Bacterial hybridization reagents and their use

Abstract

The present application relates to the field of bacterial detection, and discloses a bacterial hybridization reagent and application thereof.The reagent contains citrate buffer, disaccharide blocking agent, guanidine protein denaturant, sulfobetaine type zwitterionic surfactant, polyoxyethylene sorbitan ester and polyoxyethylene (5-30) alkyl ether.The reagent is safe, efficient and suitable for clinical diagnosis of bacteria.

Description

Technical Field

[0001] This invention relates to the field of bacterial strain detection, specifically to bacterial hybridization reagents and their applications. Background Technology

[0002] Bacterial infections are among the infectious diseases with the highest morbidity and mortality rates worldwide, with Gram-positive bacteria (such as Staphylococcus aureus) being the most prevalent. Staphylococcus aureus ), Enterococcus faecalis ( Enterococcus faecalis ), saprophytic Staphylococcus ( Staphylococcus saprophyticus ) and Streptococcus pneumoniae ( Streptococcus pneumoniae )) and Gram-negative bacteria (such as Escherichia coli ( Escherichia coli ), Klebsiella pneumoniae ( Klebsiella pneumoniae Acinetobacter baumannii ( Acinetobacter baumannii ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa These bacteria account for over 85% of clinical bacterial infection cases. The spread of drug resistance in these bacteria has become a global public health crisis and a significant cause of clinical treatment failure. Precision diagnosis and treatment of these bacteria relies on fluorescence in situ hybridization (FISH) technology. This technology utilizes fluorescently labeled DNA probes to target and bind to bacterial ribosomal RNA (16S / 23S rRNA). Labeled bacterial cells are precisely captured by magnetic particles and rapidly migrate to the imaging focal plane under the influence of a magnetic field. Signals are acquired and images are analyzed by a fluorescence imaging system, allowing for bacterial detection and counting within 30 minutes—more than 40 times more efficient than traditional culture methods (24-48 hours). This technology eliminates the need for bacterial culture, directly detects live bacteria in clinical samples, is simple to operate, and provides accurate results. It has become one of the mainstream technologies for rapid bacterial detection and drug resistance gene analysis and is widely used in the clinical diagnosis of pathogenic bacteria.

[0003] Hybridization solution is the core reagent of this technology and must simultaneously meet three specific requirements: first, it must be mildly permeable to the bacterial cell wall to ensure probe entry into the cell; second, it must protect the bacterial cell morphology to avoid loss of in situ information; and third, it must efficiently remove free probes to reduce background fluorescence. However, existing hybridization reagents have shortcomings in performance, safety, and stability (reproducibility), becoming a technical bottleneck for rapid infection detection. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems of poor safety and stability in the existing technology, and to provide a bacterial hybridization reagent and its application.

[0005] To achieve the above objectives, the first aspect of the present invention provides a bacterial hybridization reagent comprising a citrate buffer, a disaccharide blocker, a guanidine protein denaturant, a sulfobetaine-type zwitterionic surfactant, polyoxyethylene sorbitan ester, and polyoxyethylene (5-30) alkyl ether, wherein, relative to 10g of the disaccharide blocker, the content of the guanidine protein denaturant is 8-12g, the content of the sulfobetaine-type zwitterionic surfactant is 2-4g, the content of the polyoxyethylene sorbitan ester is 2-4g, and the content of the polyoxyethylene (5-30) alkyl ether is 1.5-2.5g.

[0006] A second aspect of the invention provides the use of the reagents described above in the detection of bacteria for non-diagnostic purposes.

[0007] The third method of the present invention provides a method for detecting bacteria using the reagents described above.

[0008] The bacterial hybridization reagent of this invention can gently penetrate the bacterial cell wall, ensuring successful probe insertion into the cell; simultaneously, it can perfectly protect the bacterial cell morphology, prevent loss of in situ information, and efficiently remove free probes and reduce background fluorescence. This hybridization reagent does not use toxic reagents such as formamide, which are carcinogenic and reproductively toxic (safe), exhibits good stability, and demonstrates excellent overall performance. It effectively solves the problems of insufficient performance and poor stability of traditional hybridization solutions, breaking through the technical bottleneck of rapid bacterial detection and significantly improving detection results. Attached Figure Description

[0009] Figure 1 This is the standard curve obtained in Example 1.

[0010] Figure 2 This is the standard curve obtained in Example 2.

[0011] Figure 3 This is the standard curve obtained in Example 3.

[0012] Figure 4 This is the standard curve obtained in Example 4.

[0013] Figure 5 This is a standard curve obtained when the amount of guanidine hydrochloride used in Reference Example 1 was 7g.

[0014] Figure 6 This is a standard curve obtained when the amount of guanidine hydrochloride used in Reference Example 1 was 8g.

[0015] Figure 7 This is a standard curve obtained when the amount of guanidine hydrochloride used in Reference Example 1 was 10g.

[0016] Figure 8This is a standard curve obtained when the amount of Tween-80 used in Reference Example 2 was 1.5g.

[0017] Figure 9 This is the standard curve obtained when the amount of Tween-80 used in Reference Example 2 was 2g.

[0018] Figure 10 This is a standard curve obtained when the amount of Tween-80 used in Reference Example 2 was 3g.

[0019] Figure 11 This is the standard curve obtained when the amount of Tween-80 used in Reference Example 2 was 3.5g. Detailed Implementation

[0020] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0021] This invention provides a bacterial hybridization reagent containing a citrate buffer, a disaccharide blocker, a guanidine protein denaturant, a sulfobetaine-type zwitterionic surfactant, polyoxyethylene sorbitan ester, and polyoxyethylene (5-30) alkyl ether. The content of the guanidine protein denaturant is 5-15g per 10g of the disaccharide blocker, the content of the sulfobetaine-type zwitterionic surfactant is 1-5g, the content of the polyoxyethylene sorbitan ester is 1-5g, and the content of the polyoxyethylene (5-30) alkyl ether is 1-8g.

[0022] According to a preferred embodiment of the present invention, the content of the guanidine protein denaturant is 8-12g relative to every 10g of disaccharide blocker, such as 8g, 8.5g, 9g, 9.5g, 9.7g, 9.76g, 10g, 10.5g, 11g, 12g or any value or range between the above values, more preferably 9.7-10.5g.

[0023] According to a preferred embodiment of the present invention, the content of the sulfobetaine-type zwitterionic surfactant is 2-4g relative to every 10g of disaccharide blocker, such as 2g, 2.2g, 2.4g, 2.6g, 2.7g, 2.8g, 2.82g, 3g, 3.07g, 3.1g, 3.2g, 3.4g, 3.6g, 3.8g, 4g, or any value or range between the above values, more preferably 2.8-3.2g.

[0024] According to a preferred embodiment of the present invention, the content of the polyoxyethylene sorbitan ester is 2-4g relative to each 10g of disaccharide blocker, such as 2g, 2.2g, 2.4g, 2.6g, 2.7g, 2.8g, 2.82g, 3.07g, 3g, 3.1g, 3.2g, 3.4g, 3.6g, 3.8g, 4g, or any value or range between the above values, more preferably 2.8-3.2g.

[0025] According to a preferred embodiment of the present invention, the content of the polyoxyethylene (5-30) alkyl ether is 1.5-2.5g relative to every 10g of disaccharide blocker, such as 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 1.92g, 2g, 2.09g, 2.1g, 2.2g, 2.3g, 2.4g, 2.5g, or any value or range between the above values, more preferably 1.9-2.1g.

[0026] The inventors of this invention have discovered that preparing the bacterial hybridization reagent according to a preferred ratio can further improve the linear fit and further enhance the accuracy of the hybridization signal.

[0027] According to a preferred embodiment of the present invention, the disaccharide blocker is β-lactose. The main function of β-lactose in the hybridization reagent is to reduce non-specific binding and synergistically improve the signal-to-noise ratio of the hybridization reaction with other components.

[0028] According to a preferred embodiment of the present invention, the guanidine protein denaturant can be a guanidine protein denaturant commonly used in the art, preferably guanidine hydrochloride.

[0029] According to a preferred embodiment of the present invention, the sulfobetaine-type zwitterionic surfactant has a carbon number of C20-C40, more preferably 3-[(3-cholamidopropyl)dimethylammonium]-2-hydroxy-1-propanesulfonate (CHAPSO, structure shown below).

[0030] According to a preferred embodiment of the present invention, the polyoxyethylene sorbitan ester is polyoxyethylene sorbitan monooleate (i.e., Tween-80).

[0031] According to a preferred embodiment of the present invention, the polyoxyethylene (5-30) alkyl ether is a polyoxyethylene (10-20) alkyl ether, and more preferably a polyoxyethylene (20) hexadecyl ether (trade name Brij 58, CAS: 9004-95-9).

[0032] In this invention, the citrate can be a common citrate buffer in the art. According to a preferred embodiment of the invention, the citrate buffer is a combination of sodium citrate (2-hydroxypropane-1,2,3-tricarboxylic acid trisodium, C6H5Na3O7) and monosodium citrate (2-hydroxypropane-1,2,3-tricarboxylic acid monosodium, C6H7NaO7). The weight ratio of sodium citrate to monosodium citrate is preferably 2-6, such as 2g, 2.1g, 2.5g, 3g, 3.1g, 3.2g, 3.3g, 3.6g, 3.5g, 4g, 4.1g, 4.2g, 4.5g, 4.8g, 5g, 6g, or any value or range between the above values, more preferably 3-5. In this invention, the sodium citrate can be used in anhydrous form or in dihydrate form.

[0033] According to a preferred embodiment of the present invention, the citrate buffer content can be 2-6g relative to every 10g of disaccharide blocker, such as 2g, 2.1g, 2.5g, 3g, 3.1g, 3.2g, 3.3g, 3.5g, 4g, 4.1g, 4.2g, 4.5g, 5g, 6g or any value or range between the above values, preferably 3-4g.

[0034] In this invention, to further increase bacterial cell permeability by hydrolyzing the cell wall without damaging cell morphology, the reagent may further include an enzyme preparation. The enzyme preparation can be a common enzyme used in the art to increase cell wall permeability (cell wall lysing enzyme), and is generally stored independently. The enzyme preparation is preferably a peptidoglycan hydrolase, more preferably at least one or at least two of achromopeptidases, lysozymes, and Labiases. Achromopeptidases are enzymes derived from bacteria (such as...) Lysobacter species Lysozyme is a lysyl endopeptidase that can cleave proteins at specific locations within the polypeptide chain (such as after lysine residues). Lysozyme can disrupt the β-1,4 glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in bacterial cell walls and in chitodextrin. Rabiazymes are typically produced by Streptomyces (such as Streptomyces f. davidii). Streptomyces fulvissimusIt is prepared by culturing TU-6 and mainly contains multiple enzyme activities such as β-N-acetyl-D-glucosidase and lysozyme. The content of lysozyme relative to 100U of achromopeptidase is preferably 0.05-1mg, such as 0.05mg, 0.15mg, 0.2mg, 0.25mg, 0.35mg, 0.45mg, 0.55mg, 0.65mg, 0.75mg, 0.85mg, 0.95mg, 1mg, or any value or range between the above values, more preferably 0.1-0.3mg. The content of the labia enzyme relative to 100U of achromopeptidase is preferably 0.05-1mg, such as 0.05mg, 0.08mg, 0.1mg, 0.15mg, 0.2mg, 0.25mg, 0.35mg, 0.45mg, 0.55mg, 0.65mg, 0.75mg, 0.85mg, 0.95mg, 1mg or any value or range between the above values, more preferably 0.08-0.2mg. The content / dosage of achromopeptidase relative to each gram of disaccharide blocker can be 500-5000U, such as 500U, 600U, 700U, 800U, 1000U, 1200U, 1290U, 1300U, 1600U, 1900U, 2200U, 2500U, 2800U, 3000U, 3500U, 4000U, 4500U, 5000U, or any value or range between the above values.

[0035] In this invention, to further maintain system stability, the reagent may also contain urea. The urea content / amount per gram of disaccharide blocker can be 10-30g, such as 10g, 12g, 14g, 16g, 18g, 20g, 22g, 23g, 24g, 26g, 28g, 30g, or any value or range between the above.

[0036] In this invention, for ease of direct use, the reagent may also contain water. The water content relative to each gram of disaccharide blocker can be 8-200g, such as 8g, 10g, 15g, 20g, 28g, 48g, 68g, 88g, 100g, 108g, 128g, 148g, 168g, 188g, 200g, or any value or range between these. To further ensure the accuracy of the detection, the water is molecular biological grade water (USP grade distilled pure water).

[0037] This invention also provides the application of the reagents described above in the detection of bacteria (for non-diagnostic purposes). The bacteria can be Gram-negative bacteria (such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa) or Gram-positive bacteria. Existing hybridization reagents often fall short in targeting Gram-positive bacteria with thick cell walls; however, the reagents or methods of this invention are particularly suitable for the detection of Gram-positive bacteria with thick cell walls (especially those greater than or equal to 20 nm (e.g., 20-100 nm thick), such as at least one of Staphylococcus aureus (SA), Enterococcus faecalis (EF), Staphylococcus saprophyticus (SS), and Streptococcus pneumoniae (SP).

[0038] The present invention also provides a method for detecting bacteria (for non-diagnostic purposes), the method comprising: S1. In the presence of the reagents described above, the probe, magnetic beads and the sample to be tested are co-incubated. S2. Perform magnetic separation on the co-incubated product; S3. Fluorescence detection.

[0039] In step S1, the probe is capable of hybridizing with the target sequence within the target bacteria. The surface of the magnetic beads is modified with a probe or antibody that recognizes and binds to the target bacteria. Those skilled in the art can select the probe, the probe on the surface of the magnetic beads, or the antibody according to the type of target bacteria, which will not be elaborated here. The incubation temperature can be 35-40℃. The incubation time can be 20-40 minutes.

[0040] In step S1, the co-incubation is typically performed in a microplate. The amount of reagent used can be 5-15 μL / well. To further increase bacterial cell permeability by hydrolyzing the cell wall without disrupting cell morphology, the method may also include adding an enzyme preparation (as described above) to the system. The amount of the enzyme preparation used can be 5-15 μL / well. The amount of the achromopeptidase used can be 0.4-0.5 U / well.

[0041] In step S1, to further stabilize the system, the method may further include adding urea to the system. The amount of urea used may be 15-20 mg / well.

[0042] In step S2, magnetic separation can be accomplished with the help of an external magnetic field.

[0043] In step S3, fluorescence signals can be observed using a fluorescence microscope or an automated imaging system to perform bacterial labeling and quantitative analysis.

[0044] Understandably, to achieve quantitative detection, the method may further include using standards to detect fluorescence signals, performing linear regression analysis, and obtaining a regression equation. When detecting unknown samples, the fluorescence signal is measured; substituting it into the regression equation, the concentration of the target bacteria in the sample can be deduced.

[0045] In a preferred embodiment of the present invention, the method of the present invention does not include the operation of ethanol dehydration, thereby avoiding problems such as poor repeatability caused by ethanol dehydration.

[0046] The reagents of this invention can be used as hybridization solutions (to promote the binding of probes to target bacteria) or as washing solutions (to remove excess probes that failed to bind to target bacteria).

[0047] The present invention will be described in detail below through examples. All reagents used in the following experiments were of molecular biological purity.

[0048] Example 1 I. Prepare the hybridization reagent according to the following steps: Weigh out 8.5±0.3g of β-lactose; 2.5±0.1g of sodium citrate dihydrate; 0.6±0.01g of monosodium citrate; 2.5±0.01g of CHAPSO; 8.6±0.01g of guanidine hydrochloride; 2.5±0.01g of Tween-80; 1.7±0.01g of BriJ-58; and 78.5±0.15g of molecular biological grade water. Place the solution in a 60℃ oven and dissolve for 15±5 min. After cooling, add 25μL of a universal probe (probe concentration of 10) to the solution. 5 μg / L), the sequence of the universal probe is: 5'-ACGACAACCATGCACCACCTG-3' (SEQ ID NO:1, the 5' end of the probe sequence is coupled with Cy5 far-infrared fluorescent dye via N-hydroxysuccinimide ester (NHS), water is added to make the total mass of the system 110g, and it is left at room temperature for 15±2min.

[0049] II. Prepare the three-enzyme mixture according to the following steps: 1. A three-enzyme mixture was prepared by discoloration peptidase (Sigma-Aldrich A3422), lysozyme (Sigma-Aldrich L7651), and labiazyme (Sigma-Aldrich L1414) at concentrations of 100 U / mL, 0.2 mg / mL, and 0.1 mg / mL, respectively.

[0050] III. Test for bacteria according to the following steps: 1. Prepare a mid-log phase bacterial culture of Staphylococcus aureus and incubate for 1-2 hours. Use OD200 to measure the bacterial growth. 600The concentration of bacterial culture was determined by optical density method, based on the standard formula "OD". 600 =1 corresponds to 8×10 8 The initial bacterial concentration was calculated using "CFU / mL". Multiple bacterial solutions covering low, medium, and high concentration ranges were obtained by diluting the bacterial solutions with PBS buffer, at concentrations of 1×10⁻⁶. 7 1×10 6 2×10 5 4×10 4 A blank control group was used, and each group was repeated 4 times.

[0051] 2. Take a transparent 96-well plate and add the above-mentioned gradient concentration bacterial solutions and blank control PBS to specific wells of the 96-well plate, with the sample volume per well controlled at 30 μL.

[0052] 3. Add 10 μL of the three-enzyme mixture to each well of the plate.

[0053] 4. Add 10 μL of the hybridization reagent containing the probe prepared in step one to the plate and mix well. Place the 96-well plate with the sample added in a 37°C incubator and react for 40 min. Add 20 μL of 15M urea and 5 μL of MP-CT lyophilized beads (fluidMAG-CT(4122)) to the plate. Mix by pipetting and aspirating, then transfer the liquid to the plate containing the dye pad and incubate at 37°C for 30 min.

[0054] 5. Transfer the plate to the magnet and incubate for 4 minutes.

[0055] 6. MultiPath imager imaging, image analysis, and recording of linear fitting data are performed. The results are as follows: Figure 1 As shown.

[0056] Examples 2-4 Bacterial detection was performed according to the method in Example 1, except that Staphylococcus aureus was replaced with Enterococcus faecalis (EF), Staphylococcus saprophyticus (SS), and Streptococcus pneumoniae (SP), respectively. The obtained standard curves are shown in Figure 1. Figure 2-4 As shown.

[0057] like Figure 1-4 As shown, in the detection experiments of different bacteria, the R² of the present invention is close to 1, indicating that the linear fit between concentration and hybridization signal is extremely high, proving that the system is stable and effective and can accurately capture the hybridization signal of each bacterium.

[0058] Reference Example 1 Bacterial detection was performed according to the method described in Example 1, except that the amount of guanidine hydrochloride was adjusted to 7g, 8g, and 10g (relative to 8.5g of β-lactose), and the obtained standard curves are shown below. Figures 5-7 As shown.

[0059] Reference Example 2 Bacterial detection was performed as in Example 1, except that the amount of Tween-80 was adjusted to 1.5g, 2g, 3.0g, and 3.5g (relative to 8.5g of β-lactose), respectively. The resulting standard curve is shown below. Figures 8-11 As shown.

[0060] Reference Example 3 Bacterial detection was performed as in Example 1, except that BriJ-58 was replaced with E-1007 (isotridecyl alcohol polyoxyethylene ether-7). The obtained regression equation and R0 were... 2 As shown in Table 1 below.

[0061] Reference Example 4 Bacterial detection was performed as described in Example 1, except that the sodium citrate buffer system (sodium citrate + monosodium citrate) was replaced with PBS buffer. The obtained regression equation and R... 2 As shown in Table 1 below.

[0062] Reference Example 5 Enterococcus faecalis was detected according to the method described in Example 1, except that the concentrations of achromopeptidase, lysozyme, and rapyzase in the three-enzyme mixture were adjusted to 50 U / mL, 0.1 mg / mL, and 0.05 mg / mL, respectively. The obtained regression equation and R0 were then analyzed. 2 As shown in Table 1 below.

[0063] Reference Example 6 Staphylococcus aureus detection was performed as described in Example 1, except that achromopeptidase was not used. The obtained regression equation and R... 2 As shown in Table 1 below.

[0064] Reference Example 7 Staphylococcus aureus detection was performed as in Example 1, except that lysozyme was not used. The obtained regression equation and R0 were... 2 As shown in Table 1 below.

[0065] Reference Example 8 Staphylococcus aureus detection was performed as in Example 1, except that Labja enzyme was not used. The obtained regression equation and R0 were... 2 As shown in Table 1 below.

[0066] Table 1

[0067] Application Example 1 The detection limits for Staphylococcus aureus (SA), Enterococcus faecalis (EF), Staphylococcus saprophyticus (SS), and Streptococcus pneumoniae (SP) were performed according to Examples 1-4, except that the above bacteria were diluted to 5E3 (5 × 10⁻⁶). 3 CFU / mL), E3 (10 3 The concentrations (CFU / mL) were calculated, with three replicates for each concentration. The data obtained are shown in Table 2 below.

[0068] Table 2

[0069] Therefore, when the bacterial culture is diluted to E3, the visible signal does not exceed 200, and no effective signal can be detected. The detection limit of this invention is 5E3, which is lower than the detection limit of traditional FISH technology (10). 4 (CFU / mL).

[0070] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A bacterial hybridization reagent, characterized in that, The reagent contains a citrate buffer, a disaccharide blocker, a guanidine protein denaturant, a C20-C40 sulfobetaine-type zwitterionic surfactant, polyoxyethylene sorbitan ester, and polyoxyethylene (5-30) alkyl ether. The content of the guanidine protein denaturant is 8-12g per 10g of disaccharide blocker, the content of the sulfobetaine-type zwitterionic surfactant is 2-4g, the content of the polyoxyethylene sorbitan ester is 2-4g, and the content of the polyoxyethylene (5-30) alkyl ether is 1.5-2.5g.

2. The reagent according to claim 1, characterized in that, The content of the guanidine protein denaturant is 9.7-10.5g relative to every 10g of disaccharide blocker; And / or, relative to each 10g of disaccharide blocker, the content of the sulfobetaine-type zwitterionic surfactant is 2.8-3.2g; And / or, relative to 10g of disaccharide blocker, the content of the polyoxyethylene sorbitan ester is 2.8-3.2g; And / or, relative to 10g of disaccharide blocker, the content of the polyoxyethylene (5-30) alkyl ether is 1.9-2.1g.

3. The reagent according to claim 1, characterized in that, The polyoxyethylene (5-30) alkyl ether is a polyoxyethylene (10-20) alkyl ether.

4. The reagent according to any one of claims 1-3, characterized in that, The disaccharide blocker is β-lactose; And / or, the guanidine protein denaturant is guanidine hydrochloride; And / or, the sulfobetaine-type zwitterionic surfactant is 3-[(3-cholamidopropyl)dimethylammonium]-2-hydroxy-1-propanesulfonate; And / or, the polyoxyethylene sorbitan ester is polyoxyethylene sorbitan monooleate; And / or, the polyoxyethylene (5-30) alkyl ether is polyoxyethylene (20) hexadecyl ether.

5. The reagent according to any one of claims 1-3, characterized in that, The citrate buffer is sodium citrate and monosodium citrate, and the weight ratio of sodium citrate to monosodium citrate is 2-6; And / or, the citrate buffer content is 2-6g relative to every 10g of disaccharide blocker.

6. The reagent according to claim 5, characterized in that, The weight ratio of sodium citrate to monosodium citrate is 3-5; And / or, the citrate buffer content is 3-4g per 10g of disaccharide blocker.

7. The reagent according to any one of claims 1-3, characterized in that, The reagent also contains cell wall lysing enzyme; And / or, the reagent also contains urea; And / or, the reagent also contains water, in the form of 8-200g of water per gram of disaccharide blocker.

8. The reagent according to claim 7, characterized in that, The cell wall lysing enzymes include achromopeptidase, lysozyme, and rapiazase.

9. The use of the reagent according to any one of claims 1-8 in the detection of bacteria for non-diagnostic purposes.

10. A method for detecting bacteria for non-diagnostic purposes, characterized in that, The method includes: S1. In the presence of the reagent described in any one of claims 1-8, the probe, magnetic beads and the sample to be tested are co-incubated. S2. Perform magnetic separation on the co-incubated product; S3. Fluorescence detection.

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