Rapid and visual pathogen detection method and portable detection device

Abstract

The invention belongs to the technical field of pathogenic bacteria detection, discloses a rapid and visual pathogenic bacteria detection method and a portable detection device, and particularly discloses a sensor for detecting pathogenic bacteria, and the sensor is a1) or a2); (a1) comprises bfDNA-metal nano particles, S1-CuO and S2-CuO; (a1, a2) comprises ssDNA-metal nanoparticles, S1-CuO and S2-CuO. According to the preparation method, bifunctional nucleic acid is modified on the surface of metal nanoparticles to obtain bfDNA-metal nanoparticles, S1-CuO and S2-CuO are sequentially connected with the bfDNA-metal nanoparticles through complementary base pairing, S1-CuO and S2-CuO can be sequentially connected through loop chain hybridization to form a polymer with a plurality of CuO nanoparticles, and a sensor for detecting pathogenic bacteria is obtained. The method can be used for effectively detecting pathogenic bacteria and can also be used for detecting the sensitivity of antibacterial drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of pathogen detection, and in particular relates to a rapid and visual pathogen detection method and a portable detection device. Background Art

[0002] Urinary tract infection (UTI) is one of the most common infectious diseases in the community. Approximately 150 million people are affected by UTIs each year worldwide, accounting for more than 15% of all antibiotic prescriptions in primary healthcare institutions. Previous studies have shown that only about 20% of urine samples from patients suspected of uremia test positive for infection, while the remaining 80% of patients do not actually require antimicrobial treatment. Currently, traditional methods for identifying pathogenic microorganisms and performing antimicrobial susceptibility testing (AST) rely on culture methods, which usually take several days. Given the risks of delayed treatment, doctors can only prescribe broad-spectrum antibiotics based on experience. Empirical antimicrobial treatment of these non-UTI patients accelerates antibiotic resistance, which is more serious in underdeveloped areas.

[0003] The traditional methods for UTI identification and AST are disk diffusion or broth microdilution. However, this method requires bacterial culture, which usually takes 24 to 72 hours. At present, various methods for identifying pathogens and AST have been developed. Pathogen identification mainly includes amplification technology based on specific nucleic acid sequences (polymerase chain reaction, PCR), nucleic acid sequence-based amplification (NASBA) and loop-mediated isothermal amplification (LAMP). AST analysis is mainly divided into two categories: genotypic methods and phenotypic methods. Among them, genotypic AST provides AST results by identifying whether there are drug-resistant genes in bacteria, such as hairpin structure magnetic surface enhanced Raman scattering (SERS) sensors, multiplexed photoelectrochemical bioanalysis, fluorescent biosensors, PCR and commercial T2Resistance TM panel17. However, genotypic testing is often inconsistent with the results of phenotypic AST, and samples require complex steps to extract DNA. Phenotypic AST assesses drug resistance by culturing bacteria in antibiotic culture medium, such as PCR and high-resolution melting, LAMP, electrochemiluminescence biosensors, biochemiluminescence sensors, deep learning analysis of SERS sensors, deep learning video of microscopes, capillaries, paper chips, and hydrogel sensors. However, the above methods all require specific algorithms or expensive equipment. Therefore, it is crucial to develop a simple, rapid, easy-to-use, and low-cost technology to distinguish UTI from non-UTI and perform AST. Summary of the Invention

[0004] The first aspect of the present invention aims to provide a sensor for detecting pathogens.

[0005] The purpose of the second aspect of the present invention is to provide an application of the sensor of the first aspect of the present invention.

[0006] The third aspect of the present invention is to provide a kit.

[0007] The fourth aspect of the present invention aims to provide a method for detecting pathogens.

[0008] The fifth aspect of the present invention aims to provide a method for detecting antimicrobial drug sensitivity.

[0009] The sixth aspect of the present invention aims to provide a portable detection device.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] A first aspect of the present invention provides a sensor for detecting pathogens, wherein the sensor is a1) or a2);

[0012] a1) includes bfDNA (bifunctional deoxyribonucleic acid)-metal nanoparticles, S1-CuO and S2-CuO; a2) includes ssDNA-metal nanoparticles, S1-CuO and S2-CuO; wherein, the bfDNA-metal nanoparticles are obtained by coupling bfDNA and metal nanoparticles; the ssDNA-metal nanoparticles are obtained by coupling ssDNA and metal nanoparticles; the S1-CuO and the S2-CuO are obtained by coupling S1 or S2 with CuO respectively; the nucleotide sequence of the bfDNA includes A1, A2 and A3, and the A3 specifically targets and binds to the 16S rRNA / 18S rRNA; the nucleotide sequence of the S1 includes B1 and B2, and the B2 is reverse complementary to the A2 portion; the nucleotide sequence of the S2 includes C1 and C2, the C1 is reverse complementary to the B1, and the C2 is reverse complementary to B2; the nucleotide sequence of the ssDNA includes D1 and D2, and the D2 is identical to the A2 portion.

[0013] In some embodiments of the present invention, A1 in the nucleotide sequence of the bfDNA is a random sequence.

[0014] In some embodiments of the present invention, the S1-CuO is connected through complementary base pairing between S1 and the bfDNA in the bfDNA-metal nanoparticles, and the S2-CuO is connected through complementary base pairing between S2 and S1 in S1-CuO.

[0015] In some embodiments of the present invention, the bfDNA-metal nanoparticles, S1-CuO, S2-CuO and ssDNA-metal nanoparticles are synthesized by a high temperature drying method.

[0016] In some embodiments of the present invention, the preparation process of the bfDNA-metal nanoparticles includes the following steps: mixing bfDNA with metal nanoparticles, heating on a heating device at 70-90°C to evaporate excess water, thereby obtaining bfDNA-metal nanoparticles.

[0017] In some embodiments of the present invention, the mixed system contains bfDNA with a final concentration of 0.9 to 1.1 μM and 0.5 to 2 mg mL -1 of metal nanoparticles.

[0018] In some embodiments of the present invention, the preparation process of the S1-CuO includes the following steps: mixing S1, CuO and a solvent (water), heating on a heating device at 70 to 90° C. to evaporate excess water, thereby obtaining S1-CuO.

[0019] In some embodiments of the present invention, the mixed system contains S1 with a final concentration of 0.9-1.1 μM and 0.5-2 mg mL -1 of CuO.

[0020] In some embodiments of the present invention, the preparation process of S2-CuO includes the following steps: mixing S2, CuO and solvent (water), heating on a heating device at 70-90°C to evaporate excess water, thereby obtaining S1-CuO.

[0021] In some embodiments of the present invention, the mixed system contains S2 with a final concentration of 0.9 to 1.1 μM and 0.5 to 2 mg mL -1 of CuO.

[0022] In some embodiments of the present invention, the preparation process of the ssDNA metal nanoparticles includes the following steps: mixing ssDNA with metal nanoparticles, heating on a heating device at 70-90° C. to evaporate excess water, thereby obtaining bfDNA-metal nanoparticles.

[0023] In some embodiments of the present invention, the mixed system contains ssDNA with a final concentration of 0.9 to 1.1 μM and 0.5 to 2 mg mL -1 of metal nanoparticles.

[0024] In some embodiments of the present invention, the S1-CuO is connected through complementary base pairing between S1 and the ssDNA in the ssDNA-metal nanoparticles, and the S2-CuO is connected through complementary base pairing between S2 and S1 in the S1-CuO.

[0025] In some embodiments of the present invention, the preparation method of the sensor described in a1) includes the following steps: bfDNA-metal nanoparticles, S1-CuO and S2-CuO are respectively dissolved in 0.5×SSC solution to obtain bfDNA-metal nanoparticle solution, S1-CuO solution and S2-CuO solution, respectively; the bfDNA-metal nanoparticle solution and S1-CuO solution are mixed, reacted at room temperature for 0.5 to 1.5 hours, S2-CuO solution is added, and the mixture is reacted for 0.5 to 1.5 hours to obtain the sensor.

[0026] In some embodiments of the present invention, the method for preparing the sensor described in a2) comprises the following steps: dissolving ssDNA-metal nanoparticles, S1-CuO, and S2-CuO in 0.5×SSC solution to obtain ssDNA-metal nanoparticle solution, S1-CuO solution, and S2-CuO solution, respectively; mixing the ssDNA-metal nanoparticle solution and the S1-CuO solution, reacting at room temperature for 0.5 to 1.5 hours, adding the S2-CuO solution, and mixing and reacting for 0.5 to 1.5 hours to obtain the sensor.

[0027] In some embodiments of the present invention, the bfDNA is A1, A2 and A3 from the 5' end to the 3' end.

[0028] In some embodiments of the present invention, the random sequence does not contain more than 6 consecutive bases that are complementary and / or reverse complementary to the bfDNA, S1, and S2 sequences.

[0029] In some embodiments of the present invention, the nucleotide sequence of the A1 random sequence is 5'-CCGGATCG-3'.

[0030] In some embodiments of the present invention, the bfDNA includes a 5' end modification.

[0031] In some embodiments of the present invention, the 5' end modification includes at least one of thiol modification, phosphorylation modification, phosphorylation analog modification, amino modification, dideoxy modification, spacer modification, 3'-O-propylene modification or 3'-O-azidomethyl modification; preferably thiol modification.

[0032] In some embodiments of the present invention, the 3' ends of S1 and S2 contain tail sequences.

[0033] In some embodiments of the present invention, the nucleotide sequence of the tail sequence is 5'-TTTTTT-3'.

[0034] In some embodiments of the present invention, the S1 includes a 5' terminal modification; the 5' terminal modification includes at least one of thiol modification, phosphorylation modification, phosphorylation analog modification, amino modification, dideoxy modification, spacer modification, 3'-O-propylene modification or 3'-O-azidomethyl modification; preferably thiol modification.

[0035] In some embodiments of the present invention, the S2 includes a 3' terminal modification; the 3' terminal modification includes at least one of thiol modification, phosphorylation modification, phosphorylation analog modification, amino modification, dideoxy modification, spacer modification, 3'-O-propylene modification or 3'-O-azidomethyl modification; preferably, thiol modification.

[0036] In some embodiments of the present invention, the nucleotide sequence of the bfDNA includes at least one of SEQ ID NO: 1 to SEQ ID NO: 27.

[0037] In some embodiments of the present invention, the nucleotide sequence of S1 is shown in SEQ ID NO: 28.

[0038] In some embodiments of the present invention, the nucleotide sequence of S2 is shown in SEQ ID NO: 29.

[0039] In some embodiments of the present invention, D1 in the nucleotide sequence of the ssDNA is a random sequence.

[0040] In some embodiments of the present invention, the nucleotide sequence of the ssDNA is shown in SEQ ID NO: 32.

[0041] In some embodiments of the present invention, the metal nanoparticles are magnetic nanoparticles.

[0042] In some embodiments of the present invention, the metal nanoparticles include at least one of Au@Fe3O4 and polystyrene microspheres.

[0043] In some embodiments of the present invention, the pathogenic bacteria include at least one of Gram-positive bacteria, Gram-negative bacteria and fungi.

[0044] In some embodiments of the present invention, the Gram-positive bacteria include at least one of Staphylococcus, Enterococcus faecalis, Enterococcus faecium, Pneumococcus, Bacillus anthracis, Corynebacterium diphtheriae, Listeria monocytogenes, Vibrio parahaemolyticus, and Clostridium tetani.

[0045] In some embodiments of the present invention, the Gram-negative bacteria include at least one of Klebsiella pneumoniae, Pseudomonas aeruginosa, Shigella dysenteriae, Salmonella typhi, Escherichia coli, Proteus, Bordetella pertussis, Salmonella, and Vibrio cholerae.

[0046] In some embodiments of the present invention, the fungus includes at least one of Candida albicans, Leptotrichia vaginalis, and Cryptococcus.

[0047] In some embodiments of the present invention, when the pathogen is a Gram-negative bacterium, the nucleotide sequence of the bfDNA is SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0048] In some embodiments of the present invention, when the pathogen is a Gram-positive bacterium, the nucleotide sequence of the bfDNA is SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0049] In some embodiments of the present invention, when the pathogen is a fungus, the nucleotide sequence of the bfDNA is SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

[0050] In some embodiments of the present invention, when the pathogen is E. coli, the nucleotide sequence of the bfDNA is SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0051] In some embodiments of the present invention, when the pathogen is K. pneumoniae, the nucleotide sequence of the bfDNA is SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

[0052] In some embodiments of the present invention, when the pathogen is P. aeruginosa, the nucleotide sequence of the bfDNA is SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.

[0053] In some embodiments of the present invention, when the pathogen is S. aureus, the nucleotide sequence of the bfDNA is SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21.

[0054] In some embodiments of the present invention, when the pathogen is E. faecalis, the nucleotide sequence of the bfDNA is SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24.

[0055] In some embodiments of the present invention, when the pathogen is E. faecium, the nucleotide sequence of the bfDNA is SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27.

[0056] The second aspect of the present invention provides the use of the sensor in any one of (1) to (6):

[0057] (1) Detection of pathogens; (2) Preparation of products for detection of pathogens; (3) Identification of pathogenic bacteria; (4) Preparation of products for identification of pathogenic bacteria; (5) Antimicrobial susceptibility testing; (6) Preparation of products for antimicrobial susceptibility testing.

[0058] In some embodiments of the present invention, the product includes but is not limited to reagents and kits.

[0059] In some embodiments of the present invention, the pathogenic bacteria include at least one of Gram-positive bacteria, Gram-negative bacteria and fungi.

[0060] In some embodiments of the present invention, the Gram-positive bacteria include at least one of Staphylococcus, Enterococcus faecalis, Enterococcus faecium, Pneumococcus, Bacillus anthracis, Corynebacterium diphtheriae, Listeria monocytogenes, Vibrio parahaemolyticus, and Clostridium tetani.

[0061] In some embodiments of the present invention, the Gram-negative bacteria include at least one of Klebsiella pneumoniae, Pseudomonas aeruginosa, Shigella dysenteriae, Salmonella typhi, Escherichia coli, Proteus, Bordetella pertussis, Salmonella, and Vibrio cholerae.

[0062] In some embodiments of the present invention, the fungus includes at least one of Candida albicans, Leptotrichia vaginalis, and Cryptococcus.

[0063] The third aspect of the present invention provides a kit comprising the sensor according to the first aspect of the present invention.

[0064] In some embodiments of the present invention, when the sensor is a1), the kit further comprises exonuclease, DNA-AuNPs whose 3' end is modified with C≡C-, DNA-AuNPs whose 3' end is modified with N3-, and a reducing agent.

[0065] In some embodiments of the present invention, the exonuclease comprises at least one of 5' phosphate-dependent exonuclease, Saccharomyces cerevisiae XrnI exoribonuclease, Exo-I, Exo-II or Exo-III, preferably Exo-III.

[0066] In some embodiments of the present invention, the reducing agent includes at least one of ascorbic acid, sodium borohydride, vitamin E, iron powder, and zinc powder.

[0067] In some embodiments of the present invention, the kit further comprises an acidic solvent, such as hydrochloric acid, phosphoric acid, sulfuric acid, etc.

[0068] In some embodiments of the present invention, the nucleotide sequence of the DNA in the DNA-AuNPs with the 3' end modified with C≡C- is as shown in SEQ ID NO: 30.

[0069] In some embodiments of the present invention, the nucleotide sequence of the DNA in the DNA-AuNPs modified with N3- at the 3' end is as shown in SEQ ID NO: 31.

[0070] In some embodiments of the present invention, the preparation method of the DNA-AuNPs modified with C≡C- at the 3' end comprises the following steps: mixing the DNA modified with C≡C- at the 3' end (denoted as C≡C-DNA) with AuNPs and sodium citrate solution, and incubating at room temperature for 2 to 5 minutes to obtain the DNA-AuNPs modified with C≡C- at the 3' end (denoted as C≡C-DNA@AuNPs).

[0071] In some embodiments of the present invention, the molar ratio of the C≡C-DNA to AuNPs is 180-250:1, preferably 210-240:1.

[0072] In some embodiments of the present invention, the method for preparing DNA-AuNPs with N3 modified at the 3' end comprises the following steps: mixing DNA with N3 modified at the 3' end (denoted as N3-DNA) with AuNPs and sodium citrate solution, and incubating at room temperature for 2 to 5 minutes to obtain DNA-AuNPs with N3 modified at the 3' end (denoted as N3-DNA@AuNPs).

[0073] In some embodiments of the present invention, the molar ratio of N3-DNA to AuNPs is 180-250:1, preferably 210-240:1.

[0074] In some embodiments of the present invention, when the sensor is a2), the kit further comprises Cas protein, crRNA, cDNA, ds DNA, H2O2 and tetramethylbenzidine (TMB).

[0075] In some embodiments of the present invention, the kit further comprises an acidic solvent, such as hydrochloric acid, phosphoric acid, acetic acid, and the like.

[0076] In some embodiments of the present invention, the kit further comprises sodium acetate buffer.

[0077] In some embodiments of the invention, the Cas protein is selected from Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d and Cas14, including any recombinant variants thereof, and in particular selected from Cas9, including any recombinant variants thereof.

[0078] In some embodiments of the present invention, the crRNA includes an anchor sequence and a guide sequence, the anchor sequence specifically binds to the Cas protein, and the guide sequence targets ds DNA.

[0079] In some embodiments of the invention, the cDNA is at least partially reverse complementary to the ds DNA.

[0080] In some embodiments of the present invention, the cDNA at least partially targets 16S rRNA / 18S rRNA of pathogenic bacteria.

[0081] In some embodiments of the present invention, the nucleotide sequence of the cr RNA includes at least one of SEQ ID NO: 51 to SEQ ID NO: 59.

[0082] In some embodiments of the present invention, the nucleotide sequence of the cDNA comprises at least one of SEQ ID NO: 33 to SEQ ID NO: 41.

[0083] In some embodiments of the present invention, the nucleotide sequence of the dsDNA comprises at least one of SEQ ID NO: 42 to SEQ ID NO: 50.

[0084] The fourth aspect of the present invention provides a method for detecting pathogens, comprising the step of detecting a sample using the kit according to the third aspect of the present invention.

[0085] In some embodiments of the present invention, the method further comprises a fluorescence signal reading step or observing the color change of the reaction system after the detection is completed.

[0086] In some embodiments of the present invention, when the kit selects a1) sensor, the method comprises the following steps:

[0087] (1) Pretreatment of the sample to be tested: extracting the 16S rRNA / 18S rRNA of the sample to be tested; (2) mixing the 16S rRNA / 18S rRNA obtained in step (1) with an acidic solvent, an exonuclease, and the sensor a1) and reacting for 20 to 80 minutes. The reaction supernatant is mixed with an acidic solvent, a reducing agent, N3-DNA-AuNPs, and C≡C-DNA-AuNPs to perform a click reaction; (3) reading the fluorescence signal or visually observing the color change of the reaction system and the visible light signal (when the target molecule is present in the sample to be tested, the AuNPs solution changes from a red free state to a purple aggregated state).

[0088] In some embodiments of the present invention, the final concentration of the exonuclease in the mixture of 16S rRNA / 18S rRNA, acidic solvent, exonuclease and sensor a1) in step (2) is 10 to 500 U L -1 ; preferably 10 to 200 U L -1 .

[0089] In some embodiments of the present invention, the time for the mixed reaction of 16S rRNA / 18S rRNA with the acidic solvent, the exonuclease and the sensor a1) in step (2) is 20 to 70 minutes; further 40 to 60 minutes.

[0090] In some embodiments of the present invention, the N3-DNA-AuNPs and C≡C-DNA-AuNPs are used in equimolar concentrations, and the final concentrations in the reaction system are both 1 to 8 nM; further 2 to 8 nM.

[0091] In some embodiments of the present invention, a portable detection device and / or a microplate reader is used to read the fluorescent signal or visible light signal.

[0092] When the portable detection device reads the signal, the light intensity of 405-425nm / 435-455nm / 470-490nm / 505-525nm / 545-565nm / 580-600nm / 620-640nm / 670-690nm is used to determine whether the sample to be tested contains pathogens; when the microplate reader reads the signal, the absorbance value at 630nm and the absorbance value at 530nm are read, and the ratio of the absorbance at 630nm to the absorbance at 530nm is used to determine whether the sample to be tested contains pathogens.

[0093] In some embodiments of the present invention, the portable detection device includes a display screen, a light intensity knob, a light intensity display screen, a radiator, a switch, a constant voltage power supply, a detector and a sample holder, and the sample holder can hold commercially available 8 / 12 enzyme label strips (8 / 12 strip tubes).

[0094] The portable testing equipment is a portable device designed by Solidworks software ( Figure 1 (C) The main body (184×150×112mm) consists of a screen, display, light intensity knob, light intensity display, heat sink, switch, and constant voltage power supply. The constant voltage power supply can be powered by either a wall-mounted power supply or a battery, overcoming the limitations of wall-mounted power supplies. The display and light intensity knob are located on the top surface of the device, the heat sink and light intensity display are located on the front, and the switch and charging port are located on the back. After adding samples to the 8 / 12 enzyme strip (8 / 12 tube strip), the signal can be automatically detected and read directly on the display. In addition, for ease of use, two operating modes, automatic and manual, are designed and implemented through a dynamic layout interface. Automatic operation can read the signal values ​​of 12 samples in 36 seconds, while manual operation allows selective detection of specific samples.

[0095] In some embodiments of the present invention, when the kit selects a2) sensor, the method comprises the following steps:

[0096] 1) Pretreatment of the test sample: extracting 16S rRNA / 18S rRNA from the test sample; 2) mixing the 16S rRNA / 18S rRNA obtained in step 1) with an acidic solvent, Cas protein, crRNA, and the sensor in step a2) and reacting for 5 to 50 minutes. The reaction supernatant was mixed with an acidic solvent, sodium acetate buffer, tetramethylbenzidine, and H2O2 and reacted for 5 to 40 minutes. The reaction was terminated by adding sulfuric acid.

[0097] 3) Signal reading or visual observation of the color change of the reaction system. In some embodiments of the present invention, the mixed solution of 16S rRNA / 18S rRNA, acidic solvent, Cas protein, crRNA, and ssDNA-Au@Fe3O4 in step 2) contains Cas protein at a final concentration of 0.5 nM to 20 nM; further at a final concentration of 0.5 nM to 15 nM; and further at a final concentration of 10 nM to 15 nM.

[0098] In some embodiments of the present invention, in step 2), the 16S rRNA / 18S rRNA is mixed with the acidic solvent, Cas protein, crRNA, and a2) sensor and reacted for 10 to 40 minutes; further reacted for 20 to 40 minutes; and further reacted for 30 to 40 minutes.

[0099] In some embodiments of the present invention, in step 2), the final concentration of tetramethylbenzidine in the reaction system is 0.05-10 mM; further the final concentration is 0.05-10 mM; further the final concentration is 1-5 mM.

[0100] In some embodiments of the present invention, the reaction supernatant in step 2) is mixed with an acidic solvent, sodium acetate buffer, tetramethylbenzidine and H2O2 and reacted for 5 to 30 minutes; further reacted for 10 to 25 minutes; and further reacted for 15 to 20 minutes.

[0101] In some embodiments of the present invention, a portable detection device and / or a microplate reader is used to read the fluorescent signal or visible light signal, and further read the absorbance value at 450 nm to determine whether the sample contains pathogens.

[0102] In some embodiments of the present invention, the sample includes urine, blood, plasma, bacterial fluid or tissue.

[0103] In some embodiments of the present invention, the sample can be pretreated as follows: the sample is mixed with a 0-1 M alkaline solution (e.g., sodium hydroxide solution) and boiled for 5-15 minutes to obtain 16S rRNA / 18S rRNA in the sample. Extraction can also be performed using commonly available methods, such as commercially available kits.

[0104] The fifth aspect of the present invention provides a method for detecting antimicrobial drug sensitivity, comprising the step of detecting a sample using the kit of the third aspect of the present invention.

[0105] In some embodiments of the present invention, the method further comprises a fluorescence signal reading step or observing the color change of the reaction system after the detection is completed.

[0106] In some embodiments of the present invention, the sample to be tested is a sample treated with different antimicrobial drugs, and the rest of the detection process is the same as the method of the fourth aspect of the present invention.

[0107] In some embodiments of the present invention, the antibacterial drug is selected from β-lactams (such as penicillins, cephalosporins, carbapenems, β-lactams containing enzyme inhibitors and monocyclic amides (aztreonam), etc.), aminoglycosides (gentamicin, streptomycin, tobramycin, netilmicin, amikacin, isopamicin, ethidium, spectinomycin), tetracyclines (minocycline, doxycycline, tetracycline and oxytetracycline, etc.), fluoroquinolones (nalidixic acid, pyridinic acid, enoxacin, ofloxacin, pefloxacin, ciprofloxacin, lomefloxacin, etc.), folate pathway inhibitors, chloramphenicol (chloramphenicol), glycopeptides (vancomycin and teicoplanin, etc.) and macrolides (erythromycin, erythromycin ethylsuccinate, erythromycin stearate, erythromycin ethyl carbonate, erythromycin acetate stearate, erythromycin lactobionate, erythromycin sirolimus, tacrolimus, sirolimus, amphotericin B, pentamycin and fidaxomicin, etc.).

[0108] The sixth aspect of the present invention provides a portable device comprising a display screen, a light intensity knob, a light intensity display screen, a radiator, a switch, a constant voltage power supply, a detector, a sample holder, and a linear stepper motor guide slide (T-type lead screw linear rail electric slide module), wherein the portable device is used to read the fluorescent signal or visible signal described in the method of the fourth or fifth aspect of the present invention. The linear stepper motor guide slide is used to advance the sample holder; a sample placement port is provided in the middle of the sample holder; the sample holder includes a placement port for 8 or 12 enzyme label strips (8 or 12 tube strips). The sample holder is rectangular and has a sample placement port with a hollow structure in the middle. The placement port is 7 to 12 cm long and 0.5 to 1.5 cm wide.

[0109] The portable device provided by the present invention has higher accuracy and sensitivity in reading fluorescence signals than that of an enzyme marker.

[0110] The beneficial effects of the present invention are:

[0111] The present invention modifies bfDNA on the surface of metal nanoparticles (such as Au@Fe3O4) to obtain bfDNA-metal nanoparticles (such as bfDNA-Au@Fe3O4), and sequentially connects S1-CuO and S2-CuO to the bfDNA-metal nanoparticles through base complementary pairing. S1-CuO and S2-CuO can be orderly connected through ring hybridization to form a polymer with multiple CuO nanoparticles, thereby obtaining a sensor that can be used to detect pathogens, effectively detect pathogens (such as pathogens in urinary tract infections), and can also be used for antimicrobial drug sensitivity detection.

[0112] The present invention is based on bfDNA sensor and click chemistry induced nanogold aggregation (using a1) sensor) or Cu 2+ By inducing H2O2 to decompose and change the color of TMB (using a2) sensor), a rapid and visual pathogen detection method was established. When pathogens are present, 16S rRNA or 18S rRNA is released (first signal amplification), hybridized with bfDNA, and activated exonuclease III (Exonuclease III, Exo-III) (second signal amplification), releasing S1-CuO and S2-CuO polymers into the solution, and dissolving CuO to reduce it to Cu +(Third signal amplification), through the click reaction, the signal molecules C≡C-DNA-AuNPs and N3-DNA-AuNPs are hybridized to trigger the aggregation of AuNPs. The entire experimental scheme includes a triple signal amplification process, which can achieve specific and rapid detection, and the test results can be seen with the naked eye. When using a2) sensor, when the pathogen is lysed and 16S rRNA / 18S rRNA is released to achieve the first signal amplification, 16S rRNA / 18S rRNA binds to cDNA and releases ds DNA. ds DNA binds to cr RNA to activate the trans-cutting activity of Cas 12a, cuts the 5' end single strand of ss DNA, releases CuO nanoparticles, and achieves the second signal amplification. Under acidic conditions, the CuONPs concatemer in the solution dissolves and is reduced to Cu 2+ , to achieve the third signal amplification, followed by the addition of sodium acetate buffer (pH 5.0), TMB and H2O2, Cu 2+ Induces H2O2 decomposition and changes TMB color, allowing detection down to 20 CFU mL -1 The pathogens can be detected and the test results can be seen with the naked eye.

[0113] The experiment proved that the method has a wide detection range (using a1) sensor, with a detection range of 500~10 8 CFU mL -1 , using a2) sensor, the detection range is 20~10 7 CFU mL -1 ), and the correlation coefficients of the portable device and the microplate reader were r = 0.9985 and r = 0.9944, respectively, showing good linear correlation. The method has good specificity, requires few reagents and samples, and is low in cost. Pathogen detection can be completed within 55 minutes, and AST can be completed within 145 minutes. It does not require a wall power supply and can be used in the wild. In addition, the identification threshold of urinary tract infection (UTI) (10 4 CFU mL -1 ), the color of the solution will change significantly, and the color can be observed by the naked eye to assess whether the urine sample is from a UTI patient (bacteria level ≥10 4 CFU mL -1 The method provided by the present invention has application potential in pathogen detection.

[0114] This invention innovatively combines bfDNA, CuO nanoparticles, and click chemistry / TMB to develop a portable detection device with direct readout. The new method provided by this invention is simple, rapid, visual, and wall-power-independent, making it easy to operate and meeting the requirements of pathogen detection in specialized environments. The device is portable and suitable for use in multiple healthcare settings, providing clinicians with timely laboratory support for real-time pathogen identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 Schematic diagram of the bfDEC-AA method; among them, a is a schematic diagram of the preparation of the bfDNA-Au@Fe3O4 sensor, b is a schematic diagram of the detection principle of the bfDEC-AA method, and c is a design diagram of the appearance of the portable device.

[0116] Figure 2 Figure 2 is the workflow for UTI identification, strain identification, and AST; a is the currently commonly used detection scheme, and b is the detection flowchart of the bfDEC-AA method.

[0117] Figure 3 Effects of different Exo-III concentrations (a) and reaction times (b) on the bfDEC-AA method.

[0118] Figure 4 Effects of the molar ratio of N3-DNA to AuNPs (a) and the concentration of N3-DNA-AuNPs (b) on the bfDEC-AA method.

[0119] Figure 5 The effect of different Cas 12a concentrations (a) and reaction times (b) on the pathogen detection method of method 2.

[0120] Figure 6 The effects of different TMB concentrations (a) and reaction times (b) on the pathogen detection method of method 2.

[0121] Figure 7 is the specific detection result of bfDEC-AA method; where a is G - The test results of bacteria (including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes), b is G + The detection results of bacteria (including Staphylococcus aureus, Enterobacter faecium, Enterobacter faecium, Streptococcus pneumoniae, and Streptococcus agalactiae) are shown in Table 1. C is the detection result of fungi (Candida glabrata), and d is the recognition efficiency of different types of microorganisms at the same concentration. The concentration of all bacteria was 10 4 CFU mL -1 .

[0122] Figure 8The sensitivity and accuracy of the bfDEC-AA method are tested, where a is the calibration curve using the portable device. The curve includes the complementary probe (G - -bfDNA sensor) and non-complementary probes (G + -bfDNA sensor), b is the detection of 10 using a portable device 3 , 10 5 、107CFU mL -1 coli, c is the UV-visible absorption spectrum of E. coli at different concentrations of AuNPs, and d is the calibration curve of the microplate reader, which includes the complementary probe (G - -bfDNA sensor) and non-complementary probes (G + -bfDNA sensor), e is the detection of 10 using a microplate reader 3 , 10 5 、107CFU mL -1 coli recovery rate.

[0123] Figure 9 The sensitivity and accuracy test results of the pathogen detection method of method 2 are shown in FIG. 1 , where a is the UV-visible absorption spectrum of Escherichia coli at different concentrations, and b is the calibration curve using an enzyme reader, which includes complementary probes and non-complementary probes.

[0124] Figure 10 is the result of antimicrobial sensitivity test; a is the concentration of sensitive E. coli solution and 8, 16, and 32 μg mL -1 AST results after co-incubation with AMP. b is the AST results after co-incubation with sensitive E. coli and 2, 4, and 8 μg mL -1 AST results after co-incubation with OFX, c is ESBL-E.coli solution and 8, 16, 32 μg mL -1 AST results after co-incubation with AMP, d is ESBL-E.coli solution and 2, 4, 8 μg mL -1 AST results after co-incubation with OFX. DETAILED DESCRIPTION

[0125] The present invention is further described in detail below through specific examples.

[0126] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0127] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0128] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0129] Example 1

[0130] 1. Preparation of AuNPs

[0131] This experiment used a sodium citrate reduction method to prepare 13nm AuNPs. The process is as follows: Soak the three-necked flask, magnetic rotor, and condenser required for AuNP synthesis in aqua regia overnight, rinse repeatedly with ultrapure water, and dry. Mix 1.7mL of 1% AuCl4H2O and 48.3mL of ultrapure water, heat and stir in an oil bath to 140°C (after the liquid in the condenser refluxes). Quickly add 5mL of freshly prepared 38.8mM C6H5Na3O7 and continue stirring until the color changes from light yellow to purple-red. After the color changes, continue heating and stirring for 20 minutes. Turn off the heater and continue stirring until the reaction solution cools to room temperature. Aliquot and store at 4°C in the dark until ready for use.

[0132] 2. Preparation of Au@Fe3O4

[0133] Fe₃O₄ magnetic nanoparticles were prepared using a solvothermal synthesis method. The procedure was as follows: 0.65 g of FeCl₃ was dissolved in 20 mL of ethylene glycol and stirred until the solution became orange and transparent. 0.30 g of sodium citrate was dissolved in the solution and stirred for 1 hour. 1.20 g of sodium acetate was dissolved in the solution and stirred for 40 minutes. The suspension was transferred to a stainless steel autoclave lined with polytetrafluoroethylene (50 mL capacity), heated at 200°C for 10 hours, and then cooled to room temperature. The precipitate was washed three times with ethanol and three times with deionized water, and then dried in a vacuum at 60°C to obtain a black powder, which was the Fe₃O₄ magnetic nanoparticles.

[0134] AuNPs@Fe3O4 were synthesized by electrostatic adsorption of polydimethyldiamine (PDDA) on the Fe3O4 surface. The procedure was as follows: 10 mg of Fe3O4 magnetic nanoparticles were dissolved in 10 mL of a 3% PDDA solution and stirred for 30 minutes. The solution was then centrifuged at 10,000 rpm for 5 minutes and washed three times with deionized water to remove excess PDDA. This solution was then dispersed in 60 mL of an AuNPs solution and stirred at room temperature for 8 hours. The mixture was then magnetically separated and washed three times with deionized water. The precipitate was then suspended in 10 mL of deionized water to obtain Au@Fe3O4.

[0135] 3. Preparation of bfDNA Sensors

[0136] G - For example, the preparation of the bfDNA sensor was performed for a single use. The specific steps for the single use were as follows: G was synthesized by high temperature drying method. - -bfDNA-Au@Fe3O4, S1-CuO and S2-CuO. Take 0.5μL of 100μM G - -bfDNA 1, G - -bfDNA 2 and G - -bfDNA3 (specific sequence see Table 1) was added to 49.5 μL of a 1 mg mL -1 1 μL of 100 μM S1 aqueous solution and 1 μL of 100 μM S2 aqueous solution were added to 99 μL of 1 mg mL -1 The above solution was mixed and transferred to a beaker and heated on a hot plate at 80°C to evaporate excess water. Subsequently, 100 μL ddH2O was added to each glass to resuspend the dried NPs (G - -bfDNA-AuNPs@Fe3O4, S1-CuO or S2-CuO) and transferred to a centrifuge tube. Centrifuge at 10,000 rpm / min for 5 min to remove the DNA not loaded on the NPs. After discarding the supernatant, G - 50 μL of 0.5×SSC solution (75 mM NaCl solution, 7.5 mM sodium citrate solution, pH 7.0) was added to the bfDNA-AuNPs@Fe3O4, and 100 μL of 0.5×SSC solution was added to S1-CuO and S2-CuO respectively. Finally, 50 μL of G - -bfDNA-AuNPs@Fe3O4 and 100μL S1-CuO were mixed and reacted (hybridized) for 1 hour, and then 100μL S2-CuO was added and mixed and reacted (hybridized) for 1 hour. The unhybridized H1-CuO and S2-CuO were removed by magnetic separation, and the precipitate was S2-CuO-H1-CuO-G - -bfDNA-Au@Fe3O4(denoted as G - -bfDNA sensor).

[0137] G + The preparation methods for the -bfDNA sensor, F-bfDNA sensor, and bfDNA sensors for the corresponding strains were the same as above, with only the corresponding bfDNA being replaced. The sequence information of the aforementioned bfDNA, S1, and S2 is shown in Table 1.

[0138] Table 1 Sequence information of bfDNA, S1 and S2

[0139]

[0140]

[0141] Note: The double-underlined portion in the table represents the random sequence A1, the italicized portion represents the A2 sequence, and the single-underlined portion (A3) is complementary to the 16S rRNA / 18S rRNA sequences of corresponding Gram-positive bacteria, Gram-negative bacteria, fungi, E. coli, K. pneumoniae, P. aeruginosa, S. aureus, E. faecalis, and E. faecium. The italicized portion in bfDNA is the reverse complement of the italicized bold portion in S1, and the bold and underlined portions in S1 and S2 are reverse complements. The bold and underlined portion in the S1 sequence represents the B1 sequence, and the black italicized bold portion represents the B2 sequence. The bold and underlined portion in the S2 sequence represents the C1 sequence, and the black italicized bold portion represents the C2 sequence.

[0142] 4. Preparation of ssDNA-bfDNA Sensor

[0143] ssDNA-Au@Fe3O4, S1-CuO and S2-CuO were synthesized by high temperature drying method. Specifically, 0.5 μL of 100 μM ssDNA (sequence shown in Table 2) aqueous solution was added to 49.5 μL of 1 mg mL -1 At the same time, take 1 μL of 100 μM S1 aqueous solution and 1 μL of 100 μM S2 aqueous solution, and add them to 99 μL of 1 mg mL -1The solution was mixed and transferred to a beaker and heated on an 80°C hot plate to evaporate excess water. Subsequently, 100 μL of ddH2O was added to each glass to resuspend the dried NPs. The mixture was then transferred to a centrifuge tube and centrifuged at 10,000 rpm for 5 minutes to remove DNA not loaded onto the NPs. The supernatant was discarded to obtain ssDNA-Au@Fe3O4. 50 μL of 0.5× SSC solution (75 mM NaCl, 7.5 mM sodium citrate, pH 7.0) was added to the ssDNA-Au@Fe3O4, followed by 100 μL of 0.5× SSC solution to each of S1-CuO and S2-CuO. Finally, 50 μL of ssDNA-Au@Fe3O4 and 100 μL of S1-CuO were mixed and reacted (hybridized) at room temperature for 1 hour, followed by the addition of 100 μL of S2-CuO and the reaction (hybridization) for another hour. Unhybridized H1-CuO and S2-CuO were removed by magnetic separation, and the precipitate was S2-CuO-H1-CuO-ssDNA-Au@Fe3O4 (denoted as ssDNA-bfDNA sensor).

[0144] Table 2 Sequences used in ssDNA-bfDNA sensors

[0145]

[0146]

[0147] Note: The bold portion of the ssDNA in the table is the D1 sequence, the italic portion (D2 sequence) is reverse complementary to the italic bold portion in S1 in Table 1, the underlined portion in the cDNA sequence is reverse complementary to its corresponding ds DNA sequence, and the ds DNA sequence is reverse complementary to its corresponding crRNA guide sequence (italic bold portion). The crRNA anchor sequence (double underlined portion) specifically binds to the Cas12a protein.

[0148] 5. Preparation of C≡C-DNA@AuNPs and N3-DNA@AuNPs

[0149] C≡C-DNA@AuNPs and N3-DNA@AuNPs were prepared using a low-pH method. 1.2-1.92 μL of a 100 μM C≡C-DNA (sequence shown in Table 1) and 1.2-1.92 μL of a 100 μM N3-DNA (sequence shown in Table 1) solution were mixed with 99 μL of an 8 nM AuNP solution. 2 μL of sodium citrate solution (500 mM, pH 3.0) was then added to each solution. The mixture was incubated at room temperature for 3 minutes, centrifuged at 13,000 rpm for 15 minutes, washed three times, and resuspended in water for later use.

[0150] Example 2

[0151] A method for detecting pathogens includes method one (bfDNA-ExoIII-CuO-AA (bfDEC-AA) method) and method two.

[0152] Among them, method 1 includes the following steps:

[0153] (1) Pretreatment of the test sample: Mix the test sample (bacterial solution, urine sample) with 25 μL of 1 M NaOH solution, boil for 10 minutes, and set aside;

[0154] (2) Add 0-24.5 μL 1M HCl, 125.5 μL UL -1 Exo-III and bfDNA sensors were mixed and reacted for 40 minutes; magnetic separation was performed, and 60 μL of the supernatant solution was mixed with 20 μL of 1-20 mM hydrochloric acid, 20 μL of 1-20 mM ascorbic acid solution, and 100 μL of a mixture consisting of 50 μL of N3-DNA-AuNPs and 50 μL of C≡C-DNA-AuNPs (the final concentrations of N3-DNA-AuNPs and C≡C-DNA-AuNPs in the reaction system were both 2 nM) for a click reaction;

[0155] (3) Use a portable device / microplate reader to detect the signal and read the signal. The signal value is the ratio of the absorbance at 630 nm to the absorbance at 530 nm. Determine the pathogen of the sample based on the signal value.

[0156] The portable device is a portable device designed by Solidworks software ( Figure 1 (C) The main body (184 × 150 × 112 mm) consists of a screen, display, light intensity knob, light intensity display, heat sink, switch, and constant voltage power supply. The constant voltage power supply can be powered by either a wall-mounted power supply or batteries, overcoming the limitations of wall-mounted power supplies. After adding samples to 8- or 12-link enzyme strips (8 or 12 tube strips), the signal is automatically detected and read directly on the display. In addition, for ease of use, two operating modes, automatic and manual, are designed and implemented through a dynamic layout interface. Automatic operation can read the signal values ​​of 12 samples in 36 seconds, while manual operation allows selective detection of specific samples.

[0157] When the bfDNA sensor used is G - -bfDNA sensor, G +-bfDNA sensor, Fungus-bfDNA sensor, then determine whether the sample to be tested is / contains Gram-positive bacteria, Gram-negative bacteria, or fungi based on the signal value. Furthermore, if the bfDNA sensor used is the specific pathogen it corresponds to, the corresponding specific pathogen can be determined.

[0158] The detection principle of method 1 is as follows: bfDNA is coupled to Au@Fe3O4 to form bfDNA-Au@Fe3O4, and S1 or S2 is coupled to CuO nanoparticles, forming S1-CuO or S2-CuO, respectively. S1-CuO hybridizes with bfDNA and immobilizes on the Au@Fe3O4 surface. The remaining bases of S1 are then linked to S2-CuO to form the detection sensor. To improve detection sensitivity, three cDNAs with different sequences were designed for each conserved region of the 16S rRNA or 18S rRNA gene based on the National Center for Biotechnology Information (NCBI) database. After the pathogens in the sample are lysed (each bacterium has over 10,000 copies of 16S rRNA, and each fungus has over 10,000 copies of 18S rRNA), the 3' end of the bfDNA is paired with the 16S rRNA / 18S rRNA fragments, enabling target recognition and achieving the first-level signal amplification. Exo-III is activated, cutting bfDNA and releasing CuO nanoparticles. When bfDNA is completely cut by Exo-III, 16S rRNA / 18S rRNA is released again and hybridizes with other bfDNA, leading to a new round of release of CuO nanoparticle concatemers, achieving a second signal amplification. Finally, under acidic and ascorbic acid reducing conditions, the CuO nanoparticle concatemers in the solution dissolve and are reduced to Cu + , realizing the third signal amplification, and triggering the click chemistry between C≡C-DNA@AuNPs and N3-DNA@AuNPs, triggering nanogold hybridization (AuNPs agglutination, AA), detecting the signal value, then the experiment detects the pathogens. This method is recorded as bfDNA-ExoIII-CuO-AA (bfDEC-AA) method (schematic diagram as shown) Figure 1 As shown, the detection process is as follows Figure 2 shown).

[0159] Using the bfDEC-AA method, the identification threshold of urinary tract infection (UTI) was (10 4 CFU mL -1 ), the color of the solution will change significantly, and the color can be observed by the naked eye to assess whether the urine sample is from a UTI patient (bacteria level ≥10 4 CFU mL-1 positive samples).

[0160] Method 2 includes the following steps:

[0161] (1) The pretreatment of the sample to be tested is the same as step (1) of method 1;

[0162] (2) Add 24.5 μL 1M HCl, Cas12a (final concentration of 15 nM) buffer and ssDNA-bfDNA sensor to the treated sample, as well as cDNA with a final concentration of 50 nM, dsDNA with a final concentration of 50 nM, and crRNA with a final concentration of 30 nM, and mix and react for 30 minutes; perform magnetic separation, take 180 μL supernatant solution and 20 μL HCl (final concentration of 3 mM), 400 μL sodium acetate buffer (pH 5.0, final concentration of 50 mM), TMB (tetramethylbenzidine) (final concentration in the reaction system is 5 mM) and 100 μL H2O2 (final concentration is 125 mM), react for 15 minutes, and then add 100 μL sulfuric acid (final concentration is 62.5 mM) to terminate the reaction;

[0163] (3) Use a portable device / microplate reader to detect the signal and read the signal. The signal value is the absorbance value at 450nm. Determine whether the sample to be tested is a pathogen based on the signal value.

[0164] The detection principle of method 2 is as follows: when the pathogen is lysed and 16S rRNA / 18S rRNA is released to achieve the first signal amplification, 16S rRNA / 18S rRNA hybridizes with the cDNA in the cDNA and ds DNA hybrid chain, releasing ds DNA. The free ds DNA hybridizes with the free cr RNA to activate the trans-cleavage activity of Cas 12a, cutting the 5' end single strand of ssDNA-Au@Fe3O4, releasing S1-CuO and S2-CuO concatemers, and achieving the second signal amplification. Finally, under acidic conditions, the CuO nanoparticle concatemers in the solution dissolve and are reduced to Cu 2+ , to achieve the third signal amplification, then add sodium acetate buffer (pH 5.0), TMB and H2O2, react for 15 minutes, add sulfuric acid to terminate the reaction, and detect the absorbance at 450nm to achieve the detection of pathogens.

[0165] Example 3

[0166] The bfDEC-AA method in Example 2 was further optimized as follows:

[0167] 1. Exo-III concentration and time

[0168] The cells were cultured with different concentrations of Escherichia coli (0, 10 4CFU mL -1 ) was used as the positive sample to optimize the concentration of Exo-III in the reaction system. The results were as follows Figure 3 As shown in (a), as the concentration of Exo-III increased from 0 U L -1 Increased to 200U L -1 , the signal value increased, but with the further increase of Exo-III concentration, the background signal value (NC) was too large, resulting in a decrease in the signal-to-noise ratio. Therefore, 200U L was selected in subsequent experiments. -1 By optimizing the time, the results are as follows Figure 3 As shown in Figure b, it reaches a relatively stable state after 40 minutes, so a 40-minute exposure time was selected in subsequent experiments. To ensure high throughput, this optimization will use a microplate reader for signal reading, with the signal value being the ratio of the absorbance at 630 nm to the absorbance at 530 nm.

[0169] 2. Molar ratio of N3-DNA to AuNPs and concentration of N3-DNA-AuNPs

[0170] The molar ratio of DNA to AuNPs is a key factor affecting the degree of Cu-triggered AuNPs aggregation, so the molar ratios of N3-DNA to AuNPs ranging from 150:1 to 240:1 were investigated. Figure 4 As shown in a, A 630 / A 530 The molar ratio reaches a relatively stable value at 210:1.

[0171] The concentration of N3-DNA-AuNPs is the main factor that determines the color intensity in the reaction system and also affects the difficulty of visual observation. The effect of different N3-DNA-AuNPs concentrations in the reaction system on the detection results was investigated. Figure 4 As shown in b, the optimal concentration of N3-DNA-AuNPs in the reaction system is 2 nM.

[0172] Since C≡C-DNA-AuNPs and N3-DNA-AuNPs are a one-to-one click chemistry reaction, the molar ratio of C≡C-DNA to AuNPs and the concentration of C≡C-DNA-AuNPs were also selected as 210:1 and 2 nM, respectively.

[0173] Method 2 in Example 2 is further optimized as follows:

[0174] 1. Effect of Cas 12a concentration and time

[0175] Using Escherichia coli (10 3 CFU mL -1) was used as a positive sample to optimize the concentration of Cas 12a in the reaction system. The results were as follows Figure 5 As shown. As the concentration of Cas12a increases from 0.5nM to 15nM, the signal value increases. However, as the concentration of Cas12a further increases, the background signal value (NC) is too large, resulting in a decrease in the signal-to-noise ratio ( Figure 5 Therefore, 15 nM Cas12a was selected in subsequent experiments. Figure 5 Figure b shows the effect of Cas12a time, which reaches a relatively stable state in 30 minutes, so a 30-minute action time was selected in subsequent experiments.

[0176] 2. Effect of TMB concentration and time

[0177] Using Escherichia coli (10 3 CFU mL -1 ) was used as the positive sample to optimize the TMB concentration in the reaction system. Figure 6 As shown in Figure 2, the signal value increased as the TMB concentration increased from 0.05mM to 5mM, but as the TMB concentration further increased, the background signal value (NC) was too large, resulting in a decrease in the signal-to-noise ratio ( Figure 6 Therefore, a TMB concentration of 5 mM was selected in subsequent experiments. Figure 6 Figure b shows the effect of TMB time, which reached a relatively stable state at 15 minutes, so a 15-minute action time was selected in subsequent experiments.

[0178] Example 4 Specificity of the detection method

[0179] To evaluate the specificity of the bfDEC-AA method in Example 2, this example used conserved sequence cDNA of pathogens to analyze three different types of bacterial samples, including G - (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes), G + (Staphylococcus aureus, Enterobacter faecalis, Enterobacter faecium, Streptococcus pneumoniae, Streptococcus agalactiae) and fungi (Candida glabrata). 630 / A 530 The ratio of the two groups was used to evaluate the ability of the detection method to selectively detect pathogens.

[0180] The results are as follows Figure 7 As shown. The corresponding bfDNA sensor is used to detect pathogens (i.e. G - -bfDNA sensor for detection of Gram-negative bacteria, G + -bfDNA sensor detects Gram-positive bacteria, and Fungus-bfDNA sensor detects fungi). 630 / A 530The ratio was significantly higher than that of samples of other bacterial categories ( Figure 7 a~c), which shows that the method has high specificity. Furthermore, at the same concentration, there is no significant difference in the recognition efficiency of different strains, indicating that the signal response of different strains is consistent ( Figure 7 (d)

[0181] Example 5 Detection Sensitivity and Accuracy of the Detection Method

[0182] The detection sensitivity and accuracy of the bfDEC-AA method in Example 2 were evaluated. This example tested the linearity and spiked recovery of the method.

[0183] Since there is no significant difference in the recognition efficiency of different strains at the same concentration ( Figure 7 d), so this example selects Escherichia coli at 500~10 8 CFU mL -1 The linear relationship is established within the range of 1:1, and the specific process is the same as in Example 2. At the same time, a non-complementary probe (G + -bfDNA sensor) were used to measure seven different concentrations (500, 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 CFU mL -1 ) of the Escherichia coli standard solution.

[0184] The results are as follows Figure 8 and Table 3. E. coli standard solutions with different concentrations and A 630 / A 530 There is a strong correlation between the ratio of portable devices ( Figure 8 a) and microplate reader ( Figure 8 The correlation coefficients of c and d) were r = 0.9985 and r = 0.9944, respectively. The curves include the complementary probe (G - -bfDNA sensor) and non-complementary probes (G + -bfDNA sensor), non-complementary probe pairs up to 10 8 CFU mL -1 The response of the standard E. coli solution was low (less than 3SD). The detection limit (LOD) of the microplate reader (S / N=3) was 331.8 CFU mL -1 , the LOD of the portable device was 119.1 CFU mL -1 , indicating high sensitivity. Figure 8 As shown in the upper right corner of a, the aggregation degree of AuNPs is positively correlated with the bacterial concentration, and 4CFU mL -1 In addition, different concentrations of E. coli samples were detected by portable devices ( Figure 8 b) and microplate reader ( Figure 8 The recoveries in (e) were 94.2-101.2% and 95.0-112.7%, respectively, indicating that the bfDEC-AA method has high accuracy.

[0185] Table 3 Recovery of Escherichia coli in spiked samples

[0186]

[0187]

[0188] The detection sensitivity and accuracy of the second method in Example 2 were evaluated. This example tested the linearity of the method.

[0189] Select Escherichia coli between 20 and 10 7 CFU mL -1 The linear relationship was established within the range of 20, 10, and 20 μg / mL. The specific process was the same as in Example 2. At the same time, non-complementary probes were used to measure the concentrations of seven different 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 CFU mL -1 ) of the Escherichia coli standard solution.

[0190] The results showed that different concentrations of E. coli standard solution and A 450 The values ​​have a strong correlation, with correlation coefficients of r = 0.9906 ( Figure 9 ), and non-complementary probe pairs up to 10 7 CFU mL -1 The reaction of the standard E. coli solution was low (less than 3SD), indicating that the detection method has high sensitivity. Figure 9 As shown in the upper right, the color change is positively correlated with the bacterial concentration.

[0191] Example 6 Antimicrobial drug sensitivity

[0192] In order to evaluate whether the bfDEC-AA method in Example 2 is applicable to antimicrobial drug sensitivity, in this example, bacteria were co-incubated with different concentrations of ampicillin (AMP) or ofloxacin (OFX), and the bfDEC-AA method was used to quantitatively detect the growth of bacteria. 4 CFU mL -1The effects of AMP or OFX on sensitive E. coli or extended-spectrum β-lactamase (ESBL)-producing E. coli were evaluated. The details are as follows:

[0193] The initial concentration is 10 4 CFU mL -1 coli (sensitive E. coli or ESBL-E. coli) were added to 4 mL of MHB culture medium in a centrifuge tube. Tube No. 1 did not contain any drug, and tubes No. 2 to 4 contained drugs with a final concentration of 8, 16, and 32 μg mL -1 Ampicillin (AMP) was added to test tubes No. 5 to 7 at final concentrations of 2, 4, and 8 μg mL -1 Ofloxacin (OFX) was cultured at 37°C and 250 rpm / min for 0, 1, 1.5, 2, and 2.5 hours, respectively. The bacterial suspension was collected and assayed using the bfDEC-AA method described in Example 2.

[0194] The results are as follows Figure 10 Compared with the control group, the antibiotic group (8 μg mL -1 AMP / 2μg mL -1 OFX) significantly inhibited the growth of sensitive E. coli, and the difference was statistically significant ( Figure 10 a and b); while the use of high concentrations of antibiotics (32 μg mL -1 AMP / 8μg mL -1 OFX) does not inhibit the growth of ESBL-producing E. coli ( Figure 10 c and d), indicating that the bfDEC-AA method of Example 2 can be used for antimicrobial drug sensitivity detection.

[0195] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A sensor for detecting pathogens, wherein the sensor is a1) or a2); a1) including bfDNA-metal nanoparticles, S1-CuO and S2-CuO; a2) including ssDNA-metal nanoparticles, S1-CuO and S2-CuO; in, The bfDNA-metal nanoparticles are obtained by coupling bfDNA and metal nanoparticles; The ssDNA-metal nanoparticles are obtained by coupling ssDNA and metal nanoparticles; The S1-CuO and the S2-CuO are obtained by coupling S1 or S2 with CuO respectively; The nucleotide sequence of the bfDNA includes A1, A2 and A3, and the A3 specifically targets and binds to the 16SrRNA / 18S rRNA of the pathogen; The nucleotide sequence of S1 includes B1 and B2, and B2 is partially reverse complementary to A2; The nucleotide sequence of S2 includes C1 and C2, wherein C1 is reverse complementary to B1, and C2 is reverse complementary to B2; The nucleotide sequence of the ssDNA includes D1 and D2, and the D2 is identical to the A2 portion.

2. The sensor according to claim 1, characterized in that The S1-CuO is connected through complementary base pairing between S1 and the bfDNA in the bfDNA-metal nanoparticle, and the S2-CuO is connected through complementary base pairing between S2 and S1 in S1-CuO; and / or, the S1-CuO is connected through complementary base pairing between S1 and the ssDNA in the ssDNA-metal nanoparticle, and the S2-CuO is connected through complementary base pairing between S2 and S1 in S1-CuO.

3. The sensor according to claim 1 or 2, characterized in that: The nucleotide sequence of the bfDNA also includes a random sequence; Preferably, the bfDNA is A1, A2 and A3 from the 5' end to the 3' end; Preferably, the bfDNA includes a 5' end modification; Preferably, the 5' end modification includes at least one of thiol modification, phosphorylation modification, phosphorylation analog modification, amino modification, dideoxy modification, spacer modification, 3'-O-propylene modification or 3'-O-azidomethyl modification; Preferably, the 3' ends of S1 and S2 contain tail sequences; Preferably, the nucleotide sequence of the tail sequence is 5'-TTTTTT-3'.

4. The sensor according to claim 1 or 2, characterized in that: The ssDNA also includes a random sequence; Preferably, the nucleotide sequence of the ssDNA is as shown in SEQ ID NO:

32.

5. The sensor according to claim 3, characterized in that The nucleotide sequence of the bfDNA includes at least one of SEQ ID NO: 1 to SEQ ID NO: 27; Preferably, the nucleotide sequence of S1 is as shown in SEQ ID NO: 28; Preferably, the nucleotide sequence of S2 is as shown in SEQ ID NO: 29; Preferably, the metal nanoparticles are magnetic nanoparticles; Preferably, the metal nanoparticles include at least one of Au@Fe3O4 and polystyrene microspheres.

6. The sensor according to claim 5, characterized in that The pathogenic bacteria include at least one of Gram-positive bacteria, Gram-negative bacteria and fungi; Preferably, the Gram-positive bacteria include at least one of Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Pneumococcus, Bacillus anthracis, Corynebacterium diphtheriae, Listeria monocytogenes, Vibrio parahaemolyticus, and Clostridium tetani; Preferably, the Gram-negative bacteria include at least one of Klebsiella pneumoniae, Pseudomonas aeruginosa, Shigella dysenteriae, Salmonella typhi, Escherichia coli, Proteus, Bordetella pertussis, Salmonella, and Vibrio cholerae; Preferably, the fungus includes at least one of Candida albicans, Leptotrichia vaginalis, and Cryptococcus; Preferably, when the pathogen is a Gram-negative bacterium, the nucleotide sequence of the bfDNA is SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; Preferably, when the pathogen is a Gram-positive bacterium, the nucleotide sequence of the bfDNA is SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; Preferably, when the pathogen is a fungus, the nucleotide sequence of the bfDNA is SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:

9.

7. Use of the sensor according to any one of claims 1 to 6 in any one of (1) to (6): (1) Detection of pathogens; (2) Preparation of products for detecting pathogens; (3) Identification of pathogenic bacteria; (4) Preparation of products for identifying pathogenic bacteria; (5) Antimicrobial susceptibility testing; (6) Prepare products for antimicrobial susceptibility testing.

8. A kit comprising the sensor according to any one of claims 1 to 6; Preferably, when the sensor is a1), the kit further comprises a nuclease, a DNA-AuNPs modified with C≡C- at the 3' end, a DNA-AuNPs modified with N3- at the 3' end, and a reducing agent; Preferably, the nucleotide sequence of the DNA in the DNA-AuNPs with the 3' end modified with C≡C- is as shown in SEQ ID NO: 30; Preferably, the nucleotide sequence of the DNA in the DNA-AuNPs modified with N3- at the 3' end is as shown in SEQ ID NO: 31; Preferably, when the sensor is a2), the kit further comprises Cas protein, cr RNA, cDNA, ds DNA, H2O2 and tetramethylbenzidine; Preferably, the cr RNA comprises an anchor sequence and a guide sequence, the anchor sequence specifically binds to the Cas protein, and the guide sequence targets ds DNA; Preferably, the cDNA is at least partially reverse complementary to the ds DNA; Preferably, the cDNA at least partially targets 16S rRNA / 18S rRNA of pathogenic bacteria; Preferably, the nucleotide sequence of the cr RNA includes at least one of SEQ ID NO:51 to SEQ ID NO:59; Preferably, the nucleotide sequence of the cDNA includes at least one of SEQ ID NO:33 to SEQ ID NO:41; Preferably, the nucleotide sequence of the ds DNA comprises at least one of SEQ ID NO:42 to SEQ ID NO:

50.

9. A method for detecting pathogens, comprising the step of using the kit according to claim 8 to detect a sample to be detected; preferably, the method further comprises a step of reading a fluorescent signal or observing the color change of a reaction system after the detection is completed; Preferably, a portable detection device and / or an ELISA reader is used to read the fluorescent signal or the visible light signal; Preferably, the portable detection device comprises a display screen, a light intensity knob, a light intensity display screen, a heat sink, a switch, a constant voltage power supply, a detector, a sample placement plate and a linear stepper motor guide rail slide; Preferably, the linear stepper motor guide slide is used to advance the sample placement plate; Preferably, a sample placement opening is provided in the middle of the sample placement plate.

10. A portable testing device, comprising a display screen, a light intensity knob, a light intensity display screen, a heat sink, a switch, a constant voltage power supply, a detector, a sample placement rack and a linear stepper motor guide rail slide; Preferably, the linear stepper motor guide slide is used to advance the sample placement rack; Preferably, the sample placement rack is rectangular, with a sample placement opening of a hollow structure in the middle, the placement opening is 7 to 12 cm long and 0.5 to 1.5 cm wide; Preferably, the sample placement rack can hold commercially available 8 or 12-link enzyme label strips (8 or 12-link tubes).

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