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

Abstract

The primer probe combination, kit and application for simultaneously detecting four kinds of tomato pathogens belong to the technical field of molecular biology detection, and can solve the technical problems that the existing molecular biology detection methods for tomato diseases cannot simultaneously detect multiple tomato pathogens, or cannot simultaneously detect bacterial pathogens and viral pathogens of tomatoes, and are time-consuming, etc.The present application provides a primer probe combination for detecting tomato or pepper crops, a one-piece microfluidic chip kit containing the primer probe combination, and a detection method.The method for detecting pathogens in tomato or pepper crops by using the primer probe combination and the kit provided by the present application has high sensitivity and accuracy, and is simple, fast and time-saving.The primer probe combination, kit and method provided by the present application can simultaneously detect the four kinds of pathogens mentioned above, and play an important role in the on-site rapid detection of pathogens in tomato or pepper 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 tomato pathogens, a kit and application thereof. BACKGROUND

[0002] Tomato is an important economic crop in the world and plays an important role in agricultural production. However, the frequent occurrence of tomato diseases seriously affects the yield and quality of tomatoes. Among them, Tomato leaf curl New Delhi virus (ToLCNDV), Tomato spotted wilt virus (TSWV), Tomato brown rugose fruit virus (ToBRFV) and Xanthomonas campestris pv. vesicatoria (Xcv) are the four main pathogens that harm tomatoes and peppers. These pathogens can be transmitted through seeds, soil, vectors and other means, and the symptoms are easy to be confused with other diseases, increasing the difficulty of early diagnosis and control. The four pathogens have a long survival time and cause serious damage. Seed-borne pathogens are the main initial infection source for long-distance transmission, so it is important to do a good job of disease supervision. Clavibacter michiganensis subsp. michiganenis , Cmm ))are the four main pathogens that harm tomatoes and peppers. These pathogens can be transmitted through seeds, soil, vectors and other means, and the symptoms are easy to be confused with other diseases, increasing the difficulty of early diagnosis and control. The four pathogens have a long survival time and cause serious damage. Seed-borne pathogens are the main initial infection source for long-distance transmission, so it is important to do a good job of disease supervision.

[0003] Plant quarantine disease diagnosis and detection technology mainly includes physiological and biochemical detection, serological detection and molecular biology detection. Among them, 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) and other methods. For example, the national standard GB / T 29431-2012 of the People's Republic of China issued a method for quarantine identification of Xanthomonas campestris pv. vesicatoria; the national standard GB / T 28982-2012 of the People's Republic of China issued a PCR detection method for Tomato spotted wilt virus. However, there is no method for simultaneously detecting ToLCNDV, TSWV, ToBRFV and Xcv at present. Cmm, Multiplex fluorescent PCR detection method of ToLCNDV, TSWV and ToBRFV. In addition, the existing molecular biology detection system or method of tomato diseases generally requires pre-treatment of tomato samples before detection, and most detection methods cannot simultaneously detect multiple pathogens, or cannot simultaneously detect bacterial and viral pathogens of tomatoes, the operation of the detection method or system is complicated, and it takes a long time. Therefore, it is urgent to establish a rapid field detection method for tomato pathogens which can simultaneously detect multiple tomato pathogens, has high sensitivity, strong specificity and simple operation. SUMMARY

[0004] To solve the technical problems that the existing molecular biology detection system or method of tomato diseases generally requires pre-treatment of tomato samples before detection, and most detection methods cannot simultaneously detect multiple tomato pathogens, or cannot simultaneously detect bacterial and viral pathogens of tomatoes, the operation of the detection method or system is complicated, and it takes a long time, the present application provides a primer probe combination for detecting tomato or pepper crops, an integrated closed microfluidic chip kit containing the primer probe combination and a method. The primer probe combination, integrated closed microfluidic chip kit and detection method provided by the present application can simultaneously detect four pathogens of tomato canker bacteria, New Delhi tomato leaf curl virus, tomato spotted wilt virus and tomato brown rugose fruit virus, which plays an important role in the rapid field detection of pathogens in tomato and pepper planting.

[0005] 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 tomato pathogens, which comprises a primer probe combination for detecting tomato canker bacteria, a primer probe combination for detecting New Delhi tomato leaf curl virus, a primer probe combination for detecting tomato spotted wilt virus, a primer probe combination for detecting tomato brown rugose fruit virus and an IPC primer probe combination for detecting internal process control (IPC).

[0007] The primer probe combination for detecting tomato canker bacteria 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;

[0008] The primer probe combination for detecting New Delhi Tomato Leaf Curl Virus consists of an upstream primer having a nucleotide sequence as shown in SEQ ID NO. 4, a downstream primer having a nucleotide sequence as shown in SEQ ID NO. 5 and a probe having a nucleotide sequence as shown in SEQ ID NO. 6;

[0009] The primer probe combination for detecting Tomato Spotted Wilt Virus consists of an upstream primer having a nucleotide sequence as shown in SEQ ID NO. 7, a downstream primer having a nucleotide sequence as shown in SEQ ID NO. 8 and a probe having a nucleotide sequence as shown in SEQ ID NO. 9;

[0010] The primer probe combination for detecting Tomato Brown Rugose Fruit Virus consists of an upstream primer having a nucleotide sequence as shown in SEQ ID NO. 10, a downstream primer having a nucleotide sequence as shown in SEQ ID NO. 11 and a probe having a nucleotide sequence as shown in SEQ ID NO. 12;

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

[0012] Preferably, the 5' end of the above 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 FAM, ROX, HEX, CY5, VIC, TET, JOE, CY3, CY7, RED610, Texas Red, RED670, NED, AMCA, Pacific Blue, ATTO 425, BODIPY FL, Alexa Fluor 488, Yakima Yellow, Quasar 570, Aqua Phluor 593, ATTO 590 and CY5.5.

[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] Preferably, the quencher group is a fluorescent quencher group.

[0016] Further preferably, the fluorescent quencher 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 fluorescence quenching group is selected from one or a combination of two or more 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 for preparing a reagent, a kit or a chip for simultaneously detecting Clavibacter michiganensis, Tomato leaf curl New Delhi virus, Tomato spotted wilt virus and Tomato brown rugose fruit 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 for simultaneously detecting four tomato pathogens, Clavibacter michiganensis, Tomato leaf curl New Delhi virus, Tomato spotted wilt virus and Tomato brown rugose fruit virus.

[0020] A fourth object of the present application is to provide a kit for detecting tomato 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 lyophilized internal process quality control.

[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 lyophilized internal process quality control is an RNA pseudovirus.

[0026] Further preferably, the lyophilized internal process quality control is packaged in the form of a lyophilized 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 in 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, which is to simultaneously detect four tomato pathogens, i.e. Clavibacter michiganensis, Tomato leaf curl New Delhi virus, Tomato spotted wilt virus and Tomato brown rugose fruit virus.

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

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

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

[0034] The tomato pathogens are Clavibacter michiganensis, Tomato leaf curl New Delhi virus, Tomato spotted wilt virus and Tomato brown rugose fruit virus.

[0035] In an embodiment of the present application, the sample to be tested in step (1) is a tomato seed, a tomato leaf, a tomato fruit, a pepper seed, a pepper leaf or a pepper fruit.

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

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

[0038] (1) preparing the above-mentioned primer probe combination into an integrated closed microfluidic chip kit;

[0039] (2) adding a tomato seed, a tomato seed powder, a tomato leaf, a tomato fruit, a pepper seed, a pepper leaf or a pepper fruit sample into a sample processing tube for processing to obtain a liquid to be tested;

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

[0041] (4) placing the integrated closed microfluidic chip with the added liquid to be tested into a detection device for detection and analysis.

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

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

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

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

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

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

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

[0049] 4) Run the detection;

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

[0051] As an additional option, the present application provides a method for on-site detection of pathogens of tomato or pepper crops, the method comprising the following steps:

[0052] (1) Prepare an integrated closed microfluidic chip kit for the primer probe combination mentioned above;

[0053] (2) Use a sample processing tube to rapidly pre-treat the sample;

[0054] (3) Use the purification reagent on the chip to perform sample lysis and nucleic acid extraction and purification;

[0055] (4) Use the primer probe combination mentioned above to rapidly amplify the sample nucleic acid obtained by extraction and purification in step (3);

[0056] (5) Analyze the amplification results.

[0057] The present application has the following advantages:

[0058] The application provides a primer probe combination for detecting tomato canker disease bacteria, New Delhi tomato leaf curl virus, tomato spotted wilt virus and tomato brown rugose fruit virus in tomato or pepper crops, an integrated closed microfluidic chip kit containing the primer probe combination and a method. 2 CFU / reaction, the minimum detection limit of the New Delhi tomato leaf curl virus is 1.0 x 10 1 copies / reaction, the minimum detection limit of the tomato spotted wilt virus is 1.0 x 10 1 copies / reaction, and the minimum detection limit of the tomato brown rugose fruit virus is 1.0 x 10 1 copies / reaction. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 is a negative detection result graph of a real tomato sample;

[0060] Figure 2 is a detection result graph of tomato seeds infected with tomato canker disease bacteria;

[0061] Figure 3 is a detection result graph of tomato fruits infected with New Delhi tomato leaf curl virus;

[0062] Figure 4 is a detection result graph of tomato leaves infected with tomato spotted wilt virus;

[0063] Figure 5 is a detection result graph of tomato leaves infected with tomato brown rugose fruit virus;

[0064] Figure 6 is a detection result graph of tomato leaves infected with both tomato canker disease bacteria and tomato brown rugose fruit virus;

[0065] Figure 7A result chart for detecting tomato fruits infected with tomato canker, tomato leaf curl New Delhi virus and tomato spotted wilt virus at the same time;

[0066] Figure 8 A physical map of the chip device used in the present application for nucleic acid detection;

[0067] Figure 9 A physical map of the sample processing tube used in the present application. DETAILED DESCRIPTION

[0068] In order to make the purpose, technical solutions and advantages of the present application clearer, 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 examples are only used to explain the present application, but not to limit the scope of the present application. The following examples are only a part of the embodiments of the present application, 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 considered to be included in the present application. The methods and applications of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present application, to realize and apply the present application technology. In the art, other technicians will not make creative efforts, and the examples they obtain are protected by the present application.

[0069] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and instruments used are conventional materials, reagents and instruments in the art unless otherwise specified, and 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.

[0070] 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, the overall equipment, i.e. the PCR reaction device, uses CarryOn P1000F rapid nucleic acid detection equipment, and the details are disclosed in Chinese Invention Patent No. CN116200514A. Among them, the physical map of the above-mentioned chip device is shown in Figure 8 The physical map of the above-mentioned sample processing tube is shown in Figure 9 .

[0071] Tomato canker pathogen, New Delhi tomato leaf curl virus, tomato spotted wilt virus and tomato brown rugose fruit virus in the following examples were provided by Plant Inspection and Quarantine Institute of China National Institute of Standardization.

[0072] Example 1: Primer probe combination for simultaneous detection of tomato canker pathogen, New Delhi tomato leaf curl virus, tomato spotted wilt virus and tomato brown rugose fruit virus

[0073] According to the specific sequences of tomato canker pathogen, New Delhi tomato leaf curl virus, tomato spotted wilt virus and tomato brown rugose fruit virus, the primer probe sequences for amplifying each pathogen were designed respectively.

[0074] Among them, the primer probe combination for detecting tomato canker pathogen is as follows:

[0075] Cmm-F: 5'-GCAGACGACCAGAAGAAGTTGTT-3' (SEQ ID NO. 1);

[0076] Cmm-R: 5'-CCGAGGCTCGTAGTCATTGAC-3' (SEQ ID NO. 2);

[0077] Cmm-P: 5'-ACTTCGGCCGGCATACGCTGTACTC-3' (SEQ ID NO. 3);

[0078] The primer probe combination for detecting New Delhi tomato leaf curl virus is as follows:

[0079] ToLA-F: 5'-GGTTACCTCTGCAGCATCGT-3' (SEQ ID NO. 4);

[0080] ToLA-R: 5'-CGCTGCGCTTCCTAAATGTC-3' (SEQ ID NO. 5);

[0081] ToLA-P: 5'-CCCACCCACTCCCAGACCCA-3' (SEQ ID NO. 6);

[0082] The primer probe combination for detecting tomato spotted wilt virus is as follows:

[0083] TSWV-F: 5'-GCTTGATCAGGGTCAGGCTT-3' (SEQ ID NO. 7);

[0084] TSWV-R: 5'-TGGCAAGCCTCACAGACTTT-3' (SEQ ID NO. 8);

[0085] TSWV-P: 5'-TGCTTCTCACCCTCTGATTCAAGCC-3' (SEQ ID NO. 9);

[0086] The primer probe combination for detecting Tomato brown rugose fruit virus is as follows:

[0087] ToBRFV-F: 5'-ATTCCACATGGCAACGGCTA-3' (SEQ ID NO. 10);

[0088] ToBRFV-R: 5'-TTGACTAGGTTCGACACCGC-3' (SEQ ID NO. 11);

[0089] ToBRFV-P: 5'-CGATTGTGTACACGGGCCCCA-3' (SEQ ID NO. 12);

[0090] The primer probe combination for detecting internal process quality control product is as follows:

[0091] IPC-F: 5'-CTCTAAGTTAGCGAAATTGATGGTATTG-3' (SEQ ID NO. 13);

[0092] IPC-R: 5'-ATAATTATCCCAGGGCCTCCT-3' (SEQ ID NO. 14);

[0093] IPC-P: 5'-ATTACTTGCGCTGCCACATTGCTG-3' (SEQ ID NO. 15).

[0094] The primer probe combination for detecting tomato pathogens provided in the embodiment is used for jointly or individually detecting Tomato canker, Tomato leaf curl New Delhi virus, Tomato spotted wilt virus and Tomato brown rugose fruit virus.

[0095] In the embodiment, the probe for detecting Tomato canker is labeled with ATTO 425, and the quenching group is SQ1; the probe for detecting Tomato leaf curl New Delhi virus is labeled with FAM, and the quenching group is SQ1; the probe for detecting Tomato spotted wilt virus is labeled with HEX, and the quenching group is SQ1; the probe for detecting Tomato brown rugose fruit virus is labeled with CY5, and the quenching group is BHQ3; the IPC probe is labeled with CY5.5, and the quenching group is BHQ3.

[0096] Example 2: Integrated closed microfluidic chip kit for rapid simultaneous detection of four tomato pathogens in the field

[0097] The kit provided in the embodiment comprises the following substances:

[0098] (1) Chip device for nucleic acid detection;

[0099] (2) Further comprising purification reagent, air-dry reagent and lyophilized internal process control added in the chip device.

[0100] 1) Purification reagent

[0101] The purification reagent includes 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 reagent is added to the reagent compartment in the chip.

[0102] 2) Air-dry reagent

[0103] The reaction system of the air-dry reagent 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 probe in a 50 μL amplification system is shown in Table 2. The reagent in Table 1 is added to the reaction layer of the chip, the chip is placed in a forced air drying oven, and the air-dry operation is performed according to the instructions of 4x Air-Dryable qPCR Mix. Finally, the air-dry reagent is dissolved with 50 μL of sample grinding solution for reaction.

[0104] Table 1 Air-dry reagent reaction system

[0105]

[0106] Table 2 Final concentration of each primer probe

[0107]

[0108] 3) Lyophilized internal process control

[0109] The preparation of the lyophilized internal process control includes the preparation of IPC pseudovirus and the preparation of internal process control lyophilized balls.

[0110] 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.

[0111] Using the lyophilization system (Table 3) and the lyophilization program (Table 4), 1×10 6The IPC pseudovirus with 100 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 be freeze-dried according to the freeze-drying program of the freeze dryer shown in Table 4 to obtain a freeze-dried ball. The freeze-dried ball is placed on the chip where the reagents have been dried, and assembled into an integrated closed microfluidic chip kit.

[0112] Table 3 Freeze-dried IPC reagent system

[0113]

[0114] Table 4 Freeze-drying program

[0115]

[0116] (3) Sample processing tube

[0117] The integrated closed microfluidic chip kit provided in this embodiment can also contain a sample processing tube, which is assembled as follows: 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 is packaged with aluminum foil at 160°C.

[0118] Example 3: Method for simultaneously detecting four tomato pathogens using an integrated closed microfluidic chip kit

[0119] (1) Sample processing: The aluminum foil protective film of the sample processing tube is torn off, and the sample processing tube is placed vertically. The tomato leaf, seed or fruit sample (100-500 mg) to be detected is added to the sample processing tube, the tube cap is tightened, the tube body is kept vertical, and the sample is rubbed for 5-10 times to ensure that the grinding material and the sample are in full contact, and the grinding is sufficient to obtain a sample grinding solution;

[0120] (2) Chip assembly: The reagent compartment and the reaction layer of the chip are taken out, the sealing tape on the back of the reagent compartment is torn off, the reagent compartment and the reaction layer are held, the head of the reaction layer is vertically upward, and the reagent compartment is pressed to pierce the reagent compartment to the upper and lower layers of the chip to be seamlessly connected, and the assembly is completed;

[0121] (3) Sample addition: The sample compartment cover is unscrewed, 2-3 drops (50 μL) of the sample grinding solution obtained in step (1) are added to the sample compartment, and the sample compartment cover is tightened;

[0122] (4) Nucleic acid detection: the chip obtained in step (3) is placed into a CarryOn P1000F rapid nucleic acid detection device for nucleic acid detection, and the operation method of the nucleic acid detection device is as follows: turn on the device for more than 3 s, click the "detection" button on the home page, wait for 3 s, and then scan the chip two-dimensional code when the red light of the scanning window flashes; according to the screen prompt, scan the sample information or skip; according to the screen prompt, open the door, insert the chip, and close the door; click the "run" button to start detection; after the detection is completed, the detection result is viewed.

[0123] In the detection method provided in the embodiment, the sample processing tube takes about 1 min to process the sample; in the nucleic acid detection process, 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, and the total time is 23 min. The whole process time of the detection of tomato crop diseases from sample processing, nucleic acid extraction and purification to amplification detection result is about 36 min.

[0124] Example 4: Specificity evaluation of the method for simultaneously detecting four kinds of tomato pathogens by using the integrated closed microfluidic chip kit

[0125] The negative quality control (deionized water) and Cmn bacterial liquid, ToLCNDV plasmid, TSWV plasmid and ToBRFV plasmid are detected by using the detection method described in Example 3. The pathogenic bacteria and pathogenic fungi used in this experiment are provided by the Plant Inspection and Quarantine Institute of China Institute for Quality Inspection and Testing Science; the virus plasmid is synthesized by Beijing Qikexin Biotechnology Co., Ltd. The specificity detection results are shown in Table 5.

[0126] Determination of target detection results: IPC is internal process control, and the IPC of each sample is amplified, indicating that the sample nucleic acid extraction and amplification process are normal; the negative sample has no amplification; the positive samples of tomato canker bacteria, New Delhi tomato curly leaf virus, tomato spotted wilt virus and tomato brown rugose fruit virus all have amplification, while other pathogen samples have no amplification.

[0127] From the specificity 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.

[0128] Table 5 Specificity test results of primer probe combination for detecting four kinds of tomato diseases

[0129]

[0130] Example 5: Sensitivity evaluation of the method for simultaneously detecting four kinds of tomato pathogens by using the integrated closed microfluidic chip kit

[0131] The bacterial solution of Clavibacter michiganensis was prepared, and after gradient dilution, the dilution solution with different concentrations was used as a sample, and the kit provided in Example 2 and the detection method provided in Example 3 were used for detection. The pseudo-virus of Tomato leaf curl New Delhi virus, Tomato spotted wilt virus and Tomato brown rugose fruit virus was prepared, and after gradient dilution, the dilution solution with different concentrations was used as a sample, and the kit provided in Example 2 and the detection method provided in Example 3 were used for detection. The detection results are shown in Table 6 below, the minimum detection limit of Clavibacter michiganensis is 9.0 x 10 2 CFU / reaction, the minimum detection limit of Tomato leaf curl New Delhi virus is 1.0 x 10 1 copies / reaction, the minimum detection limit of Tomato spotted wilt virus is 1.0 x 10 1 copies / reaction, and the minimum detection limit of Tomato brown rugose fruit virus is 1.0 x 10 1 copies / reaction.

[0132] From the sensitivity detection results, it can be seen that the primer probe combination provided in the present application, the kit containing the primer probe combination and the detection method have good sensitivity, and can be used for field rapid detection of tomato diseases.

[0133] Table 6 Sensitivity test results of primer probe combinations for detecting four kinds of tomato diseases

[0134]

[0135] Experimental Example 6: Detection of real samples of tomato leaves, seeds and fruits by using integrated closed microfluidic chip kit

[0136] The real samples were detected by the method of Example 3. Tomato seed, leaf, fruit and other materials were collected from tomato planting areas and farmer planting fields in Sanya City, Hainan Province. The kit provided in Example 2 was used, and the method provided in Example 3 was used to detect the collected samples. The detection results of 52 tomato samples (12 seed samples, 25 leaf samples and 15 fruit samples) were as follows: 37 samples were negative (not all results are listed), 2 seed samples were infected with Clavibacter michiganensis, 3 fruit samples were infected with Tomato leaf curl New Delhi virus, 2 leaf samples were infected with Tomato spotted wilt virus, 1 leaf sample and 5 fruit samples were infected with Tomato brown rugose fruit virus, 1 leaf sample was infected with Clavibacter michiganensis and Tomato brown rugose fruit virus at the same time, and 1 fruit sample was infected with Clavibacter michiganensis, Tomato leaf curl New Delhi virus and Tomato spotted wilt virus at the same time, a total of 15 tomato samples were detected (not all results are listed). Figure 1 Figures 2-7 ​The results of the detection of the real samples show that the primer probe composition, the kit and the detection method for simultaneously detecting the four tomato pathogens provided by the application can detect the real samples of tomato leaves, seeds and fruits, and can be used for rapid identification of tomato diseases in the field and the like.

[0137] In addition, since tomato canker bacteria, New Delhi tomato leaf curl virus, tomato mottle virus and tomato brown rugose fruit virus can also infect peppers, the kit provided in Example 2 is used to detect the seed, leaf, fruit and the like of the pepper with the method provided in Example 3. The detection results show that the primer probe composition, the kit and the detection method provided by the application can detect the real samples of pepper leaves, seeds and fruits, and can be used for rapid identification of pepper diseases in the field and the like.

[0138] Comparative example:

[0139] The difference between the comparative example and Examples 1-3 is only that the primer probe combination for simultaneously detecting tomato canker bacteria, New Delhi tomato leaf curl virus, tomato mottle virus and tomato brown rugose fruit virus is different, and the specific difference is as follows:

[0140] The primer probe combination for detecting tomato canker bacteria is as follows:

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

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

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

[0144] The primer probe combination for detecting New Delhi tomato leaf curl virus is as follows:

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

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

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

[0148] The primer probe combination for detecting tomato spotted wilt virus is as follows:

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

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

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

[0152] The primer probe combination for detecting tomato brown rugose fruit virus is as follows:

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

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

[0155] ToBRFV-P: 5'-CGATTGTGTACACGGGCCCCA-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] An integrated closed microfluidic chip kit was prepared by using the primer probe combination provided in the comparative example, in the same manner as in Example 2, and the detection method provided in Example 3 was used to detect Figure 2 、 Figure 3 、 Figure 4 、 Figure 6 and Figure 7The real samples involved in the examples were detected respectively, and then the detection results were compared with those obtained by the comparative examples. The comparison results of the amplification Ct values are shown in Table 7. As can be seen from the results in Table 7, the amplification Ct values obtained by the primer probe combination in the comparative examples are obviously delayed compared with the primer probe combination in the present application, and the amplification fluorescence values also have different degrees of reduction. In the comparative examples, the tomato canker bacteria were not detected in the mixed sample containing tomato canker bacteria and tomato brown rugose fruit virus; the new delhi tomato leaf curl virus was not detected in the mixed sample containing tomato canker bacteria, new delhi tomato leaf curl virus and tomato spotted wilt virus.

[0161] Table 7 Comparison results of amplification Ct values of examples and comparative example 1

[0162]

[0163] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, 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 tomato pathogens, characterized in that, The primer probe combination comprises a primer probe group for detecting Clavibacter michiganensis, a primer probe group for detecting Tomato leaf curl New Delhi virus, a primer probe group for detecting Tomato spotted wilt virus, a primer probe group for detecting Tomato brown rugose fruit virus, and an IPC primer probe group for detecting internal process control; The primer probe group for detecting Clavibacter michiganensis 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 group for detecting Tomato leaf curl New Delhi virus 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 group for detecting Tomato spotted wilt virus 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 group for detecting Tomato brown rugose fruit virus 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 group 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 Clavibacter michiganensis, Tomato leaf curl New Delhi virus, Tomato spotted wilt virus and Tomato brown rugose fruit virus.

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 tomato pathogens, namely Clavibacter michiganensis, Tomato leaf curl New Delhi virus, Tomato spotted wilt virus and Tomato brown rugose fruit virus.

4. A kit for detecting a tomato pathogen in situ, characterized in that, The kit comprises the primer probe combination of claim 1.

5. The kit of claim 4, wherein The kit is an integrated closed microfluidic chip kit, and the integrated closed microfluidic chip contains the primer probe combination.

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 tomato pathogens, namely Clavibacter michiganensis, Tomato leaf curl New Delhi virus, Tomato spotted wilt virus and Tomato brown rugose fruit virus.

9. A method for detecting tomato pathogens on site using the kit according to any one of claims 5-7, characterized in that, 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 an integrated closed microfluidic chip in the kit, and the pretreated sample is detected on the microfluidic chip by using a CarryOn P1000F rapid nucleic acid detection device; The tomato pathogens are Clavibacter michiganensis, Tomato leaf curl New Delhi virus, Tomato mottle virus and Tomato brown rugose fruit virus.

10. The method of claim 9, wherein, The sample to be detected in step (1) is tomato seeds, tomato leaves, tomato fruits, pepper seeds, pepper leaves or pepper fruits.

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