Corn dwarf mosaic virus polygene joint detection kit and application thereof

Abstract

The invention discloses a corn dwarf mosaic virus polygene joint detection kit and application thereof. Specific primers are respectively designed by taking a CP gene and a CI gene of the maize dwarf mosaic virus as targets, and two pairs of specific primer combinations for detecting the CP gene and the CI gene are screened out after repeated tests and verification. And the optimal reaction system and reaction conditions for the multi-gene joint detection of the maize dwarf mosaic virus are determined after numerous times of optimization of parameters such as annealing temperature, primer concentration and the like, so that the multi-gene joint detection method of the maize dwarf mosaic virus is successfully established. Experiments prove that compared with conventional RT-PCR (Reverse Transcription-Polymerase Chain Reaction) taking a single gene as a target, the detection method is higher in accuracy and higher in reliability. The invention provides a more accurate and more reliable method for MDMV detection, and the method has very wide application prospects in entry and exit port quarantine and agricultural production.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a multi-gene combined detection kit for corn dwarf mosaic virus and an application thereof. Background Art

[0002] Corn is an annual herbaceous plant belonging to the Poaceae family. It is widely planted throughout the world due to its important economic value. It is an important food crop, cash crop and industrial raw material in my country. Viral diseases of corn are one of the most serious diseases in corn production. Among them, maize dwarf mosaic virus (MDMV) is an important virus on corn. In 2021, it was added as a quarantine pest for imported plants in my country. The MDMV virus particles are curved and linear, about 750nm long and 12-15nm in diameter. Wind wheel-shaped and scroll-shaped inclusion bodies can be observed in plant host cells infected with the virus by electron microscopy. The genome of MDMV encodes a polyprotein of approximately 338kD, which is cleaved by the three proteases it contains to form 10 proteins with multiple functions. MDMV has a narrow host range and only infects some grass crops, such as corn and sorghum. After the virus infects the host, many oval chlorotic spots initially appear between the fine veins at the base of the new leaves of the seedlings, arranged in dotted lines, and then gradually form solid lines, forming mosaic symptoms, the leaves turn yellow, brown, purple or dry, and the plants become dwarfed.

[0003] At present, the main methods for MDMV detection include serological and molecular biological methods. The commercial ELISA kit is used to detect MDMV, which has the advantages of simple operation and large sample volume. However, due to the limitation of sensitivity, the ELISA method is often unable to accurately detect samples with low MDMV content, resulting in false negative results. Compared with the ELISA method, the molecular biological method has the significant advantages of fast time, high sensitivity and strong specificity in detecting MDMV. MDMV belongs to RNA virus, which mutates rapidly. A single gene fragment is only a part of the whole genome of the virus. If the fragment undergoes a large mutation, it cannot be accurately detected. Multi-gene joint detection targets two or more target genes of the virus, which can effectively overcome the problem of inaccurate detection caused by mutation of a single gene fragment. Therefore, the multi-gene joint detection method has higher accuracy and reliability. There has been no relevant report on the multi-gene joint detection of MDMV, let alone a special multi-gene joint detection kit. Summary of the invention

[0004] The purpose of the present invention is to provide a corn dwarf mosaic virus multi-gene combined detection kit and a detection method thereof which are simple to operate, have strong specificity, high sensitivity and good reproducibility, so as to overcome the defects and shortcomings of the prior art, such as poor specificity, low accuracy (false positive or false negative results), easy to produce nonspecific amplification, easy to contaminate, and complicated operation, so as to realize the rapid and accurate identification of corn dwarf mosaic virus at entry and exit ports and agricultural production.

[0005] In order to achieve the above object, the present invention first provides a primer set for detecting corn dwarf mosaic virus.

[0006] The primer set for detecting corn dwarf mosaic virus provided by the present invention comprises a primer pair for detecting the CP gene of corn dwarf mosaic virus and a primer pair for detecting the CI gene of corn dwarf mosaic virus;

[0007] The primer pair for detecting the CP gene of maize dwarf mosaic virus is composed of primer MDMV-F1 and primer MDMV-R1;

[0008] The primer pair for detecting the CI gene of maize dwarf mosaic virus is composed of primer MDMV-F2 and primer MDMV-R2;

[0009] The primer MDMV-F1 is a single-stranded DNA molecule shown in sequence 1;

[0010] The primer MDMV-R1 is a single-stranded DNA molecule shown in sequence 2;

[0011] The primer MDMV-F2 is a single-stranded DNA molecule shown in sequence 3;

[0012] The primer MDMV-R2 is a single-stranded DNA molecule shown in Sequence 4.

[0013] In the above primer set, the molar ratio of the primer MDMV-F1, the primer MDMV-R1, the primer MDMV-F2 and the primer MDMV-R2 is 1:1:4:4.

[0014] In order to achieve the above object, the present invention also provides a kit for detecting corn dwarf mosaic virus.

[0015] The kit for detecting corn dwarf mosaic virus provided by the present invention contains the above primer set; the function of the kit is any one of the following a1)-a3):

[0016] a1) Identifying or assisting in identifying whether the virus to be tested is maize dwarf mosaic virus;

[0017] a2) Detecting or assisting in detecting whether the sample to be tested contains maize dwarf mosaic virus;

[0018] a3) Differentiate or assist in differentiating maize dwarf mosaic virus from other viruses.

[0019] Furthermore, the kit also includes reverse transcription reagents (such as RT Mix, 5×RT Buffer) and PCR amplification reagents (such as 2×Multiplex Buffer, Multiplex DNA Polymerase).

[0020] Furthermore, the kit also includes a negative control (such as a corn leaf sample that does not contain corn dwarf mosaic virus) and a positive control (such as a corn leaf sample that contains corn dwarf mosaic virus).

[0021] In order to achieve the above object, the present invention also provides a new use of the above primer set or the above kit.

[0022] The present invention provides the above primer set or the above kit in any one of the following b1)-b6):

[0023] b1) Identifying or assisting in the identification of whether the virus to be tested is maize dwarf mosaic virus;

[0024] b2) Detecting or assisting in detecting whether the sample to be tested contains maize dwarf mosaic virus;

[0025] b3) Differentiate or assist in differentiating maize dwarf mosaic virus from other viruses;

[0026] b4) preparing a product for identifying or assisting in identifying whether the virus to be tested is maize dwarf mosaic virus;

[0027] b5) preparing a product for detecting or assisting in detecting whether a sample to be tested contains corn dwarf mosaic virus;

[0028] b6) Prepare products that can distinguish or assist in distinguishing corn dwarf mosaic virus from other viruses.

[0029] In order to achieve the above object, the present invention also provides a method for detecting or assisting in detecting whether a sample to be tested contains corn dwarf mosaic virus.

[0030] The method for detecting or assisting in detecting whether a sample to be tested contains corn dwarf mosaic virus provided by the present invention comprises the following steps: using the cDNA of the sample to be tested as a template, adopting the above primer set to perform PCR amplification to obtain a PCR product; and judging whether the sample to be tested contains corn dwarf mosaic virus by detecting the size of the PCR product.

[0031] In the above method, the method of judging whether the sample to be tested contains corn dwarf mosaic virus by detecting the size of PCR products is: if PCR amplification obtains a target fragment with a size of 343bp and / or 490bp, the sample to be tested contains corn dwarf mosaic virus, otherwise the sample to be tested does not contain corn dwarf mosaic virus.

[0032] In order to achieve the above objectives, the present invention finally provides a method for distinguishing or assisting in distinguishing corn dwarf mosaic virus from other viruses.

[0033] The method for distinguishing or assisting in distinguishing corn dwarf mosaic virus from other viruses provided by the present invention comprises the following steps: using the cDNA of the sample to be tested as a template, using the above primer set to perform PCR amplification to obtain a PCR product; and determining whether the virus to be tested is corn dwarf mosaic virus or other viruses by detecting the size of the PCR product.

[0034] In the above method, the method of judging whether the virus to be tested is corn dwarf mosaic virus or other viruses by detecting the size of PCR products is: if PCR amplification obtains a target fragment with a size of 343bp and / or 490bp, the virus to be tested is corn dwarf mosaic virus, otherwise the virus to be tested is other viruses.

[0035] In some embodiments, the PCR amplification obtains target fragments with sizes of 343 bp and 490 bp.

[0036] In some embodiments, the nucleotide sequence of the target fragment with a size of 343 bp is shown in Sequence 5.

[0037] In some embodiments, the nucleotide sequence of the target fragment with a size of 490 bp is shown in Sequence 6.

[0038] In any of the above methods, the cDNA of the sample to be tested is obtained by reverse transcription using RNA extracted from the sample to be tested as a template.

[0039] In any of the above methods, the final concentrations of the primer MDMV-F1 and the primer MDMV-R1 in the PCR amplification system are both 0.2 μmol / L.

[0040] The final concentrations of the primer MDMV-F2 and the primer MDMV-R2 in the PCR amplification system are both 0.8 μmol / L.

[0041] In some embodiments, the reaction system for PCR amplification is as follows: 2 μL of cDNA, 10.5 μL of MDMV-F1 with a concentration of 10 μmol / L, 10.5 μL of MDMV-R1 with a concentration of 10 μmol / L, 2 μL of MDMV-F2 with a concentration of 10 μmol / L, 2 μL of MDMV-R2 with a concentration of 10 μmol / L, 12.5 μL of 2×Multiplex buffer, 1 μL of Multiplex DNA Polymerase, and 4.5 μL of RNase free ddH2O.

[0042] In any of the above methods, the annealing temperature of the PCR amplification is 47°C.

[0043] In some embodiments, the reaction conditions of the PCR amplification are as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 47°C for 90 s, extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for 10 min.

[0044] Any of the other viruses mentioned above is at least one of the following viruses: sugarcane mosaic virus (SCMV), Johnson grass mosaic virus (JGMV), wheat streak mosaic virus (WSMV), maize chlorotic mottle virus (MCMV), barley streak mosaic virus (BSMV), tobacco mosaic virus (TMV), southern rice black-streaked dwarf virus (SRBSDV), and cucumber mosaic virus (CMV).

[0045] Any of the above-mentioned samples to be tested may be samples from different tissue parts (such as leaves) of gramineous crops (such as sorghum, barley, and corn).

[0046] In some embodiments, the sample to be tested is a corn leaf.

[0047] The present invention takes the CP gene and CI gene of maize dwarf mosaic virus as targets, designs specific primers respectively, and screens out two pairs of specific primer combinations for detecting the CP gene and the CI gene after repeated tests and verifications, and determines the optimal reaction system and reaction conditions for the combined detection of maize dwarf mosaic virus multi-genes after numerous optimizations of parameters such as annealing temperature and primer concentration, and successfully establishes a combined detection method for maize dwarf mosaic virus multi-genes. Experiments have shown that the detection method is more accurate and more reliable than conventional RT-PCR with a single gene as a target. The present invention provides a more accurate and more reliable method for the detection of MDMV, and has a wide range of application prospects in entry and exit port quarantine and agricultural production.

[0048] Compared with the prior art, the multi-gene joint detection method of maize dwarf mosaic virus provided by the present invention has the following beneficial effects:

[0049] (1) The present invention is a special dual RT-PCR detection method, comprising two sets of specific primers, one set targeting the CP gene of maize dwarf mosaic virus, and the other set targeting the CI gene of maize dwarf mosaic virus. Therefore, the present invention can detect both the CP and CI genes of maize dwarf mosaic virus at one time. When the variation of maize dwarf mosaic virus is small, the detection results of the two sets of primers are both positive; when the variation of maize dwarf mosaic virus is large, the detection result of at least one set of primers is positive. Compared with the conventional RT-PCR molecular detection method targeting a single gene, the present invention has higher accuracy and better reliability.

[0050] (2) The present invention gives full play to the advantages of high specificity and sensitivity of multi-gene detection. On the one hand, it avoids non-specific amplification. The detection results of sugarcane mosaic virus (SCMV), Johnson grass mosaic virus (JGMV), wheat streak mosaic virus (WSMV), maize chlorotic mottle virus (MCMV), barley streak mosaic virus (BSMV), tobacco mosaic virus (TMV), southern rice black streak dwarf virus (SRBSDV) and cucumber mosaic virus (CMV) are all negative, and there is no specific amplification. On the other hand, the sensitivity reaches the minimum and can detect 10 -4 times the maize dwarf mosaic virus (MDMV) sample.

[0051] (3) The present invention has good repeatability and can be effectively used for the detection of corn dwarf mosaic virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The results of multi-gene combined detection and single gene detection in Example 2. Wherein, M: DNA molecular weight standard; 1: CP single gene detection; 2: CI single gene detection; 3: multi-gene combined detection; 4: negative control of CP single gene detection; 5: negative control of CI single gene detection; 6: negative control of multi-gene combined detection.

[0053] Figure 2 The annealing temperature of the multi-gene joint detection in Example 2 is optimized. Wherein, M: DNA molecular weight standard; 1-8: annealing temperatures are 45°C, 47°C, 49°C, 51°C, 53°C, 55°C, 57°C, and 59°C respectively; 9: negative control.

[0054] Figure 3 The primer concentrations for the multi-gene combined detection in Example 2 are optimized. Wherein, M: DNA molecular weight standard; 1-8: combination G1-G8; 9: negative control.

[0055] Figure 4 It is the specificity determination of Example 3. Wherein, M: DNA molecular weight standard; 1: maize dwarf mosaic virus (MDMV); 2-9: sugarcane mosaic virus (SCMV), Johnson grass mosaic virus (JGMV), wheat streak mosaic virus (WSMV), maize chlorotic mottle virus (MCMV), barley streak mosaic virus (BSMV), tobacco mosaic virus (TMV), southern rice black streaked dwarf virus (SRBSDV), cucumber mosaic virus (CMV); 10: negative control.

[0056] Figure 5The sensitivity test of Example 4. a: multi-gene joint detection. b: PCR detection based on CI gene. c: PCR detection based on CP gene. Wherein, M: DNA molecular weight standard; 1-8: 10 0 , 10 -1 times, 10 -2 times, 10 -3 times, 10 -4 times, 10 -5 times, 10 -6 Times and 10 -7 times; 9: negative control.

[0057] Figure 6 This is the repeatability determination of Example 5. Wherein, M: DNA molecular weight standard; 1-5: MDMV positive samples; 6: negative control.

[0058] Figure 7 The results of multi-gene joint detection in Example 6. a: 1-15 are corn samples imported from Fuzhou Port, Fujian. b: 1-6 are barley samples imported from Fuzhou Port, Fujian, and 7-15 are sorghum samples imported from Fuzhou Port, Fujian. c: 1-10 are sorghum samples imported from Xiamen Port, Fujian, and 11-15 are corn samples imported from Xiamen Port, Fujian. d: 1-15 are sorghum samples imported from Shanghai Port. Among them, M: DNA molecular weight standard; 16: negative control; 17: blank control (RNase freeddH2O).

[0059] Figure 8 The results of conventional RT-PCR detection in Example 6. a: 1-15 are corn samples imported from Fuzhou Port, Fujian. b: 1-6 are barley samples imported from Fuzhou Port, Fujian, and 7-15 are sorghum samples imported from Fuzhou Port, Fujian. c: 1-10 are sorghum samples imported from Xiamen Port, Fujian, and 11-15 are corn samples imported from Xiamen Port, Fujian. d: 1-15 are sorghum samples imported from Shanghai Port. Among them, M: DNA molecular weight standard; 16: negative control; 17: blank control (RNase freeddH2O). DETAILED DESCRIPTION

[0060] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0061] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. The negative controls in the following examples, unless otherwise specified, are all corn leaf samples that do not contain corn dwarf mosaic virus.

[0062] Example 1. Primer set and kit for combined detection of multi-genes of maize dwarf mosaic virus

[0063] 1. Maize Dwarf Mosaic Virus Multi-Gene Joint Detection Primer Set

[0064] According to the genome sequence of maize dwarf mosaic virus, after a large number of sequence comparisons and analyses, different specific primers are designed for target genes, and the specific sequences are shown in Table 1. After repeated tests and optimizations, a pair of specific amplification primers MDMV-F1 / MDMV-R1 designed for detecting the coat protein (CP) gene of maize dwarf mosaic virus and a pair of specific amplification primers MDMV-F2 / MDMV-R2 designed for detecting the columnar inclusion protein (CI) gene of maize dwarf mosaic virus were finally screened out.

[0065] Table 1. Primer sequences

[0066]

[0067] 2. Maize Dwarf Mosaic Virus Multi-Gene Joint Detection Kit (10 times detection)

[0068] The corn dwarf mosaic virus multi-gene joint detection kit of the present invention comprises the following reagents:

[0069] 1. Primer MDMV-F1 solution with a concentration of 10 μmol / L, 1 tube (30 μL);

[0070] 2. Primer MDMV-R1 solution with a concentration of 10 μmol / L, 1 tube (30 μL);

[0071] 3. Primer MDMV-F2 solution with a concentration of 10 μmol / L, 1 tube (30 μL);

[0072] 4. Primer MDMV-R2 solution with a concentration of 10 μmol / L, 1 tube (30 μL);

[0073] 5. RT Mix (Genstar; ZA230-101), 1 tube (20 μL);

[0074] 6. 5×RT Buffer (Genstar; ZA230-102), 1 tube (50 μL);

[0075] 7. 2×Multiplex Buffer (Vazyme; PM202-01-AA), 1 tube (200 μL);

[0076] 8. Multiplex DNA Polymerase (Vazyme; PM202-01-AB) at a concentration of 10 U / μL, 1 tube (20 μL);

[0077] 9. Positive control sample containing maize dwarf mosaic virus, 1 tube (100 μL);

[0078] 10. Negative control sample without maize dwarf mosaic virus, 1 tube (100 μL);

[0079] 11. RNase free ddH2O, 1 tube (1mL).

[0080] Example 2: Detection method of the multi-gene combined detection kit for maize dwarf mosaic virus

[0081] 1. Establishment of a multi-gene joint detection method for maize dwarf mosaic virus

[0082] 1. Reverse transcription reaction: add 3μL total RNA of the sample to be tested, 1μL RT Mix, 4μL 5×RT Buffer, and 12μL RNase free ddH2O into a PCR tube; then react in a PCR instrument under the following conditions: 37℃ for 2min, 50℃ for 15min, and 85℃ for 2min, then cool to room temperature to synthesize cDNA.

[0083] 2. PCR reaction: add 2 μL of cDNA synthesized in step 1, 12.5 μL of 2×Multiplex Buffer, 1 μL of Multiplex DNA Polymerase at a concentration of 10 U / μL, 0.5 μL of MDMV-F1 at a concentration of 10 μmol / L, 0.5 μL of MDMV-R1 at a concentration of 10 μmol / L, 2 μL of MDMV-F2 at a concentration of 10 μmol / L, 2 μL of MDMV-R2 at a concentration of 10 μmol / L, and 4.5 μL of RNase free ddH2O into a PCR tube to make the total reaction volume 25 μL; then react in a PCR instrument under the following reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 47°C for 90 s, and extension at 72°C for 1 min, for a total of 35 cycles; after the last cycle, continue extension at 72°C for 10 min, the reaction is completed, and the PCR product is obtained.

[0084] 3. Electrophoresis detection of PCR products: Take 5 μL of the PCR product obtained in step 2 and perform detection by 1.5% agarose gel electrophoresis. Observe and record the experimental results on a gel imaging system: If the PCR amplification obtains target fragments of 343 bp (nucleotide sequence as shown in Sequence 5) and 490 bp (nucleotide sequence as shown in Sequence 6), the sample to be tested contains corn dwarf mosaic virus; otherwise, the sample to be tested does not contain corn dwarf mosaic virus.

[0085] 2. Optimization of the multi-gene joint detection method for maize dwarf mosaic virus

[0086] 1. Comparison of the effects of combined gene testing and single gene testing

[0087] RNA was extracted from corn leaves containing maize dwarf mosaic virus and reverse transcribed to obtain cDNA. Using cDNA as a template, PCR amplification was performed with two pairs of primers MDMV-F1 / MDMV-R1 and MDMV-F2 / MDMV-R2 to obtain PCR products. 5 μL of PCR product was tested by 1.5% agarose gel electrophoresis to observe the gel electrophoresis results. At the same time, PCR amplification was performed with a single pair of primers MDMV-F1 / MDMV-R1 or MDMV-F2 / MDMV-R2 as a control.

[0088] The PCR reaction system for combined gene detection (total volume 25 μL): cDNA 2 μL, MDMV-F1 1 μL at a concentration of 10 μmol / L, MDMV-R1 1 μL at a concentration of 10 μmol / L, MDMV-F2 1 μL at a concentration of 10 μmol / L, MDMV-R2 1 μL at a concentration of 10 μmol / L, 2×Multiplex buffer 12.5 μL, Multiplex DNA Polymerase 1 μL, RNasefree ddH2O 5.5 μL. The PCR reaction conditions were as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 49°C annealing for 90 s, 72°C extension for 1 min, for a total of 35 cycles; 72°C extension for 10 min.

[0089] The PCR reaction system (total volume 25 μL) for single gene detection (used to detect the CP gene of maize dwarf mosaic virus) was as follows: 2 μL of cDNA, 1 μL of MDMV-F1 at a concentration of 10 μmol / L, 1 μL of MDMV-R1 at a concentration of 10 μmol / L, 12.5 μL of 2×PCR Mix (Promega, M7122), and 8.5 μL of RNase free ddH2O. The PCR reaction conditions were as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 51°C annealing for 45 s, 72°C extension for 1 min, for a total of 35 cycles; 72°C extension for 10 min.

[0090] The PCR reaction system (total volume 25 μL) for single gene detection (used to detect CI gene of maize dwarf mosaic virus) was as follows: 2 μL of cDNA, 1 μL of MDMV-F2 at a concentration of 10 μmol / L, 1 μL of MDMV-R2 at a concentration of 10 μmol / L, 12.5 μL of 2×PCR Mix (Promega, M7122), and 8.5 μL of RNase free ddH2O. The PCR reaction conditions were as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 51°C annealing for 45 s, 72°C extension for 1 min, for a total of 35 cycles; and 72°C extension for 10 min.

[0091] The results are as follows Figure 1 As shown in the figure, the results show that the multi-gene joint detection system amplified two fragments of the CP gene and CI gene with the expected size, which were 343bp and 490bp respectively, which was consistent with the amplification effect of the two genes alone, and there was no non-specific amplification. In addition, the two target fragments were significantly different in size, which was convenient for separation and observation in the multi-gene joint detection, and the amplification effect was good.

[0092] 2. Optimization of annealing temperature

[0093] RNA was extracted from corn leaves containing maize dwarf mosaic virus, and cDNA was obtained by reverse transcription. Using cDNA as a template, PCR amplification was performed according to the multi-gene joint detection method in step 1, and the annealing temperature was set to 8 temperature gradients of 45°C, 47°C, 49°C, 51°C, 53°C, 55°C, 57°C, and 59°C, respectively. The optimal annealing temperature was selected according to the gel electrophoresis results.

[0094] The results are as follows Figure 2 As shown, the results show that clear and specific target fragments can be obtained under 8 temperature gradients, among which 47°C is relatively good.

[0095] 3. Primer concentration optimization

[0096] RNA was extracted from corn leaves containing maize dwarf mosaic virus, and cDNA was obtained by reverse transcription. Using cDNA as a template, PCR amplification was performed according to the multi-gene joint detection method in step 1. On the basis of optimizing the annealing temperature reaction conditions, in order to ensure that the brightness and effect of the amplified fragments of the two pairs of primers were basically the same, the dosage of the two pairs of primers was adjusted, and different primer concentration combinations were set (Table 2). According to the gel electrophoresis results, the optimal concentration combination of the two pairs of primers was found.

[0097] Table 2. Primer combinations with different concentration gradients

[0098]

[0099]

[0100] The results are as follows Figure 3 The results showed that the G2 combination had the best effect, that is, when the amount of MDMV-F1 / MDMV-R1 was 0.5 μL each and the amount of MDMV-F2 / MDMV-R2 was 2 μL each, the brightness and effect of the two amplified target fragments were basically the same.

[0101] After the above optimization, the optimal reaction system of the multi-gene joint detection method of maize dwarf mosaic virus of the present invention is as follows: cDNA 2μL, MDMV-F10.5μL with a concentration of 10μmol / L, MDMV-R10.5μL with a concentration of 10μmol / L, MDMV-F22μL with a concentration of 10μmol / L, MDMV-R22μL with a concentration of 10μmol / L, 2×Multiplex buffer 12.5μL, Multiplex DNA Polymerase 1μL, RNase free ddH2O4.5μL. Among them, the final concentrations of MDMV-F1 and MDMV-R1 in the reaction system are both 0.2μmol / L, and the final concentrations of MDMV-F2 and MDMV-R2 in the reaction system are both 0.8μmol / L.

[0102] The optimal reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 47°C for 90 s, and extension at 72°C for 1 min, for a total of 35 cycles; and extension at 72°C for 10 min.

[0103] Example 3: Specificity determination of the multi-gene combined detection kit for maize dwarf mosaic virus

[0104] Corn leaf samples containing maize dwarf mosaic virus (MDMV), sugarcane mosaic virus (SCMV), Johnson grass mosaic virus (JGMV), wheat streak mosaic virus (WSMV), maize chlorotic mottle virus (MCMV), barley streak mosaic virus (BSMV), tobacco mosaic virus (TMV), southern rice black-streaked dwarf virus (SRBSDV), and cucumber mosaic virus (CMV) were used as materials, RNA was extracted and then reverse transcribed to obtain cDNA, and then the cDNA was used as a template, and the kit in Example 1 was used to perform detection according to the multi-gene joint detection method optimized in Example 2 to determine the specificity of the multi-gene joint detection kit.

[0105] The results are as follows Figure 4As shown, the results show that the kit of the present invention can only simultaneously amplify the corresponding two target fragments on the positive sample containing MDMV, and no non-specific amplification occurs, while the corresponding target fragments are not amplified in the positive samples containing SCMV, JGMV, WSMV, MCMV, BSMV, TMV, SRBSDV or CMV. At the same time, after purifying and sequencing the PCR products that amplified the corresponding target fragments, it was found that the measured sequence results were compared with the reported MDMV sequence, and the consistency was as high as more than 94%, further indicating that the kit of the present invention has strong specificity.

[0106] Example 4: Sensitivity determination of the multi-gene combined detection kit for maize dwarf mosaic virus

[0107] RNA was extracted from corn leaves containing maize dwarf mosaic virus and reverse transcribed to obtain cDNA. The cDNA was diluted in 10-fold gradients, and the original solution was diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 times, and then using the cDNA solutions diluted at different times as templates, respectively, using the kit in Example 1, according to the optimized multi-gene joint detection method in Example 2 to perform detection, the sensitivity of the multi-gene joint detection kit was determined, and conventional RT-PCR was performed on a single gene to determine the sensitivity of single gene detection, and the sensitivities of the two were compared.

[0108] The results are as follows Figure 5 The results show that the sensitivity of the kit of the present invention is 10 -4 times cDNA ( Figure 5 a) The sensitivity of the conventional RT-PCR detection method using CI gene is 10 -5 times cDNA ( Figure 5 b) The sensitivity of the conventional RT-PCR detection method of CP gene is 10 -3 times cDNA ( Figure 5 c). This indicates that the kit of the present invention has high sensitivity.

[0109] Example 5: Repeatability determination of the multi-gene combined detection kit for maize dwarf mosaic virus

[0110] RNA was extracted from corn leaves containing maize dwarf mosaic virus, and cDNA was obtained by reverse transcription. Using cDNA as a template, the kit in Example 1 was used to perform 5 repeated tests according to the optimized multi-gene joint detection method in Example 2 to determine the repeatability of the multi-gene joint detection kit.

[0111] The results are as follows Figure 6 As shown, the results show that the results of 5 repeated tests are consistent, and two target fragments with expected sizes can be amplified simultaneously, indicating that the kit of the present invention has good repeatability.

[0112] Example 6: Application of the multi-gene joint detection method for maize dwarf mosaic virus

[0113] Corn samples, sorghum samples and barley samples imported from Fuzhou, Fujian, Xiamen and Shanghai ports were used as samples to be tested (a total of 60 samples), RNA of the samples to be tested was extracted, and cDNA was obtained by reverse transcription. Then, cDNA was used as a template, and the kit in Example 1 was used to perform detection according to the optimized multi-gene joint detection method in Example 2. Conventional RT-PCR and DAS-ELISA methods were used for verification.

[0114] The specific steps of the conventional RT-PCR method are as follows:

[0115] 1. Reverse transcription reaction: Plant RNA Kit was used to extract total RNA from the sample. The extraction method was carried out according to the instructions of the kit. 3 μL of total RNA of the sample to be tested, 1 μL of RT Mix, 4 μL of 5×RTBuffer, and 12 μL of RNase free ddH2O were added to the PCR tube and reacted in a PCR instrument. The reaction conditions were as follows: 37°C for 2 min, 50°C for 15 min, and 85°C for 2 min. The reaction was cooled to room temperature and cDNA was synthesized.

[0116] 2. PCR reaction: Add 2 μL of cDNA synthesized in step 1, 1 μL of 10 μmol / L forward primer CP482-F (5'-YGCATCTCCAACTTTCAGACA-3'), 1 μL of 10 μmol / L reverse primer (5'-CCTCACTCACTTGCVGACA-3'), 12.5 μL of 2×PCR Mix, and 8.5 μL of RNase free ddH2O to the PCR tube. Conventional PCR amplification reaction conditions are as follows: 94°C, 3 min; 94°C 30 s, 49°C 45 s, 72°C 1 min, 35 cycles in total; 72°C extension for 10 min. After the reaction is completed, take 5 μL of PCR product and detect it by 1.5% agarose gel electrophoresis. If the PCR amplification obtains a target fragment of 482 bp, the sample to be tested contains corn dwarf mosaic virus, otherwise the sample to be tested does not contain corn dwarf mosaic virus.

[0117] The specific steps of DAS-ELISA are as follows:

[0118] 1. Coating antibody: dilute the MDMV antibody to the working concentration (200 times) with coating buffer, add to the wells of the ELISA plate, 100 μL / well, and incubate at 37°C for 2 h.

[0119] 2. Add samples: Pour out the coated antibody solution in the ELISA plate and wash three times with PBST buffer; add 100 μL of sample extract to the wells of the ELISA plate, 100 μL / well, and incubate at 37°C for 2 hours or 4°C overnight.

[0120] 3. Add enzyme-labeled antibody: dilute the enzyme-labeled antibody to the working concentration (200 times) with enzyme-labeled antibody buffer, add it to the wells of the ELISA plate, 100 μL / well, and incubate at 37°C for 2 hours.

[0121] 4. Add substrate: Pour out the enzyme-labeled antibody solution in the ELISA plate and wash three times with PBST buffer; prepare the substrate pNPP with substrate buffer to a final concentration of 1 mg / mL (prepare immediately before use), add it to the wells of the ELISA plate, 100 μL / well, and place it at room temperature away from light for about 30-60 minutes.

[0122] 5. Read the absorbance (OD) at 405 nm using an ELISA reader. 405nm ). If the sample OD 405nm Value / negative control OD 405nm If the value is >2, the sample to be tested contains maize dwarf mosaic virus, otherwise the sample to be tested does not contain maize dwarf mosaic virus.

[0123] The results are shown in Table 3. Figure 7 and Figure 8 The results of the multi-gene joint detection method of the present invention showed that 8 samples out of 60 samples were able to simultaneously amplify 2 specific target fragments of 343 bp and 490 bp in size, and 52 samples did not amplify the target fragments ( Figure 7 ); The results of conventional RT-PCR detection method showed that 7 samples out of 60 samples were able to amplify the target fragment of 482bp, and 53 samples did not amplify the target fragment ( Figure 8 ). Qingke Biotechnology Co., Ltd. was commissioned to sequence the PCR products of the samples that were not detected by the conventional PT-PCR method but detected by the multi-gene joint detection method of the present invention. The sequence comparison results confirmed that the sample did contain MDMV. The above results show that the multi-gene joint detection method of the present invention has a higher detection rate and can be used for the detection and identification of MDMV more accurately and efficiently.

[0124] Table 3. Test results of actual samples (corn, sorghum and barley)

[0125]

[0126]

[0127] Note: - indicates the test result is negative; + indicates the test result is positive.

[0128] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A primer set for detecting maize dwarf mosaic virus, the primer set comprising a primer pair for detecting the CP gene of maize dwarf mosaic virus and a primer pair for detecting the CI gene of maize dwarf mosaic virus; The primer pair for detecting the CP gene of maize dwarf mosaic virus is composed of primer MDMV-F1 and primer MDMV-R1; The primer pair for detecting the CI gene of maize dwarf mosaic virus is composed of primer MDMV-F2 and primer MDMV-R2; The primer MDMV-F1 is a single-stranded DNA molecule shown in sequence 1; The primer MDMV-R1 is a single-stranded DNA molecule shown in sequence 2; The primer MDMV-F2 is a single-stranded DNA molecule shown in sequence 3; The primer MDMV-R2 is a single-stranded DNA molecule shown in Sequence 4.

2. The primer set according to claim 1, characterized in that: The molar ratio of the primer MDMV-F1, the primer MDMV-R1, the primer MDMV-F2 and the primer MDMV-R2 is 1:1:4:

4.

3. A kit for detecting maize dwarf mosaic virus, the kit comprising the primer set according to claim 1 or 2; the function of the kit is any one of the following a1)-a3): a1) Identifying or assisting in identifying whether the virus to be tested is maize dwarf mosaic virus; a2) Detecting or assisting in detecting whether the sample to be tested contains maize dwarf mosaic virus; a3) Differentiate or assist in differentiating maize dwarf mosaic virus from other viruses.

4. The kit according to claim 3, characterized in that: The kit also includes a reverse transcription reagent and a PCR amplification reagent.

5. The kit according to claim 3 or 4, characterized in that: The kit also includes a negative control and a positive control.

6. The primer set according to claim 1 or 2 or the kit according to any one of claims 3 to 5 is any one of the following b1) to b6): b1) Identifying or assisting in the identification of whether the virus to be tested is maize dwarf mosaic virus; b2) Detecting or assisting in detecting whether the sample to be tested contains maize dwarf mosaic virus; b3) Differentiate or assist in differentiating maize dwarf mosaic virus from other viruses; b4) preparing a product for identifying or assisting in identifying whether the virus to be tested is maize dwarf mosaic virus; b5) preparing a product for detecting or assisting in detecting whether a sample to be tested contains corn dwarf mosaic virus; b6) Prepare products that distinguish or assist in distinguishing corn dwarf mosaic virus from other viruses.

7. A method for detecting or assisting in detecting whether a sample to be tested contains corn dwarf mosaic virus, comprising the following steps: using the cDNA of the sample to be tested as a template, using the primer set described in claim 1 or 2 to perform PCR amplification to obtain a PCR product; and determining whether the sample to be tested contains corn dwarf mosaic virus by detecting the size of the PCR product.

8. A method for distinguishing or assisting in distinguishing corn dwarf mosaic virus from other viruses, comprising the following steps: using the cDNA of the sample to be tested as a template, using the primer set described in claim 1 or 2 to perform PCR amplification to obtain a PCR product; and determining whether the virus to be tested is corn dwarf mosaic virus or other viruses by detecting the size of the PCR product.

9. The method according to claim 7 or 8, characterized in that: The final concentrations of the primer MDMV-F1 and the primer MDMV-R1 in the PCR amplification system are both 0.2 μmol / L; The final concentrations of the primer MDMV-F2 and the primer MDMV-R2 in the PCR amplification system are both 0.8 μmol / L.

10. The method according to claim 8 or 9, characterized in that: The annealing temperature of the PCR amplification was 47°C.

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