A rapid, visual pathogen detection method and portable detection device

Abstract

The application belongs to the technical field of pathogenic bacteria detection, and discloses a rapid and visual pathogenic bacteria detection method and a portable detection device, and specifically discloses a sensor for detecting pathogenic bacteria, wherein the sensor is a1) or a2); a1) comprises bfDNA-metal nanoparticles, S1-CuO and S2-CuO; and a2) comprises ssDNA-metal nanoparticles, S1-CuO and S2-CuO. In the application, 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 base complementary pairing, S1-CuO and S2-CuO can be sequentially connected through ring chain hybridization, a polymer with multiple CuO nanoparticles is formed, the sensor for detecting pathogenic bacteria is obtained, pathogenic bacteria can be effectively detected, and the sensor can also be used for antibacterial drug sensitivity detection.

Description

Technical Field

[0001] This invention belongs to the field of pathogen detection technology, specifically relating to a rapid, visual pathogen detection method and a portable detection device. Background Technology

[0002] Urinary tract infections (UTIs) are among the most common infectious diseases in communities. Globally, approximately 150 million people are affected by UTIs each year, accounting for more than 15% of all antibiotic prescriptions in primary care settings. Previous studies have shown that only about 20% of urine samples from suspected uremia patients test positive for infection, while the remaining 80% of patients do not actually require antibiotic treatment. Currently, traditional methods for identifying pathogens and performing antimicrobial susceptibility testing (AST) rely on culture methods, which typically take several days. Given the risks of delayed treatment, physicians often prescribe broad-spectrum antibiotics based on experience. Empirical antibiotic treatment of these non-UTI patients accelerates antibiotic resistance, a problem exacerbated in underdeveloped regions.

[0003] Traditional methods for UTI identification and AST analysis include disc diffusion or broth microdilution, which require bacterial culture, typically 24–72 hours. Currently, various methods for identifying pathogens and performing AST have been developed. Pathogen identification primarily utilizes specific nucleic acid sequence-based amplification techniques (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. Genotypic AST provides AST results by identifying the presence of drug resistance genes in bacteria, such as hairpin-structured magnetic surface-enhanced Raman scattering (SERS) sensors, multiplex photoelectrochemical bioassays, fluorescent biosensors, PCR, and commercial T2Resistance. TM Panel 17. However, genotypic detection often yields inconsistent results with phenotypic AST, and samples require complex DNA extraction procedures. Phenotypic AST assesses antibiotic resistance by culturing bacteria in antibiotic media, using methods such as PCR and high-resolution melting, LAMP, electrochemiluminescence biosensors, biochemiluminescence sensors, deep learning analysis of SERS sensors, deep learning video microscopy, capillaries, paper chips, and hydrogel sensors. However, these methods all require specific algorithms or expensive equipment. Therefore, developing a simple, rapid, easy-to-use, and low-cost technique to distinguish between UTI and non-UTI and perform AST is crucial. Summary of the Invention

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

[0005] The second aspect of this invention aims to provide an application of the sensor of the first aspect of this invention.

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

[0007] The fourth aspect of this invention is to provide a method for detecting pathogens.

[0008] The fifth aspect of this invention aims to provide a method for detecting antimicrobial drug susceptibility.

[0009] The sixth aspect of this invention aims to provide a portable testing device.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] In a first aspect, the present invention provides a sensor for detecting pathogens, said sensor being a1) or a2);

[0012] a1) comprises bfDNA (bifunctional deoxyribonucleic acid)-metal nanoparticles, S1-CuO, and S2-CuO; a2) comprises 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, wherein A3 specifically targets and binds to the 16S rRNA / 18S rRNA of the pathogen. rRNA; the nucleotide sequence of S1 includes B1 and B2, wherein B2 is anticomplementary to the A2 portion; the nucleotide sequence of S2 includes C1 and C2, wherein C1 is anticomplementary to B1, and C2 is anticomplementary to B2; the nucleotide sequence of ssDNA includes D1 and D2, wherein 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 linked to the bfDNA base in the bfDNA-metal nanoparticle through complementary pairing of S1 and S2-CuO through complementary pairing of S1 base 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 and 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 at a final concentration of 0.9–1.1 μM and 0.5–2 mg / mL. -1 Metal nanoparticles.

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

[0019] In some embodiments of the present invention, the mixed system contains S1 at a final concentration of 0.9–1.1 μM and 0.5–2 mg / mL. -1 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), and 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 at a final concentration of 0.9–1.1 μM and 0.5–2 mg / mL. -1 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 and 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 at a final concentration of 0.9–1.1 μM and 0.5–2 mg / mL. -1 Metal nanoparticles.

[0024] In some embodiments of the present invention, the S1-CuO is linked to the ssDNA base in the ssDNA-metal nanoparticle through complementary pairing of S1 and S2-CuO through complementary pairing of S1 base in 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 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 and reacted at room temperature for 0.5 to 1.5 h; S2-CuO solution is added and the mixture is reacted for 0.5 to 1.5 h to obtain the sensor.

[0026] In some embodiments of the present invention, the preparation method of the sensor described in a2) includes the following steps: ssDNA-metal nanoparticles, S1-CuO and S2-CuO are dissolved in 0.5×SSC solution to obtain ssDNA-metal nanoparticle solution, S1-CuO solution and S2-CuO solution respectively; the ssDNA-metal nanoparticle solution and S1-CuO solution are mixed and reacted at room temperature for 0.5 to 1.5 h; S2-CuO solution is added and the mixture is reacted for 0.5 to 1.5 h 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 to and / or anticomplementary 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, interarm modification, 3'-O-propenyl 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, S1 includes 5' end modification; the 5' end modification includes at least one of thiol modification, phosphorylation modification, phosphorylation analog modification, amino modification, dideoxy modification, interarm modification, 3'-O-propenyl modification or 3'-O-azidomethyl modification; preferably thiol modification.

[0035] In some embodiments of the present invention, S2 includes 3' end modification; the 3' end modification includes at least one of thiol modification, phosphorylation modification, phosphorylation analog modification, amino modification, dideoxy modification, interarm modification, 3'-O-propenyl 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, Streptococcus pneumoniae, 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, Ciliophora 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] A second aspect of the invention provides the application of the sensor in any one of (1) to (6):

[0057] (1) Detecting pathogens; (2) Preparing products for detecting pathogens; (3) Identifying pathogen species; (4) Preparing products for identifying pathogen species; (5) Testing for susceptibility to antimicrobial drugs; (6) Preparing products for testing for susceptibility to antimicrobial drugs.

[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, Streptococcus pneumoniae, 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, Ciliophora vaginalis, and Cryptococcus.

[0063] A third aspect of the present invention provides a reagent kit including the sensor of the first aspect of the present invention.

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

[0065] In some embodiments of the present invention, the exonuclease includes at least one of 5' phosphate-dependent exonuclease, Saccharomyces cerevisiae XrnI ribonuclease, 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 includes 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 C≡C- modified at the 3' end is shown in SEQ ID NO:30.

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

[0070] In some embodiments of the present invention, the preparation method of the DNA-AuNPs with C≡C- modified at the 3' end includes the following steps: mixing DNA with C≡C- modified 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 DNA-AuNPs with C≡C- modified at the 3' end (denoted as C≡C-DNA@AuNPs).

[0071] In some embodiments of the present invention, the molar ratio of 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 includes 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 includes Cas protein, crRNA, cDNA, dsDNA, H2O2, and tetramethylbenzidine (TMB).

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

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

[0077] In some embodiments of the present 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 particularly 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 binding to the Cas protein and the guide sequence targeting ds DNA.

[0079] In some embodiments of the present invention, the cDNA and the dsDNA are at least partially reverse complementary.

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

[0081] In some embodiments of the present invention, the nucleotide sequence of the crRNA 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 includes 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 includes at least one of SEQ ID NO:42 to SEQ ID NO:50.

[0084] A fourth aspect of the present invention provides a method for detecting pathogens, comprising the step of detecting a sample to be tested using a reagent kit from the third aspect of the present invention.

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

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

[0087] (1) Pretreatment of the sample to be tested: 16S rRNA / 18S rRNA of the sample to be tested is extracted; (2) The 16S rRNA / 18S rRNA obtained in step (1) is mixed with acidic solvent, exonuclease and a1) sensor, and reacted for 20-80 min. The reaction supernatant is then mixed with acidic solvent, reducing agent, N3-DNA-AuNPs and C≡C-DNA-AuNPs and click reaction is performed; (3) Fluorescence signal reading or visual observation of the color change of the reaction system and visible light signal reading (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 step (2) in the mixture of 16S rRNA / 18S rRNA, acidic solvent, exonuclease, and a1) sensor is 10–500 U / L. -1 Preferably 10–200 μL -1 .

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

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

[0091] In some embodiments of the present invention, portable detection devices and / or enzyme-linked immunosorbent assay (ELISA) readers are used to read fluorescence signals or visible light signals.

[0092] Portable detection devices use light intensity at wavelengths of 405-425nm, 435-455nm, 470-490nm, 505-525nm, 545-565nm, 580-600nm, 620-640nm, and 670-690nm to determine whether the sample contains pathogens. Microplate readers read absorbance values ​​at 630nm and 530nm, and the ratio of the absorbance at 630nm to 530nm is used to determine whether the sample 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 heat sink, a switch, a constant voltage power supply, a detector, and a sample rack, wherein the sample rack can hold commercially available 8 / 12-linked enzyme-linked labeling strips (8 / 12-linked tubes).

[0094] The portable testing device is a portable device designed using Solidworks software. Figure 1 (c) The main body (184×150×112mm) consists of a screen, display screen, light intensity knob, light intensity display screen, 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 screen and light intensity knob are located on the top of the device, the heat sink and light intensity display screen are on the front, and the switch and charging interface are on the back. After adding a sample to the 8 / 12-linked ELISA strip (8 / 12-linked tube), the signal can be automatically detected and read directly on the display screen. Furthermore, for ease of use, two operating modes are designed: automatic and manual, implemented through a dynamically laid-out interface. Automatic operation can read the signal values ​​of 12 samples within 36 seconds, while manual operation allows for selective detection of specific samples.

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

[0096] 1) Pretreatment of the test sample: Extract the 16S rRNA / 18S rRNA of the test sample; 2) Mix the 16S rRNA / 18S rRNA obtained in step 1) with acidic solvent, Cas protein, crRNA and a2) sensor, react for 5-50 min, take the reaction supernatant and mix with acidic solvent, sodium acetate buffer, tetramethylbenzidine and H2O2, react for 5-40 min, and add sulfuric acid to terminate the reaction.

[0097] 3) Signal reading or visual observation of color changes in the reaction system. In some embodiments of the present invention, the mixed solution of 16S rRNA / 18S rRNA with 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, the final concentration is 0.5 nM to 15 nM; and even further, the final concentration is 10 nM to 15 nM.

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

[0099] In some embodiments of the present invention, the final concentration of tetramethylbenzidine in the reaction system in step 2) is 0.05-10 mM; further, the final concentration is 0.05-10 mM; and even 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 an ELISA reader is used to read the fluorescence signal or visible light signal; the absorbance value at 450 nm is further read. The absorbance value at 450 nm is used to determine whether the sample contains pathogens.

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

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

[0104] A fifth aspect of the present invention provides a method for detecting antimicrobial susceptibility, comprising the step of detecting a sample to be tested using a kit from a third aspect of the present invention.

[0105] In some embodiments of the present invention, the method further includes a fluorescence signal reading step or an observation of 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 antibacterial drugs, and the remaining 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, isipamicin, etilmicin, spectinomycin), tetracyclines (minocycline, doxycycline, tetracycline, and oxytetracycline, etc.), and fluoroquinolones. (Neidinic acid, pyridinic acid, enoxacin, ofloxacin, pefloxacin, ciprofloxacin, lomefloxacin, etc.), folic acid pathway inhibitors, chloramphenicol, glycopeptides (vancomycin and teicoplanin, etc.) and macrolides (erythromycin, erythromycin ethylsuccinate, erythromycin stearate, erythromycin ethyl carbonate, erythromycin acetate, erythromycin lactobionate, erythromycin etoritize, tacrolimus, sirolimus, amphotericin B, pentathromycin and fenadine, etc.).

[0108] A sixth aspect of the present invention provides a portable 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 holder, and a linear stepper motor guide slide (T-type lead screw and linear rail electric slide module). The portable device is used to read the fluorescence 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; the sample holder has a sample placement opening in the middle; the sample holder includes an opening for placing 8 or 12-strand ELISA strips (8 or 12-strand tubes). The sample holder is rectangular, with a hollow sample placement opening in the middle, the opening being 7–12 cm long and 0.5–1.5 cm wide.

[0109] The portable device provided by this invention offers higher accuracy and sensitivity for reading fluorescence signals compared to an ELISA reader.

[0110] The beneficial effects of this invention are:

[0111] This invention modifies bfDNA onto the surface of metal nanoparticles (such as Au@Fe3O4) to obtain bfDNA-metal nanoparticles (such as bfDNA-Au@Fe3O4). S1-CuO and S2-CuO are sequentially linked to the bfDNA-metal nanoparticles through base complementary pairing. S1-CuO and S2-CuO can be linked in an orderly manner through ring hybridization to form a polymer with multiple CuO nanoparticles, resulting in a sensor that can be used to detect pathogens. This sensor can effectively detect pathogens (such as pathogens in urinary tract infections) and can also be used for antimicrobial drug sensitivity testing.

[0112] This invention is based on a bfDNA sensor and click chemistry-induced gold nanoparticle aggregation (using an a1 sensor) or Cu. 2+ A rapid and visualized method for pathogen detection was established by inducing H2O2 decomposition and causing TMB color change (using the a2 sensor). When using the a1 sensor, the presence of pathogens releases 16S or 18S rRNA (first-level signal amplification), which hybridizes with bfDNA, activating exonuclease III (Exo-III) (second-level signal amplification), releasing S1-CuO and S2-CuO polymers into solution, and dissolving and reducing CuO to Cu. +(Third signal amplification) By using a click reaction, signal molecules C≡C-DNA-AuNPs and N3-DNA-AuNPs hybridize, triggering AuNP aggregation. The entire experimental protocol includes a triple signal amplification process, enabling specific and rapid detection with visually perceptible results. When using the a2) sensor, after the pathogen is lysed and releases 16S rRNA / 18S rRNA (achieving the first signal amplification), the 16S rRNA / 18S rRNA binds to cDNA, releasing dsDNA. The dsDNA binds to crRNA, activating the trans-cleavage activity of Cas 12a, cleaving the 5' end of the ssDNA single strand, releasing CuO nanoparticles, achieving the second signal amplification. Under acidic conditions, the CuONPs tandem in solution dissolves and is reduced to Cu. 2+ This achieves third-level signal amplification, followed by the addition of sodium acetate buffer (pH 5.0), TMB, and H2O2, Cu. 2+ Inducing H2O2 decomposition and causing TMB to change color, detectable down to 20 CFU / mL. -1 The pathogens can be detected by visual means.

[0113] Experiments have demonstrated that this method has a wide detection range (using an a1 sensor), with a detection range of 500–10. 8 CFU mL -1 Using an a2) sensor, the detection range is 20–10. 7 CFU mL -1 The correlation coefficients between the portable device and the ELISA reader were r = 0.9985 and r = 0.9944, respectively, showing good linear correlation. The method has good specificity, requires fewer reagents and samples, and is low-cost. Pathogen detection can be completed within 55 minutes, and AST within 145 minutes. Furthermore, it does not require a wall-mounted power source, making it suitable for field applications. Additionally, the threshold for identifying urinary tract infection (UTI) is 10... 4 CFU mL -1 When the solution changes color, the color can be visually observed to assess whether the urine sample originated from a UTI patient (bacterial level ≥10). 4 CFU mL -1 (positive samples). The method provided by this invention has the potential for application in pathogen detection.

[0114] This invention innovatively combines bfDNA, CuO nanoparticles, and click chemistry / TMB to develop a portable detection device with directly readable results. The novel method provided by this invention is simple, rapid, visual, and can operate independently of a wall power source, making it easy to use and meeting the requirements for pathogen detection in special environments. The portable device is suitable for multi-level medical institutions, providing clinicians with timely laboratory evidence for real-time pathogen identification. Attached Figure Description

[0115] Figure 1 The diagram shows the bfDEC-AA method; where 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 portable device.

[0116] Figure 2 This diagram illustrates the workflow for UTI identification, strain identification, and AST. Here, 'a' represents the commonly used detection scheme, and 'b' is the detection flowchart for the bfDEC-AA method.

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

[0118] Figure 4 The 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 effect of different TMB concentrations (a) and reaction times (b) on the pathogen detection method of Method 2.

[0121] Figure 7 This represents the specificity detection result of the bfDEC-AA method; where a is G - The test results for bacteria (including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter aerogenes) are given, with b representing Gram-positive bacteria. + The results for bacteria (including Staphylococcus aureus, Enterobacter faecalis, Enterobacter faecium, Streptococcus pneumoniae, and Streptococcus agalactiae) are shown in Figure 1; c represents the results for fungi (Candida glabrata); and d represents the recognition efficiency of different types of microorganisms at the same concentration. All bacterial concentrations are 10-1. 4 CFU mL -1 .

[0122] Figure 8The results show the sensitivity and accuracy of the bfDEC-AA method, where 'a' represents the calibration curve using a portable device. The curve includes the complementary probe (G... - -bfDNA sensor) and non-complementary probe (G + -bfDNA sensor), b for detecting 10 using a portable device. 3 10 5 107 CFU / mL -1 The recovery rate of *E. coli* is given by denoted as c, where c represents the UV-Vis absorption spectra of *E. coli* at different concentrations of AuNPs, and d represents the calibration curve using a microplate reader, including the complementary probe (G...). - -bfDNA sensor) and non-complementary probe (G + -bfDNA sensor), e is for detecting 10 using an enzyme-linked immunosorbent assay (ELISA) reader. 3 10 5 107 CFU / mL -1 The recovery rate of Escherichia coli.

[0123] Figure 9 The results show the sensitivity and accuracy of the pathogen detection method in Method 2. Among them, a is the UV-Vis absorption spectrum of Escherichia coli at different concentrations, and b is the calibration curve using an ELISA reader, which includes complementary and non-complementary probes.

[0124] Figure 10 This represents the results of antimicrobial susceptibility testing; where 'a' represents sensitive E. coli cultures at concentrations of 8, 16, and 32 μg / mL. -1 AST results after co-incubation with AMP, b represents sensitive E. coli culture with 2, 4, and 8 μg mL -1 AST results after co-incubation with OFX, c represents ESBL-E. coli culture with 8, 16, and 32 μg mL. -1 AST results after co-incubation with AMP, d represents ESBL-E. coli culture with 2, 4, and 8 μg mL -1 AST results after co-incubation with OFX. Detailed Implementation

[0125] The present invention will be further described in detail below through specific embodiments.

[0126] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0127] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

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

[0129] Example 1

[0130] 1. Preparation of AuNPs

[0131] This experiment used the sodium citrate reduction method to prepare 13nm AuNPs. The procedure was as follows: The three-necked flask, magnetic rotor, and condenser required for AuNP synthesis were soaked overnight in aqua regia, rinsed repeatedly with ultrapure water, and dried. 1.7 mL of 1% AuCl4H and 48.3 mL of ultrapure water were mixed and heated and stirred in an oil bath to 140 °C (after reflux of the liquid in the condenser). 5 mL of freshly prepared 38.8 mM C6H5Na3O7 was quickly added, and stirring continued until the color changed from pale yellow to purplish-red. After the color change, heating and stirring were continued for 20 minutes. The heating device was then turned off, and stirring continued until the reaction solution cooled to room temperature. The solution was dispensed and stored at 4 °C in the dark for later use.

[0132] 2. Preparation of Au@Fe3O4

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

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

[0135] 3. Preparation of bfDNA sensor

[0136] With G - Taking the preparation of the -bfDNA sensor as an example, the specific steps for a single use are as follows: G was synthesized using a high-temperature drying method. - -bfDNA-Au@Fe3O4, S1-CuO, and S2-CuO. Take 0.5 μL of 100 μM G... - -bfDNA 1, G - -bfDNA 2 and G - A mixed aqueous solution of -bfDNA3 (specific sequence shown in Table 1) was added to 49.5 μL of a solution containing 1 mg / mL. -1 In an AuNPs@Fe3O4 solution, 1 μL of a 100 μM S1 aqueous solution and 1 μL of a 100 μM S2 aqueous solution were simultaneously added to 99 μL of a 1 mg / mL solution. -1 The CuONPs were dissolved in an aqueous solution (Aladdin, Lot#G2131134). After mixing the solution, it was transferred to beakers and heated on an 80°C hot plate to evaporate excess water. Subsequently, 100 μL of ddH2O was added to each beaker to resuspend the dried NPs (G... - -bfDNA-AuNPs@Fe3O4, S1-CuO, or S2-CuO) and transfer to a centrifuge tube. Centrifuge at 10,000 rpm for 5 minutes to remove DNA not loaded onto the NPs. Discard the supernatant. G - Add 50 μL of 0.5×SSC solution (75 mM NaCl solution, 7.5 mM sodium citrate solution, pH 7.0) to -bfDNA-AuNPs@Fe3O4, and add 100 μL of 0.5×SSC solution to S1-CuO and S2-CuO respectively. Finally, add 50 μL of G to the solution at room temperature. - -bfDNA-AuNPs@Fe3O4 and 100 μL S1-CuO were mixed and reacted (hybridized) for 1 hour, followed by the addition of 100 μL S2-CuO and reacted (hybridized) for another hour. 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 each corresponding strain are the same as above, only the corresponding bfDNA is replaced. The sequence information of the bfDNA, S1, and S2 mentioned above is shown in Table 1.

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

[0139]

[0140]

[0141] Note: In the table, the double-underlined portion represents the random sequence A1, the italicized portion represents the sequence A2, and the single-underlined portion (A3) is complementary to the corresponding 16S rRNA / 18S rRNA sequences of 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 inversely complementary to the bold italicized portion in S1, and the bolded and underlined portions in S1 and S2 are inversely complementary. In the S1 sequence, the bolded and underlined portion is the B1 sequence, and the bolded black italicized portion is the B2 sequence; in the S2 sequence, the bolded and underlined portion is the C1 sequence, and the bolded black italicized portion is the C2 sequence.

[0142] 4. Preparation of ssDNA-bfDNA sensor

[0143] ssDNA-Au@Fe3O4, S1-CuO, and S2-CuO were synthesized using a high-temperature drying method. Specifically, 0.5 μL of a 100 μM ssDNA aqueous solution (sequence shown in Table 2) was added to 49.5 μL of a 1 mg / mL solution. -1 The AuN@Fe3O4 solution was added to the solution. Simultaneously, 1 μL of a 100 μM S1 aqueous solution and 1 μL of a 100 μM S2 aqueous solution were added to 99 μL of a 1 mg / mL solution, respectively. -1The CuONPs were dissolved in an aqueous solution. The solution was mixed thoroughly and transferred to a beaker, then heated on an 80°C hot plate to evaporate excess water. Subsequently, 100 μL of ddH2O was added to each beaker to resuspend the dried NPs, and the mixture was transferred to a centrifuge tube and centrifuged at 10,000 rpm for 5 min 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 solution, 7.5 mM sodium citrate solution, pH 7.0) was added to ssDNA-Au@Fe3O4, and 100 μL of 0.5×SSC solution was added to S1-CuO and S2-CuO respectively. Finally, 50 μL of ssDNA-Au@Fe3O4 and 100 μL of S1-CuO were mixed and reacted (hybridized) at room temperature for 1 h, followed by the addition of 100 μL of S2-CuO and reacted (hybridized) for another 1 h. Unhybridized H1-CuO and S2-CuO are removed by magnetic separation, and the precipitate is S2-CuO-H1-CuO-ssDNA-Au@Fe3O4 (denoted as ssDNA-bfDNA sensor).

[0144] Table 2 shows the sequences used in the ssDNA-bfDNA sensor.

[0145]

[0146]

[0147] Note: In the table, the bolded portion of ssDNA represents the D1 sequence, and the italicized portion (D2 sequence) is anticomplementary to the italicized and bolded portion of S1 in Table 1. The underlined portion of the cDNA sequence is anticomplementary to its corresponding dsDNA sequence, and the dsDNA sequence is anticomplementary to the guide sequence (italicized and bolded portion) of its corresponding crRNA. The anchoring sequence of the crRNA (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 100 μM C≡C-DNA (sequences shown in Table 1) and 1.2–1.92 μL of 100 μM N3-DNA (sequences shown in Table 1) were respectively mixed with 99 μL of 8 nM AuNPs solution. Then, 2 μL of sodium citrate solution (500 mM, pH 3.0) was added to each solution. The mixtures were incubated at room temperature for 3 minutes, centrifuged at 13000 rpm for 15 minutes, washed three times, and resuspended in water for later use.

[0150] Example 2

[0151] A method for detecting pathogenic bacteria includes Method 1 (bfDNA-ExoIII-CuO-AA(bfDEC-AA) method) and Method 2.

[0152] Method 1 includes the following steps:

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

[0154] (2) Add 0–24.5 μL of 1M HCl and 125.5 μL of UL to the treated sample. -1 Exo-III and bfDNA sensors were mixed and reacted for 40 min; magnetic separation was then performed, and 60 μL of the supernatant 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 click reaction.

[0155] (3) Use a portable device / ELISA reader to detect the signal and read the signal value. 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 using Solidworks software. Figure 1 (c) The main body (184×150×112mm) consists of a screen, display screen, light intensity knob, light intensity display screen, heat sink, switch, and constant voltage power supply. The constant voltage power supply can be powered by a wall-mounted power supply or a battery, overcoming the limitations of wall-mounted power supplies. After adding samples to 8 or 12-strip ELISA strips (8 or 12-strip tubes), the signal can be automatically detected and read directly on the display screen. Furthermore, for ease of use, two operating modes are designed: automatic and manual, implemented through a dynamically laid-out interface. Automatic operation can read the signal values ​​of 12 samples within 36 seconds, while manual operation allows for selective detection of specific samples.

[0157] When the bfDNA sensor used is G - -bfDNA sensor, G +The -bfDNA sensor and the Fungus-bfDNA sensor determine whether the sample contains Gram-positive bacteria, Gram-negative bacteria, or fungi based on the signal value. Furthermore, if the bfDNA sensor used corresponds to a specific pathogen, the specific pathogen can be identified.

[0158] The detection principle of Method 1 is as follows: bfDNA is coupled with Au@Fe3O4 to form bfDNA-Au@Fe3O4; S1 or S2 is coupled with CuO nanoparticles to form 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 with S2-CuO to form the detection sensor. To improve detection sensitivity, three cDNA sequences with different sequences are designed based on the conserved regions of each 16S rRNA or 18S rRNA gene according to the National Center for Biotechnology Information (NCBI) database. When the pathogen in the sample is lysed (each bacterium has more than 10,000 copies of 16S rRNA, and each fungus has more than 10,000 copies of 18S rRNA), the 3' end of the bfDNA is paired with the 16S rRNA / 18S rRNA fragment, achieving target recognition and realizing the first signal amplification. Activation of Exo-III cleaves bfDNA and releases CuO nanoparticles. Once the bfDNA is completely cleaved by Exo-III, 16S rRNA / 18S rRNA is re-released and hybridizes with other bfDNA molecules, leading to a new round of release of CuO nanoparticle tandems, achieving a second layer of signal amplification. Finally, under acidic and ascorbic acid-reducing conditions, the CuO nanoparticle tandems in solution dissolve and are reduced to Cu. + This achieves a third level of signal amplification and triggers click chemistry between C≡C-DNA@AuNPs and N3-DNA@AuNPs, initiating gold nanoparticle hybridization (AuNPs agglutination, AA). The signal value is then detected, allowing for the experimental detection of pathogens. This method is denoted as the bfDNA-ExoIII-CuO-AA (bfDEC-AA) method (the schematic diagram is shown below). Figure 1 As shown, the detection process is as follows: Figure 2 (As shown).

[0159] The bfDEC-AA method was used to identify the threshold (10) for urinary tract infection (UTI). 4 CFU mL -1 When the solution changes color, the color can be visually observed to assess whether the urine sample originated from a UTI patient (bacterial 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 one;

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

[0163] (3) Use a portable device / ELISA reader to read the signal. The signal value is the absorbance value at 450 nm. Determine the type of pathogen in the sample based on the signal value.

[0164] The detection principle of Method 2 is as follows: After the pathogen is lysed and releases 16S rRNA / 18S rRNA to achieve the first level of signal amplification, the 16S rRNA / 18S rRNA hybridizes with cDNA in the cDNA and dsDNA hybridization strands, releasing dsDNA. The free dsDNA hybridizes with free cRNA, thereby activating the trans-cleavage activity of Cas 12a, cleaving the 5' end single strand of ssDNA-Au@Fe3O4, releasing S1-CuO and S2-CuO tandem strands, achieving the second level of signal amplification. Finally, under acidic conditions, the CuO nanoparticle tandem strands in the solution dissolve and are reduced to Cu. 2+ This process amplifies the signal a third time. Then, sodium acetate buffer (pH 5.0), TMB, and H2O2 are added, and the reaction is allowed to proceed for 15 minutes. The reaction is then terminated by adding sulfuric acid, and the absorbance at 450 nm is measured to detect the pathogen.

[0165] Example 3

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

[0167] 1. Exo-III concentration and time

[0168] Using different concentrations of E. coli (0, 10) 4CFU mL -1 As a positive sample, the concentration of Exo-III in the reaction system was optimized, and the results are as follows: Figure 3 As shown in Figure a, as the Exo-III concentration decreases from 0 U / L... -1 Increased to 200U L -1 The generated signal value increased, but with further increases in Exo-III concentration, the background signal value (NC) became too large, leading to a decrease in the signal-to-noise ratio. Therefore, 200 U / L was selected in subsequent experiments. -1 The Exo-III. Through time optimization, the results are as follows... Figure 3 As shown in Figure b, the reaction time reached a relatively stable state after 40 minutes, so 40 minutes was chosen for subsequent experiments. To ensure high throughput, this optimization will use a microplate reader for signal reading, with the signal value being the ratio of absorbance at 630 nm to absorbance at 530 nm.

[0169] 2. The molar ratio of N3-DNA to AuNPs and the concentration of N3-DNA-AuNPs.

[0170] The molar ratio of DNA to AuNPs is a key factor affecting the degree of Cu-triggered AuNP aggregation; therefore, molar ratios of N3-DNA to AuNPs ranging from 150:1 to 240:1 were investigated. Figure 4 As shown in Figure 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 determining the color intensity in the reaction system and also affects the ease of visual observation. This study investigated the effect of different concentrations of N3-DNA-AuNPs in the reaction system on the detection results. The results are as follows: Figure 4 As shown in Figure 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 one-to-one click chemistry reactions, the molar ratio of C≡C-DNA to AuNPs and the concentration of C≡C-DNA-AuNPs are selected as 210:1 and 2nM, respectively.

[0173] The second method in Example 2 is further optimized as follows:

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

[0175] Use E. coli (10) 3 CFU mL -1As a positive sample, the concentration of Cas 12a in the reaction system was optimized, and the results are as follows: Figure 5 As shown, the signal value increases with the Cas12a concentration from 0.5 nM to 15 nM. However, with further increases in Cas12a concentration, the background signal value (NC) becomes too large, leading to a decrease in the signal-to-noise ratio. Figure 5 Therefore, Cas12a at 15 nM was chosen in subsequent experiments (a). Figure 5 b represents the effect of time on Cas12a, which reaches a relatively stable state at 30 min. Therefore, a treatment time of 30 min was chosen in subsequent experiments.

[0176] 2. Effects of TMB concentration and time

[0177] Use E. coli (10) 3 CFU mL -1 As a positive sample, the concentration of TMB in the reaction system was optimized, and the results are as follows: Figure 6 As shown, the signal value increases with increasing TMB concentration from 0.05 mM to 5 mM. However, with further increases in TMB concentration, the background signal value (NC) becomes too large, leading to a decrease in the signal-to-noise ratio. Figure 6 Therefore, a TMB concentration of 5 mM was chosen in subsequent experiments, as indicated in a). Figure 6 In the figure, b represents the effect of TMB time, which reaches a relatively stable state at 15 min. Therefore, a time of 15 min was chosen 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 analyzed three different types of bacterial samples using conserved cDNA sequences of the pathogen, including G... - (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes), G + (Staphylococcus aureus, Enterobacter faecalis, Streptococcus pneumoniae, Streptococcus agalactiae) and fungi (Candida glabrata). Measurement A 630 / A 530 The ratio is used to evaluate the ability of a detection method to selectively detect pathogens.

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

[0181] Example 5: Detection sensitivity and accuracy of the detection method

[0182] To evaluate the detection sensitivity and accuracy of the bfDEC-AA method in Example 2, this example tested the linearity and spike recovery rate of the method.

[0183] Since there was no significant difference in recognition efficiency among different strains at the same concentration ( Figure 7 Therefore, in this embodiment, Escherichia coli was selected at a concentration of 500–100 mg / L. 8 CFU mL -1 A linear relationship is established within the specified range, following the same process as in Example 2. Additionally, a non-complementary probe (G) is used. + The -bfDNA sensor measured seven different concentrations (500, 10...). 3 10 4 10 5 10 6 10 7 10 8 CFU mL -1 Escherichia coli standard solution.

[0184] The results are as follows Figure 8 And Table 3. Escherichia coli standard solutions of different concentrations and A 630 / A 530 There is a strong correlation between the ratios, and portable devices ( Figure 8 a) and ELISA reader ( Figure 8 The correlation coefficients for c and d are r = 0.9985 and r = 0.9944, respectively. The curves include complementary probes (G... - -bfDNA sensor) and non-complementary probe (G + -bfDNA sensor), non-complementary probe pairs up to 10 8 CFU mL -1 The reaction rate of the standard E. coli solution was low (below 3 SD). The limit of detection (LOD) of the microplate reader (S / N = 3) was 331.8 CFU / mL. -1 The LOD of the portable device is 119.1 CFU / mL. -1 This indicates very high sensitivity. At the same time, such as Figure 8 As shown in the upper right of image a, the aggregation degree of AuNPs is positively correlated with bacterial concentration, and this correlation is particularly strong at concentrations ≥10. 4CFU mL -1 The color change can be discerned with the naked eye. Furthermore, different concentrations of E. coli samples were analyzed using a portable device (…). Figure 8 (b) and ELISA reader ( Figure 8 The recoveries of 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 rate of Escherichia coli in spiked samples

[0186]

[0187]

[0188] To evaluate the detection sensitivity and accuracy of Method 2 in Example 2, this example tested the linearity of the method.

[0189] Select Escherichia coli with a concentration of 20–10. 7 CFU mL -1 A linear relationship was established within the specified range, following the same procedure as in Example 2. Additionally, non-complementary probes were used to measure seven different concentrations of 20 and 10... 2 10 3 10 4 10 5 10 6 10 7 CFU mL -1 Escherichia coli standard solution.

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

[0191] Example 6 Antimicrobial Susceptibility

[0192] To evaluate whether the bfDEC-AA method in Example 2 can be used for antimicrobial susceptibility testing, bacteria were co-cultured with different concentrations of ampicillin (AMP) or ofloxacin (OFX), and the bfDEC-AA method was used to quantitatively detect bacterial growth by adding 10... 4 CFU mL -1The effects of AMP or OFX on two E. coli species were evaluated using either a sensitive E. coli solution or an E. coli solution producing extended-spectrum β-lactamase (ESBL) solution. Details are as follows:

[0193] The initial concentration was 10. 4 CFU mL -1 Escherichia coli (sensitive E. coli or ESBL-E. coli) was added to centrifuge tubes containing 4 mL of LHB medium. Tube NO.1 contained no drug, while tubes NO.2-4 contained final concentrations of 8, 16, and 32 μg / mL, respectively. -1 Ampicillin (AMP) was added to test tubes NO.5–7, respectively, to final concentrations of 2, 4, and 8 μg / mL. -1 Ofloxacin (OFX) was cultured at 37°C and 250 rpm for 0, 1, 1.5, 2, and 2.5 hours, respectively. The bacterial suspensions were collected and analyzed using the bfDEC-AA method described in Example 2.

[0194] The results are as follows Figure 10 As shown. Compared with the control group, after 1.5 hours, 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 using high-concentration antibiotics (32 μg / mL) -1 AMP / 8μg mL -1 OFX does not inhibit the growth of ESBL-E. coli. Figure 10 (c and d) suggest that the bfDEC-AA method of Example 2 can be used for antimicrobial susceptibility testing.

[0195] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A reagent kit, characterized in that, Including sensors a1 and a2 for detecting pathogens. a1) includes bfDNA-metal nanoparticles, S1-CuO, and S2-CuO; a2) includes ssDNA-metal nanoparticles, S1-CuO and S2-CuO; 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, wherein A3 specifically targets and binds to the 16S rRNA / 18S rRNA of the pathogen. The nucleotide sequence of S1 includes B1 and B2, wherein B2 is inversely complementary to the portion of A2. The nucleotide sequence of S2 includes C1 and C2, wherein C1 is anticomplementary to B1; and C2 is anticomplementary to B2. The nucleotide sequence of the ssDNA includes D1 and D2, wherein D2 is identical to the A2 portion; When the sensor is a1), the kit further includes an exonuclease, DNA-AuNPs with C≡C- modified at the 3' end, DNA-AuNPs with N3- modified at the 3' end, and a reducing agent; When the sensor is a2), the kit also includes Cas protein, crRNA, cDNA, dsDNA, H2O2, and tetramethylbenzidine; The crRNA includes an anchor sequence and a guide sequence, wherein the anchor sequence specifically binds to the Cas protein and the guide sequence targets dsDNA; The cDNA and the dsDNA are at least partially reverse complementary; The cDNA at least partially targets the 16S rRNA / 18S rRNA of the pathogen. The nucleotide sequence of the cr RNA includes at least one of SEQ ID NO:51 to SEQ ID NO:59; The nucleotide sequence of the cDNA includes at least one of SEQ ID NO:33 to SEQ ID NO:41; The nucleotide sequence of the dsDNA includes at least one of SEQ ID NO:42 to SEQ ID NO:50; The bfDNA is named A1, A2, and A3 from the 5' end to the 3' end; The nucleotide sequence of the bfDNA is a random sequence in A1; the random sequence does not contain more than 6 consecutive bases that are complementary to and / or anticomplementary to the bfDNA, S1, and S2 sequences.

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

3. The kit according to claim 1 or 2, characterized in that, The bfDNA includes a 5' end modification.

4. The reagent kit according to claim 3, characterized in that, The 5' end modification includes at least one of the following: thiol modification, phosphorylation modification, phosphorylation analog modification, amino modification, dideoxy modification, interarm modification, 3'-O-propenyl modification, or 3'-O-azidomethyl modification.

5. The kit according to claim 1 or 2, characterized in that, The 3' ends of S1 and S2 contain tail sequences; The nucleotide sequence of the tail sequence is 5'-TTTTTT-3'.

6. The kit according to claim 1 or 2, characterized in that, The nucleotide sequence of the ssDNA is shown in SEQ ID NO:32; The nucleotide sequence of the bfDNA includes at least one of SEQ ID NO:1 to SEQ ID NO:

27.

7. The reagent kit according to claim 6, characterized in that, The pathogens include at least one of Gram-positive bacteria, Gram-negative bacteria, and fungi.

8. The use of the kit according to any one of claims 1 to 7 in any one of (1) to (2): (1) Prepare products for detecting pathogens; (2) Prepare products for antimicrobial susceptibility testing.

9. A method for detecting pathogens for non-disease diagnostic purposes, comprising the step of detecting a sample to be tested using the kit described in any one of claims 1 to 8.

10. The method according to claim 9, characterized in that, The method also includes a fluorescence signal reading step or observation of the color change of the reaction system after detection.

11. The method according to claim 9, characterized in that, Use portable detection devices and / or ELISA readers to read fluorescence or visible light signals; The portable testing device includes 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. The linear stepper motor guide slide is used to advance the sample placement plate; The sample placement plate has a sample placement opening in the middle.

Images