A method for detecting a hibiscus mosaic virus (hiv) by rt-pcr

Abstract

The application provides a RT-PCR detection method of Hippeastrum hybridum mosaic virus (HiMV), and is characterized by comprising the following steps: 1) extracting total RNA of Hippeastrum hybridum plant by using a kit; 2) synthesizing cDNA by reverse transcription; 3) designing specific detection primers; 4) performing PCR reaction; and 5) performing electrophoresis detection. The collected Hippeastrum hybridum plant tissues can be conveniently detected, and whether the Hippeastrum hybridum plant tissues are infected with HiMV can be analyzed, so that whether the Hippeastrum hybridum tissues are infected with mosaic virus can be found as early as possible, reliable technical support for preventing the spread of mosaic virus is provided, normal growth of the Hippeastrum hybridum plant is ensured, and the quality of the seed ball is improved. The detection method is designed according to the specific sequence of the HiMV conservation, specific primers are designed without specific antibodies, the detection period is short, the sensitivity is high, low-load and symptomless samples can be detected, and the detection method can be used for mass screening of seed balls and tissue culture seedlings.

Description

Technical Field

[0001] This invention relates to an RT-PCR detection method for amaryllis mosaic virus (HiMV), belonging to the field of molecular biology detection technology. Background Technology

[0002] Amaryllis boasts numerous varieties, diverse flower shapes, rich colors, high ornamental value, simple care, and easy cultivation, making it a favorite among consumers and gradually becoming one of the most important bulbous flowers. In recent years, it has become a best-selling variety in the Lunar New Year flower market. In production, amaryllis is mainly propagated asexually through bulb division, scale propagation, and tissue culture. During asexual propagation, viruses can continuously spread through infected parent plants or propagation materials. As the number of generations increases, viruses accumulate and spread within the seedling population, leading to a higher virus carriage rate. This directly affects plant growth, flowering quality, and bulb quality, ultimately impacting the economic benefits for planting companies and farmers.

[0003] Hippeastrum mosaic virus (HiMV) is one of the important viruses that infect plants in the Amaryllidaceae family, such as amaryllis and spider lily. After infection, HiMV can cause symptoms such as mosaic patterns, yellow-green mottling, streaks, and chlorosis on leaves. In severe cases, it can weaken plant growth, cause leaf deformities, reduce flowering quality, and hinder bulb growth and development. However, the symptoms of HiMV infection are not stable; the severity of symptoms varies greatly among different varieties, at different growth stages, and under different environmental conditions. Some plants may not even show obvious symptoms in the early stages of infection or at low viral loads, exhibiting a certain degree of latent infection or occult infection. Furthermore, HiMV can be preserved long-term with vegetative propagation materials and vertically transmitted, allowing seemingly normal infected mother plants, bulbs, or tissue culture materials to potentially be used as propagation material for the next generation of propagation systems.

[0004] Currently, the main detection methods for HiMV include electron microscopy and ELISA serological testing. Electron microscopy requires specialized equipment and a high level of operational skill; sample preparation and observation are cumbersome, resulting in high costs and long testing cycles. Furthermore, its ability to identify low-concentration virus samples is limited, making it difficult to meet the needs of large-scale, rapid, and efficient screening in production processes. While ELISA serological testing has a certain capacity for batch detection, its effectiveness largely depends on the quality and specificity of the antibody and the viral load in the sample. When the viral load is low or the virus is unevenly distributed in tissues, weak positives or false negatives are likely to occur. In addition, some universal antibodies against Potato viruses can cross-react with other viruses in the same genus, making it difficult to accurately distinguish HiMV from other Potato Y virus viruses based solely on serological results. The high cost and long development cycle of preparing high-quality specific antibodies against HiMV also limit the widespread application of this method in ordinary production enterprises and grassroots planting stages.

[0005] Due to the characteristics of HiMV, such as its ability to continuously spread through asexually propagated materials, atypical or unstable symptoms after infection, latent infection in some materials, difficulty in visual identification at low viral loads, and potential differences in viral content and distribution in different tissues and at different growth stages, early detection in actual production is quite challenging. Especially during bulb propagation, rapid tissue culture propagation, and mother plant selection, materials carrying the virus but not yet showing obvious symptoms, if not detected in time, are highly likely to continue to be used as breeding stock or production bulbs in the propagation system, leading to the continuous accumulation and spread of the virus during subsequent asexual reproduction.

[0006] Current HiMV detection technologies suffer from drawbacks such as long detection cycles, heavy reliance on specialized instruments or antibodies, and limitations in rapid batch testing. These limitations make it difficult to simultaneously meet the practical needs of early HiMV diagnosis, seed bulb screening, tissue culture material testing, and large-scale production monitoring. Therefore, it is necessary to improve existing HiMV detection technologies and establish a detection method that is highly sensitive, specific, has a short detection cycle, is relatively simple to operate, and is suitable for practical production applications. Summary of the Invention

[0007] This invention aims to provide an RT-PCR detection method for Amaryllis mosaic virus (HiMV) to detect HiMV infection in amaryllis tissues as early as possible. It addresses the problems of existing detection methods, such as long detection cycles, easy missed detection in low-load samples, insufficient specificity, and difficulty in adapting to rapid production testing. This provides reliable technical support for the screening of amaryllis bulbs, detection of tissue culture materials, virus monitoring during seedling propagation, and the production of virus-free seedlings. Ultimately, it effectively prevents the spread of HiMV in asexual reproduction systems, ensures the normal growth of amaryllis plants, and improves bulb quality and commercial value.

[0008] This invention is achieved through the following technical solution: an RT-PCR detection method for amaryllis mosaic virus (HiMV), comprising the following steps: 1) Extract total RNA from amaryllis plants using a kit. Take amaryllis plant tissue and extract total RNA using a specialized RNA extraction kit. 2) Reverse transcription to synthesize cDNA: Use a specialized reverse transcription kit to remove gDNA and first-strand cDNA in one step to synthesize cDNA, and store it at -20 ℃ for later use. 3) Specific detection primer design: Primers were designed based on the conserved sequence of the HiMV CP protein of amaryllis mosaic virus as follows: The forward primer is: 5'-AAGGATGTGGACGCAGGAAC-3'. The reverse primer is: 5'-TCTGAATTGCATATCGCGGC-3'; 4) PCR reaction 4.1) The PCR reaction system is as follows: PCR SuperMix 10μL 0.4 μL of 10 μM HiMV forward primer 0.4 μL of 10 μM HiMV reverse primer 2 μL of cDNA 5μL of sterile water Total reaction volume: 20 μL; 4.2) The PCR reaction procedure is as follows: After mixing all components of the PCR reaction system, pre-denature at 94℃ for 5 min, then denature at 94℃ for 30 s, anneal at 58℃ for 30 s, and extend at 72℃ for 20 s; repeat this cycle 35 times, and finally extend at 72℃ for 5 min to obtain the PCR reaction product. 5) Electrophoresis detection: The PCR reaction products from step 4) are electrophoresed in a 1×TBE buffer system using 1% agarose gel to detect whether there is a specific CP protein gene fragment of amaryllis mosaic virus HiMV. If so, it indicates that the tested amaryllis plant tissue is infected with amaryllis mosaic virus HiMV.

[0009] The total RNA extraction from Amaryllis plants in step 1) is as follows: 1.1) Take amaryllis plant tissue and put it into a grinding tube containing small steel balls. Treat it with liquid nitrogen, then place it in a grinder at 60 Hz and grind for 60 seconds until it becomes powder. Repeat three times. 1.2) Transfer the powder sample to a centrifuge tube, add 1 ml / 100g TransZol Up and 0.2 ml / 100g RNA Extraction Agent to the powder sample, place it on a vortex mixer, and vortex at room temperature for 5 min; then place it in a centrifuge column and centrifuge at 10,000 xg, 2-8℃ for 15 min. The sample is separated into: an upper colorless aqueous phase, a middle layer, and a lower pink organic phase, wherein the volume of the colorless aqueous phase containing RNA is 50-60% of the volume of the TransZol Up reagent used; 1.3) Take the colorless aqueous phase from step 1.2) and place it in a new centrifuge tube. Add an equal volume of anhydrous ethanol and mix well to obtain a mixture. 1.4) Place the mixture obtained in step 1.3) into a centrifuge column and centrifuge at 12,000 x g at room temperature for 30 s to obtain centrifuge stock, discarding the eluent; add 500 μL CB9 to the centrifuge stock, place it into a centrifuge column, and centrifuge at 12,000 x g at room temperature for 30 s to obtain centrifuge stock, discarding the eluent; repeat this step once to obtain centrifuge stock, discarding the eluent; add 500 μL WB9 to the obtained centrifuge stock, place it into a centrifuge column, and centrifuge at 12,000 x g at room temperature for 30 s to obtain centrifuge stock, discarding the eluent; repeat this step once to obtain centrifuge stock, discarding the eluent; place the obtained centrifuge stock into a centrifuge column and centrifuge at 12,000 x g at room temperature for 2 min to remove residual ethanol, obtaining centrifuge stock; 1.5) Place the centrifuged material obtained in step 1.4) into the prepared RNase-free tube in the kit, add 50 μL of RNase-free water, place it in the center of the centrifuge column, and let it stand at room temperature for 1 minute to obtain the settled material; 1.6) Place the static material obtained in step 1.5) into a centrifuge column, centrifuge at 12,000 xg at room temperature for 1 min to elute RNA, obtain total RNA, and store at -80℃ for later use.

[0010] Step 1) uses a specialized RNA extraction kit, specifically the TransZol UpPlus RNA Kit high-purity RNA extraction kit manufactured by Beijing TransGen Biotech Co., Ltd.

[0011] Step 2) uses a specialized reverse transcription kit, namely the EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix EasyScript Reverse Transcription Kit manufactured by Beijing TransGen Biotech Co., Ltd.

[0012] The reverse transcription system used for cDNA synthesis in step 2) is as follows: Anchored Oligo(dT)18 Prime 1μL, 2xES Reaction Mix 2μL EasyScript RT / RI Enzyme Mix 1μL, gDNA Remover 1μL, RNase-free Water 2μL Total RNA 5μL, Total reaction volume: 20 μL; When synthesizing cDNA by reverse transcription, mix the above components, incubate at 42°C for 30 min, heat at 85°C for 2 min to obtain the synthesized cDNA, and store it in a -20°C refrigerator for later use.

[0013] The specific detection primer design in step 3) is based on the conserved sequence of the HiMV CP protein of amaryllis mosaic virus with NCBI accession number MN068006.1. The primers designed were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0014] Compared with existing technologies, this invention has the following advantages and effects: Using the above technical solution, collected amaryllis plant tissues can be conveniently detected and analyzed for the presence of HiMV (Amaryllis Mosaic Virus), thus enabling early detection of HiMV infection in amaryllis tissues. This provides reliable technical support for preventing the spread of HiMV, ensuring normal growth of amaryllis plants, and improving bulb quality. The detection method of this invention uses specific primers designed for the conserved HiMV sequence, eliminates the need for specific antibody preparation, has a short detection cycle, high sensitivity, can detect low-load and asymptomatic samples, and can be used for large-scale screening of bulbs and tissue culture seedlings. Attached Figure Description

[0015] Figure 1 This is an electrophoresis result of the HiMV virus detected by the present invention.

[0016] In the figure: M is the DL2000 Marker, lanes 1-4 are amaryllis leaf samples with HiMV virus, and lane 5 is a negative control of sterile water. Detailed Implementation

[0017] The present invention will be further described below with reference to embodiments. Example 1

[0018] The amaryllis leaves provided in Example 1 were collected from the base of second-generation amaryllis plants grown in Dounan Flower Planting Area, Yunnan Province, and were used to detect whether the amaryllis plants were infected with Amaryllis Mosaic Virus (HiMV). The specific steps included: 1) Extract total RNA from Amaryllis plants using a kit. The TransZol UpPlus RNA Kit (Beijing TransGen Biotech Co., Ltd.) was used to extract total RNA from Amaryllis leaves. The following procedures were performed according to the kit's instructions: 1.1) Take 100 mg of amaryllis leaves, place them in a grinding tube containing small steel balls, treat with liquid nitrogen, and then grind them in a grinder at 60 Hz for 60 seconds until the leaves are powdered. Repeat three times. 1.2) Transfer the ground powder to a centrifuge tube, add 1 ml of TransZol Up and 0.2 ml of RNA Extraction Agent to 100 mg of sample, place on a vortex mixer, and vortex at room temperature for 5 min; then place in a centrifuge column and centrifuge at 10,000 x g and 2-8 °C for 15 min. The sample is separated into: an upper colorless aqueous phase, a middle layer, and a lower pink organic phase, wherein the volume of the colorless aqueous phase containing RNA is 50-60% of the volume of the TransZol Up reagent used; 1.3) Take 500 μL of the colorless aqueous phase from step 1.2) and place it in a new centrifuge tube. Add an equal volume of anhydrous ethanol and mix well to obtain a mixture. 1.4) Place the mixture obtained in step 1.3) into a centrifuge column and centrifuge at 12,000 x g at room temperature for 30 s to obtain centrifuges, discarding the eluent; add 500 μL of CB9 to the centrifuges, place it into a centrifuge column, and centrifuge at 12,000 x g at room temperature for 30 s to obtain centrifuges, discarding the eluent; repeat this step once to obtain centrifuges, discarding the eluent; add 500 μL of WB9 to the obtained centrifuges, place it into a centrifuge column, and centrifuge at 12,000 x g at room temperature for 30 s to obtain centrifuges, discarding the eluent; repeat this step once to obtain centrifuges, discarding the eluent; place the obtained centrifuges into a centrifuge column and centrifuge at 12,000 x g at room temperature for 2 min to remove residual ethanol, obtaining centrifuges; 1.5) Place the centrifuged material obtained in step 1.4) into the prepared RNase-free tube in the kit, add 50 μL of RNase-free water, place it in the center of the centrifuge column, and let it stand at room temperature for 1 minute to obtain the settled material; 1.6) Place the static material obtained in step 1.5) into a centrifuge column, centrifuge at 12,000 xg at room temperature for 1 min to elute RNA, obtain total RNA, and store at -80℃ for later use; 2) Reverse transcription to synthesize cDNA: The EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix EasyScript Reverse Transcription Kit (manufactured by Beijing TransGen Biotech Co., Ltd.) was used. After removing gDNA and first-strand cDNA in one step, the RNA obtained in step 1) was synthesized into cDNA according to the kit's instructions. The reverse transcription system is as follows: Anchored Oligo(dT)18 Prime 1μL, 2xES Reaction Mix 2μL EasyScript RT / RI Enzyme Mix 1μL, gDNA Remover 1μL, RNase-free Water 2μL Total RNA 5μL, Total reaction volume: 20 μL; After gently mixing the above components, incubate at 42°C for 30 min and heat at 85°C for 2 min to obtain the synthesized cDNA, and store it in a -20°C freezer for later use. Specific detection primers were designed using the conserved sequence of the HiMV CP protein of amaryllis mosaic virus (GenBank accession number: MN068006.1) as a reference sequence. These primers were synthesized by Beijing Qingke Biotechnology Co., Ltd., as detailed below: The forward primer is: 5'-AAGGATGTGGACGCAGGAAC-3'. The reverse primer is: 5'-TCTGAATTGCATATCGCGGC-3'; 4) PCR reaction 4.1) The PCR reaction system is as follows: PCR SuperMix 10μL 0.4 μL of 10 μM HiMV forward primer 0.4 μL of 10 μM HiMV reverse primer 2 μL of cDNA 5μL of sterile water Total reaction volume: 20 μL; Take 4 reaction tubes, add the above PCR reaction system to each tube, mix well, and use an equal volume of sterile water instead of the target RNA as a negative control. 4.2) The PCR reaction procedure is as follows: The PCR reaction system of step 4.1) was pre-denatured at 94℃ for 5 min, then denatured at 94℃ for 30 s, annealed at 58℃ for 30 s, and extended at 72℃ for 20 s; after 35 cycles, the final extension was performed at 72℃ for 5 min, and the reaction was terminated at 4℃ to obtain the PCR reaction product. 5) Electrophoresis detection: The PCR reaction products from step 4) were subjected to electrophoresis on a 1% agarose gel in a 1×TBE buffer system. A 445bp CP protein gene fragment was detected in all four reaction tubes, indicating that the 100mg of amaryllis leaves taken in step 1) were infected with Amaryllis mosaic virus HiMV. (See attached image) Figure 1 .

Claims

1. An RT-PCR detection method for Amaryllis Mosaic Virus (HiMV), characterized in that: Includes the following steps: 1) Extract total RNA from amaryllis plants using a kit. Take amaryllis plant tissue and extract total RNA using a specialized RNA extraction kit. 2) Reverse transcription to synthesize cDNA: Use a specialized reverse transcription kit to remove gDNA and first-strand cDNA in one step to synthesize cDNA, and store it at -20 ℃ for later use. 3) Specific detection primer design: Primers were designed based on the conserved sequence of the HiMV CP protein of amaryllis mosaic virus as follows: The forward primer is: 5'-AAGGATGTGGACGCAGGAAC-3'. The reverse primer is: 5'-TCTGAATTGCATATCGCGGC-3'; 4) PCR reaction 4.1) The PCR reaction system is as follows: PCR SuperMix 10μL 0.4 μL of 10 μM HiMV forward primer 0.4 μL of 10 μM HiMV reverse primer 2 μL of cDNA 5μL of sterile water Total reaction volume: 20 μL; 4.2) The PCR reaction procedure is as follows: After mixing all components of the PCR reaction system, pre-denature at 94℃ for 5 min, then denature at 94℃ for 30 s, anneal at 58℃ for 30 s, and extend at 72℃ for 20 s; repeat this cycle 35 times, and finally extend at 72℃ for 5 min to obtain the PCR reaction product. 5) Electrophoresis detection: The PCR reaction products from step 4) are electrophoresed in a 1×TBE buffer system using 1% agarose gel to detect whether there is a specific CP protein gene fragment of amaryllis mosaic virus HiMV. If so, it indicates that the tested amaryllis plant tissue is infected with amaryllis mosaic virus HiMV.

2. The RT-PCR detection method for Amaryllis Mosaic Virus (HiMV) according to claim 1, characterized in that: The total RNA extraction from Amaryllis plants in step 1) is as follows: 1.1) Take amaryllis plant tissue and put it into a grinding tube containing small steel balls. Treat it with liquid nitrogen, then place it in a grinder at 60 Hz and grind for 60 seconds until it becomes powder. Repeat three times. 1.2) Transfer the powder sample to a centrifuge tube, add 1 ml / 100g TransZol Up and 0.2 ml / 100g RNA Extraction Agent to the powder sample, place it on a vortex mixer, and vortex at room temperature for 5 min; then place it in a centrifuge column and centrifuge at 10,000 xg, 2-8℃ for 15 min. The sample is separated into: an upper colorless aqueous phase, a middle layer, and a lower pink organic phase, wherein the volume of the colorless aqueous phase containing RNA is 50-60% of the volume of the TransZol Up reagent used; 1.3) Take the colorless aqueous phase from step 1.2) and place it in a new centrifuge tube. Add an equal volume of anhydrous ethanol and mix well to obtain a mixture. 1.4) Place the mixture obtained in step 1.3) into a centrifuge column and centrifuge at 12,000 x g at room temperature for 30 s to obtain centrifuge stock, discarding the eluent; add 500 μL CB9 to the centrifuge stock, place it into a centrifuge column, and centrifuge at 12,000 x g at room temperature for 30 s to obtain centrifuge stock, discarding the eluent; repeat this step once to obtain centrifuge stock, discarding the eluent; add 500 μL WB9 to the obtained centrifuge stock, place it into a centrifuge column, and centrifuge at 12,000 x g at room temperature for 30 s to obtain centrifuge stock, discarding the eluent; repeat this step once to obtain centrifuge stock, discarding the eluent; place the obtained centrifuge stock into a centrifuge column and centrifuge at 12,000 x g at room temperature for 2 min to remove residual ethanol, obtaining centrifuge stock; 1.5) Place the centrifuged material obtained in step 1.4) into the prepared RNase-free tube in the kit, add 50 μL of RNase-free water, place it in the center of the centrifuge column, and let it stand at room temperature for 1 minute to obtain the settled material; 1.6) Place the static material obtained in step 1.5) into a centrifuge column, centrifuge at 12,000 xg at room temperature for 1 min to elute RNA, obtain total RNA, and store at -80℃ for later use; Step 1) uses a specialized RNA extraction kit, specifically the TransZol UpPlus RNA Kit high-purity RNA extraction kit manufactured by Beijing TransGen Biotech Co., Ltd.

3. The RT-PCR detection method for Amaryllis Mosaic Virus (HiMV) according to claim 1, characterized in that: The reverse transcription system used for cDNA synthesis in step 2) is as follows: Anchored Oligo(dT)18 Prime 1μL, 2xES Reaction Mix 2μL EasyScript RT / RI Enzyme Mix 1μL, gDNA Remover 1μL, RNase-free Water 2μL Total RNA 5μL, Total reaction volume: 20 μL; When synthesizing cDNA by reverse transcription, mix the above components, incubate at 42℃ for 30 min, heat at 85℃ for 2 min to obtain the synthesized cDNA, and store it in a -20℃ refrigerator for later use. The specific detection primer design in step 3) is based on the conserved sequence of the HiMV CP protein of amaryllis mosaic virus with NCBI accession number MN068006.

1. The primers designed were synthesized by Beijing Qingke Biotechnology Co., Ltd.

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