CN116103298A - Aptamer for detecting nitrofurantoin metabolite, kit using method thereof - Google Patents

Abstract

The invention relates to the technical field of biological detection, in particular to a nucleic acid aptamer for detecting nitrofurantoin metabolites and a kit using method thereof. A nucleic acid aptamer for detecting nitrofurantoin metabolites, wherein the sequence of the nucleic acid aptamer is AHD-No.1 recorded in a nucleotide sequence table: AHD8, AHD-No.2: AHD12, AHD-No.3: AHD33, AHD-No.4: AHD50, and an aptamer derivative obtained by modifying or transforming the nucleic acid aptamer; the aptamer consisted of single-stranded DNA, and the 5' end was labeled with a FAM fluorophore.

Description

Aptamer for detecting nitrofurantoin metabolite, kit using method thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a nucleic acid aptamer for detecting nitrofurantoin metabolites and a kit using method thereof.

Background

Nitrofurantoin belongs to nitrofurans, is an important broad-spectrum antibacterial drug, has a killing effect on most pathogens such as gram bacteria, fungi, protozoa and the like, has been widely used for preventing and treating infectious diseases and gastrointestinal diseases of animals such as aquatic products, poultry and the like, and is also used as a feed additive for promoting animal growth. Along with the deep research, the half-life of parent medicines such as nitrofurantoin, furacilin, furazolidone and the like in animal bodies is very short, and the parent medicines can be rapidly metabolized, but metabolites can be tightly combined with proteins in animal bodies to form stable residues of 1-aminohydantoin, and when people eat foods containing nitrofurans, a series of adverse reactions can be generated on the bodies, and the products have potential teratogenicity and mutagenicity. In order to ensure the safety of animal-derived foods, the use of such drugs has been banned in many countries in the field of animal husbandry. Because nitrofurantoin has the advantages of broad antibacterial spectrum, low cost and the like, some breeders still have illegal use under the drive of economic benefit and the like. In order to control veterinary drug residues and ensure food safety, it is necessary to establish a rapid and effective analysis method for detecting 1-aminohydantoin.

The detection method of the 1-aminohydantoin is more, and comprises high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, capillary zone electrophoresis, chemiluminescent enzyme immunoassay and enzyme-linked immunosorbent assay. High performance liquid chromatography is the most commonly used AHD detection method at present, and has higher sensitivity and specificity. However, high performance liquid chromatography and liquid chromatography-tandem mass spectrometry require expensive experimental equipment and require specialized operators to perform, which is disadvantageous for detection of large amounts of samples. Capillary zone electrophoresis also requires expensive experimental equipment and is cumbersome to operate. Chemiluminescent enzyme immunoassays and enzyme-linked immunosorbent assays are limited by the preparation of antibodies, the properties and specificity of which determine the accuracy of the assay. The common methods can not meet the detection requirements of wider, simpler and more economical, so that the detection method with high sensitivity and high specificity meeting the food detection requirements is researched, and the method has very urgent practical significance for detecting and controlling the drug residues.

In recent years, a novel nucleic acid probe has emerged in the detection field: nucleic acid aptamer (aptamer for short). Aptamers, also known as "chemical antibodies", have a number of advantages over traditional antibodies, such as: stable chemical property, easy modification, short screening time, no need of animal experiment, etc. Aptamers, obtained by exponential enrichment of ligand system evolution (SELEX), with high affinity and high specificity, capable of binding specifically to target molecules, have been used to detect a variety of substances, such as: metal ions, antibiotics, toxins, cells, viruses, proteins, and the like. The aptamer is widely applied to the fields of biological medicine industry, food environment detection and the like. It has now been demonstrated that aptamers can fold into unique spatial structures under specific buffer systems that can specifically bind tightly to target molecules through van der waals forces, hydrogen bonding and hydrophobic interactions.

There is currently no report on detection of AHD by an aptamer sensor, and the aptamer is usually obtained from Graphene Oxide (GO) -SELEX, MB-SELEX, cell-SELEX, and there is currently no report on enrichment of small molecule aptamer by MC-SELEX.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a technical scheme capable of solving the problems.

A nucleic acid aptamer for detecting nitrofurantoin metabolites, wherein the sequence of the nucleic acid aptamer is AHD-No.1 recorded in a nucleotide sequence table: AHD8, AHD-No.2: AHD12, AHD-No.3: AHD33, AHD-No.4: AHD50, and an aptamer derivative obtained by modifying or transforming the nucleic acid aptamer; the aptamer consisted of single-stranded DNA, and the 5' end was labeled with a FAM fluorophore.

Further, the nucleic acid aptamer is prepared by the following method:

s11: obtaining a candidate sequence of AHD through a non-fixed MC-SELEX screening method;

s12: analyzing the primary structure, the secondary structure and the tertiary structure of the candidate sequence to obtain candidate nucleic acid aptamer;

s13: the core recognition region of the aptamer is obtained through fluorescence measurement, molecular docking technology and molecular dynamics simulation technology.

A method for using a detection kit based on the above-mentioned aptamer for detecting nitrofurantoin metabolite, comprising the following steps:

s21: setting a kit containing a nucleic acid aptamer;

s22: obtaining a metabolite sample and preprocessing the metabolite sample;

s23: putting the treated nitrofurantoin metabolite sample into a kit, and shaking uniformly for 60 minutes;

s24: adding graphene oxide into the kit, and continuously shaking for 10min;

s25: after centrifugation and standing, the supernatant was aspirated and the fluorescence value was measured.

Further, the kit set in step S21 includes the following components: fluorescent-labeled aptamer, binding buffer.

Further, the metabolite sample obtained in step S22 is culture water.

Compared with the prior art, the invention has the beneficial effects that:

(1) The aptamer constructed based on the aptamer is used for detecting AHD, and has the advantages of high sensitivity, simplicity in operation, strong selectivity, high speed and low price, and has a good application prospect in AHD detection.

(2) According to the invention, the aptamer sequence obtained through non-immobilized MC-SELEX screening is firstly provided for analysis and optimization, the core recognition region of the aptamer is obtained, and the aptamer with high affinity and specificity to AHD is synthesized through in vitro cloning.

(3) According to the invention, a fluorescent aptamer sensor is constructed based on an AHD aptamer and Graphene Oxide (GO). When AHD exists in the detection system, the aptamer specifically binds to the AHD and cannot be adsorbed by Graphene Oxide (GO) through pi-pi stacking. When the concentration of AHD is different, the fluorescence value detected is different, and when the detection system does not contain AHD, the fluorescent-labeled aptamer is adsorbed by Graphene Oxide (GO) through pi-pi accumulation. Based on the principle, the quantitative detection of AHD is realized.

(4) Aiming at the problems that a large-scale sample cannot be detected by a traditional detection method, the operation is complicated, and a long-time animal experiment is required for preparing an antibody in an immunoassay method, the invention establishes a fluorescent sensor rapid detection method based on Graphene Oxide (GO), can be used for rapid and sensitive quantitative detection of AHD residues in an actual sample, and overcomes the defects of the method. Provides a new detection method for detecting antibiotics in samples.

(5) According to the invention, the AHD aptamer is screened and sequence optimized based on Graphene Oxide (GO), so that the fluorescent aptamer sensor based on Graphene Oxide (GO) is constructed, the fluorescent aptamer sensor is successfully detected in actual sample culture water and lake water, the linear relationship is good, and a foundation is laid for detecting and developing products of AHD.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1 is a schematic diagram of the working principle of the nucleic acid aptamer of the invention;

FIG. 2 is a graph showing the relationship between concentration and fluorescence intensity in the present invention

FIG. 3 shows the results of specific comparison of an aptamer with AHD in accordance with the present invention;

fig. 4 is a schematic representation of a standard curve in accordance with the present invention.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

A nucleic acid aptamer for detecting nitrofurantoin metabolites, wherein the sequence of the nucleic acid aptamer is AHD-No.1 recorded in a nucleotide sequence table: AHD8, AHD-No.2: AHD12, AHD-No.3: AHD33, AHD-No.4: AHD50, and an aptamer derivative obtained by modifying or transforming the nucleic acid aptamer; the aptamer consisted of single-stranded DNA, and the 5' end was labeled with a FAM fluorophore.

Further, the nucleic acid aptamer is prepared by the following method:

s11: obtaining a candidate sequence of AHD through a non-fixed MC-SELEX screening method;

s12: analyzing the primary structure, the secondary structure and the tertiary structure of the candidate sequence to obtain candidate nucleic acid aptamer;

s13: the core recognition region of the aptamer is obtained through fluorescence measurement, molecular docking technology and molecular dynamics simulation technology.

A method for using a detection kit based on the above-mentioned aptamer for detecting nitrofurantoin metabolite, comprising the following steps:

s21: setting a kit containing a nucleic acid aptamer;

s22: obtaining a metabolite sample and preprocessing the metabolite sample;

s23: placing the treated nitrofurantoin metabolite sample into a kit, and shaking uniformly for 60min;

s24: adding graphene oxide into the kit, and continuously shaking for 10min;

s25: after centrifugation and standing, the supernatant was aspirated and the fluorescence value was measured.

Further, the kit set in step S21 includes the following components: fluorescent-labeled aptamer, binding buffer.

Further, the metabolite sample obtained in step S22 is culture water.

Specifically, the technical scheme of the invention further comprises the following specific embodiments:

first embodiment establishment of AHD aptamer detection System

1. Non-immobilized magnetic bead-SELEX process for screening AHD aptamer

(1) Library and primer processing: 200. Mu.L of a 100nM aptamer library (5 '-FAM-CACCTAATACGACTCACTATAGCGGATCCGA-N40-CTGGCTCGAACAAGCTTG C-3', N random sequence), 600. Mu.L of a 15. Mu.M forward primer (5'-CACCTAATACGACTCACTATAGCGGA-3') and 600. Mu.L of a 1. Mu.M reverse primer (5'-GCAAGCTTGTTCGAGCCAG-3') and 600. Mu.L of a 10. Mu.M Biotin-reverse primer (Biotin-5'-GCAAGCTTGTTCGAGCCAG-3') were placed in a 95℃metal bath for 10 minutes, followed by 30 minutes on ice, followed by removal from the light and 10 minutes at room temperature, and stored at 4℃to allow a large amount of ssDNA in the library to fold into a unique tertiary structure, and the fluorescence values were determined, as described herein. Mu. L, mL in volume units, and. Mu. M, nM in concentration units.

(2) Preparation of carboxyl magnetic beads and streptavidin magnetic beads:

(1) carboxyl magnetic bead activation: 100 mu L of ddH for 10mg/mL carboxyl magnetic beads ₂ O was washed three times, then DMF was washed four times, and collected using a magnet. The beads were reacted with 50. Mu.L of 5. Mu.M HATU and 20. Mu.L of 3. Mu.M DIPEA at 25℃for 2 hours, followed by the use of binding buffer (100 mM NaCl, 2mM MgCl) ₂ 、20mM Tris-HCl、1mM CaCl ₂ 5mM KCl and 0.02% Tween20, ph=7.4) the beads were washed 4 times. The resulting beads were stored at 4℃for use.

(2) Streptavidin magnetic beads: to obtain purified magnetic beads, 0.6mL of 1mg/mL streptavidin magnetic beads in PBS (ph=7.4) was reacted with 10 μl of 10 μΜ biotin-reverse primer at 25 ℃ for 1 hour and washed 3 times with PBS (ph=7.4). The collected beads were stored in PBS (ph=7.4) at 4 ℃ for later use.

(3) AHD aptamer is obtained through non-immobilized magnetic bead-SELEX screening:

100. Mu.L of carboxyl magnetic beads were taken to remove the supernatant and 200. Mu.L of 100nM random library was added to incubate at 25℃for 2 hours, and the supernatant from the magnetic separation was collected and used as a new library for forward selection.

mu.L of 1mg/mL AHD was incubated with 200. Mu.L of 100nM new library collected in the first step for 2 hours, and then added to the deactivated supernatant activated carboxyl beads and incubated at 25℃for 2 hours. Washing 4 times with binding buffer, the resulting supernatant was removed entirely. Subsequently, the magnetic beads were washed with 200. Mu.L of elution buffer (40 mM Tris-HCl, 3.5m urea, 10mM EDTA, 0.02% Tween20, pH=8.0), incubated at 80℃for 10 minutes, and then the eluate was collected by magnetic separation. This elution procedure was repeated 3 times.

The collected 600 μl of eluate and streptavidin magnetic beads were incubated at 25 ℃ for 1 hour, after which the supernatant was washed 3 times with PBS (ph=7.4) and removed, then 100 μl of 0.05M NaOH was added for elution, and after incubation at 25 ℃ for 10 minutes the eluate (100 μl) was collected by magnetic separation, the pH was adjusted to 7.4 using 0.05M HCl. Then using 2 Xbinding buffer (175 mM NaCl, 4mM MgCl) ₂ 、40mM Tris-HCl、2mM CaCl ₂ 10mM KCl and 0.04% Tween20, ph=7.6) equilibrates the buffer system in which the aptamer was located.

(4) The ssDNA library obtained in the eluate was used for PCR amplification:

PCR procedure: mu.L of ssDNA library, 2. Mu.L of 15. Mu.M forward primer, 2. Mu.L of 1. Mu.M reverse primer, 8. Mu.L of 2X taq PCR MasterMix II and 36. Mu.L of ddH were mixed ₂ O-mixing, denaturation at 95℃for 30s, renaturation at 58℃for 30 seconds, and extension at 72℃for 0.06 seconds. The amplification process was repeated 25 rounds. The amplified product was purified using the same procedure as in step (3) above and used as an aptamer library for the next round.

(5) And in the seventh round, nitrofurans technical stock, metabolites and an aptamer library are used for incubation, and then are added into magnetic beads for reverse screening, and then AHD is used for forward screening. Ultraviolet-visible spectrum UV-VIS spectrum was used to determine the concentration of aptamer at the end of each round of screening.

(6) Determination of recovery: (concentration of ssDNA recovered at the end of each round of purification/concentration of ssDNA to be put into each round).

2. Cloning and sequencing

And (3) carrying out nine rounds of screening, amplifying the obtained product by PCR, and selecting the cloned product for sequencing.

3. Aptamer sequence analysis and optimization

Secondary structure of the obtained aptamer was predicted by dancan, RNA structure, vienna RNA Web Services and UNA Fold Web Server; the sequences meeting the requirements are selected and shown in Table 1. The affinity of the candidate aptamer is then determined by an affinity assay. Sequence cutting is carried out on the aptamer with highest affinity, so that a new aptamer is obtained: SME4-1, sequences are shown in Table 2.

TABLE 1

TABLE 2

4. Affinity assay

To assess the affinity of candidate aptamers, 30 μl of AHD was mixed with different concentrations of FAM-labeled aptamers (i.e. 0, 25, 50, 100, 150 and 200 nM) at 200 μl, the mixture was reacted at 25 ℃ in the dark for 2 hours, and then the reaction mixture was added to carboxyl-activated magnetic beads, which were washed three times with binding buffer. Subsequently, 100. Mu.L of the eluate was added to the obtained magnetic beads, and incubated at 80℃for 10 minutes. Finally, the supernatant from the reaction mixture was collected and applied to a fluorometer at λ _ex =492 nm and λ _em Its fluorescence intensity was measured at=518 nm. K representing binding affinity _d Values were analyzed using Origin 2019 software and according to the nonlinear regression equation y=b _max ×X÷(K _d +X) calculation, wherein X represents the aptamer concentration, Y represents the relative fluorescence intensity, B _max Representing the most binding sites, a concentration-fluorescence intensity relationship graph as shown in FIG. 2 was established. As shown in Table 3, the affinities of AHD8, AHD12, AHD33, AHD50, and AHD50-1 were 209, 161, 235, 104, and 86nM, respectively.

TABLE 3 Table 3

5. Preparation of Graphene Oxide (GO)

1mL of 2mg/mL graphene oxide dispersion is taken, 1mL of binding buffer solution is added, 13000rpm and 25 ℃ are used for centrifugation for 10min, the supernatant is taken, 1mL of binding buffer solution is added, 13000rpm and 25 ℃ are used for centrifugation for 10min, until no solid particles are visible, the operation is repeated twice, 1mL of binding buffer solution is added, ultrasound is carried out for 15min, and the mixture is placed in a refrigerator at 4 ℃ for standby.

6. Aptamer and AHD specificity analysis

To assess the aptamer specificity for AHD, 30 μl of AHD was mixed with other antibiotics (furacilin metabolite, furazolidone metabolite, nitrofurantoin, calicheamicin, penicillin, sulfonamide core, etc.) with 200 μl of 100nM FAM-labeled aptamer, the mixture was reacted in the dark at 25 ℃ for 2 hours, and then the reaction mixture was added to carboxyl-activated magnetic beads, and washed three times with binding buffer. Subsequently, 100. Mu.L of the eluate was added to the obtained magnetic beads, and incubated at 80℃for 10 minutes. Finally, the supernatant from the reaction mixture was collected and applied to a fluorometer at λ _ex =492 nm and λ _em Its fluorescence intensity was measured at=518 nm. The specificity of the aptamer was evaluated using the relative fluorescence intensity, the formula being fluorescence intensity relative= (measured fluorescence intensity of other antibiotics/measured fluorescence intensity of AHD) ×100%. The results of specific comparison of the aptamer with AHD are shown in fig. 2, with the relative fluorescence intensity of other antibiotics and structural analogs of AHD less than 25%. These results indicate that AHD50-1 has a higher specificity for AHD.

7. Establishment of a Standard Curve

The standard curve was constructed using AHD standards, FAM fluorescent-labeled aptamer (100 nM) was incubated with a series of AHD concentrations ranging from 0.25-150ng/mL in 200. Mu.L of binding buffer at 25℃in the dark for 1h, GO was added to the mixture, incubated at room temperature for 10min in the dark followed by 13000rmp, centrifugation for 5min and supernatant was removed, and the fluorescence intensity was measured by an enzyme-labeling instrument at an emission wavelength of 520nM and the standard curve was plotted (as shown in FIG. 3).

Actual sample AHD determination

The aptamer sensor was validated using a culture water sample taken locally, 10mL of water sample was taken, centrifuged at 10000rpm for 10min, the supernatant was taken and filtered with a 0.22 μm filter membrane for use.

The accuracy and precision of the aptamer sensor are expressed in terms of recovery and coefficient of variation, respectively. Samples were added with known concentrations of AHD (25, 75 and 125 μg/kg) for aptamer sensor analysis. The average recovery is calculated by the following equation: (measured concentration/standard concentration). Times.100%. The coefficient of variation was determined by analyzing the above-described samples with three different levels of sulfadiazine added. The calculation formula of the variation coefficient is as follows: coefficient of variation cv= (standard deviation/average value) ×100%. Each concentration level was tested five times and the correlation of the aptamer sensor in terms of AHD detection in the labeled sample was calculated.

TABLE 4 Table 4

As shown in Table 4, the recovery rate of the samples was between 89.14% and 118.29%, and the coefficient of variation was between 0.04% and 0.09%. Positive correlation of aptamer sensor results was also observed (R2 > 0.987). The results indicate that the proposed aptamer sensor is used to detect the reliability of AHD.

The invention provides a detection kit for detecting AHD:

the detection kit comprises the following components:

(1) FAM-AHD50-1 recorded in a 100nMFAM fluorescent labeling nucleotide sequence table;

(2) 2mg/mL Graphene Oxide (GO);

(3) Binding buffer (100 mM NaCl, 2mM MgCl) ₂ 、20mM Tris-HCl、1mM CaCl ₂ 5mM KCl and 0.02% Tween20, ph=7.4);

the detection is carried out according to the following steps:

1) Processing the sample according to the requirement;

2) The treated sample was dissolved in the binding buffer and diluted 10-fold. Adding 100nM FAM-AHD50-1, mixing and shaking uniformly for 1 hour;

3) Add 4. Mu.L 2mg/mL GO and continue shaking for 10 minutes;

4) After 13000rmp,5min centrifugation, the supernatant was aspirated and the fluorescence value was measured.

The specific inspection procedure was the same as in example 2.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (5)

1. A nucleic acid aptamer for detecting nitrofurantoin metabolites, which is characterized in that the sequence of the nucleic acid aptamer is AHD-No.1 recorded in a nucleotide sequence table: AHD8, AHD-No.2: AHD12, AHD-No.3: AHD33, AHD-No.4: AHD50, and an aptamer derivative obtained by modifying or transforming the nucleic acid aptamer; the aptamer consisted of single-stranded DNA, and the 5' end was labeled with a FAM fluorophore.

2. A nucleic acid aptamer for detecting a nitrofurantoin metabolite according to claim 1, wherein the nucleic acid aptamer is prepared by the following method:

s11: obtaining a candidate sequence of AHD through a non-fixed MC-SELEX screening method;

s12: analyzing the primary structure, the secondary structure and the tertiary structure of the candidate sequence to obtain candidate nucleic acid aptamer;

s13: the core recognition region of the aptamer is obtained through fluorescence measurement, molecular docking technology and molecular dynamics simulation technology.

3. A method of using a kit based on a aptamer for detecting a nitrofurantoin metabolite according to claim 1 or 2, comprising the steps of:

s21: setting a kit containing a nucleic acid aptamer;

s22: obtaining a metabolite sample and preprocessing the metabolite sample;

s23: placing the treated nitrofurantoin metabolite sample into a kit, and shaking uniformly for 60min;

s24: adding graphene oxide into the kit, and continuously shaking for 10min;

s25: after centrifugation and standing, the supernatant was aspirated and the fluorescence value was measured.

4. The method of claim 3, wherein the kit set in step S21 comprises the following components: fluorescent-labeled aptamer, binding buffer.

5. The method of claim 3, wherein the sample of the nitrofurantoin is culture water.

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