CN112575064A

CN112575064A - Aac- (6') -Ib-cr gene detection kit

Info

Publication number: CN112575064A
Application number: CN202010859995.2A
Authority: CN
Inventors: 明德松; 王梦鹤
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-08-24
Filing date: 2020-08-24
Publication date: 2021-03-30

Abstract

The invention discloses an aac- (6') -Ib-cr gene detection kit, which comprises a capture probe and K⁺Exo III and ThT. The capture probe is a hairpin structure containing a G-quadruplex sequence, and under the condition that a target gene exists, the target gene and the hairpin probe can form a more stable double-stranded structure, so that a first sequence is revealed; exo III cleaves a second sequence in the hairpin probe that is complementary to the gene of interest to release the gene of interest and the first sequence, thereby amplifying the signal; the first sequence is at K⁺Can be folded into a G-quadruplex under the action of the action; after ThT is added, the G-quadruplex can enhance the fluorescent signal of ThT, can realize simple, quick, high-sensitivity and good-specificity detection on the aac- (6') -Ib-cr gene, and is expected to be widely applied to the detection of clinical drug-resistant genes.

Description

Aac- (6') -Ib-cr gene detection kit

Technical Field

The invention belongs to the technical field of drug-resistant gene detection, and particularly relates to an aac- (6') -Ib-cr gene detection kit.

Background

The aac- (6 ') -Ib-cr is a mutant of aac- (6') -Ib. Although aac- (6 ') -Ib mediate mainly resistance to aminoglycosides, aac- (6') -Ib-cr may mediate not only resistance to aminoglycosides but also resistance to quinolones. Especially, aac- (6') -Ib-cr can be transmitted not only by plasmid-mediated transmission but also by integration between chromosomes, thereby causing the drug resistance rate of bacteria to increase year by year. Therefore, the drug resistance gene brings great pressure to clinical treatment.

Clinically, methods such as PCR, electrophoresis, sequencing and the like are usually adopted for analyzing the drug-resistant genes, and the methods are relatively time-consuming and high in cost. Therefore, a simpler and more efficient nucleic acid detection method can be adopted. Nucleic acid detection is mainly focused on the use of DNA biosensors, and among such sensors, fluorescent sensors are widely used. Fluorescent sensors are well known for their advantages of high sensitivity, high specificity, simplicity and ease of operation. Fluorescence sensors mainly use labeled fluorescent probes, but they are often expensive. Therefore, there is a need to select a more economical and efficient fluorescent probe for detecting a target. In response to this requirement, a large number of dyes that fluoresce have been discovered. Among them, the DNA dyes which are used more commonly include Propidium Iodide (PI), 4', 6-diamidino-2-phenylindole (DAPI), etc., and the RNA dyes used include thiazole orange, acridine orange, etc. These are still not satisfactory.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides an aac- (6') -Ib-cr gene detection kit.

The technical scheme of the invention is as follows:

an aac- (6') -Ib-cr gene detection kit, which comprises a capture probe and K⁺Exo III and ThT; wherein the content of the first and second substances,

a capture probe having a first sequence at its 5 ' end and a second sequence at its 3 ' end that is fully complementary to the aac- (6 ') -Ib-cr gene, the first and second sequences being linked by a buffer sequence; the first sequence forms a partially interrupted complementary double strand with the second sequence such that the capture probe is in a hairpin structure and, when free, the first sequence is capable of forming a hairpin at K⁺Forming a G-quadruplex;

the reaction specifically comprises the following steps: the reaction is at a pH of 7.3 to 7.5, and when the presence of the aac- (6 ') -Ib-cr gene in the test sample is detected, the aac- (6') -Ib-cr gene competes for binding to the second sequence to form a double-stranded structure, the first sequence is detached from the 3 'end of the capture probe, the double-stranded structure is cleaved by Exo III to release the aac- (6') -Ib-cr gene, the buffer sequence prevents Exo III cleavage of the first sequence, the first sequence is free and is in K⁺Under the action of (a) to form a G-quadruplexUpon binding to ThT, fluorescence from ThT is enhanced.

In a preferred embodiment of the invention, the first sequence is as shown in SEQ ID NO. 01.

In a preferred embodiment of the invention, the second sequence is as shown in SEQ ID NO. 02.

In a preferred embodiment of the invention, the buffer sequence is shown in SEQ ID NO. 03.

In a preferred embodiment of the invention, the sequence of the capture probe is shown in SEQ ID NO. 04.

In a preferred embodiment of the invention, the temperature of the reaction is between 25 and 41 ℃.

In a preferred embodiment of the present invention, the reaction time is 10 to 40 min.

In a preferred embodiment of the present invention, the concentration of the ThT in the system of the reaction is 2 to 6. mu.M.

In a preferred embodiment of the present invention, the Exo III is used in an amount of 1 to 2.5U in 20. mu.L of the system of the reaction.

In a preferred embodiment of the present invention, the temperature of the reaction is 37 ℃ for 30min, the concentration of ThT in the system of the reaction is 5. mu.M, and the amount of Exo III in the system of 20. mu.L of the reaction is 2U.

The invention has the beneficial effects that:

1. in the invention, the capture probe of the hairpin structure containing the G-quadruplex sequence can form a more stable double-chain structure with the hairpin probe in the presence of the target gene, thereby revealing a first sequence; exo III cleaves a second sequence in the hairpin probe that is complementary to the gene of interest to release the gene of interest and the first sequence, thereby amplifying the signal; the first sequence is at K⁺Can be folded into a G-quadruplex under the action of the action; after ThT is added, the G-quadruplex can enhance the fluorescent signal of ThT, can realize simple, quick, high-sensitivity and good-specificity detection on the aac- (6') -Ib-cr gene, and is expected to be widely applied to the detection of clinical drug-resistant genes.

2. The kit of the invention presents good linear relation in the range of 1nM-200nM, and the detection limit can reach 0.69 nM.

3. The invention does not need other marks, reduces the detection cost and has high cost performance.

Drawings

FIG. 1 is an experimental schematic of the present invention.

Fig. 2 is a graph of experimental results of feasibility verification in example 1 of the present invention, in which a: HP; b: HP (200nM) + target (200 nM); c: HP + Exo III (200 nM); d: HP (200nM) + target (200nM) + Exo III (2U).

FIG. 3 is a diagram of agarose gel electrophoresis in example 1 of the present invention, wherein Lane 1: HP (3. mu.M); lane 2: HP (3. mu.M) + target (0.5. mu.M); lane 3: HP (3. mu.M) + Exo III (9U); lane 4: HP (3. mu.M) + target (0.5. mu.M) + Exo III (9U).

FIG. 4 is a graph showing the effect of different reaction temperatures on fluorescence intensity in example 1 of the present invention, wherein the reaction conditions are: HP (200nM), target (300nM), pH (7.4), reaction time 30min, Exo III (2U).

FIG. 5 is a graph showing the effect of different reaction times on fluorescence intensity in example 1 of the present invention, wherein the reaction conditions are: HP (200nM), Target (300nM), T (37 ℃), pH (7.4), Exo III (2U).

FIG. 6 is a graph showing the effect of the amounts of different exonucleases III on the fluorescence intensity in example 1 of the present invention, wherein the reaction conditions: HP (200nM), Target (300nM), T (37 ℃), pH (7.4).

FIG. 7 is a graph showing the effect of different thioflavin T concentrations on fluorescence intensity in example 1 of the present invention, wherein the reaction conditions are as follows: HP (200nM), Target (300nM), T (37 ℃), pH (7.4), Exo III (2U).

FIG. 8 is a graph showing the results of the quantitative analysis in example 1 of the present invention.

FIG. 9 is a graph showing the results of the experiment for examining the specificity in example 1 of the present invention.

Detailed Description

The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.

Example 1

1 materials of the experiment

1.1 Primary reagents and materials

1.2 oligonucleotide sequences

The oligonucleotide sequences used in this example were synthesized by Biotechnology engineering (Shanghai) Inc., as shown in Table 1 below:

TABLE 1 oligonucleotide sequences used in this example

The underlined stretch of nucleic acid sequence in the capture probe (HP, SEQ ID No.04) is a second sequence (SEQ ID No.02) which is complementary to the underlined stretch of nucleic acid sequence in the gene of interest (Target, SEQ ID No. 05); the bold nucleotide sequence is the first sequence Pu22(SEQ ID NO.01) at K⁺In the presence of a G-quadruplex which can form a two-dimensional structure, the first and second sequences are linked by a buffer sequence (AAAA, SEQ ID NO. 03). The nucleotide sequences of T1, T2 and T3(SEQ ID NO.06-08) are a single-base mutant sequence, a double-base mutant sequence and a triple-base mutant sequence, respectively, wherein underlined bases are mutation sites.

1.3 solution preparation:

(1)0.01mol/L TE (pH 8.0) buffer solution: 0.2422g Tris, 1.1688g NaCl and 0.0585g EDTA were weighed out accurately and dissolved in 200mL ultrapure water, mixed well and the pH was adjusted to 8.0 with 1M NaOH and HCl.

(2)0.01mol/LTris-HCl (pH 7.4) reaction buffer: 0.2422g Tris and 1.1688g NaCl were weighed out accurately and dissolved in 200mL ultrapure water, mixed well and the pH adjusted to 7.4 with 1M NaOH and HCl.

(3)3mol/L KCl: 44.73g of KCl was accurately weighed and dissolved in 200mL of Tris-HCl (pH 7.4) buffer.

(4) Preparation of 1m mol/L Thioflavin T (ThT): 0.0318g of thioflavine T is accurately weighed, dissolved in 100mL of ultrapure water, and contained in a brown glass bottle. After fully dissolving, storing at 4 ℃. The application method comprises diluting sulfur T (1: 20) with the above concentration.

(5)5 × TBE buffer: 54g Tris, 4.04g EDTA and 27.5g boric acid were accurately weighed out and dissolved in 1000mL ultrapure water, mixed well and the pH was adjusted to 7.4 with 1M NaOH and HCl. The following experiment was diluted with ultrapure water to 0.5 XTBE buffer and used.

(6) Use and dispensing of DNA probes: the cryovial filled with the purified oligonucleotide sequence lyophilized powder is centrifuged in a high-speed centrifuge (4000rpm, 4 ℃, 10min), and then TE buffer solution with corresponding volume is transferred into the cryovial according to the instructions to dissolve the oligonucleotide sequence to 100 mu M. The mother liquor is divided into a plurality of tubes according to the requirement and stored in a refrigerator at the temperature of minus 20 ℃. When in use, the mother liquor after subpackaging is taken out and diluted to the corresponding concentration by Tris-HCl (pH 7.4) buffer solution for use.

2 method of experiment

2.1 Experimental procedures

(1) The dispensed hairpin capture probe (HP) was diluted to 10. mu.M with Tris-HCl (pH 7.4) buffer, heated at 95 ℃ for 5min before use and slowly cooled to room temperature before being stored in a refrigerator at 4 ℃.

(2) HP (2. mu.L 10. mu.M), Tris-HCl (14. mu.L), 10 XNEB buffer 1 (2. mu.L) and target genes (2. mu.L) at various concentrations were added to a 20. mu.L reaction system, blown and mixed well, and reacted at 37 ℃ for 30 min. The reacted solution, KCl (2.5. mu.L, 3M), Tris-HCl (67.5. mu.L), ThT (10. mu.L, 50. mu.M) were added to a brown EP tube, and mixed by pipetting. Subsequently, the mixture was left standing at 4 ℃ for 15min in the dark.

(3) And (4) detecting the fluorescence intensity after the reaction is finished.

2.2 fluorescence detection of samples

The spectrofluorometer used in this experiment was an Agilent Carry Eclipse spectrofluorometer. The sample cell used for sample detection is a 100 mu L trace quartz cuvette. In the experiment, the voltage adopted by the fluorescence spectrophotometer is 570V, the excitation slit and the emission slit are both 10nm, the scanning speed is 1200nm/min, the excitation wavelength is 441nm, the emission waveband is 460-600nm for scanning, and the fluorescence value from 460nm to 600nm is recorded.

2.3 agarose gel electrophoresis

After 20. mu.L of the reaction solution was blown and mixed well, the mixture was reacted at 37 ℃ for 30min and then heated at 75 ℃ for 25 min. 5ul of each reaction solution and 1ul of Loading buffer are blown and uniformly mixed, and then the mixture is added into a pore channel corresponding to the prepared 3.5 percent agarose gel. The gel was run at a constant voltage of 110V for 75min and finally photographed with a gel imager.

2.4 simulation of sample processing

After the normal human serum (Solebao biotechnology) is diluted 1000 times, the target gene with corresponding concentration is added to prepare a simulation sample with corresponding concentration. Storing at 4 ℃.

A clinical bacterial infection sample is taken for culture, and a well-grown bacterial colony is taken to be prepared into suspension in physiological saline. Taking 1 XPBS to dissolve the suspension to 0.5 McLeod unit, adding the target gene with corresponding concentration to prepare a simulation sample with corresponding concentration. Storing at 4 ℃.

3 results of the experiment

3.1 principle of the experiment

As shown in FIG. 1, this example designs a hairpin-structured capture probe. The capture probe comprises three parts, wherein the 5 'end of the hairpin probe is provided with a nucleic acid sequence (Pu22), the nucleic acid sequence and a sequence which is completely complementary with the target gene at the 3' end of the hairpin probe form a partially discontinuous complementary double strand, and the binding force between the nucleic acid sequence and the sequence is relatively weak; the middle part next to Pu22 is provided with 4 adenine (A) rich nucleic acid sequences, and the sequences are buffer sequences which can prevent Exo III from shearing Pu22 and reduce steric hindrance; the 3' end is a sequence completely complementary to the target gene. When the target gene exists, the target gene competes to bind with the complete complementary sequence to form a double-stranded structure with stronger binding force and more stability, Pu22 competes by the target gene to be separated from the 3' end of the probe to be exposed, and the complementary sequence is cut by Exo III. After one round of shearing, the target gene can be released into the next round of shearingThereby amplifying the signal. Released Pu22 nucleic acid sequence at K⁺In the presence of a G-quadruplex. When ThT is added, the G-quadruplex binds to ThT and enhances its fluorescence intensity. This change in fluorescence intensity can be recorded by a fluorescence spectrophotometer. The method adopts the Exo III, G-quadruplex/ThT compound to detect the target gene, so that the detection method has simplicity and high sensitivity and specificity.

3.2 feasibility verification of the method

To verify the feasibility of this example for detecting aac- (6 ') -Ib-cr, a fluorescence spectrum was obtained upon quantification of aac- (6') -Ib-cr, with the results shown in FIG. 2. As shown, very weak signals were monitored in the absence of the gene of interest (curves a, c), indicating that Hp did not bind any single strand and that no G-quadruplexes were formed; after the target gene is added, the monitored signal intensity is higher than that without the target gene, because the target gene and the hairpin probe are complementarily paired, the hairpin probe is opened, the G-quadruplex sequence is exposed, and the G-quadruplex is folded to form a G-quadruplex which is combined with ThT to enhance the signal intensity (curve b); when the target gene is present together with Exo III, the signal detected (curve d) is much higher than when the target gene is present alone (curve c). From the fluorescence spectra, the signal intensity was significantly enhanced in the wavelength range of 460-600nm, with the peak occurring at 490 nm. Thus, the subsequent correlation measurements were taken at 490 nm. The results of the above comparative experiments show that EGTF fluorescence sensors based on the Exo III, G-quadruplex/ThT complex can be used to detect the aac- (6') -Ib-cr gene.

3.3 characterization of gel electrophoresis

This example demonstrates the feasibility of the fluorescence sensor by 3.5% agarose gel electrophoresis. As can be seen from the electropherogram of FIG. 3, lane1 is a hairpin probe. Lane 2 has two bands, the front band is the hairpin capture probe, the rear band is the dsDNA bound by the capture probe and the target gene pair. Lane 3 shows that the hairpin capture probe and Exo III are reacted at the same time, and the band corresponding to the hairpin capture probe is not changed significantly. Lane 4 shows the simultaneous reaction of the hairpin capture probe, the target gene, and Exo III. Wherein, the band corresponding to the capture probe of the hairpin structure disappears, which indicates that the capture probe of the hairpin structure is sheared; the front band is the ssDNA generated. The above comparative experiments show that hairpin probes can bind to the target gene after addition of the target gene, revealing Pu22, while Exo III cleaves complementary sequences to amplify the signal. Therefore, this example can be used to detect the drug-resistant gene aac- (6') -Ib-cr.

3.4 optimization of the conditions

In order to improve the sensitivity of the detection system, the present example was next optimized for 4 experimental conditions, such as the reaction temperature, the amount of exonuclease III, the reaction time, and the thioflavin T concentration.

3.4.1 optimization of the temperature of the reaction

The detection system of the invention is developed by means of base complementary pairing, the base complementary pairing has a temperature suitable for the base complementary pairing, and meanwhile, the exonuclease has an optimal enzyme cutting temperature. Therefore, it is necessary to examine the reaction temperature. Therefore, this example optimizes the reaction temperature of the detection system. In this example, the effect of different reaction temperatures on fluorescence intensity was analyzed, 4 ℃ was set for examining the effect of different reaction temperatures on detection at intervals, and different reaction temperatures of 25 ℃, 29 ℃, 33 ℃, 37 ℃ and 41 ℃ were selected for experiments. As is clear from FIG. 4, in the presence of the target DNA, the fluorescence intensity increased with the temperature increase from 25 ℃. The fluorescence signal is strongest at a temperature of 37 ℃. When the temperature is higher than 37 ℃ (41 ℃), the signal intensity slightly decreases. It can be seen that 37 ℃ is the optimal reaction temperature for the detection system. Therefore, in this example, 37 ℃ was selected as the optimal reaction temperature for detecting the target DNA.

3.4.2 optimization of reaction time

Because the DNA hybridization capacity is strong, the efficiency of the Exo III action is relatively quick, and if the reaction time is set to be too short, the shearing is incomplete; if the reaction time is set too long, excessive shearing occurs, and nonspecific adsorption increases and the detection time is prolonged. Likewise, Exo III requires appropriate reaction time to cleave hairpin probes in order to amplify the signal as much as possible. Therefore, it is important to select an appropriate reaction time to improve the performance of the detection system. Therefore, in the present embodiment, 10min is set as an interval to examine the influence of different reaction times on the detection effect, and 10min, 20min, 30min and 40min are respectively selected as different reaction times to be studied. As can be seen from FIG. 5, the signal intensity has reached a higher level since 10min, but the fluorescence intensity still increases in a small range as time increases. Until the reaction time reached 30min, the signal intensity reached a maximum. As the reaction time continued to increase, the signal intensity did not change more significantly. It can be seen that the reaction reached the optimum state after 30min of reaction. Therefore, 30min was selected as the optimal reaction time for detecting the target gene.

3.4.3 optimization of the amount of exonuclease III

The use of the amount of Exo III may have an effect on the magnitude of the fluorescence intensity. When the amount of use is too low, the shearing is limited, and thus the signal cannot be amplified to the maximum. When the concentration is too high, the background signal is high, and the target with lower concentration cannot be detected, so that the selection of proper amount of Exo III is very important for improving the performance of the detection system. Therefore, in this embodiment, 0.5U is set as an interval to examine the influence of different amounts of Exo III on the detection effect, and 1U, 1.5U, 2U and 2.5U are selected as different amounts of Exo III for research. As can be seen from FIG. 6, the signal intensity increased with the increase in the amount of enzyme from 1U. The signal intensity reached a maximum until the enzyme dosage reached 2U. When the enzyme dosage is continuously increased, the signal intensity is not obviously changed, namely, the reaction reaches the maximum state after the enzyme dosage reaches 2U. Therefore, 2U was selected as the optimum amount of exonuclease III reaction.

3.4.4 optimization of Thioflavin T concentration

The concentration of thioflavin T used may have an effect on the magnitude of the fluorescence intensity. When the concentration is too low, the difference in the generated signal is small, and the target gene cannot be detected to the maximum extent. When the concentration is too high, the background signal is higher, and the target with lower concentration cannot be detected, so that the selection of proper reaction concentration is very important for improving the performance of a detection system. Therefore, in this example, the effect of different thioflavin T concentrations on the detection effect was examined by setting 1. mu.M as the interval, and 2. mu.M, 3. mu.M, 4. mu.M, 5. mu.M and 6. mu.M were selected as different reaction concentrations for the study. As can be seen from FIG. 7, the signal intensity increased with increasing concentration from 2. mu.M. Until the thioflavin T concentration reached 5 μ M, the signal intensity reached a maximum. As the concentration continues to increase, the signal intensity does not change more significantly. From this, it was found that the reaction reached the optimum state after the thioflavin T concentration reached 5. mu.M. Therefore, 5. mu.M was selected as the optimum reaction concentration of thioflavin T.

3.5 quantitative analysis of the target DNA (aac (6') -Ib-cr)

Under the optimal experimental conditions, the quantitative analysis is carried out on the target genes (aac- (6') -Ib-cr) with different concentrations in the example, so as to detect the analytical performance of the EGTF fluorescent sensor. As can be seen from FIG. 8(A), different concentrations of the target produced different fluorescence signals. FIG. 8(B) is a graph showing the scatter plot between the fluorescence signal and the target gene. The signal intensity increases with the increase in the concentration of the target gene aac- (6') -Ib-cr, showing a linear correlation. This is because the hairpin probe is specifically recognized in the presence of the gene of interest, and the exposed Pu22 is at K⁺Folding into a two-dimensional structure in the presence to obtain a significantly enhanced fluorescence signal. FIG. 8(B) shows that the concentration of the target gene (aac- (6') -Ib-cr) was gradually increased from 1nM to 300nM, and the fluorescence intensity increased with the increase of the target gene. When the target gene is increased between 1nM and 200nM, the fluorescence intensity shows linear correlation, and when the concentration of the target gene is increased, the fluorescence intensity still increases, but the increase amplitude begins to decrease. In the range of 1nM to 200nM, the fluorescence intensity and the concentration of the target gene show good linear relation, and the linear fitting equation is that Y is 2.278X +37.34, wherein Y is the fluorescence signal intensity, X is the concentration (nM) of the target gene, and the linear correlation coefficient is R²0.9860, the limit of detection was 0.69nM according to the 3. sigma./s principle.

The performance of this protocol was analyzed by comparing the detection methodology presented in this example with the relevant DNA detection methodologies already established. As shown in Table 2, it was found that compared to the two-enzyme-assisted fluorescence sensor (Lee I J, Goo NI, Kim D. Label/queue-free detection of single-nucleotide changes in DNA using fluorescence amplification and G-quadraples [ J ]. analysis, 2016, 141 (24): 6503-.

And

zhang X F, Xu H M, Han L, Li N B, Luo H Q.A Thioflavin T-induced G-quadrupulex Fluorescent Biosensor for targetdnadetection [ J ]. Anal sci, 2018, 34 (2): 149-: 1088e1094.), this example has a better detection range and a lower detection limit. Therefore, the invention has great potential to be applied to the detection of the aac- (6') -Ib-cr gene.

TABLE 2 comparison with other methods for detecting DNA

3.6 investigation of specificity

In order to evaluate the specificity of the EGTF fluorescence sensor for detecting the aac- (6') -Ib-cr drug resistance gene, three nucleic acid sequences T1, T2 and T3, which are different from the target gene, were designed and the target gene and the three nucleic acid sequences were analyzed under the same conditions. As can be seen from FIG. 9, when the gene of interest is present in the reaction system, the fluorescence signal intensity can be significantly increased. Only one nucleic acid sequence of T1 was altered relative to the target gene, and its fluorescence signal intensity was 1/4 of the signal generated by the target gene. Since this study distinguishes aac- (6 ') -Ib-cr from aac- (6 ') -Ib based on a single base mutation at position 102 of the aac- (6 ') -Ib coding region, T1, which has been single base mutated on the basis of the gene of interest, was the main subject of investigation. As can be seen from the figure, the signal value of T1 is significantly different from that of the target gene, and therefore, the detection method can sufficiently distinguish aac- (6 ') -Ib-cr from aac- (6') -Ib in principle. The slightly increased intensity of the fluorescent signal corresponding to T2, modified by adding a single nucleic acid site to T1, is due to the fact that when two mutation sites are added, the binding of the gene of interest to the 5' end of the hairpin probe is reduced, the steric hindrance is reduced, and the partially opened hairpin probe is more likely to fold into a G-quadruplex and bind ThT. When the number of mutations is increased to 3, the signal is reduced on the basis of T2. Although the change in fluorescence values among T1, T2, and T3 was small, it was also large compared to the target gene. In conclusion, the invention has higher specificity.

4 simulated sample analysis

TABLE 3 serum spiking experiments

TABLE 4 bacteria liquid labeling experiment

In order to examine the practical use effect of the invention, the serum and the bacterial liquid are taken as the simulation samples, the operation is carried out under the optimal condition by using the standard addition method, and the signal generated by the target object is examined. The results are shown in tables 3 and 4. As can be seen from the table, the recovery rate of the target gene in the serum is between 99.50% and 105.21%, and the recovery rate in the bacterial liquid is between 101.62% and 108.13%. Also, the invention has better anti-interference capability. Therefore, the detection method established by the invention is expected to be clinically used.

Evaluation of economic analysis

TABLE 5 cost and Performance of two different solutions

TABLE 6 CEA and CUA comparison of two protocols

Since the variety of the types of drug-resistant gene sensors is examined, the criteria for evaluating the technical scheme also show the variety. In view of the specificity of clinical application, the technical scheme with lower cost and higher cost performance has more practical application value. Therefore, it is necessary to select a technical solution with higher cost performance. It is worth noting that the technical scheme adopted by the invention has higher advantage in cost performance. In this example, a double-enzyme-assisted fluorescence sensor for detecting single-nucleotide mutant DNA (comparative technical scheme: Lee I J, Goo N I, Kim D E. Label/query-free detection of single-nucleotide changes in DNA using isothermal amplification and G-quadruplexes [ J ] analysis, 2016, 141 (24): 6503-.

As can be seen from a combination of tables 5 and 6, the present invention is simpler and more convenient than the comparative technical solutions in terms of the steps. In terms of time, the present invention takes a short time. In terms of cost, the invention has lower cost. Compared with the comparison technical scheme, the invention has lower C/E and C/U values, namely the invention is far superior to the comparison technical scheme in the aspects of CEA and CUA. Therefore, the invention has higher cost performance from the point of view of economic analysis.

The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.

Sequence listing

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Claims

1. An aac- (6') -Ib-cr gene detection kit, characterized in that: comprising a capture probe, K⁺Exo III and ThT; wherein the content of the first and second substances,

the reaction specifically comprises the following steps: the reaction is at a pH of 7.3 to 7.5, and when the presence of the aac- (6 ') -Ib-cr gene in the test sample is detected, the aac- (6') -Ib-cr gene competes for binding to the second sequence to form a double-stranded structure, the first sequence is detached from the 3 'end of the capture probe, the double-stranded structure is cleaved by Exo III to release the aac- (6') -Ib-cr gene, the buffer sequence prevents Exo III cleavage of the first sequence, the first sequence is free and is in K⁺To form a G-quadruplex which, upon binding to ThT, enhances ThT fluorescence.

2. The kit for detecting the aac- (6') -Ib-cr gene according to claim 1, wherein: the first sequence is shown as SEQ ID NO. 01.

3. The kit for detecting the aac- (6') -Ib-cr gene according to claim 1, wherein: the second sequence is shown as SEQ ID NO. 02.

4. The kit for detecting the aac- (6') -Ib-cr gene according to claim 1, wherein: the buffer sequence is shown as SEQ ID NO. 03.

5. The kit for detecting the aac- (6') -Ib-cr gene according to claim 1, wherein: the sequence of the capture probe is shown as SEQ ID NO. 04.

6. The kit for detecting an aac- (6') -Ib-cr gene according to any one of claims 1 to 5, wherein: the temperature of the reaction is 25-41 ℃.

7. The kit for detecting an aac- (6') -Ib-cr gene according to any one of claims 1 to 5, wherein: the reaction time is 10-40 min.

8. The kit for detecting an aac- (6') -Ib-cr gene according to any one of claims 1 to 5, wherein: the concentration of the ThT in the system of the reaction is 2 to 6. mu.M.

9. The kit for detecting an aac- (6') -Ib-cr gene according to any one of claims 1 to 5, wherein: the amount of the Exo III in 20. mu.L of the reaction system is 1-2.5U.

10. The kit for detecting an aac- (6') -Ib-cr gene according to any one of claims 1 to 5, wherein: the reaction temperature was 37 ℃ and the reaction time was 30min, the concentration of ThT in the reaction system was 5. mu.M, and the amount of Exo III used in 20. mu.L of the reaction system was 2U.