CN113549626B - Aptamer Pr-A08 specifically bound with procymidone and application thereof - Google Patents

Abstract

The present invention discloses a nucleic acid aptamer Pr-A08 specifically binding to procymidone, the nucleotide sequence of which is shown in SEQ ID NO: 1, the secondary structure has protruding loops and stems, and has G- Quadruplex structure, Gibbs free energy DG=‑12.18; aptamer specificity and affinity were evaluated based on colloidal gold spectrophotometry. The experimental results showed that the aptamer Pr‑A08 can be specific to procymidone Binding, has the characteristics of high specificity and high affinity, the nucleic acid aptamer has the characteristics of high specificity and high affinity, it can be used in the preparation of nucleic acid aptamers in the preparation of reagents for recognizing procymidyl or in the preparation and detection of procymidone In the kit, it is of great significance in reducing the damage of procymidone to the human body.

Description

Nucleic acid aptamer Pr-A08 specifically binding to procymidone and its application

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to a nucleic acid aptamer Pr-A08 specifically binding to procymidone and its application.

Background technique

Procymidone, the chemical name is N-(3,5-dichlorophenyl)-1,2-dimethylcyclopropane-1,2-diformyl imide Bad luck. It is easily soluble in acetone, chloroform, xylene, dimethylformamide, slightly soluble in ethanol, and insoluble in water. It is a systemic fungicide with both protective and therapeutic effects, and it has obvious effects under low temperature and high humidity conditions. Used for rape, radish, eggplant, cucumber, cabbage, tomato, sunflower, watermelon, strawberry, onion, peach, cherry, flower, grape and other crops to control gray mold and sclerotinia, gray star disease, flower rot, Brown rot, vine blight, etc., can also be used for bacteria resistant to thiophanate-methyl and carbendazim, but the strains are easy to develop resistance to them. In recent years, it has been widely used in vegetables and fruits, and it is also one of the pesticide varieties with high detection rate and excessive rate of pesticide residues in the quality and safety monitoring of agricultural products at all levels. The MRLs in leeks are 0.2mg/kg, vegetable oils are 0.5mg/kg, tomatoes and cucumbers are 2mg/kg, eggplants, peppers, grapes, mushrooms (fresh), fresh corn are 5mg/kg, and strawberries are 10mg/kg. A small amount of procymidone has no effect on the human body, but if it is consumed in excess of the standard for a long time, it will affect human health. It may irritate the eyes and skin, and it may be quantitatively deposited in the human body. cause harm. The traditional detection technology of procymidone is usually through gas chromatography-mass spectrometry and antibody test strip method. Gas chromatography-mass spectrometry requires complex equipment and expensive cost, and the antibody has disadvantages such as long synthesis cycle and instability.

Aptamer is an aptamer that is screened from an artificially constructed oligonucleotide library by the in vitro ligand exponential enrichment system evolution technology (Systematicevolution of ligands by exponential enrichment, SELEX) with high affinity for the target substance. , and can specifically recognize single-stranded oligonucleotide sequences. Aptamers can bind target substances in specific two-dimensional or three-dimensional conformations, including stem-loop, hairpin, G-quadruplex and other structures, so as to realize the detection of target substances. The unique advantage of aptamers is that aptamers have strong specificity and high affinity for target substances, and the dissociation constant between aptamers and target molecules is generally 10 ^-9 ~10 ^-12 mol/L; A wide range of aptamers can be combined not only with molecules such as enzymes and antibodies, but also with small molecules such as metal ions, biotoxins, and drugs; aptamers can be chemically synthesized in large quantities and rapidly in vitro, and are easily modified by functional groups; The molecular weight of aptamers is small, generally composed of single-stranded oligonucleotide fragments of 25-100 bases, and the steric hindrance formed when binding to the target substance is small; the stability and refoldability of aptamers are good, Repeatable denaturation and renaturation. Therefore, the advantages of nucleic acid aptamers make them widely used in analytical chemistry, medical fields, environmental monitoring and food safety control.

SUMMARY OF THE INVENTION

The present invention provides a nucleic acid aptamer Pr-A08 specifically binding to procymidone, the nucleotide sequence of which is shown in SEQ ID NO: 1, the secondary structure has protruding loops and stems, and has G- Quadruplet structure, Gibbs free energy DG=-12.18.

Another object of the present invention is to apply the nucleic acid aptamer that specifically binds to procymidone in the preparation of a probe molecule for recognizing procymidone or in the preparation of a procymidone detection kit.

The object of the present invention is achieved through the following technical solutions:

Step 1. Filter

Use SELEX technology to screen out the aptamer population that can specifically bind to procymidyl through a column capable of coupling hydroxyl groups;

Step 2. Cloning

Design primers for PCR amplification, ligation and transformation, pick single clones, use the PMD 19-T vector to ligate the PCR products into competent cells, and ligate the transformed competent cells in the ampicillin-containing cells by streaking. Cultured on a medium plate overnight at 37°C to obtain a single colony;

The nucleic acid aptamer amplification primers are: Aptamer Fw: GACATATTCAGTCTGACAGCG; Aptamer Rv: GCTAGACGATATTCGTCCATC;

Step 3. Identify positive clones and sequence

Randomly select a single colony on the solid medium, cultivate it in a liquid medium containing ampicillin resistance to turbidity, carry out PCR amplification of the bacterial liquid, and then use agarose gel electrophoresis to verify the PCR product, and the selection is consistent with the target size. Sequencing the fragments of , and the sequencing results show that the nucleic acid aptamer (Pr-A08) that specifically binds to procymidone is composed of 82 nucleotides, and its sequence (5' end to 3' end) is:

gacatattcagtctgacagcgcgtaagaaatcaaggctgctcaccgttgacgggctggatagctagacgatattcgtccatc;

Step 4. Characterization of the secondary structure of single-stranded DNA of nucleic acid aptamer (Pr-A08)

Using MFOLD software to predict the secondary structure of aptamer Pr-A08 single-stranded DNA molecule, the results show that its secondary structure has prominent loops and stems, and there is a G-quadruplex structure, Gibbs free energy DG =-12.84, its secondary structure is as follows:

；

;

Step 5. Affinity, specificity and sensitivity to procymidyl of nucleic acid aptamer (Pr-A08)

The affinity, specificity and sensitivity of nucleic acid aptamers were identified by colloidal gold spectrophotometry. The results showed that the affinity constant of Pr-A08 was 27.01±6.519 nmol/L, and Pr-A08 had high specificity and could detect The lowest concentration to procymidone is 50ng/mL.

The beneficial effects of the present invention are: compared with the existing procymidon detection technology on the market, the nucleic acid aptamer Pr-A08 screened by the SELEX technology can recognize and bind to procymidone with high affinity and high specificity, The detection technology based on the nucleic acid aptamer can realize the direct detection of procymidone in food; the specificity and affinity of the nucleic acid aptamer Pr-A08 ensure the accuracy of the detection results of the residual procymidone in food, The method of the invention is simple and easy to operate, and is suitable for industrial production and market promotion and application.

Description of drawings

Fig. 1 is the secondary structure schematic diagram of nucleic acid aptamer Pr-A08 of the present invention;

Fig. 2 is the affinity analysis result of nucleic acid aptamer Pr-A08 of the present invention;

Fig. 3 is the specificity analysis result of nucleic acid aptamer Pr-A08 of the present invention;

Fig. 4 is the analysis result of the sensitivity of the nucleic acid aptamer Pr-A08 of the present invention to procymidone.

Detailed ways

The substantive content of the present invention will be further described below in conjunction with the accompanying drawings and examples. The examples are only for better understanding of the present invention but are not intended to limit the scope of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used Unless otherwise specified, all reagents are conventional commercially available reagents or reagents prepared by conventional methods.

Example 1: Screening, cloning, isolation and sequencing of nucleic acid aptamer Pr-A08 and prediction of secondary structure of single-stranded DNA

1. Coupling of procymidone and immunoaffinity column

1. Dilute Procymidil to 50μg/mL, and the total volume is 1.5mL;

2. Take 3mL of the immunoaffinity column Sepharose 6B (preserved in acetone) solution and the diluted procymidone solution and mix them in a 15mL centrifuge tube;

3. Incubate for 48 h at 37°C and 20rpm/min;

4. After 48 hours, add 5 mL of 1 mol/L aminoethanol to block the unbound sites of procymidon, mix gently and place on a shaker (37°C, 20 rpm/min) to incubate for 24 hours;

5. Centrifuge at 4000rpm/min for 2min and discard the supernatant;

6. Add pH8.0 buffer, pH4.0 buffer, and coupling buffer in sequence to wash the immunoaffinity column, add 5 mL of each wash solution, centrifuge at 4000rpm/min for 2min, and discard the supernatant;

7. Finally, add 6 mL of coupling buffer, transfer the entire mixed solution to the chromatography column to form a filter column (to prevent the immunoaffinity column from drying out); place it in a 4°C refrigerator, seal it with a parafilm, and place it upright.

2. PCR of nucleic acid aptamer library (ssDNA)

Use the ssDNA aptamer library synthesized by TaKaRa company;

1. Preheat the PCR instrument at 94°C;

2. Add 5 μL ssDNA, 28.6 μL deionized water, 5 μL 10×Buffer, 3 μL MgCl ₂ , 4 μL dNTP mixture (each 2.5 μmol/L), 2 μL forward amplification primer (concentration is 10 mol/L), 2 μL reverse amplification primer (concentration is 10mol/L) and 0.4 μL Taq centrifuge tube for reaction; the forward amplification primer sequence is 5'-GACATATTCAGTCTGACAGCG-3', and the reverse amplification primer sequence is 5'-GCTAGACGATATTCGTCCATC-3'.

3. Amplify in the PCR machine according to the following procedure

(1) Pre-denaturation: 94°C, 5min; (2) 30 cycles: 94°C, 45s; 58°C, 30s; 72°C, 30s; (3) Post-amplification: 72°C, 7min.

3. Purification, loading and elution of ligand library

1. Purification of the ligand library

(1) The PCR product of the nucleic acid aptamer library is directly mixed with the solution in the gel recovery kit at a volume ratio of 1:1 to recover DNA fragments;

(2) After recovery of DNA fragments, water bath at 95°C for 10 minutes and ice bath for 10 minutes; after this denaturation treatment, double-stranded DNA becomes single-stranded DNA.

2. Affinity chromatography

(1) Add single-stranded DNA to the chromatography column prepared by coupling, and dilute the entire solution to 5 mL with binding buffer (10 mmol/L KCl); place on a shaker for incubation at 37 °C and gently rotate at 40 rpm/min for 45 min ;

(2) Discard the binding buffer in the immunochromatographic column, add ultrapure water to wash, and wash 3 times, 2 mL each time;

(3) Then carry out linear gradient elution with 3-4 column volumes of elution buffer (0.1mol/L NaCl, 0.1mol/L acetate, pH 4.0), and collect elution fractions;

(4) Centrifuge at 12000rmp/min for 2min in the centrifuge, and spin dry the liquid in the collection tube;

(5) Add 50 μL of LTE buffer to the collection tube, centrifuge at 12000 rmp/min for 1 min in a centrifuge, and transfer the liquid in the collection tube to the PCR tube.

4. PCR optimization and mass amplification of nucleic acid aptamers

Using the liquid prepared in step 3 as a template, follow the steps below:

1. Preheat the PCR instrument at 94°C;

2. Put 5 μL template, 28.6 μL deionized water, 5 μL 10×Buffer, 3 μL MgCl ₂ , 4 μL dNTP mixture (2.5 μmol/L each), 2 μL forward amplification primer, 2 μL reverse amplification primer, 0.4 μL Taq enzyme in PCR The reaction was carried out in a centrifuge tube. The forward amplification primer sequence is 5'-GACATATTCAGTCTGACAGCG-3', and the reverse amplification primer sequence is 5'-GCTAGACGATATTCGTCCATC-3';

3. Amplify in the PCR machine according to the following procedure:

(1) Pre-denaturation: 94°C, 5min; (2) 30 cycles: 94°C, 45s; 58°C, 30s; 72°C, 30s; (3) Post-amplification: 72°C, 7min;

4. After the cycle is over, the PCR product is extracted and recovered with the DNA product purification kit of TIANGEN Company. The steps are as follows:

(1) Invert and mix the PCR product with an equal volume of membrane binding buffer, then transfer the mixture to the affinity chamber, let it stand at room temperature for 5 minutes to fully bind the DNA to the silica membrane, centrifuge at 12,000 rpm/min for 1 minute, and discard the collection tube waste liquid in;

(2) Add 700µL of rinsing solution (containing ethanol) to the centrifugal purification column, centrifuge at 12000rpm/min for 1min, and discard the waste liquid in the collection tube;

(3) repeat step (2);

(4) Centrifuge at 12000rpm/min for 3min;

(5) place the centrifugal purification column in a new centrifuge tube;

(6) Add 30µL of ultrapure water and let stand for 5min at room temperature;

(7) Centrifuge at 12000 rpm for 1 min, and the solution at the bottom of the tube is the PCR product of the purified nucleic acid aptamer.

5. Cloning, isolation and sequencing of nucleic acid aptamers and prediction of the secondary structure of single-stranded DNA

1. Preparation of Escherichia coli DH5α competent cells

(1) Pick a single DH5α colony, inoculate it in 3mL of LB medium without ampicillin, cultivate overnight at 37°C, take the above bacterial liquid at a ratio of 1:100 the next day and inoculate it in 50mL of liquid LB medium at 37°C. Shake for 2h; when the OD600 value reaches 0.35, harvest the bacterial culture;

(2) Transfer the bacterial culture to a 50 mL pre-cooled sterile polypropylene tube and place on ice for 10 min to cool the culture;

(3) Centrifuge at 4000rpm/min for 10min at 4°C, discard the culture solution, and invert the tube for 1min to drain the remaining culture solution;

(4) Add 150 μL ice-precooled 0.1 mmol/L CaCl ₂ solution to each, combine the two tubes, and take an ice bath for 10 min;

(5) Centrifuge at 4000rpm/min for 10min at 4°C, discard the supernatant, and invert the tube for 1 min to drain the remaining liquid;

(6) First add 800 μL of ice-cold 0.1 mol/L CaCl ₂ solution to resuspend the cells, then add 25 μL of pre-cooled 75% glycerol, and store at -80°C for later use.

2. Ligation and transformation of ligation products

(1) Add 1 μL Takara pMD19-T simple vector, 4 μL nucleic acid aptamer PCR product and 5 μL ligase buffer mixture to a microcentrifuge tube;

(2) Incubate the reaction at 16°C overnight;

(3) Put the competent cells on ice for 15 minutes immediately after taking them out of the -80°C refrigerator;

(4) The system (10 μL) of the connected carrier was added to 100 μL of DH5α competent cells, and placed on ice for 30 min;

(5) After heating at 42°C for 90s, place in ice for 5min;

(6) Add 890 μL of LB liquid medium (without ampicillin) that has been warmed at 37°C, and culture with slow shaking at 37°C for 60 minutes;

(7) 200 μL was spread on LB solid medium containing ampicillin, and cultured at 37° C. for 16 h to form a single colony.

3. Cloning screening and sequencing of nucleic acid aptamers and prediction of secondary structure of single-stranded DNA

Pick the above single colony in the LB medium containing ampicillin, shake and cultivate to turbidity under the conditions of 37 ° C and 150rpm/min, and carry out PCR amplification of the bacterial liquid; the amplification primers and amplification conditions are the same as those of the aforementioned nucleic acid aptamers. Amplification conditions. After plasmid extraction of the positive clones confirmed by PCR, the nucleotide sequence was determined by the American Applied Biosystems 3730A automatic nucleotide sequencer; the sequencing results showed that the nucleic acid aptamer (Pr- A08) is composed of 82 nucleotides, and its sequence (5' end to 3' end) is:

gacatattcagtctgacagcgcgtaagaaatcaaggctgctcaccgttgacgggctggatagctagacgatattcgtccatc;

Sequence length of the nucleic acid aptamer Pr-A08 that specifically binds to procymidyl: 82 bases, sequence type: nucleic acid, number of strands: single-stranded, topology: linear, sequence type: ssDNA.

The temperature was set at 26 °C, Na ⁺ concentration at 150 mmol/L, ^Mg concentration at 1 mmol/L (http://mfold.rna.albany.edu/q=mfold/DNA-Folding-Form) and QGRS by MFOLD software Map (http://bioinformatics.ramapo.edu/QGRS/analyze.php) to predict the secondary structure of the nucleic acid aptamer Pr-A08 single-stranded DNA molecule that specifically binds to procymidoid; the results show that the aptamer Containing protruding loops and stems, its Gibbs free energy DG=-12.18, the structure has high stability (see Figure 1).

Example 2: Affinity, specificity and sensitivity detection of nucleic acid aptamer Pr-A08

1. Affinity detection of nucleic acid aptamer Pr-A08

Colloidal gold spectrophotometry uses colloidal gold as a marker, and uses the characteristics of colloidal gold to aggregate in a high-salt environment, which causes the red shift of the colloidal gold wavelength and the right shift of the characteristic peak to determine the absorbance value of the colloidal gold solution at a specific wavelength to identify .

(1) Preparation of 25nm colloidal gold

1) Prepare 1% sodium citrate solution, weigh 0.1g sodium citrate and add 10mL ultrapure water;

2) Take 25μL of chloroauric acid solution (containing 49% of Au) and dilute it by 45 times to 1125μL (this step should be carried out in the dark);

3) Take 99mL of purified water and add it to a clean conical flask. Purified water, sodium citrate solution and chloroauric acid solution all need to pass through a 45μm filter membrane;

4) Add 1 mL of chloroauric acid solution to the conical flask and mix well with an electric heating mantle to adjust to 80°C and heat to boiling (magnetic stirring is performed with a rotor during this process);

5) When it is about to boil, adjust the temperature to 100 °C and heat it to complete boiling, then add 1.5 mL of 1% sodium citrate solution;

6) Adjust the temperature to 0°C, the solution changes from colorless-black-purple-red, after the color is stable, adjust the temperature to 70°C and heat for 10min;

7) After cooling to room temperature, transfer to 4°C and store in the dark.

(2) Dilute Procymidil to 1 μg/mL with ultrapure water, 50 μL per tube;

(3) The nucleic acid aptamer Pr-A08 was sent to Beijing Qingke Biotechnology Co., Ltd. for synthesis; when using, a brief centrifugation was performed first to make all the nucleic acid aptamers gather at the bottom of the test tube. According to the instructions, fully dissolve the nucleic acid aptamer into a storage solution with a concentration of 10 ^-4 mol/L in sterilized water. In order to avoid repeated freezing and thawing, it can be divided into small portions; The ligand was diluted to 50 nmol/L, 100 nmol/L, 200 nmol/L, 400 nmol/L, 800 nmol/L, 1600 nmol/L, and added to step (2) procymidyl solution, adding 50 μL to each tube, Incubate at room temperature for 30 minutes; at the same time, set a blank control without adding nucleic acid aptamers and add 50 μL of 1×PBS, and repeat 3 parallel experiments for each sample;

(4) Concentrate the colloidal gold prepared in step (1) by 4 times, centrifuge 4 mL of colloidal gold at 4°C and 8000 rpm/min for 12 min, discard 3 mL of supernatant, and pipette the remaining 1 mL with a pipette to uniformly prepare for use;

(5) Add 50 μL of concentrated colloidal gold to each tube in step (3), and incubate at room temperature for 30 minutes in the dark;

(6) In step (5), add NaCl with a final concentration of 40 mmol/L to each tube, and after mixing, use a microplate reader to measure the absorbance value at 520 nm, and calculate (A'-A ₀ )/A ₀ , where A' is the absorbance value corresponding to different concentrations of aptamer, A ₀ is the absorbance value without adding aptamer; with (A'-A ₀ )/A ₀ as the ordinate and the nucleic acid aptamer concentration as the abscissa, use GraphPad Prism8 software Analysis and calculation of the affinity constant show that the Kd of the nucleic acid aptamer Pr-A08 is 27.01±6.519nmol/L (see Figure 2), which indicates that the aptamer has affinity. The smaller the affinity constant, the higher the affinity of the aptamer. The higher the affinity the aptamer has.

2. Specificity detection of nucleic acid aptamer Pr-A08

(1) Dilute procymidil to 1 μg/mL with ultrapure water, add 50 μL to each tube, and set non-specific target control samples with the same concentration as procymidone: dimethomorph, pentachloronitrobenzene, glyphosate Phosphine, set a blank control without adding procymidyl but adding 50 μL of ultrapure water, and repeat 3 parallel experiments for each target;

(2) Dilute the aptamer to 400 nmol/L with 1×PBS, add 50 μL to each tube in step (1), and incubate at room temperature for 30 min;

(3) Concentrate the colloidal gold prepared in step 1 by 4 times, centrifuge 4 mL of colloidal gold at 4°C and 8000 rpm/min for 12 min, discard 3 mL of supernatant, and pipette the remaining 1 mL with a pipette to homogeneously prepare for use;

(4) Add 50 μL of concentrated colloidal gold to each tube in step (2), and incubate at room temperature for 30 minutes in the dark;

(5) Add NaCl with a final concentration of 40 mmol/L to each tube in step (4), use a microplate reader to measure the absorbance values at 520 nm and 620 nm, calculate A620nm/A520nm, and calculate (A'-A' ₀ )/A ' ₀ , where A' is the ratio of the absorbance at 620 nm to the absorbance at 520 nm corresponding to different concentrations of aptamer, where A' ₀ is the ratio of the absorbance at 620 nm to the absorbance at 520 nm when no target is added; the results are shown in Fig. 3. The results show that the value of (A'-A' ₀ )/A' ₀ in the tube added with procymidone is much larger than that of other non-specific targets, and the aptamer Pr-A08 can be specific to procymidone. sexual union.

3. Sensitivity detection of nucleic acid aptamer Pr-A08

(1) Dilute procymidyl with ultrapure water to different concentration gradients of 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 600ng/mL, 800ng/mL, and add 50 μL to each tube; set no addition Procymid was added to ultrapure water as a blank control, and three parallel experiments were repeated for each concentration;

(2) Dilute the aptamer to 400 nmol/L with 1×PBS, add 50 μL per tube to step (1), and incubate at room temperature for 30 min;

(3) Concentrate the colloidal gold prepared in step 1 by 4 times, centrifuge 4 mL of colloidal gold at 4°C and 8000 rpm/min for 12 min, discard 3 mL of supernatant, and pipette the remaining 1 mL with a pipette to homogeneously prepare for use;

(4) Add 50 μL of concentrated colloidal gold to each tube in step (2), and incubate at room temperature for 30 minutes in the dark;

(5) In step (4), add NaCl with a final concentration of 40 mmol/L to each tube, use a microplate reader to measure the absorbance values at wavelengths of 520 nm and 620 nm, and calculate A=A620nm/A520nm; When the concentration reaches 50ng/mL, the A620 nm/A520 nm value is significantly higher than that of the blank control. With the increase of the procymid concentration, the A620 nm/A520 nm value gradually increases, and the aptamer Pr-A08 has higher sensitivity (see Figure 4). ).

The aptamer Pr-A08 of the present invention exhibits good affinity, specificity and sensitivity, and the aptamer can be used for the detection of procymidyl in subsequent actual samples.

sequence listing

<110> Kunming University of Science and Technology

<120> Nucleic acid aptamer Pr-A08 specifically binding to procymidone and its application

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<170> SIPOSequenceListing 1.0

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<212> DNA

<213> Artificial Sequence (Artificial)

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gacatattca gtctgacagc gcgtaagaaa tcaaggctgc tcaccgttga cgggctggat 60

agctagacga tattcgtcca tc 82

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<212> DNA

<213> Artificial Sequence (Artificial)

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gacatattca gtctgacagc g 21

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<212> DNA

<213> Artificial Sequence (Artificial)

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gctagacgat attcgtccat c 21

Claims (2)

1. An aptamer Pr-A08 specifically combined with procymidone has a nucleotide sequence shown in SEQ ID NO. 1.

2. The use of the aptamer Pr-A08 of claim 1 in the preparation of a reagent for identifying procymidone or a kit for detecting procymidone.

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