Cucumber leaf spot virus and cucumber bulgaricus latent virus detection primer group and detection method
By designing a dual RT-PCR detection method, using specific primers to detect cucumber leaf spot virus and cucumber Bulgarian latent virus, the problems of insufficient detection sensitivity and long time in the existing technology are solved, and fast and accurate virus detection is achieved, ensuring the safety of vegetable production.
Patent Information
- Application Number
- CN202510293836.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-13
- Publication Date
- 2025-05-16
AI Technical Summary
Cucumber leaf spot virus and cucumber Bulgarian latent virus have serious impacts on cucumber planting. The existing detection methods have problems such as insufficient sensitivity and long detection time, making it difficult to quickly and accurately detect the existence of these viruses.
A dual RT-PCR detection method was designed, using specific primers to detect cucumber leaf spot virus and cucumber Bulgarian latent virus. Through reverse transcription and PCR amplification technology, specific DNA bands were rapidly amplified to determine whether the sample carries viruses.
The rapid, specific and high sensitivity detection of cucumber leaf spot virus and cucumber Bulgarian latent virus was achieved, with short detection time and reliable results, avoiding the impact of the outbreak of viral diseases on vegetable production.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of molecular biology, and in particular to a cucumber leaf spot virus and cucumber Bulgarian latent virus detection primer set and a detection method. Background Art
[0002] cucumber( Cucumis sativus L.) has a large planting area in my country. According to statistics, by the end of 2021, my country's cultivation area has reached more than 19 million mu, with a yield of 75.6 million tons, accounting for 80% of the world's total cucumber production. With global climate change and the adjustment of agricultural planting structure, cucumber virus diseases have become more serious year by year in various cucumber producing areas. At present, more than 700 plant viruses have been found worldwide, harming 147 crops, and more than 80 species have been found on vegetables alone. In recent years, viruses such as Watermelon silver mottle virus (WSMoV), Cucurbit chlorotic yellows virus (CCYV), Melon necrotic spot virus (MNSV), and Cucumber green mottle mosaic virus (CGMMV) have been reported to cause damage to cucumbers in China, causing serious impacts and economic losses.
[0003] In 2024, during the investigation of cucumber virus disease in Wuqing, Beichen and other areas of Tianjin, it was found that cucumber leaf deformity, wrinkling, chlorosis, yellowing, mosaic and other virus disease symptoms were serious. Based on this, the types of virus sources of cucumber virus disease and their combined infection were tested. It was found that the combined infection of cucumber virus disease was serious, and the test found that cucumbers were infected by Cucumber leaf spot virus (CLSV) and Cucumber bulgariaenselatent virus (CBLV) (see Figure 1 ).
[0004] Cucumber leaf spot virus (CLSV) belongs to Tombusviridae Science AureusvirusThe virus is of the genus Cucumber leaf spot virus, which causes local and systemic infections in plants, sometimes resulting in local necrotic lesions on leaves. Cucumber leaf spot virus is usually detected using a virus-specific double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). The presence of the virus can be detected by collecting leaf samples and performing an enzyme-linked assay. When plant sap is used to mechanically inoculate healthy plants, lesions, sometimes with necrotic centers, are observed on the leaves within 20 to 25 days after inoculation. Cucumber leaf spot virus disease has been reported in countries and regions such as Iran and Poland. For example, the presence of cucumber leaf spot virus has been detected in cucumbers, melons and zucchini in Iran. In addition, the virus has also been found in greenhouse cucumbers in Poland, causing symptoms such as stunting and delayed flowering in cucumber plants.
[0005] Cucumber bulgariaense latent virus (CBLV) belongs to Tombusviridae Science Tombusvirus CBLV was first reported in Bulgaria in 2003 and was identified as Tombusvirus In 2015, CBLV was found again in Iran. In 2020, CBLV was detected in Taiwan, China. Currently, all these CBLV isolates are from cucumbers. At the same time, CBLV was found to be co-infected with Cucurbitaceae Chlorotic Virus (CCYV) and Watermelon Silver Mottle Virus (WSMoV).
[0006] Vegetables are easily infected by pests and diseases, which pose a serious threat to production. Viral diseases have latent infection phenomena. Once symptoms appear, they are difficult to prevent and control, resulting in outbreaks of viral diseases that have a serious impact on vegetable production. Viruses can be transmitted through seeds, juice, virus vectors, soil, and diseased residues. Therefore, it is crucial to identify whether plants, seeds, virus vectors, soil, and diseased residues carry viruses at an early stage. Based on this, we have developed a primer set and detection method for cucumber leaf spot virus and cucumber Bulgarian latent virus. The detection primer set and detection method are simple to determine the results, easy to operate, and have the characteristics of rapidity, specificity, and high sensitivity, and have broad application prospects. Summary of the invention
[0007] The purpose of the present invention is to provide a cucumber leaf spot virus and cucumber Bulgaria latent virus detection primer set and detection method. By designing specific primers of cucumber leaf spot virus and cucumber Bulgaria latent virus genes, a double RT-PCR detection method of cucumber leaf spot virus and cucumber Bulgaria latent virus is established, providing technical support for conveniently, quickly and effectively detecting whether a sample to be tested carries cucumber leaf spot virus and cucumber Bulgaria latent virus.
[0008] To achieve the above object, the present invention adopts the following technical solution: The present invention first provides a dual RT-PCR detection primer set for cucumber leaf spot virus and cucumber Bulgarian latent virus, characterized in that: the detection primer set is a CLSV-Pri-F / R primer pair and a CBLV-Pri-F3904 / R4573 primer pair: CLSV-Pri-F / R Primer Pair: Forward primer sequence CLSV-Pri-F: 5′-ATAAGACAACCAAAGCCATACTACC-3′, SEQ ID NO. 1; Reverse primer sequence CLSV-Pri-R: 5′-CATAACAGACTTCACTCTGGACAAC-3′, SEQ ID NO. 2; CBLV-Pri-F3904 / R4573 Primer Pair: Forward primer sequence CBLV-Pri-F3904: 5′-ATCCCCTTGGTTTCAAGGAAA -3′, SEQ ID NO. 3; Reverse primer sequence CBLV-Pri-R4573: 5'-CTGCCTTTCGGCAATGTTCCGG-3', SEQ ID NO.4.
[0009] At the same time, the present invention also stipulates a dual RT-PCR detection primer genome, the molar concentration ratio of the CLSV-Pri-F / R primer pair and the CBLV-Pri-F3904 / R4573 primer pair is 4: (4~6) or 5: (4~6) or 6: (4~6), respectively, and proposes a detection kit for detecting cucumber leaf spot virus and cucumber Bulgarian latent virus, comprising any of the dual RT-PCR detection primer sets.
[0010] Secondly, the present invention also provides a dual RT-PCR detection method for cucumber leaf spot virus and cucumber Bulgarian latent virus: first, the total RNA of the sample to be tested is extracted and converted into cDNA by reverse transcription, and then the reverse transcribed cDNA is used as a template and the detection primer set described in claim 1 is used as a primer for dual RT-PCR amplification, the amplification product is detected by gel electrophoresis, and the electrophoresis result is judged to determine whether the sample to be tested carries cucumber leaf spot virus and cucumber Bulgarian latent virus.
[0011] The double RT-PCR detection method for cucumber leaf spot virus and cucumber Bulgarian latent virus specifically comprises the following steps: S1. Extract total RNA from the sample to be tested; S2. Reversely transcribing the total RNA of the sample to be tested extracted in step S1 into cDNA; S3. Using the cDNA obtained in step S2 as a template, perform double RT-PCR amplification using the double RT-PCR detection primer set described in any one of claims 1 to 4, and detect the amplified products by gel electrophoresis.
[0012] S4. The results are judged according to the DNA map detected by gel electrophoresis. If a specific DNA band of 333bp is amplified, it means that the sample to be tested carries cucumber leaf spot virus. If a specific DNA band of 669bp is amplified, it means that the sample to be tested carries cucumber Bulgarian latent virus. If no specific DNA band is amplified, it means that the sample to be tested does not carry cucumber leaf spot virus and cucumber Bulgarian latent virus.
[0013] The total volume of the double RT-PCR reaction system contains the following components: 1 to 3 parts cDNA template, 12.5 parts TransTaq Mix, 0.8 to 1.2 parts 10 μmoL / LCLSV-Pri-F, 0.8 to 1.2 parts 10 μmoL / LCBLV-Pri-F3904, 0.8 to 1.2 parts 10 μmoL / LCLSV-Pri-R, 0.8 to 1.2 parts 10 μmoL / LCBLV-Pri-R4573, and 4.5 to 8.3 parts ddH2O.
[0014] The reaction procedure of double RT-PCR was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 56°C-58°C for 30 sec, extension at 72°C for 45 sec, 30-35 cycles; extension at 72°C for 10 min.
[0015] Finally, the test samples applicable to dual RT-PCR were specified, including but not limited to seeds of cucumber and other melon crops, all parts of the plant such as leaves and stalks of cucumber and other melon crops, virus vectors such as aphids, whiteflies and thrips, and samples obtained from soil and diseased residues.
[0016] The present invention further discloses a dual RT-PCR detection method for cucumber leaf spot virus and cucumber Bulgarian latent virus, which is used for rapid and effective detection of whether a sample to be tested carries cucumber leaf spot virus and cucumber Bulgarian latent virus. Experimental results show that the detection method has negative amplification results for five common melon viruses, including cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), cucumber green mottle virus (CGMMV), melon necrotic spot virus (MNSV), and melon chlorotic yellows virus (CCYV), and has good specificity and sensitivity. Compared with the prior art, the cucumber leaf spot virus and cucumber Bulgarian latent virus detection primer set and detection method disclosed in the present invention have the following beneficial effects: The cucumber leaf spot virus and cucumber Bulgarian latent virus detection primer genome and detection method provided by the present invention have the advantages of high sensitivity, short detection time, reliable detection results, easy judgment, and good repeatability compared with electron microscopy virus detection and enzyme-linked immunosorbent assay virus detection methods, as follows: 1. Detection of viruses using an electron microscope. This method is the most classic, effective, and reliable, but it is difficult to separate virus particles and requires expensive equipment, which is not conducive to rapid detection in ordinary laboratories.
[0017] 2. Enzyme-linked immunosorbent assay (ELISA) is used to detect viruses. Although this method has the advantages of high sensitivity and good specificity, when the concentration of the extracted virus is low, the specificity is relatively poor, and there are certain limitations in its application.
[0018] 3. The dual RT-PCR detection method established with the genome of cucumber leaf spot virus and cucumber Bulgarian latent virus provided by the present invention has the advantages of short detection time, high sensitivity, reliable detection results, easy judgment, good repeatability, etc. Genes are the material basis of heredity and are not easily affected by external factors. At the same time, the obtained detection spectrum has a specific band: 333bp or 669bp, and its dominant marker judgment is clear at a glance.
[0019] The present invention can detect, but is not limited to, seeds of melon crops such as cucumbers, leaves of melon crops such as cucumbers, all parts of plants such as melon strips, virus vectors such as aphids, whiteflies and thrips, soil and diseased and residual samples to be tested, and can determine whether they carry cucumber leaf spot virus and cucumber Bulgarian latent virus through detection. Healthy seedlings and cultivation environment are the basis of production, and the quality of seedlings and the like is related to the success or failure of planting. The present invention detects cucumber leaf spot virus and cucumber Bulgarian latent virus, which can avoid the use of seedlings carrying cucumber leaf spot virus and cucumber Bulgarian latent virus in production, and ensure planting safety; the detection technology has great application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 Cucumber plants are infected with the virus, showing symptoms such as mottled leaves, yellowing, and partial deformity; Figure 2 Cucumber plants are infected with viruses, showing symptoms such as deformed cucumbers and yellowing, which affects the quality; Figure 3 The electrophoresis patterns of cucumber leaf spot virus and cucumber Bulgaria latent virus detection are shown in Figure 1. Lane 1 is a cucumber leaf spot virus plasmid sample, lane 2 is a cucumber Bulgaria latent virus plasmid sample, lane 3 is a cucumber leaf spot virus positive sample, lane 4 is a cucumber Bulgaria latent virus positive sample, lane 5 is a cucumber leaf spot virus and cucumber Bulgaria latent virus positive sample, and lane 5 is a negative control. M indicates a DNA MARK with a weight of DL2000. DETAILED DESCRIPTION
[0021] In order to make the contents of the present invention easier to understand, the technical scheme of the present invention is further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form and do not represent the scope of rights protection defined by the present invention. The scope of rights protection of the present invention is subject to the claims. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
[0022] Unless otherwise specified, the reagents and materials used in the following examples are commercially available. Gene Touch BIOER, a universal PCR instrument, was purchased from Hangzhou Bioray Technology Co., Ltd.; the sample to be tested was extracted using a Quick RNA Isolation Kit huayueyang, purchased from Beijing Huayueyang Biotechnology Co., Ltd.; chloroform was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; and the reverse transcription kit (PerfectStart® Uni RT&qPCRKit) was purchased from Beijing Quanshijin Biotechnology Co., Ltd.
[0023] Example 1: Design of Molecular Detection Primers for Cucumber Leaf Spot Virus and Cucumber Bulgarian Latent Virus Collect cucumber virus disease leaf samples (the overall symptoms are leaf deformity, wrinkling, chlorosis, yellowing, mosaic, etc.), pre-treat the collected cucumber virus disease leaves, perform tissue crushing and homogenization, remove tissue and cell fragments by low-speed centrifugation, and purify and concentrate the virus particles in the solution by ultracentrifugation after membrane filtration. Extract RNA from the purified and concentrated samples, and reverse transcribe RNA to obtain cDNA. Send the samples to Guangdong Meige Gene Technology Co., Ltd. for library construction and high-throughput sequencing. The qualified cDNA is randomly interrupted by an ultrasonic disruptor, and the short fragments obtained after interruption are used for sequencing library construction. The library that passes the quality inspection is sequenced using the Illumina platform or the BGISeq platform, and the sequencing read length is PE150.
[0024] Fastp software was used to perform quality control on the data, remove some sequences that would affect subsequent assembly, and obtain high-quality clean reads. Clean reads were aligned with ribosome and host genomes using BWA software and other software to remove ribosome and host sequences and avoid the influence of host and ribosome on subsequent analysis. Megahit software was used to assemble clean reads after removing contamination to obtain longer contigs sequences, and BWA software was used to align clean reads with assembly results and calculate reads utilization.
[0025] CheckV software was used to annotate the assembled contigs, identify the genes of microorganisms and viruses, identify potential viral sequences through sequence alignment and other methods, predict their complete viral structures, and evaluate the quality of the sequences. RNA viral sequences were annotated, viral abundance was counted, genes were predicted, UniProt functional annotation and KEGG functional annotation were performed to obtain the functional information of the virus.
[0026] According to the analysis of the genome data of cucumber leaf spot virus and cucumber Bulgaria latent virus, Primer5 was used to design RNA virus-specific primers for the obtained sequences, and the detection primer set of cucumber leaf spot virus and cucumber Bulgaria latent virus was obtained. The detection primer set is characterized by specific amplification sites for cucumber leaf spot virus and cucumber Bulgaria latent virus genes, and the obtained fragment sizes are 333bp and 669bp, while the amplification results of five common melon viruses, including cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), cucumber green mottle virus (CGMMV), melon necrotic spot virus (MNSV), and melon chlorotic yellows virus (CCYV), are all negative, with good specificity and sensitivity. The detection primer genomes of cucumber leaf spot virus and cucumber Bulgaria latent virus designed are CLSV-Pri-F / R primer pair and CBLV-Pri-F3904 / R4573 primer pair.
[0027] CLSV-Pri-F / R Primer Pair: Forward primer sequence CLSV-Pri-F: 5'-ATAAGACAACCAAAGCCATACTACC-3'; SEQ ID NO.1 Reverse primer sequence CLSV-Pri-R: 5'-CATAACAGACTTCACTCTGGACAAC-3'; SEQ ID NO.2 CBLV-Pri-F3904 / R4573 Primer Pair: Forward primer sequence CBLV-Pri-F3904: 5′-ATCCCCTTGGTTTCAAGGAAA -3′, SEQ ID NO. 3; Reverse primer sequence CBLV-Pri-R4573: 5′-CTGCCTTTCGGCAATGTTCCGG-3′, SEQ ID NO.4.
[0028] Example 2: Detection of cucumber disease tissue samples (1) Extraction of total RNA and acquisition of cDNA from cucumber disease tissue samples Diseased cucumber leaves and cucumber strips were collected from the field. 0.05-0.15 g of diseased tissue was taken and total RNA was extracted using a plant RNA extraction kit. The specific operation steps were based on the product instructions. The extracted RNA was reverse transcribed using a reverse transcription kit (Beijing Quanshijin Biotechnology Co., Ltd.) to obtain cDNA.
[0029] (2) Detection of cucumber disease tissue samples PCR amplification reaction system: cDNA template 1μL, Trans Taq Mix 12.5μL, 10μmoL / LCLSV-Pri-F 1μL, 10μmoL / LCBLV-Pri-F3904 1μL, 10μmoL / LCLSV-Pri-R 1μL, 10 μmoL / LCBLV-Pri-R45731μL, ddH2O 7.5μL.
[0030] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec, extension at 72°C for 45 sec, 30 cycles; and extension at 72°C for 10 min.
[0031] (3) Electrophoresis detection of PCR products Take 2 μL of PCR product and spot it on 1% agarose gel. Use DL2000 DNA MARK as a reference for fragment size. Perform electrophoresis at 110V for 20 min to 30 min in 1×TAE buffer. After the electrophoresis is completed, take a photo on a gel imager. Figure 3 As shown, lane 1 is a cucumber leaf spot virus plasmid sample, lane 2 is a cucumber Bulgaria latent virus plasmid sample, lane 3 is a cucumber leaf spot virus positive sample, lane 4 is a cucumber Bulgaria latent virus positive sample, lane 5 is a cucumber leaf spot virus and cucumber Bulgaria latent virus positive sample, and lane 5 is a negative control. M represents a DNA MARK with a weight of DL2000.
[0032] Example 3: Detection of cucumber seed samples (1) Extraction of total RNA and acquisition of cDNA from cucumber seed samples Cucumber seed samples 1, 2, 3, and 4 were collected, and 0.05-0.15 g of seeds were crushed and total RNA was extracted using a plant RNA extraction kit. The specific operation steps were based on the product instructions. The extracted RNA was reverse transcribed using a reverse transcription kit (Beijing Quanshijin Biotechnology Co., Ltd.) to obtain cDNA.
[0033] (2) Testing of cucumber seed samples PCR amplification reaction system: cDNA template 3μL, Trans Taq Mix 12.5μL, 10μmoL / LCLSV-Pri-F 1μL, 10μmoL / LCBLV-Pri-F3904 1μL, 10μmoL / LCLSV-Pri-R 1μL, 10 μmoL / LCBLV-Pri-R45731μL, ddH2O 5.5μL.
[0034] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 57°C for 30 sec, extension at 72°C for 45 sec, 35 cycles; and extension at 72°C for 10 min.
[0035] (3) Electrophoresis detection of PCR products Take 4 μL of PCR product and spot it on 1% agarose gel. Use DL2000 DNA MARK as a reference for fragment size. Perform electrophoresis at 110 V for 20 min to 30 min in 1×TAE buffer. After electrophoresis, read the result with a gel imager. It was found that cucumber seed samples 1, 2, 3, and 4 did not carry cucumber leaf spot virus and cucumber Bulgarian latent virus.
[0036] Example 4: Detection of virus vector samples (1) Extraction of total RNA and cDNA from virus vector samples Aphid, whitefly and thrips samples were collected, and 0.05-0.15 g of each aphid, whitefly and thrips were crushed and total RNA was extracted using an RNA extraction kit. The specific operation steps were based on the product instructions. The extracted RNA was reverse transcribed using a reverse transcription kit (Beijing Quanshijin Biotechnology Co., Ltd.) to obtain cDNA.
[0037] (2) Detection of virus vector samples PCR amplification reaction system: cDNA template 3μL, Trans Taq Mix 12.5μL, 10μmoL / LCLSV-Pri-F1.2μL, 10μmoL / LCBLV-Pri-F3904 1.2μL, 10μmoL / LCLSV-Pri-R 1.2μL, 10 μmoL / LCBLV-Pri-R4573 1.2μL, ddH2O 4.7μL.
[0038] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 45 sec, 35 cycles; and extension at 72°C for 10 min.
[0039] (3) Electrophoresis detection of PCR products Take 4 μL of PCR product and spot it on 1% agarose gel. Use DL2000 DNA MARK as a reference for fragment size. Perform electrophoresis at 110 V for 20 min to 30 min in 1×TAE buffer. After electrophoresis, read the results with a gel imager. The results showed that aphid, whitefly and thrips samples did not carry cucumber leaf spot virus and cucumber Bulgarian latent virus.
[0040] Example 5: Detection of soil and diseased body samples (1) Total RNA extraction and cDNA acquisition from soil and diseased body samples Soil and diseased body samples 1, 2, and 3 were collected. 0.05-0.15 g of soil and diseased body were crushed and total RNA was extracted using an RNA extraction kit. The specific operation steps were based on the product instructions. The extracted RNA was reverse transcribed using a reverse transcription kit (Beijing Quanshijin Biotechnology Co., Ltd.) to obtain cDNA.
[0041] (2) Detection of virus vector samples PCR amplification reaction system: cDNA template 3μL, Trans Taq Mix 12.5μL, 10μmoL / LCLSV-Pri-F1.2μL, 10μmoL / LCBLV-Pri-F3904 1.2μL, 10μmoL / LCLSV-Pri-R 1.2μL, 10 μmoL / LCBLV-Pri-R4575 1.2μL, ddH2O 4.7μL.
[0042] The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 45 sec, 35 cycles; and extension at 72°C for 10 min.
[0043] (3) Electrophoresis detection of PCR products 4 μL of the PCR product was taken and spotted on a 1% agarose gel. The DNA MARK of DL2000 was used as a reference for the fragment size. Electrophoresis was performed in 1×TAE buffer at 110 V for 20 min to 30 min. After the electrophoresis, the results were read using a gel imager. The results showed that soil and diseased body samples 1 and 2 did not carry cucumber leaf spot virus and cucumber Bulgarian latent virus. Soil and diseased body sample 3 carried cucumber Bulgarian latent virus but did not carry cucumber leaf spot virus.
Claims
1. A double RT-PCR detection primer genome for cucumber leaf spot virus and cucumber Bulgarian latent virus, characterized in that: The detection primer group is a CLSV-Pri-F / R primer pair and a CBLV-Pri-F3904 / R4573 primer pair: CLSV-Pri-F / R Primer Pair: Forward primer sequence CLSV-Pri-F: 5′-ATAAGACAACCAAAGCCATACTACC-3′, SEQ ID NO. 1; Reverse primer sequence CLSV-Pri-R: 5′-CATAACAGACTTCACTCTGGACAAC-3′, SEQ ID NO. 2; CBLV-Pri-F3904 / R4573 Primer Pair: Forward primer sequence CBLV-Pri-F3904: 5′-ATCCCCTTGGTTTCAAGGAAA -3′, SEQ ID NO. 3; Reverse primer sequence CBLV-Pri-R4573: 5′-CTGCCTTTCGGCAATGTTCCGG-3′, SEQ ID NO.
4.
2. The dual RT-PCR detection primer genome for cucumber leaf spot virus and cucumber Bulgarian latent virus according to claim 1, characterized in that The molar concentration ratio of the CLSV-Pri-F / R primer pair and CBLV-Pri-F3904 / R4573 is 4:(4~6).
3. The dual RT-PCR detection primer genome for cucumber leaf spot virus and cucumber Bulgarian latent virus according to claim 1, characterized in that The molar concentration ratio of the CLSV-Pri-F / R primer pair and CBLV-Pri-F3904 / R4573 is 5:(4~6).
4. The dual RT-PCR detection primer genome for cucumber leaf spot virus and cucumber Bulgarian latent virus according to claim 1, characterized in that The molar concentration ratio of the CLSV-Pri-F / R primer pair and CBLV-Pri-F3904 / R4573 is 6:(4~6).
5. A detection kit for detecting cucumber leaf spot virus and cucumber Bulgarian latent virus, characterized in that: Contains the dual RT-PCR detection primer genome described in any one of claims 1 to 4.
6. A dual RT-PCR detection method for cucumber leaf spot virus and cucumber Bulgarian latent virus, characterized in that: First, the total RNA of the sample to be tested is extracted and converted into cDNA by reverse transcription. Then, the reverse transcribed cDNA is used as a template and the detection primer genome described in claim 1 is used as a primer for double RT-PCR amplification. The amplification product is detected by gel electrophoresis, and the electrophoresis result is judged to determine whether the sample to be tested carries cucumber leaf spot virus and cucumber Bulgarian latent virus.
7. The dual RT-PCR detection method for cucumber leaf spot virus and cucumber Bulgarian latent virus according to claim 6, characterized in that: The following steps are involved: S1. Extract total RNA from the sample to be tested; S2. Reversely transcribing the total RNA of the sample to be tested extracted in step S1 into cDNA; S3. Using the cDNA obtained in step S2 as a template, performing dual RT-PCR amplification using the dual RT-PCR detection primer set according to any one of claims 1 to 4, and detecting the amplified product by gel electrophoresis; S4. The results are determined based on the DNA map detected by gel electrophoresis. If a specific DNA band of 333 bp is amplified, it means that the sample to be tested carries cucumber leaf spot virus. If a specific DNA band of 669 bp is amplified, it means that the sample to be tested carries cucumber Bulgarian latent virus. If no specific DNA band is amplified, it means that the sample to be tested does not carry cucumber leaf spot virus and cucumber Bulgarian latent virus. The total volume of the double RT-PCR reaction system in step S3 contains the following components: 1 to 3 parts of cDNA template, 12.5 parts of Trans Taq Mix, 0.8 to 1.2 parts of 10 μmoL / LCLSV-Pri-F, 0.8 to 1.2 parts of 10 μmoL / LCBLV-Pri-F3904, 0.8 to 1.2 parts of 10 μmoL / LCLSV-Pri-R, 0.8 to 1.2 parts of 10 μmoL / LCBLV-Pri-R4573, and 7.5 to 9 parts of ddH2O.
8. The dual RT-PCR detection method for cucumber leaf spot virus and cucumber Bulgarian latent virus according to claim 7, characterized in that: The reaction procedure of the double RT-PCR in step S3 is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 56°C-58°C for 30 sec, extension at 72°C for 45 sec, 30-35 cycles; extension at 72°C for 10 min.
9. The dual RT-PCR detection method for cucumber leaf spot virus and cucumber Bulgarian latent virus according to claims 7 to 8, wherein the samples to be tested include but are not limited to seeds of cucumber and other melon crops, all parts of the plant such as leaves and stalks of cucumber and other melon crops, virus vectors such as aphids, whiteflies and thrips, and samples obtained from soil and diseased residues.
10. Use of the dual RT-PCR detection method for cucumber leaf spot virus and cucumber Bulgarian latent virus according to claim 6 for quickly and effectively detecting whether a sample to be tested carries cucumber leaf spot virus and cucumber Bulgarian latent virus.