A primer set for amplifying the whole genome sequence of maize chlorotic mottle virus and application thereof

Abstract

The application discloses a primer set for amplifying a whole genome sequence of maize chlorotic mottle virus and application thereof. The primer set comprises 10 primer pairs. By using each primer pair in the primer set, one-step RT-PCR amplification and sequencing are carried out on a maize chlorotic mottle virus or a sample containing the maize chlorotic mottle virus as a template, and comparison analysis and sequence splicing are carried out on the sequencing result, so that the whole genome sequence of the maize chlorotic mottle virus can be obtained. The primer set provided by the application can be used for detection and identification of the maize chlorotic mottle virus, is simple in operation method, and is accurate and reliable in result. The application not only has guiding significance for rapid and accurate detection and identification of the maize chlorotic mottle virus in China and scientific prevention and control of maize lethal necrosis disease, but also provides a powerful tool for evolution analysis and molecular epidemiology research of the maize chlorotic mottle virus.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a primer set for amplifying the whole genome sequence of maize chlorotic mottle virus and its application. Background Technology

[0002] Maize chlorotic mottle virus belongs to the Tomato Cluster Dwarf Virus Family ( Tombusviridae Maize chlorotic mottle virus (Machlomovirus) is a member of the genus Machlomovirus, whose natural hosts include maize (Maize). Zea mays ) and sugarcane ( Saccharum officinarum Under experimental conditions, it can also infect wheat ( Triticum aestivum ),barley( Hordeum vulgare ),oat( Avena sativa ), sorghum ( Sorghum bicolor It infests grasses such as Gramineae. The genome of maize chlorotic mottle virus is a positive-sense single-stranded RNA, approximately 4.4 kb in length, with a methylated nucleotide cap at the 5′ end and no PolyA tail at the 3′ end. The genome contains four major open reading frames, encoding a total of seven proteins: P32, P50, P111, P7a, P7b, P31, and the capsid protein (CP).

[0003] Maize chlorotic mottle virus is a quarantine pest for imported plants in my country, mainly distributed in countries such as Argentina, Mexico, Peru, and the United States. Infecting maize alone causes symptoms such as chlorosis of leaves and the appearance of yellow-green mottled stripes parallel to the leaf veins, resulting in yield losses of 10%–15%. However, when maize chlorotic mottle virus is combined with the virus from the Potato Virus Y family (…),… Potyviridae When viruses such as maize dwarf mosaic virus, wheat streak mosaic virus, sugarcane mosaic virus, or Johnson's grass mosaic virus cause co-infection, a synergistic effect can occur, leading to a significant increase in the accumulation of viruses in maize plant tissues. This can induce a devastating viral disease—maize lethal necrosis disease (MLND), which can cause yield losses exceeding 90%. MLND presents different symptoms at different growth stages: during the seedling stage, maize plants are stunted, and leaves develop chlorotic spots that merge to form streaks and die from the leaf margins inward, leading to seedling wilting and necrosis in severe cases; during the jointing stage, maize leaves show chlorotic mottling and necrosis, which can cause premature plant death and ear deformities in severe cases; during the milk stage, the entire maize plant dies, and the kernels become shriveled.

[0004] Maize chlorotic mottle virus (MCMV) is easily transmitted through mechanical friction inoculation and can also be transmitted via seeds and insect vectors such as leaf beetles. Currently, there are no effective control agents against this virus; strengthening quarantine remains the most effective way to prevent its introduction. Traditional detection and identification methods, including host identification inoculation, electron microscopy, and serological testing, all have limitations, making rapid and accurate identification of MCMV difficult and prone to false negatives or false positives. Reverse transcription polymerase chain reaction (RT-PCR) has the advantages of high specificity and sensitivity, but existing methods mainly target single gene fragments such as the MCMV coat protein (CP), P111 protein (P111), and P32 protein (P32) for detection and analysis. Given that a single gene fragment is only a part of the viral genome, significant mutations in that fragment can still lead to inaccurate detection results. Therefore, identification methods based on the whole MCMV genome sequence offer higher accuracy and reliability. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to obtain the whole genome sequence of maize chlorotic mottle virus in order to achieve rapid and accurate detection and identification of maize chlorotic mottle virus.

[0006] In a first aspect, the present invention claims protection for a primer set for amplifying the whole genome sequence of maize chlorotic mottle virus.

[0007] The primer set claimed in this invention for amplifying the whole genome sequence of maize chlorotic mottle virus includes primer pair 1 consisting of MCMV-1-F and MCMV-1-R, primer pair 2 consisting of MCMV-2-F and MCMV-2-R, primer pair 3 consisting of MCMV-3-F and MCMV-3-R, primer pair 4 consisting of MCMV-4-F and MCMV-4-R, primer pair 5 consisting of MCMV-5-F and MCMV-5-R, primer pair 6 consisting of MCMV-6-F and MCMV-6-R, primer pair 7 consisting of MCMV-7-F and MCMV-7-R, primer pair 8 consisting of MCMV-8-F and MCMV-8-R, primer pair 9 consisting of MCMV-9-F and MCMV-9-R, and primer pair 10 consisting of MCMV-10-F and MCMV-10-R. The MCMV-1-F is the single-stranded DNA shown in sequence 1; The MCMV-1-R is the single-stranded DNA shown in sequence 2; The MCMV-2-F is the single-stranded DNA shown in sequence 3; The MCMV-2-R is the single-stranded DNA shown in sequence 4; The MCMV-3-F is the single-stranded DNA shown in sequence 5; The MCMV-3-R is the single-stranded DNA shown in sequence 6; The MCMV-4-F is the single-stranded DNA shown in sequence 7; The MCMV-4-R is the single-stranded DNA shown in sequence 8; The MCMV-5-F is the single-stranded DNA shown in sequence 9; The MCMV-5-R is the single-stranded DNA shown in sequence 10; The MCMV-6-F is the single-stranded DNA shown in sequence 11; The MCMV-6-R is the single-stranded DNA shown in sequence 12; The MCMV-7-F is the single-stranded DNA shown in sequence 13; The MCMV-7-R is the single-stranded DNA shown in sequence 14; The MCMV-8-F is the single-stranded DNA shown in sequence 15; The MCMV-8-R is the single-stranded DNA shown in sequence 16; The MCMV-9-F is the single-stranded DNA shown in sequence 17; The MCMV-9-R is the single-stranded DNA shown in sequence 18; The MCMV-10-F is the single-stranded DNA shown in sequence 19; The MCMV-10-R is the single-stranded DNA shown in sequence 20.

[0008] Secondly, the present invention claims protection for a kit for amplifying the whole genome sequence of maize chlorotic mottle virus.

[0009] The kit provided by this invention for amplifying the whole genome sequence of maize chlorotic mottle virus contains the above-mentioned primer set.

[0010] Furthermore, the kit also contains reagents for extracting RNA from maize chlorotic mottle virus and other reagents for one-step RT-PCR amplification of the whole genome sequence of maize chlorotic mottle virus.

[0011] Furthermore, the reagents used for extracting maize chlorotic mottle virus RNA include TrizoL reagent.

[0012] Other reagents used in the one-step RT-PCR amplification of the whole genome sequence of maize chlorotic mottle virus include 2 × OneStep Mix (Dye Plus) and One Step Enzyme Mix.

[0013] Thirdly, the present invention claims protection for new uses of the above-described primer set or the above-described reagent kit.

[0014] This invention claims protection for the use of the above-described primer set or kit in any of the following: a1) Obtain the complete genome sequence of maize chlorotic mottle virus; a2) Identify whether the virus to be tested is maize chlorotic mottle virus; a3) Detect whether the sample to be tested contains maize chlorotic mottle virus; a4) Evolutionary analysis of maize chlorotic mottle virus; a5) Molecular epidemiological study of maize chlorotic mottle virus.

[0015] This invention also claims protection for the use of the above-described primer set or the above-described kit in any of the following: b1) Prepare a product for which the complete genome sequence of maize chlorotic mottle virus is obtained; b2) Prepare products to identify whether the virus to be tested is maize chlorotic mottle virus; b3) Prepare products for detecting whether the sample to be tested contains maize chlorotic mottle virus; b4) Prepare products for evolutionary analysis of maize chlorotic mottle virus; b5) Prepare products for molecular epidemiological studies of maize chlorotic mottle virus.

[0016] Fourthly, the present invention claims protection for any of the following methods: c1) A method for obtaining the whole genome sequence of a maize chlorotic mottle virus, comprising the following steps: Using the RNA of maize chlorotic mottle virus or samples containing maize chlorotic mottle virus as templates, one-step RT-PCR amplification was performed using the above primer pairs to obtain amplification products; the amplification products were sequenced, and the sequencing results were compared and analyzed and the sequences were assembled to obtain the whole genome sequence of the maize chlorotic mottle virus. c2) A method for identifying whether a virus to be tested is maize chlorotic mottle virus, comprising the following steps: obtaining the whole genome sequence of the virus to be tested according to the method described in c1); performing BLAST analysis on the whole genome sequence to determine whether the virus to be tested is maize chlorotic mottle virus; c3) A method for detecting whether a sample to be tested contains maize chlorotic mottle virus, comprising the following steps: obtaining the whole genome sequence of the sample to be tested according to the method described in c1); performing BLAST analysis on the whole genome sequence to determine whether the sample to be tested contains maize chlorotic mottle virus.

[0017] In c1) above, the sequencing method is bidirectional sequencing.

[0018] In c1) above, the alignment analysis and sequence assembly are performed using DNAMAN software.

[0019] In c1) above, the reaction system for the one-step RT-PCR amplification is as follows: 3 μL total RNA, 12.5 μL 2 × One Step Mix (Dye Plus), 1.25 μL One Step Enzyme Mix, 1 μL upstream primer (10 μmol / L) and 1 μL downstream primer (10 μmol / L), and 6.25 μL ddH2O. The upstream and downstream primers are respectively MCMV-1-F and MCMV-1-R in primer pair 1, MCMV-2-F and MCMV-2-R in primer pair 2, MCMV-3-F and MCMV-3-R in primer pair 3, MCMV-4-F and MCMV-4-R in primer pair 4, MCMV-5-F and MCMV-5-R in primer pair 5, MCMV-6-F and MCMV-6-R in primer pair 6, MCMV-7-F and MCMV-7-R in primer pair 7, MCMV-8-F and MCMV-8-R in primer pair 8, MCMV-9-F and MCMV-9-R in primer pair 9, and MCMV-10-F and MCMV-10-R in primer pair 10. The final concentration of each primer in each primer pair in the one-step RT-PCR amplification system is 0.4 μmol / L.

[0020] The reaction conditions for the one-step RT-PCR amplification are as follows: reverse transcription at 50℃ for 30 min, pre-denaturation at 94℃ for 3 min, denaturation at 94℃ for 30 s; annealing at 51-59℃ for 30-50 s; extension at 72℃ for 60-90 s, 35 cycles, followed by 72℃ for 7 min.

[0021] In some preferred embodiments, the annealing temperatures for one-step RT-PCR amplification using primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5, primer pair 6, primer pair 7, primer pair 8, primer pair 9, and primer pair 10 are 56℃, 55℃, 55℃, 54℃, 51℃, 54℃, 53℃, 53℃, and 59℃, respectively.

[0022] In some preferred embodiments, the annealing time for one-step RT-PCR amplification using primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5, primer pair 6, primer pair 7, primer pair 8, primer pair 9, and primer pair 10 is 30 seconds.

[0023] In some preferred embodiments, the extension times for one-step RT-PCR amplification using primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5, primer pair 6, primer pair 7, primer pair 8, primer pair 9, and primer pair 10 are 60s, 60s, 60s, 60s, 60s, 60s, 60s, 60s, and 90s, respectively.

[0024] In some specific implementations, the amplification product sizes of primer pair 1, primer pair 2, primer pair 3, primer pair 4, primer pair 5, primer pair 6, primer pair 7, primer pair 8, primer pair 9, and primer pair 10 are 247bp, 630bp, 572bp, 533bp, 512bp, 531bp, 589bp, 720bp, 567bp, and 1034bp, respectively.

[0025] In some specific embodiments, the whole genome sequence of the maize chlorotic mottle virus is shown in sequence 21, which includes seven protein-coding genes: p32, replicate, replicate-associated protein, p31, p7a, p7b, and coat protein.

[0026] In c2) and c3) above, the method for identifying whether the virus to be tested is maize mottle virus or for detecting whether the sample to be tested contains maize mottle virus can be carried out as follows: if the nucleotide sequence of the whole genome of the virus to be tested or the sample to be tested has a similarity of more than 96.55% with the whole genome nucleotide sequence of a maize mottle virus in GenBank, then the virus to be tested is maize mottle virus or the sample to be tested contains maize mottle virus.

[0027] The application of the above-mentioned maize chlorotic mottle virus whole genome sequence in any of the following is also within the scope of protection of this invention: d1) Identify whether the virus to be tested is maize chlorotic mottle virus; d2) Detect whether the sample to be tested contains maize chlorotic mottle virus; d3) Evolutionary analysis of maize chlorotic mottle virus; d4) Molecular epidemiological study of maize chlorotic mottle virus; d5) Prepare a product to identify whether the virus to be tested is maize chlorotic mottle virus; d6) Prepare a product for detecting whether the sample to be tested contains maize chlorotic mottle virus; d7) Prepare products for evolutionary analysis of maize chlorotic mottle virus; d8) Prepare products for molecular epidemiological research on maize chlorotic mottle virus.

[0028] In any of the methods described above, the sample or test sample containing maize chlorotic mottle virus may be maize or sugarcane or material derived from different tissue parts thereof, such as maize seeds.

[0029] In some implementations, the sample or test sample containing maize chlorotic mottle virus is maize seed imported from Thailand.

[0030] This invention provides a primer set for amplifying the whole genome sequence of maize chlorotic mottle virus and its application in the detection and identification of maize chlorotic mottle virus. The primer set includes 10 primer pairs. By using each primer pair in this set, one-step RT-PCR amplification and sequencing are performed using maize chlorotic mottle virus or samples containing maize chlorotic mottle virus as templates. The sequencing results are then compared and analyzed, and the sequence is assembled to obtain the whole genome sequence of maize chlorotic mottle virus. This invention was used to detect imported maize samples from Thailand, obtaining a 4436 bp whole genome sequence of a Thai isolate of maize chlorotic mottle virus, which includes seven protein-coding genes: p32, replicate, replicate-associated protein, p31, p7a, p7b, and coat protein. The primer set provided by this invention for amplifying the whole genome sequence of maize chlorotic mottle virus can be used for the detection and identification of maize chlorotic mottle virus; it is not only simple to operate but also provides accurate and reliable results. This invention not only provides guidance for the rapid and accurate detection and identification of maize chlorotic mottle virus and the scientific prevention and control of maize lethal necrosis in my country, but also provides a powerful tool for the evolutionary analysis and molecular epidemiological research of maize chlorotic mottle virus. Attached Figure Description

[0031] Figure 1 The images show agarose gel electrophoresis results of the amplified fragments from each primer pair. M represents the 100bp DNA Ladder (DyePlus); 1-10 represent the amplified fragments from primer pairs MCMV-1-F / MCMV-1-R, MCMV-2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6-R, MCMV-7-F / MCMV-7-R, MCMV-8-F / MCMV-8-R, MCMV-9-F / MCMV-9-R, and MCMV-10-F / MCMV-10-R, respectively.

[0032] Figure 2Agarose gel electrophoresis images of amplified fragments of maize chlorotic mottle virus isolated from Thailand using various primer pairs. M: 100bp DNA Ladder (Dye Plus); 1-10 represent primer pairs MCMV-1-F / MCMV-1-R, MCMV-2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6-R, and MCMV-7-F / MCMV-7-R, respectively. 2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6 Amplified fragments of -R, MCMV-7-F / MCMV-7-R, MCMV-8-F / MCMV-8-R, MCMV-9-F / MCMV-9-R and MCMV-10-F / MCMV-10-R.

[0033] Figure 3 This is the complete genome structure map of the maize chlorotic mottle virus isolate from Thailand. Except for the UTR region, the numbers on the vertical lines indicate the start and end nucleotide positions of the gene. Detailed Implementation

[0034] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0035] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the experimental methods in the following embodiments are performed at least three times.

[0036] Example 1: Design of primer sets for amplifying the whole genome sequence of maize chlorotic mottle virus Based on the existing maize chlorotic mottle virus gene sequence, the inventors of this application designed primers for amplifying the whole genome sequence of maize chlorotic mottle virus. After repeated testing and optimization, a primer set for amplifying the whole genome sequence of maize chlorotic mottle virus was finally selected. This primer set consists of the following 10 pairs of specific primers: MCMV-1-F / MCMV-1-R, MCMV-2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6-R, MCMV-7-F / MCMV-7-R, MCMV-8-F / MCMV-8-R, MCMV-9-F / MCMV-9-R, and MCMV-10-F / MCMV-10-R. The amplified fragments of each primer pair have varying degrees of overlap. The primer sequence information is shown in Table 1.

[0037] Table 1. Primer sequences for amplifying the MCMV whole genome sequence

[0038] Note: F represents the upstream primer, and R represents the downstream primer.

[0039] Example 2: Optimization of amplification conditions for primer sets used to amplify the whole genome sequence of maize chlorotic mottle virus. 1. Weigh 0.1g of Thai maize seeds infected with maize chlorotic mottle virus and extract total RNA using TrizoL reagent.

[0040] 2. Using the RNA obtained in step 1 as a template, perform one-step RT-PCR amplification using the 10 pairs of primers from step 1.

[0041] The reaction volume for one-step RT-PCR amplification was 25 μL, specifically including 3 μL of total RNA and 2 × One Step Mix (Dye Plus) (Nanjing Novizan Biotechnology Co., Ltd., catalog number 7E1352B5). ） 12.5 μL, One Step Enzyme Mix (Nanjing Novizan Biotechnology Co., Ltd., catalog number 7E1352B5) 1.25 μL, upstream primer (10 μmol / L) 1 μL and downstream primer (10 μmol / L) 1 μL, ddH2O 6.25 μL.

[0042] The reaction conditions for one-step RT-PCR amplification were as follows: reverse transcription at 50℃ for 30 min, pre-denaturation at 94℃ for 3 min, denaturation at 94℃ for 30 s, annealing at 51-59℃ for 30-50 s, extension at 72℃ for 60-90 s, 35 cycles, followed by a final extension at 72℃ for 7 min. Each primer pair was amplified under different annealing temperatures (51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃), different annealing times (30 s, 35 s, 40 s, 45 s, 50 s), and different extension times (60 s, 70 s, 80 s, 90 s).

[0043] 3. After PCR amplification, 5 μL of the PCR product was analyzed by 2% agarose gel electrophoresis. The results were observed and recorded using a gel imaging system. The results are as follows: Figure 1 As shown in the figure. Among them, the amplified fragment sizes of primer pairs MCMV-1-F / MCMV-1-R, MCMV-2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6-R, MCMV-7-F / MCMV-7-R, MCMV-8-F / MCMV-8-R, MCMV-9-F / MCMV-9-R, and MCMV-10-F / MCMV-10-R are 247bp, 630bp, 572bp, 533bp, 512bp, 531bp, 589bp, 720bp, 567bp, and 1034bp, respectively.

[0044] After optimization, the optimal annealing temperatures for primer pairs MCMV-1-F / MCMV-1-R, MCMV-2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6-R, MCMV-7-F / MCMV-7-R, MCMV-8-F / MCMV-8-R, MCMV-9-F / MCMV-9-R, and MCMV-10-F / MCMV-10-R were determined to be 56℃, 55℃, 55℃, 55℃, 54℃, 51℃, 54℃, 53℃, 53℃, and 59℃, respectively.

[0045] After optimization, the optimal annealing time for primer pairs MCMV-1-F / MCMV-1-R, MCMV-2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6-R, MCMV-7-F / MCMV-7-R, MCMV-8-F / MCMV-8-R, MCMV-9-F / MCMV-9-R, and MCMV-10-F / MCMV-10-R was determined to be 30 s.

[0046] After optimization, the optimal extension times for primer pairs MCMV-1-F / MCMV-1-R, MCMV-2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6-R, MCMV-7-F / MCMV-7-R, MCMV-8-F / MCMV-8-R, MCMV-9-F / MCMV-9-R, and MCMV-10-F / MCMV-10-R were determined to be 60 s, 60 s, 60 s, 60 s, 60 s, 60 s, 60 s, 60 s, 60 s, and 90 s, respectively.

[0047] Example 3: Detection and identification method of whole genome sequence of maize chlorotic mottle virus 1. Extract total RNA from the virus or sample to be tested.

[0048] 2. Using the RNA obtained in step 1 as a template, one-step RT-PCR amplification was performed using the 10 primer pairs MCMV-1-F / MCMV-1-R, MCMV-2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6-R, MCMV-7-F / MCMV-7-R, MCMV-8-F / MCMV-8-R, MCMV-9-F / MCMV-9-R, and MCMV-10-F / MCMV-10-R from Example 1.

[0049] The one-step RT-PCR amplification detection system and reaction conditions are the same as in Example 2, except that each primer pair uses the optimal annealing temperature, optimal annealing time and optimal extension time from Example 2.

[0050] 3. After PCR amplification, the PCR products were collected and detected by agarose gel electrophoresis.

[0051] 4. The amplified fragments of each primer pair were sequenced using the cloning sequencing method. The determined sequences were compared, analyzed, and assembled to obtain the complete genome sequence of maize chlorotic mottle virus.

[0052] Example 4: Application of the whole genome sequence detection and identification method for maize chlorotic mottle virus in actual sample testing. 1. Using corn seeds suspected of being infected with maize chlorotic mottle virus from a batch of imported Thai corn as the test sample, 0.1g of Thai corn seeds were weighed and total RNA was extracted using TrizoL reagent.

[0053] 2. Using the RNA obtained in step 1 as a template, one-step RT-PCR amplification was performed using the 10 primer pairs MCMV-1-F / MCMV-1-R, MCMV-2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6-R, MCMV-7-F / MCMV-7-R, MCMV-8-F / MCMV-8-R, MCMV-9-F / MCMV-9-R, and MCMV-10-F / MCMV-10-R from Example 1.

[0054] The one-step RT-PCR amplification detection system and reaction conditions are the same as in Example 2, except that each primer pair uses the optimal annealing temperature, optimal annealing time and optimal extension time from Example 2.

[0055] 3. After PCR amplification, take 5 μL of PCR product and detect it by 2% agarose gel electrophoresis. Observe and record the experimental results using a gel imaging system.

[0056] The results are as follows Figure 2 As shown in the figure. Among them, the amplified fragment sizes of primer pairs MCMV-1-F / MCMV-1-R, MCMV-2-F / MCMV-2-R, MCMV-3-F / MCMV-3-R, MCMV-4-F / MCMV-4-R, MCMV-5-F / MCMV-5-R, MCMV-6-F / MCMV-6-R, MCMV-7-F / MCMV-7-R, MCMV-8-F / MCMV-8-R, MCMV-9-F / MCMV-9-R, and MCMV-10-F / MCMV-10-R are 247bp, 630bp, 572bp, 533bp, 512bp, 531bp, 589bp, 720bp, 567bp, and 1034bp, respectively.

[0057] 4. The amplified fragments of each primer pair were sequenced using cloning sequencing. All fragment sequences were bidirectionally sequenced. The determined sequences were aligned and assembled to obtain the full genome sequence of the Thai isolate of maize chlorotic mottle virus, which is 4436 bp in length. Its nucleotide sequence is as follows:

[0058] The complete genome sequence of the maize chlorotic mottle virus isolated from Thailand includes seven protein-coding genes: p32, replicate, replicate-associated protein, p31, p7a, p7b, and coat protein. The composition and map of its complete genome structure are shown in Tables 2 and 3, respectively. Figure 3 As shown.

[0059] Table 2. Genome structure of the Thai isolate of maize chlorotic mottle virus

[0060] 5. The whole genome sequence of the maize chlorotic mottle virus isolate from Thailand obtained in step 4 was analyzed by BLAST in NCBI. The results showed that its nucleotide sequence identity with the whole genome of the maize chlorotic mottle virus isolate from Yunnan, China (GenBank accession number: JQ982468) published in GenBank was over 98.63%. Through the detection and identification of the whole genome sequence of the maize chlorotic mottle virus, it was confirmed that the above-mentioned Thai maize samples carried the maize chlorotic mottle virus.

[0061] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A primer set for amplifying the whole genome sequence of maize chlorotic mottle virus, the primer set comprising primer pair 1 consisting of MCMV-1-F and MCMV-1-R, primer pair 2 consisting of MCMV-2-F and MCMV-2-R, primer pair 3 consisting of MCMV-3-F and MCMV-3-R, primer pair 4 consisting of MCMV-4-F and MCMV-4-R, primer pair 5 consisting of MCMV-5-F and MCMV-5-R, primer pair 6 consisting of MCMV-6-F and MCMV-6-R, primer pair 7 consisting of MCMV-7-F and MCMV-7-R, primer pair 8 consisting of MCMV-8-F and MCMV-8-R, primer pair 9 consisting of MCMV-9-F and MCMV-9-R, and primer pair 10 consisting of MCMV-10-F and MCMV-10-R; The MCMV-1-F is the single-stranded DNA shown in sequence 1; The MCMV-1-R is the single-stranded DNA shown in sequence 2; The MCMV-2-F is the single-stranded DNA shown in sequence 3; The MCMV-2-R is the single-stranded DNA shown in sequence 4; The MCMV-3-F is the single-stranded DNA shown in sequence 5; The MCMV-3-R is the single-stranded DNA shown in sequence 6; The MCMV-4-F is the single-stranded DNA shown in sequence 7; The MCMV-4-R is the single-stranded DNA shown in sequence 8; The MCMV-5-F is the single-stranded DNA shown in sequence 9; The MCMV-5-R is the single-stranded DNA shown in sequence 10; The MCMV-6-F is the single-stranded DNA shown in sequence 11; The MCMV-6-R is the single-stranded DNA shown in sequence 12; The MCMV-7-F is the single-stranded DNA shown in sequence 13; The MCMV-7-R is the single-stranded DNA shown in sequence 14; The MCMV-8-F is the single-stranded DNA shown in sequence 15; The MCMV-8-R is the single-stranded DNA shown in sequence 16; The MCMV-9-F is the single-stranded DNA shown in sequence 17; The MCMV-9-R is the single-stranded DNA shown in sequence 18; The MCMV-10-F is the single-stranded DNA shown in sequence 19; The MCMV-10-R is the single-stranded DNA shown in sequence 20.

2. A kit for amplifying the whole genome sequence of maize chlorotic mottle virus, comprising the primer set as described in claim 1.

3. The use of the primer set of claim 1 or the kit of claim 2 in any of the following: a1) Obtain the complete genome sequence of maize chlorotic mottle virus; a2) Identify whether the virus to be tested is maize chlorotic mottle virus; a3) Detect whether the sample to be tested contains maize chlorotic mottle virus; a4) Evolutionary analysis of maize chlorotic mottle virus; a5) Molecular epidemiological study of maize chlorotic mottle virus.

4. The use of the primer set of claim 1 or the kit of claim 2 in any of the following: b1) Prepare a product for which the complete genome sequence of maize chlorotic mottle virus is obtained; b2) Prepare products to identify whether the virus to be tested is maize chlorotic mottle virus; b3) Prepare products for detecting whether the sample to be tested contains maize chlorotic mottle virus; b4) Prepare products for evolutionary analysis of maize chlorotic mottle virus; b5) Prepare products for molecular epidemiological studies of maize chlorotic mottle virus.

5. Any of the following methods: c1) A method for obtaining the whole genome sequence of a maize chlorotic mottle virus, comprising the following steps: Using the RNA of maize chlorotic mottle virus or a sample containing maize chlorotic mottle virus as a template, one-step RT-PCR amplification was performed using the primer pairs described in claim 1 to obtain amplification products; the amplification products were sequenced, and the sequencing results were compared and analyzed and the sequences were assembled to obtain the whole genome sequence of the maize chlorotic mottle virus. c2) A method for identifying whether a virus to be tested is maize chlorotic mottle virus, comprising the following steps: obtaining the whole genome sequence of the virus to be tested according to the method described in c1); performing BLAST analysis on the whole genome sequence to determine whether the virus to be tested is maize chlorotic mottle virus; c3) A method for detecting whether a sample to be tested contains maize chlorotic mottle virus, comprising the following steps: obtaining the whole genome sequence of the sample to be tested according to the method described in c1); performing BLAST analysis on the whole genome sequence to determine whether the sample to be tested contains maize chlorotic mottle virus.

6. The method according to claim 5, characterized in that: The annealing temperatures for one-step RT-PCR amplification using primer pairs 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 were 56℃, 55℃, 55℃, 54℃, 51℃, 54℃, 53℃, 53℃, and 59℃, respectively.

7. The method according to claim 5, characterized in that: The annealing time for one-step RT-PCR amplification using primer pairs 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 was 30 s.

8. The method according to claim 5, characterized in that: The extension times for one-step RT-PCR amplification using primer pairs 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 were 60 s, 60 s, 60 s, 60 s, 60 s, 60 s, 60 s, 60 s, 60 s, and 90 s, respectively.

9. The method according to any one of claims 5-8, characterized in that: The complete genome sequence of the maize chlorotic mottle virus is shown in sequence 21.

10. The use of the whole genome sequence of the maize chlorotic mottle virus as described in claim 9 in any of the following: d1) Identify whether the virus to be tested is maize chlorotic mottle virus; d2) Detect whether the sample to be tested contains maize chlorotic mottle virus; d3) Evolutionary analysis of maize chlorotic mottle virus; d4) Molecular epidemiological study of maize chlorotic mottle virus; d5) Prepare a product to identify whether the virus to be tested is maize chlorotic mottle virus; d6) Prepare a product for detecting whether the sample to be tested contains maize chlorotic mottle virus; d7) Prepare products for evolutionary analysis of maize chlorotic mottle virus; d8) Prepare products for molecular epidemiological research on maize chlorotic mottle virus.

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