A microbial immunochromogenic reagent for fluorescent staining of vaginal secretion wet mounts

Abstract

The present invention relates to a microbial immunochromogenic reagent for fluorescent staining of vaginal secretion wet mounts, in particular to a microbial immunochromogenic reagent and a preparation method and application thereof. The microbial immunochromogenic reagent of the present invention comprises a fluorescent stain, a buffer solution, a permeabilizing agent and an antibacterial agent. The microbial immunochromogenic reagent and the microbial sample are mixed, and wet mount detection can be performed under a fluorescence microscope. This operation is simple, fast and convenient, greatly reduces the workload of operators, and can achieve rapid detection of a large number of samples. It also has high sensitivity and accuracy, and a wide range of applications.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent staining liquids, and in particular relates to a microbial immunochromogenic reagent for fluorescent staining of vaginal secretion wet mounts, in particular to a microbial immunochromogenic reagent and a preparation method and application thereof. Background Art

[0002] Traditional detection methods include wet mount microscopy and Gram staining. However, both methods have great limitations. Wet mount microscopy often cannot accurately observe bacterial morphology for clue cells with smaller bacterial morphology, and is not effective in observing fungal blastospores. When Trichomonas is inactivated or dead, its morphology is almost the same as that of white blood cells and difficult to distinguish. Gram staining is relatively accurate, but time-consuming and has a certain relationship with the operating techniques of each test worker, so it is not suitable for promotion in outpatient testing. Therefore, rapid and accurate vaginal secretion detection methods have also received more and more attention, especially in outpatient testing.

[0003] Fluorescent staining is a recently developed staining method that can directly examine fluorescently labeled samples with the help of fluorescent staining fluid and a fluorescent microscope. The method can also save images and send them out together with the report for direct clinical review. In addition to being used in pathology departments, this method has also been used to a certain extent in the testing of antinuclear antibodies and fungi. Summary of the invention

[0004] The purpose of the present invention is to provide a microbial immunochromogenic reagent for fluorescent staining of vaginal secretion wet mounts, in particular a microbial immunochromogenic reagent and a preparation method and application thereof, in view of the problems existing in the prior art.

[0005] The microbial immunochromogenic reagent of the present invention comprises a fluorescent stain, a buffer solution, a permeabilizing agent and an antibacterial agent. The microbial immunochromogenic reagent and the microbial sample are mixed, and wet-mount detection can be performed under a fluorescence microscope. This operation is simple, fast and convenient, greatly reduces the workload of operators, and can achieve rapid detection of a large number of samples. In addition, the sensitivity and accuracy are high, and the application range is wide.

[0006] The purpose of the present invention can be achieved by the following scheme:

[0007] The invention provides a microbial immunochromogenic reagent for fluorescent staining of vaginal secretion wet mounts, comprising a fluorescent agent, an antibacterial agent, a permeabilizing agent, a buffer solution and water.

[0008] The fluorescent agent includes acridine orange and propidium iodide, and the mass ratio is 1:2 to 7:6.

[0009] The antibacterial agent includes ProClin TM 300. One or more of sodium azide.

[0010] The permeabilizing agent includes one or more of PVP-k90, PVA, Tween-20, Triton-100, and S9, preferably PVP-k90 and PVA, and more preferably PVP-k90. PVP-k90 is a water-soluble polymer with good biological stability, which is chemically stable, low in toxicity, and good in biocompatibility. The present invention is directed to wet-staining, and PVP-k90 is added. After cell staining, the morphology of the cell nucleus and cytoplasm is clearer, and the effect is better than other permeabilizing agents conventionally used for dry staining.

[0011] The buffer solution includes one or more of phosphate buffer, tris(hydroxymethylaminomethane) buffer, acetic acid-sodium acetate buffer, preferably tris(hydroxymethylaminomethane) buffer. The concentration of the buffer solution is 10-100 mM.

[0012] The usage ratio of the fluorescent agent, the antibacterial agent, the permeation agent, the buffer solution and the water is 1-18 mg: 250-2500 μL: 0.1-1 g: 2000-4000 ml: 1000-1600 ml.

[0013] The present invention also provides a method for preparing the microbial immunochromogenic reagent, which comprises the following steps:

[0014] Step 1, weigh each component, and evenly dissolve the fluorescent agent, antibacterial agent, and permeabilizing agent in water to obtain raw material solutions of each component: fluorescent agent solution, antibacterial agent solution, and permeabilizing agent solution;

[0015] Step 2: Add the raw material solutions of each component into the buffer solution according to the amount to obtain the microbial immunochromogenic reagent.

[0016] In step 1, the concentration of the fluorescent agent in the fluorescent agent solution is 0.02-10 mg / L.

[0017] In step 1, the concentration of the antibacterial agent in the antibacterial agent solution is 0.1 to 10 μL / mL.

[0018] In step 1, the mass fraction of the permeabilizing agent in the permeabilizing agent solution is 0.01% to 1%.

[0019] In step 2, the concentration of the buffer solution is 10 mM to 100 mM.

[0020] The present invention also provides an application of the microbial immunochromogenic reagent in wet slide staining.

[0021] The present invention also provides a method for using the microbial immunochromogenic reagent, the steps of which are as follows: method 1 or method 2:

[0022] Method 1: Mix the sample and the microbial immunochromogenic reagent, add them to a wet slide, and observe the staining morphology under a microscope;

[0023] Method 2: Add the sample to the coverslip, then add the microbial immunochromogenic reagent, and observe the staining morphology under a microscope.

[0024] In method 1, the volume ratio of the sample to the microbial immunochromogenic reagent is 1:4-6, preferably 1:5.

[0025] In method 1, the volume ratio of the sample to the microbial immunochromogenic reagent is 1:0.5-2, preferably 1:1.

[0026] The samples include vaginal secretion samples and cervical exfoliated cell samples.

[0027] The specific usage is:

[0028] After mixing the sample and the microbial immunochromogenic reagent, take a 200μL centrifuge tube, add 10μL of the sample, then add 50μL of the microbial immunochromogenic reagent, blow or mix, take 20μL of the mixed solution and add it to the wet slide (Chongqing Tianhai Medical Equipment Co., Ltd.), wipe the overflowed liquid on the cover glass, and observe the staining morphology under a microscope; (commonly used)

[0029] Or, after mixing the sample and staining solution, take a wet slide (Chongqing Tianhai Medical Equipment Co., Ltd.), add 10μL of sample to the right side of the cover glass, then add 10μL of microbial immunochromogenic reagent, wipe the liquid overflowing from the cover glass, and observe the staining morphology under a microscope. (Some samples on the left side of the cover glass may not be stained or missed due to edge effects)

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The cells and microorganisms in clinical samples (such as vaginal secretion samples and cervical exfoliated cell samples) are specially stained by microbial immunochromogenic reagents, which solves the problem of missed detection in wet mounts. The wet mount fluorescent staining can show the specific morphology of cells and microorganisms, and effectively distinguish epithelial cells, white blood cells, red blood cells, bacteria, fungi and protozoa.

[0032] (2) By adopting the method of the present invention, a microbial immunochromogenic reagent staining method is prepared. The microbial immunochromogenic reagent can simultaneously stain DNA and RNA in cells to display fluorescence of different colors. It can observe the exfoliated epithelial cells, white blood cells, bacteria, fungi, Trichomonas and mixed infections in vaginal secretions, thereby improving the detection rate of pathogens; making the detection not affected by the inactivation of Trichomonas, reducing the missed diagnosis rate.

[0033] (3) The product prepared by this method is a microbial immunochromogenic reagent. This invention does not need to dry the diluent on the glass slide during the staining operation, but converts it into a smear for staining. This invention mixes the diluent and the microbial immunochromogenic reagent liquid and directly drips it onto the glass slide, and then performs wet-slide fluorescence staining observation under a biological microscope. The morphology of exfoliated epithelial cells, white blood cells, red blood cells, bacteria, fungi, and protozoa can be clearly presented in the field of vision. The operation is simple, the amount of staining used is extremely small, and the time and money costs are greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0035] Figure 1 It is a process flow state diagram of the microbial immunochromogenic reagent of the present invention;

[0036] Figure 2 This is a state diagram after the microbial immunochromogenic reagent of Example 3 of the present invention is used to stain the standard strain of Candida albicans;

[0037] Figure 3 This is a state diagram after the microbial immunochromogenic reagent of Example 3 of the present invention is used to stain the standard strain of Staphylococcus aureus;

[0038] Figure 4 This is a state diagram after the microbial immunochromogenic reagent of Example 3 of the present invention is used to stain the standard strain of Escherichia coli;

[0039] Figure 5 This is a state diagram of the clue cells in vaginal secretions after staining with the microbial immunochromogenic reagent of Example 3 of the present invention;

[0040] Figure 6 This is a state diagram of the mycelium in vaginal secretions after being stained by the microbial immunochromogenic reagent of Example 3 of the present invention;

[0041] Figure 7 This is a state diagram of the microbial immunochromogenic reagent of Example 3 of the present invention after staining spores in vaginal secretions;

[0042] Figure 8 This is a state diagram of epithelial cells in vaginal secretions after staining with the microbial immunochromogenic reagent of Example 3 of the present invention;

[0043] Fig. 9 This is a morphological diagram of the VVC sample after staining with the staining solution of Example 2;

[0044] Fig.10 This is a morphological diagram of the VVC sample after staining with the staining solution of Example 5;

[0045] Fig.11 This is a morphological diagram of a VVC sample stained with the staining solution of Example 6;

[0046] Fig.12 This is a morphological diagram of the VVC sample after staining with the staining solution of Example 7;

[0047] Fig.13 This is a morphological diagram of the VVC sample after staining with the staining solution of Example 8;

[0048] Fig.14 This is a morphological diagram of a VVC sample stained with the staining solution of Example 9;

[0049] Fig.15 The morphological diagram of the VVC sample after staining with the staining solution using 0.01% PVP-k90 in Example 10;

[0050] Fig.16 The morphological diagram of the VVC sample after staining with the staining solution using 0.05% PVP-k90 in Example 11;

[0051] Fig.17 The morphological diagram of the VVC sample after staining with the staining solution using 0.01% PVA in Example 12;

[0052] Fig.18 This is a single-stain morphology diagram when the permeabilizing agent is PVA;

[0053] Fig.19 This is the single staining morphology when the permeabilizing agent is PVP-k90;

[0054] Fig. 20 This is the single-staining morphology when the permeabilizing agent is Tx-100;

[0055] Fig.21 This is the single-staining morphology when the permeabilization agent is Tween-20;

[0056] Fig. 22 This is the single-staining morphology when the permeabilizing agent is S9;

[0057] Fig.23 This is a single-stained morphology diagram without adding permeabilizing agent. DETAILED DESCRIPTION

[0058] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, provide detailed implementation methods and specific operation processes, and will help those skilled in the art to further understand the present invention. It should be pointed out that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made under the premise of the concept of the present invention all belong to the protection scope of the present invention.

[0059] The present invention provides a microbial immunochromogenic reagent, comprising the following components: a fluorescent agent, a permeabilizing agent, an antibacterial agent, and a buffer. Figure 1 As shown, it includes: dissolving the fluorescent agent, the antibacterial agent and the permeabilizing agent uniformly in water respectively to obtain the fluorescent agent solution, the antibacterial agent solution and the permeabilizing agent solution; adding them into the prepared buffer solution to obtain the microbial immunochromogenic reagent, and then through inner packaging and outer packaging, the final product is obtained.

[0060] Example 1

[0061] In this embodiment, a microbial immunochromogenic reagent is prepared, including the following components: a fluorescent agent, a permeabilizing agent, an antibacterial agent, and a buffer. The fluorescent agent is acridine orange and propidium iodide, the permeabilizing agent is PVP-k90, and the antibacterial agent is ProClin TM 300, the buffer solution is phosphate buffer (concentration is 100mM).

[0062] The preparation method is as follows:

[0063] (1) Dissolve 4.5 mg of acridine orange in 0.45 L of ultrapure water (concentration of 10 mg / L), 7.5 mg of propidium iodide in 0.75 L of ultrapure water (concentration of 10 mg / L), and the antibacterial agent ProClin TM 300 500 μL was dissolved in 49.5 ml ultrapure water (concentration of 10 μL / mL), 0.5 g permeabilizing agent PVP-k90 was dissolved in 50 ml ultrapure water (mass fraction of 1%), and the components were mixed to obtain a raw material solution.

[0064] (2) Weigh 7.2 g of NaH2PO4 (sodium dihydrogen phosphate), dissolve it in 300 mL of ultrapure water and mix it to prepare a 0.2 M NaH2PO4 solution; weigh 71 g of Na2HPO4 (disodium hydrogen phosphate), dissolve it in 2500 mL of ultrapure water and mix it to prepare a 0.2 M Na2HPO4 solution; take 262.5 mL of 0.2 M NaH2PO4, add 2237.5 mL of 0.2 M Na2HPO4, and mix it in 2500 mL of ultrapure water to obtain a buffer solution.

[0065] (3) Add 3800 ml of phosphate buffer (concentration of 100 mM) to the raw material solution obtained in step (1) to obtain a microbial immunochromogenic reagent.

[0066] Example 2

[0067] In this embodiment, a microbial immunochromogenic reagent is prepared, including the following components: a fluorescent agent, a permeabilizing agent, an antibacterial agent, and a buffer. The fluorescent agent is acridine orange and propidium iodide, the permeabilizing agent is PVP-k90, and the antibacterial agent is ProClin TM300, the buffer solution is Tris buffer (concentration is 10 mM).

[0068] The preparation method is as follows:

[0069] (1) Dissolve 4.5 mg of acridine orange in 0.45 L of ultrapure water, 7.5 mg of propidium iodide in 0.75 L of ultrapure water, and the antibacterial agent ProClin TM 300 5 ml was dissolved in 45 ml ultrapure water (concentration was 1 mL / 10 mL), 0.5 g permeabilizing agent PVP-k90 was dissolved in 50 ml ultrapure water (mass fraction was 1%), and the components were mixed to obtain a raw material solution.

[0070] (2) Weigh 12.11 g of tris(hydroxymethyl)aminomethane (Tris), add 1000 mL of ultrapure water to dissolve and mix, then adjust the pH to 9.0±0.05 (temperature is 25°C) with concentrated hydrochloric acid to prepare 100 mM Tris (pH=9.0), take 400 mL of 100 mM Tris (9.0) buffer solution, add 3600 mL of ultrapure water and mix to obtain a buffer solution.

[0071] (3) The raw material solution obtained in step (1) is first added with 3750 ml of tris(hydroxymethyl)aminomethane buffer (concentration: 10 mM) to obtain a microbial immunochromogenic reagent.

[0072] Example 3

[0073] In this embodiment, a microbial immunochromogenic reagent is prepared, including the following components: a fluorescent agent, a permeabilizing agent, an antibacterial agent, and a buffer. The fluorescent agent is acridine orange and propidium iodide, and the antibacterial agent is ProClin TM 300, the permeabilizing agent is PVP-k90, and the buffer solution is Tris buffer.

[0074] The preparation method is as follows:

[0075] (1) Dissolve 4.5 mg of acridine orange in 0.45 L of ultrapure water, 7.5 mg of propidium iodide in 0.75 L of ultrapure water, and the antibacterial agent ProClin TM 300 500 μL was dissolved in 49.5 ml ultrapure water (concentration of 10 μL / mL), 0.5 g permeabilizing agent PVP-k90 was dissolved in 50 ml ultrapure water (mass fraction of 1%), and the components were mixed to obtain a raw material solution.

[0076] (2) Weigh 12.11 g of tris(hydroxymethyl)aminomethane (Tris), add 1000 mL of ultrapure water to dissolve and mix, then adjust the pH to 9.0±0.05 (temperature is 25°C) with concentrated hydrochloric acid to prepare 100 mM Tris (pH=9.0), take 400 mL of 100 mM Tris (9.0) buffer solution, add 3600 mL of ultrapure water and mix to obtain a buffer solution.

[0077] (3) The raw material solution obtained in step (1) is first added with 3700 ml of tris(hydroxymethyl)aminomethane buffer (concentration: 10 mM) to obtain a microbial immunochromogenic reagent.

[0078] Example 4

[0079] After mixing different samples and the microbial immunochromogenic reagent prepared in Example 3, take a 200 μL centrifuge tube, add 10 μL of sample, then add 50 μL of microbial immunochromogenic reagent, mix well, take 20 μL of the mixed solution and add it to a wet slide (Chongqing Tianhai Medical Equipment Co., Ltd.), wipe the overflowed liquid on the cover glass, and observe the staining morphology under a microscope. Screenshots were taken and saved using Imageview software.

[0080] Sample 1 is a standard strain of Candida albicans (BNCC 186382), the volume ratio of bacterial solution to ultrapure water is 3:17, and the state of the standard strain of Candida albicans after staining with the microbial immunochromogenic reagent is shown in the figure below: Figure 2 As shown;

[0081] Sample 2 is a standard strain of Staphylococcus aureus (BNCC 186335). The volume ratio of bacterial solution to ultrapure water is 1:3. The state of the standard strain of Staphylococcus aureus after staining with the microbial immunochromogenic reagent is shown in the figure below. Figure 3 As shown;

[0082] Sample 3 is a standard strain of Escherichia coli (BNCC 185253). The volume ratio of bacterial solution to ultrapure water is 1:3. The state of the standard strain of Escherichia coli after staining with the microbial immunochromogenic reagent is shown in the figure below. Figure 4 As shown;

[0083] Sample 4 is a BV sample (bacterial vaginosis). The state of the clue cells in the vaginal secretions stained by the microbial immunochromogenic reagent is shown in the figure below. Figure 5 As shown;

[0084] Sample 5 is a VVC sample (vulvovaginal candidiasis). The microbial immunochromogenic reagent used to stain the hyphae in vaginal secretions is shown in the figure below. Figure 6 As shown;

[0085] Sample 6 is a VVC sample (vulvovaginal candidiasis). The microbial immunochromogenic reagent used to stain the spores in vaginal secretions is shown in the figure below. Figure 7 As shown;

[0086] Sample 7 is a negative sample (normal human sample). The microbial immunochromogenic reagent used to stain the epithelial cells in vaginal secretions is shown in the figure below. Figure 8 shown.

[0087] Example 5-12

[0088] The composition and preparation method of the microbial immunochromogenic reagent of this embodiment are basically the same as those of Embodiment 2, except that different permeabilizing agents and dosages are used, as shown in the following table.

[0089] Example Permeabilization agent Dosage The staining morphology is shown in the figure Example 2 PVP-k 90 0.5g Fig. 9 Example 5 PVA 0.5g Fig.10 Example 6 Tween-20 0.5g Fig.11 Example 7 TX-100 0.5g Fig.12 Example 8 TX-100 2.5g Fig.13 Example 9 S9 0.5g Fig.14 Example 10 PVP-k90 0.005 Fig.15 Embodiment 11 PVP-k90 0.025 Fig.16 Example 12 PVA 0.005 Fig.17

[0090] The microbial immunochromogenic reagents of Examples 2, 5-10 were used to observe the staining morphology according to the method of Example 4. The samples used were: the same tube of VVC samples (vulvovaginal candidiasis).

[0091] Example 2 The morphology after dyeing with the dyeing solution is as follows Fig. 9 As shown, using PVP-90, the spore morphology is clear and the color is bright, the bacillus morphology is clear, the cells are clear, and the morphology and color of the cell nucleus and cytoplasm are clearly distinguished, making them easy to observe.

[0092] Example 5 The morphology after dyeing with the dyeing solution is as follows Fig.10 As shown, when using PVA, the spore morphology is clear and the color is bright, the bacillus morphology is clear, the overall cell morphology is clear, and the color of the cell nucleus covers the cytoplasm. When observing the morphology, the green color of the cytoplasm will be blocked by the orange color of the cell nucleus, making the specific morphology of the cell nucleus difficult to observe.

[0093] Example 6 The morphology after staining with the staining solution is as follows Fig.11 As shown, using Tween-20, the cell morphology is more blurred.

[0094] Example 7 The morphology after dyeing with the dyeing solution is as follows Fig.12 As shown, when TX-100 (0.1%, mass / volume) was used, there was background and the cell morphology was blurred.

[0095] Example 8 The morphology after dyeing with the dyeing solution is as follows Fig.13 As shown, when TX-100 (0.5%, mass / volume) was used, the spore color was not bright, there was a background, and the blurred degree of cell morphology was less than that of 0.1% TX-100.

[0096] Example 9: The morphology after dyeing with the dyeing solution is as follows Fig.14As shown, using S9, the spores are bright in color, clear in morphology, and the bacilli are clear, but the cells are not as clear as PVP-k90 and PVA.

[0097] In summary, the cell morphology is clearer in the staining field when PVP-k90 and PVA are added, and the spore morphology is clear and bright, without missing any. The morphology of living cells is blurred when the other three staining solutions are added.

[0098] The microbial immunochromogenic reagents of Examples 10-12 were used to observe the staining morphology according to the method of Example 4. The samples used were: the same tube of VVC samples (vulvovaginal candidiasis).

[0099] Example 10 uses 0.01% PVP-k90, and the morphology after staining with the staining solution is as follows Fig.15 shown.

[0100] Example 11 uses 0.05% PVP-k90, and the morphology after staining with the staining solution is as follows Fig.16 shown.

[0101] Example 12 uses 0.01% PVA, and the morphology after staining with the staining solution is as follows Fig.17 shown.

[0102] Observe the staining effect. When low concentration PVP-k90 and PVA are used to prepare the staining solution, the cells are stained more clearly. Compared with the same concentration of PVP-k90 and PVA, 0.01% PVP-k90 in the staining solution can show the cytoplasm and nuclear morphology and color most clearly, without the influence of background color, and the boundary between the cytoplasm and the nuclear can be clearly seen.

[0103] The present invention also conducted a single staining experiment, with only 50 μL of the staining agent Sybr Green I, 850 μL of the buffer solution, and 10 mg of the permeabilizing agent.

[0104] When the permeabilization agent is PVA, the single staining morphology is as follows Fig.18 As shown, the morphology is incomplete;

[0105] When the permeabilization agent is PVP-k90, the single staining morphology is as follows Fig.19 As shown, the morphology is complete;

[0106] When the permeabilization agent is Tx-100, the single staining morphology is as follows Fig. 20 As shown, the morphology is not particularly complete and the field of view is not very clear;

[0107] When the permeabilization agent is Tween-20, the single staining morphology is as follows Fig.21 As shown, the morphology is incomplete and the field of vision is clear;

[0108] When the permeabilization agent is S9, the single staining morphology is as follows Fig. 22 As shown, the morphology is incomplete and the visual field is not clear;

[0109] When no permeabilization agent is added (control group, only buffer and stain), the single staining morphology is as follows Fig.23 As shown, the morphology is incomplete and the field of vision is not clear.

[0110] After adding PVA and PVA-k90, the fluorescence quenching time of single-stained yeast was about 30s, and the fluorescence maintenance time was 10s longer than that of ultrapure water as buffer solution before; the fluorescence maintenance time after TX-100, Tween-20, and S9 staining was about 20s, which was no different from that of ultrapure water as buffer solution before; comparing the staining results of the control group and the experimental group, the effect of the control group was slightly worse.

[0111] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. Use of a microbial immunochromogenic reagent in wet mount staining, characterized in that: The microbial immunochromogenic reagent includes a fluorescent agent, an antibacterial agent, a permeabilizing agent, a buffer solution, and water; The permeabilizing agent is PVP-k90; The fluorescent agent includes acridine orange and propidium iodide, with a mass ratio of 1:2 to 7:6; The usage ratio of the fluorescent agent, the antibacterial agent, the permeation agent, the buffer solution and the water is 1-18 mg: 250-2500 μL: 0.1-1 g: 2000-4000 ml: 1000-1600 ml.

2. The use according to claim 1, characterized in that: The antibacterial agent includes ProClin TM 300. One or more of sodium azide.

3. The use according to claim 1, characterized in that: The buffer solution includes one or more of a phosphate buffer, a tris buffer, and an acetic acid-sodium acetate buffer solution.

4. A method for preparing the microbial immunochromogenic reagent for use as claimed in claim 1, characterized in that: The preparation method comprises the following steps: Step 1, weigh each component, and evenly dissolve the fluorescent agent, antibacterial agent, and permeabilizing agent in water to obtain raw material solutions of each component: fluorescent agent solution, antibacterial agent solution, and permeabilizing agent solution; Step 2: Add the raw material solutions of each component into the buffer solution according to the amount to obtain the microbial immunochromogenic reagent.

5. The preparation method according to claim 4, characterized in that: The concentration of the fluorescent agent in the fluorescent agent solution is 0.02-10 mg / L; the concentration of the antibacterial agent in the antibacterial agent solution is 0.1-10 μL / mL; the mass fraction of the permeabilizing agent in the permeabilizing agent solution is 0.01%-1%; and the concentration of the buffer solution is 10mM-100mM.

6. A method for using the microbial immunochromogenic reagent in the application as claimed in claim 1, characterized in that: The steps are as follows: Method 1 or Method 2: Method 1: Mix the sample and the microbial immunochromogenic reagent, add them to a wet slide, and observe the staining morphology under a microscope; Method 2: Add the sample to the wet slide, then add the microbial immunochromogenic reagent, and observe the staining morphology under a microscope.

7. The method of use according to claim 6, characterized in that: The samples include vaginal secretion samples and cervical exfoliated cell samples.

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