A multiplex RT-PCR primer set, kit and detection method for simultaneously detecting WSMV, MDMV, MCMV and WHPV

Abstract

The present invention discloses a multiplex RT-PCR primer set, kit and detection method for simultaneously detecting WSMV, MDMV, MCMV and WHPV, belonging to the technical field of plant quarantine. The primer set, kit and detection method of the present invention can simultaneously detect four viruses, namely WSMV, MDMV, MCMV and WHPV at one time. Through a series of condition exploration and optimization, an optimal reaction condition is developed, which greatly improves the accuracy and sensitivity of detection, shortens the detection cycle from the original 4 days to 1-2 days, speeds up the detection process, provides technical support for shortening the detection cycle of inspection and quarantine departments, and lays a foundation for accurately and rapidly identifying pathogens in the wild.

Description

Technical Field

[0001] The present invention relates to the technical field of plant quarantine, and more particularly to a multiplex RT-PCR primer set, a kit and a detection method for simultaneously detecting WSMV, MDMV, MCMV and WHPV. Background Art

[0002] In recent years, viral diseases of grains such as corn and wheat have occurred severely, causing huge economic losses. Among them, there are reports of the harms caused by 4 kinds of viruses, namely Wheat streak mosaic virus (WSMV), Maize dwarf mosaicvirus (MDMV), Machlomovirus zeae (MCMV) and wheatmosaic virus (WHPV). Since the occurrence of viral diseases is difficult to detect, they generally start to cause harm in the early stage, are difficult to control, and are extremely likely to cause large outbreaks in food crops. In severe cases, the crops may have no harvest at all.

[0003] Both WSMV and MDMV belong to the viruses of the family Potyviridae. WSMV can cause a reduction of up to 64% in the yield of a single wheat plant, and a reduction of 50 - 91% or even 100% in the grain yield, which once caused losses of 5,000 to 20,000 hectares of crops in Australia and losses of 76.8 million US dollars in Kansas. The sterility rate of crops infected by MDMV can reach 25% or even higher, and the 1000-grain weight can be reduced by 26 - 42%. MCMV belongs to the family Tombusviridae. Infection with this virus can cause plant deformity and even death. Relevant reports show that it causes an annual loss of about 500,000 tons of corn yield in Kenya, and the average loss of wheat and sweet corn in Peru is between 10% - 15%. When it is co-infected with the viruses of the family Potyviridae, the corn yield loss can be as high as 90%. WHPV is a member of the genus Emaravirus in the order of negative-strand RNA viruses, which has not been classified into a family yet. It can infect gramineous plants such as wheat, corn, barley, rye, oats and bromegrass, and has a wide range of hosts. It is a virus with a greater economic impact. At present, there are no reports of WSMV, MDMV, MCMV and WHPV in China. Among them, 3 kinds such as WSMV, MDMV and MCMV have been included in the list of quarantine pests in China, and WHPV is under revision. They are all key detection objects.

[0004] Currently, the detection methods for plant viruses mainly include electron microscopy inspection methods, ELISA methods, RT-PCR methods, real-time fluorescence PCR, etc. Since grain kernels contain a large amount of substances such as starch and enzymes that interfere with electron microscopy inspection methods and ELISA methods, the application of these two detection methods in grain virus detection is greatly restricted; although the real-time fluorescence PCR method has the advantages of being fast and accurate, its equipment is expensive and it is difficult to carry out in grass-roots laboratories, fields, and field surveys, which is not conducive to its popularization and development to a large extent; the RT-PCR method, as a mature technical means, has been widely used.

[0005] Currently, the detection methods for these four quarantine viruses are mainly single or double RT-PCR methods. There is no method or literature report that can detect these 4 viruses simultaneously. Therefore, the current laboratory still detects them according to traditional single viruses, resulting in an overly long detection cycle and being unable to meet the current detection cycle requirements, especially affecting the customs clearance time.

[0006] Therefore, how to establish a detection method that can simultaneously identify WSMV, MDMV, MCMV, and WHPV is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a multiplex RT-PCR primer set, kit, and detection method for simultaneously detecting WSMV, MDMV, MCMV, and WHPV, and for the first time establishes a multiplex RT-PCR method for WSMV, MDMV, MCMV, and WHPV, greatly shortening the detection time.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A multiplex RT-PCR primer set for simultaneously detecting WSMV, MDMV, MCMV, and WHPV, and the primer sequences of the primer set are as follows:

[0010] WSMV-F: 5'-CGACAATCAGCAAGAGACCA-3', SEQ ID NO.1;

[0011] WSMV-R: 5'-TGAGGATCGCTGTGTTTCAG-3', SEQ ID NO.2;

[0012] MDMV-F: 5'-TGCATCTCCATCTTTCAGACAA-3', SEQ ID NO.3;

[0013] MDMV-R: 5'-AACCTCACTCTCTTGCAGACA-3', SEQ ID NO.4;

[0014] MCMV-F: 5'-TCCAGAGCAATTCGAGCCA-3', SEQ ID NO.5;

[0015] MCMV-R: 5'-TATTGTAGCAGAGGGCACG-3', SEQ ID NO.6;

[0016] WHPV-F: 5'-CATAGCAATTACCTCAGCAGGA-3', SEQ ID NO.7;

[0017] WHPV-R: 5'-ACCTGGTTTRAAGCCTAAYAAGT-3', SEQ ID NO.8.

[0018] Another object of the present invention is to provide: a multiplex RT-PCR detection kit for simultaneously detecting WSMV, MDMV, MCMV and WHPV, comprising the above-mentioned multiplex RT-PCR primer set.

[0019] Another object of the present invention is to provide: a multiplex RT-PCR detection method for simultaneously detecting WSMV, MDMV, MCMV and WHPV, comprising the following steps:

[0020] (1) Extraction of RNA from the sample to be tested;

[0021] (2) Using the above-mentioned multiplex RT-PCR primer set to perform RT-PCR amplification with the RNA obtained in step (1) as a template. The amplification reaction system is as follows: The total volume of the reaction system is 25 μL, One step enzyme Mix 1 μL, 2×One-step reaction solution 12.5 μL, the final concentrations of MCMV, WSMV, MDMV, and WHPV primers are 0.2 μmol / L, 0.2 μmol / L, 0.2 μmol / L, and 0.32 μmol / L respectively, Total RNA 2 μL, and RNase free water is added to make up to 25 μL;

[0022] (3) Amplification reaction program: 50°C for 30 min; 94°C for 2 min; 94°C for 30 s, 59°C for 30 s, 66°C for 1 min, 35 cycles; 72°C for 2 min;

[0023] (4) Detecting the amplification products by agarose gel electrophoresis.

[0024] Another object of the present invention is to provide: the application of the above-mentioned multiplex RT-PCR primer set in the detection of WSMV, MDMV, MCMV and WHPV.

[0025] Another object of the present invention is to provide: the application of the above-mentioned multiplex RT-PCR detection kit in the detection of WSMV, MDMV, MCMV and WHPV.

[0026] Another object of the present invention is to provide: the application of the above-mentioned multiplex RT-PCR detection method in the detection of WSMV, MDMV, MCMV and WHPV.

[0027] As can be seen from the above technical solutions, compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The primer set, kit and detection method of the present invention can simultaneously detect four viruses, namely WSMV, MDMV, MCMV and WHPV, at one time. Compared with single PCR, multiplex RT-PCR reaction is not simply the superposition of multiple pairs of specific primers. It is necessary to consider the pairing between primers, competitive amplification, reaction conditions, nucleic acid concentration and purity on the amplification effect. Through a series of condition exploration and optimization, the present invention has developed an optimal reaction condition, which greatly improves the accuracy and sensitivity of detection, shortens the detection cycle from the original 4 days to 1-2 days, speeds up the detection process, provides technical support for shortening the detection cycle of inspection and quarantine departments, and lays a foundation for accurate and rapid identification of pathogens in the wild;

[0029] (2) The sizes of the amplified fragments of the four viruses selected in the present invention are in a gradient shape, avoiding similar or identical amplified fragment sizes, making the detection results look simple and clear; and applying the method of the present invention to 103 actual samples for detection, it is found that there are 48 samples that detect one or more viruses, indicating the accuracy of the method of the present invention. At the same time, for the first time, it is found that in addition to the phenomenon of co-infection of the three viruses reported in the literature, there is also a phenomenon of co-infection of four viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to the provided drawings.

[0031] Figure 1 It is: the RT-PCR amplification result under the condition of annealing temperature of 55-60 °C;

[0032] Among them, M: marker DL2000; 1 - 4: annealing temperature 60°C, final concentration of each primer 0.24 - 0.36 μmol / L; 5 - 8: annealing temperature 59°C, final concentration of each primer 0.24 - 0.36 μmol / L; 9 - 12: annealing temperature 58°C, final concentration of each primer 0.24 - 0.36 μmol / L; 13 - 16: annealing temperature 57°C, final concentration of each primer 0.24 - 0.36 μmol / L; 17 - 20: annealing temperature 56°C, final concentration of each primer 0.24 - 0.36 μmol / L; 21 - 24: annealing temperature 55°C, final concentration of each primer 0.24 - 0.36 μmol / L;

[0033] Figure 2 are: RT-PCR amplification results under the conditions of extension temperature 66 - 72°C and primer concentration 0.2 - 0.24 μmol / L;

[0034] Among them, M: marker DL2000; the final concentration of each primer for 1, 2, 3, 4, 5, 6, 7 is 0.2 μmol / L, and the extension temperatures are 66 - 72°C respectively; the final concentration of each primer for 8, 9, 10, 11, 12, 13, 14 is 0.24 μmol / L, and the extension temperatures are 66 - 72°C respectively;

[0035] Figure 3 are: WHPV amplification results at different extension temperatures and concentrations;

[0036] Among them, M: marker DL2000; for 1 - 3, the extension temperature is 66°C, and the final concentrations of WHPV primers are 0.24, 0.28, and 0.32 μmol / L; for 4 - 6, the extension temperature is 67°C, and the final concentrations of WHPV primers are 0.24, 0.28, and 0.32 μmol / L; for 7 - 9, the extension temperature is 68°C, and the final concentrations of WHPV primers are 0.24, 0.28, and 0.32 μmol / L; for 10 - 12, the extension temperature is 69°C, and the final concentrations of WHPV primers are 0.24, 0.28, and 0.32 μmol / L; for 13 - 15, the extension temperature is 70°C, and the final concentrations of WHPV primers are 0.24, 0.28, and 0.32 μmol / L; for 16 - 18, the extension temperature is 71°C, and the final concentrations of WHPV primers are 0.24, 0.28, and 0.32 μmol / L; for 19 - 21, the extension temperature is 72°C, and the final concentrations of WHPV primers are 0.24, 0.28, and 0.32 μmol / L;

[0037] Figure 4 are: multiplex RT-PCR sensitivity detection results;

[0038] Among them, M: marker DL2000; 1 - 10: the template concentrations of WHPV, MDMV, WSMV, and MCMV are 1 ng to 10^-9 ng respectively; CK: blank;

[0039] Figure 5 It is: the amplification results of 103 samples to be tested. Specific implementation manners

[0040] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0041] The materials required in the embodiments are specifically sourced as follows:

[0042] Four viruses, namely WSMV, MDMV, MCMV, and WHPV, were all intercepted and obtained by the Huangpu Customs Technical Center from imported gramineous grains and are currently held by the Huangpu Customs Technical Center. For the purpose of meeting the requirements of full disclosure of the patent, they can be provided to the public;

[0043] Among them, the original taxonomic name of the maize chlorotic mottle virus (Machlomovirus zeae, MCMV) was the maize chlorotic mottle virus (Maize chlorotic mottle virus, MCMV);

[0044] The wheat mosaic virus (wheatmosaic virus, WHPV) is the same virus as the wheat mosaic virus (wheatmosaic virus, WMoV) mentioned in the article "Detection of Wheat Mosaic Virus in Imported Maize Seeds and Full - length Amplification and Analysis of Its RNA3 Sequence" by Tian Yimin et al., Plant Quarantine, the first issue in 2022.

[0045] Example 1

[0046] Design of multiplex RT - PCR primer sets

[0047] According to the gene sequences of WSMV polyprotein gene, MCMV coat protein (CP), MDMV coat protein (CP), and WHPV nucleocapsid (N) in NCBI, sequence alignment analysis was carried out, and the corresponding primer sequences were designed using the primer design software Oligo Primer Analysis Software version 7.60. At the same time, Primer BLAST (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / / ) in NCBI was used to verify the designed primers to ensure their specificity.

[0048] The relevant information of the primers is shown in Table 1:

[0049] Table 1 Specific primer sequences

[0050]

[0051] Example 2

[0052] Obtaining virus-positive plasmids:

[0053] (1) Extraction of total RNA: Weigh 10 g of the grain seeds to be tested, freeze them in liquid nitrogen, and grind them into powder in a grinder. Weigh 0.1 g of the small sample, add PBS buffer (137 mmol / L NaCl, 12.7 mmol / L KCl, 10 mmol / L Na 2 HPO 4 ·12H 2 O, 2 mmol / L KH 2 PO 4 ) in a mass-to-volume ratio of 1:2, mix well and let stand for 15 - 30 min, and take 1 mL of the supernatant for standby. Extract the total RNA of the grain powder supernatant according to the instructions of the total RNA extraction kit and Tianlong T016, and store it at -80°C after extraction;

[0054] (2) RT-PCR Amplification: Using WSMV, MDMV, MCMV, and WHPV RNA as templates and their respective corresponding primers for RT-PCR amplification verification. The total volume of the reaction system is 25 μL, including 1 μL of One step enzyme Mix, 12.5 μL of 2×One-step reaction solution, 1 μL each of forward and reverse primers (10 μmol / L), 2 μL of Total RNA, and 8.5 μL of RNase-free water; the reaction conditions are: 50 °C for 30 min; 94 °C for 2 min; 94 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min, for 35 cycles; 72 °C for 2 min. The PCR products are electrophoresed in a 2% agarose gel at 125 V for 30 min, and then observed and photographed in a gel imager;

[0055] (3) The PCR products are sequenced and compared by Sangon Biotech (Shanghai) Co., Ltd. Then, the amplified product sequences of each virus are ligated into the pUC57 cloning vector, transformed into Escherichia coli competent cells, positive clones are obtained by colony PCR, plasmids are extracted after shaking culture in LB liquid medium for 12 h, and the plasmids are sequenced and compared. Finally, 4 kinds of virus positive plasmids are obtained.

[0056] Optimization of the annealing temperature for multiplex RT-PCR

[0057] Based on the reaction system and reaction program in step (2), the annealing temperature is optimized, and different annealing temperatures are set as follows: 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, and different final primer concentrations (0.24 μmol / L, 0.28 μmol / L, 0.32 μmol / L, 0.36 μmol / L) are set. RNase-free-water is used as the template for amplification, and the amplification results are as Figure 1 shown.

[0058] Result analysis: Under this reaction condition, no primer dimers are generated at 59 °C and 60 °C. Under other temperature conditions, a small amount of primer dimers are generated, which will affect the test results to a certain extent and is not conducive to the accuracy of the test. Therefore, 59 °C is selected as the annealing temperature for this experiment.

[0059] Optimization of the extension temperature and primer concentration for multiplex RT-PCR

[0060] Based on the above reaction system and reaction program, the extension temperature is further optimized, and different extension temperatures are set as follows: 66 °C, 67 °C, 68 °C, 69 °C, 70 °C, 71 °C, 72 °C, and different final primer concentrations (0.2 μmol / L, 0.24 μmol / L) are set. The mixed positive plasmid of the four viruses is used as the template for amplification, and the amplification results are asFigure 2 as shown

[0061] Result analysis: The results show that under the condition of an extension temperature of 66 °C, the test has the best effect. When the final concentration of the primers for MCMV, WSMV, and MDMV is 0.2 μmol / L, relatively clear bands can already be amplified, so it is determined that 0.2 μmol / L is the optimized final concentration of the primers for MCMV, WSMV, and MDMV. At the same time, it is also found that when the final concentration of the primers is 0.2 μmol / L, the amplification result of WHPV is not good. Therefore, the final concentration of its primers is optimized separately, and the final concentrations of 0.24 μmol / L, 0.28 μmol / L, and 0.32 μmol / L are set to obtain better experimental results. The experimental results are as Figure 3 shown. When the reaction concentration of WHPV is 0.32 μmol / L, the amplification effect is the best.

[0062] In summary, in the reaction system, the final concentrations of the primers for MCMV, WSMV, MDMV, and WHPV are 0.2 μmol / L, 0.2 μmol / L, 0.2 μmol / L, and 0.32 μmol / L respectively, the annealing temperature is 59 °C, and the extension temperature is 66 °C, and the test effect is the best.

[0063] Example 3

[0064] Sensitivity detection test

[0065] Configure the reaction system according to the primer concentrations optimized in Example 2, and establish a multiplex RT-PCR detection system with the optimized annealing temperature and extension temperature. The optimized reaction system and procedure are as follows:

[0066] Reaction system: 1 μL of One step enzyme Mix, 12.5 μL of 2×One-step reaction solution, the final concentrations of the primers for MCMV, WSMV, MDMV, and WHPV are 0.2 μmol / L, 0.2 μmol / L, 0.2 μmol / L, and 0.32 μmol / L respectively, 2 μL of Total RNA, and make up to 25 μL with RNase free water; the concentrations of the above forward and reverse primers are both 10 μmol / L;

[0067] Amplification reaction procedure: 50 °C for 30 min; 94 °C for 2 min; 94 °C for 30 s, 59 °C for 30 s, 66 °C for 1 min, 35 cycles; 72 °C for 2 min;

[0068] Using the mixed positive plasmid of the four viruses as a template, dilute it to 1 ng, 10 -1 ng, 10 -2 ng, 10 -3 ng, 10- 4 ng, 10 -5 ng, 10 -6 ng, 10 -7 ng, 10 -8 ng, 10 -9 ng were used for the sensitivity test, and the experimental results are as Figure 4 shown.

[0069] Result analysis: By implementing the above reaction conditions, it was found that the lower limit for detecting the target virus by multiplex RT-PCR was 10 - 6 ng; when the concentration of the viral positive plasmid template was lower than 10 -7 ng, the target virus could not be detected. Therefore, the detection threshold of this test was 10 -7 ng.

[0070] Example 4

[0071] Clinical trial

[0072] According to the reaction system and reaction degree of Example 3, 103 collected samples were tested to evaluate the detection effect of multiplex RT-PCR on actual samples. The experimental results are as Figure 5 shown.

[0073] Result analysis: There were 48 samples that detected more than 1 type of target virus. Among them, 28 samples detected WSMV, 18 samples detected WHPV, 5 samples detected MCMV, and 11 samples detected MDMV; 13 samples detected more than 2 types of viruses at the same time; 1 sample detected 3 types of viruses at the same time, and 1 sample detected 4 types of viruses at the same time. This demonstrates the accuracy of the present invention. At the same time, for the first time, it was found that in addition to the phenomenon of simultaneous infection by 3 viruses reported in the literature, there is also a phenomenon of simultaneous infection by 4 viruses.

[0074] In this specification, each example is described in a progressive manner. The key point of each example is to illustrate the differences from other examples. For the same and similar parts among the examples, reference can be made to each other.

[0075] The above description of the disclosed examples enables those skilled in the art to implement or use the present invention. Various modifications to these examples will be obvious to those skilled in the art. The general principles defined herein can be implemented in other examples without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these examples shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A multiplex RT-PCR primer set for simultaneous detection of WSMV, MDMV, MCMV and WHPV, characterized in that: The primer sequences of the primer set are as follows: WSMV-F: 5'-CGACAATCAGCAAGAGACCA-3', SEQ ID NO.1; WSMV-R: 5'-TGAGGATCGCTGTGTTTCAG-3', SEQ ID NO.2; MDMV-F: 5'-TGCATCTCCATCTTTCAGACAA-3', SEQ ID NO.3; MDMV-R: 5'-AACCTCACTCTTCTGCAGACA-3', SEQ ID NO.4; MCMV-F: 5'-TCCAGAGCAATTCGAGCCA-3', SEQ ID NO.5; MCMV-R: 5'-TATTGTAGCAGAGGGCACG-3', SEQ ID NO.6; WHPV-F: 5'- CATAGCAATTACCTCAGCAGGA-3', SEQ ID NO.7; WHPV-R: 5'-ACCTGGTTTRAAGCCTAAYAAGT-3', SEQ ID NO.8; Among them, the primer set of MCMV-F / R was constructed based on the MCMV coat protein gene.

2. A multiplex RT-PCR detection kit for simultaneously detecting WSMV, MDMV, MCMV and WHPV, characterized in that: The method comprises the multiplex RT-PCR primer set according to claim 1.

3. A multiplex RT-PCR detection method for simultaneously detecting WSMV, MDMV, MCMV and WHPV, characterized in that: The detection is performed using the multiplex PT-PCR primer set described in claim 1, specifically comprising the following steps: (1) Extraction of RNA from plant samples to be tested; (2) Using the multiplex RT-PCR primer set and the RNA obtained in step (1) as a template, RT-PCR amplification was performed. The amplification reaction system was as follows: the total volume of the reaction system was 25 μL, One step enzyme mix was 1 μL, 2×One-step reaction solution was 12.5 μL, the final concentrations of MCMV, WSMV, MDMV, and WHPV primers were 0.2 μmol / L, 0.2 μmol / L, 0.2 μmol / L, and 0.32 μmol / L, respectively, Total RNA was 2 μL, and RNase free water was added to 25 μL. (3) Amplification reaction program: 50°C for 30 min; 94°C for 2 min; 94°C for 30 s, 59°C for 30 s, 66°C for 1 min, 35 cycles; 72°C for 2 min; (4) Detect the amplified product by agarose gel electrophoresis.

4. Use of the multiplex RT-PCR primer set according to claim 1 in detecting WSMV, MDMV, MCMV and WHPV in plants.

5. Use of the multiplex RT-PCR detection kit according to claim 2 in detecting WSMV, MDMV, MCMV and WHPV in plants.

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