A rapid method for detecting microorganisms

Abstract

The present application relates to the technical field of microorganism detection, and discloses a rapid microorganism detection method. The method comprises the following steps: lysing a to-be-detected sample to release pathogenic bacteria target nucleic acid; contacting the target nucleic acid with a DNAzyme recognition system, so that the DNAzyme responds to the pathogenic bacteria target nucleic acid to generate a cleavage reaction and release a copper-based signal carrier; after the copper-based signal carrier is treated, the copper-based signal carrier participates in a copper-catalyzed click reaction to form a signal conversion product; the signal conversion product is detected, and the presence and / or content of pathogenic bacteria in the to-be-detected sample are determined according to a detection signal; and according to detection requirements, the nucleic acid signal can be further amplified before detection. The method is suitable for samples such as whole blood, plasma, serum, urine, biological products, drugs and medical device eluate, and can be used for pathogenic bacteria detection, infection auxiliary diagnosis, antibacterial drug sensitivity analysis and drug sterility detection.

Description

Technical Field

[0001] This invention relates to the field of microbial detection technology, and in particular to a rapid method for detecting microorganisms. Background Technology

[0002] Bloodstream infection (BSI) is an acute infectious disease caused by pathogenic microorganisms such as bacteria and fungi invading the circulatory system and multiplying rapidly. It is a major cause of sepsis in clinical practice. BSI has a rapid onset and progression, and can quickly lead to serious complications such as septic shock. Early and accurate identification of the pathogen and timely antimicrobial susceptibility testing (AST) are crucial for implementing targeted antimicrobial therapy, reducing patient mortality, and improving prognosis. However, the pathogen load in the early stages of BSI is extremely low, as low as 1 CFU / mL, far below the detection limit of conventional detection methods. At the same time, the complex composition of the blood matrix, with interfering substances such as hemoglobin and immunoglobulins, significantly increases the difficulty of detection and the risk of false negatives.

[0003] In the field of pharmaceutical manufacturing and quality control, sterility testing of drugs is a crucial legal requirement for ensuring medication safety. According to pharmacopoeia standards of various countries, current drug sterility testing primarily employs membrane filtration and direct inoculation methods, relying on microbial culture for determination, with testing cycles as long as 14 days. Due to the excessively long result turnaround time, companies cannot promptly detect contaminated batches before production release. They typically have to release the contaminated batches first, allowing them to enter warehousing, distribution, and even clinical use. If subsequent positive results are detected, companies face large-scale recalls, production halts, destruction, and substantial compensation claims, resulting in extremely severe economic losses and safety risks.

[0004] Therefore, whether for rapid clinical BSI diagnosis or rapid screening of drug sterility, there is an urgent need for a detection technology that combines speed, high sensitivity, anti-interference ability, and automation.

[0005] However, current testing methods in the clinical and pharmaceutical industries all have significant limitations. Specifically, existing technologies mainly suffer from the following shortcomings: (1) Traditional detection methods are too time-consuming. The clinically common blood culture method requires 24-48 hours to determine the growth of pathogenic bacteria, and the subsequent identification of bacterial species and AST still require an additional 24-72 hours. The entire process often takes several days to complete. The sterility test of drugs, according to the pharmacopoeia standard, takes up to 14 days, which cannot meet the time requirements of emergency treatment for critically ill patients and the production needs of pharmaceutical companies for rapid quality control.

[0006] (2) Insufficient detection capability of low-abundance pathogens. Whether it is the trace pathogens in the blood samples of patients with early infection or the contaminants that may remain in the samples of sterile drugs, the content of pathogens is extremely low. However, the sensitivity of existing conventional detection methods is insufficient, which can easily lead to missed detections and make it impossible to achieve stable and accurate detection of trace pathogens.

[0007] (3) Sample matrix interference seriously affects detection accuracy. Human whole blood samples contain endogenous impurities such as hemoglobin, immunoglobulins, lipids and various proteins. Drug test samples also contain a variety of pharmaceutical excipients and matrix components. All of these substances can interfere with the normal operation of the nucleic acid reaction system and the effective acquisition of fluorescence signals, resulting in high detection background and significantly reducing the accuracy and stability of the detection.

[0008] (4) Existing detection modes have limited functionality. Most rapid detection methods can only perform preliminary screening of pathogens, cannot simultaneously conduct phenotypic AST, cannot provide drug sensitivity results that can be directly referenced in clinical practice, cannot effectively guide the rational selection of antimicrobial drugs in clinical practice, and cannot form a complete detection system that integrates screening, identification, and drug sensitivity.

[0009] (5) The testing process is complex and has a low degree of automation. Most existing testing methods are labor-intensive and require high professional skills from operators. This is not only unfavorable for widespread use in primary healthcare institutions, but also difficult to meet the needs of pharmaceutical companies for large-scale, automated, aseptic testing.

[0010] In summary, existing technologies cannot simultaneously meet the multi-dimensional requirements of rapid, highly sensitive, interference-resistant, simple, and compatible clinical pathogen detection and drug sterility detection. Therefore, developing a new method that can integrate these functions has become a key technical problem urgently needing to be solved in this field. Summary of the Invention

[0011] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of this invention is to provide a rapid microbial detection method. The method of this invention, based on a self-developed dedicated lysis buffer, enables rapid detection of various microbial strains and is applicable to multiple sample systems, including clinical blood samples and drug samples. It can be used for infection diagnosis and AST (assay-assay), and can also be extended to sterile testing scenarios for other samples such as urine and medical devices, demonstrating excellent versatility.

[0012] In a first aspect, the present invention provides a method for detecting pathogenic microorganisms, comprising the following steps: S1, lyse the sample to be tested to release the target nucleic acid of the pathogen; S2, the target nucleic acid is brought into contact with the DNAzyme recognition system, so that the DNAzyme responds to the pathogen target nucleic acid to cleave and releases a copper-based signal carrier; S3, the copper-based signal carrier is acidified and reduced to generate catalytically active copper ions, which catalyze a copper-catalyzed click reaction to form signal conversion products; S4, detect the signal conversion product, and determine the presence and / or content of pathogens in the sample to be tested based on the detection signal.

[0013] In some embodiments of the present invention, the detection method further includes: performing nucleic acid signal amplification after S3 before detection.

[0014] In some embodiments of the present invention, the nucleic acid signal amplification includes: performing nucleic acid signal amplification using hybridization chain reaction (HCR).

[0015] In some embodiments of the present invention, the DNAzyme recognition system is capable of recognizing conserved sequences of bacterial 16S rRNA and / or fungal 18S rRNA.

[0016] In some embodiments of the present invention, the DNAzyme recognition system includes: a probe sequence or a nucleic acid sequence having the same pathogen target recognition and cleavage function, a substrate chain (Sub), copper oxide tandem linking chains C1 and C2, a click reaction complex and a hybridization chain reaction complex.

[0017] In some embodiments of the present invention, the probe sequence includes at least one of SEQ ID NO: 1-20.

[0018] In some embodiments of the present invention, the probe sequence may be reasonably designed and adjusted based on the target analyte, including but not limited to at least one of SEQ ID NO: 1-20.

[0019] In some embodiments of the present invention, the substrate chain is as shown in SEQ ID NO: 18.

[0020] In some embodiments of the present invention, the substrate chain can be reasonably adjusted based on specific usage scenarios and usage requirements, including but not limited to using the substrate chain shown in SEQ ID NO: 18.

[0021] In some embodiments of the present invention, the copper oxide tandem connecting chains C1 and C2 are as shown in SEQ ID NO: 19-20.

[0022] In some embodiments of the present invention, the click-reactive complex chain comprises or is composed of: SEQ ID NO: 21 and 22.

[0023] In some embodiments of the present invention, the cascade reaction reagent includes several pairs of hybrid chain reaction complexes.

[0024] In some embodiments of the present invention, the cascade reaction reagent includes two pairs of hybrid chain reaction complexes.

[0025] In some embodiments of the present invention, the hybridization chain reaction complex includes: SEQ ID NO: 23-24 and 25-26.

[0026] In some embodiments of the present invention, SEQ ID NO: 23-24 are used in combination (to form a complex), and SEQ ID NO: 25-26 are used in combination (to form a complex).

[0027] In some embodiments of the present invention, the copper-based signal carrier includes: copper oxide magnetic beads, copper oxide nanoparticles, cuprous oxide nanoparticles, or other copper-based materials capable of participating in click reactions.

[0028] In some embodiments of the present invention, the pyrolysis includes pyrolysis using a mild pyrolysis solution or a rapid pyrolysis solution.

[0029] In some embodiments of the present invention, the mild lysis buffer is used to lyse microorganisms in blood samples.

[0030] In some embodiments of the present invention, the blood sample includes: whole blood sample, serum sample or plasma sample.

[0031] In some embodiments of the present invention, the rapid lysis buffer is used to lyse microorganisms in non-whole blood samples.

[0032] In some embodiments of the present invention, the mild lysis buffer includes EDTA, lysozyme, RNase inhibitor and sucrose.

[0033] In some embodiments of the present invention, the mild lysis solution further includes a solvent.

[0034] In some embodiments of the present invention, the solvent includes: buffer solution or water.

[0035] In some embodiments of the present invention, the buffer solution comprises: Tris-HCl buffer solution.

[0036] In some embodiments of the present invention, the mild lysis buffer consists of EDTA, lysozyme, RNase inhibitor, sucrose, and Tris-HCl buffer.

[0037] In some embodiments of the present invention, the mild lysis buffer comprises, based on final concentration: EDTA: 5-40 mM; lysozyme: 0.2-2 mg / mL; RNase inhibitor: 0.2-2 U / μL; sucrose: 0.5%-4%.

[0038] In some embodiments of the present invention, the mild lysis buffer comprises, by final concentration: 10 mM EDTA, 1 mg / mL lysozyme; 1 U / μL RNase inhibitor, 3% sucrose; and 50 mM buffer.

[0039] In some embodiments of the present invention, the rapid lysis solution includes EDTA, sodium hydroxide, and Triton X-100.

[0040] In some embodiments of the present invention, the rapid lysis solution comprises, by final concentration: EDTA: 5-40 mM; sodium hydroxide: 0.05-1 M; Triton X-100: 0.1-1%.

[0041] In some embodiments of the present invention, the rapid lysis solution comprises, by final concentration: 10 mM EDTA, 0.1 M sodium hydroxide, and 0.5% Triton X-100.

[0042] The samples to be tested include one or more of the following: whole blood, plasma, serum, urine, cerebrospinal fluid, ascites, pleural and peritoneal effusions, biological products, pharmaceuticals, and medical device eluents.

[0043] In some embodiments of the present invention, the microorganisms include bacteria and fungi.

[0044] In some embodiments of the present invention, the bacteria include Gram-negative bacteria and Gram-positive bacteria.

[0045] In some embodiments of the present invention, the microorganisms include: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, and Candida albicans.

[0046] In a second aspect, the present invention provides the application of the above-mentioned detection methods in pathogen detection, antimicrobial susceptibility analysis, and drug sterility testing.

[0047] In some embodiments of the present invention, the applications do not involve the diagnosis of diseases.

[0048] A third aspect of the present invention provides a microbial detection product comprising: the lysis buffer and DNAzyme recognition system described above.

[0049] In some embodiments of the present invention, the microbial detection product further includes a solvent.

[0050] In some embodiments of the present invention, the microbial detection product may be in the form of a detection reagent, a detection kit, or a kit.

[0051] A fourth aspect of the present invention provides the application of the microbial detection products described above in microbial detection.

[0052] A fifth aspect of the present invention provides the application of the microbial detection products described above in the construction of an antimicrobial drug screening platform.

[0053] A sixth aspect of the present invention provides the application of the microbial detection products described above in strain screening.

[0054] In some embodiments of the present invention, the strains include sensitive strains and resistant strains.

[0055] The beneficial effects of this invention are: 1. This invention pioneers a novel detection method, effectively improving detection sensitivity and timeliness. The method breaks through the limitations of traditional single-signal amplification strategies, innovatively constructing a multi-stage cascaded signal amplification system of "target recognition – DNAzyme cleavage – click chemical coupling – hybridization chain reaction." In this system, each stage of the reaction is tightly coupled and driven step-by-step according to a preset sequence, achieving deep integration and synergistic effect of signal transduction, chemical linkage, and nucleic acid amplification. Based on this cascade logic, this invention can transform weak target recognition events into significant and precisely quantifiable fluorescence signals, thereby achieving stable detection of pathogens down to 1 CFU / mL. Simultaneously, thanks to efficient sample pretreatment and the efficient synergy of the cascaded amplification reaction, the overall time for BSI and drug sterility testing is reduced to within 2 hours, and the drug sensitivity testing time is reduced to within 5 hours, fundamentally solving the problems of timeliness in clinical emergency care and excessively long sterility quality control cycles in pharmaceutical companies.

[0056] 2. The method of this invention integrates pathogen identification, phenotypic drug susceptibility testing, and sterility detection into a unified system. This method not only provides highly sensitive qualitative / quantitative detection of pathogens, but also enables simultaneous AST (assay-assay) detection of pathogenic bacteria on a single platform, forming an integrated technical solution from screening, identification, drug susceptibility testing to sterility detection. Compared to molecular biology methods that only provide nucleic acid detection results, the method of this invention better meets the actual needs of clinical medication decision-making, and a single system can simultaneously meet the needs of multiple application scenarios, including clinical diagnosis, pharmaceutical and medical device quality control.

[0057] 5. This invention provides two dedicated lysis systems for different application scenarios: a mild lysis buffer with low hemolysis and low background, enabling direct and complete lysis of pathogens in whole blood samples, eliminating complex pretreatment steps; and a rapid lysis buffer that can efficiently lyse bacteria in non-whole blood samples (such as sterile drug testing samples) within 2 minutes without heating, offering simple operation and strong versatility. The synergistic effect of these two solutions ensures the method's high efficiency in processing complex samples and excellent anti-interference capabilities, further improving the accuracy and reliability of the detection results.

[0058] 6. The core cascade logic in this invention has good replaceability and scalability. The multi-stage cascade amplification logic in this invention does not rely on a single specific enzyme or reaction. It can be adaptively adjusted based on the target substance to its pre-stage cleavage recognition system (such as DNAzyme, copper-based nanomaterials) and post-stage nucleic acid amplification system (such as hybridization chain reaction). While ensuring that the core logic of "target recognition → cleavage trigger → click coupling → chain amplification" remains unchanged, each functional module has rich replaceability, which significantly improves the application scenarios of the method in this invention. Attached Figure Description

[0059] Figure 1 The lysis effects of the mild lysis buffer and the rapid lysis buffer in this invention are shown. A represents the hemolysis of the system based on absorbance; B and C represent the effects of Escherichia coli and Staphylococcus aureus on lysis with mild lysis buffer and heating, respectively; D and E represent the comparison of detection signals of Escherichia coli and Staphylococcus aureus in different matrices; and F represents the comparison of signal recovery before and after the addition of lysis buffer to the drug sample.

[0060] Figure 2 The results of the composition optimization experiment for the mild lysis buffer are shown, where AE corresponds to the optimization results of EDTA concentration, lysozyme concentration, RNase inhibitor concentration, sucrose concentration, and lysis time, respectively.

[0061] Figure 3 The results of the composition optimization experiments for the rapid lysis buffer are shown, where AD corresponds to the optimization results of EDTA concentration, sodium hydroxide concentration, Triton X-100 concentration, and lysis time, respectively.

[0062] Figure 4 This is a schematic diagram illustrating the principle of the detection method of the present invention.

[0063] Figure 5 This is a schematic diagram of the detection process of the detection method of the present invention.

[0064] Figure 6 The results represent the specific detection results of the detection method of the present invention, wherein AC corresponds to Gram-negative bacteria, Gram-positive bacteria, and fungi, respectively.

[0065] Figure 7 The results show the sensitivity (A), accuracy (BC), and recovery rate (D) of the detection method of this invention.

[0066] Figure 8 The effects of probe (13PD1) concentration (A), the number of bases bound to the target by the probe (B), and enzyme digestion time (C) on the detection effect of the detection method of the present invention are shown.

[0067] Figure 9 The effects of the molar ratio (A) of the click reaction component (C3N3) and the concentration (B) of the click reaction component (C3N3) on the detection effect of the detection method of the present invention are shown.

[0068] Figure 10 The effects of the molar ratio (A) of hybridization chain reaction component (P1T1), the concentration (B) of hybridization chain reaction component (P1T1), the molar ratio (C) of hybridization chain reaction component (A1A2), and the concentration (D) of hybridization chain reaction component (A1A2) on the detection effect of the detection method of the present invention are shown.

[0069] Figure 11 The effects of catalyst (THPAT) concentration (A) and cascade reaction time (B) on the detection effect of the detection method of the present invention are shown.

[0070] Figure 12 The results are based on the method of the present invention for antimicrobial susceptibility testing, where AB represents the detection results of ceftazidime in susceptible and resistant Escherichia coli, respectively; and CD represents the detection results of meropenem in susceptible and resistant Escherichia coli, respectively.

[0071] Figure 13 The results of the detection of pathogenic bacteria in different samples based on the method of the present invention are as follows: No. 1-20 are blood samples, No. 21-28 are antibiotic injections, No. 29-39 are nutrients and homeostasis regulators, No. 40-48 are biological products, and No. 49-50 are traditional Chinese medicine injections. Detailed Implementation

[0072] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0073] Example 1 This embodiment provides two bacterial lysis buffers, which are specially designed to address the matrix differences of whole blood samples and sterile drug samples, respectively, to meet the pretreatment requirements of different samples. Neither of the two lysis buffers requires heating during use.

[0074] (1) Mild lysis buffer suitable for whole blood samples: This mild lysis buffer is used for whole blood sample pretreatment. Its components include: 10 mM EDTA, 1 mg / mL lysozyme, 1 U / μL RNase inhibitor (purchased from Shenzhen Kangti Biomedical Technology Co., Ltd.), 3% sucrose, and 50 mM buffer (based on final concentration). The buffer is Tris-HCl (pH 7.5). The lysis buffer has an incubation time of 5-25 minutes (typically 20 minutes) at room temperature. It exhibits low hemolysis and low background, effectively lysing pathogens and releasing targets while preserving blood cell integrity. It is suitable for direct bacterial lysis of whole blood samples.

[0075] (2) Rapid lysis buffer suitable for sterile drug samples: This rapid lysis buffer is used for the pretreatment of sterile pharmaceutical samples. Its components include, by final concentration: 10 mM EDTA, 0.1 M sodium hydroxide, and 0.5% Triton X-100. The lysis buffer reacts at room temperature for 0.5–4 minutes (typically 2 minutes). It rapidly breaks down the cell walls and membranes of pathogenic bacteria, making it suitable for rapid lysis of bacteria in non-whole blood samples.

[0076] The specific usage methods for the two bacterial lysis buffers are as follows: For mild lysis buffer: Take 500 μL of whole blood sample, add 500 μL of mild lysis buffer and 4 mL of physiological saline, lyse at room temperature for 20 minutes, centrifuge for 5 minutes, and take 150 μL of supernatant for subsequent identification reaction.

[0077] For the rapid lysis buffer, mix 500 μL of the drug sample to be tested with 500 μL of rapid lysis buffer and lyse at room temperature for 2 minutes. Then, add 500 μL of 1M Tris-HCl buffer (pH 7.4) to neutralize and take 150 μL of the supernatant for subsequent identification reaction.

[0078] The effectiveness of the bacterial lysis buffer was tested.

[0079] For the mild lysis buffer, after processing the whole blood sample according to the above steps, take 150 μL of supernatant and add it to the reaction system containing 30 μL of the corresponding 500 nM 13PD1 probe sequence and 120 μL of 13PD1 buffer (composed of 50 mM hydroxyethylpiperazine ethanesulfonic acid (Tris) buffer, 100 mM NaCl, 20 mM MgCl2 and 2 mM MnCl2, pH 7.0) containing a copper oxide magnetic bead sensor. The total reaction volume is 300 μL. React at room temperature for 40 min. After magnetic separation, 100 μL of supernatant was collected and 20 μL of 43 mM HCl solution was added, followed by 180 μL of cascade reaction buffer (composed of 20 μL of 45 mM ascorbic acid, 30 μL of 700 nM MC3-N3 complex mixture, 30 μL of 200 nM P1-T1 complex mixture, 30 μL of 100 nM A1-A2 complex mixture, and 70 μL of Tris-T buffer, pH 7.5). The reaction was carried out in the dark for 30 min. The Tris-T buffer was prepared by mixing 20 mM Tris and 1 mM trihydroxypropyltriazolylmethylamine (THPTA). Finally, fluorescence intensity was detected and quantitatively analyzed using a multi-functional microplate reader or automated device.

[0080] To evaluate the hemolytic effect of the lysis buffer components on whole blood, the following steps were performed to verify the effect. Specifically, the following experimental groups were designed: Blank control group (BC group): 500 μL lysis buffer + 4.5 mL physiological saline, mix well.

[0081] Negative control (no lysis buffer, group S): Take 500 μL of whole blood + 500 μL of physiological saline + 4 mL of physiological saline and mix well.

[0082] Positive control (complete lysis, PW group): Take 500 μL of whole blood + 500 μL of distilled water + 4 mL of physiological saline and mix well.

[0083] Experimental group (L group): Take 500 μL of whole blood + 500 μL of lysis buffer + 4 mL of physiological saline and mix well.

[0084] Subsequently, each group of samples was incubated at room temperature for 20 min, then centrifuged at 4000 rpm / min for 5 min to precipitate unruptured cells. The supernatant contained hemoglobin released from ruptured cells. The supernatant was transferred to a quartz cuvette, and the absorbance (OD) of each group's supernatant was measured. 414 ).

[0085] To evaluate the lysis efficiency of the mild lysis buffer, it was compared with the traditional alkaline pyrolysis method to verify its lysis effect. Specifically, Escherichia coli and Staphylococcus aureus bacterial suspensions were diluted with physiological saline to the required final concentration (10-100 CFU / mL), and then lysed using both the alkaline pyrolysis method and the mild lysis buffer. Subsequent detection steps were the same as described above. The specific steps for the alkaline pyrolysis method were as follows: 500 μL of bacterial suspension was added to 500 μL of 1 M NaOH, boiled for 5 min, neutralized with 499.5 μL of HCl, and then diluted with 3.5 mL of physiological saline. 150 μL of the bacterial lysis buffer was then taken and detected according to the above detection steps.

[0086] To evaluate the anti-interference ability of the mild lysis buffer in whole blood, a control group (physiological saline) and an experimental group (whole blood) were designed. Both groups used Escherichia coli and Staphylococcus aureus bacterial suspensions, spiked to the final bacterial concentration (10 CFU / mL), and lysed according to the sample treatment steps of the mild lysis buffer described above. 150 μL of supernatant was collected and detected according to the detection steps described above.

[0087] To evaluate the overall signal recovery capability of mild lysis buffer in whole blood, a control group (without lysis buffer) and an experimental group (with lysis buffer) were designed. Both groups used high-concentration Escherichia coli bacterial suspension. Whole blood was spiked to a final bacterial concentration of 100 CFU / mL and lysed according to the sample treatment steps described above for mild lysis buffer (the control group was not lysed with lysis buffer). 150 μL of supernatant was collected and detected according to the detection steps described above.

[0088] The results are as follows Figure 1 As shown.

[0089] For the rapid lysis buffer, process the drug sample as described above. Take 150 μL of supernatant and add 30 μL of the corresponding 500 nM 13PD1 probe sequence and 120 μL of 13PD1 buffer containing copper oxide magnetic beads to the reaction system, for a total reaction volume of 300 μL. Incubate at room temperature for 40 min. After magnetic separation, take 100 μL of supernatant and add 20 μL of 43 mM HCl solution, followed by 180 μL of cascade reaction solution (composed of 20 μL of 45 mM ascorbic acid, 30 μL of 700 nM C3-N3 complex mixture, 30 μL of 200 nM P1-T1 complex mixture, 30 μL of 100 nM A1-A2 complex mixture, and 70 μL of Tris-T buffer, pH 7.5). Incubate in the dark for 30 min. Finally, use a multi-functional microplate reader or automated device to detect fluorescence intensity and perform quantitative analysis. The 13PD1 probe sequence used was determined based on the corresponding target objects in Table 1 below.

[0090] The copper oxide magnetic bead sensor used in this invention was prepared according to the method disclosed in the inventor's prior patent CN119799857A and prior literature "CRISPR-Programmed CuO Nanocatalyst Release for Ultrasensitive Detection of Pathogens Analytical Chemistry 2025, 97 (40)". The specific preparation process is as follows.

[0091] 1. Preparation of AuNPs In this embodiment, AuNPs with a diameter of 13 nm were prepared using the sodium citrate reduction method. Specifically, before the experiment, the three-necked flask, condenser, and magnetic stirrer were soaked in aqua regia, then rinsed three times with ultrapure water and dried. 1.7 mL of 1% HAuCl4·4H2O was added to the three-necked flask, followed by 48.3 mL of ultrapure water, and the mixture was stirred and heated to 140 °C. Once liquid reflux began to appear in the condenser, 5 mL of 38.8 mM sodium citrate was quickly added, and stirring continued for 30 min. Heating was then stopped, and the mixture was allowed to cool naturally to room temperature. The resulting solution was aliquoted and stored at 4 °C.

[0092] 2. Preparation of Au@Fe3O4 In this embodiment, Fe3O4 magnetic nanoparticles were synthesized using a solvothermal method. First, 0.65 g of FeCl3 was weighed and added to 20 mL of ethylene glycol, and the mixture was stirred continuously until the solution became clear. Then, 0.30 g of sodium citrate was weighed and added to the solution, and the mixture was stirred for 1 h. Next, 1.20 g of sodium acetate was added, and the mixture was stirred for another 40 min. The mixture was then placed in a 100 mL stainless steel high-pressure reactor lined with polytetrafluoroethylene (PTFE) and reacted at 200 °C for 10 h. After the reactor cooled to room temperature, the product was collected using magnetic separation. After washing with anhydrous ethanol and ultrapure water, the product was vacuum-dried at 60 °C for 6 h to obtain the Fe3O4 magnetic nanoparticles.

[0093] Weigh 10 mg of the Fe3O4 magnetic nanoparticles obtained in the above steps and add them to 10 mL of an aqueous solution containing 3% (v / v) polydiallyldimethylammonium chloride (PDDA). React on a shaker for 20 min, followed by a reaction at 10000 rpm·min. -1 Centrifuge for 5 min to remove excess PDDA solution. After three washes, disperse the product in 50 mL of the AuNPs solution prepared in the above steps, and continue stirring for 8 h to obtain Au@Fe3O4 magnetic nanoparticles. Perform magnetic separation on the Au@Fe3O4 magnetic nanoparticles in the system, wash three times with ultrapure water, disperse in 10 mL of ultrapure water, and store at 4 ℃ for later use.

[0094] 3. Fabrication of copper oxide magnetic bead sensors After reacting the thiol-modified Sub, C1, and C2 with 20 mM TCEP for 1 h, 1 μL of 100 μM Sub was added to 100 μL of 1 mg·mL⁻¹ -1 In an Au@Fe3O4 solution, 2 μL of 100 μM C1 and C2 were added to 200 μL of 1 mg·mL⁻¹ solution, respectively. -1 The sample was dried in a CuONPs solution at 80 °C. It was then resuspended in 100 μL of ultrapure water and heated at 10,000 rpm·min⁻¹. -1 Centrifuge for 5 min and wash 3 times. Finally, mix 100 μL Sub-Au@Fe3O4 with 200 μL Cl-CuO and react for 1 h, then add 200 μL C2-CuO and react for another 1 h. Perform magnetic separation and washing to obtain the copper oxide magnetic bead sensor.

[0095] The relevant sequence information of the above probes and copper oxide magnetic bead sensors is shown in the table below.

[0096] Table 1. Sequence information of the 13PD1 probe and copper oxide magnetic bead sensor in this embodiment.

[0097] It should be understood that the sequences listed in the above embodiments are merely preferred embodiments. Nucleic acid sequences with the same recognition and cleavage functions obtained by those skilled in the art based on the same design principles are all within the scope of protection of this invention.

[0098] The results are as follows Figure 1 As shown.

[0099] It can be observed that the mild lysis buffer can achieve complete lysis of pathogens at room temperature while minimizing red blood cell rupture, and can be directly applied to whole blood sample testing. The rapid lysis buffer system can rapidly release target nucleic acids in a short time, meeting the needs of large-scale, rapid, and sterile screening of drugs.

[0100] Example 2 In this embodiment, the optimization of the content of each component in the mild lysis solution is explored.

[0101] In this embodiment, single-factor experiments were conducted according to the requirements for EDTA concentration (5-40 mM), lysozyme concentration (0.2-2 mg / mL), RNase inhibitor concentration (0.2-2 U / μL), sucrose concentration (0.5-4%), and lysis time (5-25 min). The detection methods were the same as in Example 1 above.

[0102] The results are as follows Figure 2 As shown.

[0103] It was found that the optimal choice was: 10 mM EDTA, 1 mg / mL lysozyme; 1 U / μL RNase inhibitor, 3% sucrose, and treatment for 20 minutes. Therefore, the optimal formulation of the mild lysis buffer is: 10 mM EDTA, 1 mg / mL lysozyme; 1 U / μL RNase inhibitor (purchased from Shenzhen Kangti Biomedical Technology Co., Ltd.), 3% sucrose, 50 mM buffer, and treatment for 20 minutes.

[0104] Example 3 In this embodiment, the optimization of the content of each component in the rapid lysis solution is explored.

[0105] In this embodiment, single-factor tests were conducted on EDTA concentration (5-40 mM), sodium hydroxide concentration (0.05-1 M), Triton X-100 concentration (0.1-1%), and pyrolysis time (0.5-4 min) according to the single-factor experimental requirements. The detection method was the same as in Example 1 above.

[0106] The results are as follows Figure 3 As shown.

[0107] It can be found that the optimal choice is: 10 mM EDTA, 0.1 M sodium hydroxide, 0.5% Triton X-100, treated for 2 minutes. Therefore, the optimal formulation of the rapid lysis buffer is: 10 mM EDTA, 0.1 M sodium hydroxide, 0.5% Triton X-100, treated for 2 minutes.

[0108] Example 4 This embodiment provides a rapid detection method for pathogenic bacteria. The method includes sequentially coupling a cleavage reaction, a click reaction, and a hybridization chain reaction triggered by the target rRNA to form a cascaded amplification detection system.

[0109] Its technical principle is as follows: Figure 4As shown, this method primarily uses bacterial 16S rRNA and fungal 18S rRNA as broadly conserved recognition targets. After lysis pretreatment, the target ribonucleic acid is released from the sample and undergoes complementary pairing with a specific DNAzyme probe, activating the enzyme's specific cleavage activity. This cleaves a specific sequence on the sensor, releasing the loaded copper oxide nanoparticles. The released copper oxide is dissolved by acidification and then reduced to generate monovalent copper ions. These monovalent copper ions efficiently catalyze click chemistry reactions, generating intermediates that initiate subsequent nucleic acid assembly. These intermediates further trigger a hybridization chain reaction, initiating the self-assembly and stacking of nucleic acid hairpin probes, achieving a stepwise amplification of the fluorescence signal, and ultimately quantifying trace amounts of pathogenic bacteria through fluorescence intensity.

[0110] A detailed flowchart is shown below. Figure 5 As shown, the specific steps include: lysing and centrifuging the sample using the lysis buffer described in the above examples; adding 150 μL of the supernatant to a 13PD1 buffer solution containing 30 μL of 500 nM 13PD1 solution (DNAzyme probe) and 120 μL of 13PD1 buffer containing copper oxide magnetic beads; and reacting at room temperature for 40 minutes. For magnetic separation, 100 μL of the supernatant is added to a reaction system containing 20 μL of 43 mM HCl, followed by the sequential addition of 20 μL of 45 mM ascorbic acid, 30 μL of 700 nM MC3-N3 complex, 30 μL of 200 nM P1-T1 complex, 30 μL of 100 nM A1-A2 complex, and 70 μL of Tris-T buffer, bringing the final volume to 300 μL. The reaction is then carried out in the dark for 30 minutes. Finally, the fluorescence intensity is detected and quantitatively analyzed according to the steps described in the above examples.

[0111] In this embodiment, the 13PD1, the 13PD1 buffer containing the copper oxide magnetic bead sensor, the 30 μL 700 nMC3-N3 complex, the 30 μL 200 nM P1-T1 complex, the 30 μL 100 nM A1-A2 complex, and the Tris-T buffer are all the same as in Example 1 above.

[0112] The samples to be tested included pathogens at a concentration of 100 CFU / mL, including Gram-positive bacteria, Gram-negative bacteria, and fungi. In this embodiment, the pathogens were: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 19434), Enterococcus faecium (ATCC 29212), Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 700603), Pseudomonas aeruginosa (ATCC 27853), Acinetobacter baumannii (ATCC 19606), Enterobacter cloacae (ATCC 13047), and Candida albicans (ATCC 10231). All of the above pathogens were donated by the Guangdong Provincial Microbial Culture Collection Center.

[0113] The specific testing steps are the same as those in the above embodiments.

[0114] Meanwhile, different concentrations of Escherichia coli (1, 5, 10, 50, 100, 500 and 10) were used to... 3 Using CFU / mL as an example, the sensitivity, accuracy, and recovery rate of this method were tested.

[0115] The results are shown in 6-7.

[0116] It can be observed that the method of this invention can accurately detect Gram-negative bacteria, Gram-positive bacteria, and fungi. Taking *Escherichia coli* as an example, the detection limit is as low as 0.34 CFU / mL, and the recovery rate is 96.9%-103.6%.

[0117] In addition, the inventors have also explored and optimized the various reaction conditions in the method to improve the cascade amplification efficiency while reducing the background signal, thereby ensuring that the method still has high sensitivity and high stability in whole blood matrix. These include: probe (13PD1) concentration (5-500 nM), number of bases bound to the target by the probe (12-28 nt), enzyme digestion reaction time (10-50 min), click reaction component (C3N3) molar ratio (2:1-1:2), click reaction component (C3N3) concentration (10-110 nM), hybridization chain reaction component (P1T1) molar ratio (3:1-1:3), hybridization chain reaction component (P1T1) concentration (5-60 nM), hybridization chain reaction component (A1A2) molar ratio (2:1-1:2), hybridization chain reaction component (A1A2) concentration (1-40 nM), catalyst THTTA concentration (0.05-5 mM), and cascade reaction time (10-60 min).

[0118] The results are as follows Figure 8-11 As shown.

[0119] Therefore, the optimal conditions can be determined as follows: probe (13PD1) concentration 50 nM, number of bases bound to the target by the probe 20 nt, enzyme digestion reaction time 40 min, click reaction component (C3N3) molar ratio C3:N3=1:1, click reaction component (C3N3) concentration 70 nM, hybridization chain reaction component (P1T1) molar ratio P1:T1=1:2, hybridization chain reaction component (P1T1) concentration 20 nM, hybridization chain reaction component (A1A2) molar ratio A1:A2=1:1, hybridization chain reaction component (A1A2) concentration 10 nM, catalyst (THPAT) concentration 1 nM, and cascade reaction time 30 min.

[0120] Example 5 In this embodiment, the effects of ceftazidime and meropenem on validated susceptible and resistant Escherichia coli were tested using the methods described in the previous embodiments. Specifically, the detection method involved incubating 500 CFU / mL of susceptible and resistant Escherichia coli in Mueller-Hinton broth containing different concentrations (4-16 μg / mL) of ceftazidime and meropenem for 3 h. Lysis was then performed using a mild lysis buffer, followed by centrifugation after 20 min. The supernatant was then analyzed using the method described above to obtain the fluorescence detection signal. If the fluorescence signal in the drug-treated group was significantly lower than that in the control group, it indicated that the pathogen was sensitive to the antimicrobial drug; conversely, a significantly higher fluorescence signal suggested drug resistance.

[0121] Test results as follows Figure 12 As shown.

[0122] Furthermore, the inventors verified the versatility of the method. Specifically, following the above method, various samples (including blood samples, antibiotic injections, nutrients and homeostasis regulators, biological products, and traditional Chinese medicine injections) were lysed using the above lysis buffer (a mild and then a rapid lysis buffer). After lysis, 500 μL of 1M Tris-HCl buffer was added for neutralization, and 150 μL of the supernatant was taken for detection.

[0123] The results are as follows Figure 13 As shown.

[0124] It can be seen that the method in this invention can also be adapted to the detection of sterile drugs. Therefore, it can be said that it has good versatility and scenario adaptability. It can flexibly match the lysis system and pretreatment scheme according to different types of test samples, and can realize a set of detection system to adapt to diverse detection scenarios.

[0125] Meanwhile, the inventors compared the detection effects of various inspection methods currently applicable to the field with the method in the embodiments of the present invention, and the results are shown in the table below.

[0126] Table 2 Comparison of the effects of existing methods and the present invention

[0127] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for detecting pathogenic microorganisms, characterized in that, Includes the following steps: S1, lyse the sample to be tested to release the target nucleic acid of the pathogen; S2, the target nucleic acid is brought into contact with the DNAzyme recognition system, so that the DNAzyme responds to the pathogen target nucleic acid to cleave and releases a copper-based signal carrier; S3, the copper-based signal carrier is acidified and reduced to generate catalytically active copper ions, which catalyze a copper-catalyzed click reaction to form signal conversion products; S4, detect the signal conversion product, and determine the presence and / or content of pathogens in the sample to be tested based on the detection signal; Optionally, the detection method further includes: performing nucleic acid signal amplification after S3 before detection.

2. The method according to claim 1, characterized in that, The nucleic acid signal amplification includes: using hybridization chain reaction (HCR) to amplify the nucleic acid signal.

3. The method according to claim 1 or 2, characterized in that, The DNAzyme recognition system can recognize conserved sequences of bacterial 16S rRNA and / or fungal 18S rRNA. Preferably, the DNAzyme recognition system includes: a probe sequence or a nucleic acid sequence having the same pathogen target recognition and cleavage function, a substrate chain (Sub), copper oxide tandem linking chains C1 and C2, a click reaction complex, and a hybridization chain reaction complex.

4. The method according to claim 1, characterized in that, The copper-based signal carriers include: copper oxide magnetic beads, copper oxide nanoparticles, cuprous oxide nanoparticles, or other copper-based materials capable of participating in click reactions.

5. The method according to claim 4, characterized in that, The click reaction is a copper-catalyzed azide-alkyne click reaction, and the resulting click product is used to trigger nucleic acid signal amplification or to directly detect the signal.

6. The method according to claim 1, characterized in that, The pyrolysis includes: pyrolysis using a mild pyrolysis solution or a rapid pyrolysis solution; The mild lysis buffer includes EDTA, lysozyme, RNase inhibitor and sucrose. The rapid lysis buffer includes EDTA, NaOH, and Triton X.

100.

7. The method according to claim 1, characterized in that, The detection signal includes signal detection based on at least one of the following methods: Fluorescence, colorimetry, scattering, Raman spectroscopy, chemiluminescence, or electrochemical signals.

8. The method according to claim 1, characterized in that, The samples to be tested include one or more of the following: whole blood, plasma, serum, urine, cerebrospinal fluid, ascites, pleural and peritoneal effusions, biological products, pharmaceuticals, and medical device eluents.

9. The method according to claim 1, characterized in that, The pathogens include one or more of Gram-positive bacteria, Gram-negative bacteria, and fungi.

10. The application of the detection method according to any one of claims 1-9 in pathogen detection, antimicrobial susceptibility analysis, and drug sterility testing; None of the applications mentioned involve the diagnosis of diseases.

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