Method for rapidly detecting sensitivity of bacterial antibiotics

Abstract

The invention relates to the technical field of bacterial drug sensitivity detection, and discloses a method for rapidly detecting bacterial antibiotic sensitivity, which comprises the following steps: (1) preparing a mixed solution from a bacterial strain liquid to be detected, antibiotics and glucose, and incubating to obtain a mixed reagent; (2) adding a viable bacterium coloring agent and a dead bacterium coloring agent with different fluorescence colors into the mixed reagent for dyeing; and (3) respectively detecting the fluorescence intensity corresponding to the viable bacterium coloring agent and the dead bacterium coloring agent, and judging the antibiotic sensitivity of the bacteria according to the fluorescence intensity. By adopting the method disclosed by the invention, the reaction of the to-be-detected strain to the antibiotics can be accelerated, and the time required for detecting the antibiotic sensitivity of the bacteria is shortened.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial drug sensitivity detection, and in particular to a method for rapidly detecting bacterial antibiotic sensitivity. Background Art

[0002] Bacterial resistance is a global problem. The abuse of antibiotics has led to an increasing number of resistant bacteria, and even the emergence of super bacteria that are resistant to multiple antibiotics. In clinical practice, it is necessary to quickly identify resistant bacteria.

[0003] Timely detection of bacterial resistance or sensitivity to antibiotics in clinical practice is crucial for effective treatment of patients, especially critically ill patients (such as sepsis and pneumonia). At present, mainstream hospitals and institutions all use traditional phenotypic drug sensitivity tests, namely the disc diffusion method (KB value) or the microbroth dilution method (MIC value). This traditional clinical drug sensitivity test relies on bacterial growth. It takes 1 day for the drug sensitivity results of common fast-growing bacteria, while it takes several weeks for slow-growing bacteria, which makes it impossible for patients to receive accurate and effective treatment, resulting in drug resistance and increased mortality. However, molecular drug sensitivity detection technology (mass spectrometry, NGS) has great limitations and cannot be applied on a large scale in clinical practice. In addition, although the automatic drug sensitivity testers used in clinical laboratories, such as the BioMérieux VITEK-2 and BD Phoenix M50 drug sensitivity instruments, are simple, they require more than ten hours of overnight culture to obtain drug sensitivity results. The detection time of slow-growing bacteria such as Mycobacterium tuberculosis is as long as 2-4 weeks, which is time-consuming and easily affected by the operator.

[0004] In view of this, in recent years, many research institutions and enterprises want to make breakthroughs and seek innovations at the technical level. Nowadays, most of the automated drug sensitivity testers on the market use photoelectric colorimetry and fluorescence methods. The former measures the bacterial viability by measuring the changes in pH and transmittance caused by the growth of bacteria using substrates, while the latter adds enzyme substrates to the small holes of the test card to combine with the enzymes produced by bacteria to generate fluorescent substances. However, both methods cannot avoid the occurrence of false negatives; BGI uses molecular biological methods to develop detection kits to identify drug-resistant bacteria by detecting bacterial resistance gene mutations. This method has high accuracy, but the cost is high and the universality is low. It cannot cover all phenotypic resistance (gold standard); in addition, there are kits that use biochemical methods to detect drug-resistant bacteria, such as the Antu Biological Mycoplasma Drug Sensitivity Kit and the Merieux Pneumococcal Drug Sensitivity Kit. These kits are generally small in scope and can only detect one or several common drug-resistant bacteria. In addition, although methods for detecting bacterial metabolic states (such as Raman spectroscopy) and detecting morphological changes of single bacteria under the action of antibiotics (such as Xinke Formula) can shorten the drug sensitivity time to 2-3 hours, these technologies are expensive, have low throughput, and their accuracy needs to be improved or verified.

[0005] Therefore, it is of great significance to develop a rapid bacterial resistance detection method that does not require bacterial growth, which can quickly detect resistant bacteria in a shorter time and provide patients with personalized, high-end precision medical services. Summary of the invention

[0006] In order to solve the technical problem that the existing bacterial antibiotic sensitivity detection methods are time-consuming, the present invention provides a method for quickly detecting bacterial antibiotic sensitivity. The method of the present invention can accelerate the reaction of the strain to be tested to antibiotics, shorten the time required for bacterial antibiotic sensitivity detection, and improve the accuracy of bacterial antibiotic sensitivity detection.

[0007] The specific technical scheme of the present invention is: A method for rapidly detecting bacterial antibiotic sensitivity comprises the following steps: (1) preparing a mixed solution of a bacterial liquid of the strain to be tested, antibiotics and glucose, and incubating the mixture to obtain a mixed reagent; (2) adding live bacteria stains and dead bacteria stains of different fluorescent colors into the mixed reagent for staining; (3) Detect the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain respectively, and judge the antibiotic sensitivity of the bacteria accordingly.

[0008] The present invention uses live bacteria stains and dead bacteria stains of different fluorescent colors to stain live bacteria and dead bacteria respectively. No bacterial growth is required during the detection process, thus greatly shortening the time required for detection and obtaining the test results within 1 hour. In addition, the method has a small amount of operation throughout the experiment, which can avoid errors caused by different personnel operations to the greatest extent.

[0009] On this basis, the team of the present invention found that when glucose is not added, it is easy to be difficult to accurately distinguish between antibiotic-resistant strains and sensitive strains, that is, within 1 hour, the survival rates of antibiotic-resistant strains and sensitive strains cannot show a regular and obvious difference; by adding glucose, the above problem can be solved, and the resistant strains can be distinguished from the sensitive strains more accurately. Therefore, the present invention can improve the accuracy of bacterial antibiotic sensitivity detection by adding glucose.

[0010] Preferably, step (3) specifically comprises the following steps: (3.1) At a preset time point, the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain is detected, which are recorded as G1 and R1 respectively; the V1 value is calculated according to the following formula: V1 = (G1-3R1) / (G1+3R1); (3.2) Repeat steps (1) and (2), except that no antibiotics are added; at a preset time point, the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain is detected, which are recorded as G2 and R2, respectively; and the V2 value is calculated according to the following formula: V2 = (G2-3R2) / (G2+3R2); (3.3) Substitute V1 and V2 into the standard curve of bacterial survival rate and V value respectively, calculate the survival rates S1 and S2, and obtain the relative survival rate RS based on S1 / S2; when RS≥the preset resistance breakpoint value, the strain to be tested is an antibiotic-resistant bacteria; when RS≤the preset sensitivity breakpoint value, the strain to be tested is an antibiotic-sensitive bacteria.

[0011] Preferably, step (3) specifically comprises the following steps: (3.1) At a preset time point, the fluorescence intensities of the live bacteria stain and the dead bacteria stain are detected, recorded as G1 and R1 respectively, and the G1 / R1 value is calculated; (3.2) Repeat steps (1) and (2), except that no antibiotics are added; at a preset time point, the fluorescence intensities of the live bacteria stain and the dead bacteria stain are detected, recorded as G2 and R2, respectively, and the G2 / R2 value is calculated; (3.3) Substitute G1 / R1 and G2 / R2 into the standard curve of bacterial survival rate and G / R value respectively, calculate the survival rates S1 and S2, and obtain the relative survival rate RS based on S1 / S2; when RS ≥ the preset resistance breakpoint value, the test strain is an antibiotic-resistant bacteria; when RS ≤ the preset sensitivity breakpoint value, the test strain is an antibiotic-sensitive bacteria.

[0012] Preferably, step (3) specifically comprises the following steps: (3.1) At a preset time point, the fluorescence intensities of the live bacteria stain and the dead bacteria stain are detected and recorded as G1 and R1, respectively, and the D1 value is calculated according to the following formula: D1 = G1 / R1; (3.2) Repeat steps (1) and (2), except that no antibiotics are added; at a preset time point, the fluorescence intensities of the live bacteria stain and the dead bacteria stain are detected, which are recorded as G2 and R2, respectively, and the D2 value is calculated according to the following formula: D2 = G2 / R2; (3.3) The relative survival rate RS is obtained according to D1 / D2; when RS≥the preset resistance breakpoint value, the strain to be tested is an antibiotic-resistant bacterium; when RS≤the preset sensitivity breakpoint value, the strain to be tested is an antibiotic-sensitive bacterium.

[0013] In the above three specific methods of step (3), a suitable method can be selected according to the species to which the strain to be tested belongs, so as to improve the accuracy of judging the antibiotic sensitivity.

[0014] When the antibiotic type and concentration are different, and the strain to be tested belongs to a different taxonomic species, the values ​​of the "preset time point", "preset resistance breakpoint value" and "preset sensitivity breakpoint value" mentioned in the above process are different, but can be preset as follows: within the species, take a plurality of known antibiotic-resistant strains and antibiotic-sensitive strains, and according to steps (1) to (3), obtain the relative viability at different time points after staining, draw a curve of the relationship between relative viability and time, and determine the preset time point and the corresponding preset resistance breakpoint value and preset sensitivity breakpoint value based on the preset time point, so that at the preset time point, the relative viability of the antibiotic-resistant strain is higher than the preset resistance breakpoint value, and the relative viability of the antibiotic-sensitive strain is lower than the preset sensitivity breakpoint value, and the preset resistance breakpoint value minus the preset sensitivity breakpoint value>0.2.

[0015] Furthermore, in step (3.3), the method for obtaining the standard curve comprises the following steps: taking live bacteria and dead bacteria within the species to which the strain to be tested belongs, mixing them in different proportions and preparing them into bacterial liquids, and calculating the bacterial survival rate in each bacterial liquid according to the ratio between live bacteria and dead bacteria; staining each bacterial liquid with the live bacteria stain and the dead bacteria stain, and then detecting the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain, which are recorded as G and R respectively, and calculating the V value according to V=(G-3R) / (G+3R), or calculating the G / R value; and drawing a relationship curve between the bacterial survival rate and the V value or the G / R value.

[0016] Preferably, in step (1), the concentration of glucose in the mixed solution is not less than 10 mM.

[0017] Preferably, in step (1), the concentration of the antibiotic in the mixed solution is 50 to 1600 μg / mL.

[0018] Preferably, in step (1), the incubation temperature is 35-37° C. and the incubation time is 0.5-1 h.

[0019] Preferably, in step (1), the process of preparing a mixed solution from the bacterial liquid of the test strain, antibiotics and glucose comprises the following steps: dissolving glucose in water to prepare a glucose solution; dissolving glucose and antibiotics in water respectively to prepare a glucose solution and an antibiotic solution; and mixing the bacterial liquid of the test strain, the glucose solution and the antibiotic solution to obtain a mixed solution.

[0020] Furthermore, the OD value of the bacterial solution of the strain to be tested is 0.1-0.5.

[0021] Preferably, in step (2), the live bacteria stain is SYBR Green I, and the dead bacteria stain is propidium iodide.

[0022] Preferably, in step (2), the dyeing temperature is 35-37° C. and the dyeing time is 20-30 min.

[0023] Preferably, in step (2), the process of adding live bacteria stains and dead bacteria stains of different fluorescent colors into the mixed reagent comprises the following steps: mixing the live bacteria stain, the dead bacteria stain and water to prepare a stain solution, and then mixing the stain solution with the mixed reagent.

[0024] Preferably, the strain to be tested belongs to Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Acinetobacter baumannii, Stenotrophomonas maltophilia, Candida albicans, Streptococcus pneumoniae, Haemophilus influenzae, Oligomyces maltophilia, Burkholderia cepacia, Aspergillus or Mycobacterium tuberculosis.

[0025] Preferably, the antibiotic is an aminoglycoside antibiotic.

[0026] Compared with the prior art, the present invention has the following advantages: (1) The present invention adopts a method of staining live bacteria and dead bacteria separately, which can eliminate the need for bacterial growth during the bacterial antibiotic sensitivity test, thereby effectively shortening the time required for the test.

[0027] (2) The present invention can improve the accuracy of bacterial antibiotic sensitivity detection by adding glucose before incubating bacteria with antibiotics.

[0028] (3) The method of the present invention is simple to operate and can largely avoid errors caused by operations by different personnel. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the standard curve of S value-bacterial survival rate of Klebsiella pneumoniae.

[0030] Figure 2 This is the dynamic curve of the relative activity of Klebsiella pneumoniae at a gentamicin concentration of 50 μg / mL (without glucose).

[0031] Figure 3 This is the relative activity curve of Klebsiella pneumoniae at a gentamicin concentration of 100 μg / mL (without glucose).

[0032] Figure 4 This is the relative activity curve of Klebsiella pneumoniae at a gentamicin concentration of 200 μg / mL (without glucose).

[0033] Figure 5 This is the relative activity curve of Klebsiella pneumoniae at a gentamicin concentration of 400 μg / mL (without glucose).

[0034] Figure 6 This is the relative activity curve of Klebsiella pneumoniae at a gentamicin concentration of 50 μg / mL (with glucose).

[0035] Figure 7 This is the relative activity curve of Klebsiella pneumoniae at a gentamicin concentration of 100 μg / mL (with glucose).

[0036] Figure 8 This is the relative activity curve of Klebsiella pneumoniae at a gentamicin concentration of 200 μg / mL (with glucose).

[0037] Fig. 9 This is the relative activity curve of Klebsiella pneumoniae at a gentamicin concentration of 400 μg / mL (with glucose).

[0038] Fig.10 This is the relative activity curve of Klebsiella pneumoniae at a gentamicin concentration of 800 μg / mL (with glucose).

[0039] Fig.11 This is the relative activity curve of Klebsiella pneumoniae at a gentamicin concentration of 1600 μg / mL (with glucose).

[0040] Fig.12 This is the S value-bacterial survival rate standard curve of Staphylococcus aureus.

[0041] Fig.13 This is the dynamic curve of the relative activity of Staphylococcus aureus at a gentamicin concentration of 32 μg / mL (without glucose).

[0042] Fig.14 This is the dynamic curve of the relative activity of Staphylococcus aureus at a gentamicin concentration of 16 μg / mL (without glucose).

[0043] Fig.15 This is the dynamic curve of the relative activity of Staphylococcus aureus at a gentamicin concentration of 8 μg / mL (without glucose).

[0044] Fig.16 This is the dynamic curve of the relative activity of Staphylococcus aureus at a gentamicin concentration of 32 μg / mL (with glucose).

[0045] Fig.17 This is the dynamic curve of the relative activity of Staphylococcus aureus at a gentamicin concentration of 16 μg / mL (with glucose).

[0046] Fig.18 This is the dynamic curve of the relative activity of Staphylococcus aureus at a gentamicin concentration of 8 μg / mL (with glucose).

[0047] Fig.19This is the dynamic curve of the relative activity of Pseudomonas aeruginosa at a gentamicin concentration of 4 μg / mL (without glucose).

[0048] Fig. 20 This is the dynamic curve of the relative activity of Pseudomonas aeruginosa at a gentamicin concentration of 4 μg / mL (with glucose).

[0049] Fig.21 This is the dynamic curve of the relative activity of Pseudomonas aeruginosa at an amikacin concentration of 16 μg / mL (without glucose).

[0050] Fig. 22 This is the dynamic curve of the relative activity of Pseudomonas aeruginosa at an amikacin concentration of 16 μg / mL (with glucose). DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with the embodiments.

[0052] Overall embodiment A method for rapidly detecting bacterial antibiotic sensitivity comprises the following steps: (1) preparing a mixed solution of a bacterial liquid of the strain to be tested, antibiotics and glucose, and incubating the mixture to obtain a mixed reagent; (2) adding live bacteria stains and dead bacteria stains of different fluorescent colors into the mixed reagent for staining; (3) Detect the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain respectively, and judge the antibiotic sensitivity of the bacteria accordingly.

[0053] As a specific embodiment, the strain to be tested belongs to Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Acinetobacter baumannii, Stenotrophomonas maltophilia, Candida albicans, Streptococcus pneumoniae, Haemophilus influenzae, Oligomyces maltophilia, Burkholderia cepacia, Aspergillus or Mycobacterium tuberculosis.

[0054] As a specific embodiment, the antibiotic is an aminoglycoside antibiotic.

[0055] As a specific implementation, in step (1), in the mixed solution, the concentration of glucose is not less than 10 mM, and the concentration of antibiotics is 50 to 1600 μg / mL.

[0056] As a specific embodiment, in step (1), the incubation temperature is 35-37° C. and the time is 0.5-1 h.

[0057] As a specific implementation, in step (1), the process of preparing a mixed solution with the bacterial liquid of the test strain, antibiotics and glucose comprises the following steps: dissolving glucose and antibiotics in water respectively to prepare a glucose solution and an antibiotic solution; mixing the bacterial liquid of the test strain with an OD value of 0.1 to 0.3 with the glucose solution and the antibiotic solution to obtain a mixed solution.

[0058] As a specific embodiment, in step (2), the live bacteria stain is SYBR Green I, and the dead bacteria stain is propidium iodide.

[0059] As a specific implementation, in step (2), the dyeing temperature is 35-37° C. and the dyeing time is 20-30 min.

[0060] As a specific implementation, in step (2), the process of adding live bacteria stains and dead bacteria stains of different fluorescent colors into the mixed reagent includes the following steps: mixing the live bacteria stain, the dead bacteria stain and water to prepare a stain solution, and then mixing the stain solution with the mixed reagent.

[0061] As a specific implementation, step (3) specifically includes the following steps: (3.1) At a preset time point, the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain is detected, which are recorded as G1 and R1 respectively; the V1 value is calculated according to the following formula: V1 = (G1-3R1) / (G1+3R1); (3.2) Repeat steps (1) and (2), except that no antibiotics are added; at a preset time point, the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain is detected, which are recorded as G2 and R2, respectively; and the V2 value is calculated according to the following formula: V2 = (G2-3R2) / (G2+3R2); (3.3) Substitute V1 and V2 into the standard curve of bacterial survival rate and V value respectively, calculate the survival rate S1 and S2, and obtain the relative survival rate RS according to S1 / S2; when RS≥preset resistance breakpoint value, the strain to be tested is antibiotic-resistant; when RS≤preset sensitivity breakpoint value, the strain to be tested is antibiotic-sensitive. The method for obtaining the standard curve includes the following steps: take live bacteria and dead bacteria within the species to which the strain to be tested belongs, mix them in different proportions and prepare them into bacterial liquid, calculate the bacterial survival rate in each bacterial liquid according to the ratio between live bacteria and dead bacteria; use the live bacteria stain and the dead bacteria stain to stain each bacterial liquid, and then detect the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain, which are recorded as G and R respectively, and calculate the V value according to V=(G-3R) / (G+3R); draw the relationship curve between bacterial survival rate and V value.

[0062] As another specific implementation, step (3) specifically includes the following steps: (3.1) At a preset time point, the fluorescence intensities of the live bacteria stain and the dead bacteria stain are detected, recorded as G1 and R1 respectively, and the G1 / R1 value is calculated; (3.2) Repeat steps (1) and (2), except that no antibiotics are added; at a preset time point, the fluorescence intensities of the live bacteria stain and the dead bacteria stain are detected, recorded as G2 and R2, respectively, and the G2 / R2 value is calculated; (3.3) Substitute G1 / R1 and G2 / R2 into the standard curve of bacterial survival rate and G / R value respectively, calculate the survival rates S1 and S2, and obtain the relative survival rate RS according to S1 / S2; when RS≥the preset resistance breakpoint value, the strain to be tested is an antibiotic-resistant bacterium; when RS≤the preset sensitivity breakpoint value, the strain to be tested is an antibiotic-sensitive bacterium. The method for obtaining the standard curve includes the following steps: take live bacteria and dead bacteria within the species to which the strain to be tested belongs, mix them in different proportions and prepare them into bacterial liquids, calculate the bacterial survival rate in each bacterial liquid according to the ratio between live bacteria and dead bacteria; use the live bacteria stain and the dead bacteria stain to stain each bacterial liquid, and then detect the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain, record them as G and R respectively, and calculate the G / R value; draw a relationship curve between the bacterial survival rate and the G / R value.

[0063] As another specific implementation, step (3) specifically includes the following steps: (3.1) At a preset time point, the fluorescence intensities of the live bacteria stain and the dead bacteria stain are detected and recorded as G1 and R1, respectively, and the D1 value is calculated according to the following formula: D1 = G1 / R1; (3.2) Repeat steps (1) and (2), except that no antibiotics are added; at a preset time point, the fluorescence intensities of the live bacteria stain and the dead bacteria stain are detected, which are recorded as G2 and R2, respectively, and the D2 value is calculated according to the following formula: D2 = G2 / R2; (3.3) The relative survival rate RS is obtained according to D1 / D2; when RS≥the preset resistance breakpoint value, the strain to be tested is an antibiotic-resistant bacterium; when RS≤the preset sensitivity breakpoint value, the strain to be tested is an antibiotic-sensitive bacterium. Specific embodiments The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0065] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional raw materials and equipment in the field and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the field.

[0066] Example 1: Detection of sensitivity of Klebsiella pneumoniae to gentamicin 1.1 Drawing the V value-bacterial survival rate standard curve 1.1.1 Preparation of dead and live bacterial cultures The dead bacteria solution of Klebsiella pneumoniae is prepared by the following steps: (1) Centrifuge 1 mL of overnight culture (8000 rpm, 5 min) and discard the supernatant; (2) First add 1 mL of isopropanol (70%, diluted with ddH2O) and quickly resuspend after adding isopropanol (you can vortex); (3) Place the bacterial solution treated with isopropanol on a shaker for 20 min; (4) Centrifuge the bacterial solution after 20 min of treatment (8000 rpm, 5 min) and discard the supernatant; (5) After removing the supernatant, add 1 mL of PBS to resuspend the bacterial solution.

[0067] The live bacterial liquid of Klebsiella pneumoniae was directly used after shaking overnight without any treatment.

[0068] 1.1.2 Preparation of bacterial solution with different live bacteria percentages Prepare bacterial solutions with different live bacteria percentages according to the following formula: (1) 0% live bacteria: 0 μL live bacteria, 400 μL dead bacteria; (2) 20% live bacteria: 80 μL live bacteria, 320 μL dead bacteria; (3) 50% live bacteria: 200 μL live bacteria, 200 μL dead bacteria; (4) 80% live bacteria: 320 μL live bacteria, 80 μL dead bacteria; (5) 100% live bacteria: 400 μL live bacteria, 0 μL dead bacteria.

[0069] 1.1.3 Dye preparation The dye solution was prepared according to the following recipe: SYBR-Green (10000×) 6 μL, PI (20 mM) 2 μL, ddH2O 192 μL.

[0070] 1.1.4 Sample addition and determination Add 10 μL dye solution to each well of the 96-well plate, and then add 100 μL of bacterial solution with different proportions of live bacteria. Make 3 parallel duplicate wells for each group of bacterial solution and place it in 37°C away from light for 20 minutes. Finally, use 485 / 538 / 612 (the excitation wavelength and emission wavelength of SYBRGreen I are 485nm and 538nm, respectively, and the excitation wavelength and emission wavelength of PI are 485nm and 612nm, respectively) to measure the green (G) and red (R) fluorescence intensities. In order to reduce the effect of cell concentration on fluorescence intensity, the V value is introduced: V = (G-3R) / (G + 3R). Through analysis, the regression curve of bacterial survival rate and V value is obtained (the horizontal axis is the bacterial survival rate, and the vertical axis is the V value), and this curve is used as the standard curve.

[0071] The obtained standard curve is as follows Figure 1 shown.

[0072] 1.2 Experiment without glucose concentration gradient 1.2.1 Experimental methods Two gentamicin-resistant strains (referred to as R-Q1 and R-Q3) and one sensitive strain (referred to as S-XY) of Klebsiella pneumoniae were used for antibiotic sensitivity testing. The specific steps are as follows: (1) Weigh gentamicin powder, add sterile deionized water to fully dissolve, prepare 20000 μg / mL gentamicin stock solution, filter and sterilize with a 0.22 μm filter membrane, dilute the gentamicin stock solution with sterile water to 500, 1000, 2000, and 4000 μg / mL to obtain gentamicin solution; take the cultured Klebsiella pneumoniae resistant and sensitive strains, and adjust the OD to 0.2 with liquid culture medium; take a 96-well plate, add 90 μL of the adjusted bacterial solution to each well, and then add 10 μL of gentamicin solution to make the final concentration of gentamicin 50, 100, 200, and 400 μg / mL, and set up a control well without antibiotics. Each group has three replicates and incubate at 37°C for 30 minutes.

[0073] (2) Prepare SG / PI mixed dye in a dark environment: Mix SYBR Green I (10000×), PI (20mM) and sterile water in a volume ratio of 3:1:96. Take the plate after incubation and add 10μL of the mixed dye to each well. Incubate at 37°C for 20 minutes and read the data. Read every 10 minutes from zero for a total of seven times and record the data. Calculate the V values ​​of the experimental group (with antibiotics added) and the blank control (without antibiotics) based on the measured green and red fluorescence intensities, denoted as V1 and V2, respectively. The formulas are as follows: V1 = (G1-3R1) / (G1+3R1), V2 = (G2-3R2) / (G2+3R2), where G1 and R1 are the green fluorescence intensity and red fluorescence intensity measured in the experimental group, respectively, and G2 and R2 are the green fluorescence intensity and red fluorescence intensity measured in the control group, respectively. Substitute V1 and V2 into Figure 1 Calculate the bacterial survival rates S1 and S2 from the standard curve. Calculate the relative survival rate RS according to RS = S1 / S2. Draw a 1h dynamic curve with time as the horizontal axis and RS as the vertical axis.

[0074] 1.2.2 Experimental results The dynamic curves measured within 0 to 1 hour under different gentamicin concentrations and without glucose are shown in Figures 2 to 5 .

[0075] 1.3 Experiment with low glucose concentration gradient 1.3.1 Experimental methods Two gentamicin-resistant strains (referred to as R-Q1 and R-Q3) and one sensitive strain (referred to as S-XY) of Klebsiella pneumoniae were used for antibiotic sensitivity testing. The specific steps are as follows: (1) Weigh glucose, dissolve it fully with sterile deionized water, prepare a 100 mM glucose solution, filter it with a 0.22 μm filter membrane for sterilization and set aside; weigh gentamicin powder, add sterile water to dissolve it fully, prepare a 20000 μg / mL gentamicin stock solution, filter it with a 0.22 μm filter membrane for sterilization, dilute the gentamicin stock solution with sterile water to 500 and 1000 μg / mL to obtain a gentamicin solution; take the cultured Klebsiella pneumoniae resistant and sensitive strains, and adjust the bacterial OD to 0.2 with liquid culture medium; take a 96-well plate, add 90 μL of the adjusted bacterial solution to each well, then add 10 μL of gentamicin solution and 10 μL of glucose solution, so that the final concentration of gentamicin is about 50 and 100 μg / mL, and the final concentration of glucose is about 10 mM, and set up a control well without antibiotics, and repeat three times for each group, and incubate at 37°C for 30 min.

[0076] (2) Prepare SG / PI mixed dye in a dark environment: Mix SYBR Green I, PI and sterile water in a ratio of 3:1:96. Take the plate after incubation and add 10 μL of mixed dye to each well. Incubate at 37°C for 20 minutes and read the data. Read every 10 minutes from zero for a total of seven times and record the data. Calculate the V values ​​of the experimental group (with antibiotics added) and the blank control (without antibiotics) based on the measured green and red fluorescence intensities, denoted as V1 and V2, respectively. The formulas are as follows: V1 = (G1-3R1) / (G1+3R1), V2 = (G2-3R2) / (G2+3R2), where G1 and R1 are the green fluorescence intensity and red fluorescence intensity measured in the experimental group, respectively, and G2 and R2 are the green fluorescence intensity and red fluorescence intensity measured in the control group, respectively. Substitute V1 and V2 into Figure 1 Calculate the bacterial survival rates S1 and S2 from the standard curve. Calculate the relative survival rate RS according to RS = S1 / S2. Draw a 1h dynamic curve with time as the horizontal axis and RS as the vertical axis.

[0077] 1.3.2 Experimental Results Under the conditions of different gentamicin concentrations and the addition of glucose, the dynamic curves measured within 0 to 1 hour are shown in Figure 6 and Figure 7 .

[0078] 1.4 Experiment with high glucose concentration gradient 1.4.1 Experimental methods Two gentamicin-resistant strains (referred to as R-Q1 and R-Q3) and one sensitive strain (referred to as S-XY) of Klebsiella pneumoniae were used for antibiotic sensitivity testing. The specific steps are as follows: (1) Weigh glucose, fully dissolve it with sterile deionized water to prepare a 100 mM glucose solution, filter it with a 0.22 μm filter membrane to sterilize it and set it aside; weigh gentamicin powder, add sterile water to fully dissolve it, prepare a 20,000 μg / mL gentamicin mother solution, filter it with a 0.22 μm filter membrane to sterilize it, and dilute the gentamicin mother solution with sterile water to 2,000, 4,000, 8,000, and 16,000 μg / mL to obtain a gentamicin solution. ; Take the cultured resistant and sensitive strains of Klebsiella pneumoniae, and use liquid culture medium to adjust the bacterial OD to 0.2; Take a 96-well plate, add 90 μL of the adjusted bacterial solution to each well, and then add 10 μL of gentamicin solution and 10 μL of glucose solution, so that the final concentration of gentamicin is about 200, 400, 800, and 1600 μg / mL, and the final concentration of glucose is about 10 mM. At the same time, set up a control well without antibiotics, and repeat three times in each group. Incubate at 37°C for 30 minutes.

[0079] (2) Prepare SG / PI mixed dye in a dark environment: Mix SYBR Green I, PI and sterile water in a ratio of 3:1:96. Take the plate after incubation and add 10 μL of mixed dye to each well. Incubate at 37°C for 20 minutes and read the data. Read every 10 minutes from zero for a total of seven times and record the data. Calculate the V values ​​of the experimental group (with antibiotics added) and the blank control (without antibiotics) based on the measured green and red fluorescence intensities, denoted as V1 and V2, respectively. The formulas are as follows: V1 = (G1-3R1) / (G1+3R1), V2 = (G2-3R2) / (G2+3R2), where G1 and R1 are the green fluorescence intensity and red fluorescence intensity measured in the experimental group, respectively, and G2 and R2 are the green fluorescence intensity and red fluorescence intensity measured in the control group, respectively. Substitute V1 and V2 into Figure 1 Calculate the bacterial survival rates S1 and S2 from the standard curve. Calculate the relative survival rate RS according to RS = S1 / S2. Draw a 1h dynamic curve with time as the horizontal axis and RS as the vertical axis.

[0080] 1.4.2 Experimental Results Under the conditions of different gentamicin concentrations and the addition of glucose, the dynamic curves measured within 0 to 1 hour are shown in Figures 8 to 11 .

[0081] 1.5 Experimental results analysis and conclusions Figures 2 to 11 It can be seen that: without adding glucose, when the concentration of gentamicin is 400 μg / mL, in the dynamic curve ( Figure 5 ) could not distinguish the resistant strains (R-Q1, R-Q3) from the sensitive strains (S-XY). However, when glucose was added, both low and high concentrations of gentamicin were detected in the dynamic curve ( Figures 6 to 11 ) can better separate the resistant strains (R-Q1, R-Q3) from the sensitive strains (S-XY). The above results show that the addition of glucose can help to better distinguish between antibiotic-resistant strains and sensitive strains, thereby improving the accuracy of bacterial antibiotic sensitivity detection.

[0082] 1.6 Determination of preset time points and preset breakpoint values According to the method in 1.4.1, 30 gentamicin-resistant strains of Klebsiella pneumoniae and 20 gentamicin-sensitive strains of Klebsiella pneumoniae were tested at a gentamicin concentration of 400 μg / mL, and a dynamic curve of relative viability over time was drawn. According to the dynamic curves of all strains, the preset time point was determined to be 50 minutes, the preset sensitive breakpoint was 1.37, and the preset resistance breakpoint was 1.71. The relative viabilities of the 30 resistant strains at 50 minutes were all greater than 1.71, and the relative viabilities of the 20 sensitive strains at 50 minutes were all less than 1.37. When testing the gentamicin sensitivity of the strain to be tested, if the measured relative viability is ≤1.37 50 minutes after the staining is completed, the strain to be tested is a gentamicin-sensitive strain, and if the measured relative viability is ≥1.71, the strain to be tested is a gentamicin-resistant strain.

[0083] Example 2: Detection of the sensitivity of Staphylococcus aureus to gentamicin 2.1 Drawing the G / R value-bacterial survival rate standard curve The standard curve of Staphylococcus aureus is G / R value-bacterial survival rate, the horizontal axis is G / R value (ratio of green fluorescence intensity to red fluorescence intensity), and the vertical axis is bacterial survival rate. Fig.12 shown.

[0084] 2.2 Experiment without glucose concentration gradient 2.2.1 Experimental methods The antibiotic sensitivity test was performed using a gentamicin-resistant strain (1 strain, denoted as R-60) and a sensitive strain (2 strains, denoted as S-25923 and S-25) of Staphylococcus aureus. The specific steps are as follows: (1) Weigh gentamicin powder, add sterile deionized water to fully dissolve, prepare 20000 μg / mL gentamicin stock solution, filter and sterilize with a 0.22 μm filter membrane, dilute the gentamicin stock solution with sterile water to 80, 160, and 320 μg / mL, and obtain gentamicin solution; take the cultured resistant and sensitive Staphylococcus aureus strains, and adjust the OD to 0.2 with liquid culture medium; take a 96-well plate, add 90 μL of the adjusted bacterial solution to each well, and then add 10 μL of gentamicin solution to make the final concentration of gentamicin 8, 16, and 32 μg / mL, and set up a control well without antibiotics. Each group has three replicates and incubate at 37°C for 30 min.

[0085] (2) Prepare SG / PI mixed dye in a dark environment: Mix SYBR Green I (10000×), PI (20mM) and sterile water in a volume ratio of 3:1:96. Take the plate after incubation and add 10μL of mixed dye to each well. Incubate at 37℃ for 20min and read the data. Read every 10min from zero for a total of seven times and record the data. Calculate the G / R value (ratio of green fluorescence intensity to red fluorescence intensity) of the experimental group (adding antibiotics) and the blank control (not adding antibiotics) based on the measured green and red fluorescence intensities, and then substitute the results into Fig.12 Calculate the bacterial survival rates S1 and S2 from the standard curve. Calculate the relative survival rate RS according to RS = S1 / S2. Draw a 1h dynamic curve with time as the horizontal axis and RS as the vertical axis.

[0086] 2.2.2 Experimental results The dynamic curves measured within 0 to 1 hour under different gentamicin concentrations and without glucose are shown in Figures 13-15 .

[0087] 2.3 Experiment with glucose concentration gradient 2.3.1 Experimental methods The antibiotic sensitivity test was performed using a gentamicin-resistant strain (1 strain, denoted as R-60) and a sensitive strain (2 strains, denoted as S-25923 and S-25) of Staphylococcus aureus. The specific steps are as follows: (1) Weigh glucose, dissolve it fully with sterile deionized water, prepare a 100 mM glucose solution, filter it with a 0.22 μm filter membrane for sterilization and set aside; weigh gentamicin powder, add sterile water to dissolve it fully, prepare a 20000 μg / mL gentamicin stock solution, filter it with a 0.22 μm filter membrane for sterilization, dilute the gentamicin stock solution with sterile water to 80, 160, and 320 μg / mL to obtain a gentamicin solution; take the cultured resistant and sensitive Staphylococcus aureus strains, and adjust the bacterial OD to 0.2 with liquid culture medium; take a 96-well plate, add 90 μL of the adjusted bacterial solution to each well, then add 10 μL of gentamicin solution and 10 μL of glucose solution, so that the final concentration of gentamicin is about 8, 16, and 32 μg / mL, and the final concentration of glucose is about 10 mM, and set up a control well without antibiotics, and repeat three times for each group, and incubate at 37°C for 30 min.

[0088] (2) Prepare SG / PI mixed dye in a dark environment: Mix SYBR Green I, PI and sterile water in a ratio of 3:1:96. Take the plate after incubation and add 10 μL of mixed dye to each well. Incubate at 37°C for 20 min and read the data. Read every 10 min from zero for a total of seven times and record the data. Calculate the G / R value (ratio of green fluorescence intensity to red fluorescence intensity) of the experimental group (adding antibiotics) and the blank control (not adding antibiotics) based on the measured green and red fluorescence intensities, and then substitute the results into Fig.12 Calculate the bacterial survival rates S1 and S2 from the standard curve. Calculate the relative survival rate RS according to RS = S1 / S2. Draw a 1h dynamic curve with time as the horizontal axis and RS as the vertical axis.

[0089] 2.3.2 Experimental Results Under the conditions of different gentamicin concentrations and the addition of glucose, the dynamic curves measured within 0 to 1 hour are shown in Figures 16 to 18 .

[0090] 2.4 Experimental results analysis and conclusions from Figures 13 to 18 It can be seen that when the concentration of gentamicin was 8 μg / mL without the addition of glucose, the trends of the resistant strain R-60 and the sensitive strain S-25923 overlapped ( Fig.15 ), so it is impossible to accurately distinguish between resistant strains and sensitive strains. However, when glucose is added, resistant strains and sensitive strains can be well distinguished at all gentamicin concentrations. The above results show that the accuracy of bacterial antibiotic sensitivity detection can be improved by adding glucose.

[0091] Example 3: Detection of sensitivity of Pseudomonas aeruginosa to gentamicin 3.1 Experiment without glucose concentration gradient 3.1.1 Experimental methods The antibiotic sensitivity test was performed using gentamicin-resistant strains (2 strains, denoted as R-1 and R-2) and sensitive strains (5 strains, denoted as S-27853, S-10104, S-135, S-38 and S-22) of Pseudomonas aeruginosa. The specific steps are as follows: (1) Weigh gentamicin powder, add sterile deionized water to fully dissolve, prepare 20000 μg / mL gentamicin stock solution, filter and sterilize with a 0.22 μm filter membrane, dilute the gentamicin stock solution to 40 μg / mL with sterile water to obtain a gentamicin solution; take the cultured resistant and sensitive strains of Pseudomonas aeruginosa, adjust the OD to 0.2 with liquid culture medium; take a 96-well plate, add 90 μL of the adjusted bacterial solution to each well, and then add 10 μL of gentamicin solution to make the final concentration of gentamicin 4 μg / mL, and set up a control well without antibiotics. Each group has three replicates and incubate at 37°C for 30 min.

[0092] (2) Prepare SG / PI mixed dye in a dark environment: Mix SYBR Green I (10000×), PI (20mM) and sterile water in a volume ratio of 3:1:96. Take the plate after incubation and add 10μL of mixed dye to each well. Incubate at 37℃ for 20min and read the data. Read every 10min from zero for a total of seven times and record the data. Calculate the G / R value (ratio of green fluorescence intensity to red fluorescence intensity) of the experimental group (adding antibiotics) and the blank control (no antibiotics) based on the measured green and red fluorescence intensities, and record them as D1 and D2. Calculate the relative activity RS based on RS=D1 / D2. Draw a 1h dynamic curve with time as the horizontal axis and RS as the vertical axis.

[0093] 3.1.2 Experimental Results The dynamic curves measured within 0 to 1 hour under different gentamicin concentrations and without glucose are shown in Fig.19 .

[0094] 3.2 Experiment with glucose concentration gradient 3.2.1 Experimental methods The antibiotic sensitivity test was performed using gentamicin-resistant strains (2 strains, denoted as R-1 and R-2) and sensitive strains (5 strains, denoted as S-27853, S-10104, S-135, S-38 and S-22) of Pseudomonas aeruginosa. The specific steps are as follows: (1) Weigh glucose, dissolve it with sterile deionized water to prepare a 100 mM glucose solution, filter it with a 0.22 μm filter membrane for sterilization and set aside; weigh gentamicin powder, add sterile water to dissolve it, prepare a 20000 μg / mL gentamicin stock solution, filter it with a 0.22 μm filter membrane for sterilization, dilute the gentamicin stock solution to 40 μg / mL with sterile water to obtain a gentamicin solution; take the cultured resistant and sensitive strains of Pseudomonas aeruginosa, adjust the bacterial OD to 0.2 with liquid culture medium; take a 96-well plate, add 90 μL of the adjusted bacterial solution to each well, then add 10 μL of gentamicin solution and 10 μL of glucose solution, so that the final concentration of gentamicin is about 4 μg / mL and the final concentration of glucose is about 10 mM, and set up a control well without antibiotics, and repeat three times in each group, and incubate at 37°C for 30 min.

[0095] (2) Prepare SG / PI mixed dye in a dark environment: Mix SYBR Green I, PI and sterile water in a ratio of 3:1:96. Take the plate after incubation and add 10 μL of mixed dye to each well. Incubate at 37°C for 20 minutes and read the data. Read every 10 minutes from zero, for a total of seven times, and record the data. Calculate the G / R value (ratio of green fluorescence intensity to red fluorescence intensity) of the experimental group (adding antibiotics) and the blank control (no antibiotics) based on the measured green and red fluorescence intensities, and record them as D1 and D2. Calculate the relative activity RS based on RS=D1 / D2. Draw a 1h dynamic curve with time as the horizontal axis and RS as the vertical axis.

[0096] 3.2.2 Experimental Results Under the conditions of different gentamicin concentrations and the addition of glucose, the dynamic curves measured within 0 to 1 hour are shown in Fig. 20 .

[0097] 3.3 Experimental results analysis and conclusions from Fig.19 and Fig. 20 It can be seen that by adding glucose, the difference between resistant strains and sensitive strains can be made more significant, which will help improve detection accuracy.

[0098] Example 4: Detection of sensitivity of Pseudomonas aeruginosa to amikacin 4.1 Experiment without glucose concentration gradient 4.1.1 Experimental methods The antibiotic sensitivity test was performed using amikacin-resistant strains (1 strain, denoted as R-117) and sensitive strains (4 strains, denoted as S-27853, S-10104, S-135, and S-38) of Pseudomonas aeruginosa. The specific steps are as follows: (1) Weigh amikacin powder, add sterile deionized water to fully dissolve, prepare 20000 μg / mL amikacin stock solution, filter and sterilize with a 0.22 μm filter membrane, dilute the amikacin stock solution to 160 μg / mL with sterile water to obtain an amikacin solution; take the cultured Pseudomonas aeruginosa resistant and sensitive strains, adjust the OD to 0.2 with liquid culture medium; take a 96-well plate, add 90 μL of the adjusted bacterial solution to each well, and then add 10 μL of the amikacin solution to make the final concentration of amikacin 16 μg / mL, and set up a control well without antibiotics. Each group has three replicates and incubate at 37°C for 30 min.

[0099] (2) Prepare SG / PI mixed dye in a dark environment: Mix SYBR Green I (10000×), PI (20mM) and sterile water in a volume ratio of 3:1:96. Take the plate after incubation and add 10μL of mixed dye to each well. Incubate at 37℃ for 20min and read the data. Read every 10min from zero for a total of seven times and record the data. Calculate the G / R value (ratio of green fluorescence intensity to red fluorescence intensity) of the experimental group (adding antibiotics) and the blank control (no antibiotics) based on the measured green and red fluorescence intensities, and record them as D1 and D2. Calculate the relative activity RS based on RS=D1 / D2. Draw a 1h dynamic curve with time as the horizontal axis and RS as the vertical axis.

[0100] 4.1.2 Experimental Results The dynamic curves measured within 0 to 1 hour under different amikacin concentrations and without glucose are shown in Fig.21 .

[0101] 4.2 Experiment with glucose concentration gradient 4.2.1 Experimental methods The antibiotic sensitivity test was performed using amikacin-resistant strains (1 strain, denoted as R-117) and sensitive strains (4 strains, denoted as S-27853, S-10104, S-135, and S-38) of Pseudomonas aeruginosa. The specific steps are as follows: (1) Weigh glucose, dissolve it fully with sterile deionized water to prepare a 100 mM glucose solution, filter it with a 0.22 μm filter membrane for sterilization and set aside; weigh amikacin powder, add sterile water to dissolve it fully, prepare a 20000 μg / mL amikacin stock solution, filter it with a 0.22 μm filter membrane for sterilization, dilute the amikacin stock solution to 160 μg / mL with sterile water to obtain an amikacin solution; take the cultured Pseudomonas aeruginosa resistant and sensitive strains, and adjust the bacterial OD to 0.2 with liquid culture medium; take a 96-well plate, add 90 μL of the adjusted bacterial solution to each well, then add 10 μL of amikacin solution and 10 μL of glucose solution, so that the final concentration of amikacin is about 16 μg / mL and the final concentration of glucose is about 10 mM, and set up a control well without antibiotics, and repeat three times in each group, and incubate at 37°C for 30 min.

[0102] (2) Prepare SG / PI mixed dye in a dark environment: Mix SYBR Green I, PI and sterile water in a ratio of 3:1:96. Take the plate after incubation and add 10 μL of mixed dye to each well. Incubate at 37°C for 20 minutes and read the data. Read every 10 minutes from zero, for a total of seven times, and record the data. Calculate the G / R value (ratio of green fluorescence intensity to red fluorescence intensity) of the experimental group (adding antibiotics) and the blank control (no antibiotics) based on the measured green and red fluorescence intensities, and record them as D1 and D2. Calculate the relative activity RS based on RS=D1 / D2. Draw a 1h dynamic curve with time as the horizontal axis and RS as the vertical axis.

[0103] 4.2.2 Experimental Results Under the conditions of different amikacin concentrations and the addition of glucose, the dynamic curves measured within 0 to 1 hour are shown in Fig. 22 .

[0104] 4.3 Experimental results analysis and conclusions from Fig.21 and Fig. 22 It can be seen that compared with the case without adding glucose, the difference between resistant strains and sensitive strains is greater after adding glucose, which will help improve detection accuracy.

[0105] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for rapidly detecting bacterial antibiotic sensitivity, characterized in that: The following steps are involved: (1) preparing a mixed solution of the bacterial strain to be tested, antibiotics and glucose, and incubating the mixture to obtain a mixed reagent; (2) adding live bacteria stains and dead bacteria stains of different fluorescent colors into the mixed reagent for staining; (3) Detect the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain respectively, and judge the antibiotic sensitivity of the bacteria accordingly.

2. The method according to claim 1, characterized in that Step (3) specifically includes the following steps: (3.1) At the preset time point, the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain is detected, which are recorded as G1 and R1 respectively; the V1 value is calculated according to the following formula: V1=(G1-3R1) / (G1+3R1); (3.2) Repeat steps (1) and (2), except that no antibiotics are added; at a preset time point, the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain is detected, which are recorded as G2 and R2, respectively; and the V2 value is calculated according to the following formula: V2 = (G2-3R2) / (G2+3R2); (3.3) Substitute V1 and V2 into the standard curve of bacterial survival rate and V value respectively, calculate the survival rates S1 and S2, and obtain the relative survival rate RS based on S1 / S2; when RS ≥ the preset resistance breakpoint value, the test strain is antibiotic-resistant; when RS ≤ the preset sensitivity breakpoint value, the test strain is antibiotic-sensitive.

3. The method according to claim 1, characterized in that Step (3) specifically includes the following steps: (3.1) At the preset time point, the fluorescence intensity of the live bacteria stain and the dead bacteria stain is detected, recorded as G1 and R1 respectively, and the G1 / R1 value is calculated; (3.2) Repeat steps (1) and (2), except that no antibiotics are added; at a preset time point, the fluorescence intensities of the live bacteria stain and the dead bacteria stain are detected, recorded as G2 and R2, respectively, and the G2 / R2 value is calculated; (3.3) Substitute G1 / R1 and G2 / R2 into the standard curve of bacterial survival rate and G / R value respectively, calculate the survival rates S1 and S2, and obtain the relative survival rate RS based on S1 / S2; when RS ≥ the preset resistance breakpoint value, the test strain is antibiotic-resistant; when RS ≤ the preset sensitivity breakpoint value, the test strain is antibiotic-sensitive.

4. The method according to claim 1, characterized in that: Step (3) specifically includes the following steps: (3.1) At the preset time point, the fluorescence intensity corresponding to the live bacteria stain and the dead bacteria stain is detected, recorded as G1 and R1 respectively, and the D1 value is calculated according to the following formula: D1=G1 / R1; (3.2) Repeat steps (1) and (2), except that no antibiotics are added; at a preset time point, the fluorescence intensities of the live bacteria stain and the dead bacteria stain are detected, recorded as G2 and R2, respectively, and the D2 value is calculated according to the following formula: D2 = G2 / R2; (3.3) The relative survival rate RS is obtained based on D1 / D2; when RS≥the preset resistance breakpoint value, the strain to be tested is an antibiotic-resistant bacterium; when RS≤the preset sensitivity breakpoint value, the strain to be tested is an antibiotic-sensitive bacterium.

5. The method according to claim 1, characterized in that In step (1), the concentration of glucose in the mixed solution is not less than 10 mM.

6. The method according to claim 1, characterized in that In step (1), the concentration of the antibiotic in the mixed solution is 50-1600 μg / mL.

7. The method according to claim 1, characterized in that In step (1), the incubation temperature is 35-37°C and the time is 0.5-1 h.

8. The method according to claim 1, characterized in that In step (1), the process of preparing a mixed solution from the bacterial liquid of the test strain, antibiotics and glucose comprises the following steps: dissolving glucose and antibiotics in water respectively to prepare a glucose solution and an antibiotic solution; and mixing the bacterial liquid of the test strain, the glucose solution and the antibiotic solution to obtain a mixed solution.

9. The method according to claim 8, characterized in that The OD value of the bacterial solution of the strain to be tested is 0.1-0.

5.

10. The method according to claim 1, characterized in that In step (2), the live bacteria stain is SYBRGreen I, and the dead bacteria stain is propidium iodide.

Images