Electrochemical gene sensor based on Ti3C2atAuNP as well as preparation and application of electrochemical gene sensor

Abstract

The invention discloses an electrochemical gene sensor based on Ti3C2-coated AuNP as well as preparation and application of the electrochemical gene sensor, and belongs to the technical field of biosensing. According to the electrochemical DNA sensor based on Ti3C2 (at) AuNP, the Ti3C2 (at) AuNP is dispensed on the surface of a working electrode, then a hairpin HP-Sul2 probe marked by methylene blue and modified by sulfydryl is self-assembled on the surface of the electrode, and the ratio type electrochemical DNA sensor capable of detecting Sul2 antibiotic resistance genes is prepared. The sensor has the advantages of being simple in preparation process, low in cost, high in sensitivity, good in reproducibility and storage stability and the like, and a powerful tool is provided for sensitivity and specific detection of antibiotic resistance genes in the water environment.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors, and in particular relates to a DNA electrochemical sensor, a preparation method and an application thereof in detecting antibiotic resistance genes in a water environment. Background Art

[0002] The global spread of environmental biological pollutants, such as antibiotic-resistant bacteria and their antibiotic resistance genes (ARGs), has become a public health issue of great concern in recent years. ARGs in bacteria are ancient genes that existed long before the use of antibiotics. Bacteria can acquire ARGs through horizontal gene transfer via conjugation (cell-to-cell contact), transduction (phage infection), and transformation (direct uptake of extracellular DNA), ultimately causing difficult-to-cure resistant bacterial infections. Nowadays, with the overuse of antibiotics in aquaculture and health care, antibiotic resistance genes have been detected in various environments such as water, atmosphere, and soil. Among them, urban sewage treatment plants are important repositories of ARGs, and hundreds of resistance genes have been detected. As a key node in the water cycle, sewage from sewage treatment plants removes ARGs through ultraviolet irradiation and other methods, resulting in a large amount of cell-free DNA (including ARGs) flowing into natural water bodies, which again forms new antibiotic-resistant bacteria through horizontal gene transfer. Limited by the detection methods, most current studies only focus on traditional intracellular resistance genes (iARGs), and there is little research on extracellular resistance genes (eARGs). eARGs are produced by active secretion of cells or released after death. They can survive independently for a long time without being degraded, and re-enter the cell to express drug resistance under appropriate conditions. More and more studies have shown that eARGs are also widely present in the water environment, with equally severe or even more hidden health risks. Therefore, it is necessary to develop reliable analytical techniques to quickly and accurately quantify eARGs in the water environment.

[0003] At present, the analysis of eARGs in water environment is mainly based on molecular biology techniques such as PCR, qPCR, and metagenomic sequencing. Although these methods can provide accurate and complete information, they are very time-consuming and costly. In contrast, electrochemical biosensor technology can make up for the above shortcomings due to its fast response speed, high sensitivity, low cost, and the advantages of providing real-time data collection and multi-channel analysis in a single device. It can be used as an effective supplement to traditional detection methods and can meet the needs of rapid detection on site.

[0004] Isothermal strand displacement polymerization reaction (ISDPR) has been used as a signal amplification in biosensors due to its dispensable requirements for repeated thermal cycling, specific targets, strict temperature regulation, and complex sequence modeling, combined with the designed single-label dual-signal ratiometric sensing strategy to further improve the sensitivity and detection range.

[0005] As a two-dimensional material, MXene has been widely used in the field of biosensing due to its good conductivity, rich surface functional groups and excellent hydrophilicity. 3 C 2 @AuNP can significantly improve its conductivity and provide a better fixation for DNA probes than Ti synthesized by self-reduction method. 3 C 2 @AuNP composite materials have more active sites. At present, there is no Ti synthesized by seed growth method at home and abroad. 3 C 2 @Reports on the application of AuNP in electrochemical DNA sensors. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a simple, rapid and efficient electrochemical DNA sensor for detecting antibiotic resistance genes in water environments, as well as a preparation method and application thereof.

[0007] The object of the present invention is achieved by the following methods:

[0008] The present invention provides a two-dimensional single-layer Ti 3 C 2 @AuNP modified ratiometric electrochemical DNA sensor and its preparation and application, the two-dimensional monolayer Ti 3 C 2 @AuNP is synthesized by seed growth method and has high specific surface area and good conductivity. It not only provides more active sites for the fixation of hairpin probes, but also promotes the electron transfer between electrochemical indicators and electrode surfaces. Using ISDPR signal amplification strategy, KF exo - The enzyme catalyzes primer extension, releases the target Sul2 gene, triggers the next polymerization cycle, and enhances the change in the response signal of the electrochemical indicator marked on the probe; the second indicator is combined with the groove surface of DNA to introduce the second signal to construct a cheap dual-signal ratiometric sensing platform; the electrochemical DNA sensor designed by the present invention uses the sulfonamide antibiotic resistance gene Sul2, which is widely distributed in the water environment, as an eARGs model to achieve quantitative analysis of antibiotic resistance genes with high sensitivity and a wide detection range.

[0009] The present invention provides a two-dimensional Ti 3 C 2 The preparation method of the ratiometric electrochemical DNA sensor of @AuNP mainly includes the following steps:

[0010] (1) 0.1-1 mg / mL Ti 3 C 2 The nanosheet solution was added dropwise to 1 to 3 times the volume of 1 to 5 mg / mL polyallylamine hydrochloride solution, ultrasonically treated for 10 to 60 min, then stirred for 1 to 5 h, centrifuged, washed with water, and dispersed in water equal to the volume of the nanosheet solution to obtain Ti 3 C 2 -PAH solution;

[0011] (2) The Ti prepared in step (1) 3 C 2 -PAH solution was added dropwise to 2 to 10 times the volume of gold seed solution, ultrasonically treated for 10 to 40 minutes, stirred for 1 to 5 hours, centrifuged, washed with water, and added with Ti 3 C 2 -PAH solution was dispersed in water with an equal volume to obtain Ti 3 C 2 -Au seed solution;

[0012] (3) The Ti prepared in step (2) 3 C 2 -Au seed solution is added to 10-40 times the volume of gold growth solution, stirred for 1-3 hours, then the reducing agent solution is added dropwise, stirred for 0.5-3 hours, centrifuged, washed with water, and dispersed with water to obtain Ti 3 C 2 @AuNP solution;

[0013] (4) The Ti prepared in step (3) 3 C 2 @AuNP solution was added dropwise to the cleaned electrode surface and dried to obtain Ti 3 C 2 @AuNP-covered electrodes;

[0014] (5) modifying the electrochemical indicator-labeled and thiol-modified single-stranded DNA probe to the Ti prepared in step (4) 3 C 2 @AuNP modified electrode surface, single-stranded DNA probe modified Ti 3 C 2 @AuNP electrode;

[0015] (6) The Ti modified single-stranded DNA probe prepared in step (5)3 C 2 The @AuNP electrode is immersed in an aqueous solution containing a sealing agent and reacted for 0.5 to 2 hours to obtain an electrochemical DNA sensor.

[0016] Based on the above technical solution, further, the gold seed solution in step (2) is: 5-20 μL of 80% tetrakishydroxymethylammonium phosphate (THPC) and 0.1-1 mL of 1-3 M sodium hydroxide are added to 30-50 mL of ultrapure water, stirred evenly, and then 1-3 mL of 0.5-5% HAuCl is quickly added. 4 , stir at room temperature in the dark overnight.

[0017] Based on the above technical solution, further, the gold growth solution in step (3) is: 10-50 mg potassium carbonate is added to 100 mL containing 0.5-5 mL 0.5-5% HAuCl 4 The reducing agent solution is a formaldehyde solution with a concentration of 10-30%.

[0018] Based on the above technical solution, further, the electrode described in step (4) includes a glassy carbon electrode, a gold electrode, a paper electrode, a polymer electrode, Ti 3 C 2 @The concentration of AuNP solution is 0.1-2 mg / mL, and the drop volume is 1-10 μL.

[0019] Based on the above technical solution, further, the structure of the electrochemical indicator-labeled and thiol-modified single-stranded DNA probe in step (5) is as follows: 5'-electrochemical indicator-CAAGCGCCAGCAATCCCGACGCCGCGCCTGTTTCCTGGCGCTTG-(CH 2 ) 6 -SH; the electrochemical indicator includes but is not limited to methylene blue and ferrocene; the concentration of the diluted solution of the electrochemical indicator-labeled and thiol-modified single-stranded DNA probe is 0.1 to 10 μM, and the drop volume is 2 to 10 μL.

[0020] Based on the above technical solution, further, the blocking agent described in step (6) includes mercaptohexanol and mercaptoethanol, and the concentration of the blocking agent is 1 to 10 mM.

[0021] Another aspect of the present invention provides a two-dimensional Ti-based 3 C 2 Ratiometric electrochemical DNA sensor based on @AuNP.

[0022] Based on the above technical solution, further, the two-dimensional Ti 3 C2 Application of ratiometric electrochemical DNA sensor based on @AuNP for detection of antibiotic resistance genes in aquatic environments.

[0023] Based on the above technical solution, further, the antibiotic resistance gene includes sulfonamide, fluoroquinolone and tetracycline antibiotic resistance genes.

[0024] The present invention also provides the above-mentioned two-dimensional Ti 3 C 2 The detection method of @AuNP ratiometric electrochemical DNA sensor for detecting antibiotic resistance genes in water environment mainly includes the following steps:

[0025] (a) Place the target antibiotic resistance gene, primers, KF exo - , adding a polymerase buffer of dNTPs to the surface of the electrochemical DNA sensor electrode and incubating at 35-39° C. for 1-4 hours to obtain a sensor after amplification by reaction with the antibiotic resistance gene;

[0026] (b) immersing the electrochemical DNA sensor obtained in step (a) into a buffer solution containing another electrochemical indicator, performing differential pulse voltammetry scanning to record the current intensity values ​​of the two electrochemical indicators; wherein, when the electrochemical indicator marked on the single-stranded DNA probe is methylene blue, the other electrochemical indicator is ferrocene; when the electrochemical indicator marked on the single-stranded DNA probe is ferrocene, the other electrochemical indicator is methylene blue;

[0027] (c) establishing a standard curve using the ratio of the current value of the electrochemical indicator in the buffer to the current value of the electrochemical indicator labeled on the single-stranded DNA probe and the concentration of the target antibiotic resistance gene;

[0028] (d) testing the sample according to steps (a) to (c), substituting the test result into the standard curve, and then determining the content of the target antibiotic resistance gene in the sample.

[0029] The beneficial effects of the present invention compared with the prior art are as follows:

[0030] 1. Ti prepared by the seed growth method of the present invention 3 C 2 @AuNP has a very high specific surface area, which is beneficial to increase the loading capacity of DNA probes. 3 C 2 @AuNP's good conductivity can enhance the current response, thereby effectively improving the sensitivity of the sensor.

[0031] 2. The detection results of the DNA electrochemical sensor prepared by the present invention are reliable and accurate. The DNA sensor has better stability than protein (enzyme) sensors. The thermal stability of in vitro DNA is better than that of most protein (enzyme) molecules. The prepared sensor can be stored for a long time.

[0032] 3. The DNA electrochemical sensor prepared by the present invention has good specificity. The double strands of DNA molecules have very high specific recognition through specific base pairing. The change of a single base can cause a significant change in the electrical signal.

[0033] 4. The DNA electrochemical sensor prepared by the present invention is low-cost, simple to operate and has a fast reaction speed.

[0034] 5. The DNA electrochemical sensor prepared by the present invention has the advantages of high sensitivity, fast detection speed and low detection limit for the detection of antibiotic resistance genes in water environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0036] Figure 1 Ti prepared in Example 1 of the present invention 3 C 2 @Transmission electron microscopy (TEM) image of AuNP;

[0037] Figure 2 This is a schematic diagram of preparing a DNA electrochemical sensor according to Example 2 of the present invention;

[0038] Figure 3 is the logarithm of the concentration of the Sul2 resistance gene in Example 3 of the present invention and the peak current I of the DNA electrochemical sensor Fc / I MB Linear correlation graph of the ratio;

[0039] Figure 4 The specificity of the electrochemical DNA sensor in Example 4 of the present invention in detecting the Sul2 resistance gene;

[0040] Figure 5 The reproducibility of the electrochemical DNA sensor in detecting the Sul2 resistance gene in Example 4 of the present invention;

[0041] Figure 6 The stability of the Sul2 resistance gene detected by the electrochemical DNA sensor in Example 4 of the present invention. DETAILED DESCRIPTION

[0042] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0043] The following are the main instruments and equipment used in the examples of the present invention. Other experimental conditions not specifically noted are carried out according to conventional conditions or conditions recommended by the instrument manufacturer.

[0044] The instrument used for electrochemical detection was a portable electrochemical workstation PalmSens4, a three-electrode system; DNA sequences (including Sul2 gene fragments) were purchased from Sangon Biotech (Shanghai) Co., Ltd. The DNA sequences in the examples are shown in Table 1.

[0045] Table 1. Oligonucleotide base sequences

[0046]

[0047] Example 1Ti 3 C 2 Preparation of @AuNPs

[0048] 2 mL Ti 3 C 2 The nanosheets (1 mg / mL) were added dropwise to 4 mL of polyallylamine hydrochloride (PAH) solution (Mw = 15,000, 2 mg / mL), and after ultrasonic treatment for 30 minutes, the TiO2 was stirred for 4 hours to obtain a TiO2 solution. 3 C 2 -PAH solution, then centrifuged (14800 rpm, 5 minutes), washed with water 3 times, and then dispersed evenly with 2 mL of water. 3 C 2 -PAH solution was added dropwise to 10 mL of gold seed solution, ultrasonically treated for 30 minutes, stirred for 4 hours, centrifuged and purified twice, and dispersed evenly with 2 mL of water to obtain Ti 3 C 2 -Au seed solution. Finally, 1 mL Ti 3 C 2 -Au seed solution (1 mg / mL Ti 3 C 2 ) solution was added to 20 mL of the gold growth solution and stirred for 2 hours, followed by dropwise addition of reducing agent formaldehyde (HCHO, 29%), stirring for 1 hour, and repeated washing with water to obtain Ti 3 C 2 @AuNP composites.

[0049] Preparation of gold seed solution: 12 μL of 80% tetrakishydroxymethylammonium phosphate (THPC) and 0.25 mL of 2 M sodium hydroxide solution were added to 45 mL of ultrapure water. After stirring for 5 minutes, 2 mL of 1% HAuCl was quickly added. 4 , and stirred overnight at 25 °C in the dark.

[0050] Preparation of gold growth solution: Add 25 mg of potassium carbonate to 100 mL of 1% HAuCl 4 The solution was stored in ultrapure water for one day before use.

[0051] Example 2 Preparation of electrochemical DNA sensor

[0052] The preparation method of the DNA electrochemical biosensor of the present invention mainly comprises the following steps:

[0053] (1) Surface area is 0.02 cm 2 The gold electrode was placed in piranha solution (H 2 O 2 :H 2 SO 4 =1:3, v / v) for 15 minutes, then ultrasonically cleaned 3 times in anhydrous ethanol and deionized water for 3 minutes each time; polished with aluminum oxide powder with particle sizes of 1μm, 0.3μm, and 0.05μm, respectively, and ultrasonically cleaned 3 times in anhydrous ethanol and deionized water for 3 minutes each time; placed in 0.5M sulfuric acid solution and activated by cyclic scanning between 0 and 1.7V. When the scanning curves completely overlap, the activation is completed. After taking out the electrode, rinse it with deionized water and blow dry it with nitrogen;

[0054] (2) The Ti prepared in Example 1 3 C 2 @AuNP was prepared to a concentration of 1 mg / mL, ultrasonicated for 30 minutes, 4 μL was dripped onto the surface of the gold electrode, and dried naturally;

[0055] (3) diluting a methylene blue-labeled and thiol-modified single-stranded DNA probe HP-Sul2 solution (obtained by reacting a hairpin probe HP-Sul2 containing a disulfide bond between the chains with a reducing agent tri(2-carboxyethyl)phosphine at a molar ratio of 1:100) to 1 μM, dripping 8 μL onto the electrode surface obtained in step (2), reacting at 4°C for 16 hours, repeatedly rinsing with deionized water and 10 mM PBS buffer solution (PH 7.0), and drying with nitrogen gas to obtain HP-Sul2 / TiO2. 3 C 2 @AuNP / Au electrode.

[0056] (3) HP-Sul2 / Ti prepared in step (2) 3 C2 The AuNP / Au electrode was immersed in a 1 mM aqueous solution of mercaptohexanol (blocking agent) and reacted at room temperature for 1 hour to obtain an electrochemical DNA sensor.

[0057] Example 3 Electrochemical DNA sensor for detecting the Sul2 antibiotic resistance gene

[0058] The target DNA, primers (5 μL, 500 nM), KF exo - 50 μL of polymerase buffer (5 U) and dNTPs (2.5 μL) were added dropwise to HP-Sul2 / Ti 3 C 2 The surface of the AuNP / Au electrode was incubated at 37°C for 2.5 h, and then the electrode was washed three times with a large amount of ultrapure water, and then placed in a 10 mM PBS buffer solution containing 0.2 μg / mL ferrocene, and the differential pulse voltammetry curve was scanned between -0.5 and 0.5 V. Fc / I MB The relationship between the ratio and the logarithm of the Sul2 target gene concentration is as follows Figure 3 shown.

[0059] Example 4 Evaluation of specificity, reproducibility and storage stability of electrochemical DNA sensor

[0060] The specificity of the electrochemical DNA sensor of the present invention was studied by differential pulses, and the prepared electrochemical DNA sensor was exposed to PBS buffer solution (a), completely non-complementary genes (b), three-base mismatched gene TBM-Sul2 (c), single-base mismatched gene SBM-Sul2 (d) and completely complementary target gene sul2 (e). The results are shown in Figure 2. Figure 4 As shown, the significant change in the response signal of the sul2 gene indicated that the sensor had good specificity.

[0061] In order to evaluate the reproducibility of the electrochemical DNA sensor of the present invention between different electrodes of the same batch, 10 sensors were prepared independently under the same conditions, and 5 sensors each were used to detect the Sul2 resistance gene at concentrations of 10 pM and 100 fM, respectively. The results are shown in FIG. Figure 5 As shown, the results show that the RSDs are 1.83% and 2.24%, respectively, indicating that the electrodes prepared from different batches have good reproducibility.

[0062] In order to evaluate the storage stability of the DNA electrochemical biosensor of the present invention, the same electrode as the one used to detect 10 pM Sul2 resistance gene was stored at 4°C for 7 days and then the Sul2 resistance gene was detected. Figure 6 As shown, the I of the 5 electrodes Fc / I MBThe value (RSD=1.87%) changed by only 1.97% compared with the result one week ago, which proved that the DNA sensor developed by the present invention has good stability within the allowable error range.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a ratiometric electrochemical DNA sensor based on two-dimensional Ti3C2@AuNP, characterized in that: The main steps are as follows: (1) adding a 0.1-1 mg / mL Ti3C2 nanosheet solution dropwise into a 1-3 times volume of a 1-5 mg / mL polyallylamine hydrochloride solution, ultrasonically treating for 10-60 min, stirring for 1-5 h, centrifuging, washing with water, and adding water equal in volume to the nanosheet solution to obtain a Ti3C2-PAH solution; (2) adding the Ti3C2-PAH solution prepared in step (1) dropwise into 2 to 10 times the volume of the gold seed solution, ultrasonically treating for 10 to 40 minutes, stirring for 1 to 5 hours, centrifuging, washing with water, and adding water equal in volume to the Ti3C2-PAH solution to obtain a Ti3C2-Auseed solution; (3) adding the Ti3C2-Au seed solution prepared in step (2) to 10 to 40 times the volume of the gold growth solution, stirring for 1 to 3 hours, then dropping the reducing agent solution, stirring for 0.5 to 3 hours, centrifuging, washing with water, and dispersing with water to obtain a Ti3C2@AuNP solution; (4) adding the Ti3C2@AuNP solution prepared in step (3) dropwise to the cleaned electrode surface and drying to obtain a Ti3C2@AuNP-covered electrode; (5) modifying the single-stranded DNA probe labeled with an electrochemical indicator and modified with a thiol group onto the surface of the Ti3C2@AuNP modified electrode prepared in step (4) to obtain a Ti3C2@AuNP electrode modified with a single-stranded DNA probe; (6) Immersing the Ti3C2@AuNP electrode modified with the single-stranded DNA probe prepared in step (5) in an aqueous solution containing a blocking agent and reacting for 0.5 to 2 hours to obtain an electrochemical DNA sensor.

2. The preparation method according to claim 1, characterized in that: The gold seed solution in step (2) is: 5-20 μL of 80% tetrakishydroxymethylammonium phosphate (THPC) and 0.1-1 mL of 1-3 M sodium hydroxide are added to 30-50 mL of ultrapure water, stirred evenly, and then 1-3 mL of 0.5-5% HAuCl4 is quickly added, and stirred overnight at room temperature in the dark.

3. The preparation method according to claim 1, characterized in that: The gold growth solution in step (3) is: 10-50 mg potassium carbonate is added to 100 mL ultrapure water containing 0.5-5 mL 0.5-5% HAuCl4, and aged for 1 day to obtain the gold growth solution; the reducing agent solution is a formaldehyde solution with a concentration of 10-30%.

4. The preparation method according to claim 1, characterized in that: The electrodes described in step (4) include glassy carbon electrodes, gold electrodes, paper electrodes, and polymer electrodes. The concentration of the Ti3C2@AuNP solution is 0.1-2 mg / mL, and the drop volume is 1-10 μL.

5. The preparation method according to claim 1, characterized in that: The structure of the electrochemical indicator-labeled and thiol-modified single-stranded DNA probe in step (5) is as follows: 5'-electrochemical indicator-CAAGCGCCAGC AATCCCGACGCCGCGCCTGTTTCCTGGCGCTTG-(CH2)6-SH; the electrochemical indicator includes but is not limited to methylene blue and ferrocene; the concentration of the dilution solution of the electrochemical indicator-labeled and thiol-modified single-stranded DNA probe is 0.1 to 10 μM, and the drop volume is 2 to 10 μL.

6. The preparation method according to claim 1, characterized in that: The blocking agent described in step (6) includes mercaptohexanol and mercaptoethanol, and the concentration of the blocking agent is 1 to 10 mM.

7. A ratiometric electrochemical DNA sensor based on two-dimensional Ti3C2@AuNP prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the ratiometric electrochemical DNA sensor based on two-dimensional Ti3C2@AuNP as described in claim 7 in the detection of antibiotic resistance genes in water environment.

9. The use according to claim 8, characterized in that: The antibiotic resistance genes include, but are not limited to, sulfonamide, fluoroquinolone and tetracycline antibiotic resistance genes.

10. The method for detecting antibiotic resistance genes in water environment using a ratiometric electrochemical DNA sensor based on two-dimensional Ti3C2@AuNP as claimed in claim 7, characterized in that: The main steps are as follows: (a) Place the target antibiotic resistance gene, primers, KF exo - , adding a polymerase buffer of dNTPs to the surface of the electrochemical DNA sensor electrode and incubating at 35-39° C. for 1-4 hours to obtain a sensor after amplification by reaction with the antibiotic resistance gene; (b) immersing the electrochemical DNA sensor obtained in step (a) into a buffer solution containing another electrochemical indicator, performing differential pulse voltammetry scanning to record the current intensity values ​​of the two electrochemical indicators; wherein, when the electrochemical indicator marked on the single-stranded DNA probe is methylene blue, the other electrochemical indicator is ferrocene; when the electrochemical indicator marked on the single-stranded DNA probe is ferrocene, the other electrochemical indicator is methylene blue; (c) establishing a standard curve using the ratio of the current value of the electrochemical indicator in the buffer to the current value of the electrochemical indicator labeled on the single-stranded DNA probe and the concentration of the target antibiotic resistance gene; (d) testing the sample according to steps (a) to (c), substituting the test result into the standard curve, and then determining the content of the target antibiotic resistance gene in the sample.

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