System, kit and detection method for detecting microorganisms

Abstract

The invention belongs to the technical field of biology, and particularly relates to a reagent for detecting pathogenic genes as well as a preparation method and application thereof. The invention finds that a biological sample is enriched through broad-spectrum captured protein, the captured protein is labeled with biotin, pathogenic microorganisms are separated from the biological sample through high-density streptavidin coated magnetic beads, the pathogenic microorganisms are split after pure culture, pathogenic genes are detected after nucleic acid is extracted, and the turnover time is shortened to 4 hours. The precise distribution of nucleic acid is realized through an annular array reaction cavity preloaded with 32 freeze-dried LAMP reagents in combination with centrifugal force driving. The single chip synchronously detects more than or equal to 30 major pathogen genes / drug-resistant genes, covers common bloodstream infection pathogens, and breaks through the existing POCT flux bottleneck. According to the present invention, with the kit, the blood matrix inhibition can be eliminated, the pathogen concentration can be rapidly increased (the detection threshold value can be reached after culture), the positive detection rate can be increased to the clinical requirement level (gt, 90%), and the pathogen detection limit in the blood sample can meet the clinical critical infection early diagnosis requirement of less than or equal to 10 CFU / mL;

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a detection product and a detection method. Background Technology

[0002] Infectious diseases are a major cause of high morbidity and mortality rates worldwide. Especially among respiratory, bloodstream, and central nervous system infections, infectious diseases have become a significant challenge in global public health. They are pathophysiological responses such as activation of inflammatory cascades, tissue and cell damage, and multiple organ dysfunction caused by pathogenic microorganisms invading the human body through the respiratory tract, digestive tract, or bodily fluids. In particular, traditional etiological methods often fail to identify the causative agents of complex infectious diseases, resulting in poor anti-infective treatment efficacy and a high risk of progression to severe illness. The "Review of Antimicrobial Resistance" predicts that by 2050, the number of deaths caused by drug-resistant bacterial infections may exceed ten million annually. The problem of resistance to existing anti-infective drugs has become a major threat to human health (Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis [J]. Lancet, 2022, 399(10325): 629-655.; Houldcroft CJ, Underdown S. Infectious disease in the Pleistocene: Old friends or old foes? [J]. Am JBiol Anthropol, 2023, 182(4): 513-531.). Due to unclear pathogen diagnosis and difficulties in clinical decision-making, there is an overuse of broad-spectrum antibiotics and the emergence of selectively resistant pathogens. Therefore, there is an urgent need for rapid and accurate identification and resistance analysis of pathogens. Early diagnosis of critical infectious diseases such as sepsis highly depends on rapid and accurate identification of pathogens and drug sensitivity analysis. Establishing an efficient pathogen diagnosis system is of great significance for optimizing clinical treatment strategies and reducing drug resistance to anti-infective drugs.

[0003] Current clinical testing technologies mainly include three types of protocols. The mainstream clinical testing protocol for bloodstream infections is "blood culture + pathogen identification + biochemical drug sensitivity analysis." This protocol is used to solve the problems of pathogen culture and phenotypic drug sensitivity analysis. However, this process has significant shortcomings: According to the CLSI M47-A standard, a positive blood culture report usually takes less than 10 hours. After that, Gram staining and pure culture are required, followed by pathogen identification using MALDI-TOF mass spectrometry. The overall testing time is 1-2 days. If slow-growing pathogens (such as fastidious bacteria and fungi) are encountered, the entire testing process can be extended to 2-3 days, and the overall positive rate is less than 10%, which cannot meet the needs of rapid diagnosis of critical infections.

[0004] Molecular diagnostics can be used for the rapid detection of pathogenic microorganisms, such as bioMérieux's FilmArray™ (PCR microfluidic chip) and Ceprit's Gene Expert product. While these products can achieve multiplex nucleic acid detection of pathogens, the overall time from blood culture-positive samples to results is usually over 15 hours. These products use PCR amplification, which requires cyclical reaction steps such as denaturation, annealing, and extension. The detection is complex and relies on precise temperature-controlled equipment. The high cost hinders widespread adoption. Existing POCT devices share common shortcomings: First, low throughput, capable of detecting only 3-5 target genes per test, failing to cover the spectrum of common bloodstream infection pathogens; second, insufficient sensitivity, affected by blood matrix interference, with pathogen detection limits generally >100 CFU / mL, far exceeding the ≤10 CFU / mL standard required for early diagnosis of clinically critical infections; third, efficiency bottlenecks, while FilmArray™, Gene Expert, and iPonatic™ simplify equipment through isothermal amplification, separate nucleic acid extraction and amplification steps, requiring manual sample transfer, increasing the risk of contamination, and lacking end-to-end integration, making it difficult to achieve integrated closed-loop detection of "sample in - result out." All of these issues, such as fragmented processes and equipment dependence, prevent rapid point-of-care diagnosis; limited throughput also prevents the simultaneous processing of multiple samples, and the enrichment, extraction, amplification, and drug sensitivity steps are not fully integrated into a closed loop.

[0005] The impact of diagnostic delay on patient prognosis has been clinically confirmed: the international guidelines for the treatment of sepsis (Singer M, JAMA 2016) clearly state that for every hour the diagnosis is delayed, the mortality rate of sepsis patients increases by 7.6% (OR=1.32); for every 6 hours the treatment is delayed, the mortality rate increases by 58%. Other clinical studies have shown that if pathogen identification and intervention are completed 16 hours earlier, patient survival rates can be significantly improved, the risk of organ failure significantly reduced, and per capita medical expenditure greatly decreased. (Marta Rolo, María Carmen Martín-Higuera, Esther Viedma, Jennifer Villa, Mikel Mancheño-Losa, Jaime Lora-Tamayo, Fernando Chaves, María Ángeles Orellana, Raúl Recio, Clinical impact of time-to-positivity of blood cultures on mortality inpatients with Pseudomonas aeruginosa bacteremia, Journal of GlobalAntimicrobial Resistance,Volume 30,2022,Pages 269-275,) Rapid and accurate diagnosis of infectious diseases is crucial for improving clinical management and patient prognosis (THOENEN, A GROSETH, K ROSENKE, et al. Nanopore Sequencing as a Rapidly Deployable Ebola Outbreak Tool [J]. Emerging infectious diseases, 2016, 22(2): 331-4.). Therefore, there is an urgent need for rapid, accurate and multiplex diagnostic clinical protocols. Summary of the Invention

[0006] This invention targets the detection of microorganisms in biological samples. By enriching and culturing pathogens before detection, the entire detection time is shortened to 2-4 hours. The detection process includes pathogen enrichment, pathogen culture, and DNA extraction. The DNA is then amplified using a microfluidic chip, enabling the rapid detection of microorganisms in biological samples. Based on this, this invention was completed.

[0007] In a first aspect, the present invention provides a system for detecting microorganisms, the system comprising a pathogen enrichment module, a pathogen culture module, a DNA extraction module, a microfluidic gene amplification module, and a microorganism detection result presentation module; the kit detects microorganisms within 2-4 hours.

[0008] Furthermore, the pathogen enrichment module separates pathogenic microorganisms by contacting biological samples with magnetic beads containing surface-coupled capture proteins.

[0009] Furthermore, the magnetic beads are high-density streptavidin-coated carboxyl magnetic beads, preferably APOH / mannan lectin-coated magnetic beads.

[0010] Furthermore, the volume of the carboxyl magnetic beads is selected from 100µL to 300µL.

[0011] Furthermore, the content of APOH is selected from 5-50 µg.

[0012] Furthermore, the pathogen culture module uses pure culture medium to culture APOH / mannan lectin-coated magnetic beads to enrich the pathogen, and then culture the pathogen to proliferate to a detectable threshold.

[0013] Furthermore, the DNA extraction module lyses the DNA by injecting lysis buffer, adds magnetic beads to adsorb nucleic acids, and then dries the magnetic beads to release purified DNA for extraction.

[0014] Furthermore, the microfluidic gene amplification module loads the extracted DNA into the mixing chamber of the microfluidic chip, centrifuges it, and dispenses it into the LAMP microfluidic chip array for isothermal amplification.

[0015] Furthermore, the microbial detection result presentation module uses a detection curve obtained through microfluidics to identify the detected microorganisms and present the results.

[0016] Furthermore, the microorganisms include pathogens, drug resistance genes, and pathogen virulence genes.

[0017] Secondly, the present invention provides a method for detecting microorganisms, the method comprising the following steps: M1. Sample collection; M2. Magnetic bead capture and pathogen enrichment: APOH / mannan lectin-coated magnetic beads (surface-coupled capture proteins) are used for enrichment after contact with the sample; M3. Cultivate to increase pathogen concentration: Add APOH / mannan lectin-coated magnetic beads that have captured pathogens, and cultivate the pathogens to proliferate to the detectable threshold; M4. Pathogen lysis and nucleic acid extraction: Inject lysis buffer, add magnetic beads to adsorb nucleic acid, and dry the magnetic beads after adsorption to release purified DNA; M5. DNA is loaded into the mixing chamber of the microfluidic chip, centrifuged, and dispensed into the LAMP microfluidic chip array; M6. Isothermal amplification and real-time detection; the detected microorganisms can be identified through the obtained detection curve.

[0018] Furthermore, in step M1, the sample is blood or urine.

[0019] Furthermore, the blood sample is whole blood.

[0020] Furthermore, in step M2, the content of APOH is selected from 5-50 µg.

[0021] Furthermore, in step M3, the detectable threshold is greater than 2 copies.

[0022] Furthermore, in step M4, the volume of the lysis solution is selected from 100-400µL, preferably 200µL.

[0023] Furthermore, in step M5, the centrifugal speed is selected from 100-1000 rpm.

[0024] Furthermore, in step M6, the temperature for the isothermal amplification is selected from 60℃-65℃.

[0025] Furthermore, the detection method can detect microbial results within 2-4 hours.

[0026] Furthermore, the microorganisms include pathogens, drug resistance genes, and pathogen virulence genes.

[0027] Thirdly, the present invention provides a kit comprising instructions and detection reagents, wherein the detection reagents obtain microbial detection results through pathogen enrichment, pathogen culture, DNA extraction and microfluidic gene amplification; the kit detects microbial results in 2-4 hours.

[0028] Furthermore, the microorganisms include pathogens, drug resistance genes, and pathogen virulence genes.

[0029] Furthermore, the enrichment of pathogens is achieved by contacting magnetic beads with surface-coupled capture proteins with biological samples to isolate pathogenic microorganisms.

[0030] Furthermore, the magnetic beads are high-density streptavidin-coated carboxyl magnetic beads, preferably APOH / mannan lectin-coated magnetic beads.

[0031] Furthermore, the volume of the carboxyl magnetic beads is selected from 100µL to 300µL.

[0032] Furthermore, the content of APOH is selected from 5-50 µg.

[0033] Furthermore, the pathogen is cultured by culturing APOH / mannan lectin-coated magnetic beads enriched with the pathogen in pure culture medium, and the pathogen is cultured to proliferate to a detectable threshold.

[0034] Furthermore, the DNA extraction is performed by injecting lysis buffer to lyse the DNA, adding magnetic beads to adsorb the nucleic acid, drying the magnetic beads after adsorption to release purified DNA, and then extracting the DNA.

[0035] Furthermore, the microfluidic gene amplification involves loading the extracted DNA into the mixing chamber of a microfluidic chip, centrifuging, and then dispensing it into a LAMP microfluidic chip array for isothermal amplification.

[0036] Furthermore, the microbial detection result is a detection curve obtained through microfluidics, which reveals the detected microorganisms.

[0037] Beneficial effects The reagent for detecting pathogen genes provided by this invention has the following advantages: Short time: This invention uses broad-spectrum capture proteins such as APOH and mannan lectin to enrich pathogenic microorganisms from whole blood, and then transfers them to an undisturbed pure culture medium for short-term culture (about 1 hour). It eliminates blood matrix inhibition, rapidly increases pathogen concentration (reaching the detection threshold after culture), and improves the positive detection rate to the clinically required level (>90%). The entire process takes only 2-4 hours: pathogen enrichment (30 minutes) → lysis (75℃, 15 minutes) → LAMP amplification (65℃, 40 minutes).

[0038] Multiplex detection: This invention employs a microfluidic strategy with pre-loaded ring array reaction chambers containing 32 recombinant lyophilized LAMP reagents, combined with centrifugal force to achieve precise nucleic acid distribution. A single chip can simultaneously detect ≥30 pathogenic genes / drug resistance genes, covering common bloodstream infection pathogens, breaking through the throughput bottleneck of existing POCT methods.

[0039] Currently, the pathogens, drug resistance genes, and pathogen virulence genes being tested include: 24 pathogens, covering bacteria, fungi, mycoplasma, etc.; 4 drug resistance genes; and 4 virulence genes. Virulence gene testing clarifies the pathogenic risk of pathogens; drug resistance gene testing guides precision medicine and drug resistance control. The detection of these two types of genes complement each other, assessing the harm of infection from the perspective of "pathogenic potential" and guiding intervention plans from the perspective of "treatment feasibility," ultimately achieving scientific and precise management of microbial infections.

[0040] Limit of Detection: The broad-spectrum capture protein targets conserved antigens such as LPS from Gram-negative bacteria and peptidoglycan from Gram-positive bacteria, and binds strongly to biotin-avidin tetravalent antibodies, significantly reducing interference from the blood matrix by enriching and culturing pathogens from blood. The limit of detection for pathogens in blood samples meets the clinical requirements for early diagnosis of critical infections ≤10 CFU / mL. The Ct values ​​(cycle thresholds) of each detection target in this system are calculated based on a target concentration of 10 copies / reaction. This concentration corresponds to the limit of detection (LOD) of this LAMP assay, enabling sensitive detection of low-abundance targets and meeting the detection needs of trace pathogens and related genes in clinical or environmental samples.

[0041] Closed-loop automation: Integrating "enrichment-culture-lysis-nucleic acid extraction-amplification detection" modules, the microfluidic chip uses centrifugal force to drive the dispensing of lysis buffer (1200 rpm) + a pre-loaded lyophilized LAMP reagent ring array. This eliminates the risk of contamination from human intervention; the closed microfluidic design achieves "sample in - result out," avoiding contamination caused by the separation steps in nucleic acid extraction / amplification in traditional POCT equipment (such as Sansure iPonatic™, which requires manual sample transfer). Attached Figure Description

[0042] Figure 1 This is the testing process.

[0043] Figure 2 Image of carboxyl magnetic beads before modification.

[0044] Figure 3 Immunomagnetic beads modified with APOH antibody.

[0045] Figure 4 Image of carboxyl magnetic beads before modification under a 10X microscope.

[0046] Figure 5 Immunomagnetic beads modified with APOH antibody under a 10X microscope.

[0047] Figure 6 The results are for optimization of pyrolysis conditions.

[0048] Figure 7 A 20X micrograph of a pneumonia Klebsiella pneumoniae after being captured by APOH immunomagnetic beads stained with DAPI.

[0049] Figure 8 A 10X micrograph of E. coli after being captured by APOH immunomagnetic beads stained with DAPI.

[0050] Figure 9 The curves show the concentrations of Escherichia coli captured in different groups of whole blood samples.

[0051] Figure 10The curves show the concentrations of Klebsiella pneumoniae in different groups captured in whole blood samples.

[0052] Figure 11 The curves show the concentrations of Enterobacter cloacae captured in whole blood samples.

[0053] Figure 12 The curves show the concentrations of Salmonella typhimurium captured in different groups of whole blood samples.

[0054] Figure 13 The curves show the concentrations of Vibrio parahaemolyticus captured in different groups of whole blood samples. Detailed Implementation

[0055] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0056] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0057] Terminology Explanation Virulence genes (such as *E. coli* ompC and *Klebsiella pneumoniae* ureA) are key fragments encoding virulence factors (outer membrane proteins, toxins, etc.) in pathogens. Their detection directly reflects the pathogenicity of the pathogen—for example, *E. coli* carrying the ompC gene is more likely to adhere to host cells and cause infection, while the *Salmonella* sprB gene is closely related to intestinal invasiveness. By detecting virulence genes, "highly pathogenic strains" and "harmless colonizing strains" can be quickly distinguished, providing a basis for infection risk grading and clinical symptom prediction, and avoiding over-intervention in low-virulence strains.

[0058] Drug resistance genes (such as vanA, mecA, and blaKPC) are the core genetic material of bacteria that resist antibiotics, and their presence directly determines the effectiveness of antibiotic treatment. For example, Staphylococcus aureus (MRSA) with the detected mecA gene is resistant to β-lactam antibiotics, while Klebsiella pneumoniae with the detected blaKPC gene is resistant to carbapenems. By detecting drug resistance genes, the drug resistance spectrum of pathogens can be identified before medication, avoiding the blind use of ineffective antibiotics that could lead to treatment failure or the spread of drug resistance. At the same time, continuous monitoring of the prevalence of drug resistance genes can also provide data support for regional drug resistance control strategies (such as antibiotic use management). Example 1: Raw materials required for the experiment 1. Raw materials The reagents included carboxylated magnetic beads (100µL-300µL), recombinant protein mannan lectin (MBL), APOH (5-50µg), recombinant streptavidin-biotin, PBS, LB liquid medium, DNA extraction reagent, and LAMP amplification premix.

[0059] 2. Functions of each raw material Carboxyl magnetic beads: provide an antibody immobilization carrier, provide surface carboxyl groups (-COOH) for covalent coupling of streptavidin EDC / NHS activated carboxyl groups, and form amide bonds with streptavidin lysine residues to achieve high-density immobilization.

[0060] Mannan lectin, APOH recombinant protein: a broad-spectrum protein capture reagent that can recognize conserved epitopes of various pathogens (such as LPS and peptidoglycan).

[0061] Recombinant streptavidin, antibody-directed conjugation medium: Biotinylated antibodies are directionally immobilized on magnetic beads via the biotin-avidin system, exhibiting tetravalent binding properties (each molecule binds 4 biotinylate).

[0062] PBS buffer: Used as the base solution for washing magnetic beads to remove unbound antigens and impurities, maintaining the dispersion of magnetic beads. An isotonic buffer system (pH 7.4) prevents magnetic beads from agglomerating, ensuring subsequent capture efficiency.

[0063] LB liquid medium: The pathogen amplification medium provides nitrogen / carbon source (tryptone + yeast extract), and the pathogenic microorganisms grow rapidly (harvest when OD600=0.6).

[0064] DNA extraction reagent: pathogen lysis and DNA purification, containing ionizing salt (peptone hydrochloride), proteinase K, and detergent (SDS). It disrupts cell membranes / walls and releases DNA denatured by ionizing salt, proteinase K degrades nucleases, and SDS dissolves lipid membranes, working synergistically to improve DNA yield.

[0065] LAMP amplification premix: An isothermal nucleic acid amplification reaction system providing Bst DNA polymerase, dNTPs, buffer, and fluorescent dye (EvaGreen) to support multiplex target detection. The Bst enzyme remains active at 65°C and amplifies via strand displacement; the fluorescent dye intercalates into double-stranded DNA, emitting a fluorescent signal.

[0066] Example 2 Test methods in microbial detection 1. Preparation of biotinylated capture protein The NHS-activated biotin to capture protein molar ratio was 20:1, and the reaction was carried out at 4°C in the dark for 2 hours (pH 8.3 borate buffer). Recombinant protein lysine residues covalently reacted with biotin, achieving a labeling rate of 4-6 biotin / protein. Free biotin was removed using an ultrafiltration tube.

[0067] 2. Preparation of streptavidin magnetic beads Carboxyl magnetic bead activation: 10 mg magnetic beads + 10 mM EDC / 5 mM NHS (pH 5.5 MES buffer), shake at 25°C for 30 minutes to convert the carboxyl group (-COOH) to the active ester (-NHS). Streptavidin was then buffer-exchanged and dispersed in PBS. 1 mg of recombinant streptavidin (pH 7.4 PBS) was added, and the reaction was carried out at 25°C with shaking for 2 hours. Streptavidin was immobilized via amide bonds (loading > 40 μg / mg). Blocking was performed by adding 1% BSA for 1 hour. The magnetic beads were then stored at 4°C in PBS containing 0.1% BSA and 0.02% NaN3 to maintain stability (shelf life > 6 months).

[0068] 3. Extraction of pathogen DNA Thaw Lysozyme at room temperature before use; mix Buffer W1A and W2A with the specified amount of anhydrous ethanol before use; preheat Buffer EB to 55°C, and allow Proteinase K, RNase Solution, and MagExtract Suspension to return to room temperature before use. Place the magnetic bead-pathogen complex in a 1.5 mL centrifuge tube, magnetically attach for 30 seconds, and discard the supernatant; add 200 μL Buffer STE, vortex for 30 seconds to resuspend, and add 30 μL Lysozyme (100 mg / mL) and mix well; if Staphylococcus is present, add 1 μL Lysostaphin (20 mg / mL) and 5 μL RNase Solution, and incubate at 37°C for 30-60 minutes (mixing once every 15 minutes). Add 20 μL Proteinase K and 200 μL Buffer MBL, mix well, and incubate at 70°C with shaking for 10 minutes (300 rpm). If there are impurities, centrifuge at 13,000 × g for 1 minute and take 400 μL of supernatant. Add 20 μL MagExtractSuspension and 400 μL Buffer BB, mix well, and incubate at room temperature for 8 minutes (mix once every 2 minutes).

[0069] Adsorb onto a magnetic rack for 30 seconds and discard the supernatant; add 600 μL of Buffer W1A, vortex for 1 minute, adsorb and discard the supernatant; repeat the washing once with 600 μL of Buffer W2A; briefly centrifuge (5,000×g, 10 seconds), then aspirate the residual liquid and dry at room temperature for 15 minutes (or at 37°C for 5 minutes). Add 50-100 μL of preheated Buffer EB, vortex for 30 seconds, then incubate at 55°C with shaking for 10-15 minutes (200 rpm, mixing every 3-4 minutes); adsorb onto a magnetic rack for 2 minutes, then aspirate the supernatant (containing purified DNA) into an enzyme-free tube and store at -20°C for later use.

[0070] A. Test Results like Figure 2 The image shown is of the carboxyl magnetic beads before modification. Figure 3 As shown, these are immunomagnetic beads modified with APOH antibody, such as... Figure 4 As shown, this is a 10X microscope image of the carboxyl magnetic beads before modification. Figure 5 The image shown is an 10X microscope image of the immunomagnetic beads modified with APOH antibody. This demonstrates that the modification was successful.

[0071] Example 3: Optimization Experiment of Key Parameters in Microbial Detection The aim is to optimize key parameters of pathogen lysis and nucleic acid extraction steps, including lysozyme dosage, lysis buffer volume, proteinase K addition, lysis temperature, and time, to improve DNA extraction efficiency and ensure the sensitivity and accuracy of subsequent LAMP amplification.

[0072] A. Experimental Materials and Instruments 1. Reagents Lysozyme (100 mg / mL, stored at -20°C); Proteinase K (20 mg / mL, stored at -20°C); Lysis buffer; TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and magnetic beads. Pathogen sample: Klebsiella pneumoniae culture, concentration adjusted to 10. 6 CFU / mL. 2. Instruments Heated metal bath (temperature range: 37°C–80°C); centrifuge (maximum speed 12,000 rpm); vortex mixer; micro spectrophotometer (for DNA concentration detection); and real-time quantitative PCR instrument (for assessing DNA quality), etc.

[0073] B. Test methods 1. Experimental group design Seven lysis conditions (conditions 1–7) were set, and each group was repeated three times to screen for the optimal parameters: Conditions 1–2: Focus on the synergistic effect of lysozyme and proteinase K (screening enzyme addition combination); Conditions 3–5: Optimize pyrolysis temperature and time (screen temperature and time parameters); Conditions 6–7: Evaluate the effect of high-temperature lysis (the necessity of screening lysozymes at high temperatures).

[0074] 2. Methods (1) Pathogen sample preparation: Take Klebsiella pneumoniae culture (10 6 1 mL of lysate (CFU / mL) was centrifuged (8,000 rpm, 5 minutes), the supernatant was discarded, and the mixture was resuspended in PBS to the original volume as a standard lysate sample. (2) Lysis reaction: Take 100 μL of pathogen sample from each group, add reagents and lyse under the following conditions: Condition 1: Add 20 μL of lysozyme (100 mg / mL) and 400 μL of lysis buffer, vortex to mix, and incubate in a 37°C water bath for 10 minutes; Condition 2: Same as condition 1, after lysis, add 10 μL proteinase K (20 mg / mL) and continue incubation at 37°C for 10 minutes; Condition 3: Add 20 μL of lysozyme (100 mg / mL) and 400 μL of lysis buffer, vortex to mix, and incubate in a 37°C water bath for 1 minute; Condition 4: Add 20 μL of lysozyme (100 mg / mL) and 400 μL of lysis buffer, vortex to mix, and incubate in a 50°C water bath for 10 minutes; Condition 5: Add 20 μL of lysozyme (100 mg / mL) and 400 μL of lysis buffer, vortex to mix, incubate in a 50°C water bath for 20 minutes, and then add 10 μL of proteinase K (20 mg / mL). Condition 6: Add only 400 μL of lysis buffer, vortex to mix, and incubate in an 80°C water bath for 10 minutes; Condition 7: Add 20 μL of lysozyme (100 mg / mL) and 400 μL of lysis buffer, vortex to mix, and incubate in an 80°C water bath for 10 minutes.

[0075] (3) Nucleic acid extraction: After lysis, add 50 μL of magnetic bead suspension to each group, vortex for 10 minutes, and discard the supernatant after magnetic separation. Wash twice with 500 μL Wash Buffer (70% ethanol), dry, and then elute DNA with 50 μL TE buffer; (4) DNA quantification and quality assessment: DNA concentration (ng / μL) was detected using a micro spectrophotometer, and Ct value was determined by real-time quantitative PCR to assess amplification efficiency.

[0076] C. Experimental Results DNA extraction efficiency: The impact of lysis conditions on nucleic acid yield and quality was evaluated by comparing DNA concentration and Ct values ​​in each group. For example: Condition 5 (50°C, 20 min + proteinase K) showed the highest DNA concentrations due to prolonged incubation and enhanced cell wall degradation by proteinase K.

[0077] Conditions 6 and 7 (80°C) yielded lower DNA yields due to nucleic acid degradation caused by high temperatures, but condition 7 may show slight improvement due to lysozyme pretreatment.

[0078] Optimal condition screening: Based on the subsequent LAMP amplification results (such as amplification curves and detection limits), condition 5 was determined to be the optimal lysis scheme for use in subsequent examples.

[0079] like Figure 6 As shown, by systematically optimizing the lysis parameters, it was found that the combined use of lysozyme and proteinase K and medium-temperature long-term lysis (50°C, 20 minutes) can significantly improve DNA extraction efficiency, providing a reliable basis for pathogen gene detection.

[0080] Example 4 Real-time dual microbial detection The immunomagnetic bead technology was used to specifically enrich and capture typical pathogens (Klebsiella pneumoniae and Escherichia coli) in complex samples, verifying the functional effectiveness of APOH protein as a broad-spectrum capture protein, and laying the foundation for subsequent rapid pathogen detection methods based on magnetic beads.

[0081] A. Experimental Materials and Instruments 1. Materials Capture element: APOH immunomagnetic beads (prepared according to the method of Example 2 of this invention, consisting of streptavidin-coated carboxyl magnetic beads coupled with biotinylated APOH protein).

[0082] Pathogens: Klebsiella pneumoniae and Escherichia coli.

[0083] Staining reagent: DAPI (4',6-diamidinyl-2-phenylindole) fluorescent dye, working concentration 1 µg / mL.

[0084] Buffer: 1× PBS (phosphate buffer, pH 7.4).

[0085] 2. Instruments Fluorescence microscopes, centrifuges, and vortex mixers, etc.

[0086] B. Test methods 1. Bacterial culture and staining (1) Klebsiella pneumoniae and Escherichia coli were inoculated into LB liquid medium and cultured at 37°C and 200 rpm until the logarithmic growth phase (OD600 ≈ 0.6). (2) Take 1 mL of bacterial culture, centrifuge (8,000 rpm, 5 minutes) to collect the bacterial cells, and discard the supernatant; (3) Wash the bacterial cells once with 1 mL PBS buffer to remove residual culture medium; (4) After centrifugation again, resuspend the bacterial cells in 1 mL of PBS solution containing 1 µg / mL DAPI, incubate at room temperature in the dark for 15 minutes, and then fluorescently label the bacterial genomic DNA. (5) After incubation, centrifuge to discard the DAPI staining solution, and wash twice with 1 mL PBS buffer to completely remove unbound free dye. (6) Finally, resuspend the stained bacterial cells in PBS and adjust the bacterial concentration to approximately 10. 8 CFU / mL was used as the target bacterial solution for the capture experiment.

[0087] 2. Immunomagnetic bead capture (1) Take 50 µL of the prepared APOH immunomagnetic bead suspension into a 1.5 mL centrifuge tube, place it on a magnetic rack and let it stand for 1 minute. After the magnetic beads are completely adsorbed, carefully remove the supernatant storage solution. (2) Remove the magnetic rack and add 100 µL of the DAPI-stained bacterial suspension (10 1 CFU / mL ~10 6 (CFU / mL) (3) Use a vortex mixer to thoroughly mix the mixture, and then gently shake and incubate at room temperature for 30 minutes to ensure that the APOH immunomagnetic beads have sufficient contact and binding time with the target bacteria.

[0088] 3. Washing and film preparation (1) After incubation, place the centrifuge tube on a magnetic rack and let it stand for 2 minutes to allow the magnetic beads that have bound the bacteria to be adsorbed onto the tube wall. (2) Carefully aspirate the supernatant containing unbound bacteria; (3) Add 200 µL of PBS buffer, gently shake to rinse the magnetic bead-bacterial complex, place it on the magnetic rack again to separate, discard the supernatant, and repeat this washing step twice to remove non-specifically bound bacteria; (4) After the final wash, resuspend the magnetic bead-bacterial complex in 50 µL PBS buffer; (5) Take 10 µL of the resuspension and drop it onto the glass slide, cover it with a coverslip, and prepare a sample for microscope observation.

[0089] C. Experimental Results like Figure 7 The image shown is a 20X microscope image of Klebsiella pneumoniae captured by APOH immunomagnetic beads after being stained with DAPI. A large number of DAPI-stained bacteria are visible clustered around the magnetic beads (black circular particles in the image), exhibiting bright blue fluorescence. The bacteria are tightly bound to the magnetic beads, forming distinct "magnetic bead-bacteria" complex clusters, indicating that the APOH protein can effectively recognize and bind to conserved antigenic epitopes on the surface of Klebsiella pneumoniae.

[0090] like Figure 8 The image shown is a 10X microscope image of APOH immunomagnetic beads after capturing DAPI-stained E. coli. A large number of blue fluorescent bacteria were observed adsorbed on the surface and surrounding area of ​​the magnetic beads, and were evenly distributed. This result further confirms that APOH protein has a broad-spectrum and efficient capture ability for Gram-negative bacteria (represented by E. coli).

[0091] Therefore, the APOH immunomagnetic beads prepared in this invention can efficiently and specifically capture two common bloodstream pathogens, Klebsiella pneumoniae and Escherichia coli. DAPI staining results clearly demonstrate the successful formation of the "magnetic bead-bacteria" complex, providing crucial visual evidence for the pathogen enrichment strategy based on broad-spectrum capture proteins in this invention, and proving the feasibility and reliability of this reagent for sample pretreatment in subsequent detection procedures.

[0092] Example 5: Real-time detection of 32 microorganisms A. Test Samples In the 32-fold LAMP detection system of this invention, the detection targets for all pathogens (including bacteria, fungi, and mycoplasma), virulence genes, and drug resistance genes are plasmid standards synthesized by Shanghai Sangon Biotech Co., Ltd.; the nucleotide sequences of the plasmid standards are all derived from publicly published literature on microbial genomics or molecular diagnostics, ensuring the accuracy and traceability of the target sequences. The pathogens, drug resistance genes, and pathogen virulence genes detected are as follows: 1. Number of pathogens: 24, covering bacteria, fungi, mycoplasma, etc., specifically including: Bacteria (19 species): Escherichia coli ( E. coli Listeria monocytogenes ( ) L. monocytogenes ), Salmonella typhi ( S. typhimurium ), Vibrio parahaemolyticus ( V. parahaemolyticus Staphylococcus aureus ( S. aureus Staphylococcus epidermidis ( S. epidermidis ), Pseudomonas aeruginosa ( P. aeruginosa ), Klebsiella pneumoniae ( K. pneumoniae Streptococcus pneumoniae () S. pneumoniae ), Enterococcus faecalis ( E. faecalis Acinetobacter baumannii ( A. baumannii Serratia marcescens ( ), S. marcescens Stenotrophomonas maltophilia ( S. maltophilia ), hemolytic staphylococci ( S. haemolyticus Staphylococcus aureus ( S. hominis ), Proteus mirabilis ( P. mirabilis Enterobacter cloacae () E. cloacae ), Propionibacterium acnes ( P. acnes ), methicillin-resistant Staphylococcus aureus (MRSA) MRSA According to pathogen classification, their drug resistance genes mecA (Statistics are counted separately) Fungi (4 species): Candida albicans ( C. albicans ), Candida glabrata ( C. glabrata ), Candida parapsilosis ( C. parapsilosis ), Tropical Candida ( C. tropicalis Aspergillus flavus A. flavus Aspergillus niger ( ) A. niger (Note: The original statistical logic listed 6 types of fungi here. The aforementioned "24 pathogens" already includes all fungi. The aforementioned "19 types of bacteria" is correct. The total of 24 pathogens is 19 bacteria + 6 fungi - 1 duplicate count.) MRSA (Already classified as a variant of Staphylococcus aureus, counted without duplication), the final count was revised to 24 species, all of which were independently detected microorganisms in the document. Other microorganisms (1 type): Mycoplasma ( Mycoplasma spp. ).

[0093] 2. Number of drug resistance genes: 4, all of which are drug resistance-related genes carried by bacteria, specifically including: vanA Vancomycin-resistant Enterococci E. faecium ); vanB Vancomycin-resistant Enterococci E. faecium ); blaKPC KPC-producing Klebsiella pneumoniae K. pneumoniae )and mecA (Methicillin-resistant Staphylococcus aureus) S. aureus ); 3. Number of virulence genes: 4, covering virulence-related genes of Salmonella, Klebsiella pneumoniae, and Escherichia coli, specifically including: sprB Salmonella virulence island SPI ); ureA (Klebsiella pneumoniae virulence gene); ompC (E. coli virulence gene) and ompF (E. coli virulence gene).

[0094] B. Test methods 1. Magnetic bead capture and enrichment (30 minutes) Dissolve the plasmid in PBS, take 10 copies, and inject into the "mixing enrichment chamber". Initiate shaking to mix (950 rpm), ensuring that the 50 μL APOH / mannan lectin-coated magnetic beads (surface-coupled capture protein) pre-placed in the chamber are in full contact with the blood. After the magnetic beads are enriched, transfer them to PBS for washing.

[0095] 2. Short-term incubation to increase pathogen concentration (time 120 minutes) Pre-fill the "enrichment culture chamber" of a 96-well plate with 200 μL of LB liquid culture medium, add 50 μL of LAPOH / mannan lectin-coated magnetic beads after pathogen capture, and incubate at 37°C with shaking (200 rpm) for 60 minutes to accelerate pathogen proliferation to the detectable threshold. 3. Pathogen lysis and nucleic acid extraction (15 minutes) Inject the pathogen-containing culture medium into centrifuge tubes, add lysozyme (100 mg / mL), and incubate at 37°C for 5-30 minutes. Then add 20 μL Proteinase K and 200 μL Buffer MBL, and incubate at 70°C with shaking for 10 minutes (300 rpm). Next, add 20 μL MagExtract Suspension kit and 400 μL Buffer BB, and incubate at room temperature for 8 minutes. After adsorbing the magnetic beads onto the magnetic rack, discard the supernatant. Wash with 600 μL Buffer W1A for 1 minute, and repeat once with 600 μL Buffer W2A. Dry the magnetic beads at room temperature for 15 minutes. Finally, inject 50 μL Buffer EB (preheated to 55°C), incubate at 55°C with shaking for 10 minutes (200 rpm), and collect the supernatant after magnetic adsorption. This is the purified DNA.

[0096] 4. Centrifuge and dispense into the LAMP microfluidic chip array (takes 5 minutes) Transfer the purified DNA solution to the microfluidic chip array and start the centrifuge (1200 rpm, 30 seconds). Use centrifugal force to evenly distribute the DNA solution into the circular microfluidic reaction array (each well is pre-loaded with lyophilized LAMP reagent).

[0097] 5. Isothermal amplification and real-time detection (takes 60 minutes) The chip was heated to a constant temperature of 65℃, and LAMP amplification was initiated for 40 minutes. An integrated optical system monitored the fluorescence signal of each well in real time (excitation / emission: 485 / 520nm), acquiring data every 30 seconds. Results output: Automatic generation of melting / amplification curves, determining the positivity / negativeness of 32 pathogen genes / drug resistance targets.

[0098] C. Experimental Results Table 1 shows the real-time results of 32 microbial detections.

[0099] Table 1. Real-time detection results of 32 microorganisms Example 6 Detection of pathogenic microorganisms in whole blood / urine samples A. Test Samples Escherichia coli in whole blood and urine samples respectively E. coli ), Klebsiella pneumoniae ( K. pneumoniae Enterobacter cloacae () E. cloacae Salmonella typhimurium ( S.typhimurium ) and Vibrio parahaemolyticus ( V. parahaemolyticus ) to conduct testing.

[0100] The blood samples used in this study were anticoagulated whole blood from patients suspected of having sepsis, and the urine samples were midstream urine from patients with urinary tract infections. These included 30 whole blood samples and 28 urine samples, all provided by the Department of Laboratory Medicine at Xinhua Hospital affiliated with Shanghai Jiao Tong University School of Medicine. Sample collection and processing complied with clinical ethical guidelines and hospital biosample management requirements. Both types of samples were stored in a refrigerator after collection for later use.

[0101] The pathogen concentration gradient was set as follows: 3 CFU / mL, 30 CFU / mL, 300 CFU / mL, 10 3 CFU / mL, with a blank control set up, and 6 biological replicates for each concentration.

[0102] B. Test methods 1. Magnetic bead capture and enrichment (30 minutes) Inject anticoagulated whole blood or urine samples into the "mixing enrichment chamber". Initiate shaking to mix (950 rpm), ensuring the 50 μL APOH / mannan lectin-coated magnetic beads (surface-coupled capture protein) pre-placed in the chamber are in full contact with the blood. After enrichment, transfer the beads to PBS for washing.

[0103] 2. Short-term incubation to increase pathogen concentration (time 120 minutes) Pre-fill 200 μL LB liquid culture medium into the "enrichment culture chamber" of a 96-well plate, add 50 µL LAPOH / mannan lectin-coated magnetic beads that have captured pathogens, and incubate at 37°C with shaking (200 rpm) for 60 minutes to accelerate pathogen proliferation to the detectable threshold.

[0104] 3. Pathogen lysis and nucleic acid extraction (15 minutes) Inject the pathogen-containing culture medium into centrifuge tubes, add lysozyme (100 mg / mL), and incubate at 37°C for 5-30 minutes. Then add 20 μL Proteinase K and 200 μL Buffer MBL, and incubate at 70°C with shaking for 10 minutes (300 rpm). Next, add 20 μL MagExtract Suspension kit and 400 μL Buffer BB, and incubate at room temperature for 8 minutes. After adsorbing the magnetic beads onto the magnetic rack, discard the supernatant. Wash with 600 μL Buffer W1A, and repeat with 600 μL Buffer W2A. Dry the magnetic beads at room temperature for 15 minutes. Finally, inject 50 μL Buffer EB (preheated to 55°C), incubate at 55°C with shaking for 10 minutes (200 rpm), and collect the supernatant after magnetic adsorption. This is the purified DNA.

[0105] 4. Centrifuge and dispense into the LAMP microfluidic chip array (takes 5 minutes) Transfer the purified DNA solution to the microfluidic chip array and start the centrifuge (1200 rpm, 30 seconds). Use centrifugal force to evenly distribute the DNA solution into the circular microfluidic reaction array (each well is pre-loaded with lyophilized LAMP reagent).

[0106] 5. Isothermal amplification and real-time detection (takes 60 minutes) The chip was heated to a constant temperature of 65℃, and LAMP amplification was initiated for 40 minutes. An integrated optical system monitored the fluorescence signal of each well in real time (excitation / emission: 485 / 520nm), acquiring data every 30 seconds. Results output: Automatic generation of melting / amplification curves, determining the positivity / negativeness of 32 pathogen genes / drug resistance targets.

[0107] C. Experimental Results Capture common clinical bloodstream infection pathogens—Escherichia coli (E. coli) E. coli ), Klebsiella pneumoniae ( K. pneumoniae Enterobacter cloacae () E. cloacae Salmonella typhimurium ( S.typhimurium ) and Vibrio parahaemolyticus ( V. parahaemolyticusAfterwards, LAMP amplification detection was performed. The detection system for the five bacteria was uniformly set with amplification time (in minutes) on the x-axis and fluorescence signal intensity on the y-axis. The curves for each concentration group (3000 CFU, 300 CFU, 30 CFU, 3 CFU, BLANK) are shown in the figure. Figure 9 The figure shows the corresponding concentration curves of E. coli in different groups captured in whole blood samples; as shown... Figure 10 As shown, the curves correspond to the concentrations of Klebsiella pneumoniae captured in different groups of whole blood samples are presented; Figure 11 The figure shows the corresponding concentration curves of different groups of Enterobacter cloacae captured in whole blood samples; as shown. Figure 12 As shown, the curves correspond to the concentrations of Salmonella typhimurium captured in different groups of whole blood samples; Figure 13 The figure shows the concentration curves of Vibrio parahaemolyticus captured in different groups in whole blood samples. Figure 13 As shown, all bacteria in the 3000 CFU and 300 CFU groups exhibited typical "S-shaped" amplification curves, with the 3000 CFU group showing the earliest inflection point (2-3 minutes earlier than the 300 CFU group), and exhibiting higher and steeper fluorescence signal peaks. This indicates that the higher the pathogen concentration, the faster the LAMP amplification reaction starts and the stronger the fluorescence signal accumulation efficiency. Combined with the characteristics of the LAMP amplification premix containing Bst DNA polymerase (active at 65℃) and EvaGreen fluorescent dye as stated in the instructions, this demonstrates that the amplification system does not have pathogen-specific adaptation barriers for the four bacteria and can accurately identify targets through signal changes.

[0108] The 30 CFU and 3 CFU groups of the four bacteria still formed clear "S-shaped" curves, with the inflection point of the 3 CFU group being slightly later than that of the 30 CFU group (deviation ≤ 4 minutes). There were no signal attenuation or false negatives. This is consistent with the design in the instructions that "APOH / mannan lectin-coated magnetic beads can broadly capture pathogens and increase concentration through short-term culture." The effective amplification of the 3 CFU group directly confirms that the detection limit of this method can match the "≤10 CFU / mL" standard required for early diagnosis of clinical critical infections, solving the problem of missed detection of low-abundance pathogens by traditional methods.

[0109] The BLANK (blank control) group of all bacteria showed no increase in fluorescence signal throughout the process, eliminating interference from reagent contamination, non-specific amplification, etc., which is consistent with the technical advantage of the closed microfluidic design "avoiding contamination" in the instructions, and verifies the specificity and reliability of the detection system.

[0110] The total testing time was less than 4 hours, and the pathogen detection limit was less than 10 CFU, meeting the current clinical requirements for sepsis testing. Data in urine samples showed trends consistent with those in whole blood samples.

[0111] Example 7: Comparison with Commercially Available Methods A. Test methods 1. Commonly used methods in existing technologies: Traditional blood culture (BACTEC™ 9240 system) + MALDI-TOF mass spectrometry (Bruker Microflex) (mainstream clinical pathogen identification process) (1) Sample collection and inoculation: Collect anticoagulated whole blood samples from the patient (usually 8-10 mL for adults), immediately inject into BACTEC™ standard aerobic and anaerobic blood culture bottles, and mix gently.

[0112] (2) Blood culture: Place the blood culture bottle into the BACTEC™ 9240 fully automated blood culture system and culture continuously with shaking at 35-37℃; the system monitors the change in CO2 concentration through a fluorescence sensor and automatically alarms for positive results (the reporting time is usually 10-24 hours, depending on the growth rate of the pathogen).

[0113] (3) Gram staining and transculturing: After the blood culture reports a positive result, a small amount of culture medium is taken for Gram staining to preliminarily distinguish Gram-positive bacteria / negative bacteria; at the same time, the positive culture medium is streaked onto blood agar plates or chocolate plates and placed in an incubator at 35-37℃ for pure culture (18-24 hours) to obtain single colonies; (4) MALDI-TOF mass spectrometry identification: Pick a single colony from a pure culture plate, spread it on a MALDI-TOF target plate, cover it with α-cyano-4-hydroxycinnamic acid matrix solution, and dry at room temperature. Use a Bruker microflex mass spectrometer for detection, and complete the pathogen identification by comparing the protein spectrum in the database (the whole process takes about 10-15 minutes).

[0114] Testing indicators: (1) Total detection time: From blood culture to pathogen identification results, the total time is 18-72 hours (depending on the growth rate of the pathogen). Blood culture requires an average of 10-24 hours to report a positive result, pure culture requires 18-24 hours, and mass spectrometry identification requires 15 minutes.

[0115] (2) Limit of detection: The limit of detection for common bacteria (such as Escherichia coli) in blood culture systems is 10-100 CFU / mL, but the limit of detection for slow-growing pathogens (such as Klebsiella pneumoniae) may be as high as 10. 4 CFU / mL.

[0116] (3) Number of targets covered in a single test: A single blood culture can detect multiple pathogens, but MALDI-TOF mass spectrometry can only identify one pure culture pathogen at a time; the overall process has a limited number of targets covered and cannot directly detect drug resistance genes or virulence genes (requires additional experiments).

[0117] (4) Contamination rate: Due to the many steps involved (such as transplantation and staining), the risk of contamination is high, and the clinically reported contamination rate is approximately 2-5%.

[0118] 2. Commonly used methods in existing technologies: FilmArray™ Blood Culture Identification Panel (PCR microfluidic chip, bioMérieux) (1) Sample preparation: Take 200 μL of the sample liquid from the BACTEC™ blood culture positive test and inject it into the sample buffer of the FilmArray™ BCID test bag, and vortex to mix. (2) Loading the test bag: Place the test bag into the FilmArray™ fully automated molecular diagnostic instrument. The instrument will automatically seal and puncture the reagent chamber. (3) Automated detection: The instrument integrates nucleic acid extraction, reverse transcription (for RNA viruses), multiplex PCR amplification, and detection. Nucleic acid extraction: Pathogen nucleic acid was extracted using lysis buffer and magnetic beads (approximately 20 minutes). First round of PCR: Perform multiplex PCR pre-amplification to amplify the target gene (approximately 10 minutes). Second round of PCR: Perform nested PCR to improve specificity and sensitivity (approximately 30 minutes). Detection: Real-time detection of fluorescence signals through melting curve analysis to automatically identify pathogens and drug resistance genes; (4) Results output: The instrument software automatically generates a report listing the detected pathogens (bacteria, fungi) and drug resistance genes.

[0119] Testing indicators: (1) Total testing time: From loading a positive blood culture sample to outputting the results, the total time is 1.5 hours (but if calculated from the start of blood collection, including blood culture time, the total time is more than 15 hours).

[0120] (2) Limit of detection: The detection limit for common bacteria (such as Escherichia coli) is 500-1000 CFU / mL. Due to interference from blood matrix, the sensitivity is insufficient to meet the clinical critical infection requirements of ≤10 CFU / mL.

[0121] (3) Number of targets covered in a single test: A single test can detect 17 bacteria, 3 fungi and 6 drug resistance genes, but cannot cover the 32 targets mentioned in the document (such as mycoplasma and some virulence genes).

[0122] (4) Contamination rate: Due to the use of closed microfluidic design, the contamination rate is low (<1%), but there is still a risk of artificial contamination in the sample transfer step.

[0123] 3. The method of this invention: magnetic bead capture and enrichment (30 minutes) → short-term incubation (60 minutes) → lysis and nucleic acid extraction (15 minutes) → LAMP isothermal amplification and detection (40 minutes) Test samples: Examples 4-6.

[0124] Test method: (1) Bead capture and enrichment: Take a sepsis sample or a simulated sample and inject it into the "mixed enrichment chamber". Start the vibration at 950 rpm to mix the sample, so that the 50 μL APOH / mannan lectin-coated magnetic beads (prepared according to the method in Example 2) placed in the chamber can fully contact the sample for 30 minutes. Then transfer the magnetic beads to a centrifuge tube containing 1 mL PBS. After magnetic adsorption, discard the supernatant and repeat the washing twice to remove matrix impurities.

[0125] (2) Short-term culture: 200 μL of LB liquid culture medium was pre-embedded in the "enrichment culture chamber" of the 96-well plate, and 50 μL of coated magnetic beads that had captured the pathogens were added. The plate was cultured at 37°C and 200 rpm for 60 minutes to allow the pathogens to proliferate to the detectable threshold.

[0126] (3) Lysis and nucleic acid extraction: Inject lysozyme, proteinase K and lysis buffer into the culture medium containing pathogens in sequence, heat at 70°C for 10 minutes; add MagExtractSuspension from DNB671 Bacterial DNA Extraction Kit to adsorb nucleic acid, separate with a magnetic rack and wash once each with Buffer W1A and W2A from the kit, dry the magnetic beads and add 50 μL of Buffer EB from the kit to release and purify DNA.

[0127] (4) LAMP isothermal amplification detection: The purified DNA was transferred to the microfluidic chip array, centrifuged at 1200 rpm for 30 seconds and dispensed into the loop reaction chamber pre-loaded with lyophilized LAMP amplification premix (containing Bst DNA polymerase, dNTPs, and EvaGreen fluorescent dye); amplification was performed at 65℃ for 40 minutes, and fluorescence signals were collected every 30 seconds through the optical system (excitation / emission 485 / 520 nm) to automatically generate amplification curve to determine the positive and negative results of the target.

[0128] Testing indicators: (1) Total testing time: 2-4 hours.

[0129] (2) Limit of detection: Even in whole blood samples, the limit of detection is ≤10 CFU / mL, which meets the clinical critical infection requirement of ≤10 CFU / mL.

[0130] (3) Number of targets covered in a single test: 32 targets (such as mycoplasma and some virulence genes).

[0131] (4) Contamination rate: The closed microfluidic design is adopted, the steps are simple, and the contamination rate is low.

[0132] B. Test Results In terms of total detection time, for clinical samples, the method of this invention follows the procedure of "magnetic bead capture and enrichment (30 minutes) → short-term culture (60 minutes) → lysis and nucleic acid extraction (15 minutes) → LAMP isothermal amplification and detection (40 minutes)," with a total time of only 3.0 to 3.5 hours. The "blood culture + MALDI-TOF mass spectrometry" method, which is the mainstream clinical pathogen identification procedure, requires steps such as blood culture positivity reporting, Gram staining, pure culture, and mass spectrometry identification, with a total time of 18 to 24 hours. Although bioMérieux's FilmArray™ is a molecular diagnostic technology, its total time from sample processing to result output still requires 4.5 to 5.0 hours (United States Trademark No. 8,394,608 patent). This invention is significantly superior to the two commercially available methods in terms of detection efficiency.

[0133] In terms of the lowest detection limit, using clinically common Escherichia coli as the detection target, the method of this invention achieves an expected detection limit of ≤10 CFU / mL for pathogens, meeting the requirements for pathogen detection limits in the early diagnosis of critical clinical infections. The "blood culture + MALDI-TOF" method, limited by the positive rate of blood cultures and subsequent culture procedures, has an expected detection limit of 100-150 CFU / mL for Escherichia coli. The expected detection limit for Klebsiella pneumoniae is even higher, reaching 10 CFU / mL. 4 CFU / mL (Application of MALDI-TOF-MS in Direct Identification of Pathogens of Bloodstream and Urinary Tract Infections in Children, 2021); Although the FilmArray™ PCR microfluidic chip has better sensitivity than traditional blood culture methods, the expected detection limit for Escherichia coli is still 100~1000 CFU / mL (Gemoules, M., Timbrook, TT, Neuner, E., Dumm, RE, & Krekel, T. (2025)). This invention breaks through the existing technical bottleneck in detection sensitivity.

[0134] In terms of the number of targets covered in a single detection, this invention, through a microfluidic strategy with a pre-loaded ring array reaction chamber containing 32 refills of lyophilized LAMP reagent, can detect ≥32 bacteria (including Enterobacteriaceae, Staphylococcus, etc.), ≥5 fungi (including Candida, Aspergillus, etc.), and ≥7 drug resistance genes (including KPC, NDM, etc.) in a single detection, covering common bloodstream pathogens and drug resistance gene profiles. The "blood culture + MALDI-TOF" method can only detect one pathogen per detection and requires individual culture and identification of different pathogens. For drug resistance genes, an additional drug susceptibility test of ≥8 hours is required. FilmArray™ PCR microfluidic chips are limited by the manufacturer's fixed channel design and can only detect 17 bacteria, 0 fungi, and 6 drug resistance genes per detection. This invention achieves a comprehensive surpassing of existing methods in terms of multiple detection capabilities.

Claims

1. A system for detecting microorganisms, the system comprising a pathogen enrichment module, a pathogen culture module, a DNA extraction module, a microfluidic gene amplification module, and a microorganism detection result presentation module; the kit detects microorganisms within 2-4 hours.

2. The system as described in claim 1, wherein the pathogen enrichment module separates pathogenic microorganisms by contacting biological samples with magnetic beads containing surface-coupled capture proteins; the pathogen culture module cultivates APOH / mannan lectin-coated magnetic beads enriched with pathogens in pure culture medium, and cultures the pathogens to proliferate to a detectable threshold; the DNA extraction module lyses the DNA by injecting lysis buffer, adding magnetic beads to adsorb nucleic acids, drying the magnetic beads after adsorption to release purified DNA, and then extracting it; the microfluidic gene amplification module loads the extracted DNA into the mixing chamber of a microfluidic chip, centrifuges it, and dispenses it into a LAMP microfluidic chip array for isothermal amplification; and the microbial detection result presentation module presents the detection curve obtained through microfluidics to identify the detected microorganisms.

3. The system according to claim 2, wherein the magnetic beads are high-density streptavidin-coated carboxyl magnetic beads, preferably APOH / mannan lectin-coated magnetic beads; the volume of the carboxyl magnetic beads is selected from 100µL-300µL; and the content of APOH is selected from 5-50µg.

4. The system of claim 1, wherein the microorganism comprises a pathogen, a drug resistance gene, and a pathogen virulence gene.

5. A method for detecting microorganisms, the method comprising the following steps: M1. Sample collection; M2. Magnetic bead capture and pathogen enrichment: APOH / mannan lectin-coated magnetic beads (surface-coupled capture proteins) are used for enrichment after contact with the sample; M3. Cultivate to increase pathogen concentration: Add APOH / mannan lectin-coated magnetic beads that have captured pathogens, and cultivate the pathogens to proliferate to the detectable threshold; M4. Pathogen lysis and nucleic acid extraction: Inject lysis buffer, add magnetic beads to adsorb nucleic acid, and dry the magnetic beads after adsorption to release purified DNA; M5. DNA is loaded into the mixing chamber of the microfluidic chip, centrifuged, and dispensed into the LAMP microfluidic chip array; M6. Isothermal amplification and real-time detection; the detected microorganisms can be identified through the obtained detection curve.

6. The method of claim 5, wherein in step M1, the sample is blood or urine; in step M2, the APOH content is selected from 5-50 µg; in step M3, the detectable threshold is greater than 2 copies; and in step M4, the volume of the lysis buffer is selected from 100-400 µL.

7. The method of claim 5, wherein the detection method detects microbial results within 2-4 hours; the microorganisms include pathogens, drug resistance genes, and pathogen virulence genes.

8. A kit; the kit includes instructions and detection reagents, wherein the detection reagents obtain microbial detection results through pathogen enrichment, pathogen culture, DNA extraction and microfluidic gene amplification; the kit detects microbial results in 2-4 hours.

9. The kit of claim 8, wherein the microorganism comprises a pathogen, a drug resistance gene, and a pathogen virulence gene.

10. The kit of claim 8, wherein the enrichment of pathogens is achieved by contacting surface-coupled capture protein magnetic beads with biological samples to isolate pathogenic microorganisms; the magnetic beads are high-density streptavidin-coated carboxyl magnetic beads, preferably APOH / mannan lectin-coated magnetic beads; the cultivation of pathogens is achieved by culturing APOH / mannan lectin-coated magnetic beads enriched with pathogens in pure culture medium, and the pathogens are cultured to proliferate to a detectable threshold; the extraction of DNA is achieved by injecting lysis buffer for lysis, adding magnetic beads to adsorb nucleic acids, drying the magnetic beads after adsorption to release purified DNA, and then extracting it; the microfluidic gene amplification involves loading the extracted DNA into the mixing chamber of a microfluidic chip, centrifuging, and aliquoting it into a LAMP microfluidic chip array for isothermal amplification; the microbial detection result is a detection curve obtained through microfluidics, which indicates the detected microorganisms.

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