WO2022151333A1 - Mixture for enriching microorganisms, and method and application - Google Patents

Abstract

The present invention provides a mixture for enriching microorganisms, and a method and an application. The mixture comprises at least two magnetic nanomaterials, i.e., a mannose lectin-coated magnetic nanomaterial and an immunoglobulin G-coated magnetic nanomaterial; the mannose lectin-coated magnetic nanomaterial and the immunoglobulin G-coated magnetic nanomaterial are individually linked on magnetic nanomaterials, and mannose lectin and immunoglobulin G are not co-linked on the same magnetic nanomaterial; and the ratio of the mannose lectin-coated magnetic nanomaterial to the immunoglobulin G-coated magnetic nanomaterial in the mixture can be randomly adjusted.

Description

Mixtures, methods and applications for enriching microorganisms technical field

The invention relates to the field of microorganism detection and analysis, in particular to a method for enrichment and detection of microorganisms.

technical background

Microorganisms are widely distributed all over the earth's surface, and some harmful microorganisms are causing harm to human life. For example, the microbial contamination during food processing and transportation, the contamination of some enteric pathogenic bacteria in water samples, and the common clinical bloodstream infections all pose a threat to human life and health. For the diagnosis of infectious diseases, the pathogen is generally identified through microbial detection, and then targeted treatment plans are adopted through the pathogen. Bacterial culture is the gold standard for identifying bacteria, but it is time-consuming, has a low positive rate, and is susceptible to microbial contamination. Therefore, more rapid and reliable methods are needed for microbial identification.

In the past five years, the matrix-assisted laser desorption time-of-flight mass spectrometry system has been widely used in the identification of clinical microorganisms (in 2014, the French Meyer and German Bruck obtained the clinical license, in 2018, Zhengzhou Antu obtained the CFDA clinical license, in April 2019, Zhuhai Meihua obtained the clinical license. It has been approved by CFDA, and there are still many in clinical validation pending approval), the reason is that compared with traditional culture, biochemical identification mass spectrometry analysis has huge time and cost advantages, fast and accurate microbial identification (or typing) can be timely for doctors guidance on clinical treatment. At present, domestic top three hospitals are basically equipped with quality-assisted laser analysis time-of-flight mass spectrometry microbial identification systems (instruments and databases). However, mass spectrometry can only identify a single microorganism and has certain requirements for the sample size of microorganisms. For example, for common clinical bloodstream infections, due to the low content of microorganisms in the blood, it is commonly cultured in clinical blood bottles until positive, and then transferred to a specific agar medium for further 12-24 hours to achieve purification and purification. For the purpose of proliferation, the mass spectrometry identification of microorganisms can be carried out, so the time that mass spectrometry can be shortened for microorganism identification is limited. However, due to the complexity of the samples, the accuracy of mass spectrometry identification will be greatly reduced without the re-culture and purification of microorganisms. At the same time, the same is true for urinary tract infections. Before the doctor diagnoses and treats, the bacteria in the patient's urine also need to be cultured and enriched, and after a single bacteria is isolated, mass spectrometry or staining identification is performed. This process takes 3-5 time of day. Therefore, introducing an effective and reliable microbial enrichment method before mass spectrometry identification can not only skip the process of re-cultivation, but also improve the accuracy of mass spectrometry identification. This is very important for microbial infectious diseases. It not only avoids the abuse of antibiotics in the empirical treatment stage before the identification results are obtained, but also can determine the precise treatment plan for the patient in the shortest time. Infectious disease mortality.

However, there is no particularly simple and effective method for enrichment and identification of microorganisms, especially bacteria. Especially in complex systems such as blood samples and urine.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned technical problems, the present invention provides a mixture, enrichment, identification method and application that can use different ratios of magnetic beads to mix and enrich microorganisms.

In order to achieve the above object, the present invention provides the following scheme: a mixture for enriching microorganisms, comprising at least two magnetic nanomaterials, a magnetic nanomaterial wrapped with mannose lectin and a magnetic nanomaterial wrapped with immunoglobulin G; wherein mannose The lectin-coated magnetic nanomaterials and the immunoglobulin G-coated magnetic nanomaterials are individually linked to the magnetic nanomaterials, and the mannose lectin and the immunoglobulin G are not co-linked on the same magnetic nanomaterials; The ratios of the magnetic nanomaterials coated with glycolectin and the magnetic nanomaterials coated with immunoglobulin G can be adjusted arbitrarily.

Further, the mannose lectin-encapsulated magnetic nanomaterials linked the engineered mannose lectin to ferric oxide particles through the action of biotin and streptavidin.

Further, the ferric oxide particles in the mannose lectin-encapsulated magnetic nanomaterials are modified with streptavidin with a particle size of 300 nm.

Further, the immunoglobulin G-coated magnetic nanomaterials are formed by linking the immunoglobulin G to the iron tetroxide particles in the manner of acid-base condensation. The particle size of the ferric oxide particles may also be 300 nm.

Further, the mixture also includes magnetic nanomaterials loaded with other bacterial affinity agents.

Further, the magnetic nanomaterials loaded with other bacterial affinity agents are one or both of concanavalin A and vancomycin.

The present invention also provides a method for enriching microorganisms by using the above mixture, adding the mixture for enriching microorganisms into a buffer system or a complex system to capture target microorganisms, and the complex system is blood bottle, urine and sewage one or more of.

Further, after capturing the target microorganisms and isolating the microorganisms, use matrix-assisted laser desorption ionization mass spectrometry to perform mass spectrometry analysis on the captured microorganisms, and compare the fingerprint spectra of the obtained microorganisms with the standard spectra in commercial databases to realize the identification of microorganism species. .

Further, 70% formic acid was used to cleave the captured microorganisms, and then acetonitrile was added for extraction. After magnetic separation, the extract was spotted on the target plate. After being naturally dried, a layer of matrix solution was covered. After the matrix was dried, mass spectrometry data was collected. .

The present invention also provides the application of the above-mentioned mixture for enriching microorganisms in the enrichment and identification of microorganisms.

Mannose lectin (MBL) is a calcium-dependent lectin that recognizes mannose, fucose and N-acetylglucosamine on the surface of different bacteria, fungi and viruses. The engineered MBL (Fc-MBL) is obtained by genetic engineering to remove the coagulation-promoting domain of natural MBL, and the remaining sugar-binding domain is obtained by fusion with the Fc fragment of immunoglobulin G (IgG), and its protein structure is more stable. Therefore, Fc-MBL-coated magnetic nanomaterials have become a broad-spectrum microbial capture material, which can be used for the capture of most common bacteria; IgG-coated magnetic nanomaterials are mainly used for microorganisms that produce proteins A, G, and L on the cell surface. capture. Concanavalin A is a globulin extracted from concanavalinum, which has a high affinity for mannose-rich carbohydrates, so it can also be used as a broad-spectrum bacterial recognition protein. Vancomycin is also a broad-spectrum glycopeptide antibiotic, because it can bind to the D-alanyl-D-alanine (D-ala-D-ala) group in the cell wall of most Gram-positive bacteria, so Become a broad-spectrum enrichment material for Gram-positive bacteria.

A method for enriching microorganisms by mixing magnetic beads with different ratios disclosed in this paper can not only cover the microorganism capture ability of mannose lectin and immunoglobulin G at the same time, but also realize the blood bottle by adjusting the ratio of the two kinds of magnetic beads. , urine, sewage and other complex systems of microorganisms are effectively captured, which improves the accuracy of microbial mass spectrometry identification. For microbial enrichment of other complex systems, we can also mix two or three bacterial affinity materials by adding magnetic nanomaterials loaded with other bacterial affinity agents, such as concanavalin A and vancomycin, etc. Mixed to achieve effective enrichment of microorganisms.

The previous experiments proved that the enrichment efficiency of the engineered mannose lectin-coated magnetic nanomaterials for both Gram-positive and Gram-negative bacteria in the buffer system was greater than 90%, while the immunoglobulin G-coated magnetic nanomaterials had greater than 90% enrichment efficiency. The enrichment efficiency of the magnetic nanomaterials for Gram-positive bacteria and Gram-negative bacteria can also reach more than 80%. For complex systems, the target microorganisms can be captured by adjusting the ratio of the two magnetic nanomaterials. This method not only ensures the broad-spectrum microbial capture ability of the method, but also further improves the stability of the microbial capture effect and the accuracy of mass spectrometry identification. Rate.

Furthermore, compared with single magnetic beads, the use ratio of mixed magnetic beads can be controlled manually, which is more flexible in the enrichment and mass spectrometry identification of microorganisms in many complex systems. This method is of great significance for the early diagnosis of microbial infections.

Description of drawings

Figure 1 is a graph of the enrichment efficiency of magnetic materials for Staphylococcus aureus, using engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials to capture bacteria from buffer.

Figure 2 is a graph of the enrichment efficiency of magnetic materials for Escherichia coli, using engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials to capture bacteria from buffer.

Figure 3 is a graph of the enrichment efficiency of magnetic materials for Staphylococcus cephalus, using engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials to capture bacteria from buffer.

Figure 4 is a graph showing the enrichment efficiency of magnetic materials for Klebsiella pneumoniae, using engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials to capture bacteria from buffer.

Figure 5 is a graph of the enrichment efficiency of magnetic materials for Staphylococcus aureus. Different ratios of engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials were used to capture bacteria from blood vials.

Figure 6 is a graph showing the enrichment efficiency of magnetic materials for Escherichia coli. Different ratios of engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials were used to capture bacteria from blood vials.

Figure 7 is a graph of the enrichment efficiency of magnetic materials for Staphylococcus cephalus, using different ratios of engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials to capture bacteria from blood vials.

Figure 8 is a graph showing the enrichment efficiency of magnetic materials for Klebsiella pneumoniae, using different ratios of engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials to capture bacteria from blood vials.

Figure 9 is a graph of the enrichment efficiency of magnetic materials for Staphylococcus aureus, using different ratios of engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials to capture bacteria from urine.

Figure 10 is a graph of the enrichment efficiency of magnetic materials for Escherichia coli, using different ratios of engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials to capture bacteria from urine.

Figure 11 is a graph showing the enrichment efficiency of magnetic materials for Staphylococcus aureus. Different ratios of engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials were used to capture bacteria from sewage.

Figure 12 is a graph of the enrichment efficiency of magnetic materials for Escherichia coli, using different ratios of engineered mannose lectin-coated nanomaterials and immunoglobulin G-coated magnetic nanomaterials to capture bacteria from sewage.

Detailed ways

The use of the method will be further described in detail below in conjunction with the embodiments and the accompanying drawings.

Implementation description:

In the embodiments of the present invention, unless otherwise specified, the microorganisms enriched by the nanomaterials are coated on Tryptone Soy Agar (TSA) solid medium for cultivation, and of course they can also be coated on other suitable media for cultivation identification.

In the examples of the present invention, mass spectrometry is used for data collection, and in the following examples, the M-Discover 100 microorganism mass spectrometry rapid identification system of Zhuhai Meihua Company is used (the commercial database includes more than 2000 kinds of microorganism standard spectra).

The identification method is to collect mass spectral signals in the range of 2000-20000 Daltons in the linear positive ion mode. The obtained mass spectrum was imported into the mass spectrometry data processing software for baseline calibration, smoothing and normalization, and then matched with the standard spectrum in the database to realize the identification of target microorganisms. Specifically, the obtained mass spectrum was imported into the mass spectrometry data processing software for baseline calibration, smoothing and normalization, and then matched with the standard spectrum in the commercial database. The score lower than 1.7 indicates that the identification result is unreliable, and the score is between 1.7 and 2.0. Between represents confidence at the bacterial genus level, and scores over 2.0 indicate that the identification results are reliable at the bacterial species level.

Example 1 Capture efficiency of mixture and capture method for Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 8739, Staphylococcus cephalus CICC 21722 and Klebsiella pneumoniae CICC 21519 in buffer system

Disperse 100 μg of engineered mannose lectin-coated nanomaterials (magnetic beads Fc-MBL) or immunoglobulin G-coated magnetic nanomaterials (magnetic beads IgG) into 15 μL of buffer solution (10 mM Ca ²⁺ ), and then add 10 μL of Staphylococcus aureus or Escherichia coli or Staphylococcus cephalus or Klebsiella pneumoniae culture solution (the bacterial concentration is ~10, ~100, ~1000 CFU/mL), placed on a shaking metal bath, and reacted at 37 ° C for 20 minutes, Keep the magnetic beads suspended during this time. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Add sterile water and resuspend to 10 μL, after dilution, plate coating in a biological safety cabinet. Figure 1-4 shows that the enrichment efficiency of magnetic beads Fc-MBL for the four bacteria selected in the experiment can reach more than 90%. Although the enrichment efficiency of magnetic beads IgG to the four bacteria is slightly lower than that of magnetic beads Fc-MBL, But also >80%. This result indicates that the two magnetic beads have a good ability to capture bacteria.

Example 2 Mass spectrometry identification results of the method for four selected bacteria in the buffer system

Disperse 100 μg magnetic beads Fc-MBL or magnetic bead IgG into 100 μL buffer solution (10 mM Ca ²⁺ ), and then add 100 μL of Staphylococcus aureus or Escherichia coli or Staphylococcus head or Klebsiella pneumoniae culture solution (bacteria) respectively. The concentration is ~10, ~100, ~1000 CFU/mL), placed on a shaking metal bath, and reacted at 37°C for 20 minutes, during which the magnetic beads were kept suspended. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Then add 5 μL of 70% formic acid and 5 μL of acetonitrile, and magnetically separate 1 μL of the supernatant and spot it on the target plate. After the sample is dried at room temperature, cover it with 1 μL of matrix, dry it at room temperature, and put it into the instrument for detection. Table 1 summarizes the mass spectrometry identification results of the four bacteria. The results showed that the bacterial samples were enriched with magnetic beads Fc-MBL and then subjected to mass spectrometry analysis. The matching scores of the four bacteria and commercial databases were >2.0, and the identification results were reliable at the level of bacterial species. The bacterial samples were enriched with magnetic beads IgG and then subjected to mass spectrometry analysis. , the identification results of the four bacteria can also reach the species level with confidence. Each bacterial sample has five sample points in parallel on the target plate, and the identification results of mass spectrometry have certain stability and reliability.

Table 1

Example 3 Capture efficiency of this method on Staphylococcus aureus ATCC 25923 in blood bottle system

Disperse 100 μg of magnetic beads Fc-MBL, magnetic beads Fc-MBL:IgG=4:1, magnetic beads Fc-MBL:IgG=1:1, magnetic beads Fc-MBL:IgG=1:4 or magnetic beads IgG into 15 μL In the buffer solution (10mM Ca ²⁺ ), add 10 μL of Staphylococcus aureus-positive blood culture solution (the bacterial concentration is ~10, ~100, ~1000 CFU/mL), place it on a shaking metal bath, and react at 37°C for 20 minutes , while keeping the magnetic beads in suspension. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Add sterile water and resuspend to 10 μL, after dilution, plate coating in a biological safety cabinet. Figure 5 shows that as the ratio of the two magnetic beads changes, the enrichment efficiency also changes. The enrichment efficiency of using a single magnetic bead to capture Staphylococcus aureus from blood vials was all lower than 50%, while the enrichment efficiency increased slightly when the ratio was adjusted to 1:4 or 4:1. When the ratio of the two magnetic beads is 1:1, the enrichment efficiency of bacteria can reach more than 50%.

Example 4 Capture efficiency of the method for Escherichia coli ATCC 8739 in the blood bottle system

Disperse 100 μg of magnetic beads Fc-MBL, magnetic beads Fc-MBL:IgG=4:1, magnetic beads Fc-MBL:IgG=1:1, magnetic beads Fc-MBL:IgG=1:4 or magnetic beads IgG into 15 μL In the buffer solution (10mM Ca ²⁺ ), 10 μL of E. coli positive blood culture solution (bacterial concentration of ~10, ~100, ~1000 CFU/mL) was added, placed on a shaking metal bath, and reacted at 37°C for 20 minutes, during Keep the magnetic beads suspended. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Add sterile water and resuspend to 10 μL, after dilution, plate coating in a biological safety cabinet. Figure 6 shows that with the change of the ratio of the two magnetic beads, the capture efficiency of Escherichia coli has a normal distribution, and the enrichment efficiency reaches a peak near the same amount of the two magnetic beads.

Example 5 The capture efficiency of the method for Staphylococcus cephalus CICC 21722 in the blood bottle system

Disperse 100 μg of magnetic beads Fc-MBL, magnetic beads Fc-MBL:IgG=4:1, magnetic beads Fc-MBL:IgG=1:1, magnetic beads Fc-MBL:IgG=1:4 or magnetic beads IgG into 15 μL Buffer solution (10mM Ca ²⁺ ), and then add 10 μL of Staphylococcus cephalus positive blood culture solution (bacterial concentration is ~10, ~100, ~1000 CFU/mL), placed on a shaking metal bath, and reacted at 37 ° C for 20 minutes, Keep the magnetic beads suspended during this time. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Add sterile water and resuspend to 10 μL, after dilution, plate coating in a biological safety cabinet. Figure 7 shows that different ratios of magnetic bead mixes in blood vials also have different capture efficiencies for Staphylococcus cephalus. When the magnetic beads Fc-MBL and magnetic beads IgG are mixed in equal ratio, the enrichment efficiency of bacteria can reach more than 50%.

Example 6 Capture efficiency of the method for Klebsiella pneumoniae CICC 21519 in the blood bottle system

Disperse 100 μg of magnetic beads Fc-MBL, magnetic beads Fc-MBL:IgG=4:1, magnetic beads Fc-MBL:IgG=1:1, magnetic beads Fc-MBL:IgG=1:4 or magnetic beads IgG into 15 μL In the buffer solution (10mM Ca ²⁺ ), add 10 μL of Klebsiella pneumoniae positive blood culture solution (the bacterial concentration is ~10, ~100, ~1000 CFU/mL), place it on a shaking metal bath, and react at 37°C for 20 minutes, keeping the magnetic beads in suspension. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Add sterile water and resuspend to 10 μL, after dilution, plate coating in a biological safety cabinet. Figure 8 shows that the enrichment efficiency of Klebsiella pneumoniae by mixing different ratios of magnetic beads Fc-MBL and magnetic beads IgG is similar to the above three bacteria. 50%.

Example 7 Capture efficiency of the method for Staphylococcus aureus ATCC 25923 in urine system

Disperse 100 μg of magnetic beads Fc-MBL, magnetic beads Fc-MBL:IgG=4:1, magnetic beads Fc-MBL:IgG=1:1, magnetic beads Fc-MBL:IgG=1:4 or magnetic beads IgG into 15 μL In the buffer solution (10mM Ca ²⁺ ), add 10 μL of Staphylococcus aureus urine culture solution (bacterial concentration is ~10, ~100, ~1000 CFU/mL), place it on a shaking metal bath, and react at 37°C for 20 minutes , while keeping the magnetic beads in suspension. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Add sterile water and resuspend to 10 μL, after dilution, plate coating in a biological safety cabinet. Figure 9 shows that the enrichment efficiency also changes with the change in the ratio of magnetic beads Fc-MBL and magnetic beads IgG. The enrichment efficiency of using a single magnetic bead to capture Staphylococcus aureus from urine was all lower than 50%, and the enrichment efficiency increased when the ratio was adjusted to 1:4 or 1:1 or 4:1. When the ratio of magnetic beads is 1:1 or 1:4, the enrichment efficiency is about 50%. When the ratio of the two magnetic beads is 4:1, the enrichment efficiency of bacteria can reach more than 50%.

Example 8 Capture efficiency of the method for Escherichia coli ATCC 8739 in urine system

Disperse 100 μg of magnetic beads Fc-MBL, magnetic beads Fc-MBL:IgG=4:1, magnetic beads Fc-MBL:IgG=1:1, magnetic beads Fc-MBL:IgG=1:4 or magnetic beads IgG into 15 μL In the buffer solution (10mM Ca ²⁺ ), 10 μL of Escherichia coli urine culture solution (the bacterial concentration is ~10, ~100, ~1000 CFU/mL) was added, placed on a shaking metal bath, and reacted at 37°C for 20 minutes, during Keep the magnetic beads suspended. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Add sterile water and resuspend to 10 μL, after dilution, plate coating in a biological safety cabinet. Figure 10 shows that with the change of the ratio of the two magnetic beads, the capture efficiency of E. coli also changed. The bacterial capture efficiency of a single magnetic bead was lower than 50%. When the magnetic bead ratio was Fc-MBL:IgG=1:1 or 1:4, the effect was slightly increased, but still less than 50%. When the ratio of magnetic beads was adjusted to 4:1, the enrichment efficiency was the best (>50%).

Example 9 Capture efficiency of the method for Staphylococcus aureus ATCC 25923 in sewage system

Disperse 100 μg of magnetic beads Fc-MBL, magnetic beads Fc-MBL:IgG=4:1, magnetic beads Fc-MBL:IgG=1:1, magnetic beads Fc-MBL:IgG=1:4 or magnetic beads IgG into 15 μL In the buffer solution (10mM Ca ²⁺ ), add 10 μL of Staphylococcus aureus sewage culture solution (bacterial concentration is ~10, ~100, ~1000 CFU/mL), place it on a shaking metal bath, and react at 37 ° C for 20 minutes, Keep the magnetic beads suspended during this time. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Add sterile water and resuspend to 10 μL, after dilution, plate coating in a biological safety cabinet. Figure 11 shows that as the ratio of the two magnetic beads changes, the enrichment efficiency also changes. The enrichment efficiency of using a single magnetic bead to capture Staphylococcus aureus from sewage was all less than 50%, but when the ratio was adjusted to magnetic beads Fc-MBL:IgG 1:4 or 1:1 or 4:1, the enrichment was Efficiency increased slightly, all greater than 50%.

Example 10 Capture efficiency of the method for Escherichia coli ATCC 8739 in sewage system

Disperse 100 μg of magnetic beads Fc-MBL, magnetic beads Fc-MBL:IgG=4:1, magnetic beads Fc-MBL:IgG=1:1, magnetic beads Fc-MBL:IgG=1:4 or magnetic beads IgG into 15 μL In the buffer solution (10mM Ca ²⁺ ), add 10 μL of Escherichia coli sewage culture solution (the bacterial concentration is ~10, ~100, ~1000 CFU/mL), place it on a shaking metal bath, and react at 37 ° C for 20 minutes, keep it during Magnetic bead suspension. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Add sterile water and resuspend to 10 μL, after dilution, plate coating in a biological safety cabinet. Figure 12 shows that with the change of the ratio of the two magnetic beads, the capture efficiency of E. coli is also different. The capture efficiency of a single magnetic bead and a magnetic bead ratio of 1:4 were both less than 50%. When the ratio of the two magnetic beads was adjusted to 1:4 or 1:1, the enrichment efficiency of the mixed magnetic beads for Escherichia coli did not change much, and both were greater than 50%.

Example 11 Mass spectrometry identification results of Staphylococcus aureus, Escherichia coli, Staphylococcus cephalus and Klebsiella pneumoniae by this method in the blood bottle system

Disperse 100 μg of magnetic beads Fc-MBL, magnetic beads IgG or magnetic beads Fc-MBL:IgG=1:1 into 100 μL buffer solution, and then add 100 μL of Staphylococcus aureus or Escherichia coli or Staphylococcus cephalus or Klebsiella pneumoniae respectively. Primary bacteria positive culture solution, the bacterial culture solution was first centrifuged at 10000rpm for 2min, resuspended in buffer (10mM Ca ²⁺ ), the bacterial concentration was about ~10 ⁸ CFU/mL, placed on a shaking metal bath, and reacted at 37°C The magnetic beads were kept in suspension for 20 minutes. Magnetic separation removes the supernatant, leaving the microbe-enriched nanomaterials behind. Then add 5 μL of 70% formic acid and 5 μL of acetonitrile, and magnetically separate 1 μL of the supernatant and spot it on the target plate. After the sample is dried at room temperature, cover it with 1 μL of matrix, dry it at room temperature, and put it into the instrument for detection. 5 targets were spotted in parallel for each sample. The first and second columns of Table 2 show that the four selected bacterial samples enriched with magnetic beads Fc-MBL were subjected to mass spectrometry analysis. Except for Klebsiella pneumoniae, the identification scores were all lower than 1.7, and the results were not acceptable. letter. However, the identification results of Klebsiella pneumoniae were not reliable at the species level. For Gram-negative bacteria, both Escherichia coli and Klebsiella pneumoniae can be matched to the corresponding bacterial species in the database. Mass spectrometry analysis of bacterial samples enriched with magnetic nanomaterials coated with magnetic beads IgG showed that the identification score was lower than 1.7 except for Staphylococcus cephalus, but the identification results of Staphylococcus cephalus were also unreliable at the species level. For Gram-positive bacteria, both Staphylococcus aureus and Staphylococcus cephalus were matched to the corresponding bacterial species in the database. After enriching the sample with the two magnetic nanomaterials mixed in equal proportions, the database matching score was greater than 2.0, indicating that the bacterial species level was credible.

Table 2

Therefore, preliminary studies have shown that the use of a single magnetic bead cannot effectively capture bacteria from complex systems such as blood bottles, urine, and sewage, and the enrichment efficiency is less than 50%, including Gram-positive bacteria and Gram-negative bacteria. bacteria, and the mass spectrometry identification results of blood bottle microorganisms did not reach the confidence level of bacterial species. Therefore, different proportions of two kinds of magnetic beads were used to enrich microorganisms from the complex system. The enrichment results showed that the mixing of different proportions of magnetic beads had different enrichment abilities for the selected bacteria. At the same time, for different complex systems, to achieve the best enrichment effect, the proportion of magnetic beads used is also different. For the blood bottle system, when the ratio of mixed magnetic beads is 1:1, the enrichment efficiency of the bacteria selected in the experiment can reach more than 50%. And mass spectrometry identification results show that the selected Gram-positive bacteria and Gram-negative bacteria can reach the species level credibility. For the urine system, when the ratio of mixed magnetic beads is 4:1, the enrichment efficiency of the bacteria selected in the experiment can reach more than 50%. For the sewage system, when the ratio of mixed magnetic beads is 4:1 or 1:1 or 1:4, the enrichment efficiency of Staphylococcus aureus is not much different, all can reach more than 50%; when the ratio of mixed magnetic beads is When the ratio is 4:1 or 1:1, the enrichment efficiency of Escherichia coli is not much different, and can reach more than 50%.

Claims (10)

A mixture for enriching microorganisms, characterized in that the mixture comprises at least two magnetic nanomaterials, mannose lectin-encapsulated magnetic nanomaterials and immunoglobulin G-encapsulated magnetic nanomaterials; wherein mannose lectin-encapsulated magnetic nanomaterials Magnetic nanomaterials and immunoglobulin G-encapsulated magnetic nanomaterials are individually linked to magnetic nanomaterials, while mannose lectin and immunoglobulin G are not co-linked to the same magnetic nanomaterial; mannose lectin-encapsulated in the mixture The ratio of the magnetic nanomaterials and the immunoglobulin G-coated magnetic nanomaterials can be adjusted arbitrarily. The mixture for enriching microorganisms according to claim 1, wherein the magnetic nanomaterials coated with mannose lectin are linked to the engineered mannose lectin through the action of biotin and streptavidin on ferric oxide particles. The mixture for enriching microorganisms according to claim 2, wherein the ferric oxide particles in the mannose lectin-coated magnetic nanomaterials are modified with streptavidin with a particle size of 300 nm. The mixture for enriching microorganisms according to claim 1, wherein the magnetic nanomaterials coated with immunoglobulin G are formed by linking immunoglobulin G to ferric oxide particles in the form of acid-base condensation of. The mixture for enriching microorganisms according to claim 1, characterized in that, the mixture further comprises magnetic nanomaterials loaded with other bacterial affinity agents. The mixture for enriching microorganisms according to claim 5, wherein the magnetic nanomaterials loaded by other bacterial affinity agents are one or both of concanavalin A and vancomycin. The method for enriching microorganisms by using the mixture for enriching microorganisms according to any one of claims 1-6, wherein the mixture for enriching microorganisms is added to a buffer system or a complex system to capture the target Microorganisms, the complex system is one or more of blood bottles, urine and sewage. The method for enriching microorganisms from a mixture for enriching microorganisms according to claim 7, wherein after capturing the target microorganisms and isolating the microorganisms, mass spectrometry analysis is performed on the captured microorganisms using matrix-assisted laser desorption ionization mass spectrometry, and the The obtained fingerprint spectrum of microorganisms is compared with the standard spectrum in commercial databases to realize the identification of microorganism species. The method for enriching microorganisms with a mixture for enriching microorganisms according to claim 8, wherein the captured microorganisms are cleaved with 70% formic acid, then acetonitrile is added for extraction, and the extraction liquid is spotted on the target plate after magnetic separation , and then covered with a layer of matrix solution after natural drying, and mass spectrometry data collection was performed after the matrix was air-dried. The application of the mixture for enriching microorganisms according to any one of claims 1-6 in microorganism enrichment and identification.

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