General-purpose hybridization washing composition and use thereof

Abstract

The present application relates to the field of strain detection, and discloses a universal hybrid washing composition and application thereof.The composition contains a citrate buffer, a sugar blocking agent, a halogenated alkyl quaternary ammonium salt type cationic surfactant, an alkyl betaine type surfactant, a glycoside type nonionic surfactant and a polyoxyethylene type nonionic surfactant.The composition is safe, efficient and widely applicable.

Description

Technical Field

[0001] This invention relates to the field of microbial strain detection, and more specifically to a general-purpose hybridization detergent composition and its application. Background Technology

[0002] Gram-positive bacteria (such as Enterococcus faecalis, Enterococcus faecalis ,EF) and Gram-negative bacteria (such as Escherichia coli ( Escherichia coli EC), Klebsiella pneumoniae ( Klebsiella Pneumoniae KP), Pseudomonas aeruginosa ( Pseudomonas aeruginosa PA), Proteus mirabilis ( Proteus mirabilis Microbes (PM) are the most prevalent pathogens in clinical infections, accounting for over 85% of clinical bacterial infection cases. The spread of drug-resistant bacteria has become a global public health crisis and a significant cause of clinical treatment failure. Precise diagnosis and treatment of these bacteria relies on fluorescence in situ hybridization (FISH) technology. This technology uses species-specific DNA probes to label bacterial 16 / 23S rRNA, magnetic particles to capture bacteria, and the bacteria migrate to the imaging focal plane under the influence of a magnetic field. By photographing the fluorescently labeled bacterial cells and analyzing the images, the bacterial species can be determined. This reduces the bacterial labeling and counting experiment cycle to 30 minutes, significantly improving efficiency compared to traditional culture methods (24-48 hours), enabling rapid labeling. The core reagent for FISH technology is the hybridization detergent, which mainly includes two types: traditional formamide-based hybridization detergents and formamide-free hybridization detergents specifically for Gram-positive bacteria.

[0003] Traditional formamide-based hybridization wash solutions typically employ a "high salt + high formamide" formulation, with a typical composition of 0.5×SSC (sodium citrate-salt buffer) + 50% formamide + 0.1% SDS. These wash solutions effectively dissociate non-specifically bound probes through the strong denaturing effect of formamide, while maintaining the stability of the target hybrid due to the high salt concentration. This allows for effective washing of single-type bacterial resistance gene hybrids, such as Gram-negative bacteria, meeting the signal-to-noise ratio requirements in basic resistance gene detection. However, these wash solutions have significant limitations when used for rapid bacterial labeling of mixed infection samples. Firstly, their versatility is poor: for GC-rich hybrids in Gram-positive bacteria, the washing temperature needs to be increased to 55°C to effectively remove non-specific signals, but this temperature can cause dissociation of the target hybrids in Gram-negative bacteria, affecting detection accuracy. Secondly, washing at 42°C cannot effectively remove non-specific signals in Gram-positive bacteria, leading to increased background interference and affecting detection specificity. In addition, formamide has clear reproductive toxicity, requiring special protective measures during operation; it is also prone to volatilization and degradation during storage, further affecting the repeatability and stability of experimental results.

[0004] Formamide-free hybridization wash solutions for Gram-positive bacteria use 15-20% propylene glycol as the main denaturant, combined with a 0.1×SSC and Tris-HCl buffer system (pH 8.0), and are typically used for washing at 50-55°C. This type of wash solution achieves low-toxicity denaturation through propylene glycol, effectively dissociating GC-rich non-specific hybrids from Gram-positive bacteria while maintaining the stability of the target hybrid. This significantly improves the specificity and operational safety of Gram-positive bacteria resistance gene detection, avoiding the toxicological risks associated with formamide. However, this formulation is specifically designed for the detection of resistance genes in Gram-positive bacteria and does not consider the need for simultaneous detection of genes or Gram-negative bacterial targets. Furthermore, this formulation is susceptible to background interference in complex biological samples, limiting its detection sensitivity and applicability, and making it difficult to meet the practical needs of rapid simultaneous bacterial labeling and counting in mixed infection samples. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of poor versatility and safety in the prior art, and to provide a universal hybridization washing composition and its application.

[0006] To achieve the above objectives, a first aspect of the present invention provides a general-purpose hybridization detergent composition comprising a citrate buffer, a sugar blocker, a haloalkyl quaternary ammonium salt cationic surfactant, an alkyl betaine surfactant, a glycoside nonionic surfactant, and a polyoxyethylene nonionic surfactant, wherein, relative to each gram of sugar blocker, the content of the haloalkyl quaternary ammonium salt cationic surfactant is 6-20 mg, the content of the alkyl betaine surfactant is 50-150 mg, the content of the glycoside nonionic surfactant is 10-30 mg, and the content of the polyoxyethylene nonionic surfactant is 2-15 mg.

[0007] A second aspect of the invention provides the use of the composition as described above in the detection of Gram-positive and / or Gram-negative bacteria for non-diagnostic purposes.

[0008] The third method of the present invention provides a method for detecting bacteria using the composition described above.

[0009] This invention addresses the shortcomings of existing hybridization washing systems in terms of versatility, safety, and detection performance by providing a novel, safe, efficient, and widely applicable hybridization washing composition. This composition can simultaneously accommodate Gram-positive and Gram-negative bacteria under single reagent and standard operating conditions, significantly improving the detection coverage of mixed infection samples and avoiding the operational complexity and efficiency loss caused by reagent changes. By using a low-toxicity denaturing component instead of traditional formamide, the operational safety and storage stability of the reagent are effectively improved, reducing health risks to operators. Furthermore, through optimized formulation design, this invention enhances the ability to remove non-specific signals and maintain the stability of target signals at a uniform washing temperature, significantly improving detection specificity and sensitivity. It can detect bacterial concentrations as low as 5000 CFU / mL, meeting the clinical need for rapid and accurate labeling and counting of multiple pathogenic bacteria. Attached Figure Description

[0010] Figure 1 This is the standard curve obtained in Example 1.

[0011] Figure 2 This is the standard curve obtained in Example 2.

[0012] Figure 3 This is the standard curve obtained in Example 3.

[0013] Figure 4 This is the standard curve obtained in Example 4.

[0014] Figure 5 This is the standard curve obtained in Example 5.

[0015] Figure 6 This is a standard curve obtained when the amount of CTAB used in Reference Example 1 was 0.02g.

[0016] Figure 7 This is a standard curve obtained when the amount of CTAB used in Reference Example 1 was 0.04g.

[0017] Figure 8 This is a standard curve obtained when the amount of CTAB used in Reference Example 1 was 0.08g.

[0018] Figure 9 This is a standard curve obtained when the amount of CTAB used in Reference Example 1 was 0.10g.

[0019] Figure 10 This is a standard curve obtained when the amount of Tween-80 used in Reference Example 2 was 0.01g.

[0020] Figure 11 This is the standard curve obtained when the amount of Tween-80 used in Reference Example 2 was 0.02g.

[0021] Figure 12 This is a standard curve obtained when the amount of Tween-80 used in Reference Example 2 was 0.04g.

[0022] Figure 13 This is a standard curve obtained when the amount of Tween-80 used in Reference Example 2 was 0.05g. Detailed Implementation

[0023] 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.

[0024] This invention provides a general-purpose hybrid detergent composition comprising a citrate buffer, a sugar blocker, a haloalkyl quaternary ammonium salt cationic surfactant, an alkyl betaine surfactant, a glycoside nonionic surfactant, and a polyoxyethylene nonionic surfactant, wherein, relative to each gram of sugar blocker, the haloalkyl quaternary ammonium salt cationic surfactant comprises 6-20 mg, the alkyl betaine surfactant comprises 50-150 mg, the glycoside nonionic surfactant comprises 10-30 mg, and the polyoxyethylene nonionic surfactant comprises 2-15 mg.

[0025] According to a preferred embodiment of the present invention, the content of the haloalkyl quaternary ammonium salt cationic surfactant is 7-10 mg per gram of sugar blocker, such as 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg or any value or range between the above values, more preferably 8-9 mg.

[0026] According to a preferred embodiment of the present invention, the content of the alkyl betaine-type surfactant is 70-90 mg per gram of sugar blocker, such as 70 mg, 72 mg, 74 mg, 76 mg, 78 mg, 80 mg, 82 mg, 84 mg, 85 mg, 86 mg, 87 mg, 88 mg, 90 mg or any value or range between the above values, more preferably 85-87 mg.

[0027] According to a preferred embodiment of the present invention, the content of the glycoside-type nonionic surfactant is 15-25 mg per gram of sugar blocker, such as 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg or any value or range between the above values, more preferably 17-19 mg.

[0028] According to a preferred embodiment of the present invention, the content of the polyoxyethylene nonionic surfactant is 3.5-5.5 mg per gram of sugar blocker, such as 3.5 mg, 3.7 mg, 3.9 mg, 4 mg, 4.1 mg, 4.3 mg, 4.5 mg, 4.7 mg, 4.9 mg, 5 mg, 5.1 mg, 5.3 mg, 5.5 mg or any value or range between the above values, more preferably 4-5 mg.

[0029] The inventors of this invention have discovered that formulating the general-purpose hybridization detergent composition according to a preferred ratio can further improve the linear fit and further enhance the accuracy of the hybridization signal.

[0030] According to a preferred embodiment of the present invention, the sugar blocker is a disaccharide blocker, more preferably β-lactose. The main function of β-lactose in hybridization detergents is to reduce non-specific binding and synergistically improve the specificity and signal-to-noise ratio of the hybridization reaction with other components.

[0031] According to a preferred embodiment of the present invention, the general formula of the haloalkyl quaternary ammonium salt cationic surfactant is [CH3(CH2)]. m -N + (CH3)3]X - Where m is 9-21, and X is a halogen (which can be Cl or Br), more preferably hexadecyltrimethylammonium bromide (hexadecyltrimethylammonium bromide, CTAB, [CH3(CH2)]). 15 -N + (CH3)3]Br - ).

[0032] According to a preferred embodiment of the present invention, the alkyl betaine-type surfactant is CH3(CH2). n -N + (CH3)2-CH2-COO - Wherein, n is 7-17, more preferably dodecyl dimethyl betaine (BS-12, structural formula CH3(CH2)). 11 -N + (CH3)2-CH2-COO - ).

[0033] According to a preferred embodiment of the present invention, the glycoside-type nonionic surfactant is an alkyl-β-D-glucopyranoside with 14-24 carbon atoms, more preferably decyl-β-D-glucopyranoside (DG, structural formula: ).

[0034] According to a preferred embodiment of the present invention, the polyoxyethylene nonionic surfactant is polyoxyethylene sorbitan ester, more preferably polyoxyethylene sorbitan monooleate (i.e., Tween-80).

[0035] 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 1-3, more preferably 1.2-1.5. In this invention, the sodium citrate can be used in anhydrous form or in dihydrate form.

[0036] According to a preferred embodiment of the present invention, the content of the citrate buffer relative to each gram of sugar blocker can be 1-10g, such as 1g, 1.5g, 1.9g, 2g, 2.1g, 2.5g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, or any value or range between the above values, preferably 1.5-2.5g.

[0037] In this invention, for ease of direct use, the composition also contains water. The water content relative to each gram of carbohydrate blocker can be 8-200g, such as 8g, 10g, 15g, 20g, 28g, 48g, 68g, 88g, 108g, 128g, 148g, 168g, 188g, 200g, or any value or range between these. To further ensure the accuracy of detection, the water is molecular biological grade water (USP grade distilled pure water).

[0038] The present invention also provides the use of the compositions described above in the detection of Gram-positive and / or Gram-negative bacteria (for non-diagnostic purposes).

[0039] The present invention also provides a method for detecting bacteria, the method comprising: S1. In the presence of the composition described above, the auxiliary probe, magnetic beads and the sample to be tested are co-incubated. S2. Perform magnetic separation on the co-incubated product; S3. Fluorescence detection.

[0040] In step S1, the auxiliary probe is capable of hybridizing with the target sequence within the target bacteria. The surface of the magnetic beads is modified with probes or antibodies that specifically recognize and bind to the target bacteria. Those skilled in the art can select the auxiliary probe, the probes on the surface of the magnetic beads, or the antibodies according to the type of target bacteria, which will not be elaborated further here. The incubation temperature can be 35-40℃. The incubation time can be 20-40 minutes.

[0041] In step S2, magnetic separation can be accomplished with the aid of an external magnetic field, for example, on a magnetic rack.

[0042] In step S3, fluorescence signals can be observed using a fluorescence microscope or an automated imaging system to perform bacterial labeling and quantitative analysis.

[0043] 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.

[0044] 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.

[0045] The present invention will be described in detail below through examples. All reagents used in the following experiments were of molecular biological purity.

[0046] Example 1 I. Prepare the hybrid detergent according to the following steps: 1. Zero the analytical balance, weigh the 50mL test tube using the analytical balance, and record the weight; 2. Weigh 7.2g of β-lactose using weighing paper; 3. Weigh 8.3g of sodium citrate dihydrate using weighing paper; 4. Weigh 5.5g of sodium citrate monosodium using weighing paper; 5. Weigh 0.06g of CTAB using weighing paper; 6. Weigh 0.62g of BS-12 using weighing paper; 7. Weigh 0.13g of DG using weighing paper; 8. Weigh 0.03g of Tween-80 using weighing paper; 9. Weigh out 79g of molecular biological grade water; 10. After adding the above ingredients to the test tube, place the test tube in a 60℃ oven and dissolve for 15±5 minutes; 11. After cooling, add 25 μL of auxiliary probe (probe concentration of 10 μg / L) to the solution. The sequences of the auxiliary probes for Escherichia coli (EC) are shown in Table 1 below. For other bacteria, the universal probes shown in Table 1 are used. 12. Add water to make the total mass of the system 100.84g, and obtain the hybrid detergent; 13. Let the prepared hybrid detergent stand at room temperature for 15±2 minutes.

[0047] Table 1

[0048] II. Test for bacteria according to the following steps: 1. Take Escherichia coli (EC) in the logarithmic growth phase and analyze the OD value. 600 The 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 sets of bacterial solutions covering low, medium, and high concentration ranges were obtained by diluting the bacterial solution with PBS buffer, specifically: 3 × 10⁻⁶ CFU / mL. 5 1×10 5 3×10 4 1×10 4 Blank control (pure PBS buffer).

[0049] 2. Take a transparent 96-well plate and add the hybridization detergent containing the probe prepared in step one to the plate. Add 10 μL of universal MP-PAS magnetic beads (NPFWPA-010-020F, Nanomicro Technology) to the plate. Add the above-mentioned gradient concentration bacterial solutions and blank control PBS to the corresponding wells, with a sample volume of 100 μL per well, and 4 replicates for each concentration. Mix well by pipetting, transfer the liquid to a plate containing a dye pad, and incubate at 37°C for 30 min.

[0050] 3. Transfer the flat plate to the magnetic rack for magnetic separation for 4 minutes.

[0051] 4. After separation, the magnetic beads are placed in an Imager to obtain fluorescence signals; a standard curve of fluorescence signal vs. concentration is plotted, and the results are as follows: Figure 1 As shown.

[0052] Examples 2-5 Bacterial detection was performed as in Example 1, except that Escherichia coli was replaced with Klebsiella pneumoniae (KP), Pseudomonas aeruginosa (PA), Proteus mirabilis (PM), and Enterococcus faecalis (EF), and the probes were replaced with the universal probes shown in Table 1. The obtained standard curves are shown in the figures below. Figure 2-5 As shown.

[0053] Reference Example 1 Bacterial detection was performed according to the method in Example 5, except that the amount of CTAB was adjusted to 0.02g, 0.04g, 0.08g, and 0.10g, respectively. The obtained standard curves are shown below. Figures 6-9 As shown.

[0054] Reference Example 2 Bacterial detection was performed according to the method in Example 1, except that the dosage of Tween-80 was adjusted to 0.01g, 0.02g, 0.04g, and 0.05g, respectively. The obtained standard curves are shown below. Figures 10-13 As shown.

[0055] Reference Example 3 Bacterial detection was performed as in Example 1, except that β-lactose was replaced with D-glucose. The resulting regression equation and R0 were obtained. 2 As shown in Table 2 below.

[0056] Reference Example 4 Bacterial detection was performed as in Example 1, except that Tween-80 was replaced with Tween-20. The obtained regression equation and R0 were... 2 As shown in Table 2 below.

[0057] Reference Example 5 Bacterial detection was performed as 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 2 below.

[0058] Table 2

[0059] Application Example 1 The detection limits for EC, KP, PA, PM, and EF were performed according to the methods described in Examples 1-5, 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 3 below.

[0060] Table 3

[0061] 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).

[0062] 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 general-purpose FISH hybrid detergent composition, characterized in that, The composition contains a citrate buffer, a sugar blocker, a haloalkyl quaternary ammonium salt cationic surfactant, an alkyl betaine surfactant, a glycoside nonionic surfactant, and a polyoxyethylene nonionic surfactant, wherein, relative to each gram of sugar blocker, the content of the haloalkyl quaternary ammonium salt cationic surfactant is 7-10 mg, the content of the alkyl betaine surfactant is 70-90 mg, the content of the glycoside nonionic surfactant is 15-25 mg, and the content of the polyoxyethylene nonionic surfactant is 3.5-5.5 mg; The carbohydrate blocker is β-lactose; The haloalkyl quaternary ammonium salt cationic surfactant is hexadecyltrimethylammonium bromide; The alkyl betaine-type surfactant is dodecyl dimethyl betaine; The glycoside-type nonionic surfactant is decyl-β-D-glucopyranoside; The polyoxyethylene nonionic surfactant is polyoxyethylene dehydrated sorbitan monooleate. The citrate buffer is sodium citrate and monosodium citrate, and the weight ratio of sodium citrate to monosodium citrate is 1-3; The citrate buffer content is 1-10g per gram of sugar blocker.

2. The composition according to claim 1, characterized in that, The content of the haloalkyl quaternary ammonium salt cationic surfactant is 8-9 mg per gram of sugar blocker; And / or, relative to each gram of sugar blocker, the content of the alkyl betaine-type surfactant is 85-87 mg; And / or, relative to each gram of sugar blocker, the content of the glycoside-type nonionic surfactant is 17-19 mg; And / or, the content of the polyoxyethylene nonionic surfactant is 4-5 mg per gram of sugar blocker.

3. The composition according to claim 1, characterized in that, The weight ratio of sodium citrate to monosodium citrate is 1.2-1.5; And / or, the citrate buffer content is 1.5-2.5g relative to each gram of sugar blocker.

4. The composition according to any one of claims 1-3, characterized in that, The composition also contains water, with a water content of 8-200g per gram of sugar blocker.

5. The use of the composition according to any one of claims 1-4 in the detection of Gram-positive and / or Gram-negative bacteria for non-diagnostic purposes.

6. A method for detecting bacteria for non-diagnostic purposes, characterized in that, The method includes: S1. In the presence of the composition according to any one of claims 1-4, the FISH auxiliary probe, magnetic beads and the sample to be tested are co-incubated. S2. Perform magnetic separation on the co-incubated product; S3. Fluorescence detection.

Images