A rapid detection kit for microorganisms and cells based on confocal microscopy and multiple fluorescent labeling and its preparation method

Abstract

The present invention relates to the technical field of microbial detection, and specifically to a rapid detection kit for microorganisms and cells based on confocal microscopy and multiple fluorescent labeling, and a preparation method thereof. The kit comprises liquid A, liquid B, liquid C, liquid D, liquid E, and liquid F. Each liquid has a simple composition and is easy to prepare. The kit can achieve rapid and accurate detection of bacterial infection, cell damage, and sepsis.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid microbial diagnosis, and in particular to a rapid detection kit for microorganisms and cells based on confocal microscopy and multiple fluorescent labeling, and a preparation method thereof. Background Art

[0002] Infections are local and systemic inflammatory responses caused by pathogens such as bacteria, viruses, mycoplasmas, fungi, and parasites that invade the body. These infections can cause varying degrees of harm, ranging from mild symptoms of redness, swelling, and pain to severe symptoms such as organ dysfunction, shortness of breath, and palpitations. Bacterial infectious diseases are a variety of diseases caused by pathogens with bacterial infection as the primary clinical manifestation. Common bacterial infectious diseases include infectious diarrhea and pneumonia. There are many types of bacterial infectious diseases, each with different treatments. Accurate diagnosis and appropriate treatment are key to preventing and controlling bacterial infectious diseases.

[0003] As infection progresses, sepsis develops, a dysregulated response to infection that leads to life-threatening organ dysfunction. Although its mortality rate has decreased over the past few decades, it remains a high-risk factor for hospitalized patients. Approximately 30% of patients admitted to intensive care units (ICUs) worldwide develop severe infections, with lung infection being the most common cause of sepsis. Because sepsis symptoms are often nonspecific and accompanied by organ failure, they can easily be masked by other illnesses, potentially leading to missed or delayed diagnosis. The International Guidelines for the Management of Sepsis and Septic Shock emphasize that patients suspected of sepsis should undergo prompt testing for appropriate pathogens and early initiation of antimicrobial therapy. However, the indiscriminate use of broad-spectrum antimicrobials, even with a heavy-handed approach, is bound to lead to overuse of antimicrobial agents, increase drug toxicity and side effects, and contribute to the prevalence of multidrug-resistant and extensively drug-resistant bacteria. Therefore, early and accurate etiology diagnosis is crucial for accurately identifying infection, improving prognosis, and preventing the development of drug resistance. Laboratory diagnosis of infectious diseases (bacteria) and sepsis is currently a hot topic in the care of critically ill patients.

[0004] At present, there are many different methods for clinical examination of bacterial infection. Different detection methods have different advantages and disadvantages. The detection methods of sepsis mainly include pathogen detection, biomarker detection, scoring system evaluation and multi-omics analysis. The common detection methods are as follows: (1) Pathogen detection - traditional culture method, which is considered the "gold standard" for pathogen diagnosis and can directly isolate and identify pathogens; but it takes a long time (usually takes several days), has a low detection rate, and is easily affected by the use of antibiotics and sample contamination. In molecular detection technology, PCR amplifies the nucleic acid sequence of pathogens in vitro, has a fast detection speed, and is only sensitive to common pathogens. Next-generation sequencing (NGS): It can comprehensively detect the nucleic acid sequence of pathogens and is more suitable for the detection of complex infections and unknown pathogens. (2) Biomarker detection - Procalcitonin (PCT): It is a commonly used inflammatory marker for sepsis. Its level is related to the severity of infection, but its specificity is limited; and interleukin-6 (IL-6): It can be elevated in the early stages of sepsis, but lacks specificity. The new marker S100P is highly expressed in sepsis and is associated with prognosis. It has high sensitivity and specificity, but the detection technology is demanding, lacks large-scale validation, cannot be used for diagnosis alone, and may be affected by other factors. (3) Scoring system MEWS (Modified Early Warning Score): Indicators include heart rate, respiratory rate, systolic blood pressure, body temperature, consciousness, etc. Its advantages are simplicity and ease of use, suitable for rapid screening in emergency departments; however, its predictive value for respiratory rate is limited. (4) Multi-omics analysis - technical features: Comprehensive analysis of the pathological mechanism of sepsis through multi-omics data such as genes, proteins, and metabolites; it can discover potential diagnostic and prognostic markers, but the technology is complex and the cost is high, and it is currently mainly used for research.

[0005] Among the above detection methods, the procalcitonin (PCT) detection kit is the most commonly used in clinical practice. It is a non-invasive clinical laboratory indicator for the diagnosis and treatment monitoring of severe bacterial infections and is a good marker for bacterial infection and sepsis. For bacterial infection and sepsis, PCT testing can be used for early clinical diagnosis, and can indicate the course and prognosis of the disease and guide treatment methods. PCT is also used as an effective tool for antibiotic management. Although it is simple to operate, low in cost, and has high sensitivity and specificity, relying solely on PCT testing may not be comprehensive. In clinical practice, it is often necessary to combine other indicators such as CRP and white blood cell count for comprehensive evaluation. The test results can also be affected by non-infectious factors, such as surgery, trauma, burns, acute pancreatitis, etc., as well as some special diseases such as renal insufficiency and medullary thyroid carcinoma. PCT testing requires a highly sensitive and accurate detection platform. Especially at low concentration levels, detection bias may lead to false positive or false negative results, both of which affect clinical judgment.

[0006] Therefore, there is an urgent need for a simpler, faster and more accurate detection method for bacterial infection, cell damage and sepsis in clinical practice. Summary of the Invention

[0007] To solve the above technical problems, the purpose of the present invention is to provide a rapid detection kit for microorganisms and cells based on confocal microscopy and multiple fluorescent labeling and its preparation method. The kit has a simple configuration and can achieve rapid and accurate detection of bacterial infection, cell damage and sepsis.

[0008] In order to achieve the above technical effects, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a rapid detection kit for microorganisms and cells based on confocal microscopy and multiple fluorescent labeling, the kit comprising liquid A, liquid B, liquid C, liquid D, liquid E, and liquid F, wherein:

[0010] Solution A includes calcium fluorescent white, potassium hydroxide, dimethyl sulfoxide, protective agent and water;

[0011] Solution B is phosphate buffered saline containing Hoechst;

[0012] Solution C is an aqueous solution containing wheat germ agglutinin WGA-AF488 fluorescent probe;

[0013] Solution D is sodium acetate buffer containing acridine orange;

[0014] Liquid E contains DMAO (C8H 11 NO) fluorescent probe in physiological saline solution;

[0015] Solution F is a physiological saline solution containing the EthD-Ⅲ (C46H50Cl4N8) fluorescent probe.

[0016] Preferably, each 1000 ml of liquid A contains:

[0017] Calcein 10mg-60mg, potassium hydroxide 36mg-44mg, dimethyl sulfoxide 38.4-57.6mL, and protective agent;

[0018] Preferably, the protective agent is selected from any one or more combinations of glycerol, sulfobutyl ether-β-cyclodextrin or trehalose, and the amount of glycerol added is 0.6% to 1.2% (volume ratio), and / or the amount of trehalose added is 0.5% to 1% (mass volume ratio), and / or the amount of sulfobutyl ether-β-cyclodextrin added is 0.1% to 2% (mass volume ratio).

[0019] Preferably, the protective agent is selected from a combination of sulfobutyl ether-β-cyclodextrin and trehalose, and each 1000 ml of solution A contains 5 g of trehalose and 5 g of sulfobutyl ether-β-cyclodextrin.

[0020] Preferably, each 1000 ml of liquid A contains:

[0021] Calcein 20 mg, potassium hydroxide 40 mg, DMSO 57.6 ml, trehalose 5 g and sulfobutyl ether-β-cyclodextrin 5 g, with the balance being water.

[0022] Preferably, in solution B, 1000 ml of solution contains 1 to 2 ml of Hoechst fluorescent probe (10 mg / ml), and the rest is phosphate buffer;

[0023] Solution C contains 5-10 ml of wheat germ agglutinin WGA-AF488 fluorescent probe (1 mg / ml) per 1000 ml of solution, and the rest is pure water;

[0024] Solution D contains 0.05g-0.2g of acridine orange per 1000ml, and the rest is sodium acetate buffer;

[0025] In liquid E, each 1000ml solution contains DMAO (C8H 11 NO) fluorescent probe (1 mg / ml) 1-5 ml, the rest is 0.85% saline;

[0026] In liquid F, each 1000 ml of solution contains 1 to 2 mg of EthD-Ⅲ (C46H50Cl4N8) fluorescent probe, and the rest is 0.85% saline.

[0027] Preferably, in the kit:

[0028] Solution B, 1000 ml of solution contains 2 ml of Hoechst fluorescent probe (10 mg / ml) and 980 ml of phosphate buffer;

[0029] Solution C, 1000ml solution contains 10ml of wheat germ agglutinin-AF488 fluorescent probe (1mg / ml) and 990ml of pure water;

[0030] Solution D, 1000ml of solution contains 0.2g of acridine orange and 999.8ml of sodium acetate buffer;

[0031] Liquid E, 1000ml of solution contains DMAO (C8H 11 NO) fluorescent probe (1 mg / ml) 5 ml, 0.85% saline 995 ml;

[0032] Solution F, 1000ml solution contains 2mg of EthD-Ⅲ (C46H50Cl4N8) fluorescent probe and 998ml of 0.85% saline.

[0033] In a second aspect, the present invention further provides a method for preparing the aforementioned kit, comprising the steps of:

[0034] Preparation of Solution A:

[0035] S1: Mix potassium hydroxide and 30% to 70% of the formula amount of purified water, stir and dissolve, and wait for the solution to cool to room temperature. This is referred to as solution a.

[0036] S1: Add the formulated amount of dimethyl sulfoxide and protective agent to solution A and stir thoroughly to mix;

[0037] S3: Measure calcium fluorescent white, add it to solution a and mix well, then dilute to volume with water to make up the balance;

[0038] Preparation of Solution B:

[0039] Take 980 ml of the formulated amount of Hoechst dye and phosphate buffer and mix thoroughly to obtain the product;

[0040] Preparation of Solution C:

[0041] Take the formula amount of wheat germ agglutinin-AF488 (5 mg / ml) and pure water, mix thoroughly, and you will get it;

[0042] Preparation of Solution D:

[0043] Weigh the formulated amount of acridine orange and dissolve it in the formulated amount of sodium acetate buffer, and mix thoroughly to obtain the product;

[0044] Preparation of E solution:

[0045] Take the required DMAO stock solution and dissolve it in the formulated amount of normal saline, and mix thoroughly to obtain the product;

[0046] Preparation of Solution F:

[0047] A formula amount of EthD-III raw material powder and a formula amount of DMSO are mixed evenly to obtain a concentrated preservation solution 1, and the concentrated preservation solution 1 is added to a formula amount of physiological saline and mixed thoroughly to obtain the product.

[0048] In a third aspect, the present invention further provides a method for using the aforementioned kit, comprising the steps of:

[0049] S1: Place the three-divided slide in a horizontal position and evenly apply the sample to the three wells of the slide;

[0050] S2: Add solution A to the first well, allowing the liquid to completely cover the entire specimen. After staining for 1 to 3 minutes, discard the excess stain. Then, add solution E and solution F to the same well, staining for 1 to 3 minutes each, and discard the excess stain.

[0051] S3: Add solution B to the second well, allowing the liquid to completely cover the entire specimen. After staining for 1 to 3 minutes, discard the excess stain. Then, add solution C to the same well, staining for 5 to 10 minutes, discard the excess stain.

[0052] S4: Take liquid D and drop it into the third well, so that the liquid completely covers the entire specimen. After staining for 1 to 3 minutes, pour off the excess stain.

[0053] S4: Cover the three wells with coverslips respectively, and place the prepared samples under a confocal fluorescence biological microscope system for observation and analysis.

[0054] Compared with the prior art, the present invention has the following beneficial effects:

[0055] First, the kit provided by the present invention includes liquids A to F. Liquid A contains calcium fluorescent white, which can label chitin and cell wall β-polysaccharides in a sample, staining all fungi in the sample, and the fungi exhibit blue-white fluorescence under UV light. Liquid B contains Hoechst, which can label all DNA and exhibit blue fluorescence under UV light. The labeled cells can be observed and counted. Liquid C contains wheat germ agglutinin WGA-AF488, which can bind to the peptidoglycan in the cell wall of Gram-positive bacteria, thereby fluorescently labeling Gram-positive bacteria. Under band B, Gram-positive bacteria appear yellow-green, while Gram-positive bacteria appear other colors. Liquid D contains acridine orange, which can bind to DNA / RNA in cells to form a fluorescent polymer. Under band B, single-stranded DNA / RNA appears red, while double-stranded DNA appears green. Liquid E contains DMAO, which can selectively stain live bacteria with intact cell membranes. Taking advantage of the relatively small cell membranes of living bacteria, the staining passes through the cell membrane, and the bacteria exhibit yellow-green fluorescence under band B. EthD-III in F solution can selectively stain dead bacteria with damaged cell membranes. This staining takes advantage of the relatively large cell membranes of dead bacteria, resulting in red fluorescence in the B band. During use, a customized three-well slide, combined with the AI ​​scanning diagnostic technology of a fully automated fluorescence biological microscope, leverages differences in fluorescence signals to simultaneously detect multiple target DNA molecules in microorganisms, enabling rapid and accurate detection of pathogen DNA. This technology also provides rapid qualitative and semi-quantitative analysis of microbial species to assist in assessing the nature of a patient's disease. When applied to human body fluids, cells, and tissue samples, it can analyze fungal and bacterial infections, perform cell classification, and assess damage. It holds considerable potential, particularly in the detection of pathogens in clinical bacterial infections (mixed infections) accompanied by sepsis or tumors. This technology boasts advantages such as high sensitivity and specificity, offering significant advantages in ensuring sample quality, providing rapid preliminary diagnoses, and providing precise treatment plans. It is of great significance for improving the efficiency and accuracy of clinical diagnostics, with its primary benefits being:

[0056] (1) Simple operation and multiple simultaneous detection capabilities: The present invention uses a variety of imported fluorescent dyes to label microbial DNA and uses the difference in fluorescent signals to achieve simultaneous detection of multiple target DNA molecules. Only a few steps of fluorescent staining are needed, and a short wait is required to observe the different colors and forms of microorganisms such as bacteria, fungi and cells. These microorganisms can be differentiated and presented under the field of view of the AI ​​fluorescent biological microscope, which greatly improves the contrast clarity of clinical samples. It can quickly and accurately detect pathogen DNA, quickly identify microbial species, and assist in assessing the nature of the patient's disease. It is particularly suitable for early detection of patients with mixed infections and multiple underlying diseases, and timely differentiated diagnosis of early diseases in patients with infection types and malignant lesions, facilitating the formulation of precise treatment plans in the later stages.

[0057] (2) Fast and efficient, suitable for rapid screening of large samples: The product of the present invention combines AI confocal technology to complete the detection of multiple samples in a relatively short period of time. Based on the high-resolution characteristics of confocal technology itself, it can clearly observe the fine structure of microorganisms such as pathogens and cells, and more comprehensively display the structure and distribution of cells and microorganisms, providing richer information for research and facilitating more accurate positioning and qualitative analysis of samples. In addition, the technical experience requirements for the testers are lower, the operation is simpler, and the test results are more accurate, which is more in line with the clinical needs for rapid and efficient testing of large numbers of samples for early screening.

[0058] (3) High sensitivity, specificity, and accuracy: Preliminary clinical trials have shown that the positive detection rate of the multiple fluorescence-labeled rapid detection kit (referred to as the multiple fluorescence method) for patients with bacterial infection or sepsis is 91.00%, respectively. Taking the culture method results as the diagnostic gold standard, the multiple fluorescence method and PCT method have a sensitivity (100%), specificity (90.00%), and accuracy (99.00%) in bacterial infection detection.

[0059] (4) The product is stable and poses low hazards to samples, the environment and personnel: The product of the present invention, especially the liquid A, has good stability through reasonable compounding of the components. Experiments have shown that the sensitivity, specificity and accuracy of sample detection of this product at 40°C for 60 days are all above 90%. In addition, the fluorescent probe used in the product of the present invention has relatively low toxicity to cells, making it suitable for further clinical promotion and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0061] Figure 1 This is a diagram showing the staining effect of the multi-fluorescence method of the present invention on fungal infection of gynecological specimens (UV band);

[0062] Figure 2 This is a staining effect diagram (UV band) of gynecological specimens infected with Trichomonas using the multiple fluorescence method of the present invention;

[0063] Figure 3 This is a diagram showing the merge analysis effect of the multiple fluorescence method of the present invention on mixed infection of gynecological specimens;

[0064] Figure 4This is the live-dead staining effect diagram of bacterial infection using the multiple fluorescence method of the present invention (B band);

[0065] Figure 5 This is a diagram showing the effect of the multiple fluorescence method of the present invention on bacterial typing and activity merging;

[0066] Figure 6 This is a diagram showing the effect of the multiple fluorescence method of the present invention on the typing, phagocytosis and merging of fungi and bacteria in specimens. DETAILED DESCRIPTION

[0067] The following examples are only used to more clearly illustrate the technical scheme of the present invention and are therefore only used as examples, and cannot be used to limit the scope of the present invention. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equalization conversions and modifications made without departing from the spirit and scope of the present invention should all be encompassed within the scope of the present invention. In the examples, if specific conditions are not indicated, they are carried out according to normal conditions or the conditions recommended by the manufacturer. All reagents or instruments are not indicated by manufacturers and are all conventional products that can be purchased commercially.

[0068] To better illustrate the present invention, numerous specific details are provided in the following detailed description. Those skilled in the art will appreciate that the present invention can be practiced without certain specific details. In other embodiments, methods, means, equipment, and steps well known to those skilled in the art are not described in detail in order to highlight the main purpose of the present invention.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable errors in industrial production.

[0070] Example 1

[0071] This embodiment provides a rapid detection kit for microorganisms and cells based on confocal microscopy and multiple fluorescent labeling, and its preparation and use methods, specifically:

[0072] 1.1 Kit Composition

[0073] Each 6000 ml of the microorganism and cell multiple fluorescent labeling rapid detection kit contains the following components:

[0074] Solution A, 1000ml solution contains 60mg of calcium fluorescent white, 44mg of potassium hydroxide, 57.6ml of dimethyl sulfoxide, 6ml of glycerol, and 832.4ml of pure water.

[0075] Solution B, 1000ml of solution contains 2ml of Hoechst fluorescent probe (10mg / ml) and 980ml of phosphate buffer.

[0076] Solution C, 1000ml solution contains 10ml of wheat germ agglutinin-AF488 fluorescent probe (1mg / ml) and 990ml of pure water.

[0077] Solution D, 1000ml of solution contains 0.2g of acridine orange and 999.8ml of sodium acetate buffer.

[0078] Liquid E, 1000ml of solution contains DMAO (C8H 11 5 ml of NO) fluorescent probe (1 mg / ml) and 995 ml of 0.85% saline.

[0079] F solution, 1000ml solution contains EthD-Ⅲ (C 46 H 50 Cl4N8) fluorescent probe 2 mg, 0.85% saline 998 ml.

[0080] The following are special instructions for the components used in this kit:

[0081] Calcein white in solution A is a known fungal cell wall stain, widely used to bind chitin and cellulose. Hoechst fluorescent probe (using Hoechst 33342) is a classic DNA dye used for cell nucleus labeling; DMAO is N,N-dimethylaniline N-oxide, with a chemical formula of C8H 11 NO is a membrane-permeable green fluorescent nucleic acid dye that can penetrate cell membranes and preferentially bind to double-stranded DNA. It is commercially available. Acridine orange is a classic nucleic acid dye used to differentiate DNA from RNA. EthD-III is an ethidium bromide analog used to label dead cells (which irreversibly bind to DNA). All of the above reagents are currently available, and their preparation methods are not detailed here.

[0082] 1.2 Kit Preparation Method

[0083] Preparation of Solution A (1000 ml):

[0084] ① Use a balance to weigh 44 mg of potassium hydroxide and add it to a 1 L beaker. Slowly add 500 ml of purified water and stir to dissolve. Let it stand for 30 minutes and wait for the solution to cool to room temperature. This is called solution A.

[0085] ② Use a measuring cylinder to measure 57.6 ml of dimethyl sulfoxide and 6 ml of glycerol and add them to solution a, stirring thoroughly to mix.

[0086] ③ Use a graduated cylinder to measure 60 mg of calcium fluorescent white, add it to solution a and mix well, then pour in purified water to make up to 1 L.

[0087] ④ Filter the prepared 1L liquid, and the filtered liquid is the semi-finished product; take a certain amount of the semi-finished product for testing. If the test results meet the requirements, the semi-finished product is qualified, otherwise re-prepare.

[0088] ⑤ After the semi-finished product passes the inspection, it is put into bottles and sealed to make the finished product.

[0089] Preparation of Solution B (1000 ml):

[0090] ① Use a pipette to draw the required 2 ml of Hoechst dye (10 mg / ml) and 980 ml of phosphate buffer, and mix thoroughly to obtain the semi-finished product.

[0091] ② Take a certain amount of semi-finished product reagents for testing. If the test results meet the requirements, the semi-finished product passes the inspection, otherwise it will be reconfigured.

[0092] ③ After the semi-finished product passes the inspection, it is placed in a reagent bottle and sealed to make the finished product.

[0093] Preparation of Solution C (1000 ml):

[0094] ① Use a pipette to draw the required 10ml of wheat germ agglutinin-AF488 (5mg / ml) and 990ml of pure water, and mix them thoroughly; the semi-finished product is obtained.

[0095] ② Take a certain amount of semi-finished product reagents for testing. If the test results meet the requirements, the semi-finished product passes the inspection, otherwise it will be reconfigured.

[0096] ③ After the semi-finished product passes the inspection, it is placed in a reagent bottle and sealed to make the finished product.

[0097] Preparation of Solution D (1000 ml):

[0098] ① Use an analytical balance to accurately weigh 0.2g of acridine orange, add it to 999.8ml of sodium acetate buffer measured in a measuring cylinder, and mix thoroughly to obtain the semi-finished product.

[0099] ② Take a certain amount of semi-finished product reagents for testing. If the test results meet the requirements, the semi-finished product passes the inspection, otherwise it will be reconfigured.

[0100] ③ After the semi-finished product passes the inspection, it is placed in a reagent bottle and sealed to make the finished product.

[0101] Preparation of Solution E (1000 ml):

[0102] ① Use a pipette to draw the required 5ml of DMAO stock solution and 995ml of 0.85% saline solution, and mix thoroughly to obtain the semi-finished product.

[0103] ② Take a certain amount of semi-finished product reagents for testing. If the test results meet the requirements, the semi-finished product passes the inspection, otherwise it will be reconfigured.

[0104] ③ After the semi-finished product passes the inspection, it is placed in a reagent bottle and sealed to make the finished product.

[0105] Preparation of Solution F (1000 ml):

[0106] ① Remove 2 mg of EthD-III raw powder from a -20 °C refrigerator and centrifuge at low speed to remove the powder on the tube cap to the bottom of the tube. Add 1.16 ml of DMSO and mix well to obtain concentrated storage solution 1. Store at 4 °C in the dark.

[0107] ② Take 11ul of concentrated preservation solution 1 and add 89ul of 0.85% NaCl solution. Mix thoroughly to obtain the semi-finished product.

[0108] ③ Take a certain amount of semi-finished product reagents for testing. If the test results meet the requirements, the semi-finished product passes the inspection, otherwise it will be reconfigured.

[0109] ④ After the semi-finished product passes the inspection, it is placed in a reagent bottle and sealed to make the finished product.

[0110] 1.3 Usage

[0111] S1: Place the three-divided slide in a horizontal position, and evenly apply the sample to the three wells of the slide;

[0112] S2: Add solution A to the first well, allowing the liquid to completely cover the entire specimen. After staining for 2 minutes, discard the excess stain. Then, add solution E and solution F to the same well, staining for 2 minutes each, and discard the excess stain.

[0113] S3: Add solution B to the second well, allowing the liquid to completely cover the entire specimen. After staining for 2 minutes, discard the excess stain. Then, add solution C to the same well, staining for 5-10 minutes, discard the excess stain.

[0114] S4: Take liquid D and drop it into the third well, so that the liquid completely covers the entire specimen. After staining for 2 minutes, pour off the excess dye;

[0115] S5: Cover the three wells with coverslips respectively, and place the prepared samples under a confocal fluorescence biological microscope system for observation and analysis.

[0116] Example 2

[0117] This embodiment provides a rapid detection kit for multiple fluorescent labeling of microorganisms and cells based on confocal microscopy, and its preparation and use method, with reference to Example 1. This embodiment differs from Example 1 in the composition of the kit. Specifically, the kit of this embodiment comprises:

[0118] 2.1 Kit Composition

[0119] Each 6000 ml of the microorganism and cell multiple fluorescent labeling rapid detection kit contains the following components:

[0120] Solution A, 1000ml solution contains 10mg of calcium fluorescent white, 36mg of potassium hydroxide, 38.4ml of dimethyl sulfoxide, 4ml of glycerol, and 911.6ml of pure water.

[0121] Solution B, 1000ml of solution contains 1ml of Hoechst fluorescent probe (10mg / ml) and 999ml of phosphate buffer.

[0122] Solution C, 1000ml solution contains 5ml of wheat germ agglutinin-AF488 fluorescent probe (1mg / ml) and 995ml of pure water.

[0123] Solution D, 1000ml of solution contains 0.05g of acridine orange and 999.95ml of sodium acetate buffer.

[0124] Liquid E, 1000ml of solution contains DMAO (C8H 11 1 ml of NO) fluorescent probe (1 mg / ml) and 999 ml of 0.85% saline.

[0125] F solution, 1000ml solution contains EthD-Ⅲ (C 46 H 50 Cl4N8) fluorescent probe 1 mg, 0.85% saline 999 ml.

[0126] The preparation method and usage method are as described in Example 1 and will not be described again here.

[0127] Example 3

[0128] This embodiment provides a rapid detection kit for multiple fluorescent labeling of microorganisms and cells based on confocal microscopy, and its preparation and use method, with reference to Example 1. This embodiment differs from Example 1 in the composition of the kit. Specifically, the kit of this embodiment comprises:

[0129] 3.1 Kit Composition

[0130] Each 6000 ml of the microorganism and cell multiple fluorescent labeling rapid detection kit contains the following components:

[0131] Solution A, 1000ml solution contains 20mg of calcium fluorescent white, 40mg of potassium hydroxide, 48ml of dimethyl sulfoxide, 5ml of glycerol, and 887ml of pure water.

[0132] Solution B, 1000ml solution contains 2ml of Hoechst fluorescent probe (10mg / ml) and 998ml of phosphate buffer.

[0133] Solution C, 1000ml solution contains 2ml of wheat germ agglutinin-AF488 fluorescent probe (1mg / ml) and 998ml of pure water.

[0134] Solution D, 1000ml of solution contains 0.1g of acridine orange and 999.9ml of sodium acetate buffer.

[0135] Liquid E, 1000ml of solution contains DMAO (C8H 11 NO) optical probe (1 mg / ml) 8 ml, 0.85% saline 992 ml.

[0136] F solution, 1000ml solution contains EthD-Ⅲ (C 46 H 50 Cl4N8) fluorescent probe 1.5 mg, 0.85% saline 998.5 ml.

[0137] The preparation method and usage method are as described in Example 1 and will not be described again here.

[0138] Test Example 1

[0139] In this experimental example, the confocal microorganism and cell multiple fluorescent labeling rapid detection kit prepared in Example 1 was used to stain various clinical specimens. The staining method was as follows:

[0140] S1: Place the three-divided slide in a horizontal position, and evenly apply the sample to the three wells of the slide;

[0141] S2: Add solution A to the first well, allowing the liquid to completely cover the entire specimen. After staining for 2 minutes, discard the excess stain. Then, add solution E and solution F to the same well, staining for 2 minutes each, and discard the excess stain.

[0142] S3: Add solution B to the second well, allowing the liquid to completely cover the entire specimen. After staining for 2 minutes, discard the excess stain. Then, add solution C to the same well, staining for 5-10 minutes, discard the excess stain.

[0143] S4: Take liquid D and drop it into the third well, so that the liquid completely covers the entire specimen. After staining for 2 minutes, pour off the excess dye;

[0144] S4: Cover the three wells with coverslips respectively, and place the prepared samples under a confocal fluorescence biological microscope system for observation and analysis.

[0145] The dyeing principle is as follows:

[0146] Step (1): Sample preparation and well processing

[0147] Each of the three wells is used to stain with a different fluorescent probe, preventing cross-interference between different fluorescent signals. This split-well process facilitates subsequent multiple staining workflows, preventing probe failures (e.g., solutions A / E / F, B / C, and D) due to differences in reaction conditions (e.g., pH and ion concentration).

[0148] Step (2): First well staining (Solution A → Solution E → Solution F)

[0149] Calcofluor White specifically binds to chitin and cellulose in the fungal cell wall, emitting blue fluorescence (UV band) for preliminary fungal labeling. DMAO (a live cell membrane permeability probe) can penetrate the cell membrane of live bacteria and react with intracellular esterases to generate green fluorescence, which is used to label live bacteria. EthD-III, on the other hand, can only enter dead bacterial cells, where it embeds into DNA and emits red fluorescence. Combined with DMAO, it can distinguish live from dead bacteria.

[0150] Step (3): Second well staining (Solution B → Solution C)

[0151] Hoechst can penetrate cell membranes, bind to double-stranded DNA (AT-rich regions), and emit blue fluorescence (UV wavelength), thereby labeling all nuclear and bacterial DNA. Wheat germ agglutinin (WGA) specifically binds to the peptidoglycan of the cell wall of Gram-positive (G+) bacteria. AF488-labeled WGA emits green fluorescence, enabling the differentiation of Gram-positive from Gram-negative bacteria (requires combination with other probes).

[0152] Step (4): Staining the third well (Solution D)

[0153] Acridine orange is a metachromatic dye that can distinguish between DNA (green fluorescence) and RNA (red fluorescence). It is sensitive to acidic structures (such as acid-fast bacteria) and can detect cell metabolic activity (RNA in live cells is red, and DNA in dead cells is green).

[0154] Step (5): Preparation and confocal analysis

[0155] The sample was sealed with a coverslip to prevent drying and contamination, maintaining a stable fluorescence signal. Observation was performed using a confocal fluorescence microscope.

[0156] Based on the above detection method, the present invention provides multiple clinical specimens and observes the staining effect. The experimental results are as follows:

[0157] (1) Please refer to Figure 1 , which is a staining effect diagram of fungal infection in gynecological specimens using the multiple fluorescence method of the present invention (UV band - fungi, 20X plane);

[0158] The experimental results were analyzed as follows: Fungal infections of gynecological vaginal discharge are primarily caused by Candida species. Under UV light, the specimen was observed for the presence of bright blue fungal fluorescence signals. Observation of fluorescent fungi indicates the presence of fungi, while the absence of fluorescent signals indicates the absence of fungi. The primary fluorescent fungal morphology is pseudohyphae: septa with distinct narrowing at the septa, resembling lotus root nodes. Spores are round or oval yeast-like in shape; blastospores are spores that develop in a gourd-shaped, budding state.

[0159] (2) Please refer to Figure 2 , which is a staining effect diagram of gynecological specimens infected with Trichomonas by the invented multiple fluorescence method (UV band - Trichomonas infection, 20X);

[0160] The experimental results are analyzed as follows: When gynecological leucorrhea is infected with Trichomonas, the morphology of Trichomonas is observed under the B band; Trichomonas is an anaerobic parasitic protozoan. The typical morphology of Trichomonas observed under a fluorescence microscope is broad pear-shaped or oval, slightly larger than white blood cells, with flagella visible on the head, and a yellow vertical or oblique rod visible at 1 / 3 of the body.

[0161] (3) Please refer to Figure 3 , which is a merge analysis effect diagram of mixed infection of gynecological specimens by the multiple fluorescence method of the present invention;

[0162] The results of the experiment are as follows: When gynecological leucorrhea is infected with both fungi and trichomonas, it will have Figure 2 、 Figure 3 The typical morphology of fungi and trichomonas is formed by fusion of images after UV and B band scanning.

[0163] (4) Please refer to Figure 4 , which is a merge analysis effect diagram of the activity analysis of bacterial infection by the multiple fluorescence method of the present invention (bacterial activity merge (live bacteria: green; dead bacteria: red), 40X);

[0164] The experimental results are analyzed as follows: When clinical samples are infected with bacteria, under the fluorescent B band, active living bacteria, whether rod-shaped or spherical, appear green; inactive dead bacteria appear red.

[0165] (5) Please refer to Figure 5, which is a diagram showing the effect of the multiple fluorescence method of the present invention on bacterial typing and activity merge (three-color bacterial typing and activity merge 40X (G+ is green, red is dead bacteria, and blue is G-);

[0166] The experimental results are analyzed as follows: When clinical samples encounter bacterial (Gram-positive / Gram-negative) infection, in the fluorescence B band, G+ bacteria are green, G- bacteria are blue, and if there are dead bacteria with no activity, they will appear red.

[0167] (6) See Figure 6 , which is a merge effect diagram of the fungal and bacterial typing and phagocytosis of the specimen by the multiple fluorescence method of the present invention (the merge effect diagram of the same section fungal typing, bacterial typing, cell classification, fungal bacteria, and activity analysis, 40X);

[0168] The experimental results are analyzed as follows: When clinical samples encounter mixed infections of fungi and bacteria, typical fungal hyphae can be seen under the fluorescent UV band, and active green bacteria can be seen under the fluorescent B band, as well as the morphology of white blood cells and neutrophils phagocytosing bacteria. Figure 6 shown.

[0169] from Figures 1-6 It can be seen that the present invention uses multiple fluorescent probes to label microbial and cellular DNA, and uses the difference in fluorescent signals to achieve simultaneous detection of multiple target DNA molecules, thereby achieving rapid and accurate detection of common pathogen DNA. At the same time, it performs rapid qualitative and semi-quantitative analysis of microbial species, distinguishes fungal and bacterial infections in specimens, interprets cell classification, and detects related pathogens and cell damage; assists in assessing the nature of the patient's disease and facilitates the formulation of accurate treatment plans in the later stage; and this product combines AI confocal correlation scanning diagnostic technology with human body fluids, cells, and tissue samples, and can complete the detection of multiple samples in a relatively short period of time. Based on the high-resolution characteristics of the confocal technology itself, it can clearly observe the fine structure of microorganisms such as pathogens and cells, and more comprehensively display the structure and distribution of cells and microorganisms, providing richer information for research, and helping to more accurately locate and qualitatively analyze samples. In addition, the technical experience requirements for the test personnel are lower, the operation is simpler, and the accuracy of the test results is higher, which is more in line with the clinical needs for rapid and efficient early screening of large numbers of samples.

[0170] Test Example 2

[0171] Based on the above Example 1, this Example found that Liquid A had the following problems:

[0172] DMSO, as a highly polar aprotic solvent, effectively dissolves hydrophobic or lipid-soluble components (calcein), ensuring their uniform dispersion in aqueous solutions. DMSO also disrupts the lipid bilayer structure of cell membranes, promoting rapid penetration of calcein. However, prolonged staining (e.g., longer than 2 minutes) with high DMSO concentrations (57.6 ml / 1000 ml, or 5.76%) may excessively damage cell membranes, leading to nonspecific binding of subsequent staining probes (e.g., EthD-III) to dead cells and false positives.

[0173] Therefore, the purpose of this test example is to provide a variety of A liquid formulas, including the control group and experimental groups 1 to 8 are as follows:

[0174] Table 1 Composition of the control group and experimental groups 1 to 8

[0175]

[0176] In the formulation of Solution A in the control group, DMSO and glycerol perform the dissolution / penetration and stabilization / protection functions, respectively. While the current concentration ratio can achieve good staining results, it also presents issues such as high cytotoxicity, insufficient protein protection, and oxidation risks. Therefore, the present inventors further conducted the following experiments based on the Solution A compositions provided by the aforementioned control group and experimental groups 1 to 8:

[0177] 2.1 Detection indicators and test methods

[0178] (1) Cell survival rate

[0179] Human HeLa cells were stained according to standard procedures and the staining time was extended by 1 min (i.e., 3 min). The survival rate was calculated by trypan blue exclusion method (n=3).

[0180] (2) Fluorescence activity retention rate

[0181] Liquid A in each group was placed at 40°C for accelerated degradation (simulating long-term storage). Samples were taken on day 60 and the fluorescence intensity at Ex / Em=488 / 515nm was measured using a fluorescence spectrophotometer (the retention rate was calculated by comparing it with the initial value).

[0182] (3) Signal-to-noise ratio (SNR)

[0183] The ratio of the calcium fluorescent white signal (mean fluorescence intensity within the ROI) to the background signal (cell-free area) after quantitative staining was measured under confocal microscopy.

[0184] 2.2 Experimental Results

[0185] The experimental results are shown in Table 2;

[0186] Table 2 Effects of cytotoxicity, stability and signal-to-noise ratio on the control group and experimental groups 1 to 8

[0187]

[0188] (▲: compared with the control group p < 0.01; ●: compared with the experimental group 4 p < 0.05);

[0189] The above experimental results show that when 57.6ml of DMSO is used alone, the cell survival rate is only 65.4%, which shows high cytotoxicity. In experimental group 1, the addition of glycerol was doubled (6ml→12ml) compared with the control group, which only increased the survival rate by 9.4%, confirming that simply increasing glycerol cannot effectively neutralize the toxicity of DMSO. However, experimental group 4 showed significant improvements in all core indicators compared with the control group. At the same time, experimental group 8 showed the best effect, indicating that when the DMSO concentration remains unchanged, the combination of trehalose 5g + sulfobutyl ether-β-cyclodextrin 5g can not only effectively neutralize high concentrations of DMSO, but also has better effects than the combination of glycerol 6ml + sulfobutyl ether-β-cyclodextrin 5g, thereby effectively improving the stability of the calcium fluorescent white signal and the detection accuracy.

[0190] In addition, although the ternary compound groups (experimental groups 9-11) were better than the control group and some experimental groups, they did not surpass experimental group 8. The experimental results suggest that experimental group 8 achieved the optimal balance in cell protection, fluorescence stability and detection sensitivity through the precise compounding of trehalose and sulfobutyl ether-β-cyclodextrin, and is an ideal A liquid formula that takes into account both low toxicity and high stability.

[0191] Experimental group 8 (DMSO + trehalose 5g + sulfobutyl ether-β-cyclodextrin 5g) was significantly superior to the other groups in survival rate (93.8%), fluorescence retention rate (93.2%) and signal-to-noise ratio (17.6). Its advantages may be due to the synergistic protective effect of trehalose and sulfobutyl ether-β-cyclodextrin, which synergistically neutralize the toxicity of DMSO and enhance stability.

[0192] Based on the above experimental group 8, the present inventors further explored the effect of the mass ratio of trehalose to sulfobutyl ether-β-cyclodextrin on the staining effect of solution A, as follows:

[0193] 2.3 1. Experimental Group Design

[0194] (1) Experimental groups:

[0195] Based on the formula of experimental group 8 (DMSO 57.6ml + trehalose 5g + sulfobutyl ether-β-cyclodextrin 5g), the total amount of trehalose and sulfobutyl ether-β-cyclodextrin was kept at 10g, and the ratio between the two was adjusted to add the following experimental groups:

[0196] Experimental group 12: trehalose 4g + sulfobutyl ether-β-cyclodextrin 6g

[0197] Experimental Group 13: Trehalose 4.5g + sulfobutyl ether-β-cyclodextrin 5.5g

[0198] Experimental group 14: trehalose 5.5g + sulfobutyl ether-β-cyclodextrin 4.5g

[0199] Experimental group 15: trehalose 6g + sulfobutyl ether-β-cyclodextrin 4g

[0200] (Original experimental group 8: trehalose 5g + sulfobutyl ether-β-cyclodextrin 5g, the original experimental data was used as the baseline control);

[0201] (2) For testing indicators and methods, please refer to 2.1

[0202] Statistical analysis: Data were expressed as mean ± standard deviation. One-way ANOVA was used for comparison among groups, and p < 0.05 was considered significant.

[0203] (3) Experimental results, please refer to Table 3

[0204] Table 3 Effect of the ratio of trehalose and sulfobutyl ether-β-cyclodextrin on the staining effect of solution A

[0205]

[0206] (▲: compared with experimental group 8, p<0.05);

[0207] The above experimental results show that when the amount of sulfobutyl ether-β-cyclodextrin exceeds 5.5g (experimental group 12) or trehalose exceeds 5.5g (experimental group 14), the protective effect decreases, suggesting that the two need to maintain a dynamic balance. Experimental group 13 (trehalose 4.5g + sulfobutyl ether-β-cyclodextrin 5.5g) achieved the optimal results in cell protection, dye stability, and detection sensitivity by adjusting the ratio of trehalose to sulfobutyl ether-β-cyclodextrin.

[0208] Test Example 3

[0209] In this experimental example, the composition of Solution A provided in Experimental Example 2 (Experimental Group 13, i.e., 20 mg of calcein + 40 mg of potassium hydroxide + 57.6 ml of DMSO + 4.5 g of trehalose + 5.5 g of sulfobutyl ether-β-cyclodextrin) was combined with Solutions B to F in Example 3 to conduct a multiple fluorescence method clinical trial. Clinical comparison tests were conducted with the detection method of the procalcitonin (PCT) detection kit. The comparison indicators included positive detection rate, sensitivity, specificity, and accuracy.

[0210] 3.1 Research subjects

[0211] 300 patients with suspected bacterial infection admitted to the hospital were selected as research subjects:

[0212] Patient inclusion criteria: (1) complete clinical data, serum procalcitonin (PCT), and blood culture indicators; (2) informed consent.

[0213] Patient exclusion criteria: (1) patients with autoimmune diseases or blood system diseases; (2) patients with hyperadrenalism; (3) patients with major organ dysfunction; (4) patients with stress reactions or drug reactions; (5) patients with a history of surgery or trauma within 3 months.

[0214] Inclusion criteria for the healthy control group: (1) aged 20 to 65 years; (2) understood and agreed to this study.

[0215] Exclusion criteria for the healthy control group: (1) those with infection found during physical examination; (2) those with immune dysfunction; (3) those with major organ dysfunction.

[0216] 3.2 Methods

[0217] (1) Serum PCT test: 2–5 ml of venous blood is collected from the patient using a coagulant tube. The sample is allowed to stand at room temperature for 15–30 min. After the blood sample has coagulated, it is transferred to a centrifuge tube and centrifuged at 3000 rpm for 10–15 min. The PCT concentration in the serum is quantitatively measured using a chemiluminescent immunoassay. If the PCT value is ≥ 0.5 μg / L, the test is positive, indicating possible infection.

[0218] (2) Multiple fluorescence method: Take three smears of appropriate specimens (alveolar lavage fluid / sputum) suspected of clinical bacterial infection, stain them according to the method in Example 1, and interpret and analyze them under a confocal fluorescence biological microscope.

[0219] (3) Blood culture: After sampling, the sample is placed in a fully automatic blood culture instrument for testing. If bacteria grow during the process, an alarm will be issued. The blood culture that has issued the alarm will be transferred and stained, and observed under a microscope to determine whether it is Gram-negative or Gram-positive bacilli. Then, a single colony will be separated and tested again.

[0220] 3.3 Observation indicators and experimental results

[0221] 3.3.1 Observation indicators

[0222] (1) Observe and analyze the test results of each method, and interpret the positive rate of each test. The experimental results are shown in Table 4;

[0223] (2) Using the culture method as the gold standard, analyze the clinical diagnostic value of each detection method and analyze and compare the various detection schemes:

[0224] Sensitivity = number of true positive cases / (true positive + false negative) cases × 100%;

[0225] Specificity = number of true negative cases / (true negative + false positive) cases × 100%;

[0226] Accuracy = (number of true positives + true negatives) / total number of cases × 100%;

[0227] Positive predictive value = number of true positive cases / (true positive + false positive) cases × 100%;

[0228] Negative predictive value = number of true negative cases / (number of true negative cases + false negative cases) × 100%. The experimental results are shown in Table 5;

[0229] The experimental results were analyzed using SPSS 25.0 software. The measurement data were described as "x ± s", and the differences between the groups were compared using the t test; the counting results were described as percentages (%), and the differences between the groups were compared using the χ2 test.

[0230] 3.3.2 Test results

[0231] Table 4 Comparative analysis of the results of bacteria detected by three detection methods

[0232]

[0233] Table 5 Diagnostic value of multiplex fluorescence method and PCT method in detecting bacterial infection

[0234]

[0235] Comparison of positive rates: The positive rates of the three methods were 91.00% for the multiple fluorescence method, 93.00% for the PCT method, and 90.00% for the culture method. There was no statistically significant difference between the three groups (χ 2 =2.16, p=0.340).

[0236] Diagnostic value analysis (with culture as the gold standard):

[0237] Sensitivity: Both the multiplex fluorescence method and the PCT method were 100% (95% CI: 98.6%-100%), indicating that both methods can fully detect infection cases that are positive by culture.

[0238] Specificity: The multiple fluorescence method (90.00%, 95%CI: 74.1%-97.2%) was significantly higher than the PCT method (70.00%, 95%CI: 52.1%-83.3%), χ 2 =10.71, p=0.001, indicating that the multiplex fluorescence method has an advantage in excluding false positives.

[0239] Accuracy and predictive value: The accuracy (99.00% vs 97.00%) and positive predictive value (98.90% vs 96.77%) of the multiplex fluorescence method were superior to those of the PCT method.

[0240] In the above experiments, culture method as the gold standard may have false negatives (such as fastidious bacteria or samples after antibiotic use), which may lead to overestimation of sensitivity.

[0241] The above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art will appreciate that the technical solutions of the present invention may be modified or replaced with equivalents without departing from the spirit and scope of the technical solutions of the present invention, and such modifications or equivalents shall be encompassed by the claims of the present invention. Any techniques, shapes, and structures not described in detail herein are well known.

Claims

1. A method for using a rapid detection kit for microorganisms and cells based on confocal microscopy and multiple fluorescent labeling, characterized in that: Including steps: S1: Place the three-divided slide in a horizontal position, and evenly apply the sample to the three wells of the slide; S2: Add solution A to the first well, allowing the liquid to completely cover the entire specimen. After staining for 1 to 3 minutes, discard the excess stain. Then, add solution E and solution F to the same well, staining for 1 to 3 minutes each, and discard the excess stain. S3: Add solution B to the second well, allowing the liquid to completely cover the entire specimen. After staining for 1 to 3 minutes, discard the excess stain. Then, add solution C to the same well, staining for 5 to 10 minutes, discard the excess stain. S4: Take liquid D and drop it into the third well, so that the liquid completely covers the entire specimen. After staining for 1 to 3 minutes, pour off the excess stain. S5: Cover the three wells with coverslips respectively, and place the prepared samples under a confocal fluorescence biological microscope for observation and analysis; The kit includes solution A, solution B, solution C, solution D, solution E, and solution F, wherein: Solution A includes calcium fluorescent white, potassium hydroxide, dimethyl sulfoxide, protective agent and water; Solution B is phosphate buffered saline containing Hoechst; Solution C is an aqueous solution containing wheat germ agglutinin WGA-AF488 fluorescent probe; Solution D is sodium acetate buffer containing acridine orange; Solution E is a physiological saline solution containing DMAO fluorescent probe; Solution F is a physiological saline solution containing EthD-III fluorescent probe; And each 1000ml of liquid A contains: Calcein 10mg-60mg, potassium hydroxide 36mg-44mg, dimethyl sulfoxide 38.4-57.6mL, and protective agent; The protective agent is selected from a combination of sulfobutyl ether-β-cyclodextrin and trehalose, and each 1000 ml of solution A contains 5 g of trehalose and 5 g of sulfobutyl ether-β-cyclodextrin.

2. The method for using the confocal microorganism and cell multiple fluorescent labeling rapid detection kit according to claim 1, characterized in that: Each 1000ml of the A solution contains: Calcein 20 mg, potassium hydroxide 40 mg, DMSO 57.6 ml, trehalose 5 g and sulfobutyl ether-β-cyclodextrin 5 g, with the balance being water.

3. The method for using the confocal microorganism and cell multiple fluorescent labeling rapid detection kit according to claim 1, characterized in that: In solution B, 1000 ml of solution contained 10 mg / ml Hoechst fluorescent probe 1, and the rest was phosphate buffer; In solution C, every 1000 ml of solution contains 5-10 ml of 1 mg / ml wheat germ agglutinin WGA-AF488 fluorescent probe, and the rest is pure water; Solution D contains 0.05g-0.2g of acridine orange per 1000ml, and the rest is sodium acetate buffer; In solution E, each 1000 ml of solution contained 1 to 5 ml of 1 mg / ml DMAO fluorescent probe, and the rest was 0.85% saline; In liquid F, each 1000 ml of solution contains 1 to 2 mg of EthD-III fluorescent probe, and the rest is 0.85% normal saline.

4. The method for using the confocal microorganism and cell multiple fluorescent labeling rapid detection kit according to claim 1, characterized in that: Solution B, 1000 ml of solution contains 2 ml of 10 mg / ml Hoechst fluorescent probe and 980 ml of phosphate buffer; Solution C, 1000ml solution contains 10ml of 1mg / ml wheat germ agglutinin-AF488 fluorescent probe and 990ml of pure water; Solution D, 1000ml of solution contains 0.2g of acridine orange and 999.8ml of sodium acetate buffer; Solution E, 1000ml of solution contains 5ml of 1mg / ml DMAO fluorescent probe and 995ml of 0.85% saline; Solution F, 1000ml of solution contains 2mg of EthD-III fluorescent probe and 998ml of 0.85% saline.

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