Whole-flow rapid diagnosis kit for blood infection bacteria

Abstract

The invention discloses a whole-process rapid diagnosis kit for blood infection bacteria, which adopts a matrix-assisted laser desorption time-of-flight mass spectrometer for detection, and comprises D-type amino acid with a labeled group and a magnetic bead of which the surface is modified with an antibody of the D-type amino acid with the labeled group, the D-type amino acid with a marking group is used for marking peptidoglycan on live bacteria in a blood sample to be detected through metabolism; and the magnetic beads are used for combining with the D-type amino acid with the marking group marked on the peptidoglycan of the living bacteria through an antibody so as to enrich and capture the living bacteria and form a sample detected by a matrix-assisted laser desorption time-of-flight mass spectrometer. According to the invention, bacteria can be marked successfully, the bacteria can be separated and enriched through magnetic attraction of the magnetic beads, the bacteria can be captured successfully, rapid and accurate identification of the bacteria can be realized through MALDI-TOF MS identification, and whole-process diagnosis of clinical samples from enrichment to identification can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of medical diagnosis, and in particular relates to a full-process rapid diagnosis kit for blood infection bacteria. Background Art

[0002] Bloodstream infections (BSIs) are caused by living microorganisms, mainly bacteria, invading the blood. Such infections are usually characterized by high morbidity and mortality. In Europe and the United States, about 200,000 people die from bloodstream infections each year. Delays in treatment can lead to a rapid increase in mortality at a rate of 7% per hour, and the patient's survival rate is inversely proportional to the time from the onset of symptoms to the use of appropriate antibiotics. Inappropriate antibiotic treatment may reduce the patient's survival rate by five times. However, the identification of bacteria in clinical blood samples by conventional methods usually requires culture and enrichment, and the entire process usually takes 2-5 days, which can easily delay the best time for treatment. Therefore, it is crucial to quickly enrich, detect and identify bacteria in clinical infection samples.

[0003] For patients with acute and severe clinical infections, rapid diagnosis of pathogens can guide clinicians to conduct timely and effective antibiotic treatment, thereby significantly reducing mortality. For slow-growing bacteria, such as Mycobacterium tuberculosis, rapid diagnosis can greatly shorten the testing cycle, reduce empirical medication, and thus reduce the emergence and spread of drug-resistant bacteria.

[0004] Matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) can identify bacteria in a short time, greatly shortening the diagnosis time. However, conventional MALDI-TOF MS identification of bacteria usually requires steps such as blood culture reporting positive and subculture, which is time-consuming. Directly enriching bacteria from positive blood cultures can save the subculture step and advance the identification time by 16-24 hours. The number of bacteria when the blood culture reports positive (10 8 CFU / ml) was higher than the detection limit of MALDI-TOF MS (10 5 -10 6CFU / ml), therefore, enriching bacteria before reporting positive can speed up identification. However, since the amount of bacteria before reporting positive is small, enriching bacteria by centrifugation and removing some non-bacterial proteins from blood cells and culture medium in the supernatant that interfere with mass spectrometry identification as much as possible may cause a large loss of bacteria, and there may still be cell membrane fragments that interfere with MALDI-TOFMS identification. The method of enriching bacteria by modifying capture groups on magnetic beads can efficiently enrich samples with small amounts of bacteria, purify them, and remove some cell membrane fragments. At present, several methods for rapid enrichment of bacteria by magnetic beads have been developed, which can capture as few bacteria per milliliter, such as magnetic nanoparticles (MNPs) coated with human mannose-binding lectin (rhMBL) for enriching bacteria and identifying them by fluorescence in situ hybridization (FISH), as well as bacterial enrichment methods based on metabolic labeling of D-amino acids (DAA) or 2-keto-3-deoxyoctanoic acid (Kdo).

[0005] Many methods for detecting bacteria in blood have been developed in the prior art. For example, CN 118130419 A discloses a method for rapid detection of pathogens in blood culture bottles based on Fourier transform infrared spectroscopy (FT-IR), which expands the scope of pathogen typing based on FT-IR technology and significantly accelerates the analysis speed and accuracy of pathogens in clinical blood culture bottles. The specific steps include: 1) selecting common pathogens that cause bloodstream infection, inoculating these pathogens on a blood plate, and using Fourier transform infrared spectroscopy technology to analyze the obtained pathogen fingerprint to identify pure bacterial strains; 2) taking a small amount of pure bacterial strains and mixing them with 8-10mL of sterile blood to obtain a simulated clinical blood infection sample, and injecting the simulated clinical blood infection sample into a blood culture bottle, transferring the blood culture bottle to a blood culture system until it is reported positive, and then taking it out to obtain a blood culture; 3) taking 30μL of the prepared blood culture and directly applying it on a calcium fluoride (CaF2) target plate, and after drying, putting it into the FT-IR typing system to collect fingerprint spectrum signals, collect, scan and average them to generate a final spectrum.

[0006] However, the existing technologies have the following defects and obstacles: 1) Antibody or lectin-based capture methods cannot selectively capture live bacteria; 2) DAA or Kdo metabolic labeling bacterial enrichment methods can only detect whether bacteria exist in the sample, but fail to further identify bacteria and provide more comprehensive information; 3) Traditional MALDI-TOF MS identification of bacteria requires blood culture, which usually takes 2-5 days and is time-consuming; 4) The method based on Fourier transform infrared spectroscopy for rapid detection of pathogens in blood culture bottles also needs to wait until the blood culture is positive (>10 8 CFU / mL) before identification can be performed, which is also time-consuming and may delay treatment. Summary of the invention

[0007] The purpose of the present invention is to overcome the defects of the prior art and provide a full-process rapid diagnosis kit for blood infection bacteria.

[0008] The technical solution of the present invention is as follows:

[0009] A full-process rapid diagnosis kit for live bacteria infection in blood, which is detected by matrix-assisted laser desorption time-of-flight mass spectrometry, comprises D-amino acids with labeling groups and magnetic beads with antibodies modified on the surface with the D-amino acids with labeling groups, wherein:

[0010] The D-amino acid with a labeling group is used to metabolically label the peptidoglycan on the living bacteria in the blood sample to be tested;

[0011] The magnetic beads are used to combine with the D-amino acids with labeling groups on the peptidoglycan labeled on the living bacteria through antibodies, thereby enriching and capturing the living bacteria to form samples for detection by matrix-assisted laser desorption time-of-flight mass spectrometry.

[0012] In a preferred embodiment of the present invention, the labeling group is FITC, azide or Cy3.

[0013] More preferably, the labeling group is FITC.

[0014] In a preferred embodiment of the present invention, the particle size of the magnetic beads is 0.3 μm.

[0015] A method for enriching and capturing live bacteria in a liquid sample to be tested comprises the following steps:

[0016] (1) using a D-amino acid having a labeling group to metabolically label the peptidoglycan on the living bacteria in the liquid sample to be tested;

[0017] (2) Using magnetic beads whose surfaces are modified with the antibody having the D-amino acid with the labeling group, the antibody binds to the D-amino acid with the labeling group labeled on the peptidoglycan of the living bacteria, thereby enriching and capturing the living bacteria.

[0018] In a preferred embodiment of the present invention, the labeling group is FITC, azide or Cy3.

[0019] More preferably, the labeling group is FITC.

[0020] In a preferred embodiment of the present invention, the particle size of the magnetic beads is 0.3 μm.

[0021] In a preferred embodiment of the present invention, the liquid in the liquid test sample is blood.

[0022] The beneficial effects of the present invention are:

[0023] 1. The present invention adopts the FDAA metabolic labeling method, which can accurately label live bacteria and can be directly used for imaging without the need for click reaction, and is simple to operate.

[0024] 2. Antibody magnetic beads are used for capture, magnetic separation and enrichment. The whole process is simple and fast, and the labeling efficiency is high.

[0025] 3. The present invention can use MALDI-TOF MS identification to achieve rapid and accurate identification of bacterial species, and realize the full-process diagnosis of clinical samples from enrichment to identification, and the diagnosis results can be obtained on the same day.

[0026] 4. The present invention does not need to independently establish a database. The ribosomal protein fingerprint of the microbial strain is obtained through MALDI-TOF MS identification, and compared with the known protein fingerprint database for analysis to achieve rapid identification of bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is one of the experimental result diagrams in Example 1 of the present invention. Among them: A is the confocal fluorescence microscopy result of DAA-FITC labeled Staphylococcus aureus, scale bar, 5 μm, providing representative data from at least three independent experiments; B is the DAA-FITC labeled Staphylococcus aureus agar plate, and the bacterial growth calculated by the plate counting method; C is the flow analysis result that statistically calculates the labeling efficiency at different concentrations of DAA-FITC and different time periods.

[0028] Figure 2 This is the second graph of the experimental results in Example 1 of the present invention, showing the capture efficiency of Staphylococcus aureus under different conditions (temperature, time, magnetic bead concentration, magnetic bead particle size).

[0029] Figure 3 This is the third figure of the experimental results in Example 1 of the present invention, showing the MALDI-TOF MS identification results and identification scores of Staphylococcus aureus with different bacterial counts.

[0030] Figure 4 This is the fourth figure of the experimental results in Example 1 of the present invention. Among them: A is the distribution of Staphylococcus aureus in Buffer under different centrifugal conditions (upper layer, middle layer, lower layer solution); B is the distribution of Staphylococcus aureus in blood under different centrifugal conditions (upper layer, middle layer, lower layer solution); C is the distribution of Staphylococcus aureus after blood+water under different centrifugal conditions (upper layer, middle layer, lower layer solution).

[0031] Figure 5This is the fifth figure of the experimental results in Example 1 of the present invention. Among them: A is the growth of Staphylococcus aureus under different blood bacterial culture methods; B is the growth of Staphylococcus aureus under different blood enrichment culture medium volumes; C is the capture efficiency of magnetic beads on Staphylococcus aureus under different concentrations of DAA-FITC labeling in blood;

[0032] Figure 6 FIG6 is the experimental result of Example 1 of the present invention, showing the results of MALDI-TOF MS identification after antibody magnetic beads captured different amounts of Staphylococcus aureus in PBS.

[0033] Figure 7 FIG7 is the experimental result of Example 1 of the present invention, showing the MALDI-TOF MS identification results of Staphylococcus aureus with different spike bacterial counts in healthy human blood cultured for 7 hours and captured by magnetic beads. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0035] Example 1

[0036] (1) DAA-FITC labeling of bacteria: To verify whether DAA-FITC can label bacteria, freshly cultured S. aureus in LB medium was diluted to OD 600 =0.1~0.2, then DAA-FITC was added and shaken at 37℃ for 3h, then anti-FITC antibody with TRICT fluorescent group was added for coupling, and confocal fluorescence microscopy was used to observe whether the bacteria were labeled with FDAA. In addition, the toxicity of DAA-FITC was verified by plate counting method. DAA-FITC with a final concentration of 0.5mM was incubated with Staphylococcus aureus for 12h, which verified that DAA-FITC was non-toxic and the labeling efficiency reached 90% in 3h. The results are shown in Figure 1 shown.

[0037] (2) Antibody magnetic beads capture bacteria: In order to further improve the capture rate of antibodies and magnetic beads for bacteria, a series of optimizations were performed on the capture conditions. By controlling the variable method, the capture efficiency of DAA-FITC-labeled Staphylococcus aureus was obtained at different temperatures (room temperature, 37°C), different capture times (0.5h, 1h, 2h), different magnetic bead concentrations (0, 5mg / mL, 1mg / mL, 2mg / mL), and different magnetic bead particle sizes (0.3μm, 2.8μm). The results are shown in Figure 2 As shown, high magnetic bead concentration is most conducive to bacterial (SA) capture (capture rate of 90%), and longer incubation time and smaller particle size (0.3 μm) are conducive to bacterial capture.

[0038] (3) Verify the detection limit of bacteria identified by MALDI-TOF MS: Set the bacterial concentration to 10 1 -10 8 CFU / mL, and then pretreated and identified by MALDI-TOF MS. The detection limit was determined by the identification score (<1.7 identification result is untrustworthy; 1.7-2.0 possible genus level identification; ≥2.0 possible species level identification). The results showed that the bacterial count was greater than 10 5 Under CFU / mL conditions, the bacterial species can be accurately identified, such as Figure 3 shown.

[0039] (4) Pretreatment of bacteria in blood: 10 3 CFU of Staphylococcus aureus spike was inoculated into 5mL of PBS buffer, plasma and blood solution respectively. After mixing, centrifugation was performed under different centrifugal conditions (1000rpm-4000rpm, 3min-15min). After that, the upper, middle and lower layers of solution were taken for plating and culture. The plate count was performed the next day to count the distribution of bacteria in the solution. The specific results are as follows: Figure 4 shown.

[0040] (5) Growth conditions of bacteria in blood

[0041] A. S. aureus (10 2 CFU) spike was cultured in 5mL healthy human blood under different culture conditions for 6h. The growth of bacteria in different ways was calculated by plate counting method to select the best culture method. The best culture method was to add water, centrifuge to enrich bacteria and then continue to culture.

[0042] B. Optimize the volume of blood enrichment medium and select the volume with faster bacterial growth and lower cost as the optimal medium volume. The optimal medium volume is 2 mL. Finally, determine the labeled concentration of DAA-FITC in the blood. 2 CFU / mL) spike was added to 5mL healthy human blood, and different concentrations of DAA-FITC were added for labeling for 5h. After 5h, it was centrifuged and washed three times, and MB was added for capture. Finally, the capture efficiency was calculated by the plate counting method. Considering the cost, a final concentration of 0.25mM DAA-FITC was finally selected for labeling. Figure 5 shown.

[0043] (6) MALDI-TOF MS identification of bacteria: 2 μL of 70% formic acid was added to the enriched bacteria and mixed thoroughly by pipetting, followed by the addition of 2 μL of acetonitrile. It was then deposited on the MALDI target plate, air-dried, and 1 μL of matrix was added to the sample spot and mixed evenly. All bacterial fingerprints were obtained on a MALDI-TOF MS system, with each sample deposited on at least 10 parallel spots on one target, and all results were randomly selected from the mass spectra obtained.

[0044] (7) Labeling, capturing, and identifying bacteria in PBS and healthy human blood, specifically comprising the following steps:

[0045] 1) Labeling, capturing, and identifying bacteria in PBS buffer

[0046] To verify the feasibility of the whole process identification, the bacteria were first labeled, captured and identified in PBS buffer. S. aureus was labeled with DAA-FITC and then incubated with 0.3 μm MB for 0.5 h. After removing the supernatant, the cells were washed twice with 0.01% PBST and resuspended in 2 μL PBS before detection and identification using MALDI-TOF MS. Figure 4 As shown in the figure, after MB captures bacteria, the bacterial species can be accurately identified by mass spectrometry, and the minimum amount of bacteria that can be accurately identified is 10 5 -10 6 CFU / mL, as follows Figure 6 shown.

[0047] 2) Labeling, capturing, and identifying spike bacteria samples in healthy human blood

[0048] First, spike 1-100 CFU bacterial samples in healthy human blood, lyse blood cells by adding water to reduce the effect of white blood cells in blood cells on bacterial proliferation, pre-enrich the bacteria in the blood under centrifugal conditions of 4000 rpm and 15 min, add 2 mL of culture medium and a final concentration of 0.25 mM DAA-FITC, label the bacteria and culture for 7 hours, then incubate with 0.3 μm MB for 0.5 hours, remove the supernatant, wash twice with 0.01% PBST, finally remove the magnetic beads, and detect and identify with MALDI-TOFMS. The results are shown in Figure 5 As shown in the figure, the bacterial species can be accurately identified by mass spectrometry within 8 hours after MB captures the bacteria, and the diagnosis result can be obtained on the same day. This shows that this embodiment can remove interference such as cell membrane fragments and realize the identification of bacteria in the blood. Figure 7 shown.

[0049] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A full-process rapid diagnosis kit for live bacteria infection in blood, characterized by: The method is detected by matrix-assisted laser desorption time-of-flight mass spectrometry, comprising a D-amino acid with a labeling group and a magnetic bead with an antibody of the D-amino acid with a labeling group modified on the surface, wherein: The D-amino acid with a labeling group is used to label the peptidoglycan on the live bacteria in the blood sample to be tested through metabolism; the magnetic beads are used to combine with the D-amino acid with a labeling group labeled on the peptidoglycan of the live bacteria through antibodies, thereby enriching and capturing the live bacteria to form a sample for detection by a matrix-assisted laser desorption time-of-flight mass spectrometer.

2. A full-process rapid diagnosis kit for blood-infected bacteria according to claim 1, characterized in that: The labeling group is FITC, azide or Cy3.

3. A full-process rapid diagnosis kit for blood-infected bacteria as claimed in claim 2, characterized in that: The labeling group is FITC.

4. A full-process rapid diagnosis kit for blood-infected bacteria according to claim 1, characterized in that: The particle size of the magnetic beads is 0.3 μm.

5. A method for enriching and capturing live bacteria in a liquid sample to be tested, characterized in that: The steps include: (1) using a D-amino acid having a labeling group to metabolically label the peptidoglycan on the living bacteria in the liquid sample to be tested; (2) Using magnetic beads whose surfaces are modified with the antibody having the D-amino acid with the labeling group, the antibody binds to the D-amino acid with the labeling group labeled on the peptidoglycan of the living bacteria, thereby enriching and capturing the living bacteria.

6. The method for enriching and capturing live bacteria according to claim 5, characterized in that: The labeling group is FITC, azide or Cy3.

7. The method for enriching and capturing live bacteria according to claim 6, characterized in that: The labeling group is FITC.

8. The method for enriching and capturing live bacteria according to claim 5, characterized in that: The particle size of the magnetic beads is 0.3 μm.

9. The method for enriching and capturing live bacteria according to any one of claims 5 to 8, characterized in that: The liquid in the liquid sample to be tested is blood.

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