Primer probe combination and kit for simultaneously detecting four tomato pathogens and application of primer probe combination and kit

Abstract

The invention discloses a primer probe combination for simultaneously detecting four tomato pathogens, a kit and application thereof, and belongs to the technical field of molecular biological detection. The invention aims to solve the technical problems that the existing molecular biological detection method for tomato diseases cannot simultaneously detect various tomato pathogens or cannot simultaneously detect bacterial pathogens and virus pathogens of tomatoes and is long in time consumption and the like. The invention provides a primer probe combination for detecting tomato canker pathogen, New Delhi tomato leaf curl virus, tomato spotted wilf virus and tomato brown wrinkled fruit virus in tomato or pepper crops, an integrated micro-fluidic chip kit containing the primer probe combination and a detection method. The method for detecting pathogens in tomato or pepper crops by adopting the primer probe combination and the kit provided by the invention has the advantages of higher sensitivity and accuracy, simplicity and convenience in operation, rapidness and time saving. The primer probe combination, the kit and the method provided by the invention can simultaneously detect the four pathogens, and play an important role in on-site rapid detection of the pathogens in tomato or pepper planting.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, specifically involving primer and probe combinations, kits, and their applications for the simultaneous detection of four tomato pathogens. Background Technology

[0002] Tomatoes, as an important global economic crop, occupy a vital position in agricultural production. However, the frequent occurrence of tomato diseases seriously affects tomato yield and quality. Among these are Tomato Leaf Curl NewDelhi Virus (ToLCNDV), Tomato Spotted Wilt Virus (TSWV), Tomato Brown Rugose Fruit Virus (ToBRFV), and *Corynebacterium miltanum* subsp. *miltanum*, the pathogen of tomato bacterial canker. Clavibacter michiganensis subsp. michiganenis , Cmm These are four major pathogens that harm tomato and pepper production. These pathogens can be transmitted through various routes, including seeds, soil, and insect vectors, and their symptoms are easily confused with other diseases, increasing the difficulty of early diagnosis and control. These four pathogens have long survival times and cause serious damage; seed-borne pathogens are the main primary source of long-distance transmission, therefore, effective disease monitoring is of great importance.

[0003] The diagnostic and detection techniques for plant quarantine diseases mainly rely on physiological and biochemical detection, serological detection, and molecular biological detection. Among these, molecular biological detection is the most commonly used method, such as polymerase chain reaction (PCR), reverse transcription-PCR (RT-PCR), and real-time quantitative PCR (qPCR). For example, the People's Republic of China National Standard GB / T 29431-2012 published the quarantine identification method for tomato bacterial canker; the People's Republic of China National Standard GB / T 28982-2012 published the PCR detection method for tomato spotted wilt virus. However, there is currently no specific method for detecting... CmmMultiplex fluorescent PCR detection methods for ToLCNDV, TSWV, and ToBRFV are needed. Furthermore, existing molecular biological detection systems or methods for tomato diseases typically require sample pretreatment before detection, and most methods cannot simultaneously detect multiple pathogens, or simultaneously detect bacterial and viral pathogens. These methods are cumbersome and time-consuming. Therefore, there is an urgent need to develop a rapid, on-site detection method for tomato pathogens that can simultaneously detect multiple pathogens, has high sensitivity and specificity, and is easy to operate. Summary of the Invention

[0004] To address the technical problems of existing molecular biological detection systems or methods for tomato diseases, which typically require sample pretreatment before detection, and the inability of most methods to simultaneously detect multiple tomato pathogens, or to simultaneously detect bacterial and viral pathogens, as well as the cumbersome and time-consuming operation of these methods or systems, this invention provides a primer-probe combination for detecting *Tomato Canker*, *New Delhi Tomato Leaf Curl Virus*, *Tomato Spotted Wilt Virus*, and *Tomato Brown Ruffle Fruit Virus* in tomatoes or peppers, along with an integrated closed microfluidic chip kit and method containing this primer-probe combination. The primer-probe combination, integrated closed microfluidic chip kit, and detection method provided by this invention can simultaneously detect four pathogens—*Tomato Canker*, *New Delhi Tomato Leaf Curl Virus*, *Tomato Spotted Wilt Virus*, and *Tomato Brown Ruffle Fruit Virus*—playing a crucial role in the rapid on-site detection of pathogens in tomato and pepper cultivation.

[0005] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a primer-probe combination for the simultaneous detection of four tomato pathogens, the primer-probe combination including a primer-probe combination for detecting tomato canker pathogen, 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 wrinkle virus, and an IPC primer-probe combination for detecting internal process control (IPC). The primer-probe combination for detecting tomato canker pathogens 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. The primer-probe combination for detecting New Delhi tomato leaf curl virus 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. The primer-probe combination for detecting tomato spotted wilt 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. The primer-probe combination for detecting tomato brown wrinkle 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. 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.

[0006] Preferably, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group.

[0007] More preferably, the fluorescent reporter group is selected from one or more combinations of FAM, ROX, HEX, CY5, VIC, TET, JOE, CY3, CY7, RED610, Texas Red, RED670, NED, AMCA, Pacific Blue, ATTO 425, BODIPY FL, Alexa Fluor488, Yakima Yellow, Quasar 570, Aqua Phluor 593, ATTO 590 and CY5.5.

[0008] More preferably, the fluorescent reporter group is selected from one or more combinations of ATTO 425, FAM, HEX, CY3, CY5, CY5.5, VIC, JOE and ROX.

[0009] Preferably, the quenching group is a fluorescence quenching group.

[0010] More preferably, the fluorescence quenching group is selected from one or more combinations of 6-TAMRA, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, QYS-7 and SQ1.

[0011] More preferably, the fluorescence quenching group is selected from one or more combinations of BHQ2, BHQ3 and SQ1.

[0012] A second objective of this invention is to provide an application of the aforementioned primer-probe combination, wherein the primer-probe combination is used to prepare reagents, kits, or chips for the simultaneous detection of Tomato Canker, New Delhi Tomato Leaf Curl Virus, Tomato Spotted Wilt Virus, and Tomato Brown Wrinkle Virus.

[0013] The third objective of this invention is to provide an application of the above-mentioned primer-probe combination, wherein the primer-probe combination is used to simultaneously detect four tomato pathogens, namely, *Tomato Canker*, *New Delhi Tomato Leaf Curl Virus*, *Tomato Spotted Wilt Virus*, and *Tomato Brown Wrinkle Virus*.

[0014] A fourth objective of this invention is to provide a kit for on-site detection of tomato pathogens, the kit comprising the primer-probe combination described above.

[0015] In one embodiment of the present invention, the reagent kit is an integrated closed microfluidic chip reagent kit.

[0016] In one embodiment of the present invention, the integrated closed microfluidic chip kit further includes purification reagents, qPCR reaction reagents, and lyophilization internal process quality control materials.

[0017] Preferably, the purification reagent includes a lysis buffer, a washing buffer, an elution buffer, and magnetic beads. The components of the lysis buffer and the washing buffer include guanidine hydrochloride, sodium acetate, Triton X-100, and 1,3-butanediol. The components of the elution buffer include Tris-HCl. The magnetic beads are dried magnetic beads.

[0018] More preferably, the lysis buffer and washing buffer consist of 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 eluent consists of 10 mM Tris-HCl (pH 8.5).

[0019] Preferably, the quality control material for the internal freeze-drying process is an RNA pseudovirus.

[0020] More preferably, the freeze-drying internal process quality control material is packaged in the form of freeze-dried balls within an integrated closed microfluidic chip.

[0021] More preferably, the kit performs nucleic acid extraction, purification, amplification, and detection of samples on an integrated closed microfluidic chip.

[0022] More preferably, the integrated closed microfluidic chip kit can be used in the CarryOn P1000F rapid nucleic acid detection device.

[0023] In one embodiment of the present invention, the integrated closed microfluidic chip kit further includes a sample processing tube containing abrasive particles and sample pretreatment solution.

[0024] The fifth objective of this invention is to provide an application of the above-mentioned kit, wherein the kit is used to simultaneously detect four tomato pathogens, namely, *Tomato rot fungus*, *New Delhi tomato leaf curl virus*, *Tomato wilt virus*, and *Tomato brown wrinkle fruit virus*.

[0025] The sixth objective of this invention is to provide a method for on-site detection of tomato pathogens, the method comprising the following steps: (1) Preprocess the sample to be tested to obtain a preprocessed sample; (2) Add the pre-processed sample obtained in step (1) to the above-mentioned integrated closed microfluidic chip kit, and use the CarryOn P1000F rapid nucleic acid detection device to detect the pre-processed sample on the microfluidic chip; The tomato pathogens mentioned are Tomato Ulcer Bacterium, New Delhi Tomato Leaf Curl Virus, Tomato Spotted Wilt Virus, and Tomato Brown Wrinkle Fruit Virus.

[0026] In one embodiment of the present invention, the sample to be tested in step (1) is tomato seed, tomato leaf, tomato fruit, chili seed, chili leaf or chili fruit.

[0027] As an optional addition, the present invention provides a method for on-site detection of pathogens in tomato or pepper crops, the method comprising the preparation of an integrated closed microfluidic chip reagent kit, sample pretreatment, and rapid device detection; Specifically, the method includes the following steps: (1) The above primer and probe combination is prepared into an integrated closed microfluidic chip kit; (2) Tomato seeds, tomato seed powder, tomato leaves, tomato fruits, pepper seeds, pepper leaves or pepper fruits are added to a sample processing tube for processing to obtain the liquid to be tested; (3) The liquid to be tested is dripped into the integrated closed microfluidic chip; (4) Place the integrated closed microfluidic chip containing the liquid to be tested into the testing equipment for testing and analysis.

[0028] Preferably, the sample processing tube in step (2) contains a sample pretreatment solution and abrasive particles; the sample pretreatment solution includes Tris-HCl (pH 8.0), NaCl, EDTA and SDS.

[0029] More preferably, the concentration range of Tris-HCl is 10 mM-100 mM, the concentration range of NaCl is 30 mM-1.4 M, the concentration range of EDTA is 1.5 mM-20 mM, and the concentration range of SDS is 0.05%-2%; the diameter of the grinding particles is 0.1 μm-1 mm.

[0030] More preferably, the concentration range of Tris-HCl (pH 8.0) in the sample pretreatment solution is 30 mM-55 mM, the concentration range of NaCl is 350 mM-700 mM, the concentration range of EDTA is 5 mM-10 mM, and the concentration range of SDS is 0.5%-1%.

[0031] Preferably, step (4) specifically includes the following steps: 1) Turn on the rapid testing equipment; 2) Scan the QR code; 3) Insert the aforementioned integrated closed microfluidic chip; 4) Runtime testing; 5) End, check the test results and amplification curve.

[0032] As an optional addition, the present invention provides a method for on-site detection of pathogens in tomato or pepper crops, the method comprising the following steps: (1) Prepare an integrated closed microfluidic chip kit by combining the above primers and probes; (2) Rapid sample pretreatment using sample processing tubes; (3) Use the purification reagents on the chip to lyse the sample and extract and purify the nucleic acid; (4) Use the above primer-probe combination to rapidly amplify the nucleic acid of the sample extracted and purified in step (3); (5) Analyze the amplification results.

[0033] The beneficial effects of this invention are: This invention provides a primer-probe combination for detecting *Tomato Canker*, *New Delhi Tomato Leaf Curl Virus*, *Tomato Spotted Wilt Virus*, and *Tomato Brown Ruffle Virus* in tomato or pepper crops, an integrated closed microfluidic chip kit containing this primer-probe combination, and a method. The method for detecting pathogens in tomato or pepper crops using the primer-probe combination and kit provided by this invention integrates sample pretreatment, nucleic acid extraction, purification, amplification, and detection, and has good sensitivity (the detection limit for *Tomato Canker* is 9.0 × 10⁻⁶). 2 The limit of detection for CFU / reaction of New Delhi tomato leaf curl virus is 1.0 × 10⁻⁶. 1 The limit of detection for tomato spotted wilt virus is 1.0 × 10⁻⁶ copies / reaction.1 The limit of detection for tomato brown wrinkle virus (CWRV) is 1.0 × 10⁻⁶ copies / reaction. 1 This detection method, using the CarryOn P1000F rapid nucleic acid detection device, can complete the on-site automated nucleic acid detection of samples within 36 minutes, achieving rapid detection with "sample in, result out." The method is simple to operate, fast, and time-saving. The primer-probe combination, integrated closed microfluidic chip kit, and detection method provided by this invention can simultaneously detect four pathogens: Tomato Canker, New Delhi Tomato Leaf Curl Virus, Tomato Spotted Wilt Virus, and Tomato Brown Wrinkle Virus. It plays an important role in the rapid on-site detection of pathogens in tomato and pepper cultivation. Attached Figure Description

[0034] Figure 1 This is a diagram showing the negative test results for a real tomato sample. Figure 2 The image shows the results of testing tomato seeds infected with Tomato Canker Bacterium tumefaciens. Figure 3 The image shows the results of testing tomato fruits infected with New Delhi Tomato Leaf Curl Virus. Figure 4 The image shows the results of testing tomato leaves infected with tomato spotted wilt virus. Figure 5 The image shows the results of testing tomato leaves infected with Tomato Brown Wrinkle Fruit Virus. Figure 6 The image shows the results of testing tomato leaves simultaneously infected with Tomato Canker Bacterium and Tomato Brown Ruffle Virus. Figure 7 The image shows the results of testing tomato fruits simultaneously infected with Tomato Canker, New Delhi Tomato Leaf Curl Virus, and Tomato Spotted Wilt Virus. Figure 8 This is a physical image of the chip device used for nucleic acid detection in this invention; Figure 9 This is a physical image of the sample processing tube used in this invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, embodiments obtained by other those skilled in the art without creative effort are all protected by this invention.

[0036] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, and instruments used are all conventional materials, reagents, and instruments in the art, which can be obtained commercially by those skilled in the art. Unless otherwise specified, the molecular biology experimental procedures involved in this invention are conventional experimental procedures in the art or can be performed according to the product instructions of the corresponding reagents.

[0037] The chip device for nucleic acid detection described in this invention is detailed in Chinese Invention Patent Publication No. CN113278509A; the preparation method of the dried magnetic beads is detailed in Chinese Invention Patent Publication No. CN113005117A; the sample processing tube is detailed in Chinese Utility Model Patent Publication No. CN220136779U; and the entire device, i.e., the PCR reaction device, uses the CarryOn P1000F rapid nucleic acid detection device, detailed in Chinese Invention Patent Publication No. CN116200514A. A physical image of the aforementioned chip device is shown below. Figure 8 As shown in the image, a physical diagram of the above sample processing tube is as follows: Figure 9 As shown.

[0038] In the following examples, the tomato canker pathogen, New Delhi tomato leaf curl virus, tomato wilt virus, and tomato brown wrinkle virus were all provided by the Institute of Plant Inspection and Quarantine, China Academy of Quality Inspection and Testing.

[0039] Example 1: Primer and probe combination for simultaneous detection of Tomato Bacterium sarcodactylis, New Delhi Tomato Leaf Curl Virus, Tomato Spotted Wilt Virus, and Tomato Brown Ruffle Virus. Based on the specific sequences of Tomato Ulcer Bacterium, New Delhi Tomato Leaf Curl Virus, Tomato Spotted Wilt Virus, and Tomato Brown Wrinkle Virus, primer and probe sequences for amplifying each pathogen were designed.

[0040] The primer-probe combination used to detect the pathogen causing tomato bacterial canker is as follows: Cmm-F: 5'-GCAGACGACCAGAAGAAGTTGTT-3' (SEQ ID NO. 1); Cmm-R: 5'-CCGAGGCTCGTAGTCATTGAC-3' (SEQ ID NO. 2); Cmm-P: 5'-ACTTCGGCCGGCATACGCTGTACTC-3' (SEQ ID NO.3); The primer-probe combination used for detecting New Delhi tomato leaf curl virus is as follows: ToLA-F: 5'-GGTTACCTCTGCAGCATCGT-3' (SEQ ID NO. 4); ToLA-R: 5'-CGCTGCGCTTCCTAAATGTC-3' (SEQ ID NO. 5); ToLA-P: 5'-CCCACCCACTCCCAGACCCA-3' (SEQ ID NO. 6); The primer-probe combination used for detecting tomato spotted wilt virus is as follows: TSWV-F: 5'-GCTTGATCAGGGTCAGGCTT-3' (SEQ ID NO. 7); TSWV-R: 5'-TGGCAAGCCTCACAGACTTT-3' (SEQ ID NO. 8); TSWV-P: 5'-TGCTTCTCACCCCTCTGATTCAAGCC-3' (SEQ ID NO.9); The primer-probe combination used for detecting tomato brown wrinkle virus is as follows: ToBRFV-F: 5'-ATTCCACATGGCAACGGCTA-3' (SEQ ID NO. 10); ToBRFV-R: 5'-TTGACTAGGTTCGACACCGC-3' (SEQ ID NO. 11); ToBRFV-P: 5'-CGATTGTGTACACGGGCCCCA-3' (SEQ ID NO. 12); The primer-probe combination used for detecting internal process quality control samples is as follows: IPC-F: 5'-CTCTAAGTTAGCGAAATTGATGGTATTG-3' (SEQ ID NO. 13); IPC-R: 5'-ATAATTATCCCAGGGCCTCCT-3' (SEQ ID NO. 14); IPC-P: 5'-ATTACTTGCGCTGCCACATTGCTG-3' (SEQ ID NO. 15).

[0041] The primer-probe combination for detecting tomato pathogens provided in this embodiment is used for the combined or individual detection of tomato canker pathogen, New Delhi tomato leaf curl virus, tomato wilt virus, and tomato brown wrinkle virus.

[0042] In the examples, the probe used to detect tomato canker pathogen was labeled with ATTO 425 with quenching group SQ1; the probe used to detect New Delhi tomato leaf curl virus was labeled with FAM with quenching group SQ1; the probe used to detect tomato spotted wilt virus was labeled with HEX with quenching group SQ1; the probe used to detect tomato brown wrinkle virus was labeled with CY5 with quenching group BHQ3; and the IPC probe was labeled with CY5.5 with quenching group BHQ3.

[0043] Example 2: An integrated closed-loop microfluidic chip reagent kit for rapid on-site simultaneous detection of four tomato pathogens. The kit provided in this embodiment includes the following substances: (1) Chip devices for nucleic acid detection; (2) It also includes purification reagents, air-drying reagents and lyophilized internal process quality control materials added to the chip device.

[0044] 1) Purification reagents The purification reagents consisted of 450 μL of lysis buffer, 450 μL of washing buffer, 850 μL of eluent, and 15 μL of dried magnetic beads. The lysis buffer and washing buffer were formulated with 4.2 M guanidine hydrochloride, 0.28 M sodium acetate (pH 4.7), 1.4% Triton X-100, and 30% 1,3-butanediol; the eluent was formulated with 10 mM Tris-HCl (pH 8.5). The purification reagents were added to the reagent compartment in the chip.

[0045] 2) Air-dried reagents The reaction system for the air-dried reagent was 15 μL, and the specific components are shown in Table 1 below. The primer-probe mixture in Table 1 is a mixture of all primers and probes from Example 1, and the final concentrations of each primer and probe in the 50 μL amplification system are shown in Table 2. The reagents in Table 1 were added to the reaction layer of the chip, and the chip was placed in a forced-air drying oven and air-dried according to the instructions for the 4×Air-Dryable qPCRMix. Finally, the air-dried reagent was dissolved in 50 μL of sample homogenate for the reaction.

[0046] Table 1. Air-dried reagent reaction system

[0047] Table 2 Final concentrations of each primer and probe

[0048] 3) Quality control products for internal freeze-drying processes The preparation of freeze-dried internal process control products includes the preparation of IPC pseudoviruses and the preparation of freeze-dried pellets for internal process quality control products.

[0049] According to the method for preparing a fake virus provided in Example 1 of Chinese Patent Application No. "202110669475.X", a fake virus targeting IPC (IPC fake virus) is prepared and obtained.

[0050] Using the lyophilization system (Table 3) and lyophilization procedure (Table 4), a concentration of 1×10⁻⁶ was obtained. 6 IPC pseudoviruses of copies / mL were prepared into lyophilized pellets. Specifically, the reagents in Table 3 were added dropwise to liquid nitrogen at 5 µL, and then placed in a vial and lyophilized according to the freeze dryer program shown in Table 4 to obtain lyophilized pellets. The lyophilized pellets were then placed on a chip containing air-dried reaction reagents to assemble an integrated closed microfluidic chip kit.

[0051] Table 3. Lyophilized IPC Reagent System

[0052] Table 4 Freeze-drying process

[0053] (3) Sample processing tube The integrated closed microfluidic chip kit provided in this embodiment may also include a sample processing tube, which is assembled as follows: 1 g of abrasive particles (diameter of 15 μm diamond abrasive) and 3 mL of sample pretreatment solution are added to the sample processing tube. The sample pretreatment solution consists of 50 mM Tris-HCl (pH 8.0), 700 mM NaCl, 10 mM EDTA, and 1% SDS. The sample processing tube is then placed in the tooling position of a precision manual press and sealed with aluminum foil at 160°C.

[0054] Example 3: A method for simultaneous detection of four tomato pathogens using an integrated closed microfluidic chip reagent kit. (1) Sample processing: Tear off the aluminum foil protective film of the sample processing tube, place it vertically, add the tomato leaf, seed or fruit sample (100-500 mg) to be tested into the sample processing tube, tighten the tube cap, keep the tube upright, rub it 5-10 times to make the sample fully contact the grinding material, ensure that the grinding is thorough, and obtain the sample grinding liquid. (2) Chip assembly: Take out the reagent chamber and reaction layer of the chip, tear off the sealing strip on the back of the reagent chamber, hold the reagent chamber and reaction layer, make the head of the reaction layer vertically upward, press hard to puncture the reagent chamber until the upper and lower layers of the chip are seamlessly connected, and complete the assembly; (3) Sample addition: Unscrew the sample compartment cap, add 2-3 drops (50 μL) of the sample grinding solution obtained in step (1) into the sample compartment, and tighten the sample compartment cap; (4) Nucleic acid testing: Place the chip obtained in step (3) into the CarryOn P1000F rapid nucleic acid testing device for nucleic acid testing. The operation method of the nucleic acid testing device is as follows: Press and hold the device power button for more than 3 seconds to turn on the device, and click the "Detection" button on the home page; wait for 3 seconds until the red light of the scanning window flashes, and scan the chip QR code; scan the sample information or skip according to the screen prompts; open the door, insert the chip, and close the door according to the screen prompts; click the "Run" button to start the test; after the test is completed, check the test results.

[0055] In the detection method provided in this embodiment, the sample processing time in the sample processing tube is approximately 1 minute; during nucleic acid detection, the nucleic acid extraction and purification time on the integrated closed microfluidic chip is approximately 12 minutes, and the real-time quantitative PCR reaction program on the integrated microfluidic chip is: 95℃ for 1 minute; (95℃ for 5 seconds, 60℃ for 8 seconds), with a total time of 23 minutes for 45 cycles. The entire process of detecting tomato crop diseases, from sample processing and nucleic acid extraction and purification to amplification and detection results, takes approximately 36 minutes.

[0056] Example 4: Specificity evaluation of a method for simultaneous detection of four tomato pathogens using an integrated closed microfluidic chip kit. The detection method described in Example 3 was used to detect negative control (deionized water), Cmn bacterial suspension, ToLCNDV plasmid, TSWV plasmid, and ToBRFV plasmid. The pathogenic bacteria and fungi used in this experiment were provided by the Institute of Plant Inspection and Quarantine, Chinese Academy of Quality Inspection and Testing; the virus plasmid was synthesized by Beijing Qingke Biotechnology Co., Ltd. The specificity detection results are shown in Table 5 below.

[0057] Judgment of target detection results: IPC is an internal process quality control. The fact that each sample's IPC showed amplification indicates that there were no problems with the nucleic acid extraction and amplification process. Negative samples showed no amplification. Positive samples for Tomato Ulcer Bacterium, New Delhi Tomato Leaf Curl Virus, Tomato Spotted Wilt Virus, and Tomato Brown Wrinkle Virus showed amplification, while other pathogen samples showed no amplification.

[0058] The specificity test results show that the primer-probe combination, the kit containing the primer-probe combination, and the detection method provided by this invention have good specificity.

[0059] Table 5. Specificity test results of primer-probe combinations used to detect four tomato diseases.

[0060] Example 5: Sensitivity evaluation of a method for simultaneous detection of four tomato pathogens using an integrated closed microfluidic chip kit. A bacterial suspension of *Tomato canker pathogen* was prepared and serially diluted. Different concentrations of the diluted solutions were used as samples, and the detection was performed using the kit provided in Example 2 and the detection method provided in Example 3. Pseudoviruses of New Delhi tomato leaf curl virus, tomato spotted wilt virus, and tomato brown wrinkle fruit virus were prepared and serially diluted. Different concentrations of the diluted solutions were used as samples, and the detection was performed using the kit provided in Example 2 and the detection method provided in Example 3. The detection results are shown in Table 6 below. The limit of detection for *Tomato canker pathogen* was 9.0 × 10⁻⁶. 2 The limit of detection for CFU / reaction of New Delhi tomato leaf curl virus is 1.0 × 10⁻⁶. 1 The limit of detection for tomato spotted wilt virus is 1.0 × 10⁻⁶ copies / reaction. 1 The limit of detection for tomato brown wrinkle virus (CWRV) is 1.0 × 10⁻⁶ copies / reaction. 1 Copies / Reaction.

[0061] The sensitivity test results show that the primer-probe combination, the kit containing the primer-probe combination, and the detection method provided by this invention have good sensitivity and can be used for rapid on-site detection of tomato diseases in the field.

[0062] Table 6. Sensitivity test results of primer-probe combinations used to detect four tomato diseases.

[0063] Experiment Example 6: Detection of real samples of tomato leaves, seeds, and fruits using an integrated closed microfluidic chip reagent kit. Real samples were tested using the method described in Example 3. Diseased tomato seeds, leaves, and fruits were collected on-site from tomato plantations and farmers' fields in Sanya City, Hainan Province. The collected samples were tested using the kit provided in Example 2 and the method described in Example 3. The test results of 52 tomato samples (12 seed samples, 25 leaf samples, and 15 fruit samples) showed that 37 samples were negative. Figure 1 (Results not fully listed) 2 seed samples were infected with *Tomato Canker*, 3 fruit samples were infected with New Delhi Tomato Leaf Curl Virus, 2 leaf samples were infected with Tomato Spotted Wilt Virus, 1 leaf sample and 5 fruit samples were infected with Tomato Brown Wrinkle Virus, 1 leaf sample was infected with both *Tomato Canker* and Tomato Brown Wrinkle Virus, and 1 fruit sample was infected with all three of *Tomato Canker*, New Delhi Tomato Leaf Curl Virus, and Tomato Spotted Wilt Virus. A total of 15 tomato samples tested positive for disease. Figures 2-7 (Results not fully listed). Based on the test results of real samples, it can be seen that the primer-probe composition, kit, and detection method provided by this invention for the simultaneous detection of four tomato pathogens can be used to detect real tomato samples (leaves, seeds, and fruits), and can be used for rapid identification of tomato diseases in the field and other on-site situations.

[0064] Furthermore, since tomato canker pathogen, New Delhi tomato leaf curl virus, tomato wilt virus, and tomato brown wrinkle virus can also infect peppers, this invention also utilizes the kit provided in Example 2 and the method provided in Example 3 to detect diseased pepper seeds, leaves, and fruits. The detection results show that the primer-probe composition, kit, and detection method provided by this invention can detect real pepper samples (leaves, seeds, and fruits) and can be used for rapid identification of pepper diseases in the field.

[0065] Comparative example: The only difference between this comparative example and Examples 1-3 is the primer and probe combination used for the simultaneous detection of tomato bacterial canker, New Delhi tomato leaf curl virus, tomato wilt virus, and tomato brown wrinkle virus, as detailed below: The primer-probe combination used to detect tomato bacterial canker is as follows: Cmm-F: 5'-CCCCACAAGGAGGCGTACTA-3' (SEQ ID NO. 16); Cmm-R: 5'-GCATGTGCACCTCTCCTCTGTA-3' (SEQ ID NO. 17); Cmm-P: 5'-CAGGCGTCTGTTCTGGCGGTGG-3' (SEQ ID NO. 18); The primer-probe combination used for detecting New Delhi tomato leaf curl virus is as follows: Tolcndv-F: 5'-TCCAAGGATTCTTATCCTTKAGAGAG-3' (SEQ ID NO. 19); Tolcndv-R: 5'-CAAGCGGAATTCACAATTCCGATC-3' (SEQ ID NO. 20); Tolcndv-P: 5'-TGAGGAAGAGTAGTAGTGCAGGTTGCART-3' (SEQ ID NO. 21); The primer-probe combination used for detecting tomato spotted wilt virus is as follows: TSWV-F: 5'-CTCTCGATGATGCAAAGTCTGTGR-3' (SEQ ID NO. 22); TSWV-R: 5'-TCTCAAAGCTATCRACTGAAGCAATAA-3' (SEQ ID NO. 23); TSWV-P: 5'-AGGTAAGCTACCTCCCAGCATTATGGCAAG-3' (SEQ ID NO. 24); The primer-probe combination used for detecting tomato brown wrinkle virus is as follows: ToBRFV-F: 5'-ATTCCACATGGCAACGGCTA-3' (SEQ ID NO. 10); ToBRFV-R: 5'-TTGACTAGGTTCGACACCGC-3' (SEQ ID NO. 11); ToBRFV-P: 5'-CGATTGTGTACACGGGCCCCA-3' (SEQ ID NO. 12); The primer-probe combination used for detecting internal process control (IPC) samples is as follows: IPC-F: 5'-CTCTAAGTTAGCGAAATTGATGGTATTG-3' (SEQ ID NO. 13); IPC-R: 5'-ATAATTATCCCAGGGCCTCCT-3' (SEQ ID NO. 14); IPC-P: 5'-ATTACTTGCGCTGCCACATTGCTG-3' (SEQ ID NO. 15).

[0066] Using the primer and probe combination provided in the comparative example, an integrated closed microfluidic chip kit was prepared according to the same method as in Example 2. The detection method provided in Example 3 was then used to detect... Figure 2 , Figure 3 , Figure 4 , Figure 6 and Figure 7 The actual samples involved were tested separately, and then compared with the test results obtained in the examples. The comparison results of 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 were significantly delayed compared to the primer-probe combination in this invention, and the amplification fluorescence values ​​were also reduced to varying degrees. In the comparative examples, *Tomato Canker* was not detected in the mixed sample containing *Tomato Canker* and *Tomato Brown Wrinkle Virus*; *Tomato Brown Wrinkle Virus* was not detected in the mixed sample containing *Tomato Canker*, New Delhi Tomato Leaf Curl Virus, and Tomato Spotted Wilt Virus.

[0067] Table 7 Comparison of amplified Ct values ​​between the examples and Comparative Example 1

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer-probe combination for simultaneous detection of four tomato pathogens, characterized in that, The primer and probe combination includes a primer and probe combination for detecting tomato canker pathogen, a primer and probe combination for detecting New Delhi tomato leaf curl virus, a primer and probe combination for detecting tomato spotted wilt virus, a primer and probe combination for detecting tomato brown wrinkle virus, and an IPC primer and probe combination for detecting internal process quality control products. The primer-probe combination for detecting tomato canker pathogens 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. The primer-probe combination for detecting New Delhi tomato leaf curl virus 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. The primer-probe combination for detecting tomato spotted wilt 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. The primer-probe combination for detecting tomato brown wrinkle 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. 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.

2. The application of the primer-probe combination according to claim 1, characterized in that, The application involves using primer-probe combinations to prepare reagents, kits, or chips for the simultaneous detection of Tomato Canker, New Delhi Tomato Leaf Curl Virus, Tomato Spotted Wilt Virus, and Tomato Brown Wrinkle Virus.

3. The application of the primer-probe combination according to claim 1, characterized in that, The application involves using primer-probe combinations to simultaneously detect four tomato pathogens: Tomato Ulcer Bacterium, New Delhi Tomato Leaf Curl Virus, Tomato Spotted Wilt Virus, and Tomato Brown Wrinkle Virus.

4. A kit for on-site detection of tomato pathogens, characterized in that, The kit comprises the primer-probe combination as described in claim 1.

5. The reagent kit according to claim 4, characterized in that, The kit is an integrated closed microfluidic chip kit.

6. The reagent kit according to claim 5, characterized in that, The integrated closed microfluidic chip kit also includes purification reagents, qPCR reaction reagents, and lyophilized internal process quality control materials.

7. The reagent kit according to claim 6, characterized in that, The integrated closed microfluidic chip kit also includes a sample processing tube containing abrasive particles and sample pretreatment solution.

8. The application of the kit according to any one of claims 4-7, characterized in that, The application involves using the kit to simultaneously detect four tomato pathogens: Tomato Ulcer Bacterium, New Delhi Tomato Leaf Curl Virus, Tomato Spotted Wilt Virus, and Tomato Brown Wrinkle Virus.

9. A method for on-site detection of tomato pathogens, characterized in that, The method includes the following steps: (1) Preprocess the sample to be tested to obtain a preprocessed sample; (2) Add the pretreated sample obtained in step (1) to the integrated closed microfluidic chip kit of any of claims 5-7, and use the CarryOn P1000F rapid nucleic acid detection device to detect the pretreated sample on the microfluidic chip; The tomato pathogens mentioned are Tomato Ulcer Bacterium, New Delhi Tomato Leaf Curl Virus, Tomato Spotted Wilt Virus, and Tomato Brown Wrinkle Fruit Virus.

10. The method according to claim 9, characterized in that, The sample to be tested in step (1) is tomato seed, tomato leaf, tomato fruit, chili seed, chili leaf or chili fruit.

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