 1 μM, and the final concentration of the nucleoside triphosphates is 60 μM.

10. The kit of claim 1, wherein The buffer comprises: Tris-HAc, potassium acetate, magnesium acetate, mercaptoethanol and ATP; and the freeze-drying protective agent is polyethylene glycol.

11. The kit of claim 10, wherein The buffer 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; and the freeze-drying protective agent is polyethylene glycol 1-3% v / v.

12. A method for the rapid detection of Cucumber Green Mottle Mosaic Virus in the field, characterized in that, The method comprises the following steps: S1, sample collection and processing: collecting a plant tissue sample and adding the sample into a lysis solution; S2, heating the lysis solution containing the sample in step S1 to complete nucleic acid release; S3, adding the sample after step S2 into the reagents of the kit according to any one of claims 1-11, mixing and obtaining a reaction solution; S4, placing the reaction mixture in step S3 into a detection device and detecting to read the result.

13. The method of claim 12, wherein, The heating instrument in step S2 is a palm metal bath, and the detection device in step S4 is a palm fluorescence detector.

14. The method according to claim 12 or 13, characterized in that, The sample is one or more of watermelon, muskmelon, cucumber, calabash, bottle gourd, pumpkin, sponge gourd, bitter gourd, amaranth, quinoa, pigweed, jimsonweed, common tobacco, bengal tobacco, thrice tobacco, shandong tobacco, or petunia.

15. Use of the kit of any one of claims 1 to 11 or the method of any one of claims 12 to 14 for the detection of cucumber mosaic virus.

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