Detection primer group of beta rhabdovirus new virus infecting Chinese fevervine herb and RT-PCR detection method

Abstract

The invention discloses a detection primer group of a beta rhabdovirus new virus infecting Chinese fevervine herb and a reverse transcription-polymerase chain reaction (RT-PCR) detection method. According to nucleotide sequences of an ORF2 gene and an RNA-dependent RNA polymerase (RdRp) gene of the new beta rhabdovirus infecting Chinese fevervine herb, two specific primer pairs for detecting the new beta rhabdovirus are respectively designed, and an RT-PCR method for detecting the new beta rhabdovirus is established by utilizing the designed specific primer pairs. According to the detection primer group of the new beta rhabdovirus and the RT-PCR detection method provided by the invention, a rapid, sensitive, specific, accurate, efficient and low-cost molecular detection method is provided for the detection of the virus in plants, so that a detection method is provided for the diagnosis, prevention and control of the virus disease.

Description

Technical Field

[0001] The invention relates to a plant virus detection method in the field of molecular biology, and in particular to a detection primer set and an RT-PCR detection method for a new virus of the genus Beta Rhabdovirus infecting Croton rotundus. Background Art

[0002] Plant virus diseases are serious and difficult to prevent and control. The use of virus-free seeds and seedlings is currently an effective prevention and control measure. Once infected with the virus, it cannot be treated with effective chemical agents or other interventions, so early detection becomes a prerequisite for the prevention and control of viral diseases, which requires the establishment of rapid, efficient and sensitive virus detection methods. At present, the detection and identification technologies of plant viruses include indicator plant biology, electron microscopy, serological detection, and molecular biology detection. However, the first three detection methods have certain limitations and shortcomings. The indicator plant detection method has a long identification cycle and is greatly restricted by the host plant and the environment; the electron microscopy technology has high professional requirements and the electron microscopy equipment is precise and expensive, and is not suitable for large-scale field sample detection; although the serological detection technology has high sensitivity, it requires high-quality and highly specific antibodies and is prone to false negative results. Molecular biological detection technologies for viruses include RT-PCR / PCR, RT-qPCR / qPCR, RT-LAMP / LAMP, gene chips, high-throughput sequencing technology, CRISPR-Cas9, etc. Among them, RT-PCR technology is widely used in the detection of RNA viruses. It has the advantages of low cost, high sensitivity, good specificity, easy operation and accurate detection. It provides strong technical support for the detection and scientific prevention and control of plant viral diseases.

[0003] In recent years, more and more new viruses have been identified using high-throughput sequencing technology combined with bioinformatics technology. Paederia scandens (Lour.) Merr. is a perennial herbaceous vine of the Rubiaceae family. Paederia scandens is a traditional Chinese medicinal plant and a flower landscape plant. The whole plant of Paederia scandens is used as medicine, which has the effects of dispelling wind, activating blood circulation, relieving pain and detoxifying. In Hainan, Guangdong and Guangxi of my country, there is a tradition of eating Paederia scandens. After processing, it has a fragrant taste and endless aftertaste, but the emergence of viral diseases affects its economic value and ornamental value. The patent applicant used high-throughput RNA-seq sequencing technology combined with bioinformatics, RT-PCR, Rapidapidaplification of cDNAend (RACE), nucleic acid sequencing and sequence blast comparison to identify a new RNA virus from Paederia scandens plants with yellow and mottled leaves. It is temporarily named a new virus of the genus Beta rhabdovirus, which belongs to the genus Betanucleorhabdovirus of the family Rhabdoviridae. After full-length amplification and sequencing, the new virus genome is a monopartite genome, a single-molecule linear negative-sense ssRNA, and the sequence is shown in SEQ ID NO.7. The total length of the virus genome is 13735bp. By comparing the open reading frame (ORF) encoded by the virus in the Protein Blast module of the NCBI website, using CDD software to predict the viral protein domain, and combining the description in the International Committee on Taxonomy of Viruses (ICTV), it is predicted that the virus genome encodes a total of 6 ORFs, ORF1 encodes a protein of 52.4kDa, ORF2 encodes a protein of 36.8kDa, ORF3 encodes a protein of 37.1kDa, ORF4 encodes a protein of 38.2kDa, ORF5 encodes a protein of 71kDa, and ORF6 encodes an RNA-dependent RNA polymerase (RdRp) of 241.8kDa.

[0004] The new virus of the genus Beta Rhabdovirus seriously affects the growth of the plant of the genus Polypodium vulgare, and there is no report on the detection method and detection primers. The present invention designs two pairs of specific primers for detecting the virus according to the nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) gene and the ORF2 gene of the new virus of the genus Beta Rhabdovirus, and establishes an RT-PCR method for detecting the new virus of the genus Beta Rhabdovirus using the designed specific primers. After Blastn comparison, the target fragments amplified by the two pairs of primers are all specific fragments of the new virus of the genus Beta Rhabdovirus, and have no similarity with the plant genes in the NCBI database. The detection primer set and RT-PCR detection method of the new virus of the genus Beta Rhabdovirus of the present invention provide a rapid, sensitive, specific, accurate, efficient and low-cost molecular detection method for the detection of the virus in plants, thereby providing a detection method for the diagnosis and prevention and control of the virus. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and to solve the technical problem of providing a detection primer set and an RT-PCR detection method for a new virus of the genus Betarhabdovirus that infects Croton rotundus.

[0006] Specifically, the present invention is achieved through the following technical solutions:

[0007] A detection primer set for a new virus of the genus Beta Rhabdovirus infecting Ternifolia scabra, the primer set comprising at least one pair of primers for amplifying the RdRp gene and ORF2 gene fragments of the new virus of the genus Beta Rhabdovirus; wherein: in the primer pair for amplifying the RdRp gene fragment of the new virus of the genus Beta Rhabdovirus, the sequence of the forward primer RdRp-F is shown as SEQ ID NO.1, and the sequence of the reverse primer RdRp-R is shown as SEQ ID NO.2; in the primer pair for amplifying the ORF2 gene fragment of the new virus of the genus Beta Rhabdovirus, the sequence of the forward primer ORF2-F is shown as SEQ ID NO.3, and the sequence of the reverse primer ORF2-R is shown as SEQ ID NO.4.

[0008] Preferably, the primer pair used to amplify the RdRp gene fragment of a new virus of the genus Beta Rhabdovirus can amplify a 486bp RdRp gene fragment, the sequence of which is shown in SEQ ID NO.5; the primer pair used to amplify the ORF2 gene fragment of a new virus of the genus Beta Rhabdovirus can amplify a 481bp ORF2 gene fragment, the sequence of which is shown in SEQ ID NO.6.

[0009] Preferably, the genome of the new virus of the genus Betarhabdovirus that infects Croton rotundus is a monopartite RNA genome with a total length of 13735 bp, and the sequence is shown in SEQ ID NO.7.

[0010] The present invention also provides an RT-PCR detection method for a new virus of the genus Beta Rhabdovirus that infects the vine of Croton foetida. The method comprises the following steps: designing and synthesizing a primer set as claimed in claim 1 according to the nucleotide sequences of the RdRp gene and the ORF2 gene of the new virus of the genus Beta Rhabdovirus that infects the vine of Croton foetida, then using the primer pair of the RdRp gene or the ORF2 gene fragment of the new virus of the genus Beta Rhabdovirus to perform RT-PCR amplification on the genome sequence of the new virus of the genus Beta Rhabdovirus in the total RNA of the plant, and finally performing 1% agarose gel electrophoresis analysis on the reaction product to determine whether the new virus of the genus Beta Rhabdovirus that infects the vine of Croton foetida is present in the plant sample.

[0011] Preferably, the reaction system, reaction parameters and reaction procedure of the RT-PCR detection method are as follows:

[0012] Step (1): According to sub-steps 1.1) to 1.8), extract plant total RNA using the Trizol method:

[0013] 1.1) Take 100 mg of plant leaves and place them in a 2.0 mL EP centrifuge tube with 3 steel balls. Precool it in liquid nitrogen and grind it into powder using a grinder. Add 1 mL of Trizol in a fume hood, vortex mix, and let it stand at room temperature for 15 min.

[0014] 1.2) In a fume hood, add 200 μL of chloroform to the EP centrifuge tube obtained in 1.1) above, shake vigorously, and let stand at room temperature for 10 min;

[0015] 1.3) Place the EP centrifuge tube obtained in 1.2) in a pre-cooled 4°C centrifuge and centrifuge at 12,000 rpm for 10 min. Use a pipette to transfer the supernatant to a new 1.5 mL RNase-free EP centrifuge tube. Add an equal volume of isopropanol, invert and mix, and let stand at room temperature for 10 min or at -30°C for more than 20 min.

[0016] 1.4) Centrifuge the RNase-free EP centrifuge tube obtained in 1.3) above at 4°C, 12000 rpm for 10 min, discard the supernatant and obtain RNA precipitate;

[0017] 1.5) Add 1 mL of 75% alcohol to the RNase-free EP centrifuge tube obtained in 1.4) to wash the RNA precipitate, suspend the RNA precipitate with a pipette, centrifuge at 4°C, 12000 rpm for 5 min, discard the supernatant and retain the RNA precipitate;

[0018] 1.6) After repeating step 1.5) once, the RNA precipitate retained in the RNase-free EP centrifuge tube was placed at room temperature to dry for 5 to 10 minutes;

[0019] 1.7) Add 30-50 μL DEPC water to dissolve the RNA precipitate dried in step 1.6) to obtain dissolved plant total RNA, and store it at -80°C for later use;

[0020] Step (2): According to sub-steps 2.1) to 2.4), reverse transcription is performed using a reverse transcription kit, and the following reverse transcription reaction system, reaction parameters and reaction procedure are performed:

[0021] 2.1) Place the PCR tube on ice to prepare a reaction mixture, which contains: 2.0 μl 4xDN Master Mix, 0.5 pg to 0.5 μg of the total plant RNA extracted in step (1), and add RNase Free water to make up to 8 μl;

[0022] 2.2) Mix the reaction mixture obtained in 2.1) and incubate at 37°C for 5 minutes to remove genomic DNA;

[0023] 2.3) Add 2 μl of 5×RT Master Mix II to the reaction product obtained in 2.2) to make the total reaction system 10 μl;

[0024] 2.4) After mixing the reaction solution obtained in 2.3), reverse transcription reaction was carried out according to the reaction program of 37°C for 15 min, 50°C for 5 min, 98°C for 5 min, and 4°C for 5 min to finally obtain reverse transcribed cDNA;

[0025] Step (3): According to sub-steps 3.1) to 3.3), perform the following PCR reaction system, parameters and reaction procedures:

[0026] 3.1) PCR reaction was performed using 2×Rapid Taq Master Mix enzyme. The following PCR reaction system was prepared in a PCR tube on ice: 12.5 μl 2×Rapid Taq Master Mix, 0.5 μl of the reverse transcribed cDNA, 10 μl sterile deionized water, 1 μl each of 10 μM RdRp-F and RdRp-R upstream and downstream primers of the new Beta Rhabdovirus genus or 1 μl each of 10 μM ORF2-F and ORF2-R upstream and downstream primers of the new Beta Rhabdovirus genus, and the total volume of the reaction system was 25 μl;

[0027] 3.2) Set the PCR reaction parameters and PCR program as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 15 s, 30-35 cycles; 72°C extension for 5 min; 4°C for 5 min to terminate the reaction;

[0028] 3.3) The above PCR procedure was performed on a PCR instrument, and the PCR products were subjected to 1% agarose gel electrophoresis to analyze whether there was a 486 bp RdRp gene fragment or a 481 bp ORF2 gene fragment of a specific new Beta Rhabdovirus. The presence of the RdRp gene fragment or the ORF2 gene fragment indicated that the tested plant tissue was infected with the new Beta Rhabdovirus.

[0029] Preferably, the reverse transcription kit is ReverTra Ace qPCR RT MasterMix with gDNARemover from Toyobo.

[0030] Preferably, the 2×Rapid Taq Master Mix enzyme is 2×Rapid TaqMaster Mix enzyme from Vazyme.

[0031] Compared with the prior art, the present invention has the following beneficial effects: 1) It is the first time to provide a detection technology for a new virus of the genus Beta Rhabdovirus that infects Croton foetida, with strong technical innovation; 2) Using the detection primer pair of the present invention to perform RT-PCR reaction can quickly and simply, sensitively and specifically, accurately and efficiently, and at low cost detect and diagnose the new virus of the genus Beta Rhabdovirus in field plants, with obvious advantages; 3) The instrument used is mainly a cheap common nucleic acid amplification instrument (PCR instrument), rather than an expensive real-time quantitative fluorescence PCR instrument of the RT-qPCR method. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Results of RT-PCR detection of new Beta Rhabdovirus in plants. Lane M is a DNA molecular weight standard; Lanes 1 and 2 are the results of RT-PCR detection of plants infected with new Beta Rhabdovirus and healthy controls using the RdRp gene fragment primer pair; Lanes 4 and 3 are the results of RT-PCR detection of plants infected with new Beta Rhabdovirus and healthy controls using the ORF2 gene fragment primer pair. DETAILED DESCRIPTION

[0033] The present invention is further described and illustrated below in conjunction with the accompanying drawings and specific embodiments.

[0034] The present invention provides a detection primer set for a new virus of the genus Beta Rhabdovirus that infects the genus Patta Rhabdovirus, wherein the new virus of the genus Beta Rhabdovirus that infects the genus Patta Rhabdovirus is a monopartite RNA genome with a total length of 13735 bp, and the sequence is shown in SEQ ID NO. 7. The detection primer set for detecting the virus includes at least one of two pairs of primers for amplifying the RdRP gene and ORF2 gene fragments of the new virus of the genus Beta Rhabdovirus; in the primer pair for amplifying the RdRp gene fragment of the new virus of the genus Beta Rhabdovirus, the sequence of the forward primer RdRp-F is shown in SEQ ID NO. 1, and the sequence of the reverse primer RdRp-R is shown in SEQ ID NO. 2; in the primer pair for amplifying the ORF2 gene fragment of the new virus of the genus Beta Rhabdovirus, the sequence of the forward primer ORF2-F is shown in SEQ ID NO. 3, and the sequence of the reverse primer ORF2-R is shown in SEQ ID NO. 4.

[0035] In the actual detection process, the primer pair shown in SEQ ID NO.1 and SEQ ID NO.2, and the primer pair shown in SEQ ID NO.3 and SEQ ID NO.4 can be used selectively.

[0036] The primer pair used to amplify the RdRp gene fragment of the new virus of the genus Beta Rhabdovirus can amplify a 486 bp RdRp gene fragment, the sequence of which is shown in SEQ ID NO. 5. The primer pair used to amplify the ORF2 gene fragment of the new virus of the genus Beta Rhabdovirus can amplify a 481 bp ORF2 gene fragment, the sequence of which is shown in SEQ ID NO. 6.

[0037] The present invention also provides an RT-PCR detection method for a new virus of the genus Beta Rhabdovirus that infects Croton rotundus. The primer set (SEQ ID NOs. 1 to 4) as described above is designed and synthesized according to the nucleotide sequences of the RdRp gene and the ORF2 gene of the new virus of the genus Beta Rhabdovirus that infects Croton rotundus. Then, the genomic sequence of the new virus of the genus Beta Rhabdovirus in the total RNA of the plant is amplified by RT-PCR using the primer pair of the RdRp gene or the ORF2 gene fragment of the new virus of the genus Beta Rhabdovirus. Finally, the reaction product is subjected to 1% agarose gel electrophoresis analysis to determine whether the new virus of the genus Beta Rhabdovirus that infects Croton rotundus is present in the plant sample.

[0038] The present invention will be further explained and illustrated below in conjunction with the embodiments and drawings, but the following embodiments do not limit the scope of the present invention.

[0039] Example 1: Design and synthesis of a primer set for detecting a new virus of the genus Betarhabdovirus that infects Croton ovata

[0040] According to the genome sequence of the new virus of the genus Beta Rhabdovirus infecting the vine of dung as shown in SEQ ID NO.7, the Primer-BLAST of the BLAST module in the NCBI website was used to select appropriate parameters, and finally two pairs of detection primer pairs with high specificity were selected. The two pairs of detection primers are sequences on the RdRp gene fragment and ORF2 gene fragment of the new virus of the genus Beta Rhabdovirus, and the nucleotide sequences of the primers are shown in SEQ ID NO.1-4. Specifically, a pair of forward and reverse primer pairs designed based on the RdRp gene sequence of the new virus of the genus Beta Rhabdovirus infecting the vine of dung are respectively recorded as RdRp-F and RdRp-R, and the sequences are SEQ ID NO.1 and SEQ ID NO.2, respectively. A pair of forward and reverse primer pairs designed based on the ORF2 gene sequence of the new virus of the genus Beta Rhabdovirus are respectively recorded as ORF2-F and ORF2-R, and the sequences are SEQ ID NO.3 and SEQ ID NO.4, respectively. The above primers are synthesized by biological companies such as Beijing Qingke Biotechnology Co., Ltd.

[0041] Example 2: RT-PCR Detection Method for New Beta Rhabdovirus Infecting Croton foetida

[0042] The PCR detection method comprises steps (1) to (3), which are respectively as follows:

[0043] Total RNA is extracted from the plant of Croton dahliae, and reverse transcription PCR is performed using the total RNA as a template to obtain cDNA, which is then amplified by PCR using the specific primer pair provided by the present invention. The obtained product is subjected to agarose gel electrophoresis, and then the presence or absence of a specific band is used to determine whether the plant is infected with the new virus of the genus Beta Rhabdovirus.

[0044] Step (1): According to sub-steps 1.1) to 1.8), extract plant total RNA using the Trizol method:

[0045] 1.1) Take 100 mg of plant leaves and place them in a 2.0 mL EP centrifuge tube with 3 steel balls. Precool it in liquid nitrogen and grind it into powder using a grinder. Add 1 mL of Trizol in a fume hood, vortex mix, and let it stand at room temperature for 15 min.

[0046] 1.2) In a fume hood, add 200 μL of chloroform to the EP centrifuge tube obtained in 1.1) above, shake vigorously, and let stand at room temperature for 10 min;

[0047] 1.3) Place the EP centrifuge tube obtained in 1.2) above in a pre-cooled 4°C centrifuge and centrifuge at 12,000 rpm for 10 min. Use a pipette to transfer the supernatant in the EP centrifuge tube to a new 1.5 mL RNase-free EP centrifuge tube. Add an equal volume of isopropanol, invert and mix, and let stand at room temperature for 10 min or at -30°C for more than 20 min.

[0048] 1.4) Centrifuge the RNase-free EP centrifuge tube obtained in 1.3) above at 4°C, 12000 rpm for 10 min, discard the supernatant and obtain RNA precipitate;

[0049] 1.5) Add 1 mL of 75% alcohol to the RNase-free EP centrifuge tube obtained in 1.4) to wash the RNA precipitate, suspend the RNA precipitate with a pipette, centrifuge at 4°C, 12000 rpm for 5 min, discard the supernatant and retain the RNA precipitate;

[0050] 1.6) After repeating step 1.5) once, the RNA precipitate retained in the RNase-free EP centrifuge tube was placed at room temperature to dry for 5 to 10 minutes;

[0051] 1.7) Add 30-50 μL DEPC water to dissolve the RNA precipitate dried in step 1.6) to obtain dissolved plant total RNA, and store it at -80°C for later use;

[0052] Step (2): According to sub-steps 2.1) to 2.4), reverse transcription was performed using Toyobo's ReverTra Ace qPCR RTMaster Mix with gDNARemover reverse transcription kit, and the following reverse transcription reaction system, reaction parameters and reaction procedure were performed:

[0053] 2.1) Place the PCR tube on ice to prepare a reaction mixture, which contains: 2.0 μl 4xDN Master Mix, 0.5 pg to 0.5 μg of the total plant RNA extracted in step (1), and add RNase Free water to make up to 8 μl;

[0054] 2.2) Mix the reaction mixture obtained in 2.1) and incubate at 37°C for 5 minutes to remove genomic DNA;

[0055] 2.3) Add 2 μl of 5×RT Master Mix II to the reaction product obtained in 2.2) to make the total reaction system 10 μl;

[0056] 2.4) After mixing the reaction solution obtained in 2.3), reverse transcription reaction was carried out according to the reaction program of 37°C for 15 min, 50°C for 5 min, 98°C for 5 min, and 4°C for 5 min to finally obtain reverse transcribed cDNA;

[0057] Step (3): According to sub-steps 3.1) to 3.3), perform the following PCR reaction system, parameters and reaction procedures:

[0058] 3.1) PCR reaction was carried out using 2×Rapid Taq Master Mix enzyme from Vazyme. The following PCR reaction system was prepared in a PCR tube on ice: 12.5 μl 2×Rapid Taq Master Mix, 0.5 μl of the reverse transcribed cDNA, 10 μl sterile deionized water, 1 μl each of 10 μM RdRp-F and RdRp-R upstream and downstream primers of the new Beta Rhabdovirus genus or 1 μl each of 10 μM ORF2-F and ORF2-R upstream and downstream primers of the new Beta Rhabdovirus genus, and the total reaction system volume was 25 μl;

[0059] 3.2) Set the PCR reaction parameters and PCR program as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 15 s, 30-35 cycles; 72°C extension for 5 min; 4°C for 5 min to terminate the reaction;

[0060] 3.3) The above PCR procedure was performed on a PCR instrument, and the PCR products were subjected to 1% agarose gel electrophoresis to analyze whether there was a 486 bp RdRp gene fragment or a 481 bp ORF2 gene fragment of a specific new Beta Rhabdovirus. The presence of the RdRp gene fragment or the ORF2 gene fragment indicated that the tested plant tissue was infected with the new Beta Rhabdovirus.

[0061] It should be noted that in the PCR reaction system prepared in 3.1) above, one of the upstream and downstream primers of RdRp-F and RdRp-R, and the upstream and downstream primers of ORF2-F and ORF2-R can be selected for each detection. However, in this embodiment, two upstream and downstream primers were used for detection to determine whether the RdRp gene fragment and the ORF2 gene fragment exist in the plant tissue. In this embodiment, for the plant tissue taken, the detection results are shown in Figure 1 Lane M in the figure is a DNA molecular weight standard; Lanes 1 and 2 are the results of RT-PCR using RdRp gene fragment primer pairs to detect plants infected with new Beta Rhabdovirus and healthy controls; Lanes 4 and 3 are the results of RT-PCR using ORF2 gene fragment primer pairs to detect plants infected with new Beta Rhabdovirus and healthy controls.

[0062] The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. Those skilled in the relevant art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A primer set for detecting a new virus of the genus Beta Rhabdovirus that infects Croton foetida, characterized in that: The primer set includes at least one pair of primers for amplifying the RdRp gene and ORF2 gene fragments of a new virus of the genus Beta Rhabdovirus; wherein: in the primer pair used to amplify the RdRp gene fragment of a new virus of the genus Beta Rhabdovirus, the sequence of the forward primer RdRp-F is shown as SEQ ID NO.1, and the sequence of the reverse primer RdRp-R is shown as SEQ ID NO.2; in the primer pair used to amplify the ORF2 gene fragment of a new virus of the genus Beta Rhabdovirus, the sequence of the forward primer ORF2-F is shown as SEQ ID NO.3, and the sequence of the reverse primer ORF2-R is shown as SEQ ID NO.

4.

2. A primer set for detecting a new virus of the genus Betarhabdovirus that infects Croton foetida as claimed in claim 1, characterized in that: The primer pair used to amplify the RdRp gene fragment of a new virus of the genus Beta Rhabdovirus can amplify a 486bp RdRp gene fragment, the sequence of which is shown in SEQ ID NO.5; the primer pair used to amplify the ORF2 gene fragment of a new virus of the genus Beta Rhabdovirus can amplify a 481bp ORF2 gene fragment, the sequence of which is shown in SEQ ID NO.

6.

3. A primer set for detecting a new virus of the genus Betarhabdovirus that infects Croton foetida as claimed in claim 1, characterized in that: The genome of the new virus of the genus Beta Rhabdovirus that infects Croton rotundus is a monopartite RNA genome with a total length of 13735 bp, and the sequence is shown in SEQ ID NO.

7.

4. A RT-PCR detection method for a new virus of the genus Beta Rhabdovirus that infects Croton foetida, characterized in that: A primer set as claimed in claim 1 is designed and synthesized based on the nucleotide sequences of the RdRp gene and ORF2 gene of a new virus of the genus Beta Rhabdovirus that infects Croton rotundus, and then the genomic sequence of the new virus of the genus Beta Rhabdovirus in the total RNA of the plant is amplified by RT-PCR using primers of the RdRp gene or ORF2 gene fragment of the new virus of the genus Beta Rhabdovirus. Finally, the reaction product is analyzed by 1% agarose gel electrophoresis to determine whether the new virus of the genus Beta Rhabdovirus that infects Croton rotundus is present in the plant sample.

5. The RT-PCR detection method for a new virus of the genus Beta Rhabdovirus infecting Croton foetida according to claim 4, characterized in that: The reaction system, reaction parameters and reaction procedures of the RT-PCR detection method are as follows: Step (1): According to sub-steps 1.1) to 1.8), extract plant total RNA using the Trizol method: 1.1) Take 100 mg of plant leaves and place them in a 2.0 mL EP centrifuge tube with 3 steel balls. Precool it in liquid nitrogen and grind it into powder using a grinder. Add 1 mL of Trizol in a fume hood, vortex mix, and let it stand at room temperature for 15 min. 1.2) In a fume hood, add 200 μL of chloroform to the EP centrifuge tube obtained in 1.1) above, shake and let stand at room temperature for 10 min; 1.3) Place the EP centrifuge tube obtained in 1.2) in a pre-cooled 4°C centrifuge and centrifuge at 12,000 rpm for 10 min. Use a pipette to transfer the supernatant to a new 1.5 mL RNase-free EP centrifuge tube. Add an equal volume of isopropanol, invert and mix, and let stand at room temperature for 10 min or at -30°C for more than 20 min. 1.4) Centrifuge the RNase-free EP centrifuge tube obtained in 1.3) above at 4°C, 12000 rpm for 10 min, discard the supernatant and obtain RNA precipitate; 1.5) Add 1 mL of 75% alcohol to the RNase-free EP centrifuge tube obtained in 1.4) to wash the RNA precipitate, suspend the RNA precipitate with a pipette, centrifuge at 4°C, 12000 rpm for 5 min, discard the supernatant and retain the RNA precipitate; 1.6) After repeating step 1.5) once, the RNA precipitate retained in the RNase-free EP centrifuge tube was placed at room temperature to dry for 5 to 10 minutes; 1.7) Add 30-50 μL DEPC water to dissolve the RNA precipitate dried in step 1.6) to obtain dissolved plant total RNA, and store it at -80°C for later use; Step (2): According to sub-steps 2.1) to 2.4), reverse transcription is performed using a reverse transcription kit, and the following reverse transcription reaction system, reaction parameters and reaction procedure are performed: 2.1) Place the PCR tube on ice to prepare a reaction mixture, which contains: 2.0 μl 4xDN MasterMix, 0.5 pg to 0.5 μg of the total plant RNA extracted in step (1), and add RNase Free water to make up to 8 μl; 2.2) Mix the reaction mixture obtained in 2.1) and incubate at 37°C for 5 minutes to remove genomic DNA; 2.3) Add 2 μl of 5×RT Master Mix II to the reaction product obtained in 2.2) to make the total reaction system 10 μl; 2.4) After mixing the reaction solution obtained in 2.3), reverse transcription reaction was carried out according to the reaction program of 37°C for 15 min, 50°C for 5 min, 98°C for 5 min, and 4°C for 5 min to finally obtain reverse transcribed cDNA; Step (3): According to sub-steps 3.1) to 3.3), perform the following PCR reaction system, parameters and reaction procedures: 3.1) PCR reaction was performed using 2×Rapid Taq Master Mix enzyme. The following PCR reaction system was prepared in a PCR tube on ice: 12.5 μl 2×Rapid Taq Master Mix, 0.5 μl of the reverse transcribed cDNA, 10 μl sterile deionized water, 1 μl each of 10 μM RdRp-F and RdRp-R upstream and downstream primers of the new Beta Rhabdovirus genus or 1 μl each of 10 μM ORF2-F and ORF2-R upstream and downstream primers of the new Beta Rhabdovirus genus, and the total volume of the reaction system was 25 μl; 3.2) Set the PCR reaction parameters and PCR program as follows: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s, 58°C annealing for 15 s, 72°C extension for 15 s, 30-35 cycles; 72°C extension for 5 min; 4°C for 5 min to terminate the reaction; 3.3) The above PCR procedure was performed on a PCR instrument, and the PCR products were subjected to 1% agarose gel electrophoresis to analyze whether there was a 486 bp RdRp gene fragment or a 481 bp ORF2 gene fragment of a specific new Beta Rhabdovirus. The presence of the RdRp gene fragment or the ORF2 gene fragment indicated that the tested plant tissue was infected with the new Beta Rhabdovirus.

6. The RT-PCR detection method according to claim 5, characterized in that: The reverse transcription kit is ReverTra Ace qPCR RT Master Mix with gDNARemover from Toyobo.

7. The RT-PCR detection method according to claim 5, characterized in that: The 2×Rapid Taq Master Mix enzyme is the 2×Rapid Taq Master Mix enzyme from Vazyme.

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