Primer group for detecting watermelon silver mottle virus, kit and application

Abstract

The invention relates to a primer group and a kit for detecting watermelon silver mottle virus and application of the primer group and the kit. The primer group is a specific primer aiming at the NSs protein gene of the watermelon silver mottle virus. The detection method is a fluorescent quantitative PCR (Polymerase Chain Reaction) detection method based on an SYBR Green dye method. By applying the primer group, the kit and the detection method, the genome of the watermelon silver mottle virus can be efficiently and accurately quantitatively detected. Whether a sample contains the watermelon silver mottle virus or not can be rapidly and accurately judged through detection, and the specific content level of the watermelon silver mottle virus is determined; when the kit is applied to detection, the infection state and information of the watermelon silver mottle virus on the plant can be more accurately obtained, so that prevention and control can be carried out in time. The method has the advantages of simplicity in result judgment, convenience and rapidness in operation, high specificity and sensitivity, wide application prospect and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology, and particularly relates to a primer set for detecting watermelon silver mottle virus, a kit and application thereof. BACKGROUND

[0002] At present, more than 700 plant viruses have been found worldwide, which harm 147 crops, and more than 80 plant viruses have been found on vegetables. In recent years, watermelon silver mottle virus (WSMoV) has been reported to harm watermelon, cucumber, tomato and other melon and solanaceous crops in China, causing serious impact and economic losses.

[0003] Watermelon silver mottle virus (WSMoV) is a virus of the genus Orthotospovirus of the family Bunyaviridae. Watermelon silver mottle virus has a serious impact on crop growth and can cause plants to be dwarfed, yellow-green interlaced color spots of different sizes to appear on leaves (( Figure 1 ), no melons to be formed or melon bars to be deformed (( Figure 2 ), the whole leaf to turn yellow or withered, and even the plant to die, causing serious economic losses. It has been proved that watermelon silver mottle virus is mainly transmitted by thrips, and can also be transmitted by sap and seeds. The virus has a wide host range, and can infect Cucurbitaceae, Solanaceae, Amaranthaceae and Chenopodiaceae. Plant virus diseases have latent infection, and it is difficult to control once symptoms appear, which can easily cause virus disease outbreaks and bring serious impact on agricultural production. Plant viruses can be transmitted through seeds, sap, transmission vectors and soil and plant residues, so it is crucial to identify whether plants, seeds, transmission vectors, soil and plant residues carry viruses in early stage.

[0004] Existing methods for detecting watermelon silver mottle virus include electron microscopy detection, enzyme-linked immunosorbent assay detection and conventional PCR detection. Electron microscopy detection has the problems of difficult virus particle separation, the need for expensive equipment and the like, which is not conducive to ordinary laboratories. When the extracted virus concentration is low, the specificity of enzyme-linked immunosorbent assay is poor, and the application has certain limitations. SUMMARY

[0005] In view of the problems in the prior art, the application provides a primer set for detecting watermelon silver mottle virus, a kit and application thereof. The watermelon silver mottle virus can be detected, and the method has the advantages of simple result determination, convenient operation, rapidness, specificity, high sensitivity, wide application prospect and the like.

[0006] To achieve the above object, the present application provides the following technical solutions.

[0007] The present application provides a primer set for detecting watermelon silver mottle virus, which comprises primer pair 1 and / or primer pair 2.

[0008] The primer pair 1 comprises WSMoV-SN-Q5F and WSMoV-SN-Q5R; WSMoV-SN-Q5F comprises the sequence shown in SEQ ID NO. 1; and WSMoV-SN-Q5R comprises the sequence shown in SEQ ID NO. 2.

[0009] The primer pair 2 comprises WSMoV-SN-700F and WSMoV-SN-700R; WSMoV-SN-700F comprises the sequence shown in SEQ ID NO. 3; and WSMoV-SN-700R comprises the sequence shown in SEQ ID NO. 4.

[0010] The primer set provided by the present application is a specific primer for the NSs protein gene of watermelon silver mottle virus. The primer pair 1 is used for fluorescent quantitative detection of the genome of watermelon silver mottle virus, and the primer pair 2 is used for amplification of the long fragment of the NSs gene of WSMoV. Using the above primer set, watermelon silver mottle virus can be detected, which has the advantages of simple result determination, convenient operation, rapidness, specificity, high sensitivity, wide application prospect, etc.

[0011] The present application also provides a kit for detecting watermelon silver mottle virus, comprising the above primer set.

[0012] Further, one or more of the SYBR Green dye method fluorescent quantitative PCR reagent, negative control, and positive control can be further included.

[0013] The positive control can be a recombinant plasmid containing the NSs gene fragment of watermelon silver mottle virus.

[0014] The negative control can be enzyme-free ddH2O.

[0015] The present application provides a method for detecting watermelon silver mottle virus for non-disease diagnosis purposes, comprising the following steps: using the above primer set or kit to perform fluorescent quantitative PCR amplification on the cDNA of the sample to be detected.

[0016] Further, the preparation method of the cDNA of the sample to be detected comprises the following steps: extracting the RNA of the sample to be detected and reverse transcribing it into cDNA.

[0017] Further, after the reaction is completed, it is judged whether the watermelon silver mottle virus is contained in the sample to be detected according to the amplification curve; the judging method comprises: if the amplification curve is a typical fluorescence amplification curve, the watermelon silver mottle virus is contained in the sample to be detected; if the amplification curve is not a typical fluorescence amplification curve, the watermelon silver mottle virus is not contained in the sample to be detected.

[0018] The application provides a specific primer set of a watermelon silver mottle virus gene, establishes a fluorescent quantitative PCR detection method and kit of the watermelon silver mottle virus, and provides technical support for conveniently, quickly and effectively detecting whether a sample to be detected carries the watermelon silver mottle virus.

[0019] The application further provides application of the primer set and the kit in detection of the watermelon silver mottle virus.

[0020] The primer set can be used for preparing the kit for detecting the watermelon silver mottle virus.

[0021] Compared with the prior art, the primer set, the kit, the method and the application have the advantages of high sensitivity, short detection time, reliable detection result, easy judgment and good repeatability, and the like. 3 The kit has good specificity and can specifically detect the watermelon silver mottle virus, and the amplification curve is good, and no specific amplification curve appears for other related pathogens that can infect melon and other crops; the sensitivity is high, the positive standard product is detected in a 10-fold gradient dilution, the detection limit is 3.45x10 BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The cucumber plant is infected with the watermelon silver mottle virus, and the leaf shows symptoms such as mottling and deformation.

[0023] Figure 2 Cucumber plants infected with watermelon silver mottle virus, cucumber strip performance deformity symptoms, affect the quality.

[0024] Figure 3 The primer pair (WSMoV-SN-Q5F / WSMoV-SN-Q5R) was used to detect WSMoV and other virus positive samples by PCR electrophoretogram, wherein M is DL2000 DNA MARKER, lane 1 is WSMoV virus positive sample, lane 2 is CMV virus positive sample, lane 3 is TMV virus positive sample, lane 4 is CGMMV virus positive sample, lane 5 is MNSV virus positive sample, lane 6 is CCYV virus positive sample, lane 7 is CLSV virus positive sample, lane 8 is CBLV virus positive sample, lane 9 is SLCCNV virus positive sample, and lane 10 is negative control (amplified with ddH2O as template).

[0025] Figure 4 The primer pair 3 was used to detect WSMoV and other virus positive samples by PCR electrophoretogram, wherein M is DL2000 DNA MARKER, lane 1 is WSMoV virus positive sample; lane 2 is CMV virus positive sample; lane 3 is TMV virus positive sample; lane 4 is CGMMV virus positive sample; lane 5 is MNSV virus positive sample; lane 6 is CCYV virus positive sample; lane 7 is CLSV virus positive sample; lane 8 is CBLV virus positive sample; lane 9 is SLCCNV virus positive sample; and lane 10 is negative control (amplified with ddH2O as template).

[0026] Figure 5 The amplification curve of SYBR Green dye fluorescence quantitative PCR detection method, wherein 1 is the amplification curve of WSMoV; 2-10 are respectively the amplification curve of CMV, the amplification curve of TMV, the amplification curve of CGMMV, the amplification curve of MNSV, the amplification curve of CCYV, the amplification curve of CLSV, the amplification curve of CBLV, the amplification curve of SLCCNV, and the amplification curve of negative control (with ddH2O as template).

[0027] Figure 6 The primer pair (WSMoV-SN-Q5F / WSMoV-SN-Q5R) was used to detect WSMoV virus corresponding to different concentrations of recombinant plasmid by PCR electrophoretogram, M is DL2000 DNA MARKER, lane 1 is 3.45×10 9 copies / μL recombinant plasmid sample, lane 2 is 3.45×10 8 copies / μL recombinant plasmid sample, lane 3 is 3.45×10 7copies / μL of recombinant plasmid sample, lane 4 is 3.45 x 10 6 copies / μL of recombinant plasmid sample, lane 5 is 3.45 x 10 5 copies / μL of recombinant plasmid sample, 6 is 3.45 x 10 4 copies / μL of recombinant plasmid sample, lane 7 is 3.45 x 10 3 copies / μL of recombinant plasmid sample, lane 8 is 3.45 x 10 2 copies / μL of recombinant plasmid sample, lane 9 is 3.45 x 10 copies / μL of recombinant plasmid sample, lane 10 is the amplification curve of the negative control (using ddH2O as template).

[0028] Figure 7 The amplification curve of the SYBR Green dye fluorescence quantitative PCR detection method specific for Watermelon Silver Mottle Mosaic Virus; the amplification curve of the SYBR Green dye fluorescence quantitative PCR detection method for Watermelon Silver Mottle Mosaic Virus; wherein each curve corresponds to the amplification curve of a different concentration of recombinant plasmid, 1 is the amplification curve of 3.45 x 10 9 copies / μL; 2 is the amplification curve of 3.45 x 10 8 copies / μL; 3 is the amplification curve of 3.45 x 10 7 copies / μL; 4 is the amplification curve of 3.45 x 10 6 copies / μL; 5 is the amplification curve of 3.45 x 10 5 copies / μL; 6 is the amplification curve of 3.45 x 10 4 copies / μL; 7 is the amplification curve of 3.45 x 10 3 copies / μL; 8 is the amplification curve of 3.45 x 10 2 copies / μL; 9 is the amplification curve of 3.45 x 10 copies / μL; 10 is the amplification curve of the negative control (using ddH2O as template).

[0029] Figure 8 The standard curve amplified by the SYBR Green dye fluorescence quantitative PCR detection method for Watermelon Silver Mottle Mosaic Virus. DETAILED DESCRIPTION

[0030] In order to make the content described in the present application more convenient to understand, the technical solutions described in the present application are further described below in combination with specific embodiments, but the embodiments do not limit the present application in any form, and do not represent the scope of the protection of the rights of the present application, and the scope of the protection of the rights of the present application is subject to the claims.

[0031] The present invention provides a primer set, a kit, a fluorescence quantitative PCR detection method, and applications thereof for detecting watermelon silver mottle virus. The primer set provided by the present invention is specific for the NSs protein gene of watermelon silver mottle virus. The detection method is a SYBR Green dye-based fluorescence quantitative PCR detection method, which may include the steps of primer design, nucleic acid extraction, standard curve preparation, melting curve preparation, and sample testing. The primer set, kit, and detection method provided by the present invention enable efficient and accurate quantitative detection of the genome of watermelon silver mottle virus. The detection method of the present invention can quickly and accurately determine whether a sample contains watermelon silver mottle virus and determine its specific content level. When used for detection, the infection status and information of a plant infected with watermelon silver mottle virus can be more accurately determined, facilitating timely prevention and control. The present invention features simple result determination, convenient operation, rapidity, specificity, and high sensitivity, and has broad application prospects.

[0032] Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art. Where specific techniques or conditions are not specified in the examples, they are all conventional methods or carried out according to the techniques or conditions described in the literature in the art, or according to the product instructions.

[0033] Unless otherwise specified, all reagents and materials used in the following examples were commercially available. Some of the instruments used included a real-time fluorescence quantitative PCR system (QuantStudio3), an electrophoresis instrument (I-PROTES TERA), a gel imaging system (Gel DOC™ XR+), and a constant temperature shaking incubator.

[0034] RNA can be extracted from the sample using a Quick RNA Isolation Kit (Huayueyang), which was purchased from Beijing Huayueyang Biotechnology Co., Ltd.; a reverse transcription kit ( UniRT&qPCR Kit) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; DL2000 DNAMARKER (i.e., Trans2K DNA Maker) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; EasyPure Quick Gel Extraction Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; T1Simple Gene Cloning Kit (Dual Antibody) -T1Simple Cloning Kit was purchased from Beijing Zoman Biotechnology Co., Ltd.;E. coli competent Trans1-T1 Phage Resistant Chemical ly Competent cell was purchased from Beijing Zoman Biotechnology Co., Ltd.;Plasmid MiniPrep Kit was purchased from Beijing Zoman Biotechnology Co., Ltd.;Universal Passive Reference Dye (50x) (optional) was purchased from Beijing Zoman Biotechnology Co., Ltd.;Ampicillin was purchased from MYM Biological Technology Company Limited;IPTG was purchased from scientific research special;X-GAL was purchased from scientific research special;Dimethylformamide was purchased from Tianjin Damao Chemical Reagent Factory.

[0035] In the examples, the positive samples include watermelon silver mottle virus (WSMoV), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), cucumber green mottle mosaic virus (CGMMV), melon necrotic spot virus (MNSV), cucumber chlorotic yellowing virus (CCYV), cucumber leaf spot virus (CLSV), cucumber bulgaria latent virus (CBLV), and Chinese squash leaf curl virus (SLCCNV), which are obtained by collecting samples in Tianjin and Shandong fields, detecting by RT-PCR, sequencing and identifying by Tianjin Academy of Agricultural Sciences Institute of Plant Protection, and are now preserved in the Seedling Disease Laboratory of Tianjin Academy of Agricultural Sciences Institute of Plant Protection, and the public can obtain a copy of the examples described in the application for non-commercial purposes only.

[0036] The primers involved in the application are synthesized by Beijing Qianke Biotechnology Co., Ltd.

[0037] The LB liquid medium is prepared according to the following proportions: 10 g / L of tryptone, 5 g / L of yeast extract, and 10 g / L of sodium chloride, prepared with water.

[0038] The LB solid medium is prepared according to the following proportions: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride, and 15-20 g / L of agar powder, prepared with water.

[0039] The following is described by specific examples.

[0040] Example 1

[0041] The watermelon silver mottle virus molecular detection primer is designed according to the conserved region of the NSs gene of the watermelon silver mottle virus.

[0042] The reference sequence of watermelon silver mottle virus (WSMoV) strain was downloaded from GeneBank, and according to the sequence alignment result, two pairs of primer pairs (primer pair 1 and primer pair 3) for detection and one pair of primer pair (primer pair 2) for amplifying the full fragment of the NSs gene of WSMoV were designed for the highly conserved fragment of the NSs gene of WSMoV by using Primer 5.

[0043] Primer pair 1 includes WSMoV-SN-Q5F / WSMoV-SN-Q5R, and primer pair 1 is used for amplifying WSMoV, and the length of the target fragment amplified by WSMoV is 161 bp. The sequence of the forward primer WSMoV-SN-Q5F is 5'-CAGCCTGCAAGAGCGGTAAGTAT-3'(SEQ ID NO. 1); and the sequence of the reverse primer WSMoV-SN-Q5R is 5'-ATTTCCTGTCTAGCAGCTTCATT-3'(SEQ ID NO. 2).

[0044] Primer pair 2 includes WSMoV-SN-700F / WSMoV-SN-700R, and primer pair 2 is used for amplifying the full fragment of the NSs gene of WSMoV, and the length of the target fragment amplified by WSMoV is 687 bp. The sequence of the forward primer WSMoV-SN-700F is 5'-GGTTAGCACTGAAGACCCTGTCCAT-3'(SEQ ID NO. 3); and the sequence of the reverse primer WSMoV-SN-700R is 5'-CTGGTGGTACAGCAGATGTTGAAAT-3'(SEQ ID NO. 4).

[0045] Primer pair 3 includes WSMoV-SN-Q1 F / WSMoV-SN-Q1R, and the length of the target fragment amplified by primer pair 3 is 150 bp. The sequence of the forward primer WSMoV-SN-Q1F is 5'-GTCGGACCTGAGGACTGGACATT-3'(SEQ ID NO. 5); and the sequence of the reverse primer WSMoV-SN-Q1R is 5'-CACATTCAACCCATAAGCAGCAA-3'(SEQ ID NO. 6).

[0046] Example 2

[0047] The RT-PCR method was used to detect WSMoV, including the following steps:

[0048] (1) Preparation of RNA template of the sample to be detected

[0049] For example, the genomic RNA of the sample can be extracted by using a quick universal plant RNA extraction kit (Quick RNA Isolation Kit huayueyang), and the specific method is as follows: ① 100 mg of sample is ground in liquid nitrogen, then added to 1 mL of lysis solution A, incubated at 52°C for 2 min, and the liquid part of the mixture is transferred to a clean 1.5 mL centrifuge tube; ② add 300 μL of deproteinization liquid B and 200 μL of chloroform, mix well after oscillation for 30 s, and stand at room temperature for 2 min; ③ centrifuge at 12000 rpm for 10 min at room temperature, and take 700 μL of supernatant to a clean 1.5 mL centrifuge tube; ④ add an equal volume of rinse solution C, mix well by inverting, and column twice, each time centrifuging for 30 s and discarding the waste liquid; ⑤ add 500 μL of column washing solution D to the adsorption column, centrifuge at 12000 rpm for 1 min at room temperature, discard the waste liquid, and repeat twice; ⑥ prepare digestion solution (6 μL of RNase-free RNase I + 44 μL of RNase buffer), mix well, and add to the center of the column membrane, and stand at room temperature for 15 min; ⑦ add 500 μL of enzyme-removing liquid E to the adsorption column, mix well by inverting, centrifuge at 12000 rpm for 30 s, and then add 500 μL of enzyme-removing liquid E to the adsorption column, mix well by inverting, and centrifuge at 12000 rpm for 2 min; ⑧ open the cover and place it on the clean bench to dry the anhydrous ethanol in the adsorption column; ⑨ put the adsorption column in a clean 1.5 mL centrifuge tube, add 30 μL of elution solution F, stand at room temperature for 3 min, centrifuge at 12000 rpm for 1 min to elute the RNA, and store at -80°C for standby.

[0050] (2) Preparation of cDNA template: the RNA prepared above is used as a template to obtain cDNA by reverse transcription reaction. Reverse transcription kit can be used for reverse transcription, and the reaction system includes: RNA (300-1500 ng / μL) 1 μL, 5x All-in-One Reaction Mix for qPCR 4 μL, Uni All-in-One Enzyme Mix 1 μL, RNase-free Water to 20 μL. The reaction program includes: 50°C reaction for 5 min, 85°C reaction for 2 min, and the cDNA obtained by reaction is stored at -20°C for standby.

[0051] (3) Using WSMoV-SN-Q5F / WSMoV-SN-Q5R primers for conventional RT-PCR amplification with cDNA as a template. The reaction system of RT-PCR amplification includes: cDNA (100-500 ng / μL) 2 μL, Forward Primer (10 μM) 1 μL, Reverse Primer (10 μM) 1 μL, 2x HiFi PCR SuperMix 25 μL, Nuclease-free Water up to 50 μL. The reaction conditions of RT-PCR amplification include: pre-denaturation 94℃, 10 min; denaturation 94℃, 30 s, annealing 56℃, 30 s, extension 72℃, 30 s, 35 cycles; final extension 72℃, 10 min. The amplification products were detected by 1% agarose gel electrophoresis.

[0052] If the specific fragment of 161 bp can be amplified, it indicates that the sample to be tested is WSMoV detection positive, otherwise, it indicates that the sample to be tested is WSMoV detection negative.

[0053] Example 3

[0054] The specificity of the WSMoV-SN-Q5F / WSMoV-SN-Q5R primer pair was detected, and the experimental method included the following steps: selecting watermelon silver mottle virus (WSMoV), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), cucumber green mottle mosaic virus (CGMMV), melon necrotic spot virus (MNSV), cucumber chlorotic yellowing virus (CCYV), cucumber leaf spot virus (CLSV), cucumber bulgaria latent virus (CBLV), Chinese pumpkin curly leaf virus (SLCCNV) and other 9 common melon viruses as positive samples. The above positive samples were detected by the method of Example 2. The negative control was amplified with ddH2O as the template, and the reaction system and reaction conditions were referred to Example 2.

[0055] The experimental results are shown in Figure 3 As can be seen, using the primer pair (WSMoV-SN-Q5F / WSMoV-SN-Q5R) to detect WSMoV and other 9 virus positive samples by PCR, only the watermelon silver mottle virus positive sample can produce specific bands (there is a clear band at 161 bp), and the purpose band is clear and single, without non-specific amplification; other 8 virus samples and negative control have no band at 161 bp, indicating that using WSMoV-SN-Q5F / WSMoV-SN-Q5R primer pair to detect watermelon silver mottle virus (WSMoV) has good specificity.

[0056] The primer pair 3 was validated for PCR specificity. Conventional RT-PCR amplification and gel electrophoresis detection were performed using cDNA and ddH2O from 9 common cucurbit virus-positive samples, including watermelon silver mottle virus (WSMoV), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), cucumber green mottle virus (CGMMV), melon necrotic mottle virus (MNSV), cucurbit chlorotic yellows virus (CCYV), cucumber leaf spot virus (CLSV), cucumber Bulgarian latent virus (CBLV), and Chinese squash leaf curl virus (SLCCNV). The experimental results are shown in Figure 2. Figure 4 As shown, it can be seen that primer pair 3 exhibits non-specific amplification.

[0057] Primer pair 2 was subjected to primer specificity verification, and the results showed that primer pair 2 had good specificity, no nonspecific amplification occurred, and had the advantage of high amplification efficiency.

[0058] Example 4

[0059] A SYBR Green dye-based fluorescence quantitative PCR detection method for WSMoV was established, comprising the following steps:

[0060] (1) Extracting viral RNA from a WSMoV sample, using the same method as step (1) of Example 2;

[0061] (2) Reverse transcription of RNA into cDNA, the experimental method refers to step (2) of Example 2;

[0062] (3) According to UniRT&qPCR Kit instructions: Use the cDNA obtained after reverse transcription as a template and WSMoV-SN-Q5F / WSMoV-SN-Q5R as primers for fluorescent quantitative PCR.

[0063] The fluorescence quantitative PCR reaction system includes: cDNA (concentration is 100-500ng / μL) 1μL, Forward Primer (10μM) 1.1μL, Reverse Primer (10μM) 1.1μL, 2x Green qPCR SuperMix 10μL, Universal Passive Reference Dye (50x) (optional) 0.4μL, Nuclease-free Water make up to 20μL.

[0064] The reaction conditions of fluorescence quantitative PCR included: 94°C (pre-denaturation) for 30 s; 94°C (denaturation) for 5 s, 63°C (annealing) for 30 s, and 40 cycles (PCR amplification).

[0065] According to the above detection system, various components are added, mixed uniformly, and then subjected to cycles according to the above fluorescence quantitative PCR reaction conditions, and the fluorescence signal is detected for 40 cycles. The fluorescence detection is used to determine the activity of the detection system. The fluorescence quantitative method is used to measure the fluorescence of the detection reaction, and in the WSMoV virus detection, the NSs gene of the WSMoV virus is amplified and detected. The results are determined by the fluorescence method, and the judgment criteria include: if the amplification curve is a typical fluorescence amplification curve, the sample to be tested contains the watermelon silver mottle virus; if the amplification curve has no typical fluorescence amplification curve, the sample to be tested does not contain the watermelon silver mottle virus. The detection of the WSMoV virus is realized by the above method.

[0066] Example 5

[0067] The specificity of the SYBR Green dye fluorescence quantitative PCR detection method is verified, and the experimental method includes the following steps:

[0068] Nine common melon viruses such as watermelon silver mottle virus (WSMoV), cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), cucumber green mottle virus (CGMMV), melon necrotic spot virus (MNSV), cucumber chlorotic yellowing virus (CCYV), cucumber leaf spot virus (CLSV), cucumber bulgaria latent virus (CBLV), and Chinese pumpkin curly leaf virus (SLCCNV) are selected as positive samples.

[0069] The positive samples are detected by the method of Example 4, and the negative control is amplified with ddH2O as the template. The reaction system and reaction conditions are referred to Example 4.

[0070] The experimental results are shown in Figure 5 It can be seen that the primer pair (WSMoV-SN-Q5F / WSMoV-SN-Q5R) is used for fluorescence quantitative PCR detection of WSMoV and other nine virus positive samples, and only the cDNA of the WSMoV virus positive sample can detect specific amplification signals. The cDNA of other virus samples and the negative control are not detected by the SYBR Green dye fluorescence quantitative PCR detection. The results show that the specific primers WSMoV-SN-Q5F / R and the detection method established have good specificity.

[0071] Example 6

[0072] The template plasmid is prepared, including the following steps:

[0073] (1) Amplification of the target fragment: The cDNA of the watermelon silver mottle virus (WSMoV) (prepared by the method of Example 2) is used as the template, and the specific primers WSMoV-SN-700F / WSMoV-SN-700R are used for PCR amplification.

[0074] The PCR amplification reaction system includes: cDNA (concentration is 100-500ng / μL) 2μL, Forward Primer (10μM) 1μL, Reverse Primer (10μM) 1μL, 2x Add 25 μL of HiFi PCR SuperMix and Nuclease-free Water to make up to 50 μL.

[0075] PCR amplification reaction conditions included: initial denaturation at 94°C for 10 min; 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s; and final extension at 72°C for 10 min.

[0076] The amplified products were detected by 1% agarose gel electrophoresis.

[0077] (2) DNA gel purification: Use a DNA gel rapid purification kit to cut the target band from the agarose gel into a 1.5 mL centrifuge tube and weigh it. After weighing, add three times the volume of gel dissolving buffer (GSB) and dissolve it in a 55°C water bath for 10 minutes. After cooling the melted gel to room temperature, add it to a centrifuge column and let it stand for 1 minute. Centrifuge it at 10,000 × g for 1 minute and discard the effluent. Add 650 μL of washing solution and centrifuge it at 10,000 × g for 1 minute and discard the effluent. Centrifuge it at 10,000 × g for 2 minutes. Place the centrifuge column in a clean centrifuge tube with the lid open and let it stand for 1 minute to allow the ethanol to evaporate completely. Add 50 μL of elution solution and let it stand at room temperature for 1 minute. Centrifuge it at 10,000 × g for 1 minute. Store the eluted product at -20°C for later use.

[0078] (3) Using T1 Simple Gene Cloning Kit (Dual Resistance) -T1 Simple Cloning Kit connection transformation: Add 1 μL of Trans1-T1 Simple Cloning Vector and 3 μL of the product obtained after DNA gel purification to a 200 μL centrifuge tube, mix gently, and react at 25°C for 10 minutes. Place 50 μL of freshly thawed Escherichia coli competent cells (Trans1-T1 Phage Resistant Chemically Competent cell) and the ligation product into a clean 1.5 mL centrifuge tube and gently flick to mix. Incubate on ice for 30 minutes, heat-shock in a 42°C water bath for 30 seconds, and immediately place on ice for 2 minutes. Add 250 μL of antibiotic-free LB liquid medium and culture at 200 rpm and 37°C for 1 hour to obtain a bacterial solution.

[0079] When the above culture is 30 min, prepare LB plate containing IPTG, X-gal, Amp+: take 8 μL 500 mM IPTG and 40 μL 20 mg / mL X-gal, mix and evenly coat on the prepared LB plate with 100 U Amp+.

[0080] After 1 h culture at 37℃, take 75 μL bacterial solution and evenly coat on the LB plate containing IPTG, X-gal, Amp+, and place in 37℃ incubator for overnight culture.

[0081] (4) Detection of WSMoV gene fragment positive clone: select white single clone into 10 μL sterile water, vortex mix, take 1 μL mixed solution as template for preparation of PCR reaction system, and identify WSMoV gene fragment positive clone with WSMoV-SN-700F / WSMoV-SN-700R primer pair.

[0082] PCR reaction system includes: Template 1 μL, 2 × High fidelity (HiFi) PCR Super Mix II (-dye) 12.5 μL, upstream primer (10 μmol / L) 0.5 μL, downstream primer (10 μmol / L) 0.5 μL, Nuclease-free Water, and make up to 25 μL.

[0083] PCR reaction program includes: pre-denaturation 94℃, 10 min; denaturation 94℃, 30 s, annealing 56℃, 30 s, extension 72℃, 30 s, 35 cycles; 72℃ final extension 10 min.

[0084] After the reaction, use 1% agarose gel electrophoresis to test the amplification result, and the electrophoretogram shows that the positive clone produces specific bands with a length of 687 bp, which meets the expected fragment size. The PCR product is sent to Beijing Qianke Biological Technology Co., Ltd. for sequencing, and after comparison, it is determined that the WSMoV gene fragment positive clone is obtained.

[0085] (6) 8 mL of LB liquid medium was transferred to a 10 mL centrifuge tube. 8 μL of 100 ng / mL Amp+ and 8 μL of the bacterial solution of the positive clone were added to the centrifuge tube. The tube was cultured at 200 rpm and 37°C for 16 h to obtain the expanded bacterial solution. The recombinant plasmid was extracted using a plasmid mini-extraction kit, comprising the following steps: centrifuging the expanded bacterial culture at 10,000 × g for 1 minute, removing the supernatant, adding resuspension solution and shaking, resuspending the bacterial pellet, adding lysis solution, inverting the cells to fully lyse them, adding neutralizing solution, gently mixing 5-6 times, letting them stand at room temperature for 2 minutes, and then centrifuging at 12,000 × g for 5 minutes; adding the supernatant to a centrifuge column, centrifuging at 12,000 × g for 1 minute, and discarding the flow-through; adding 650 μL of wash solution, centrifuging at 12,000 × g for 1 minute, and discarding the flow-through; centrifuging at 12,000 × g for 2 minutes to completely remove any remaining wash solution; placing the centrifuge column in a clean centrifuge tube, adding 30-50 μL of elution solution to the center of the column, letting it stand at room temperature for 1 minute, and centrifuging at 10,000 × g for 1 minute. The eluted product was stored at -20°C until further use. The template plasmid, namely the T1-WSMoV-NSs recombinant plasmid, was obtained by this method.

[0086] Example 7

[0087] Verifying the sensitivity of the SYBR Green dye-based fluorescent quantitative PCR detection method includes the following steps:

[0088] The T1-WSMoV-NSs recombinant plasmid (prepared using the method of Example 6) was extracted and the plasmid concentration was determined. The calculation formula is as follows:

[0089] Plasmid copy number (copies / μL) = (plasmid concentration ng / μL * 6.02 * 10 23 *10 9 ) / (number of bases*660)

[0090] 3.45×10 9 The plasmid at 10 copies / μL was diluted in a 10-fold gradient to 3.45 × 10 copies / μL. The plasmids at the above concentrations were used as templates, and conventional PCR amplification and fluorescence quantitative PCR amplification were performed using the specific fluorescence quantitative primer pair WSMoV-SN-Q5F / WSMoV-SN-Q5R as primers, respectively.

[0091] The reaction system and reaction conditions for conventional PCR amplification refer to Example 2. The reaction system and reaction conditions for fluorescent quantitative PCR amplification refer to Example 4. The negative control was amplified using ddH2O as a template, and the reaction system and reaction conditions refer to the above reaction system and reaction conditions.

[0092] The results of conventional PCR amplification are as followsFigure 6 As shown in the table, the primer pair (WSMoV-SN-Q5F / WSMoV-SN-Q5R) was used to detect the WSMoV recombinant plasmid with different concentration gradients, and only the 3.45×10 9 copies / μL sample could produce specific bands. 8 copies / μL sample could produce specific bands. 7 copies / μL sample could produce specific bands.

[0093] The results of the fluorescent quantitative PCR amplification are shown in the table. Figure 7 As shown in the table, the fluorescent quantitative PCR method provided by the application has a minimum detection limit of 3.45×10 3 copies / μL, and the detection sensitivity of the fluorescent quantitative PCR method is 10,000 times that of the ordinary PCR method.

[0094] The above results show that the sensitivity of the fluorescent quantitative PCR method provided by the application for WSMoV detection can reach 3.45×10 3 copies / μL, and the sensitivity is good, and the method can be applied to primary detection.

[0095] Example 8

[0096] The standard curve of the SYBR Green dye method fluorescent quantitative PCR detection was established, including the following steps:

[0097] The T1-WSMoV-NSs recombinant plasmid (prepared by the method of Example 6) with concentrations of 3.45×10 9 copies / μL, 3.45×10 8 copies / μL, 3.45×10 7 copies / μL, 3.45×10 6 copies / μL, 3.45×10 5 copies / μL, 3.45×10 4 copies / μL and 3.45×10 3 copies / μL was used as a template, and the specific primers WSMoV-SN-Q5F / WSMoV-SN-Q5R were used for fluorescent quantitative PCR, and the fluorescent quantitative PCR reaction system and reaction conditions were referred to Example 4.

[0098] The experimental results are shown in the table. Figure 8 As shown in the table, A is the horizontal coordinate, A is the logarithm of the initial concentration Log, B is the vertical coordinate, B is the cycle number CT, the standard curve of the WSMoV virus fluorescent quantitative PCR amplification is y=-2.92152x+43.34838, R 2= -0.99515. The standard curve has a good degree of linear fitting and is suitable for quantifying WSMoV. The copy number of virus in the sample to be tested can be quantified according to the standard curve.

[0099] Example 9

[0100] The cDNA of the field collected samples was detected by the established SYBR Green dye method fluorescence quantitative PCR detection method. The experimental method includes the following steps:

[0101] The cDNA of 24 cucumber leaf samples suspected to be infected by WSMoV collected from Shandong and Tianjin fields was respectively subjected to RT-PCR and RT-qPCR.

[0102] The RT-PCR detection method was as described in Example 2. The RT-qPCR detection method was as described in Example 4. The CT value obtained from the detection result was brought into the established standard curve y = -2.92152x + 43.34838 to calculate the copy number of WSMoV in the cDNA of the sample. The detection result is shown in Table 1.

[0103] Table 1 Detection result of cDNA of field collected samples

[0104]

[0105] Note: ND indicates that Ct is greater than 35 and is not detected

[0106] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A primer set for detecting watermelon silver mottle virus, characterized in that: The primer set includes primer pair 1 and / or primer pair 2; Primer pair 1 includes WSMoV-SN-Q5F and WSMoV-SN-Q5R; WSMoV-SN-Q5F includes the sequence shown in SEQ ID NO. 1; WSMoV-SN-Q5R includes the sequence shown in SEQ ID NO. 2; Primer pair 2 includes WSMoV-SN-700F and WSMoV-SN-700R; WSMoV-SN-700F includes the sequence shown in SEQ ID NO.3; WSMoV-SN-700R includes the sequence shown in SEQ ID NO.

4.

2. A kit for detecting watermelon silver mottle virus, characterized in that: Comprising the primer set according to claim 1.

3. The kit according to claim 2, wherein It also includes any one or more of SYBR Green dye method fluorescent quantitative PCR reagents, negative controls, and positive controls.

4. The kit according to claim 3, wherein The positive control was a recombinant plasmid containing the NSs gene fragment of watermelon silver mottle virus.

5. The kit according to claim 3 or 4, characterized in that The negative control was ddH2O without enzyme.

6. A method for detecting watermelon silver mottle virus for non-disease diagnosis purposes, characterized in that: The method comprises the following steps: using the primer set according to claim 1 or the kit according to any one of claims 2 to 5 to perform fluorescent quantitative PCR amplification on the cDNA of the sample to be detected.

7. The detection method according to claim 6, characterized in that: The method for preparing cDNA of the sample to be detected includes the following steps: extracting RNA of the sample to be detected and reverse transcribing it into cDNA.

8. The detection method according to claim 6 or 7, characterized in that: After the reaction is completed, whether the sample to be tested contains watermelon silver mottle virus is judged according to the amplification curve; the judgment method includes: if the amplification curve is a typical fluorescence amplification curve, the sample to be tested contains watermelon silver mottle virus; if the amplification curve does not have a typical fluorescence amplification curve, the sample to be tested does not contain watermelon silver mottle virus.

9. Use of the primer set according to claim 1 in detecting watermelon silver mottle virus for non-diagnostic purposes.

10. Use of the kit according to any one of claims 2 to 5 in detecting watermelon silver mottle virus for non-diagnostic purposes.

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