CN111471747A - Drug-resistant bacterium detection method based on nucleic acid molecule machine colorimetric method - Google Patents
Drug-resistant bacterium detection method based on nucleic acid molecule machine colorimetric method Download PDFInfo
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Abstract
The invention discloses a drug-resistant bacterium detection method based on a colorimetric method of a nucleic acid molecule machine, which comprises the following steps: (1) construction of dual probes for bacterial specific response: mixing the sulfhydryl-modified double-Probe chain 1 and the double-Probe chain 2 with a nano material, adjusting the pH, standing and centrifuging to obtain double-Probe probes Probe1 and Probe2 with bacteria specificity response; (2) preparing a reaction solution: storing the dual-probe with bacteria specific response and the reaction mixture in a centrifuge tube to prepare reaction liquid; wherein the reaction mixture comprises double-stranded nucleic acid (RNA1-RNA2-Aptamer) and EXOIII; (3) and (3) detecting bacteria: and (3) mixing the bacterial liquid to be tested with the reaction liquid prepared in the step (2) according to a certain volume ratio, carrying out incubation reaction, measuring ultraviolet absorbance, and determining the type of bacteria. The method circularly amplifies signals at twice enzyme digestion rate, can more quickly identify common drug-resistant bacteria in hospitals, has the whole detection process of less than 4 hours, and has sensitivity and accuracy; has the advantages of good specificity, simple operation and macroscopic effect, and lays a foundation for the research and development of the bacteria rapid diagnosis kit.
Description
Technical Field
The invention relates to the technical field of detection, in particular to a drug-resistant bacterium detection method based on a colorimetric method of a nucleic acid molecule machine.
Background
Antibiotic resistance is an imminent global health-threatening problem, and since the british bacteriologist Alexander Fleming discovered penicillin in 1928 and penicillin was first applied to the clinic in 1940, the application of antibiotics has been 77 years to date, and antibiotics have made a great contribution to human health and medical progress, but the emergence of drug-resistant bacteria has made it possible to bring humans back to the "pre-antibiotic" dark age where no drugs are available for many infections. Based on the rise in drug resistance of 6 pathogenic bacteria, it was estimated that the number of deaths from AMR can increase to 1000 million people per year by 2050 unless action is taken, and the cumulative cost to the global economy by 2050 reaches $ 100 trillion. On this basis, by 2050, the number of deaths is amazing and one death occurs every three seconds, and the per-person treatment cost can reach ten thousand dollars.
Methicillin-resistant staphylococcus aureus is a multiple antibiotic resistant "superbacteria" (a class of staphylococcus aureus resistant to β -lactam antibiotics (e.g., penicillin, methicillin, dicloxacillin, nafcillin, penicillin , cephalosporin, and the like)), methicillin-resistant staphylococcus aureus (MRSA) causes a range of diseases, infection from skin to wounds, and sepsis and death when pneumonia and blood infections become severe.
Traditional methods for detecting drug-resistant bacteria include bacterial culture, PCR (polymerase chain reaction), gene sequencing, and the like. The bacteria culture method is an absorbance method for clinical bacteria detection, but the detection process is complicated and takes longer, the culture method takes as long as 24-72 hours (the method cannot be used for bacteria which are difficult to culture or grow slowly), and meanwhile, a drug resistance mechanism cannot be revealed, so that the method is not suitable for rapid detection of bacteria. PCR (polymerase chain reaction) and genome sequencing have high sensitivity and good specificity, can achieve trace-level detection, has great significance for detecting low-content bacteria, and has the advantages that the technology depends on a known drug-resistant gene reference sequence, so unknown drug resistance cannot be analyzed, and quantitative detection cannot be generally carried out, so that the technology can only be used as an auxiliary means in general. The conventional bacteria detection methods have certain limitations in the aspects of sensitivity, specificity, detection speed, consumption and high-throughput bacteria detection and analysis, so that the establishment of a simple, quick, sensitive and visual bacteria detection method is urgently needed to have important significance.
Disclosure of Invention
Aiming at the defects, the invention provides a drug-resistant bacterium detection method based on a colorimetric method of a nucleic acid molecular machine, namely a simple, rapid and visual drug-resistant bacterium detection method.
The invention provides a drug-resistant bacterium detection method based on a colorimetry of a nucleic acid molecule machine, which comprises the steps of mixing a bacterium solution to be detected and a double Probe (Probe1 and Probe2) according to a certain volume ratio, placing the mixture in a constant temperature box at 37 ℃ for incubation, wherein the double Probe in a reaction solution is placed in the double nucleic acid molecule machine (wherein the double nucleic acid molecule machine specifically refers to RNA1+ Probe1 and RNA2+ Probe2, the RNA moves on the surface of the Probe Probe, and EXII has the function of driving the RNA to move on the surface of the Probe, so that bases on the surface of the double Probe are gradually cut under the cyclic amplification action of enzyme digestion signals of the double nucleic acid molecule machine, and gold balls in the double Probe are agglomerated due to the loss of the protection action of ssDNA, so that the color of the solution is changed from red to grey, and different types of bacteria can be rapidly identified by measuring the change of ultraviolet absorbance of the solution to be detected. The method adopts a pH regulation and control method to prepare the double probes, has short time and high combination efficiency, and is obviously higher than the traditional salt aging method. In addition, the method provided by the invention can control the whole detection time within 4 hours, the sensitivity, the accuracy, the detection time and the like are obviously superior to those of the traditional detection method, the traditional pathogenic bacteria detection standard method is to firstly culture bacteria (at least 24 hours) and then carry out drug resistance test to determine which antibiotic the bacteria has drug resistance, and the method is time-consuming, labor-consuming and large in false positive signal.
In a specific embodiment, thiol-modified double-Probe chain 1 and double-Probe chain 2 are respectively modified on a gold ball with the wavelength of 13nm to prepare double probes (Probe1 and Probe2) with specific response of drug-resistant bacteria, wherein the double probes Probe1 and Probe2 are ssDNA. Adding the double probes and the reaction liquid into a bacterial liquid to be detected according to a certain molar ratio, then incubating at a constant temperature, wherein the Aptamer dissociates from the double-stranded nucleic acid (RNA1-RNA2-Aptamer) (the Aptamer dissociates from the double-stranded nucleic acid because of stronger affinity with bacteria) and is combined on the surface of the bacteria, the RNA1 and the RNA2 are combined with the complementary Probe1 and Probe2, and under the action of a double-nucleic acid molecular machine (RNA1+ Probe1 and RNA2+ Probe2), the RNA1 and the RNA2 are subjected to enzyme digestion reaction on the double probes to shear the nucleotides on the double probes, so that the gold nanoparticles are aggregated after losing the protection effect of the ssDNA, and color change is generated, thereby realizing the detection of bacterial specificity and visualization.
The invention relates to a drug-resistant bacterium detection method based on a nucleic acid molecule machine colorimetric method, which comprises the following steps:
(1) construction of Dual Probe for bacterial specific response
Mixing the sulfhydryl-modified double-Probe chain 1 and the double-Probe chain 2 with a nano material, adjusting pH, standing and centrifuging to obtain double-Probe probes 1 and Probe2 with bacterial specific response, and then measuring the concentration of the double probes for constructing (obtaining) the double-Probe probes 1 and Probe2 with bacterial specific response.
(2) Preparation of reaction solution
Storing the dual-probe with bacteria specific response and the reaction mixture in a centrifuge tube to prepare reaction liquid; wherein the reaction mixture comprises double-stranded nucleic acid (RNA1-RNA2-Aptamer) and EXOIII.
(3) Detecting bacteria
And (3) mixing the bacterial liquid to be tested with the reaction liquid prepared in the step (2) according to a certain volume ratio, carrying out incubation reaction, measuring ultraviolet absorbance, and determining the type of bacteria.
In the step (1), the nucleotide sequences of the dual probes Probe1 and Probe2 are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2:
the sequence of the double probe in step (1) of the present invention is not limited to SEQ ID NO.1 and SEQ ID NO.2, and other sequences may be used.
In the step (1), the nano material is selected from one or more of gold spheres (AuNPs), carbon nanotubes, graphene two-dimensional materials and the like; preferably gold spheres (AuNPs).
Wherein the particle size of the nano material is 10-18 nm; preferably 13 nm.
The nano material has good optical characteristics, higher electron density, good biocompatibility, good catalytic performance, surface plasma resonance, aggregation state sensitivity and the like. When the nanomaterial is gold spheres (AuNPs), a decrease in the distance between AuNPs (d > 3.5nm) results in inter-particle surface plasmon coupling, with a visible color change from red to grey at nanoscale concentrations.
In the step (1), the molar ratio of the double-probe chain 1 to the double-probe chain 2 to the nano material is (100-; preferably, it is 120: 1.
In the step (1), the mixing temperature is 22-27 ℃; preferably, it is 25 ℃.
In the step (1), the pH value is 2.5-3.5; preferably, it is pH 3.
In the step (1), the standing is preferably performed in a dark place.
Wherein the standing time is 12-18 min; preferably, it is 15 min.
In the step (1), the centrifugation rate is 9000-12000 r; preferably 10000 r.
In the step (1), the centrifugation time is 13-18 min; preferably, it is 15 min.
In the present invention, the double Probe strand 1 is a double Probe 1; double Probe strand 2, double Probe Probe 2.
In the step (1), the concentration of the double probe is determined for the purpose of: because the double probes are prepared by ssDNA and nano materials, the concentration of the double probes is different, and the color of the nano materials (such as gold spheres) is also different, the concentration of the double probes needs to be determined so as to select a proper concentration for subsequent colorimetric experiments.
In the step (2), the volume ratio of the double-stranded nucleic acid (RNA1-RNA2-Aptamer) to the EXOIII in the reaction mixture is (25-35) to (1-5); preferably, it is 30: 2.
In the step (2), the volume ratio of the dual probes specifically responding to the bacteria to the reaction mixture is (80-120) to 50; preferably, it is 100: 50.
In the step (2), the preparation process of the double-stranded nucleic acid (RNA1-RNA2-Aptamer) comprises the following steps: mixing the Aptamer specifically responding to bacteria with RNA1 and RNA2 partially complementary to the Aptamer, and then placing the mixture in a water bath kettle to heat to 90-98 ℃ to obtain double-stranded nucleic acid (RNA1-RNA 2-Aptamer);
wherein the nucleotide sequences of the RNA1 and the RNA2 are respectively shown as SEQ ID NO. 3-4:
wherein the molar ratio of the Aptamer to the RNA1 to the RNA2 is (2: 1) - (4: 1); preferably, it is 3: 1.
Wherein the molar ratio of the Aptamer to the RNA1 to the RNA2 is (2-4) to 1: 1.
Wherein the nucleotide sequence of the Aptamer is shown as SEQ ID NO. 5:
wherein the nucleotide sequence of the double-stranded nucleic acid (RNA1-RNA2-Aptamer) is shown as SEQ ID NO. 6:
wherein the heating temperature is preferably 95 ℃.
Wherein the heating time is 2-10 min; preferably, it is 5 min.
Wherein, the heating step is followed by a cooling step, preferably to room temperature.
In the step (2), the double-stranded nucleic acid (RNA1-RNA2-Aptamer) plays a role in inhibiting the enzyme digestion reaction so as to ensure that the enzyme digestion reaction is triggered only by adding bacteria.
The double-stranded nucleic acid of the invention can inhibit enzyme digestion reaction because the exoii selected by the invention can only cut from the 3 'blunt end of the double-stranded DNA or from the 3' sunken end, and can not cut RNA. Therefore, when the target bacterium is not added to the reaction solution, the exoii does not cleave the double-stranded nucleic acid, and thus does not trigger the cleavage reaction. Only after the target bacteria are added, the Aptamer is dissociated from the double strands and is bound to the surface of the bacteria, and the dissociated RNA1 and RNA2 are bound to the surfaces of Probe1 and Probe2 to form a nucleic acid molecule machine, and shearing of ssDNA on the surface of the Probe is started under the driving action of exonuclease.
In step (2), the exoii is capable of cleaving bases at the 3' end of the blunt-end double-stranded nucleic acid to release the bases into the solution, and the nanomaterials (e.g., gold nanoparticles) are aggregated after the protection of ssDNA is lost, thereby causing the color of the solution to change.
In the step (3), the bacteria is methicillin-resistant staphylococcus aureus (MRSA).
In the step (3), the volume ratio of the bacterial liquid to be detected to the reaction solution is 50: 80-120; preferably, it is 50: 100.
In the step (3), the temperature of the incubation reaction is 35-38 ℃; preferably, the temperature is 36-38 ℃; further preferably 37 deg.c.
In the step (3), the incubation reaction time is 1-3 h; preferably, it is 2 h.
The invention relates to a method for detecting drug-resistant bacteria based on a colorimetric method of a nucleic acid molecule machine, which is characterized by comprising the following steps:
the invention firstly designs a new signal amplification system of the double nucleic acid molecule machine, and secondly applies the double nucleic acid molecule machine to the pathogen detection based on the colorimetric method. The bright points of the detection method of the invention are high detection rate (less than 4 hours) and high sensitivity (single bacterium detection) (as shown in figure 3).
The steps essential to the realization of the drug-resistant bacterium detection method comprise:
firstly, preparing a dual probe for specific response of bacteria; secondly, preparing double-stranded nucleic acid; finally, the EXOIII was added to the reaction system. Then, the double probe, the double-stranded nucleic acid and the EXOIII are mixed according to a certain volume ratio to prepare a reaction mixture. And adding the bacterial liquid to be detected in the actual detection process.
Specifically, the invention relates to a drug-resistant bacterium detection method based on a nucleic acid molecule machine colorimetric method, which comprises the following steps:
(a) construction of Dual Probe for bacterial specific response
Mixing the double-Probe chain 1 and the double-Probe chain 2 (the 5' -end is modified with sulfydryl) modified with sulfydryl with a nano material, adding citrate to adjust the pH value, standing, centrifuging, cleaning to remove unreacted single-stranded DNA (the double-Probe chain 1 and the double-Probe chain 2), re-dispersing by using a phosphate buffer solution, cleaning to obtain double probes (Probe1 and Probe2) with bacteria specificity response, and finally measuring the concentration of the double probes by using an ultraviolet spectrophotometer.
(b) Preparation of reaction solution
(b-1) preparation of double-stranded nucleic acid (RNA1-RNA 2-Aptamer):
the Aptamer specifically responding to the bacteria and RNA1 and RNA2 complementary to the Aptamer are mixed, then placed in a water bath to be heated, and then cooled, so that the double-stranded nucleic acid (RNA1-RNA2-Aptamer) is obtained.
Preparing a reaction solution:
(b-2) mixing the dual probes specifically responding to the bacteria respectively obtained in the above steps with a double-stranded nucleic acid (RNA1-RNA2-Aptamer) and EXOIII according to a certain volume ratio, and controlling the final concentration of EXOIII at the initial addition.
(c) And (3) mixing the bacterial liquid to be detected with the reaction liquid prepared in the step (2) according to a certain volume ratio, placing the mixture in a thermostat for incubation for 2 hours, and finally measuring the absorbance value by using an ultraviolet spectrophotometer to determine the number of the types of bacteria.
In the step (a), the sulfhydryl-modified double-probe chain 1 and the double-probe chain 2 are fixed on the surface of the nano material, and the pH value of the solution is regulated and controlled by quickly adding citrate to increase the binding probability of the ssDNA and the surface of the nano material.
The method comprises the following main steps of quickly adding citrate to regulate the pH value of a solution: the PH was adjusted by rapidly adding citrate and adding an appropriate amount of hydrochloric acid to finally control the overall PH of the solution to 3. The repulsion force between the ssDNA and the surface of the nano material is smaller, so that the ssDNA is favorably combined on the surface of the nano material.
In the step (a), the ratio of the sum of the molar numbers of the double-probe chain 1 and the double-probe chain 2 to the molar number of the nano material is (100-; preferably, it is 150: 1. The molar ratio of the double-probe chain 1 to the double-probe chain 2 is 1: 1.
In the step (a), the nano material is selected from gold spheres (AuNPs), carbon nanotubes, graphene two-dimensional materials and the like; preferably gold spheres (AuNPs).
Wherein the diameter of the gold spheres (AuNPs) is 10-18 nm; preferably 13 nm.
In the step (a), the dark standing time is 12-18 min; preferably, it is 15 min.
In the step (a), the rotating speed of the centrifuge is 9000-12000 r; preferably 10000 r.
In the step (a), the centrifugation time is 13-18 min; preferably, it is 15 min.
In the step (a), the number of times of cleaning is 2-4 times; preferably 3 times.
In step (a), the pH is 2.5-3.5; preferably, it is 3.
In the step (a), the gene sequences of the double-probe chain 1 and the double-probe chain 2 are shown in SEQ ID NO. 1-2:
in one embodiment, the specific steps of connecting the thiol-modified double-probe strand 1 and the double-probe strand 2 to the surface of a 13nm gold ball are as follows: mu.l of thiol-modified double probe strand 1 and double probe strand 2 were mixed with 40. mu.l of AuNPs (60nM), added 4.88. mu.l of citrate (100mM, pH 3) and vortexed rapidly. The final concentration of citrate was adjusted to 10mM, and the pH of the solution was adjusted to 3. After leaving in the dark for 15min, the mixture was centrifuged at 10000r for ten minutes, the supernatant was discarded, and the mixture was washed three times with phosphate buffer (pH 7.4) to prepare double probes (Probe1 and Probe2) specifically responding to MRSA. Finally, redispersed with phosphate buffer and stored in a refrigerator at 4 ℃. Then a small amount of solution is taken and used for measuring the concentration of the double probes by an ultraviolet spectrophotometer.
In step (b), 5 thymines extend from the 3 'end of the Aptamer, so that the double-stranded nucleic acid (RNA1-RNA2-Aptamer) formed by base complementation with RNA1 and RNA2 is not blunt-ended, and the cutting action of the EXOIII on the double-stranded nucleic acid (RNA1-RNA2-Aptamer) can be avoided (the effect of the EXOIII is to cut only from the 3' end of the blunt-ended double-stranded nucleic acid DNA).
Wherein the gene sequences of the RNA1 and the RNA2 are shown as SEQ ID NO. 3-4:
in the step (b-1), the Aptamer, the RNA1 and the RNA2 are mixed according to a molar ratio of (2-4) to 1: 1; preferably, it is 3: 1.
In the step (b-1), the Aptamer gene sequence is shown in SEQ ID NO. 5:
in the step (b-1), the gene sequence of the (RNA1-RNA2-Aptamer) is shown as SEQ ID NO. 6:
in the step (b-2), the molar ratio of the dual probe specifically responding to the bacteria to the double-stranded nucleic acid (RNA1-RNA2-Aptamer) is 1: 100-; preferably, it is 1: 200.
In the step (b-1), the heating temperature is 90-98 ℃; preferably, it is 95 ℃.
In the step (b-1), the heating time is 2-10 min; preferably, it is 5 min.
In step (b-1), the cooling is preferably to room temperature.
In the step (c), the incubation temperature is 35-38 ℃; preferably, it is 37 ℃.
In the step (c), the incubation time is 1-3 h; preferably, it is 2 h.
In step (c), the EXOIII cleaves DNA strands starting from the 3' blunt end of a double-stranded nucleic acid (RNA1-RNA 2-Aptamer).
In step (c), the final reaction concentration of the exoii is 10-20U/L, preferably 15U/L, under the current system, the concentration of the exoii is saturated, and the excessive addition of the exoii enzyme causes unnecessary waste.
In the step (c), the preparation process of the bacterial liquid to be detected is as follows: separating bacteria from a sample in a super clean bench, placing the bacteria in a culture medium, culturing in a constant temperature shaking table, taking bacteria liquid, placing the bacteria liquid in a centrifugal machine for centrifugation, removing an upper layer culture solution, re-dispersing by using a phosphate buffer solution, and repeatedly cleaning to obtain the bacteria liquid to be detected.
Wherein the conditions of the constant temperature shaking table are 37 ℃ and 150 r.
Wherein the culturing time in the constant temperature shaking table is 12-48 h; preferably 24 h.
Wherein the rotating speed of the centrifuge is 3000-4500 r; preferably 3500 r.
Wherein the centrifugation time is 5-10 min; preferably, it is 6 min.
Among them, the phosphate buffer is preferably a buffer having a pH of 7.4.
Wherein the number of times of cleaning is 2-4 times; preferably 3 times.
In one embodiment, the bacterial liquid to be tested is prepared by separating bacteria MRSA from a sample in a super clean bench, placing the bacteria MRSA in 30ml of L B culture medium, performing shake culture at the constant temperature of 37 ℃ and the rotation speed of 150r for 24h, taking 1000 mu l of bacterial liquid by using a centrifugal tube, placing the bacterial liquid in a centrifugal machine at the rotation speed of 3500r, discarding the upper layer culture liquid, re-dispersing the bacterial liquid by using phosphate buffer, and repeatedly washing the bacterial liquid for three times to obtain the bacterial liquid to be tested.
In the present invention, RNA1 and RNA2 are complementary to Probe1 and Probe2, respectively, to form double-stranded nucleic acids, and blunt ends are formed at the 3' -ends of Probe1 and Probe 2.
In one embodiment, the reaction mixture solution is constructed as follows: the bacteria, the double probes specifically responding to the bacteria, the double-stranded nucleic acid (RNA1-RNA2-Aptamer) and the EXOIII are placed in a thermostat for incubation reaction, and an ultraviolet spectrophotometer is used for detecting a blank control group (phosphate buffer solution with the same volume is added to replace bacteria liquid) and the change of ultraviolet absorbance in a reaction mixed solution before and after the reaction.
Wherein the temperature of the constant temperature box is 36-38 ℃; preferably, it is 37 ℃.
Wherein the reaction time is 1-3 h; preferably, it is 2 h.
Sequences of the dual probes Probe1 and Probe2 specifically responding to bacteria are shown in SEQ ID NO. 1-2:
in the invention, an ultraviolet spectrophotometer is used for detecting the change of ultraviolet absorbance in a reaction mixed solution, and the method specifically comprises the following steps: 200. mu.l of the reaction solution was placed in a cuvette, and the ultraviolet absorbance was set at 520nm, and then the change in the ultraviolet absorbance intensity before and after the reaction of the solution to be measured was measured.
In the present invention, the target bacterium is MRSA.
The traditional drug-resistant bacteria identification method adopted in the prior art often needs bacterial culture and drug resistance test, although molecular diagnosis methods such as PCR, &ttttranslation = L "&tttL &/ltt/T &tttamp and the like have the characteristics of good specificity, simple operation, short time consumption and the like, however, if a sample matrix is adopted, the invention provides the detection of drug-resistant bacteria by using an enzyme digestion signal circulation amplification method and a colorimetric method for the first timeContains substances that inhibit the PCR reaction, the target DNA needs to be extracted and the method is sensitive to contamination and experimental conditions, furthermore, the prior art methods are not suitable for the detection of low concentrations of bacteria (< 100CFUm L)-1) And cannot be monitored in real-time (point-of-care).
The method can realize the ultra-sensitive and visual detection of the bacterial liquid to be detected under the action of a nucleic acid molecule machine after mixing the bacterial liquid to be detected with double probes (Probe1 and Probe2), double-stranded nucleic acid (RNA1-RNA2-Aptamer) and EXOIII according to a certain volume ratio and incubating at constant temperature. The whole reaction system can be placed in a 37 ℃ thermostat for reaction for 2 hours, the bases on the surfaces of the double probes are gradually cut off by the double probes in the reaction mixed solution under the enzyme digestion signal circulation amplification effect of the RNA walker, the gold spheres are agglomerated due to the loss of the protection effect of ssDNA, the color of the solution is changed from red to grey, common drug-resistant bacteria in hospitals can be identified more quickly by determining the change of the ultraviolet absorbance of the solution to be detected, and the whole detection process is less than 4 hours.
The invention also provides application of the detection method in detection of drug-resistant strains and quantity.
Wherein the drug-resistant bacterium is MRSA.
The method for detecting the drug-resistant bacteria based on the double-nucleic-acid-molecule machine can be applied to detection of MRSA (methicillin resistant Staphylococcus), carbapenemase-resistant Escherichia coli, vancomycin-resistant Pseudomonas aeruginosa and the like, and can also realize specific detection of other drug-resistant bacteria by selecting Aptamer which specifically responds to other bacteria. In the method, the variety of the drug-resistant bacteria is determined by detecting the change of the ultraviolet absorption value before and after the solution reaction, the sensitivity is high, and the pretreatment or pre-enrichment of the sample is not needed. The whole detection process is simple, the specificity is good, the sensitivity is high, the detection speed is high, the real-time detection can be realized, the whole detection process can be completed within 4h, the advantage of macroscopic observation is achieved, and meanwhile, the foundation is laid for the research and development of the bacteria rapid diagnosis kit.
Drawings
FIG. 1 is a schematic diagram of the detection method of the present invention.
FIG. 2 is a graph showing the comparison of absorbance between a control group and MRSA according to the detection method of the present invention using PBS, Pseudomonas aeruginosa (Paeru), Shigella flexneri (Shigella), and Escherichia coli (E.coli).
FIG. 3 is an ultraviolet absorbance diagram of MRSA detection according to the present invention, which shows that the method of the present invention can realize detection of MRSA of a series of concentration gradients, and can realize detection of a single bacterium at the lowest, with high sensitivity.
FIG. 4 shows the results of the method of the present invention applied to the detection of a simulated sample.
FIG. 5 shows absorbance maps corresponding to artificial cerebrospinal fluid (CSF), Urine (Urine), saliva (Spit), and human Serum (Serum).
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, except for the contents specifically mentioned below, and the present invention is not particularly limited to the contents. The following examples are intended only to further illustrate the invention and should not be construed as limiting the invention.
EXAMPLE 1 preparation of Dual Probe
In the traditional method, DNA modified by sulfydryl is connected on the surface of a gold ball, and a salt aging method is adopted, so that time of one to two days is usually needed, and time and labor are wasted. The invention adopts a novel preparation method of double probes, which comprises the following steps: citrate with certain concentration is directly added into a dual probe with bacteria specificity response, and the pH value in the solution is controlled to be 3, so that the electrostatic repulsion between AuNPs and SH-DNA is weakened, and the combination probability of SH-DNA and AuNPs is enhanced.
In this example, 4. mu.l of the thiol-modified probe strand was mixed with 40. mu.l of AuNPs (60nM) at a volume ratio of 1: 1, added with 4.88. mu.l of citrate (100mM, pH 3) and vortexed rapidly for 2 min. And to give a final citrate concentration of 10 mM. After 15min in the dark, the mixture was centrifuged at 10000r for ten minutes, and the supernatant was discarded and washed three times with PBS (pH 7.4). Finally redispersed with PBS and stored in a refrigerator at 4 ℃.
Experimental results and effects: characterization and concentration determination of the Dual Probe
The traditional salt aging method requires preparation time of one to two days, and the bonding efficiency is only 20%; the pH regulation method only needs 15min, and the binding efficiency approximately reaches 100% (generally, the binding efficiency is quantified by ultraviolet, the concentration ratio of DNA and gold spheres is compared to determine the binding efficiency, and the difference of the binding efficiency between the two methods is compared by gel electrophoresis). Therefore, compared with the traditional salt aging method, the method for regulating and controlling the pH by adding citrate not only enhances the combination probability of ssDNA and AuNPs, but also can realize rapid double-probe preparation, and is suitable for rapid detection of bacteria in actual life.
Example 2 specificity test
MRSA was used as an experimental group, PBS, Escherichia coli (E.coli), Shigella flexneri (Shigella) and Pseudomonas aeruginosa (Paeru) were used as a control group, 5 reaction mixtures each comprising 100. mu.l of a double probe (30nM), 30. mu.l of a double-stranded nucleus (RNA1-RNA2-Aptamer), 2. mu.l of EXOIII and 20. mu.l of 10 × buffer were taken, 50. mu.l of each of the bacterial suspensions (MRSA, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa) and 50. mu.l of PBS were added to the 5 reaction mixtures to prepare samples, respectively, and the bacterial concentration in all samples was controlled to 108CFU/ml (except PBS group). And (3) measuring the initial absorbance of the reaction mixed solution by using an ultraviolet spectrophotometer, placing the reaction mixed solution in a thermostat at 37 ℃ for incubation for 2h, and measuring the absorbance of the reaction mixture by using the ultraviolet spectrophotometer.
In order to verify the reliability of the detection method of the invention, the invention is proved by a series of specific experiments. For example, MRSA was used as a test group (test sample), and PBS, escherichia coli (e.coli), Shigella flexneri (Shigella), and pseudomonas aeruginosa (Paeru) were used as a control group. The bacterial concentration of all samples was controlled to 108CFU/ml。
As a result, as shown in FIG. 2, the absorbance changes hardly before and after the reaction in the control groups except the target MRSA to be measured. The spectrogram of FIG. 2 shows that the method of the present invention can realize specific detection of MRSA.
Example 3 detection of MRSA
Taking nine 100 mu l of double probe (30nM), sequentially adding 50 mu l of bacterial suspension with different concentration gradients, 30 mu l of double-stranded nucleic acid (RNA1-RNA2-Aptamer), 2 mu l of EXOIII and 20 mu l of 10 × buffer into the nine parts, and controlling the final concentration of the EXOIII in the reaction system to be 15U/L and the final concentration of the bacteria liquid diluted in the gradient to be 0, 1, 10 respectively2，103，104，105，106，107，and 108CFU/ml, and obtaining a reaction mixed solution.
After the initial absorbance of the reaction mixture solution was measured with an ultraviolet spectrophotometer, the reaction mixture solution was incubated in a 37 ℃ incubator for 2 hours, and the absorbance of the reaction mixture was measured with an ultraviolet spectrophotometer.
The principle of detecting MRSA by the method is shown in figure 1, the nucleotide on the surface of a double probe is gradually sheared off by a double-probe chain under the shearing action of EXOIII in a reaction system of the bacterial liquid, gold nanoparticles are aggregated after the protection action of ssDNA is lost, and the ultrasensitive MRSA detection can be realized by detecting the change of absorbance before and after the reaction. The colorimetric system based on the nucleic acid molecular machine can be used for rapid and high-sensitivity detection of pathogenic bacteria. Specifically, bacterial liquid to be detected, a double Probe, exonuclease and double-stranded nucleic acid are mixed according to a certain volume ratio, the mixture is placed in a 37 ℃ thermostat to react for 2 hours, at the moment, Aptamer on the double-stranded nucleic acid is combined to the surface of pathogenic bacteria, released RNA1 and RNA2 are respectively combined to the surfaces of Probe1 and Probe2 to trigger the motion of a nucleic acid molecule machine, the double Probe in a reaction solution gradually cuts off bases on the surface of the double Probe under the enzyme cutting signal circulation amplification action of RNA walker, and a gold ball is agglomerated due to the loss of the protection action of DNA, so that the color of the solution is changed from red to grey.
As shown in FIG. 3, ultraviolet absorption peaks of a series of bacteria with different concentration gradients at 520nm can be clearly distinguished, and the minimum number of detected bacteria is 1 CFU/L, namely, the method can realize single bacteria detection.
Example 4 manual simulated sample testing:
adding the suspension into artificial cerebrospinal fluid, urine, saliva, human serum and other samples as detection groups, and detecting according to step 2.
The invention adds the pre-cultured bacteria MRSA suspension into the artificial cerebrospinal fluid, urine, saliva and human serum according to a certain volume ratio after centrifuging, and controls the concentration of pathogenic bacteria to be 108CFU/ml, then adding other reaction mixtures in sequence, mixing, taking 50 mu l of the pathogen specific response double probe (30nM), 30ul of double-stranded nucleic acid (RNA1+ RNA2+ Aptamer), 2 mu l of EXOIII and 20 mu l of 10 × buffer, controlling the final concentration of the EXOIII in the reaction system to be 15U/L, measuring the initial ultraviolet absorption peak intensity of the reaction mixture by using an ultraviolet spectrophotometer, then placing the reaction mixture in a 37 ℃ incubator for incubation for 2h, and measuring the ultraviolet absorption peak value of the reaction mixture by using the ultraviolet spectrophotometer.
And (4) taking a sample without the bacterial suspension as a control group, and comparing detection results.
In actual detection, the environment of a sample to be detected is often complex, in order to prove the detection capability of the detection method in the complex environment, the invention further designs an artificial simulation sample, bacteria liquid and artificial cerebrospinal fluid, urine, saliva, human serum and the like are respectively mixed, then the detection is carried out by the method, and the detection result is shown in fig. 4 and fig. 5, which shows that the method can be effectively applied to the detection of the complex sample environment.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalent changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.
SEQUENCE LISTING
<110> university of east China
<120> drug-resistant bacterium detection method based on nucleic acid molecule machine colorimetry
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Claims (10)
1. A drug-resistant bacterium detection method based on a nucleic acid molecule machine colorimetry is characterized in that bacteria liquid to be detected and a double probe are mixed, incubation is carried out at constant temperature, basic groups on the surface of the double probe are gradually sheared under the enzyme digestion signal circulation amplification effect of RNA walker, gold balls in the double probe are agglomerated due to the fact that the protection effect of ssDNA is lost, the color of a reaction solution changes from red to grey, and drug-resistant bacterium detection of the bacteria liquid to be detected is achieved by measuring the change of ultraviolet absorbance of the reaction solution to be detected;
the method comprises the following steps:
(1) construction of Dual Probe for bacterial specific response
Mixing the sulfhydryl-modified double-probe chain 1 and the double-probe chain 2 with a nano material, adjusting the pH to 3, standing and centrifuging to obtain a double-probe with bacteria specificity response;
(2) preparation of reaction solution
Mixing the dual probes with bacteria specificity response with the reaction mixture to prepare reaction liquid; wherein the reaction mixture comprises double-stranded nucleic acid RNA1-RNA2-Aptamer and EXOIII;
(3) detecting bacteria
And (3) mixing the bacterial liquid to be tested with the reaction liquid prepared in the step (2) according to a certain volume ratio, carrying out incubation reaction, measuring ultraviolet absorbance, and determining the type and the quantity of bacteria.
2. The method for detecting drug-resistant bacteria according to claim 1, wherein in the step (1), the nucleotide sequences of the double probe strand 1 and the double probe strand 2 are respectively shown as SEQ ID No.1 and SEQ ID No. 2:
double probe chain 1: HS-TTTTTAAAGAAAGGGATTGCT (SEQ ID NO. 1);
the double-probe chain 2: HS-TTTTTACCCCGACTCGGTTAA (SEQ ID NO. 2).
3. The method for detecting drug-resistant bacteria according to claim 1, wherein in the step (2), the bacteria contained in the bacterial suspension to be tested is MRSA.
4. The method for detecting drug-resistant bacteria according to claim 1, wherein the incubation temperature is 35 to 38 ℃.
5. The method for detecting drug-resistant bacteria according to claim 1, wherein in the step (1), the pH is adjusted by one or more of citrate, phosphate and acetate; and/or, the pH is 3-4; and/or the nano material is selected from one or more of gold ball AuNPs, carbon nano tubes and graphene two-dimensional materials; and/or the molar ratio of the double-probe chain 1 and the double-probe chain 2 to the nano material is (100-; and/or the standing time is 12-18 min; and/or the rotating speed of the centrifuge is 9000-12000 r.
6. The method for detecting drug-resistant bacteria according to claim 1, wherein in the step (2), the volume ratio of the dual probe with bacteria-specific response to the reaction mixture is (80-120) to 50; and/or the volume ratio of the double-stranded nucleic acid RNA1-RNA2-Aptamer to the EXOIII is (25-35) to (1-5).
7. The method for detecting drug-resistant bacteria according to claim 1, wherein in the step (2), the double-stranded nucleic acid RNA1-RNA2-Aptamer is prepared by mixing the Aptamer specifically responding to bacteria with the RNA1 and RNA2 complementary to the Aptamer, and heating the mixture in a water bath to 90-98 ℃.
8. The method for detecting drug-resistant bacteria according to claim 7, wherein in the step (2), the gene sequences of the RNA1, the RNA2, the Aptamer, and the RNA1-RNA2-Aptamer are as follows:
RNA1：AGCAAUCCCUUUCUUU(SEQ ID NO.3)；
RNA2：UUAACCGAGUCGGGGU(SEQ ID NO.4)；
Aptamer：ACCCCGACTCGGTTAATACAAATAAAGGGATTGCTTTTTT(SEQ ID NO.5)RNA1-RNA2-Aptamer：
AGCAAUCCCUUUCUUUUUAACCGAGUCGGGGUACCCCGACTCGGTTAATACAAATAAAGGGATTGCTTTTTT(SEQ ID NO.6)。
9. the method for detecting drug-resistant bacteria according to claim 7, wherein in the step (2), the molar ratio of the Aptamer to the RNA1 to the RNA2 is (2-4) to 1: 1.
10. The method for detecting drug-resistant bacteria according to any one of claims 1 to 9, wherein the method is applied to detection of the types and the amounts of drug-resistant bacteria.
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