A rapid detection method of specific fungal viruses by RT-PCR

Abstract

The application discloses a detection kit and an RT-PCR detection method for rapidly detecting a fungal virus RsPV2, and belongs to the field of agricultural biotechnology. The detection method is characterized in that the reverse transcription (RT) and PCR are completed in one step, and the operation is simple and convenient. The RT-PCR is performed by using a pair of specific primers, and false positive can be effectively eliminated. The test conditions required by the detection method are simple, and the test conditions can be met in general nucleic acid extraction molecular laboratories and research units, so that the application is beneficial to popularization and application in the field of fungal virus research.

Description

Technical Field

[0001] This invention relates to a rapid RT-PCR method for detecting a specific fungal virus, specifically to PCR primers for detecting the fungal virus RsV2 and an accurate and rapid RT-PCR detection kit for detecting the virus. It is also applicable to the detection of other nucleic acid viruses and belongs to the field of agricultural biotechnology. Background Technology

[0002] Sheath blight is a major global disease affecting rice and wheat. Its pathogens, *Rhizoctonia solani* Kühn and *Rhizoctonia zeae*, are widely distributed in my country, affecting over 200 crops including rice, wheat, cotton, potatoes, corn, and so on. Currently, control of sheath blight relies primarily on traditional chemical control methods. This not only pollutes the environment but also leads to drug resistance in pathogens due to repeated pesticide use, increasing the difficulty of control and contradicting sustainable agricultural and environmental development. Fungal viruses are viruses that infect fungi. While they generally do not cause obvious phenotypic abnormalities in the host fungus, they typically alter its cell growth, sporulation, pigmentation, and pathogenicity. Viruses that alter the pathogenicity of the host fungus are called attenuated viruses. The discovery of attenuated viruses in *Rhizoctonia solani* has opened up a new biological control pathway for sheath blight. Research on fungal viruses is of great significance for the biological control of sheath blight.

[0003] Fungal viruses have a wide range of hosts in nature. Infection by fungi can be categorized into single-virus infection and co-infection with two or more viruses. A single fungal virus can also infect multiple fungi, causing different changes in the morphology and biological characteristics of the host fungi. Rhizoctonia solani partitivirus 2 (RSPV2) was first isolated and identified in 2014 by a team from South China Agricultural University from the rice sheath blight strain R. solani AG-1 IA GD-11 (GenBank accession number: KF372436). This fungal virus is an attenuated virus, and its use in the biocontrol of sheath blight is of great significance. However, current research lacks a rapid and effective method for screening and identifying this fungal virus. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a detection kit and RT-PCR detection method for the rapid detection of fungal virus RsPV2.

[0005] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a detection kit for rapid detection of fungal virus RsPV2, comprising: Reagents for fungal total RNA extraction: Trizol, Tris-Phenol, isopropanol, 75% ethanol, dd H2O; cDNA synthesis reagents: Oligo(dT)18, DEPC water, 5×M-MLV Reaction Buffer, dNTPMixture, RNase Inhibitor, M-MLV Reverse Transcriptase; RT-PCR reaction system: Mg 2+ dNTPs, upstream primer: 5'-CCTCAACCAAACCGTCCCT-3', downstream primer: 5'-GACTTGATTAGGCATTCG-3', Taq DNA polymerase, ddH2O.

[0006] Secondly, the present invention provides an RT-PCR method for rapid detection of fungal virus RsPV2, using the above-mentioned detection kit, comprising the following steps: (1) Extraction of total RNA from fungi; (2) cDNA synthesis: First, prepare reaction system ① and reaction system ②; Reaction system ① Sample Amount Total RNA sample 100-500 ng Oligo(dT)18 (0.5 ug / μL) 1 μL DEPC-treated water Add to total volume 20 μL Reaction system ② Sample Amount 5x M-MLV Reaction Buffer 4 μL dNTP Mixture (10 mM each) 1 μL RNase Inhibitor (40 U / μL) 0.5 μL M-MLV Reverse Transcriptase (200 U / μL) 1 μL Mix system ① thoroughly, incubate at 65˚C for 5 min in a water bath, then immediately place on ice for 2 min to allow the random primers to bind to the RNA template; gently mix systems ① and ②, incubate at 42˚C for 30 min; treat at 85˚C for 5 min to inactivate M-MLV ReverseTranscriptase; store at -80˚C. (3) RT-PCR reaction Using the cDNA synthesized in step (2) as a template, heterogeneous PCR amplification was performed using primer pairs designed with specific viral cDNA as primers. The reaction system is as follows: RT-PCR reaction system Sample Amount cDNA sample 2 μL Mg 2+ ]]> 1 μL dNTP (2.5 mM) 1 μL Upstream primer 1 μL Downstream primer 1 μL DNA polymerase 0.2 μL ddH2O Bring the system to 25 μL The PCR reaction program was as follows: 95℃ pre-denaturation for 4 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1.5 min, for a total of 30 cycles, and finally 72℃ complete extension for 10 min, and stored at 4℃ for later use. (4) Finally, 1% agarose gel electrophoresis was performed.

[0007] Beneficial effects: (1) The fungal virus RsPV2 detection kit provided by this invention has simple components, high detection sensitivity, and accurate results. It can rapidly, sensitively, and accurately detect viruses at different growth stages of various fungal diseases, greatly reducing the workload of fungal virus research. (2) The RT-PCR detection method for rapid detection of fungal virus RsPV2 provided by this invention completes reverse transcription (RT) and PCR in one step, which is simple and convenient to operate. The use of a pair of specific primers for RT-PCR can effectively eliminate false positives. The experimental conditions required for the detection method are simple and do not require large instruments and equipment such as electron microscopes and ultracentrifuges. These experimental conditions can be met in molecular laboratories and research units with nucleic acid extraction conditions, which is conducive to its promotion and application in the field of fungal virus research. Attached Figure Description

[0008] Figure 1 This is an electrophoresis detection image. Detailed Implementation

[0009] The present invention will be further described below with reference to the embodiments. Unless otherwise specified, the experimental materials used in the embodiments are all commercially available. Unless otherwise specified, the experimental methods described are conventional experimental methods.

[0010] The following tests were conducted using rice sheath blight strains A5 and A82 as samples to detect the virus. The positive control used was the virus-carrying strain GD11, and the negative control was the virus-free strain GD118. These strains were obtained from the laboratory of the College of Plant Protection, South China Agricultural University.

[0011] The upstream and downstream primers in the RT-PCR reaction were designed by the inventors. The primers were designed with reference to the Rhizoctonia solani dispora virus 2 (RsPV2, KF372436) sequence in GeneBank and the online primer design tool Primer-BLAST provided by NCBI. The nucleotide sequences are: RsPV2-F: 5'-CCTCAACCAAACCGTCCCT-3'; RsPV2-R: 5'-GACTTGATTAGGCATTCG-3'.

[0012] The detection method is as follows.

[0013] (1) Extraction of total RNA from fungi 100 mg of bacterial cells from test strains A5, A82, the positive control, and the negative control were ground into powder in liquid nitrogen and transferred to 1.5 mL centrifuge tubes. 1 mL of Trizol reagent was added, and the mixture was vigorously shaken for 30 s. After incubating the mixture at room temperature for 10 min, it was centrifuged at 12,000 g for 15 min at 4 °C. The supernatant was mixed with an equal volume of pre-chilled Tris-Phenol and isopropanol for 2 min, and then centrifuged at 12,000 g for 5 min at 4 °C. Half a volume of pre-chilled isopropanol was added to the supernatant obtained in this step, and the mixture was incubated at room temperature for 10 min, followed by centrifugation at 12,000 g for 20 min at 4 °C to precipitate RNA. The precipitate was washed once with 75% ethanol, then dissolved in 30 μL of RNase-free ddH2O and stored at –80 °C. Total RNA was extracted from the positive and negative controls simultaneously using the same method.

[0014] (2) cDNA synthesis First, prepare the reaction system according to Table 1 and Table 2.

[0015] Table 1 Reaction System ① Sample Amount Total RNA sample 100-500 ng Oligo(dT)18 (0.5 ug / μL) 1 μL DEPC-treated water Add to total volume 20 μL Table 2 Reaction System ② Sample Amount 5x M-MLV Reaction Buffer 4 μL dNTP Mixture (10 mM each) 1 μL RNase Inhibitor (40 U / μL) 0.5 μL M-MLV Reverse Transcriptase (200 U / μL) 1 μL System ① was thoroughly mixed and incubated in a water bath at 65˚C for 5 min, followed immediately by an ice bath for 2 min to allow the random primer Oligo(dT)18 to bind to the RNA template. Systems ① and ② were then gently mixed and incubated at 42˚C for 30 min. Finally, the mixture was treated at 85˚C for 5 min to inactivate M-MLV Reverse Transcriptase. The mixture was then stored at -80˚C.

[0016] (3) RT-PCR detection Using the cDNA synthesized in (2) as a template, heterogeneous PCR amplification was performed using the aforementioned primers. The reaction system is shown in Table 3.

[0017] Table 3 RT-PCR reaction system Sample Amount cDNA sample 2 μL Mg 2+ ]]> 1 μL dNTP (2.5 mM) 1 μL RsPV2-F 1 μL RsPV2-R 1 μL DNA polymerase 0.2 μL ddH2O Bring the system to 25 μL Magnesium ions can come from magnesium chloride or magnesium sulfate; The PCR reaction procedure was as follows: 95℃ pre-denaturation for 4 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1.5 min, for a total of 30 cycles, and finally 72℃ complete extension for 10 min, and stored at 4℃ for later use.

[0018] Finally, 1% agarose gel electrophoresis was used for verification, such as... Figure 1As shown, the results indicate that strain A5 and the positive control product both showed corresponding bands, while strain A82 and the negative control product did not show corresponding bands. This suggests that strain A5 was infected by virus RsPV2, while strain A82 was not infected by virus RsPV2.

[0019] This invention relates to a detection probe for the fungal virus RsPV2 in the rice sheath blight fungus strain GD11 and its application. The invention designs a detection probe for fungal virus RsPV2, which can be used to detect whether other strains are infected with fungal virus RsPV2. This invention provides a simple and rapid method for the detection of specific viruses, improves the efficiency of virus screening and identification in fungi, and provides a method for the detection of fungal viruses in other pathogenic fungi. It is of great significance for the research of fungal viruses and the prevention and control of rice sheath blight.

Claims

1. A rapid detection kit for fungal virus RsPV2, characterized in that, include: Reagents for fungal total RNA extraction: Trizol, Tris-Phenol, isopropanol, 75% ethanol, dd H2O; cDNA synthesis reagents: Oligo(dT)18, DEPC water, 5×M-MLV Reaction Buffer, dNTP Mixture, RNase Inhibitor, M-MLV Reverse Transcriptase; RT-PCR reaction system: Mg 2+ dNTPs, upstream primer: 5'-CCTCAACCAAACCGTCCCT-3', downstream primer: 5'-GACTTGATTAGGCATTCG-3', Taq DNA polymerase, ddH2O.

2. An RT-PCR method for rapid detection of fungal virus RsPV2, using the detection kit as described in claim 1, comprising the following steps: (1) Extraction of total RNA from fungi; (2) cDNA synthesis: First, prepare reaction system ① and reaction system ②; Reaction system ① Reaction system ② Mix system ① thoroughly, incubate at 65˚C for 5 min in a water bath, then immediately place on ice for 2 min to allow the random primers to bind to the RNA template; gently mix systems ① and ②, incubate at 42˚C for 30 min; treat at 85˚C for 5 min to inactivate M-MLV ReverseTranscriptase; store at -80˚C. (3) RT-PCR reaction Using the cDNA synthesized in step (2) as a template, heterogeneous PCR amplification was performed using the primer pair as primers. The reaction system is as follows: RT-PCR reaction system The PCR reaction program was as follows: 95℃ pre-denaturation for 4 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1.5 min, for a total of 30 cycles, and finally 72℃ complete extension for 10 min, and stored at 4℃ for later use. (4) Finally, 1% agarose gel electrophoresis was performed.

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