Rapid pathogen detection method based on magnetic bead enrichment-time-of-flight mass spectrometry detection system

Abstract

The invention discloses a rapid pathogen detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system. The rapid pathogen detection method comprises the following steps: S1, expressing and purifying recombinant FcMBL protein; s2, preparation and performance evaluation of enriched functionalized magnetic beads; and S3, establishing the method for rapidly detecting the BSI pathogenic bacteria based on a magnetic bead enrichment-time-of-flight mass spectrometry nucleic acid analysis technology. The recombinant FcMBL protein is coupled with the magnetic beads, so that pathogenic microorganisms in the blood of a septicopyemia patient can be efficiently enriched, and rapid detection is completed by using a nucleic acid mass spectrum platform, so that infected pathogenic bacteria are determined. According to the method, the detection time is greatly shortened, an accurate detection result can be provided for a clinician in a shorter time, and a patient can be effectively treated in time. The method is high in sensitivity and specificity, pathogenic microorganisms can be detected more accurately, missed diagnosis and misdiagnosis are reduced, powerful support is provided for precise treatment of patients, and the method has great application value and significance in the medical field.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pathogenic microorganism detection, in particular to a pathogen rapid detection method based on magnetic bead enrichment-time-of-flight mass spectrometry detection system. BACKGROUND

[0002] Sepsis is a severe systemic inflammatory response syndrome caused by infection, with rapid disease progression and high mortality. Rapid and accurate identification of the pathogenic microorganism causing infection is crucial for developing targeted treatment plans.

[0003] Currently, blood culture is still the basic method for clinically identifying pathogenic microorganisms, but this method has obvious limitations. Blood culture has a long detection period, usually requiring 3-5 days to obtain results, during which the patient may not receive timely and effective targeted treatment, leading to delayed disease progression. Moreover, the positive rate of blood culture is not high, and is affected by various factors such as the patient's use of antibiotics before sampling, the growth characteristics of the pathogenic bacteria, etc., which makes it difficult to accurately detect the pathogenic microorganism of some sepsis patients, thereby affecting the development of subsequent treatment plans. In addition, blood culture has high requirements for operating environment and technology, requiring strict sterile operation, otherwise contamination may occur, leading to false positive results.

[0004] With the continuous progress of medical technology, there is an increasing demand for rapid and accurate detection of sepsis pathogenic microorganisms, therefore, we provide a pathogen rapid detection method based on magnetic bead enrichment-time-of-flight mass spectrometry detection system. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the existing defects and provide a pathogen rapid detection method based on magnetic bead enrichment-time-of-flight mass spectrometry detection system. The recombinant FcMBL protein is coupled with magnetic beads to efficiently enrich pathogenic microorganisms in the blood of sepsis patients, and then the nucleic acid mass spectrometry platform is used for rapid detection to determine the pathogenic bacteria causing infection, thereby partially replacing the traditional method of blood culture for identifying pathogenic microorganisms and shortening the detection time to provide accurate and rapid detection and corresponding treatment plans for patients.

[0006] To achieve the above-mentioned purpose, the present application provides the following technical solutions: A pathogen rapid detection method based on magnetic bead enrichment-time-of-flight mass spectrometry detection system, comprising the following steps: S1, expression and purification of recombinant FcMBL protein; S2, preparation and performance evaluation of functionalized magnetic beads; S3, establishment of a method for rapid detection of BSI pathogenic bacteria based on magnetic bead enrichment-time-of-flight mass spectrometry nucleic acid analysis technology.

[0007] Further, the expression and purification of recombinant FcMBL protein, including the following steps: S11, construction of Trans5α-peaSKY003-mFc-MBL engineering bacteria: using the GenBank published coding human MBL gene sequence as a template, designing and synthesizing primers; PCR amplification of MBL gene and product recovery, construction of Trans5α-pUC-mFc-MBL engineering bacteria; extraction of plasmid after double enzyme digestion and ligation with peaSKY003 vector to construct Trans5α-peaSKY003-mFc-MBL engineering bacteria; S12, construction of HEK293FF-peaSKY003-mFc-MBL expression system: transfecting the recombinant plasmid peaSKY003-mFc-MBL-MBL into HEK293F cells and identifying; S13, eukaryotic expression of MBL protein: after transfecting HEK293F cells, culture in serum-free medium and passaging, collecting the supernatant for SDS-PAGE gel electrophoresis and coomassie brilliant blue staining identification; S14, purification of MBL protein.

[0008] Further, the preparation and performance evaluation of functional magnetic beads, including the following steps: S21, preparation of streptavidin magnetic beads: after carboxylated magnetic beads are activated by EDC and sulfo-NHS method, streptavidin is added for coupling reaction, and the reaction is carried out at 37℃ under rotation condition for 2 hours to obtain streptavidin magnetic beads; S22, preparation of biotinylated MBL protein: biotin is activated, and then cross-linking reaction is carried out with MBL protein at room temperature, the molar ratio of activated biotin to MBL protein is 20:1, the reaction time is 20-40 min, and after the reaction is completed, it is purified by dialysis; S23, preparation of functional magnetic beads by mixing streptavidin magnetic beads and biotinylated MBL protein, the molar ratio of streptavidin magnetic beads to biotinylated MBL protein is 1:5, mixing 1h-2h, forming functional magnetic beads, then stored at 4℃ for standby; S24, using simulation samples to evaluate the enrichment effect of functional magnetic beads, and selecting the best reaction system.

[0009] Further, the establishment and evaluation of the method for rapid detection of BSI pathogenic bacteria based on magnetic bead enrichment-time of flight mass spectrometry nucleic acid analysis technology, including the following steps: S31, determination of 15 kinds of blood stream infection pathogen specific nucleic acid sequences: referring to the data, selecting 15 kinds of representative specific gene sites; S32, design of multiplex primer: design of multiplex PCR amplification primer and multiplex extension primer for the selected three specific sequences, so that detection can be carried out in only one reaction well; S33, establishment and optimization of detection system: determine the detection process, explore the components of the detection system, and evaluate the amplification primer and extension primer.

[0010] Further, in step S13, the culture conditions are as follows: DMEM medium containing 1% penicillin, incubation in a constant temperature incubator at 37 DEG C, 6% CO2 for 120h-168h.

[0011] Further, in step S14, the optimization of MBL protein expression conditions and the purification of MBL protein include the following steps: S141, experiment on the influence of MBL protein expression conditions on MBL protein expression to determine the optimal expression conditions; S142, mass culture under the optimal expression conditions, and preliminary extraction of MBL protein; S143, protein purification by Ni column purification method, and identification of the purified protein by SDS-PAGE gel electrophoresis to determine the purity and molecular weight of the protein; S144, dialysis concentration is performed on the identified qualified protein to obtain the target protein, and the concentration of the target protein is 2mg / ml, which is stored at-20 DEG C for standby.

[0012] Further, in step S14, the MBL protein expression conditions include but are not limited to culture medium composition, culture time, serum concentration and additives.

[0013] Further, in step S32, the three blood stream infection pathogens determined are: Escherichia coli, Staphylococcus aureus and Candida albicans.

[0014] Further, in step S33, the detection process is determined, including the following steps: S331, magnetic bead enrichment is performed on the simulated sample, nucleic acid extraction is performed, and nucleic acid mass spectrometry analysis is performed; S332, the components of the detection system are explored and optimized to determine the best components of the final detection system.

[0015] Compared with the prior art, the method has the following advantages: The present application can efficiently enrich pathogenic microorganisms in the blood of patients with sepsis by coupling recombinant FcMBL protein with magnetic beads, and then complete rapid detection by using a nucleic acid mass spectrometry platform to determine the pathogenic bacteria causing infection. Compared with the traditional blood culture method, this method has significant advantages. It greatly shortens the detection time and can provide accurate detection results for clinicians in a shorter time, so that patients can receive effective treatment in time, significantly improving the treatment effect. At the same time, the method has high sensitivity and specificity, can more accurately detect pathogenic microorganisms, reduce the occurrence of missed diagnosis and misdiagnosis, and provide strong support for precise treatment of patients, and has great application value and significance in the medical field. DETAILED DESCRIPTION

[0016] The technical solutions in the embodiments of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0017] The present embodiment provides a technical solution: A pathogen rapid detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system, comprising the following steps: S1, expression and purification of MBL recombinant protein S11, construction of Trans5a-peaSKY003-mFc-MBL engineering bacteria, comprising the following steps: S111, using the publicly available human MBL gene sequence as a template, and with the help of professional primer design software, the primer is designed and synthesized in the present embodiment by using Primer Premier 5.0; S112, PCR amplification of MBL gene, in the PCR reaction system, appropriate amount of template DNA, primer, dNTPs, Taq DNA polymerase and buffer are added, and the temperature cycle conditions of the PCR instrument are accurately controlled to realize efficient amplification of the MBL gene; S113, after amplification, the PCR product is separated by agarose gel electrophoresis, and the product is recovered by using a DNA gel recovery kit; S114, link the recovered MBL gene with pUC vector to construct Trans5a-pUC-mFc-MBL engineering bacteria; S115, extract the plasmid from the engineering bacteria, and select appropriate restriction enzymes, XhoI and EcoRI are selected in the present embodiment, and double enzyme digestion treatment is performed; S116, the product after double enzyme digestion is connected with peaSKY003 carrier under the action of T4 DNA ligase, so as to construct Trans5α-peaSKY003-mFc-MBL engineering bacteria; S12, construction of Trans5α-peaSKY003-mFc-MBL expression system: take the correct recombinant plasmid peaSKY003-Fc-MBL, use polyethyleneimine (PEI) transfection reagent, and transfect the HEK293F mammalian suspension cells (cell density is 1.5×10 6 cells / mL) in logarithmic growth phase according to the manufacturer's instructions, and the specific transfection method is as follows: according to the corresponding proportion of transfection reagent, the recombinant plasmid is mixed with liposome to form transfection complex; finally, it is added to HEK293F cell culture medium for transfection; after transfection, PCR, sequencing and other methods are used to identify the transfected cells at molecular level, and it is ensured that the recombinant plasmid has been successfully integrated into the genome of HEK293F cells; S13, MBL protein eukaryotic expression, including the following steps: S131, inoculate the transfected HEK293F cells into serum-free medium, the medium is DMEM medium containing 1% penicillin, so that the cells adapt to the culture environment, and then culture in a constant temperature incubator at 37°C, 6% CO2 for 144h, during the culture process, the growth state and morphological changes of the cells are observed regularly, the cell density is detected by cell counter, and it is ensured that the cells grow normally. 5000 rpm centrifugation for 10 minutes, collect the cell supernatant; S132, collect the cell culture supernatant and analyze it by SDS-PAGE gel electrophoresis; after electrophoresis, perform coomassie brilliant blue staining identification, and judge the expression of MBL protein by observing the position and intensity of protein bands on the gel; S14, purification of MBL protein, including the following steps: S141, filter the collected supernatant with 0.22μm filter membrane; S142, use Ni column purification method, which is based on the principle that His-Tag on MBL protein specifically binds to nickel ions on Ni column for protein purification; load the preliminary extracted protein sample into Ni column, elute with buffer containing different concentrations of imidazole, and collect the elution peak; identify the eluted protein by SDS-PAGE gel electrophoresis to determine the purity and molecular weight of the protein; S143, dialysis and concentration of the qualified protein, use dialysis bag and dialysis buffer to remove impurities and salts in the protein solution, then concentrate the protein to 2mg / ml, finally store the concentrated target protein at-20℃ for standby.

[0018] Preparation and performance evaluation of enrichment functionalized magnetic beads S21, preparation of streptavidin magnetic beads, including the following steps: S211, select carboxylated magnetic beads, activate them using EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and sulfo-NHS (N-hydroxysuccinimide sulfonate sodium salt) method. During the activation process, suspend the magnetic beads in an appropriate amount of MES (2-(N-morpholino) ethanesulfonic acid) buffer, add EDC and sulfo-NHS, and stir at room temperature for 45 min to activate the carboxyl groups on the surface of the magnetic beads. Wash the activated magnetic beads with buffer several times to remove unreacted reagents; S212, add streptavidin and react at 37°C under rotation conditions for 2 hours to allow streptavidin to couple with activated magnetic beads. After the reaction is complete, wash the magnetic beads with buffer to remove uncoupled streptavidin, and obtain streptavidin magnetic beads; S22, preparation of biotinylated MBL protein, including the following steps: S221, dissolve biotin (Biotin-NHSEster is used in this example) in an appropriate amount of DMSO (dimethyl sulfoxide) to activate it; S222, crosslink the activated biotin with MBL protein at a molar ratio of 20:1 at room temperature for 30 min. Stir constantly during the reaction to ensure complete reaction; S223, after the reaction is complete, place the biotinylated MBL protein in a dialysis bag and dialyze it in dialysis buffer to remove unreacted biotin and impurities. The dialysis time is 16 hours, and the dialysis buffer is replaced 4 times during this period; S23, mix streptavidin magnetic beads and biotinylated MBL protein to prepare enrichment functionalized magnetic beads: mix the prepared streptavidin magnetic beads and biotinylated MBL protein at a molar ratio of 1:5, and enrich them at 4°C for 1.5 hours to allow them to fully bind and form enrichment functionalized magnetic beads. Gently shake or stir during the enrichment process to ensure uniform mixing. After the enrichment is complete, wash the functionalized magnetic beads with buffer to remove unbound biotinylated MBL protein. Store the prepared enrichment functionalized magnetic beads at 4°C for future use; S24, evaluate the enrichment effect of functional magnetic beads using simulated samples, including the following steps: S241, prepare simulated samples containing different concentrations and types of pathogenic bacteria. Add pathogenic bacteria to sterile saline at a concentration gradient of 10^1-10^3 CFU / ml; S242, take 10 ml of the simulated sample, add the prepared enriched functional magnetic beads, enrich for 1 h under the condition of 37℃ and rotary oscillation, so that the functional magnetic beads are fully combined with the pathogenic bacteria; use an external magnetic field (a magnetic stand is used in this embodiment) to separate the functional magnetic beads, wash the magnetic beads with a buffer solution, and remove the uncombined impurities and pathogenic bacteria; S243, use plate counting method or real-time fluorescent quantitative PCR method to detect the number of pathogenic bacteria enriched on the magnetic beads, plate technology is used in this embodiment, the reaction conditions are changed, the reaction conditions include different ratios of magnetic beads to pathogenic bacteria, different incubation times and temperatures, the enrichment effects of the functional magnetic beads under different reaction systems are compared, and the reaction system with the best enrichment effect is selected.

[0019] S3, establishment of a method for rapid detection of BSI pathogenic bacteria based on magnetic bead enrichment-time of flight mass spectrometry nucleic acid analysis technology S31, determination of 15 blood stream infection pathogen specific nucleic acid sequences: by thoroughly searching authoritative medical databases including but not limited to PubMed and Web of Science, and microbiology literature, deeply studying the genomic information of various blood stream infection pathogens, using bioinformatics analysis tools to compare and analyze the gene sequences of different pathogens, and selecting three representative specific gene sites; the finally determined three blood stream infection pathogens are: Escherichia coli, Staphylococcus aureus, and Staphylococcus aureus; for each pathogen, the sequence information of the specific gene site, the function of the gene, and the conservation in the pathogen are recorded in detail; S32, design of multiple primers: for the selected 15 specific sequences, use primer design software to design multiple PCR amplification primers and multiple extension primers, in this embodiment, Primer3 is used; in the design process, the basic principles of primer design are followed, the compatibility between primers is considered, and it is ensured that multiple PCR amplification and extension reactions can be carried out simultaneously in one reaction well, after the design is completed, the specificity of the primers is evaluated through online tools, in this embodiment, Primer-BLAST of NCBI is used, to ensure that the primers can specifically amplify the nucleic acid sequence of the target pathogen, and the designed primers are synthesized; S33, establishment of a detection system: determine the detection process, including the following steps: S331, magnetic bead enrichment of the simulated sample, the simulated sample is added to the reaction system containing the enriched functional magnetic beads, and the enrichment and separation are carried out according to the optimized conditions, so that the magnetic beads enrich the pathogenic bacteria; then nucleic acid extraction is carried out, and a nucleic acid extraction kit is used to extract nucleic acid from the magnetic beads enriched with pathogenic bacteria; the extracted nucleic acid is subjected to nucleic acid mass spectrometry analysis, and a time-of-flight mass spectrometer is used to detect and analyze the nucleic acid; S332, the components of the detection system are optimized, and the optimal components of the final detection system are determined.

[0020] The pathogen rapid detection method based on the magnetic bead enrichment-time-of-flight mass spectrometry detection system has the following advantages relative to the prior art: 1. The detection time is greatly shortened. The traditional blood culture method takes 3-5 days to detect, while the present application can complete the entire process within a few hours from sample collection to obtaining the detection result, greatly improving the detection efficiency, saving valuable treatment time for clinicians, and enabling patients to receive timely and effective treatment at the early stage of infection, thereby avoiding disease deterioration.

[0021] 2. The detection accuracy is significantly improved. Through the carefully designed MBL recombinant protein expression and purification process and the optimized functionalized magnetic bead preparation method, pathogenic microorganisms in the blood of sepsis patients can be efficiently and specifically enriched. In the functional magnetic bead enrichment effect evaluation experiment, the detection of simulated samples containing different concentrations and types of pathogenic bacteria showed that the enrichment efficiency of the method for pathogenic bacteria was as high as 60% or more.

[0022] 3. The treatment plan is optimized. The rapid and accurate detection results enable clinicians to develop personalized and precise treatment plans in a timely manner based on the types and characteristics of pathogenic bacteria. Precise detection results also help doctors assess the severity of the patient's condition, predict the development trend of the disease, and provide more scientific basis for the prognosis of the patient.

[0023] The above is only an embodiment of the present application, and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation using the content of the present application specification, or direct or indirect application in other related technical fields, is also included in the patent protection scope of the present application.

Claims

1. A rapid pathogen detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system, characterized in that, Includes the following steps: S1. Expression and purification of recombinant FcMBL protein; S2. Preparation and performance evaluation of enriched functionalized magnetic beads; S3. Establishment of a rapid detection method for BSI pathogens based on magnetic bead enrichment-time-of-flight mass spectrometry nucleic acid analysis technology.

2. The rapid pathogen detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system according to claim 1, characterized in that, The expression and purification of recombinant FcMBL protein includes the following steps: Construction of S11 and Trans5α-peaSKY003-mFc-MBL engineered bacteria: Primers were designed and synthesized using the human MBL gene sequence published in GenBank as a template; the MBL gene was amplified by PCR and the product was recovered to construct Trans5α-pUC-mFc-MBL engineered bacteria; the plasmid was extracted, digested with double enzymes, and ligated with the peaSKY003 vector to construct Trans5α-peaSKY003-mFc-MBL engineered bacteria; Construction of the S12, HEK293FF-peaSKY003-mFc-MBL expression system: The recombinant plasmid peaSKY003-mFc-MBL-MBL was transfected into HEK293F cells and identified; S13 and MBL protein eukaryotic expression: HEK293F cells were transfected, cultured and passaged in serum-free medium, and the supernatant was collected for identification by SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining. Purification of S14 and MBL proteins.

3. The rapid pathogen detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system according to claim 1, characterized in that, The preparation and performance evaluation of enriched functionalized magnetic beads include the following steps: S21. Preparation of streptavidin magnetic beads: Carboxylated magnetic beads were activated by EDC and sulfo-NHS method, and then streptavidin was added for coupling reaction. The reaction was carried out at 37°C under rotation for 2 hours to obtain streptavidin magnetic beads. S22. Preparation of biotinylated MBL protein: Biotin is activated and then cross-linked with MBL protein at room temperature. The molar ratio of activated biotin to MBL protein is (10-50):

1. The reaction time is 20-40 min. After the reaction is completed, the protein is purified by dialysis. S23. Streptavidin magnetic beads and biotinylated MBL protein were mixed to prepare enriched functionalized magnetic beads. The molar ratio of streptavidin magnetic beads to biotinylated MBL protein was 1:(1-10). The mixture was mixed for 1-2 hours to form enriched functionalized magnetic beads, which were then stored at 4°C for later use. S24. Evaluate the enrichment effect of functional magnetic beads using simulated samples and select the reaction system with the best enrichment effect.

4. The rapid pathogen detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system according to claim 1, characterized in that, The establishment of a rapid detection method for BSI pathogens based on magnetic bead enrichment-time-of-flight mass spectrometry nucleic acid analysis technology includes the following steps: S31. Determination of specific nucleic acid sequences for three bloodstream infection pathogens: Literature review was conducted, and three representative specific gene loci were selected; S32. Design of multiplex primers: Design multiplex PCR amplification primers and multiplex extension primers for the three selected specific sequences so that detection can be performed in a single reaction well; S33. Determining the testing process and establishing the system: Determine the testing process and explore the components of the testing system.

5. The rapid pathogen detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system according to claim 2, characterized in that, In step S13, the culture conditions are: DMEM medium containing 1% penicillin, cultured in a constant temperature incubator at 37℃ and 6% CO2 for 120h-168h.

6. The rapid pathogen detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system according to claim 2, characterized in that, Step S14, the optimization of MBL protein expression conditions and the purification of MBL protein, includes the following steps: S141. Experiments were conducted to investigate the effect of MBL protein expression conditions on MBL protein expression in order to determine the optimal expression conditions. S142. Cultured in large quantities under optimal expression conditions and initially extracted MBL protein; S143. Protein purification was performed using the Ni column purification method. The purified protein was then identified by SDS-PAGE gel electrophoresis to determine the purity and molecular weight of the protein. S144. The qualified protein is concentrated by dialysis to obtain the target protein with a concentration of 2 mg / ml, and then stored at -20℃ for later use.

7. The rapid pathogen detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system according to claim 6, characterized in that, MBL protein expression conditions include, but are not limited to, culture medium composition, culture time, serum concentration, and additives.

8. The rapid pathogen detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system according to claim 4, characterized in that, In step S32, the three identified bloodstream infection pathogens are: Escherichia coli, Staphylococcus aureus, and Candida albicans.

9. The rapid pathogen detection method based on a magnetic bead enrichment-time-of-flight mass spectrometry detection system according to claim 4, characterized in that, Step S33 defines the detection process, including the following steps: S331. Enrich the sample with magnetic beads, extract nucleic acids, and then perform nucleic acid mass spectrometry analysis. S332. Explore and optimize the components of the detection system to determine the optimal components for the final detection system.