Method for evaluating sample permeabilization effect in microbial single-cell sequencing experiment

Abstract

A method for evaluating the sample permeabilization effect in a microbial single-cell sequencing experiment, which belongs to the technical field of molecular hybridization detection. The evaluation method comprises the following steps: (1) fixing a microbial cell; (2) performing permeabilization treatment on the fixed cell; (3) adding the permeabilized cell sample to a glass slide for baking; (4) adding a probe for in-situ fluorescence probe hybridization; (5) washing same, and counterstaining the cell nucleus; and (6) evaluating the cell permeabilization effect according to the fluorescence intensity of the sample under a fluorescence microscope. The method for evaluating the permeabilization effect of a microbial cell sample can detect the correlation between the permeabilization time and the sample permeabilization effect in the case of a single microbial cell or a mixed microbial cell, thereby determining the optimal permeabilization treatment time of the sample and stabilizing the rRNA proportion and data output of the sample.

Description

A method for evaluating sample permeabilization in microbial single-cell sequencing experiments Technical Field

[0001] The present invention belongs to the technical field of molecular hybridization detection, and in particular relates to a method for evaluating the permeabilization effect of a sample in a microbial single-cell sequencing experiment. Background Art

[0002] With the rapid development of single-cell sequencing technology, its application is expanding from animals and plants to microorganisms, including bacteria, fungi, and viruses. Single-cell sequencing technology applied to prokaryotes is hampered by factors such as low mRNA abundance, unstable transcripts, low copy numbers, and a lack of polyA tails. Furthermore, prokaryotic cells are small and enclosed by thick and complex cell membranes and walls, making them difficult to lyse. Therefore, a fixation and permeabilization step is required in microbial single-cell sequencing experiments.

[0003] For cell membranes, formaldehyde fixation can cross-link and fix proteins and lipid molecules on the cell membrane, thereby maintaining the integrity and stability of the cell membrane. For cell walls, it can cross-link and fix polysaccharides and proteins on the wall, increasing the stability and hardness of the cell wall without causing physical damage to the bacterial cell wall and cell membrane. Therefore, after fixation, microbial cells need to be permeabilized to facilitate rRNA removal and cell lysis.

[0004] Bacterial permeabilization uses a reagent containing lysozyme for incubation. Because different microbial cell types have varying degrees of complexity in their cell membranes and cell walls, different permeabilization parameters and conditions are required. The effectiveness of permeabilization is related to rRNA removal and mRNA capture. Therefore, only by establishing a corresponding and reasonable permeabilization quality control system can stable data output from microbial single-cell sequencing be guaranteed.

[0005] To address the problem of unstable cell permeabilization effects in existing microbial single-cell sequencing experiments, the present invention provides a method for quickly and intuitively evaluating the permeabilization effects of microbial single-cell samples. The correlation between permeabilization time and permeabilization effect is displayed through visualization results, and the optimal permeabilization treatment time is ultimately optimized.

[0006] Summary of the Invention

[0007] The present invention uses fluorescent in situ hybridization technology to design a set of probes for evaluating the permeabilization effect of samples in microbial single-cell sequencing experiments. The specificity and stability of fluorescent probes used in conventional bacterial in situ hybridization are not high. Under normal circumstances, one bacteria corresponds to one specific probe, and it often takes about 24 hours to completely hybridize and bind with the bacteria. The present invention creatively uses LNA to modify the fluorescent probe, so that the probe can more quickly and stably ensure the hybridization probe to bind to the bacteria, and it can fully bind to the bacteria in only 2 hours. The method provided by the present invention for evaluating the permeabilization effect of samples in microbial single-cell sequencing experiments has the advantage of being able to detect the correlation between the sample permeabilization time and the permeabilization effect in the case of a single microbial cell or a mixed microbial cell, thereby determining the optimal permeabilization treatment time of the sample and stabilizing the rRNA proportion and data output of the sample.

[0008] One object of the present invention is to provide a method for evaluating the permeabilization effect of samples in microbial single-cell sequencing experiments; one object of the present invention is to provide a probe for evaluating the permeabilization effect of samples in microbial single-cell sequencing experiments; another object of the present invention is to provide a kit for evaluating the permeabilization effect of samples in microbial single-cell sequencing experiments; and yet another object of the present invention is to provide the use of the probe and the kit in evaluating the permeabilization effect of samples in microbial single-cell sequencing experiments.

[0009] In a first aspect, the present invention provides a method for evaluating the permeabilization effect of a sample in a microbial single-cell sequencing experiment, the method comprising the following steps:

[0010] (1) Microbial cell fixation;

[0011] (2) Permeabilization of fixed cells;

[0012] (3) Add the permeabilized cell sample to a glass slide and bake it;

[0013] (4) adding probes for in situ fluorescent probe hybridization;

[0014] (5) washing and counterstaining the cell nuclei;

[0015] (6) Evaluate the cell permeabilization effect based on the fluorescence intensity of the sample under a fluorescence microscope.

[0016] Preferably, in step (1), formaldehyde is used to fix the cells, the formaldehyde concentration is 4%, the temperature is 4° C., and the treatment time is 6-16 h.

[0017] The permeabilization treatment in step (2) is a cell permeabilization method commonly used by those skilled in the art. In a specific embodiment of the present invention, the reagents for the cell permeabilization treatment are Tween-20 solution and lysozyme solution.

[0018] The present technicians creatively proposed probes for in situ fluorescent hybridization in step (4), which include a positive probe and a negative probe. The nucleotide sequence of the positive probe is shown in SEQ ID NO.1, and the nucleotide sequence of the negative probe is shown in SEQ ID NO.2.

[0019] In the most preferred embodiment of the present invention, the positive probe is LNA-modified.

[0020] Furthermore, counting from the 5' end, the 3rd, 6th and 14th bases of the positive probe are modified with LNA.

[0021] The 5' ends of the positive probe and the negative probe are connected to a reporter fluorescent group, and the reporter fluorescent group is selected from FAM, ROX, Cy5 or Cy3.

[0022] LNA, or locked nucleic acid, is a synthetic nucleic acid analog containing a bridged bicyclic sugar moiety. A methylene group added between the 2'-O- and 4'-positions "locks" the ribofuranose into a 3'-endo conformation.

[0023] The nucleotide sequence involved in the present invention is as follows:

[0024] In the most preferred embodiment of the present invention, the method for evaluating the permeabilization effect of a sample in a microbial single-cell sequencing experiment comprises the following steps:

[0025] (a) Microbial cells were fixed with formaldehyde by shaking, washed and resuspended in PBS-RI solution, centrifuged, resuspended in Tris-HCl-RI solution, and counted;

[0026] (b) Depending on the cell suspension concentration, cells were perforated with Tween-20 on ice for 3–4 min, then permeabilized with lysozyme solution for 1–30 min, washed with PBS-RI solution, and resuspended.

[0027] (c) Pipette the resuspended cell suspension onto a glass slide, spread it evenly into a circle with a diameter of approximately 1.5 cm, and place it on a metal bath to fix it;

[0028] (d) adding hybridization buffer to the slide, covering the bacterial area, and placing it in a wet chamber for incubation; simultaneously, denaturing the probe and hybridization buffer;

[0029] (e) adding the denatured probe to the sample and performing constant temperature hybridization;

[0030] (f) After hybridization, the samples were washed with Washing Buffer, dehydrated with ethanol solutions of different concentrations, dried at room temperature, and dark-stained with DAPI.

[0031] (g) Observe the fluorescence intensity and quantity of the samples under a fluorescence microscope, and evaluate the permeabilization effect of the cell suspension samples based on the fluorescence intensity and bacterial morphology.

[0032] Since long-term treatment with Tween-20 and lysozyme will cause the bacterial structure to rupture and the complete bacterial morphology cannot be observed, and since subsequent experiments need to ensure the integrity of the bacterial morphological structure, it is necessary to explore the permeabilization treatment time under the same input amount and select a treatment time with the strongest fluorescence intensity and the largest number of intact bacteria.

[0033] Preferably, in step (a), the sample is shaken at a frequency of 30-40 rpm / min, the sample is centrifuged at a speed of 5500-7000 xg, and the centrifugation time is 5-10 min.

[0034] The final concentration of lysozyme in step (b) is 2.5-6.25 mg / ml.

[0035] In step (c), the baking temperature is 65-85° C. and the baking time is 0.5-2 h.

[0036] In step (d), the incubation temperature in the humid chamber is 42-58° C., preferably 50-58° C., and the incubation time is 0.5-2 h.

[0037] In step (d), the probe and hybridization buffer solution are diluted at a ratio of 1:(30-200), the probe denaturation temperature is 88°C, the denaturation time is 3 minutes, and the probe is equilibrated at 37°C for 5-10 minutes.

[0038] In step (e), the probe hybridization time is 1-4 hours at a temperature of 37°C.

[0039] After hybridization in step (f), wash with room temperature Washing Buffer I for 2-5 minutes, then with 50-60°C Washing Buffer I for 2-5 minutes, and finally soak with room temperature Washing Buffer II for 2-15 minutes.

[0040] The dehydration step in step (f) is to immerse the sample in 70%, 85%, and 100% ethanol in sequence for 1-3 minutes each.

[0041] Step (f) counterstaining the cell nucleus involves adding 5-20 μl of DAPI dye for staining and placing the cells in the dark for 10-20 minutes.

[0042] The present invention has no particular limitation on the specific types of microbial cells to be detected, and can be either microbial cells from environmental samples or cultured microbial cells. In a specific embodiment of the present invention, the microbial cells are bacteria, specifically including Gram-positive bacteria and Gram-negative bacteria.

[0043] In a second aspect, the present invention provides a probe for evaluating sample permeabilization in a microbial single-cell sequencing experiment, comprising a positive probe and a negative probe. The nucleotide sequence of the positive probe is shown in SEQ ID NO. 1, and the nucleotide sequence of the negative probe is shown in SEQ ID NO. 2.

[0044] In the most preferred embodiment of the present invention, the positive probe is LNA-modified.

[0045] Furthermore, counting from the 5' end, the 3rd, 6th and 14th bases of the positive probe are modified with LNA.

[0046] The 5' ends of the positive probe and the negative probe are connected to a reporter fluorescent group, and the reporter fluorescent group is selected from FAM, ROX, Cy5 or Cy3.

[0047] The nucleotide sequence involved in the present invention is as follows:

[0048] In a third aspect, the present invention provides a kit for evaluating the permeabilization effect of a sample in a microbial single-cell sequencing experiment, wherein the kit comprises the positive probe and the negative probe described in the present invention.

[0049] The kit also includes other reagents for in situ fluorescent hybridization well known to those skilled in the art, including but not limited to hybridization buffer.

[0050] In a specific embodiment of the present invention, the kit further comprises reagents for cell fixation and permeabilization conventionally used in the art, including but not limited to formaldehyde, Tween-20, and lysozyme.

[0051] In a fourth aspect, the present invention provides a use of the above-mentioned probe and / or kit in evaluating the sample permeabilization effect in a microbial single-cell sequencing experiment.

[0052] The present invention provides a quality inspection method for evaluating the permeabilization effect of samples in microbial single-cell sequencing experiments, which has the following advantages and beneficial effects:

[0053] (1) The cell permeabilization effect quality inspection method provided by the present invention has no particular restrictions on microbial cell samples, and can be used regardless of whether the microbial cells are environmental samples or cultured microbial cells.

[0054] (2) The present invention greatly shortens the hybridization time of the probe by performing LNA modification on the positive fluorescent probe, which can quickly and effectively evaluate the microbial permeabilization effect of the microbial single-cell technology. By comparing the luminescence of the fluorescent probe with the luminescence of the DAPI-stained nucleus, the correlation between the permeabilization time and the permeabilization effect can be intuitively demonstrated.

[0055] (3) The sequence of the negative probe used in the present invention can be widely applied to negative control experiments of fluorescent in situ hybridization of various bacteria. The negative probe is washed away because it cannot hybridize with the bacteria to be tested, so the negative probe group does not fluoresce under a fluorescence microscope.

[0056] (4) The treatment method of the present invention of fixing and permeabilizing the bacterial suspension and then coating the glass slide can more intuitively show the loss of microbial cell samples, overcoming the difficulty of observation and counting caused by too little bacterial volume in the cell suspension after fixation and permeabilization and uneven dye staining. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG1 is a diagram showing the in situ hybridization effect of the Escherichia coli positive probe set observed under a fluorescence microscope in Example 1.

[0058] FIG2 is a diagram showing the DAPI staining effect of the Escherichia coli positive probe group observed under a fluorescence microscope in Example 1.

[0059] FIG3 is a diagram showing the in situ hybridization effect of the Bacillus subtilis positive probe set observed under a fluorescence microscope in Example 2.

[0060] FIG4 is a diagram showing the DAPI staining effect of the Bacillus subtilis positive probe group observed under a fluorescence microscope in Example 2.

[0061] FIG5 is a diagram showing the in situ hybridization effect of the Escherichia coli probe set observed under a fluorescence microscope in Example 3. DETAILED DESCRIPTION

[0062] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0063] The reagent components involved in the embodiments of the present invention are as follows:

[0064] Washing Buffer I components: Taking 10 ml as an example, it contains 5 ml of 2X SSC solution (0.3 M NaCl, 0.03 M sodium citrate, adjust the pH to 7.0 with 1 M HCl, and make up to volume with deionized water), and 5 ml of deionized formamide.

[0065] Washing Buffer II composition: For example, 10 ml contains 10 ml of 1X SSC solution (0.15 M NaCl, 0.015 M sodium citrate, adjust the pH to 7.0 with 1 M HCl, and make up to volume with deionized water).

[0066] Hybridization buffer was NorthernMax TM Prehybridization / hybridization buffer.

[0067] The probes used in the embodiments of the present invention are as follows:

[0068] Counted from the 5' end, the 3rd, 6th and 14th bases of the positive probe are modified with LNA.

[0069] Example 1 Quality Control Method for Permeabilization Effect of Gram-Negative Bacteria-Escherichia coli

[0070] 1. Fixation and Permeabilization of E. coli

[0071] S1: Centrifuge the coarsely filtered E. coli culture medium at 5500 x g for 10 min at 4°C. Remove the liquid medium and add fresh cold 4% formaldehyde solution to resuspend the cells. Place in a 4°C refrigerator and shake for an appropriate period of time to fix the cells.

[0072] S2: The fixed microbial cells were centrifuged at 4°C, 5500 x g for 10 min to remove the formaldehyde solution, washed with 1xPBS-RI solution, centrifuged at 4°C, 5500 x g for 10 min, and resuspended in 0.1M Tris-HCl-RI. The cell suspension was diluted to an appropriate multiple for microscopic examination and counting;

[0073] S3: Based on the cell concentration observed under a microscope, approximately 40 million E. coli were taken into new 1.5 ml centrifuge tubes and grouped. Permeabilization treatment was performed under different conditions. The permeabilization conditions, groups, and specific treatments are detailed in Table 1.

[0074] Table 1 Cell permeabilization conditions

[0075] For samples that do not require special treatment, wash the cells with 175 μl PBS-RI and resuspend the cells in 40 μl PBS (without RNase inhibitors). For samples that require Tween-20 permeabilization, centrifuge and discard the supernatant, resuspend the cells in 250 μl 0.04% Tween-20, mix evenly by pipetting, incubate on ice for 3 minutes, and immediately add 1 ml of fresh cold PBS-RI and mix evenly by pipetting. For samples that require lysozyme digestion, centrifuge and discard the supernatant, resuspend the bacteria in 200 μl of lysozyme mixture, incubate in a metal bath at 37°C for 15 minutes, and immediately add 1 ml of fresh cold PBS-RI after cell wall digestion, mix evenly by pipetting, centrifuge again and discard the supernatant, wash the cells with 175 μl PBS-RI, and resuspend the cells in 40 μl PBS (without RNase inhibitors).

[0076] II. In situ Hybridization of Fixed and Permeabilized Samples

[0077] S1: Pipette 20 μl of the washed bacterial solution onto a clean glass slide and spread it evenly into a circle with a diameter of about 1.5 cm. Place it in a metal bath and bake at 78°C for 2 hours until it is completely dry and fixed.

[0078] S2: Add 100 μl of hybridization buffer to the slide, covering the bacterial area, and place in a humidified chamber for prehybridization at 55°C for 2 h.

[0079] S3: When pre-hybridization is almost finished, dilute the negative probe or positive probe with hybridization buffer at a ratio of 1:50, mix well, denature at 88°C for 3 minutes, and equilibrate at 37°C for 5 minutes. All operations after this step should be kept away from light.

[0080] S4: After the prehybridization is completed, the prehybridization solution is aspirated and 15-20 μl of the equilibrated probe is added dropwise to cover the bacterial area. The area is then covered with a coverslip and placed in a humidified chamber for hybridization at 37°C for 2 h.

[0081] S5: Place the slide in Washing Buffer I at room temperature for 5 minutes. After gently shaking to remove the coverslip, transfer the slide to new Washing Buffer I preheated to 60°C and soak for 2 minutes. Then transfer the slide to Washing Buffer II at room temperature and soak for 10 minutes.

[0082] S6: Dehydrate the specimens in 70%, 85%, and 100% ethanol for 2 min each, and dry them at room temperature.

[0083] S7: Add 10 μl of DAPI dye, cover with a coverslip, let stand in the dark for 15 min, and observe under a fluorescence microscope;

[0084] S8: Observe the slide under a fluorescence microscope. Excitation light passing through a B filter will reveal green fluorescence from the probe. Excitation light passing through a UV filter will reveal blue fluorescence from the bacterial nucleus. Eyepiece magnification is 10X, objective magnification is 20X. The absence of fluorescence from the negative probe group confirms the validity of the result.

[0085] The results are shown in Figures 1 and 2. Figure 1 shows fluorescence images of E. coli cells hybridized with each positive probe in Table 1, and Figure 2 shows fluorescence images of E. coli nuclei stained with DAPI in each positive probe in Table 1. Comparison of the two figures shows that the fluorescence intensity was strongest when treated with lysozyme reagent for 10 minutes (Group 9), indicating the best permeabilization effect.

[0086] Example 2 Quality Control Method for Permeabilization Effect of Gram-Positive Bacillus Subtilis

[0087] 1. Fixation and Permeabilization of Bacillus subtilis

[0088] S1: Centrifuge the coarsely filtered Bacillus subtilis culture medium at 5500 x g for 10 min at 4°C. Remove the liquid medium and add fresh cold 4% formaldehyde solution to resuspend the cells. Place in a 4°C refrigerator and shake for an appropriate time to fix the cells.

[0089] S2: The fixed microbial cells were centrifuged at 4°C, 5500 x g for 10 min to remove the formaldehyde solution, washed with 1xPBS-RI solution, centrifuged at 4°C, 5500 x g for 10 min, and resuspended in 0.1M Tris-HCl-RI. The cell suspension was diluted to an appropriate multiple for microscopic examination and counting;

[0090] S3: Based on the cell concentration observed under a microscope, 40 million Bacillus subtilis were taken into new 1.5 ml centrifuge tubes and grouped. Permeabilization treatment was performed under different conditions. The permeabilization conditions, groups and specific treatments are detailed in Table 2.

[0091] Table 2 Cell permeabilization conditions

[0092] For samples that do not require special treatment, wash the cells with 175 μl PBS-RI and resuspend the cells in 40 μl PBS (without RNase inhibitors). For samples that require Tween-20 permeabilization, centrifuge and discard the supernatant, resuspend the cells in 250 μl 0.04% Tween-20, mix evenly by pipetting, incubate on ice for 3 minutes, and immediately add 1 ml of fresh cold PBS-RI and mix evenly by pipetting. For samples that require lysozyme digestion, centrifuge and discard the supernatant, resuspend the bacteria in 200 μl of lysozyme mixture, incubate in a metal bath at 37°C for 15 minutes, and immediately add 1 ml of fresh cold PBS-RI after cell wall digestion, mix evenly by pipetting, centrifuge again and discard the supernatant, wash the cells with 175 μl PBS-RI, and resuspend the cells in 40 μl PBS (without RNase inhibitors).

[0093] II. In situ Hybridization of Fixed and Permeabilized Samples

[0094] S1: Pipette 20 μl of the washed bacterial solution onto a clean glass slide, spread it evenly into a circle with a diameter of about 1.5 cm, and bake it in a metal bath at 78°C for 2 h until it is completely dry and fixed;

[0095] S2: Add 100 μl of hybridization buffer to the slide, covering the bacterial area, and place in a humidified chamber for prehybridization at 55°C for 2 h.

[0096] S3: When pre-hybridization is almost finished, dilute the negative probe or positive probe with hybridization buffer at a ratio of 1:50, mix well, denature at 88°C for 3 minutes, and equilibrate at 37°C for 5 minutes. All operations after this step should be kept away from light.

[0097] S4: After the prehybridization is completed, the prehybridization solution is aspirated and 15-20 μl of the equilibrated probe is added dropwise to cover the bacterial area. The area is then covered with a coverslip and placed in a humidified chamber for hybridization at 37°C for 2 h.

[0098] S5: Place the slide in Washing Buffer I at room temperature for 5 minutes. After gently shaking to remove the coverslip, transfer the slide to new Washing Buffer I preheated to 60°C and soak for 2 minutes. Then transfer the slide to Washing Buffer II at room temperature and soak for 10 minutes.

[0099] S6: Dehydrate the specimens in 70%, 85%, and 100% ethanol for 2 min each, and dry them at room temperature.

[0100] S7: Add 10 μl of DAPI dye, cover with a coverslip, let stand in the dark for 15 min, and observe under a fluorescence microscope;

[0101] S8: Observe the slide under a fluorescence microscope. Excitation light passing through a B filter will reveal green fluorescence from the probe. Excitation light passing through a UV filter will reveal blue fluorescence from the bacterial nucleus. Eyepiece magnification is 10X, objective magnification is 20X. The absence of fluorescence from the negative probe group confirms the validity of the result.

[0102] The results are shown in Figures 3 and 4. Figure 3 is a fluorescence image of Bacillus subtilis for each positive probe hybridization group in Table 2, and Figure 4 is a fluorescence image of DAPI-stained cell nuclei of Bacillus subtilis for each positive probe hybridization group in Table 2. Comparison of the two figures shows that the fluorescence intensity is the strongest when the lysozyme reagent is treated for 15 minutes (Group 10), indicating the best permeabilization effect.

[0103] Example 3 Comparison of the binding effect of unmodified fluorescent probe based on Escherichia coli and LNA-modified fluorescent probe

[0104] 1. Fixation and Permeabilization of E. coli

[0105] S1: Centrifuge the coarsely filtered E. coli culture medium at 5500 x g for 10 min at 4°C. Remove the liquid medium and add fresh cold 4% formaldehyde solution to resuspend the cells. Place in a 4°C refrigerator and shake for an appropriate period of time to fix the cells.

[0106] S2: The fixed microbial cells were centrifuged at 4°C, 5500 x g for 10 min to remove the formaldehyde solution, washed with 1xPBS-RI solution, centrifuged at 4°C, 5500 x g for 10 min, and resuspended in 0.1M Tris-HCl-RI. The cell suspension was diluted to an appropriate multiple for microscopic examination and counting;

[0107] S3: According to the cell concentration under microscopic examination, about 40 million E. coli were taken into a new 1.5 ml centrifuge tube. A total of 12 parallel samples were set up, that is, 12 1.5 ml centrifuge tubes each containing 40 million E. coli, and the same permeabilization treatment was performed on them: the cells were washed with 175 μl PBS-RI and resuspended in 40 μl PBS (without adding RNase inhibitor); for samples that required Tween-20 permeabilization, the supernatant was discarded by centrifugation, and the cells were resuspended in 250 μl 0.04% Tween-20, mixed evenly by pipetting, incubated on ice for 3 minutes, and immediately added to 1 ml of fresh cold PBS-RI and mixed evenly by pipetting; for samples that required lysozyme digestion, the bacteria were centrifuged and the supernatant was discarded, and then resuspended in 200 μl of lysozyme mixture, incubated in a metal bath at 37°C for 10 minutes. After cell wall digestion, 1 ml of fresh cold PBS-RI was immediately added directly, mixed evenly by pipetting, centrifuged again and the supernatant was discarded, and 175 μl The cells were washed with PBS-RI and resuspended in 40 μl of PBS (without RNase inhibitor);

[0108] II. In situ Hybridization of Fixed and Permeabilized Samples

[0109] S1: Pipette 20 μl of the washed bacterial solution onto a clean glass slide, spread it evenly into a circle with a diameter of about 1.5 cm, and bake it in a metal bath at 78°C for 2 h until it is completely dry and fixed;

[0110] S2: Add 100 μl of hybridization buffer to the slide, covering the bacterial area, and place in a humidified chamber for prehybridization at 55°C for 2 h.

[0111] S3: When prehybridization is almost finished, dilute the negative probe, the positive probe without LNA treatment, and the positive probe with LNA treatment with hybridization buffer at a ratio of 1:50. Mix well, denature at 88°C for 3 minutes, and equilibrate at 37°C for 5 minutes. All operations after this step should be kept away from light.

[0112] S4: After prehybridization, aspirate the prehybridization solution and add 15-20 μl of the equilibrated probe to cover the bacterial area. Cover with a coverslip and place in a humidified chamber for hybridization at 37°C for 2-24 hours. See Table 3 for the specific grouping of in situ hybridization time.

[0113] Table 3 Grouping of cell in situ hybridization duration

[0114] The LNA-modified positive probe in the table refers to the nucleotide sequence of SEQ ID NO. 1 of the present invention with LNA modifications at bases 3, 6, and 14. The unmodified positive probe refers to the nucleotide sequence of SEQ ID NO. 1 of the present invention without LNA modifications.

[0115] S5: After hybridization, place the slide in Washing Buffer I at room temperature for 5 minutes. After gently shaking, remove the coverslip. Then, transfer the slide to new Washing Buffer I preheated to 60°C and soak for 2 minutes. Then, transfer the slide to Washing Buffer II at room temperature and soak for 10 minutes.

[0116] S6: Dehydrate the specimens in 70%, 85%, and 100% ethanol for 2 min each, and dry them at room temperature.

[0117] S7: Add 10 μl of DAPI dye, cover with a coverslip, let stand in the dark for 15 min, and observe under a fluorescence microscope;

[0118] S8: Observe the slide under a fluorescence microscope. Excitation light passes through filter B, and the probe will emit green fluorescence. The magnification is 10X with the eyepiece and 20X with the objective lens. The absence of fluorescence in the negative probe group confirms the validity of the result.

[0119] The results are shown in Figure 5, which shows fluorescence images of E. coli from each group in Table 3. Comparing the fluorescence intensities of the probes with and without LNA modification, it can be seen that the LNA-modified fluorescent probe achieved the desired fluorescence intensity after only 2 hours of hybridization, while the unmodified fluorescent probe required 24 hours of hybridization to achieve the same effect. This indicates that the positive probes provided by the present invention with LNA modifications at positions 3, 6, or 14 hybridized with the target bacteria more quickly, while positive probes without LNA modification or with LNA modifications at sites other than positions 3, 6, or 14 were unable to complete hybridization within 2 hours.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for evaluating the permeabilization effect of a sample in a microbial single-cell sequencing experiment, the method comprising the following steps: (1) Microbial cell fixation; (2) Permeabilization of fixed cells; (3) Add the permeabilized cell sample to a glass slide and bake it; (4) adding probes for in situ fluorescent probe hybridization; (5) washing and counterstaining the cell nuclei; (6) Evaluate the cell permeabilization effect based on the fluorescence intensity of the sample under a fluorescence microscope. The probes used for in situ fluorescent hybridization in step (4) include a positive probe and a negative probe. The nucleotide sequence of the positive probe is shown in SEQ ID NO.1, and the nucleotide sequence of the negative probe is shown in SEQ ID NO.

2.

2. The method according to claim 1, characterized in that The positive probe is LNA-modified.

3. The method according to claim 1, characterized in that Counted from the 5' end, the 3rd, 6th and 14th bases of the positive probe are modified with LNA.

4. The method according to claim 1, wherein The method for evaluating the permeabilization effect of a sample in a microbial single-cell sequencing experiment comprises the following steps: (a) Microbial cells were fixed with formaldehyde by shaking, washed and resuspended in PBS-RI solution, centrifuged, resuspended in Tris-HCl-RI solution, and counted; (b) Depending on the cell suspension concentration, cells were perforated with Tween-20 on ice for 3–4 min, then permeabilized with lysozyme solution for 1–30 min, washed with PBS-RI solution, and resuspended. (c) Pipette the resuspended cell suspension onto a glass slide, spread it evenly into a circle with a diameter of approximately 1.5 cm, and place it on a metal bath to fix it; (d) adding hybridization buffer to the slide, covering the bacterial area, and placing it in a wet chamber for incubation; simultaneously, denaturing the probe and hybridization buffer; (e) adding the denatured probe to the sample and performing constant temperature hybridization; (f) After hybridization, the samples were washed with Washing Buffer, dehydrated with ethanol solutions of different concentrations, dried at room temperature, and dark-stained with DAPI. (g) Observe the fluorescence intensity and quantity of the samples under a fluorescence microscope, and evaluate the permeabilization effect of the cell suspension samples based on the fluorescence intensity and bacterial morphology.

5. A probe for evaluating the permeabilization effect of a sample in a microbial single-cell sequencing experiment, the probe comprising a positive probe and a negative probe, wherein: The nucleotide sequence of the positive probe is shown in SEQ ID NO.1, and the nucleotide sequence of the negative probe is shown in SEQ ID NO.

2.

6. The probe according to claim 5, characterized in that The positive probe is LNA-modified.

7. The probe according to claim 5, characterized in that Counted from the 5' end, the 3rd, 6th and 14th bases of the positive probe are modified with LNA.

8. A kit for evaluating the permeabilization effect of a sample in a microbial single-cell sequencing experiment, comprising the positive probe and the negative probe according to any one of claims 5 to 7.

9. The kit according to claim 8, characterized in that The kit also includes reagents for in situ fluorescent hybridization and / or reagents for cell fixation and permeabilization, which are well known to those skilled in the art.

10. Use of the probe according to any one of claims 5 to 7 and / or the kit according to any one of claims 8 to 9 in evaluating the permeabilization effect of a sample in a microbial single-cell sequencing experiment.

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