Puncher for accurately detecting nucleic acid of plant leaves and use method of puncher

Abstract

The invention belongs to the technical field of plant leaf punching and sampling devices, particularly relates to a puncher for accurately detecting nucleic acid of plant leaves and a use method, and aims to solve the problems that existing sampling devices are all provided with metal modules and are reusable sampling devices, and single sampling device is large in size and inconvenient to use. In order to solve the problem that in the prior art, in the prior art, in the prior art, the use requirements of portability and disposable use required by plant leaf pathogen collection are difficult to meet, the following scheme is provided: the device comprises a punching cylinder; the first limiting hole and the second limiting hole are formed in the outer side of the punching barrel; according to the present invention, during the use process, the portable rapid pathogen detection device has characteristics of portability, easy installation, simple sampling operation, convenient and rapid sampling process, consistent size of the cut leaf tissue, regular and uniform shape, and increase of the accuracy of the pathogen detection result, and can be used for the field rapid detection of the field plant, the method has important significance on quarantine, detection, monitoring and prevention and control of plant diseases and insect pests.

Description

Technical Field

[0001] The present invention relates to the technical field of plant leaf punching samplers, and in particular to a puncher for accurate detection of nucleic acid in plant leaves and a method of using the puncher. Background Art

[0002] When detecting gene expression in plant leaves or detecting pathogens in plant leaves, it is necessary to manually sample specific areas of the leaves, and then grind and extract nucleic acids from the sampled samples to complete plant gene or pathogen real-time fluorescence quantitative nucleic acid amplification detection (Real-Quantitative PCR Detecting System, qPCR) and immunoprotein blotting (Western Blot, WB) and other operations. The duration of plant leaf sampling operation, the representativeness of the sampling site, and the uniformity of sample sampling will affect the results of molecular biology experiments. At present, the most commonly used method for sampling plant leaves is to use metal scissors, tweezers, metal punch samplers, blades or eppendorf tube caps and other tools for sampling. When using the above tools to collect samples, it is easy to cause uneven sampling between different samples, and additional tools are required for sample transfer. Or the collection and transfer time is too long, resulting in protein or nucleic acid degradation, or the cross-use of tools, resulting in mutual contamination of samples from different detection units, or sampling errors during manual operations of different samples, affecting the accuracy and stability of the gene or pathogen detection results of the mixed sample.

[0003] The patent document with publication number CN116766323A discloses a compact and efficient plant leaf puncher, which includes: a pressing handle, a hollow grip, a punching cylinder, a spring, and a centrifuge tube. When working, first fix a suitable punching cylinder on the pressing handle. At this time, the spring is in a static state. Insert a suitable cutter head gasket into the cutter head gasket groove, insert the centrifuge tube into the centrifuge tube holder, and place the leaves to be sampled steadily below the punching cylinder and above the cutter head gasket. Press the pressing handle, the spring is in a compressed state, so that the punching knife cuts out the leaf cover. Release the pressing handle, the spring is in a balanced state, and the leaf cover falls into the centrifuge tube. This puncher has a simple structure, is compact as a whole, is easy to carry, and is labor-saving and convenient to use. The punching knife is made of Teflon-coated manganese steel material, so that the punching knife is not easy to stick to the leaves, which effectively reduces the complexity of leaf punching for test personnel, reduces the difficulty of cleaning the puncher, improves work efficiency, and can achieve sampling anytime and anywhere, so that test personnel can perform leaf punching sampling more efficiently.

[0004] The patent document with publication number CN204359554U discloses a handheld punch sampler suitable for in-situ sampling and collection of tobacco leaves, which includes: upper and lower clamp bodies connected by a hinge shaft, and the upper and lower clamp bodies both include jaws and clamp handles, characterized in that: the jaws of the upper and lower clamp bodies are correspondingly equipped with a matching upper jaw cylindrical punch and a lower jaw cylindrical punch groove, the outer diameter of the cylindrical punch is smaller than the inner diameter of the cylindrical punch groove, and a centrifuge tube for storing samples is connected to the lower end of the cylindrical punch groove, and a compression spring is connected between the upper and lower clamp handles near the hinge shaft. The utility model can be used to quickly collect tobacco leaf samples in situ in various environments, and directly collect them into a 50ml centrifuge tube. The operation is simple, convenient and fast, easy to carry, can save a lot of time, can effectively ensure the original state of the sample, and has an important auxiliary role in effectively obtaining real data of the plant body;

[0005] The patent document with publication number CN208451770U discloses a portable leaf puncher for plant leaves, which includes: a scissor body I, a scissor body II, and a hinge shaft. The scissor body I is provided with an upper pressing block, which is provided with a punching block groove, in which a replaceable punching block is embedded, and the punching block structure includes a column and a sampling head; a lower pressing block is provided at the end of the scissor body II, which is a hollow body with an opening at the bottom, and an embedding block groove is provided at the top, an embedding block is embedded in the embedding block groove, and a sampling hole is provided on the embedding block; pull-tab holes are provided at corresponding positions on both sides of the lower part of the lower pressing block, and a pull-tab is provided in the pull-tab hole. The utility model utilizes replaceable punching blocks and embedding blocks, and can collect leaf samples that meet the requirements through the sampling head of the matching punching block and the sampling hole of the corresponding embedding block. It has a simple structure and is easy to disassemble and replace, and is very suitable for laboratory use.

[0006] However, the above patent documents all have metal modules during use and are reusable samplers. Moreover, a single sampler is large in size and cannot meet the "portable, disposable" use requirements required for collecting pathogens from plant leaves. Therefore, we propose a puncher for accurate detection of nucleic acid in plant leaves and a method of use to solve the above problems. Summary of the invention

[0007] The purpose of the present invention is to solve the shortcomings of the prior art that all samplers are equipped with metal modules, are reusable samplers, and a single sampler is large in size, which makes it difficult to meet the "portable, disposable" usage requirements required for collecting plant leaf pathogens. A puncher and a method of use for accurate detection of plant leaf nucleic acids are proposed.

[0008] The present application provides a puncher and a method for using the puncher for accurate detection of nucleic acid in plant leaves, which adopts the following technical solutions:

[0009] A puncher for accurate detection of nucleic acid in plant leaves, comprising:

[0010] Perforating tube;

[0011] A first limiting hole and a second limiting hole, both of which are arranged on the outer side of the punching tube;

[0012] A resettable pressing control module, the resettable pressing control module is arranged in the punching tube;

[0013] Chamfer, the chamfer is connected to one end of the punching tube, the resettable pressing control module includes a pressable control module, the pressable control module is connected to a sample push rod, the pressable control module is connected to a first limiting spring clip buckle and a second limiting spring clip buckle, the outer side of the sample push rod is provided with a spring, an integrated hollow round hole baffle is connected to the punching tube, and the sample push rod is slidably installed in the integrated hollow round hole baffle.

[0014] The present invention also proposes a method for using a puncher for accurate nucleic acid detection in plant leaves, comprising the following steps:

[0015] S1: When collecting samples, the leaves can be laid flat. The number of plant tissue leaves used for plant pathogen detection is 1-10. Use the chamfer at the bottom of the punching tube to align with the selected area of ​​the single-layer or multi-layer plant leaves, press the punching tube down to punch holes for sampling, and the collected leaves will be collected in the inner cavity of the punching tube. The weight of a single leaf tissue is 25 mg;

[0016] S2: After the sample is collected, the pressing control module is pressed downward, and the first limit spring buckle in the pressing control module 1 moves downward from the first limit hole to the second limit hole, and drives the sample push rod to push the sample collected in the punching cylinder out of the punching cylinder, and transfer 1-10 plant tissue leaves from the punching cylinder to an equal volume of nucleic acid lysis solution. The obtained sample lysis solution is used as a template, and RT-RAA amplification is performed. The amplification product is detected using a lateral flow chromatography test strip, and the color development of the strip of the lateral flow chromatography test strip is observed to determine whether the sample contains the pathogen to be detected;

[0017] S3: Finally, press the first limiting spring clip buckle in the second limiting hole, and the spring can bounce the sample push rod back into the punching tube, and then punch holes in other parts for sampling.

[0018] In summary, this application includes the following beneficial technical effects:

[0019] The present invention is portable, easy to install and sample during use, the sampling process is convenient and fast, the cut leaf tissues are of uniform size and regular shape, and the accuracy of pathogen detection results can be increased. It can be used for on-site rapid detection of field plants and is of great significance to the quarantine, detection, monitoring and prevention and control of plant diseases and insect pests. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the main structure of a puncher for accurate nucleic acid detection in plant leaves proposed by the present invention;

[0021] Figure 2 This is a schematic diagram of the structure of a pressable control module of a puncher for accurate nucleic acid detection of plant leaves proposed by the present invention;

[0022] Figure 3 This is a schematic diagram of the structure of a punching tube of a puncher for accurate nucleic acid detection of plant leaves proposed by the present invention;

[0023] Figure 4 A schematic diagram of the structure and detection results of a single-target disposable nucleic acid detection test strip (JY0201) for a puncher and a method of using the puncher for accurate detection of nucleic acid in plant leaves proposed by the present invention;

[0024] Figure 5 This is a schematic diagram of the effect of the sampling quantity on the detection results of ToBRFV virus infection in the leaves of a puncher and a method for using the puncher for accurate detection of nucleic acid in plant leaves proposed by the present invention.

[0025] Figure numerals: 1. pressable control module; 2. first limiting spring clip buckle; 3. second limiting spring clip buckle; 4. sample push rod; 5. spring; 6. punching tube; 7. first limiting hole; 8. second limiting hole; 9. integrated hollow circular hole baffle; 10. chamfer. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0027] Reference Figure 1-Figure 5 , a puncher for accurate detection of nucleic acid in plant leaves, comprising:

[0028] Perforating tube 6;

[0029] A first limiting hole 7 and a second limiting hole 8, both of which are provided on the outside of the punching tube 6;

[0030] A resettable pressing control module, which is arranged in the punching tube 6;

[0031] Chamfer 10, chamfer 10 is connected to one end of the punching tube 6, the resettable pressing control module includes a pressable control module 1, the pressable control module 1 is connected to a sample push rod 4, the pressable control module 1 is connected to a first limiting spring clip buckle 2 and a second limiting spring clip buckle 3, the outer side of the sample push rod 4 is sleeved with a spring 5, an integrated hollow circular hole baffle 9 is connected to the punching tube 6, and the sample push rod 4 is slidably installed in the integrated hollow circular hole baffle 9.

[0032] This embodiment also proposes a method for using a puncher for accurate detection of nucleic acid in plant leaves, comprising the following steps:

[0033] S1: When collecting samples, the leaves can be laid flat. The number of plant tissue leaves used for plant pathogen detection is 1-10. Use the chamfer 10 below the punching tube 6 to align with the selected area of ​​the single-layer or multi-layer plant leaves, press the punching tube 6 downward to punch holes for sampling, and the collected leaves will be collected in the inner cavity of the punching tube 6. The weight of a single leaf tissue is 25 mg;

[0034] S2: After the sample collection is completed, the pressing control module 1 is pressed downward, and the first limiting spring clip buckle 2 in the pressing control module 1 moves downward from the first limiting hole 7 to the second limiting hole 8, and drives the sample push rod 4 to push the sample collected in the punching tube 6 out of the punching tube 6, and transfer 1-10 plant tissue leaves from the punching tube 6 to an equal volume of nucleic acid lysis solution. The obtained sample lysis solution is used as a template, and RT-RAA amplification is performed. The amplification product is detected using a lateral flow chromatography test strip, and the color development of the strip of the lateral flow chromatography test strip is observed to determine whether the sample contains the pathogen to be detected;

[0035] Collect tomato leaf tissue samples using a plant leaf punch sampler:

[0036] When tomatoes are infected with ToBRFV, the main symptoms of the leaves are mosaic, dark green protrusions, narrow leaves, yellowing veins, and necrosis in severe cases. Yellow or brown spots appear on the fruits, the fruits become smaller, wrinkled, and severe cases lead to necrosis of the fruit stalks, and the number of flowers and fruits decreases. Wear latex gloves and PE gloves, and use a plant leaf punch sampler to punch holes in the pad paper of plant leaf samples suspected of being infected with the ToBRFV virus. When sampling with a punch, avoid yellow and dry leaves. After leaf sampling, push the collected samples into a grinding bag with lysis solution;

[0037] PE gloves and sampling equipment follow the principle of "one sample, one set of equipment" to strictly avoid cross contamination. After sampling is completed, disposable consumables such as PE gloves and sampling pads must be placed in corresponding garbage bags, and punch samplers must be placed in separate storage boxes for recycling. Consumables and equipment must be uniformly professionally disinfected and then disposed of in accordance with biosafety principles;

[0038] Sample grinding, lysis and RT RAA-LFS detection:

[0039] Use a plant leaf punch sampler to collect plant leaf samples suspected of being infected with the ToBRFV virus, and grind and lyse them according to the number of leaf tissues suspected of being infected. Use the handle of scissors or a marker pen to scrape the outside of the grinding bag containing 1 suspected ToBRFV-infected plant leaf, 3 suspected ToBRFV-infected plant leaves, 5 suspected ToBRFV-infected plant leaves, 7 suspected ToBRFV-infected plant leaves, 10 suspected ToBRFV-infected plant leaves and healthy tomato plant leaf samples several times to fully lyse the samples. After grinding, the leaf tissue becomes transparent or translucent, without obvious large pieces of tissue, which means that the sample has been fully lysed;

[0040] Assume that the number of amplification tubes required is n (n=number of samples + 1 tube of negative control), and prepare the primer premix according to the number of amplification tubes n according to Table 1. The reaction premix can be stored in the dark at 4°C for 7 days. The sequence of ToBRFV-F is 5′-AACCAGACAAAAACCAAAGGAAG-3′, the sequence of ToBRFV-R is 5′-AAGCAGTAACTAGAGGATCTA-3′, and the sequence of ToBRFV-P is 5′-TCACTAGGTAATCAGTTCCAAACACAACAAG(THF)TAGAACAACCGTTCA-3′. The 5′ end of the reverse primer ToBRFV-R of ToBRFV is modified with FITC, the 5′ end of the detection probe ToBRFV-P is modified with biotin, and the 3′ end of ToBRFV-P is phosphorylated. THF is tetrahydrofuran;

[0041] Table 1 ToBRFV RT RAA-LFS detection primer premix

[0042]

[0043] use The RT RAA nucleic acid amplification kit (strip method) (JY0204) was used to prepare the ToBRFV RT RAA reaction system (single sample / reaction). The ToBRFV RT RAA reaction system is shown in Table 2:

[0044] Table 2 ToBRFV RT RAA reaction system

[0045]

[0046] * Prepare the reaction solution of the supernatant of 1, 3, 5, 7 and 10 plant leaf lysates respectively;

[0047] Take out A Buffer and B Buffer from the amplification kit, melt them at room temperature, centrifuge them instantly, and add ToBRFV Mix, A Buffer, and B Buffer to the amplification reaction tube containing dry powder in the order shown in the table below. Prepare the reaction solution in the order of negative control, sample to be tested, and positive control, and mix them thoroughly;

[0048] Use a vortex oscillator to mix for 1 to 2 seconds, centrifuge instantly, collect the solution, and the preparation of the test reaction tube is completed. The prepared reaction tube is placed on a thermostat preheated to 39°C for 16 minutes;

[0049] After the reaction is completed, pipette 5 μl of the amplified product into a 1.5 ml centrifuge tube that has been added with 245 μl of ultrapure water, mark it, and vortex to mix;

[0050] The schematic diagram of the single-target disposable nucleic acid test strip (JY0201) is as follows Figure 4 As shown, drop the dilution of the amplified product into the liquid addition area of ​​the test strip (the round hole marked with an arrow), wait for the entire reading area to be soaked (about 60 seconds), and wait for the test line (T line) to appear. Read the test results directly according to the color development of the test strip, and this process takes about 3 minutes. Observe the results within 10 minutes, and the reading will be invalid after 10 minutes;

[0051] When using this puncher to punch and sample leaf samples, only one piece of tissue is needed for accurate detection of pathogens, such as Figure 5 As shown, whether one leaf tissue or multiple leaf tissues of the same plant are used for testing does not affect the test results of the same plant. After RT RAA-LFS testing of the supernatant of the lysate from 1 leaf tissue, 3 leaf tissues, 5 leaf tissues, 7 leaf tissues and 10 leaf tissues of tomato plants suspected of being infected with ToBRFV, the T lines all showed color, indicating that the tomato plant had been infected with the ToBRFV virus. In addition, a handheld fluorescence detector was used to read the LFS quantum dot fluorescence detection values ​​of different tissues, showing that after RT RAA amplification, the quantum dot fluorescence detection value of the supernatant of leaf tissue lysate was 988.33, 1036.62 for 1 leaf, 1000.80 for 5 leaf, 999.38 for 7 leaf, and 1000.14 for 10 leaf. The handheld fluorescence detector interpreted all the results as positive. The supernatant of leaf lysate of healthy tomato plants was used as a negative control. After RT RAA amplification, the LFS quantum dot fluorescence detection value was 12.29, which was interpreted as negative by the handheld fluorescence detector. The above results are completely consistent with the visual results of the visual detection.

[0052] The virus samples involved in this example come from:

[0053] The tomato brown rugose fruit virus (ToBRFV) sample and the corresponding total RNA or cDNA thereof are stored in the virus-host interaction laboratory of the Institute of Plant Virology of Ningbo University. In the ToBRFV virus carrying detection test, tomato seedlings inoculated with ToBRFV virus by friction are used as experimental objects, and a puncher is used to sample the leaves of tomato plants. The lysate supernatant of 1, 3, 5, 7, and 10 circular leaf tissues obtained by the puncher is used as an amplification template, and the lysate supernatant of healthy plant leaf tissue and ultrapure water of equal volume are used as negative controls.

[0054] In order to evaluate whether the invented plant leaf punch sampler can be used for rapid field detection of ToBRFV infection in tomato plants, 2 μL of lysate supernatant from 1, 3, 5, 7, and 10 circular leaf tissues obtained by the plant leaf punch sampler was used as an amplification template, and an equal volume of lysate supernatant from healthy plant leaf tissues and ultrapure water were used as negative controls. The sample lysate can be used for experiments immediately or stored at -70°C for more than 3 months.

[0055] S3: Finally, the first limiting spring clip buckle 2 in the second limiting hole 8 is pressed, and the spring 4 can bounce the sample push rod 4 back into the punching tube 6, and then punch holes in other parts for sampling.

[0056] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A puncher for accurate detection of nucleic acid in plant leaves, characterized in that: include: Perforating tube (6); A first limiting hole (7) and a second limiting hole (8), wherein the first limiting hole (7) and the second limiting hole (8) are both arranged on the outer side of the punching tube (6); A resettable pressing control module, the resettable pressing control module being arranged in the punching cylinder (6); A chamfer (10) is connected to one end of the perforating tube (6).

2. A puncher for accurate detection of nucleic acid in plant leaves according to claim 1, characterized in that: The resettable press control module comprises a press control module (1), the press control module (1) is connected to a sample push rod (4), and the press control module (1) is connected to a first limit spring clip buckle (2) and a second limit spring clip buckle (3).

3. A puncher for accurate detection of nucleic acid in plant leaves according to claim 2, characterized in that: The outer side of the sample push rod (4) is sleeved with a spring (5).

4. A puncher for accurate detection of nucleic acid in plant leaves according to claim 3, characterized in that: An integrated hollow circular hole baffle (9) is connected inside the perforating cylinder (6).

5. A puncher for accurate detection of nucleic acid in plant leaves according to claim 4, characterized in that: The sample push rod (4) is slidably mounted in the integrated hollow circular hole baffle (9).

6. A method for using a puncher for accurate detection of nucleic acid in plant leaves, wherein the puncher for accurate detection of nucleic acid in plant leaves is a puncher for accurate detection of nucleic acid in plant leaves according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1: When collecting samples, the leaves can be laid flat. The number of plant tissue leaves used for plant pathogen detection is 1-10. The chamfer (10) below the punching tube (6) is used to align with the selected area of ​​the single-layer or multi-layer plant leaves, and the punching tube (6) is pressed downward to punch holes for sampling. The collected leaves will be collected in the inner cavity of the punching tube (6). The weight of a single leaf tissue is 25 mg; S2: After the sample collection is completed, the pressing control module (1) is pressed downward, and the first limit spring buckle (2) in the pressing control module (1) moves downward from the first limit hole (7) to the second limit hole (8), and drives the sample push rod (4) to push the sample collected in the punching cylinder (6) out of the punching cylinder (6), and transfer 1-10 plant tissue leaves from the punching cylinder (6) to a nucleic acid lysis solution containing an equal volume. The obtained sample lysis solution is used as a template, and RT-RAA amplification is performed. The amplification product is detected using a lateral flow chromatography test strip, and the color development of the strip of the lateral flow chromatography test strip is observed to determine whether the sample contains the pathogen to be detected; S3: Finally, the first limiting spring clip buckle (2) in the second limiting hole (8) is pressed, and the spring (4) can bounce the sample push rod (4) back into the punching tube (6), and then punch holes in other parts for sampling.

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