1. A universal colloidal gold lateral chromatography test strip is characterized by comprising an aptamer, a probe polyA-DNA and streptavidin-biotin-DNAC; the probe polyA-DNA contains a polyA fragment, a fragment complementary to DNAC, and a fragment complementary to the aptamer; the 5' end of the aptamer is labeled by biotin; the aptamer can be specifically combined with the small molecular substance to be detected.

2. The universal colloidal gold lateral chromatography test strip of claim 1, wherein the streptavidin-biotin-DNAC is prepared by mixing streptavidin with DNAC labeled with biotin at the 5' end in equal volume and incubating at 3-5 ℃ for 0.8-1.2 h; the nucleotide sequence of the DNAC is shown as SEQ ID NO. 5.

3. The universal colloidal gold lateral chromatography test strip of claim 2, wherein the streptavidin concentration is 2.5mg/mL and the DNAc concentration is 250 μ Μ.

4. The universal colloidal gold lateral flow assay strip of claim 1, wherein the small molecule substance includes but is not limited to kanamycin or OTA.

5. The universal colloidal gold lateral chromatography test strip of any one of claims 1 to 4, wherein the test strip comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane, a water absorption pad and a PVC (polyvinyl chloride) rubber plate; a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad are sequentially stuck on the PVC bottom plate; the NC membrane is sequentially provided with a detection area and a control area, and the distance between the detection area and the control area is 4-6 mm; streptavidin is arranged on the detection area, and streptavidin-biotin-DNAC is arranged on the control area; the gold label pad contains AuNPs @ polyA-DNA conjugate.

6. The universal colloidal gold lateral chromatography test strip of claim 5, wherein the AuNPs @ polyA-DNA conjugate is obtained by anchoring the probe polyA-DNA of claim 1 on a nanogold particle.

7. The universal colloidal gold lateral chromatography test strip of claim 6, wherein the particle size of the gold nanoparticles is 13-17 nm; the concentration of the polyA-DNA is 80-120 mu M.

8. A method for rapidly detecting kanamycin is characterized in that the method is to use the universal colloidal gold lateral chromatography test strip of any one of claims 1 to 7 for testing, mix and incubate a solution to be tested and a nucleic acid aptamer of a small molecular substance, then drip the mixed solution on a sample pad, and carry out qualitative analysis by naked eyes or quantitative analysis by a colloidal gold test strip quantitative analyzer according to a standard curve.

9. The method according to claim 8, wherein the standard curve is prepared by diluting a standard solution of a small molecule substance to different concentrations, mixing the diluted solutions of different concentrations with the aptamer labeled with biotin at the 5' end, dropping the mixture on a sample pad, performing quantitative analysis using a quantitative analyzer using colloidal gold test paper, and fitting the data to obtain the standard curve.

10. The method according to claim 8 or 9, wherein the time for mixing and incubating the solution to be tested and the aptamer is 15-25 min; the volume ratio of the solution to be detected to the aptamer is 99: 1; the initial concentration of the aptamer is 0.1-1.0 mu M.

Background

Lateral flow assay technology is a paper-based detection platform for detecting targets, and is of great interest to researchers because of its potential to provide results in minutes. Because of its low cost and ease of development and production, lateral flow chromatographic analysis technology has been identified for in-situ test applications and is widely used in a variety of fields, including biomedicine, food safety, quality control, and environmental hygiene. Lateral flow chromatographic techniques may be applied to a range of biological samples including urine, saliva, sweat, serum, plasma and blood. Therefore, the application value of the lateral flow chromatography analysis technology is improved. There are two modes of lateral flow chromatographic analysis, competitive and sandwich, and competitive is commonly used for low molecular weight compounds (e.g., mycotoxins, etc.). Artificial antigens and species-specific anti-immunoglobulin antibodies or aptamer complementary strands and artificially designed nucleic acid strands are typically sprayed onto a digestive cellulose membrane (NC) as T-and C-lines, respectively. The signal intensity of the T-line is inversely proportional to the concentration of the analyte. The C line has independent visible signals regardless of whether the detection target is contained in the sample to be detected or not. In recent years, more and more researchers pay attention to the detection of small molecular substances by a lateral flow chromatography analysis technology, but the kanamycin and the OTA are only detected by using the lateral flow chromatography analysis technology for a few times, so that a universal aptamer test strip for detecting the small molecular substances is designed to detect the small molecular substances, particularly the kanamycin and the OTA.

The aptamer is a small segment of structured oligonucleotide sequence (RNA or DNA) obtained by a Systematic evolution of ligands by exponential enrichment (SELEX) through an in vitro screening technology, can be combined with corresponding target molecules (protein, virus, bacteria, cells, heavy metal ions and the like) with high affinity and strong specificity, and provides a novel research platform for efficient and rapid identification for the chemical biology and the biomedical science.

The colloidal gold lateral chromatography test paper based on the aptamer label has the advantages of long-term stability, short detection time, low cost, simple operation, rapidness and the like, and is an ideal method for rapidly detecting various target objects on site. In the design of the existing nucleic acid aptamer colloidal gold lateral chromatography test strip, a nucleic acid chain-streptavidin compound is usually sprayed on a T line, and a specific compound needs to be prepared again and a nitrocellulose membrane needs to be prepared by spraying along with the change of a detection target object, so that the existing lateral chromatography test strip is poor in universality.

Currently, few studies are made on colloidal gold lateral chromatography test strips based on aptamer recognition of kanamycin or OTA. In the literature (Biosensors & Bioelectronics,2015,71(15): 230-242), DNA functionalized gold nanospheres (AuNPs-DNA) are used as probes, Magnetic Microspheres (MMS) are used to rapidly separate complementary strands (cDNA) of kanamycin aptamers, the cDNA is used as a detection target, the cDNA is applied to a test strip, and the detection concentration of the cDNA is converted into that of kanamycin, so that the qualitative and quantitative detection of kanamycin is completed, and the detection limit reaches 4.96nmol/L (S/N-3). The test strip needs MMS, and the kanamycin detection is converted into the cDNA detection, so that the test strip is complex and is not beneficial to the repeatability of stable production and application of products.

In the literature (Journal of chromatography B,2016,1022: 102-108), an aptamer-based lateral chromatography strip was developed that uses a competitive binding format for the in situ rapid detection of OTA in Astragalus membranaceus. A competition reaction occurs between the DNA probe immobilized on the strip (test zone) and the target in the sample (OTA), both competing for binding to the AuNPs-apt conjugate. The color of the test strip line T is inversely related to the concentration of OTA in the sample. Based on the method, the detection of the OTA can be completed within 15min, and the detection line is 1 ng/mL. The test strip couples the aptamer to AuNPs, so that the competition of OTA in a sample on the aptamer is not facilitated, the product stability is poor, and the design is complex and the universality is poor.

At present, the developed aptamer colloidal gold lateral chromatography test paper needs repeated optimization design of a probe on the test paper aiming at a specific target object, and has poor universality and detection sensitivity to be improved.

Disclosure of Invention

[ problem ] to

In the design of the existing aptamer colloidal gold lateral chromatography test strip, a streptavidin-nucleic acid chain compound is usually sprayed on a T line, and the streptavidin-nucleic acid chain compound and the nitrocellulose membrane are required to be prepared again and sprayed again aiming at a certain specific detection target object, so that the existing lateral chromatography test strip has poor universality and high preparation cost.

[ solution ]

The invention adopts AuNPs @ polyA-DNA conjugate as a probe, and the conjugate is prepared by anchoring polyA on a nanogold particle by adopting polyA as an anchoring block. Streptavidin is sprayed on a detection area (T line) of the nucleic acid test strip, and a nucleic acid chain which is partially complementary with DNA of the AuNPs @ polyA-DNA conjugate is sprayed on a control area (C line), so that the universal test strip is constructed. The streptavidin sprayed on the T line and the streptavidin-nucleic acid chain compound sprayed on the C line of the test strip do not need to be changed, and only the part of the polyA-DNA complementary to the aptamer is changed, so that another substance can be detected, and convenience is provided for the subsequent detection.

The aptamer used in the invention is added with 5T bases on the basis of the reported aptamer nucleic acid chain, and the 5 nucleic acid bases are used for assisting hybridization. The probe has 15 bases complementary to the aptamer nucleic acid strand. After the biotinylated aptamer nucleic acid strand is bound to the probe, the probe can further bind to streptavidin on the T-line. When the small molecular substance exists, the small molecular substance and the probe compete to combine with the aptamer nucleic acid chain, and hybridization of the probe and the aptamer nucleic acid chain is inhibited, so that color development of a T line is inhibited, and rapid detection is realized.

The nucleic acid aptamer colloidal gold lateral chromatography test strip provided by the invention is universal in preparation of a nitrocellulose membrane and design of a probe, namely when the colloidal gold test strip for different small molecular substances is prepared, a T line and a C line on the nitrocellulose membrane do not need to be changed, only the nucleic acid aptamer corresponding to the small molecular substance and an AuNPs @ polyA-DNA conjugate on a gold label pad need to be changed, and corresponding changes are made on complementary fragments of the nucleic acid aptamer and polyA-DNA in the probe and the nucleic acid aptamer for different small molecular substances.

The invention provides a universal colloidal gold lateral chromatography test strip for detecting kanamycin based on an aptamer and an AuNPs @ polyA-DNA conjugate, which generally comprises a sample pad, a binding pad (gold label pad), a nitrocellulose membrane (NC membrane), a water absorption pad and a PVC (polyvinyl chloride) rubber plate.

The detection principle of the test strip is as follows: a small molecular substance is detected by adopting a competition method, when the sample solution does not contain the small molecular substance, the aptamer with 5' end biotinylation is combined with the AuNPs @ polyA-DNA probe, and then is captured by streptavidin on a T line to form an AuNPs @ polyA-DNA-Apt compound, and due to the accumulation of AuNPs, a clear deep red band can be observed on the T line, and the detection result is negative. When the solution to be detected contains the micromolecular substance, the micromolecular substance is combined with the nucleic acid aptamer biotinylated at the 5' end, and the nucleic acid aptamer cannot be combined with the AuNPs @ polyA-DNA probe, so that the accumulation of the probe in a T line area is reduced, the T line is lighter or does not develop color, and the detection result is positive. The color intensity on the T-line region is inversely related to the concentration of the small molecule species. The AuNPs-DNA probe can be captured by DNAC in the C line area and then developed no matter whether the sample solution has small molecular substances or not.

The first purpose of the invention is to provide a universal colloidal gold lateral chromatography test strip, which contains aptamer, probe polyA-DNA and streptavidin-biotin-DNAC.

In one embodiment, the probe polyA-DNA contains a polyA fragment, a fragment complementary to DNAc, and a fragment complementary to an aptamer of a small molecule substance.

In one embodiment, the 5' end of the aptamer is labeled with biotin; the aptamer can be specifically combined with the small molecular substance to be detected.

In one embodiment, the streptavidin-biotin-DNAC is prepared by mixing streptavidin and DNAC with biotin-labeled 5' end in equal volume, and incubating at 3-5 ℃ for 0.8-1.2 h.

In one embodiment, the nucleotide sequence of the DNAc is shown as SEQ ID No. 5.

In one embodiment, the streptavidin concentration is 2.5mg/mL and the DNAC concentration is 250. mu.M.

In one embodiment, the small molecule substance includes, but is not limited to, kanamycin or OTA.

In one embodiment, the aptamer is an oligonucleotide sequence obtained by in vitro screening techniques-exponential enrichment of ligand phylogenetic techniques.

In one embodiment, the test strip comprises a sample pad, a conjugate pad (gold-labeled pad), a nitrocellulose membrane (NC membrane), a water absorbent pad, and a PVC rubber plate; a sample pad, a gold label pad, an NC film and a water absorption pad are sequentially adhered on the PVC bottom plate; the NC membrane is sequentially provided with a detection area and a control area, and the distance between the detection area and the control area is 4-6 mm; streptavidin is arranged on the detection area, and streptavidin-biotin-DNAC is arranged on the control area; the gold label pad contains AuNPs @ polyA-DNA conjugate.

In one embodiment, the length of the overlapped part between the sample pad and the gold mark pad is 1-2 mm, and the sample pad is arranged above the gold mark pad; the length of the overlapped part between the gold mark pad and the NC membrane is 1-2 mm, and the gold mark pad is arranged above the NC membrane; the length of the overlapped part between the NC membrane and the water absorption pad is 1-3 mm, and the water absorption pad is arranged above the NC membrane.

In one embodiment, the AuNPs @ polyA-DNA conjugate is obtained by anchoring a probe polyA-DNA to a nanogold particle.

In one embodiment, the gold nanoparticles have a particle size of 13 to 17 nm; the concentration of the polyA-DNA is 80-120 mu M.

The second purpose of the invention is to provide a method for rapidly detecting small molecular substances, which adopts the colloidal gold lateral chromatography test strip to test, mixes and incubates the solution to be detected and the small molecular substance aptamer, then absorbs 50-100 mu L of the solution to be detected and drips the solution on a sample pad, incubates for 3-5 min, and carries out qualitative analysis by naked eyes or quantitative analysis by a colloidal gold test strip quantitative analyzer according to a standard curve.

In one embodiment, the standard curve is prepared by diluting a standard solution of a small molecular substance to different concentrations, mixing the diluted solution with a nucleic acid aptamer with a biotin-labeled 5' end, dripping the mixture onto a sample pad, incubating for 3-5 min, performing quantitative analysis by using a colloidal gold test paper quantitative analyzer, and fitting data to obtain the standard curve.

In one embodiment, the mixed incubation time is 15-25 min.

In one embodiment, the volume ratio of the aptamer to the test solution is 1: 99.

In one embodiment, the initial concentration of the aptamer is 0.1 to 1.0. mu.M.

The third purpose of the invention is to provide a preparation method of the universal colloidal gold lateral chromatography test strip, which comprises the following specific steps:

(1) the sample pad and gold label pad were cut, soaked in PBS and dried.

(2) The probe AuNPs @ polyA-DNA was sprayed onto a gold pad and dried.

(3) And spraying streptavidin on a detection area of the NC membrane, spraying streptavidin-biotin-DNAC on a control area, fixing the distance between the detection area and the spraying area at 5mm, and drying at 35-39 ℃ for 2 h.

(4) And (3) sequentially sticking the sample pad, the gold label pad, the NC membrane and the water absorption pad which are prepared in the steps (1) to (3) on a PVC plate to obtain the colloidal gold lateral chromatography test strip for detecting kanamycin.

In one embodiment, the AuNPs @ polyA-DNA conjugate is obtained by anchoring polyA-DNA on a nanogold particle by using a probe polyA-DNA as an anchoring block; the probe polyA-DNA contains a polyA fragment, a fragment complementary to DNAC and a fragment complementary to an aptamer of a small molecule substance; the particle size of the nano gold particles is 13-17 nm.

In one embodiment, the concentration of the polyA-DNA is 80 to 120. mu.M.

In one embodiment, the nucleotide sequence of the DNAc is shown as SEQ ID No. 5.

In one embodiment, the length of the overlapped part between the sample pad and the gold mark pad is 1-2 mm, and the sample pad is arranged above the gold mark pad; the length of the overlapped part between the gold mark pad and the NC membrane is 1-2 mm, and the gold mark pad is arranged above the NC membrane; the length of the overlapped part between the NC membrane and the water absorption pad is 1-3 mm, and the water absorption pad is arranged above the NC membrane.

The invention has the beneficial effects that:

(1) the invention prepares a general colloidal gold lateral chromatography test strip based on an aptamer with high sensitivity and high specificity. The test strip is sensitive to kanamycin and OTA detection reaction (the detection limits of naked eyes are respectively 15ng/mL and 10ng/mL, and the detection limits of a reading instrument are respectively 0.3ng/mL and 0.18ng/mL), rapid (20min) and high in repeatability of detection results.

(2) The detection method provided by the invention realizes high-sensitivity colorimetric detection of small molecular substances (kanamycin and OTA in the invention). In the concentration range of 5-250ng/mL, the T/C line and the concentration of kanamycin have good linear relation, and the detection limit is 0.3 ng/mL. In the concentration range of 1-250ng/mL, the T/C line and the concentration of kanamycin have good linear relation, and the detection limit is 0.18 ng/mL.

(3) The streptavidin and the nucleic acid chain sprayed on the detection area and the control area of the test strip constructed by the invention do not need to be changed, and another substance can be detected only by changing the colloidal gold-nucleic acid conjugate when the test strip is prepared, so that the general type of the nitrocellulose membrane prepared by spraying is improved, the preparation process is simplified, and the test strip cost is reduced.

(4) The test strip can observe the color change of the detection area, the qualitative detection of small molecular substances can be realized by naked eyes, and the quantitative analysis can be realized by using a colloidal gold quantitative analyzer.

(5) In the design of the aptamer of the test strip, a plurality of T bases are added at the 3' end to increase the binding efficiency of the aptamer and the probe, improve the sensitivity, reduce the using amount of the aptamer and reduce the cost.

Drawings

FIG. 1 is a schematic structural diagram and a detection principle diagram of the test strip.

Fig. 2 is a characterization of gold nanoparticles: (a) TEM images of AuNPs; (b) absorption spectra of AuNPs.

FIG. 3 is a graph of test strips for different concentrations of kanamycin standard solution; the concentrations from left to right were 0, 0.5, 5, 15, 25, 50, 150, 250 and 400ng/mL in that order.

FIG. 4 is a standard graph of the relative intensity (T/C) of the T-line to the C-line at different concentrations of kanamycin in a standard test solution; the concentrations from left to right were 0, 1, 10, 50, 100, 250 and 500ng/mL in that order.

Fig. 5 is a test strip for different concentrations of OTA standard solutions.

FIG. 6 is a standard graph of the relative intensity (T/C) of the T-line versus the C-line at different concentrations of OTA in a standard test solution.

FIG. 7 is a graph of the test strip for honey samples with different concentrations of kanamycin.

FIG. 8 shows the effect of nucleic acid strands of different sequences on the kanamycin response signal.

Detailed Description

The first embodiment is as follows: preparation of kanamycin rapid detection test strip based on nucleic acid aptamer

The method comprises the following specific steps:

1. design of aptamer sequences and probes

To ensure that the biotinylated aptamer binds to the nucleic acid on the probe and is captured by SA in the T-line region, nucleic acid strands of different sequences (polyA-DNA) were selected₍₂₀₎，polyA-DNA₍₁₅₎，polyA-DNA₍₅₊₁₀₎，polyA-DNA₍₅₊₅₎，polyA-DNA₍₁₀₊₅₎) Coupled with AuNPs (as shown in table 1). The corresponding aptamer concentration was selected to be 0.5. mu.M, and kanamycin (150ng/mL) was added at the same concentration. As a result, as shown in FIG. 8, when a plurality of T bases were ligated to the 3' -end of the aptamer sequence, the signal intensity was significantly increased, and the signal was enhanced up to 6-fold, when a nucleic acid probe strand polyA-DNA having a nucleotide sequence of SEQ ID NO.2 was used₍₁₀₊₅₎The change in the corresponding signal (. DELTA.T/C value) was the greatest for kanamycin. Thus, the probe strand polyA-DNA was used in the experiment₍₁₀₊₅₎。

TABLE 1 nucleic acid sequences for LFS universal to kanamycin aptamer

(underlined or bold sequence indicates a complementary sequence; bold italic sequence indicates an aptamer partial sequence or a complementary sequence thereof; bold sequence indicates an aptamer extended sequence or a complementary sequence thereof.)

Design of polyA-DNA: PolyA-DNA has three functional regions. The first functional region is polyA which is used as an anchoring block for anchoring polyA-DNA on the gold nano-particles; the second functional region is a region complementary to the DNAc, which is complementary to the DNAc without specific sequence requirements; the third functional region is a region complementary to a portion of the aptamer, facilitating hybridization. At the same time, it is ensured that the polyA-DNA sequences are not complementary to one another.

Design of DNAc: as shown in tables 1 and 2, the DNAC sequence was underlined to the polyA-DNA and ligated with 5A bases at the 3' end.

TABLE 2 DNA sequence of test paper strip for rapid kanamycin detection

(underlined or bold sequence indicates a complementary sequence; bold sequence indicates an aptamer partial sequence or a complementary sequence thereof; bold italic sequence indicates an aptamer extended sequence or a complementary sequence thereof.)

TABLE 3 DNA sequence of test paper strip for rapid OTA detection

(underlined or bold sequence indicates a complementary sequence; bold sequence indicates an aptamer partial sequence or a complementary sequence thereof; bold italic sequence indicates an aptamer extended sequence or a complementary sequence thereof.)

2. Preparation and functionalization of gold nanoparticles (AuNPs)

(1) Preparation of AuNPs

Experiments glassware for synthesizing and storing nano materials is used for soaking in aqua regia (hydrochloric acid: nitric acid: 3:1) for 12h, and is used after being washed clean by ultrapure water.

The AuNPs are prepared by a sodium citrate reduction method. The method comprises the following specific steps:

1)100 mL of 0.01% HAuCl₄Adding the mixture into a 250mL conical flask, heating and stirring until the solution is boiled suddenly, and keeping the temperature for 1-2 min.

2) 2mL of a 1% trisodium citrate solution was quickly added to the flask and the heating and stirring were continued. Gradually changing the color of the solution from light yellow to dark purple and finally to wine red, keeping heating for 10min to prepare AuNPs with the particle size of 15nm, cooling to room temperature, and refrigerating at 4 ℃ for later use.

(2) AuNPs functionalization

1) mu.L of 100. mu.M polyA-DNA was added to 1mL of AuNPs (10nM) prepared in step (1) and mixed well, followed by addition of 20. mu.L of 500mM citric acid buffer (pH 3.0). After mixing well, incubate at room temperature for 3 min.

2) After completion of the incubation, 60 μ LpH 7.6.6 of 500mM HEPES buffer was added, the pH of the AuNPs solution was adjusted to neutral, and the incubation was performed at room temperature for 5-10 min.

3) And after incubation, centrifuging at 10000r/min for 20min, removing supernatant, adding a resuspension solution into the precipitate for redissolving, centrifuging at 10000r/min and 20min for three times to remove unreacted nucleic acid, finally adding 400 mu L of the resuspension solution to obtain functionalized AuNPs, namely the probe AuNPs @ polyA-DNA, and refrigerating at 4 ℃ for later use.

Respectively characterizing the AuNPs prepared in the step (1) and the AuNPs @ polyA-DNA prepared in the step (2) by using a transmission electron microscope, wherein the result is shown in figure 2, the prepared AuNPs with the particle size of 15nm have a single characteristic absorption peak at the position of 520nm, and when the AuNPs are combined with the polyA-DNA, the maximum absorption wavelength is shifted to 530nm, and the AuNPs are initially proved to be successfully modified by the polyA-DNA.

Composition of the resuspension: 20mM Na₃PO₄5% BSA, 10% sucrose, 0.25% Tween-20.

3. Preparation of streptavidin-biotin-DNAC

(1) mu.L of 2.5mg/mL streptavidin was mixed with 100. mu.L of 250. mu.M 5' -end biotin-labeled DNAC and incubated at 4 ℃ for 1h to obtain a mixture.

(2) The mixture was treated with an ultrafiltration tube (MWCO 30kDa), centrifuged three times at 6000r/min for 20min, resuspended in 300. mu.L of 10mM PBS to obtain streptavidin-biotin-DNAC, and stored at 4 ℃ for further use.

4. Assembly of aptamer test strip

(1) The sample pad and conjugate pad (gold-labeled pad) were cut to size, soaked in 10mM PBS for 30min, and then dried at 45 ℃.

(2) The probe AuNPs @ polyA-DNA prepared in the step 1 is evenly sprayed on a bonding pad and dried for 2 hours at 37 ℃.

(3) And (3) uniformly spraying streptavidin and the streptavidin-biotin-DNAC prepared in the step (2) on the NC membrane by using a three-dimensional spraying instrument at the speed of 0.9 mu L/cm, wherein the streptavidin and the streptavidin-biotin-DNAC are respectively used as a detection area (T line) and a control area (C line), the distance between the detection area and the control area is fixed at 5mm, and the streptavidin-biotin-DNAC are dried for 2 hours at the temperature of 37 ℃.

(4) And (3) sequentially adhering the sample pad, the bonding pad, the NC membrane and the water absorption pad prepared in the steps (1) to (3) to a PVC plate according to the figure 1, uniformly cutting the assembled test strip into strips with the width of 4mm, putting the strips into subpackage bags, and sealing and storing the strips.

Example two: test strip for testing kanamycin standard solution

(1) Preparation of kanamycin Standard solution

Kanamycin standard solution was diluted with Runningbuffer (4 x SSC, pH7) to final concentrations of 0.5, 5, 15, 25, 50, 150, 250 and 400ng/mL, respectively. Kanamycin aptamer having a nucleotide sequence shown in SEQ ID NO.1 was diluted to 0.5. mu.M with ultrapure water.

(2) Establishing a kanamycin nucleic acid test strip detection standard curve:

and (2) mixing and incubating 99 mu L of kanamycin standard solution with different concentrations and 1 mu L of kanamycin aptamer solution for 20min in the step (1), dropwise adding the mixed solution into a sample pad for detection after mixing reaction, measuring the relative signal intensity of (T/C) after reacting for 3min, and establishing a standard curve of the corresponding relation between the relative optical signal intensity of (T/C) and different kanamycin concentrations.

As shown in FIG. 3, when the concentration of kanamycin was 15ng/mL, the color of the T line on the test strip was significantly different from that of the T line on the test strip containing a kanamycin-free solution (kanamycin at 0 ng/mL); the color of the T line is reduced along with the increase of the kanamycin concentration when the kanamycin concentration is 5-250ng/mL, and the color of the T line is basically not changed when the kanamycin concentration is 250ng/mL, so the lower limit of visual detection is 15ng/mL, and the upper limit is 250 ng/mL.

The relative intensity of kanamycin with different concentrations is read by a colloidal gold test paper quantitative analyzer to obtain a change relation curve of (T/C) relative light signal intensity and kanamycin concentration shown in figure 4, and the lowest detection line is 0.3 ng/mL. Wherein in the concentration range of 5-250ng/mL, (T/C) has a linear relation between the intensity of the relative light signal and the concentration, and the linear regression equation is that R is-0.1637 x +0.4341²0.9819, where y is the (T/C) relative light signal intensity and x is the Log function of kanamycin concentration (ng/mL).

Example three: determination of OTA standard solution by test strip

(1) Preparation of OTA Standard solution

OTA standard solutions were diluted with Running buffer (4 XSSC, pH7) to final concentrations of 1, 10, 50, 100, 250 and 500ng/mL, respectively. The OTA aptamer having a nucleotide sequence shown in SEQ ID NO.1 was diluted to 0.5. mu.M with ultrapure water.

(2) Establishing an OTA nucleic acid test strip detection standard curve:

and (2) mixing and incubating 99 mu L of OTA standard solution with different concentrations and 1 mu L of OTA aptamer solution in the step (1) for 20min, dropwise adding the mixed solution into a sample pad for detection after mixing and reaction, measuring the relative signal intensity of the (T/C) after reaction for 3min, and establishing a standard curve of the corresponding relation between the relative optical signal intensity of the (T/C) and the different OTA concentrations.

The results are shown in FIG. 5, where the OTA concentration is 10ng/mL, the color of the T-line on the test strip can be significantly different from the color of the T-line on the test strip without the OTA solution (0ng/mL OTA); the color of the T line is weakened along with the increase of the concentration of the OTA when the concentration of the OTA is 1-250ng/mL, and the color of the T line is basically unchanged when the concentration of the OTA is 250ng/mL, so the lower limit of visual detection is 10ng/mL, and the upper limit is 250 ng/mL.

The relative intensities of OTAs with different concentrations were read by a quantitative analyzer using colloidal gold test paper to obtain a curve showing the relationship between the intensity of the relative optical signal and the concentration of OTA as shown in FIG. 6 (T/C), with the lowest detection line at 0.18 ng/mL. Wherein in the concentration range of 1-250ng/mL, the (T/C) relative light signal intensity has a linear relation with the concentration, the linear regression equation is that y is-0.151 x +0.4371, R²0.9794, where y is the (T/C) relative optical signal intensity and x is the Log function of the concentration of OTA (ng/mL).

Example four: detection of kanamycin residues in honey samples

The recovery rate is tested by using a honey simulation sample, and the steps are as follows:

(1) AuNPs @ polyA-DNA solution pretreatment: adding 0.8-1.4 mu L of AuNPs @ polyA-DNA prepared and stored to a gold label pad of a test strip, and storing at 4 ℃.

(2) Sample pretreatment: the honey sample was diluted 10 times and filtered through a 0.22 μm microfiltration membrane. Adding kanamycin (50, 150, 250ng/mL) with different concentrations into honey;

(3) determination of recovery rate of kanamycin in honey: taking 1 mu L of kanamycin aptamer (0.5 mu M) with the nucleotide sequence shown in SEQ ID NO.1 in example 1, mixing and incubating 99 mu L of honey solution containing different concentrations of kanamycin in step (2) for 20min, and detecting by using a test strip after mixing reaction.

The test paper strip for the aptamer prepared in the first example was used to detect the kanamycin content in milk, and the results are shown in table 4 and fig. 7.

TABLE 4 detection of kanamycin residues in honey samples

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> universal aptamer colloidal gold lateral chromatography test paper for detecting small molecular substances

<130> BAA210601A

<160> 13

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<212> DNA

<213> Artificial sequence

<400> 8

tgggggttga ggctaagccg a 21

<210> 9

<211> 51

<212> DNA

<213> Artificial sequence

<400> 9

aaaaaaaaaa aaaaattata ttattattat agagtgtcgg cttagcctca a 51

<210> 10

<211> 51

<212> DNA

<213> Artificial sequence

<400> 10

aaaaaaaaaa aaaaattata ttattattat agagtgtcgg cttagcctca a 51

<210> 11

<211> 51

<212> DNA

<213> Artificial sequence

<400> 11

aaaaaaaaaa aaaaattata ttattattat agagtgaaaa aaaaaatcgg c 51

<210> 12

<211> 26

<212> DNA

<213> Artificial sequence

<400> 12

tgggggttga ggctaagccg attttt 26

<210> 13

<211> 46

<212> DNA

<213> Artificial sequence

<400> 13

aaaaaaaaaa aaaaattata ttattattat agagtgaaaa atcggc 46