Primer probe combination, kit for simultaneously detecting four corn pathogens and application thereof

Abstract

The primer probe combination, kit and application for simultaneously detecting four corn pathogens belong to the technical field of molecular biology detection, and can solve the technical problems that the existing molecular biology detection methods for corn diseases cannot simultaneously detect multiple corn pathogens, or cannot simultaneously detect bacterial and viral pathogens of corn, and are complicated in operation and time-consuming. The primer probe combination for detecting corn inner state wilt fungus, corn bacterial wilt fungus, corn chlorotic mottle virus and corn dwarf mosaic virus, an integrated microfluidic chip kit containing the primer probe combination and a detection method are provided. The method for detecting pathogens in corn crops by using the primer probe combination and the kit has high sensitivity and accuracy, is simple, fast and time-saving in operation. The primer probe combination, kit and method can simultaneously detect the four pathogens, and play an important role in the on-site rapid detection of pathogens in corn planting.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular biology detection, and particularly relates to a primer probe combination for simultaneously detecting four corn pathogens, a kit and application thereof. BACKGROUND

[0002] With the rapid development of foreign trade economy, the varieties and quantities of imported and exported plants and plant products are increasing. As an important food crop, corn seed resources are also increasingly frequently transported. Some corn pathogens spread with the seeds, causing irreparable loss to corn yield. The List of Quarantine Harmful Organisms of Inbound Plants of the People's Republic of China clearly stipulates that the corn inner state wilt fungus (Gaeumannomyces graminis Clavibacter michiganensis nebraskensis Cmn Pantoea stewartii stewartii Pss subsp. Figure 1 , Figure 2 ), corn bacterial wilt fungus (Fusarium verticilliodes Figure 3 Figure 4 subsp. Figure 5 , Figure 6 ), maize chlorotic mottle virus (MCMV) and maize mottle virus (MDMV) are quarantine harmful organisms of corn. The four pathogens have a long survival time, and the related diseases caused thereby are seriously harmful to corn and can cause a substantial reduction in yield. Since the seeds carrying the four pathogens are the main initial infection source and transmission route of the related diseases, it is of great significance to detect the pathogens carried by the seeds as early as possible and find the disease symptoms for preventing, controlling, guiding production and reducing economic losses.

[0003] The detection techniques of plant quarantine pathogens mainly include physiological and biochemical detection, serological detection and molecular biology detection. Among them, the molecular biology detection is the most commonly used method, such as Polymerase Chain Reaction (PCR), Reverse Transcription-PCR (RT-PCR), Real-time Quantitative PCR (qPCR) method and the like. The prior art discloses a molecular biology detection method for detecting individually C. nigrum, C. vinnulatum, maize chlorotic mottle virus or maize dwarf mosaic virus. However, there is no multiplex real-time quantitative PCR detection method for the above four pathogens. In addition, the existing molecular biology detection system or method of corn diseases usually needs to be pretreated before detection, and most of the detection methods cannot detect multiple pathogens at the same time, or cannot detect bacterial and viral pathogens at the same time. The operation of the detection method or system is complicated, and the time is long. Therefore, it is urgent to establish a corn pathogen field rapid detection method which can detect multiple corn pathogens at the same time, has high sensitivity, strong specificity and simple operation. SUMMARY

[0004] In order to solve the technical problems that the existing molecular biology detection system or method of corn diseases usually needs to be pretreated before detection, and most of the detection methods cannot detect multiple corn pathogens at the same time, or cannot detect corn bacterial and viral pathogens at the same time, the operation of the detection method or system is complicated, and the time is long, the present application provides a primer probe combination for detecting corn pathogens including C. nigrum, C. vinnulatum, maize chlorotic mottle virus and maize dwarf mosaic virus, an integrated closed microfluidic chip kit containing the primer probe combination and a detection method. The primer probe combination, the integrated closed microfluidic chip kit and the detection method provided by the present application can detect C. nigrum, C. vinnulatum, maize chlorotic mottle virus and maize dwarf mosaic virus at the same time, which plays an important role in the field rapid detection of pathogens in corn planting.

[0005] In order to solve the above technical problems and achieve the corresponding technical effects, the present application provides the following technical solutions:

[0006] The first object of the present application is to provide a primer probe combination for simultaneously detecting four corn pathogens, which comprises a primer probe combination for detecting Gossypium hirsutum, a primer probe combination for detecting Fusarium virguliforme, a primer probe combination for detecting maize chlorotic mottle virus, a primer probe combination for detecting maize dwarf mosaic virus, and an IPC primer probe combination for detecting internal process control (IPC);

[0007] The primer probe combination for detecting Gossypium hirsutum consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 2, and a probe with a nucleotide sequence as shown in SEQ ID NO. 3;

[0008] The primer probe combination for detecting Fusarium virguliforme consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 4, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 5, and a probe with a nucleotide sequence as shown in SEQ ID NO. 6;

[0009] The primer probe combination for detecting maize chlorotic mottle virus consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 7, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 8, and a probe with a nucleotide sequence as shown in SEQ ID NO. 9;

[0010] The primer probe combination for detecting maize dwarf mosaic virus consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 10, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 11, and a probe with a nucleotide sequence as shown in SEQ ID NO. 12;

[0011] The IPC primer probe combination consists of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 13, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 14, and a probe with a nucleotide sequence as shown in SEQ ID NO. 15.

[0012] Preferably, the 5' end of the above-mentioned probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group.

[0013] Further preferably, the fluorescent reporter group is selected from one or a combination of more than two of ATTO 425, FAM, HEX, CY3, CY5, CY5.5, VIC, JOE and ROX.

[0014] Further preferably, the fluorescent reporter group is selected from one or a combination of more than two of ATTO 425, FAM, HEX, CY3, CY5, CY5.5, VIC, JOE and ROX.

[0015] Further preferably, the fluorescent quenching group is a fluorescent quenching group.

[0016] Further preferably, the fluorescent quenching group is selected from one or a combination of more than two of 6-TAMRA, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, QYS-7 and SQ1.

[0017] Further preferably, the fluorescent quenching group is selected from one or a combination of more than two of BHQ2, BHQ3 and SQ1.

[0018] A second object of the present application is to provide an application of the primer probe combination as described above, which is to use the primer probe combination to prepare a reagent, a kit or a chip for simultaneously detecting Goss' Wilt, Goss' Bacterial Wilt, Maize Chlorotic Mottle Virus and Maize Dwarf Mosaic Virus.

[0019] A third object of the present application is to provide an application of the primer probe combination as described above, which is to use the primer probe combination to simultaneously detect four maize pathogens, i.e. Goss' Wilt, Goss' Bacterial Wilt, Maize Chlorotic Mottle Virus and Maize Dwarf Mosaic Virus.

[0020] A fourth object of the present application is to provide a kit for detecting maize pathogens on site, which comprises the primer probe combination as described above.

[0021] In an embodiment of the present application, the kit is an integrated closed microfluidic chip kit.

[0022] In an embodiment of the present application, the integrated closed microfluidic chip kit further comprises a purification reagent, a qPCR reaction reagent and a freeze-dried internal process quality control product.

[0023] Preferably, the purification reagent comprises a lysis solution, a washing solution, an elution solution and magnetic beads, the components of the lysis solution and the washing solution comprise guanidine hydrochloride, sodium acetate, Triton X-100 and 1,3-butanediol, the component of the elution solution comprises Tris-HCl, and the magnetic beads are dried magnetic beads.

[0024] Further preferably, the components of the lysis solution and the washing solution comprise 4.2 M guanidine hydrochloride, 0.28 M sodium acetate (pH 4.7), 1.4% Triton X-100 and 30% 1,3-butanediol, and the component of the elution solution comprises 10 mM Tris-HCl (pH 8.5).

[0025] Preferably, the freeze-dried internal process quality control product is an RNA pseudo-virus.

[0026] Further preferably, the freeze-dried internal process quality control product is packaged in the form of a freeze-dried ball in the integrated closed microfluidic chip.

[0027] Further preferably, the kit completes the nucleic acid extraction, purification, amplification and detection of the sample on the integrated closed microfluidic chip.

[0028] Further preferably, the integrated closed microfluidic chip kit can be used for the CarryOn P1000F rapid nucleic acid detection device.

[0029] In an embodiment of the present application, the integrated closed microfluidic chip kit further comprises a sample processing tube containing grinding particles and a sample pretreatment solution.

[0030] A fifth object of the present application is to provide the use of the above-mentioned kit for simultaneously detecting four corn pathogens, i.e., the corn inner state wilt fungus, the corn bacterial wilt fungus, the corn chlorotic mottle virus and the corn dwarf mosaic virus.

[0031] A sixth object of the present application is to provide a method for detecting corn pathogens on site, which comprises the following steps:

[0032] (1) pretreating a sample to be detected to obtain a pretreated sample;

[0033] (2) adding the pretreated sample obtained in step (1) to the above-mentioned integrated closed microfluidic chip kit, and using the CarryOn P1000F rapid nucleic acid detection device to detect the sample to be detected on the microfluidic chip;

[0034] The corn pathogens are the corn inner state wilt fungus, the corn bacterial wilt fungus, the corn chlorotic mottle virus and the corn dwarf mosaic virus.

[0035] In one embodiment of the present application, the sample to be tested is corn seed, corn seed powder, corn leaf or corn fruit.

[0036] As an additional option, the present application provides a method for detecting pathogens of corn crops on site, which comprises preparation of an integrated closed microfluidic chip kit, sample pretreatment, rapid equipment detection.

[0037] Specifically, the method comprises the following steps:

[0038] (1) The primer probe combination is prepared into an integrated closed microfluidic chip kit;

[0039] (2) The corn seed, corn seed powder, corn leaf or corn fruit sample is added to the sample treatment tube for treatment to obtain a liquid to be tested;

[0040] (3) The liquid to be tested is dropped into the integrated closed microfluidic chip;

[0041] (4) The integrated closed microfluidic chip with the added liquid to be tested is placed on a detection device for detection analysis.

[0042] Preferably, the sample treatment tube in step (2) contains a sample pretreatment liquid and grinding particles; the sample pretreatment liquid comprises Tris-HCl (pH 8.0), NaCl, EDTA and SDS.

[0043] Further preferably, the concentration of Tris-HCl is in the range of 10 mM-100 mM, the concentration of NaCl is in the range of 30 mM-1.4 M, the concentration of EDTA is in the range of 1.5 mM-20 mM, and the concentration of SDS is in the range of 0.05%-2%; the diameter of the grinding particles is in the range of 0.1 μm-1 mm.

[0044] Further preferably, the concentration of Tris-HCl (pH 8.0) in the sample pretreatment liquid is in the range of 30 mM-55 mM, the concentration of NaCl is in the range of 350 mM-700 mM, the concentration of EDTA is in the range of 5 mM-10 mM, and the concentration of SDS is in the range of 0.5%-1%.

[0045] Preferably, the above step (4) specifically comprises the following steps:

[0046] 1) Turn on the rapid detection equipment;

[0047] 2) Scan the two-dimensional code;

[0048] 3) Insert the above integrated closed microfluidic chip;

[0049] 4) Run the detection;

[0050] 5) End, view the detection results and amplification curve.

[0051] As an additional solution, the present application provides a method for detecting pathogens in corn crops in situ, the method comprising the following steps:

[0052] (1) An integrated closed microfluidic chip kit is prepared by combining the primer probe combination described above;

[0053] (2) The sample is rapidly pretreated using a sample processing tube;

[0054] (3) The sample is lysed and the nucleic acid is extracted and purified using the purification reagent on the chip;

[0055] (4) The sample nucleic acid obtained by extraction and purification in step (3) is rapidly amplified using the primer probe combination described above;

[0056] (5) The amplification results are analyzed.

[0057] The beneficial effects of the present application are:

[0058] The present application provides a primer probe combination for detecting pathogens in corn crops, including corn inner state wilt fungus, corn bacterial wilt fungus, corn chlorotic mottle virus and corn dwarf mosaic virus, an integrated microfluidic chip kit containing the primer probe combination and a detection method. The method for detecting pathogens in corn crops using the primer probe combination and kit provided by the present application integrates sample pretreatment, nucleic acid extraction, purification, amplification and detection, has good sensitivity (the minimum detection limit of corn inner state wilt fungus is 9.0 x 10 2 CFU / reaction, the minimum detection limit of corn bacterial wilt fungus is 1.2 x 10 3 CFU / reaction, the minimum detection limit of corn chlorotic mottle virus is 1.0 copies / reaction, and the minimum detection limit of corn dwarf mosaic virus is 1.0 x 10 1 copies / reaction) and accuracy. The detection method uses CarryOn P1000F rapid nucleic acid detection equipment, and can complete the in-situ automatic nucleic acid detection of the sample to be detected within 36 min, realizing the rapid detection of "sample in, result out". The primer probe combination, integrated microfluidic chip kit and detection method provided by the present application can simultaneously detect four pathogens, corn inner state wilt fungus, corn bacterial wilt fungus, corn chlorotic mottle virus and corn dwarf mosaic virus, and play an important role in the in-situ rapid detection of pathogens in corn planting. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 7 Figure 2 is a negative detection result chart for a real corn sample.

[0060] Figure 8 Resulting image for detecting corn seed infected with Goss Williamsia zeae;

[0061] Figure 9 Resulting image for detecting corn leaf infected with Goss Williamsia zeae;

[0062] Figure 10 Resulting image for detecting corn fruit infected with Marmor cornellum;

[0063] Figure 9 Resulting image for detecting corn fruit infected with Maize dwarf mosaic virus;

[0064] Figure 10 Resulting image for detecting corn leaf infected with Goss Williamsia zeae and Maize dwarf mosaic virus;

[0065] Figure 1 Resulting image for detecting corn fruit infected with Marmor cornellum and Maize dwarf mosaic virus;

[0066] Figures 2-8 Resulting image for detecting corn fruit infected with Goss Williamsia zeae, Goss Williamsia zeae, Marmor cornellum and Maize dwarf mosaic virus;

[0067] Figure 2 Physical image of the chip device used for nucleic acid detection in the present application;

[0068] Figure 3 Physical image of the sample processing tube used in the present application. DETAILED DESCRIPTION

[0069] In order to make the purpose, technical solutions and advantages of the present application more clear, the present application will be further described in detail below in combination with specific embodiments and the accompanying drawings. It should be noted that the following embodiments are only used to explain the present application but not to limit the scope of the present application. The following embodiments are only a part of the embodiments of the present application but not all the embodiments. Those skilled in the art can refer to the content herein to appropriately improve the process parameters to achieve the purpose of the present application. It should be particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application described herein to realize and apply the present application technology without departing from the content and scope of the present application. In the art, other technicians will not make creative efforts, and the embodiments obtained by them are protected by the present application.

[0070] The experimental methods used in the following examples are conventional methods, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art unless otherwise specified, which can be obtained by commercial channels by those skilled in the art. The molecular biology experimental operations involved in the present application are conventional experimental operations in the art or can be performed according to the product instructions of the corresponding reagents unless otherwise specified.

[0071] The chip device for nucleic acid detection of the present application is disclosed in Chinese Invention Patent No. CN113278509A, the preparation method of the dried magnetic beads is disclosed in Chinese Invention Patent No. CN113005117A, the sample processing tube is disclosed in Chinese Utility Model Patent No. CN220136779U, and the overall equipment, i.e. the PCR reaction device, uses CarryOn P1000F rapid nucleic acid detection equipment, which is disclosed in Chinese Invention Patent No. CN116200514A. The physical diagram of the above-mentioned chip device is shown in Figure 4 The physical diagram of the above-mentioned sample processing tube is shown in Figure 7 .

[0072] The following examples of corn inner wilt fungus, corn bacterial wilt fungus, corn chlorotic mottle virus and corn dwarf mosaic virus are provided by the Plant Inspection and Quarantine Institute of China Institute for Quality Control Inspection and Scientific Research.

[0073] Example 1: Primer probe combination for simultaneous detection of corn inner wilt fungus, corn bacterial wilt fungus, corn chlorotic mottle virus and corn dwarf mosaic virus

[0074] According to the specific sequences of corn inner wilt fungus, corn bacterial wilt fungus, corn chlorotic mottle virus and corn dwarf mosaic virus, primer probe sequences for amplifying each pathogen are designed.

[0075] Among them, the primer probe combination for detecting corn inner wilt fungus is as follows:

[0076] Cmn-F: 5'-CCTCTCCGTCTACGACCTGT-3' (SEQ ID NO. 1);

[0077] Cmn-R: 5'-ACGGTGTTCCAGACCGTG-3' (SEQ ID NO. 2);

[0078] Cmn-P: 5'-CGGTGCTCTCGGTGGGCG-3' (SEQ ID NO. 3);

[0079] The primer probe combination for detecting corn bacterial wilt fungus is as follows:

[0080] Pss-F: 5'-TGCATTACATGCTTGCGTATGAC-3' (SEQ ID NO. 4);

[0081] Pss-R: 5'-TAATCATTCTGTTTTGTCTGCACTGT-3' (SEQ ID NO. 5);

[0082] Pss-P: 5'-TGTTCTGTCTGGCATCAGCGTTTTCTACC-3' (SEQ ID NO. 6);

[0083] The primer probe combination for detecting maize chlorotic mottle virus is as follows:

[0084] MCMV-F: 5'-GATTCCAGTGTGTGCAGGGA-3' (SEQ ID NO. 7);

[0085] MCMV-R: 5'-AGTGATGCGCACAGAGTTGA-3' (SEQ ID NO. 8);

[0086] MCMV-P: 5'-CCCGATACATTGGGGCGGCC-3' (SEQ ID NO. 9);

[0087] The primer probe combination for detecting maize dwarf mosaic virus is as follows:

[0088] MDMV-F: 5'-TGATGGGAATGTCGGAG-3' (SEQ ID NO. 10);

[0089] MDMV-R: 5'-GGGAGTGCATATTGCGACTG-3' (SEQ ID NO. 11);

[0090] MDMV-P: 5'-CCCACGAAAATACAGAACGCCATACAG-3' (SEQ ID NO. 12);

[0091] The primer probe combination for detecting internal process control is as follows:

[0092] IPC-F: 5'-CTCTAAGTTAGCGAAATTGATGGTATTG-3' (SEQ ID NO. 13);

[0093] IPC-R: 5'-ATAATTATCCCAGGGCCTCCT-3' (SEQ ID NO. 14);

[0094] IPC-P: 5'-ATTACTTGCGCTGCCACATTGCTG-3' (SEQ ID NO. 15).

[0095] The primer probe combination for detecting the corn pathogen provided in the embodiment is used for detecting G. ips, G. aglata, M. zeae and M. zeae separately or jointly.

[0096] The probe for G. ips in the embodiment is labeled with ATTO 425 and the quenching group is SQ1, the probe for G. aglata is labeled with FAM and the quenching group is SQ1, the probe for M. zeae is labeled with HEX and the quenching group is SQ1, the probe for M. zeae is labeled with CY5 and the quenching group is BHQ3, the probe for IPC is labeled with CY5.5 and the quenching group is BHQ3.

[0097] Example 2: Integrated closed microfluidic chip kit for rapid simultaneous detection of four corn pathogens in the field

[0098] The kit provided in the embodiment comprises the following substances:

[0099] (1) a chip device for nucleic acid detection;

[0100] (2) further comprising purification reagents, air-drying reagents and freeze-dried internal process quality control products added in the chip device.

[0101] 1) Purification reagents

[0102] The purification reagents comprise 450 μL of lysis solution, 450 μL of washing solution, 850 μL of elution solution and 15 μL of dried magnetic beads. The formula of the lysis solution and the washing solution is 4.2 M guanidine hydrochloride, 0.28 M sodium acetate (pH 4.7), 1.4% Triton X-100 and 30% 1,3-butanediol; the formula of the elution solution is 10 mM Tris-HCl (pH 8.5). The purification reagents are added to the reagent bin in the chip.

[0103] 2) Air-drying reagents

[0104] The reaction system of the air-drying reagents is 15 μL, and the specific components are shown in Table 1. The primer probe mixture in Table 1 is a mixture of all primers and probes in Example 1, and the final concentration of each primer and probe in a 50 μL amplification system is shown in Table 2. The reagents in Table 1 are added to the reaction layer of the chip, the chip is placed in an air-drying oven, and the air-drying operation is performed according to the instructions of 4x Air-Dryable qPCR Mix. Finally, the air-drying reagents are dissolved with 50 μL of sample grinding solution for reaction.

[0105] Table 1. Air-dried reagent reaction system

[0106]

[0107] Table 2. Final concentration of each primer probe

[0108]

[0109] 3) Freeze-dried internal process quality control product

[0110] The preparation of the freeze-dried internal process quality control product includes the preparation of the IPC pseudovirus and the preparation of the freeze-dried ball of the internal process quality control product.

[0111] According to the preparation method of the pseudovirus provided in Example 1 of the Chinese patent application with the application number “202110669475.X”, the pseudovirus of the IPC target (IPC pseudovirus) is prepared and obtained.

[0112] Using the freeze-drying system (Table 3) and the freeze-drying procedure (Table 4), the IPC pseudovirus with a concentration of 1×10 6 copies / mL is prepared into a freeze-dried ball, specifically, the reagents in Table 3 are dropped into liquid nitrogen at 5 μL each, and then placed into a Schlenk flask to perform freeze-drying according to the freeze-drying procedure of the freeze-drying machine shown in Table 4 to obtain a freeze-dried ball. Then, the freeze-dried ball is placed on the chip with the air-dried reaction reagents to assemble into an integrated closed microfluidic chip kit.

[0113] Table 3. Freeze-dried IPC reagent system

[0114]

[0115] Table 4. Freeze-drying procedure

[0116]

[0117] (3) Sample processing tube

[0118] The integrated closed microfluidic chip kit provided in this embodiment can also contain a sample processing tube, which is assembled in the following manner: 1 g of grinding particles (carborundum with a diameter of 15 μm) and 3 mL of sample pretreatment solution are added to the sample processing tube. The components of the sample pretreatment solution are 50 mM Tris-HCl (pH 8.0), 700 mM NaCl, 10 mM EDTA, and 1% SDS. The sample processing tube is placed in the tooling position of a precision hand press, and aluminum foil is used for packaging at 160°C.

[0119] Example 3: Method for simultaneously detecting four corn pathogens using an integrated closed microfluidic chip kit

[0120] (1) Sample processing: tear off the aluminum foil protective film of the sample processing tube, place it vertically, add the corn leaf, corn seed or corn fruit sample (100-500 mg) to be detected into the sample processing tube, tighten the tube cover, keep the tube body upright, rub for 5-10 times to make the sample fully contact with the grinding material and ensure sufficient grinding to obtain a sample grinding liquid;

[0121] (2) Chip assembly: take out the reagent compartment and reaction layer of the chip, tear off the sealing tape on the back of the reagent compartment, hold the reagent compartment and reaction layer with the head of the reaction layer vertically upwards, press the reagent compartment to pierce the chip to connect the upper and lower layers seamlessly, and complete the assembly;

[0122] (3) Sample addition: unscrew the sample compartment cover, add 2-3 drops (50 μL) of the sample grinding liquid obtained in step (1) to the sample compartment, and tighten the sample compartment cover;

[0123] (4) Nucleic acid detection: place the chip obtained in step (3) into the CarryOn P1000F rapid nucleic acid detection equipment for nucleic acid detection. The operation method of the nucleic acid detection equipment is as follows: turn on the equipment by pressing the power button for more than 3 s, click the "detection" button on the home page; wait for 3 s, and scan the chip two-dimensional code when the red light flashes in the scanning window; according to the screen prompt, scan the sample information or skip; according to the screen prompt, open the hatch, insert the chip, and close the hatch; click the "run" button to start detection; after the detection is completed, view the detection results.

[0124] In the detection method provided in this embodiment, the sample processing tube takes about 1 min to process the sample; during the nucleic acid detection, the nucleic acid extraction and purification on the integrated closed microfluidic chip takes about 12 min, and the real-time quantitative PCR reaction program on the integrated microfluidic chip is as follows: 95℃ for 1 min; (95℃ for 5 s, 60℃ for 8 s), 45 cycles, with a total time of 23 min. The whole process time of the detection of corn crop diseases from sample processing, nucleic acid extraction and purification to amplification detection results is about 36 min.

[0125] Example 4: Specificity evaluation of the method for simultaneously detecting four kinds of corn pathogens by using the integrated closed microfluidic chip kit

[0126] The negative control (deionized water) and Cmn bacterial solution, Pss bacterial solution, MCMV plasmid and MDMV plasmid were detected by using the detection method described in Example 3. The pathogenic bacteria and pathogenic fungi used in this experiment were provided by the Plant Inspection and Quarantine Institute of China Quality Inspection and Test Scientific Research Institute; the virus plasmid was synthesized by Beijing Qikexin Biotechnology Co., Ltd. The specificity detection results are shown in Table 5.

[0127] Judgment of target detection result: IPC is internal process quality control, and the IPC of each sample is amplified, which indicates that the sample nucleic acid extraction and amplification process are all normal; the negative sample has no amplification; the positive samples of Gossypium hirsutum endophytic fusarium, corn bacterial wilt, corn chlorotic mottle virus and corn mosaic virus all have amplification, while other pathogen samples have no amplification.

[0128] From the specific detection results, it can be seen that the primer probe combination, the kit containing the primer probe combination and the detection method provided by the application have good specificity.

[0129] Table 5 Specificity test results of primer probe combination for detecting four corn diseases

[0130]

[0131] Example 5: Sensitivity evaluation of the method for simultaneously detecting four corn pathogens by using the integrated closed microfluidic chip kit

[0132] The bacterial liquid of Gossypium hirsutum endophytic fusarium and corn bacterial wilt was prepared respectively, and after gradient dilution, different concentrations of diluents were used as samples, and the method provided in Example 3 was used for detection. The pseudovirus of corn chlorotic mottle virus and corn mosaic virus was prepared respectively, and after gradient dilution, different concentrations of diluents were used as samples, and the method provided in Example 3 was used for detection. The detection results are shown in Table 6 below, the minimum detection limit of Gossypium hirsutum endophytic fusarium is 9.0 x 10 2 CFU / reaction, the minimum detection limit of corn bacterial wilt is 1.2 x 10 3 CFU / reaction, the minimum detection limit of corn chlorotic mottle virus is 1.0 copies / reaction, and the minimum detection limit of corn mosaic virus is 1.0 x 10 1 copies / reaction.

[0133] From the sensitivity detection results, it can be seen that the primer probe combination, the kit containing the primer probe combination and the detection method provided by the application have good sensitivity, and can be used for field rapid detection of corn diseases.

[0134] Table 6 Sensitivity test results of primer probe combination for detecting four corn diseases

[0135]

[0136] Experimental example 6: Detection of corn leaf, seed and fruit real samples by using integrated closed microfluidic chip kit

[0137] The real samples were detected by the method of Example 3. The corn seed, leaf and other materials were collected from the corn planting area and the farmer's planting field in Sanya City, Hainan Province. The collected samples were detected by the kit provided in Example 2 and the method provided in Example 3. The detection results of 29 corn samples (5 seed samples, 10 leaf samples and 14 fruit samples) were as follows: 21 samples were negative (see ​ , and the results were not all listed), 1 seed sample was infected with C. nigrum, 1 leaf sample was infected with F. oxysporum, 2 fruit samples were infected with M. viridis, 1 fruit sample was infected with CMV, 1 leaf sample was infected with F. oxysporum and CMV, 1 fruit sample was infected with M. viridis and CMV, and 1 fruit sample was infected with 4 pathogens, a total of 8 corn samples were detected with pathogens ​ , and the results were not all listed). From the detection results of the real samples, it can be seen that the primer probe composition, kit and detection method for simultaneously detecting 4 corn pathogens provided by the application can detect real samples such as leaves, seeds and fruits, and can be used for rapid identification of corn diseases in the field.

[0138] Comparative Example

[0139] The difference between the present comparative example and Examples 1-3 is only that the primer probe combination for simultaneously detecting C. nigrum, F. oxysporum, M. viridis and CMV is different, and the specific difference is as follows:

[0140] The primer probe combination for detecting C. nigrum is as follows:

[0141] Cmn-F': 5'-TGCACCTTCATCACGACATGG-3' (SEQ ID NO. 16);

[0142] Cmn-R': 5'-ACGATGATCATGCTGGCAATG-3' (SEQ ID NO. 17);

[0143] Cmn-P': 5'-TGTTCGGTCTCGTCATCGCACGGCA-3' (SEQ ID NO. 18);

[0144] The primer probe combination for detecting F. oxysporum is as follows:

[0145] Pss-F': 5'-TGCTGATTTTAAGTTTTGCTA-3' (SEQ ID NO. 19);

[0146] Pss-R': 5'-AAGATGAGCGAGGTCAGGATA-3' (SEQ ID NO. 20);

[0147] Pss-P': 5'-TCGGGTTCACGTCTGTCCAACT-3' (SEQ ID NO. 21);

[0148] The primer probe combination for detecting corn chlorotic mottle virus is as follows:

[0149] MCMV-F': 5'-CTCTCGATGATGCAAAGTCTGTGR-3' (SEQ ID NO. 22);

[0150] MCMV-R': 5'-TCTCAAAGCTATCRACTGAAGCAATAA-3' (SEQ ID NO. 23);

[0151] MCMV-P': 5'-AGGTAAGCTACCTCCCAGCATTATGGCAAG-3' (SEQ ID NO. 24);

[0152] The primer probe combination for detecting corn chlorotic mottle virus is as follows:

[0153] MDMV-F: 5'-TGATGGGAATGTCGGAG-3' (SEQ ID NO. 10);

[0154] MDMV-R: 5'-GGGAGTGCATATTGCGACTG-3' (SEQ ID NO. 11);

[0155] MDMV-P: 5'-CCCACGAAAATACAGAACGCCATACAG-3' (SEQ ID NO. 12);

[0156] The primer probe combination for detecting internal process control (IPC) is as follows:

[0157] IPC-F: 5'-CTCTAAGTTAGCGAAATTGATGGTATTG-3' (SEQ ID NO. 13);

[0158] IPC-R: 5'-ATAATTATCCCAGGGCCTCCT-3' (SEQ ID NO. 14);

[0159] IPC-P: 5'-ATTACTTGCGCTGCCACATTGCTG-3' (SEQ ID NO. 15).

[0160] The kit prepared by using the primer probe combination provided in the comparative example and the detection method described in Example 3 was used to detect the samples involved in Examples 1-3, and the detection results were compared with the detection results obtained in Examples 1-3. The comparison results of the Ct values are shown in Table 7. As can be seen from Table 7, the amplification Ct values obtained by using the primer probe combination in the comparative example are significantly delayed compared with the primer probe combination in the present application, and the amplification fluorescence values also decrease to different degrees. In the comparative example, the corn chlorotic mottle virus was not detected in the mixed sample containing the corn chlorotic mottle virus and the corn dwarf mosaic virus. ​ 、 ​ 、 ​ and ​ The samples involved in Examples 1-3 were detected by using the kit prepared by using the primer probe combination provided in the comparative example and the detection method described in Example 3, and the detection results were compared with the detection results obtained in Examples 1-3. The comparison results of the Ct values are shown in Table 7. As can be seen from Table 7, the amplification Ct values obtained by using the primer probe combination in the comparative example are significantly delayed compared with the primer probe combination in the present application, and the amplification fluorescence values also decrease to different degrees. In the comparative example, the corn chlorotic mottle virus was not detected in the mixed sample containing the corn chlorotic mottle virus and the corn dwarf mosaic virus.

[0161] Table 7 Comparison results of amplification Ct values of Examples and comparative examples

[0162]

[0163] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A primer probe combination for simultaneous detection of 4 corn pathogens, characterized in that, The primer probe combination comprises a primer probe combination for detecting Goss' Wilt of corn, a primer probe combination for detecting Bacterial Wilt of corn, a primer probe combination for detecting Mosaic Virus of corn, a primer probe combination for detecting Dwarf Mosaic Virus of corn, and an IPC primer probe combination for detecting internal process control; The primer probe combination for detecting Goss' Wilt of corn is composed of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 2, and a probe with a nucleotide sequence as shown in SEQ ID NO. 3; The primer probe combination for detecting Bacterial Wilt of corn is composed of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 4, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 5, and a probe with a nucleotide sequence as shown in SEQ ID NO. 6; The primer probe combination for detecting Mosaic Virus of corn is composed of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 7, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 8, and a probe with a nucleotide sequence as shown in SEQ ID NO. 9; The primer probe combination for detecting Dwarf Mosaic Virus of corn is composed of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 10, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 11, and a probe with a nucleotide sequence as shown in SEQ ID NO. 12; The IPC primer probe combination is composed of an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 13, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 14, and a probe with a nucleotide sequence as shown in SEQ ID NO.

15.

2. Use of the primer probe combination according to claim 1, characterized in that The application is to use the primer probe combination to prepare a reagent, a kit or a chip for simultaneously detecting Goss' Wilt of corn, Bacterial Wilt of corn, Mosaic Virus of corn and Dwarf Mosaic Virus of corn.

3. Use of the primer probe combination according to claim 1, characterized in that The application is to use the primer probe combination to simultaneously detect four corn pathogens, i.e. Goss' Wilt of corn, Bacterial Wilt of corn, Mosaic Virus of corn and Dwarf Mosaic Virus of corn.

4. A kit for detecting a corn pathogen in the field, characterized by, The kit comprises the primer probe combination of claim 1, and the corn pathogens are Goss' Wilt of corn, Bacterial Wilt of corn, Mosaic Virus of corn and Dwarf Mosaic Virus of corn.

5. The kit of claim 4, wherein The kit is an integrated closed microfluidic chip kit.

6. The kit of claim 5, wherein The integrated closed microfluidic chip kit further comprises a purification reagent, a qPCR reaction reagent and a freeze-dried internal process control.

7. The kit of claim 6, wherein The integrated closed microfluidic chip kit further comprises a sample processing tube containing grinding particles and a sample pretreatment solution.

8. Use of a kit according to any one of claims 4 to 7, characterized in that, The application is to use the kit to simultaneously detect four corn pathogens, i.e. Goss' Wilt of corn, Bacterial Wilt of corn, Mosaic Virus of corn and Dwarf Mosaic Virus of corn.

9. A method of detecting a corn pathogen in the field, characterized by, The method comprises the following steps: (1) pretreating the sample to be tested to obtain a pretreated sample; (2) The pretreated sample obtained in step (1) is added into the integrated closed microfluidic chip kit according to any one of claims 5-7, and the pretreated sample is detected on the microfluidic chip by using a CarryOn P1000F rapid nucleic acid detection device; The corn pathogens are corn inner state wilt pathogen, corn bacterial wilt pathogen, corn chlorotic mottle virus and corn dwarf mosaic virus.

10. The method of claim 9, wherein, The sample to be detected in step (1) is corn seed, corn leaf or corn fruit.

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