A drug sensitivity detection method

Abstract

The present invention discloses a drug sensitivity detection method, including setting a microplate, culturing the microplate, and regularly collecting fluorescence signals and analyzing data. Based on the characteristics of bacterial growth, the present invention utilizes the advantages of fluorescence interpretation to achieve real-time monitoring and analysis, can obtain drug sensitivity results faster, terminate monitoring in time, and has a short time and high efficiency; and has low requirements for equipment, and can achieve monitoring and analysis using a commonly used multifunctional plate reader with a luminescence detection function.

Description

Technical Field

[0001] The present invention relates to the technical field of drug sensitivity detection and analysis, and in particular to a drug sensitivity detection method. Background Art

[0002] Minimum inhibitory concentration (MIC) refers to the amount of drug contained in 1 mL of the highest dilution concentration of the drug that completely inhibits bacterial growth when the sensitivity of bacteria to the drug is determined by the broth dilution method. It is used to indicate the sensitivity of the tested bacteria to the drug. When using different types of drugs to perform drug sensitivity tests on different types of bacteria, due to differences in bacterial resistance mechanisms, growth rates and culture conditions, the time and mathematical methods for determining the results cannot be completely unified. In traditional methods, the MIC value is often determined by visually judging the turbidity after 18 to 24 hours of culture or by the results of ultraviolet absorption. It is impossible to obtain the accurate sensitivity of bacteria to the drug in real time during the culture process; therefore, the existing methods have long detection times, low efficiency and high costs. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a drug sensitivity detection method.

[0004] Compared with the existing analysis technology, the present invention is designed for long-term instrument monitoring of bacterial growth. By combining the bacterial growth curve and fluorescence change trend, real-time data processing and analysis are performed to give a determined MIC value within 6 hours.

[0005] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0006] A drug sensitivity detection method comprises the following steps:

[0007] (1) Setting up the microplate: The microplate is equipped with positive control wells, negative control wells, and experimental wells, which are numbered with the natural number i.

[0008] Positive control well number i=0;

[0009] The experimental wells are numbered sequentially with positive integers from 1 to n, i=1, i=2, ..., i=n;

[0010] Negative control well number i=n+1;

[0011] Wherein, n is a positive integer greater than 2;

[0012] The positive control well, negative control well and experimental well all contain the same concentration of fluorescent dye. The positive control well and the experimental well also contain the same concentration of bacteria. The experimental well also contains drugs. The drug concentrations of the experimental wells increase in sequence according to the numbers i=1, i=2, ..., i=n.

[0013] (2) Incubate the microplate, collect fluorescence signals regularly and analyze the data;

[0014] The steps to analyze the data are as follows:

[0015] Step 1,

[0016] Let the microplate reaction time t = 0h;

[0017] Step 2,

[0018] The fluorescence value F of each well at time t is measured t,i ;

[0019] The relative growth fluorescence value of each well at time t is K t,i ;

[0020] Among them, the relative growth fluorescence value of the experimental well at time t is K t,i =F t,i / F t,i-1 ,i=1,i=2,…,i=n,

[0021] The relative growth fluorescence value of the positive control well at time t is K t,0 =F t,0 / F t,0 =1,

[0022] The relative growth fluorescence value of the negative control well at time t is K t,n+1 =F t,n+1 / F t,n ;

[0023] Step 3,

[0024] The relative growth fluorescence value K of each well i at time t t,i Compared with 0.85, K appears at the same time t,i The number of micropores ≤0.85 is N;

[0025] Step 4,

[0026] When N=0, t=t+(0.2~0.5)h, return to step 2 and analyze the next time point;

[0027] When N=1, take K less than or equal to 0.85 t,i Assign the minimum value K min , that is, K min =K t,i ;

[0028] When N ≥ 2, take all K values ​​less than or equal to 0.85 t,i Compare i value, i max is the maximum value among them; if imax =n+1, then K min =K t,n+1 ; if i max ≠n+1, then take K t,imax+1 Compared with 0.98; if K t,imax+1 ≥0.98, then K t,imax Assign the minimum value K min , that is, K min =K t,imax ; if K t,imax+1 <0.98 and K t+0.25,imax+1 <K t,imax+1 , then K t,imax+1 Assign the minimum value K min , that is, K min =K t,imax+1 ; if K t,imax+1 <0.98 and K t+0.25,imax+1 ≥K t,imax+1 , then let t = t + (0.2~0.5) h, return to step 2, and analyze the next time point;

[0029] Get K min Then terminate the data collection;

[0030] Step 5,

[0031] Analysis K min Corresponding i value, if i=2~n, report MIC value = [drug concentration corresponding to well i]; if i=1, report MIC value ≤ [drug concentration corresponding to well i=1]; if i=n+1, report MIC value>[drug concentration corresponding to well i=n].

[0032] Preferably, the volume of the detection solution in each well of the microplate is equal.

[0033] Preferably, the fluorescent dye is an AIE fluorescent dye; the AIE fluorescent dye includes an AIE-1 molecule, and the AIE-1 molecule is selected from at least one of Formula 1;

[0034]

[0035] Formula 1

[0036] In Formula 1, R is independently selected from hydrogen, amino, alkyl having 1 to 20 carbon atoms, unsaturated hydrocarbon having 2 to 20 carbon atoms, heteroalkyl having 1 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, heterocycloalkyl having 2 to 20 carbon atoms, aryl having 6 to 30 carbon atoms, and heteroaryl having 5 to 30 carbon atoms;

[0037] R1 is selected from one of the following structures:

[0038]

[0039]

[0040] Z is an anion;

[0041] R2 is selected from one of the following structures:

[0042]

[0043] n1 is an integer from 1 to 5.

[0044] Further preferably, the AIE fluorescent dye further comprises an AIE-2 molecule, and the AIE-2 molecule is selected from at least one of Formula 2;

[0045]

[0046] Formula 2

[0047] In Formula 2, R1, R2, R3, R4, and R5 are each independently selected from a substituted or unsubstituted straight-chain alkyl group having 1 to 20 carbon atoms, a substituted or unsubstituted branched-chain alkyl group having 3 to 20 carbon atoms, or a substituted or unsubstituted cycloalkyl group having 3 to 20 carbon atoms;

[0048] n2 is an integer from 2 to 10;

[0049] X - It is an anion.

[0050] More preferably, the molar ratio of AIE-1 molecules to AIE-2 molecules in the AIE fluorescent dye is 1:(5-10).

[0051] Further preferably, the AIE-1 molecule is selected from the following:

[0052]

[0053] The AIE-2 molecule is selected from one of the following:

[0054] .

[0055] Preferably, the final concentration of the fluorescent dye is 5-12 μM.

[0056] Preferably, the bacterium is Staphylococcus aureus, Staphylococcus sciuri, Escherichia coli, Salmonella, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, MRSA, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pyogenes, Serratia or Stenotrophomonas maltophilia.

[0057] Preferably, the final concentration of the bacteria is any concentration between 0.5 McFarland turbidity and 0.5 McFarland turbidity diluted 1000 times; more preferably, it is any concentration between 0.5 McFarland turbidity diluted 100 to 500 times.

[0058] Preferably, the drug is levofloxacin, ceftazidime, ampicillin, imipenem, cefuroxime, gentamicin, tetracycline, piperacillin / tazobactam or co-trimoxazole, and the concentration gradient of the drug is set according to the CLSI standard.

[0059] Preferably, n is a positive integer from 3 to 20 (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

[0060] Preferably, the culture temperature is 28-37°C.

[0061] Preferably, the excitation wavelength of the fluorescent signal is 460 nm, and the emission wavelength is 560 nm.

[0062] Preferably, the monitoring time interval is 0.20~0.50h.

[0063] More preferably, the monitoring time interval is 0.25-0.50h.

[0064] Preferably, the MIC value is compared with the breakpoints of sensitive, intermediate, and resistant in the CLSI standard, and a report is output.

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] The present invention is based on the characteristics of bacterial growth and utilizes the advantages of fluorescence interpretation to achieve real-time monitoring and analysis, which can obtain drug sensitivity results more quickly and terminate monitoring in a timely manner with short time and high efficiency. It also has low requirements for equipment and can achieve monitoring and analysis using a commonly used multifunctional plate reader with luminescence detection function. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 The data processing analysis flow chart of the present invention;

[0068] Figure 2a The data processing result of Staphylococcus aureus ATCC 29213 in the levofloxacin susceptibility test in Example 1;

[0069] Figure 2bThe data processing result of Escherichia coli ATCC 25922 in the levofloxacin susceptibility test in Example 1;

[0070] Figure 3a The data processing result of clinically isolated Salmonella in the ceftazidime drug sensitivity test in Example 2;

[0071] Figure 3b The data processing result of clinically isolated Enterococcus faecium in the ceftazidime drug sensitivity test in Example 2;

[0072] Figure 4a The data processing result of Staphylococcus aureus ATCC 29213 in the ceftazidime susceptibility test in Example 3;

[0073] Figure 4b The data processing result of Staphylococcus aureus ATCC 29061 in the ceftazidime susceptibility test in Example 3;

[0074] Figure 5a The data processing result of Pseudomonas aeruginosa ATCC 27853 in the ceftazidime susceptibility test in Example 4;

[0075] Figure 5b This is the data processing result of clinically isolated Klebsiella pneumoniae in the levofloxacin susceptibility test in Example 4. DETAILED DESCRIPTION

[0076] The present invention is described in detail below in conjunction with examples, but the implementation manner and protection scope of the present invention are not limited to the following examples.

[0077] The culture medium of the embodiment of the present invention adopts Mueller-Hinton broth medium (MH broth medium).

[0078] The fluorescence detection instrument used in the embodiment of the present invention is a Bioteck multifunctional microplate reader.

[0079] Embodiment 1:

[0080] A drug sensitivity detection method comprises the following steps:

[0081] (1) Cultivate the bacteria to be tested and dilute to an appropriate concentration to obtain a bacterial solution.

[0082] The bacterial culture and dilution were carried out according to conventional methods in the art.

[0083] The specific method of this embodiment is as follows:

[0084] Prepare MH (Mueller-Hinton) broth medium, sterilize it with high-pressure steam at 121.0℃ for 15 min, and cool it to room temperature for use; culture Staphylococcus aureus ATCC 29213 and Escherichia coli ATCC 25922 on flat culture plates at 37℃ for 15-20 h, pick a single colony from the plate, prepare a 0.5 McFarland turbidity bacterial solution with the culture medium, and then dilute it 100 times and refrigerate it at 4℃ for use.

[0085] (2) The wells of the 96-well microplate are assigned i, i=n+1 is the negative control group (MH), i=0 is the positive control group, and i=1, 2, ..., n are the experimental groups, representing the drug concentrations increasing in sequence. The bacterial solution and the fluorescent dye in step (1) are mixed to obtain a mixed solution 1, and the culture medium and the fluorescent dye are mixed to obtain a mixed solution 2 (the concentration of the fluorescent dye is the same as that of the mixed solution 1). An equal amount of mixed solution 1 is added to the wells i=1, 2, ..., n of the experimental group and the well i=0 of the positive control group, and an equal amount of mixed solution 2 is added to the well i=n+1 of the negative control group. Then, an equal amount of the corresponding gradient diluted antimicrobial drug solution is added to the wells i=1, 2, ..., n of the experimental group, and an equal amount of solvent is added to the well i=0 of the positive control group and the well i=n+1 of the negative control group.

[0086] The n value, drug concentration and dosage of each reagent are determined by referring to conventional methods in the art; the fluorescent staining solution can accurately detect the bacterial concentration without any special requirements.

[0087] The specific parameters of this embodiment are as follows:

[0088] The fluorescent staining solution uses AIE drug sensitivity monitoring dual fluorescent staining solution, which consists of 10mM AIE-1 molecules ( ) aqueous solution with 10 mM AIE-2 molecules ( ) water solution in a volume ratio of 1:5 to prepare the dyeing solution.

[0089] The antimicrobial drug was levofloxacin (the diluent was water);

[0090] The dosage ratio of bacterial solution and fluorescent staining solution was 1 mL:1.2 μL;

[0091] Mixed solution 1 was added at 100 μL / well, and drug solution was added at 100 μL / well;

[0092] The final drug concentrations corresponding to wells i=1, 2, …, 10 are: i=1 is 0.25 μg / mL, i=2 is 0.5 μg / mL, i=3 is 1 μg / mL, i=4 is 2 μg / mL, i=5 is 4 μg / mL, i=6 is 8 μg / mL, i=7 is 16 μg / mL, i=8 is 32 μg / mL, i=9 is 64 μg / mL, and i=10 is 128 μg / mL;

[0093] All reagents and 96-well microplates were stored at 4°C before use.

[0094] (3) The prepared 96-well microplate is placed in a fluorescence reader (such as an ELISA reader) for incubation, and the fluorescence signal is regularly monitored and collected to analyze the data.

[0095] The bacteria are cultured according to conventional methods in the art, and the optimal growth temperature of the bacteria to be tested can be referred to; the time point for collecting the fluorescence signal is set according to conventional methods in the art; the excitation wavelength and emission wavelength are set according to the requirements of the fluorescent staining solution.

[0096] The specific parameters of this embodiment are as follows:

[0097] The culture temperature was 37°C;

[0098] Fluorescence signal excitation 460nm, emission 560nm;

[0099] The fluorescence signal was collected every 15 min.

[0100] The steps for analyzing the data are as follows (see the flowchart for Figure 1 ):

[0101] Methods for determining MIC

[0102] Step 1,

[0103] Let the microplate reaction time t = 0h; that is, the time point when the culture starts after the microplate detection solution is prepared;

[0104] Step 2,

[0105] The fluorescence value of each well at time t (t is the time point of each detection, in hours) is measured as F t,i , subscript t is the time point of each detection, in hours; as shown in Table 1.

[0106] Table 1

[0107]

[0108] K t,i is the relative growth fluorescence value of each well at time t; the relative growth fluorescence value of the experimental well at time t is K t,i =F t,i / F t,i-1 (i=1, 2, ..., n), the relative growth fluorescence value of the positive control well at time t is K t,0 =F t,0 / F t,0 =1 (i=0), the relative growth fluorescence value of the negative control well at time t is K t, n+1 =F t,n+1 / Ft,n (i=n+1); as shown in Table 2.

[0109] Table 2

[0110]

[0111] Step 3,

[0112] The relative growth fluorescence value K of each well at time t t,i Compare the size with 0.85 and record the K in different wells at the same time point t,i The number of ≤0.85 is N;

[0113] Step 4,

[0114] When N=0, it means that there is no obvious growth of bacteria, t=t+0.25, return to step 2, and perform data analysis at the next time point;

[0115] When N=1, take K less than or equal to 0.85 t,i Assign the minimum value K min , that is, K min =K t,i ;

[0116] When N ≥ 2, take all K values ​​less than or equal to 0.85 t,i Compare i value, i max is the maximum value among them; if i max =n+1, then K min =K t,n+1 ; if i max ≠n+1, then take K t,imax+1 Compared with 0.98, if K t,imax+1 ≥0.98, then K t,imax Assign the minimum value K min , that is, K min =K t,imax , i=i max ; if K t,imax+1 <0.98 and K t+0.25,imax+1 <K t,imax+1 , then K t,imax+1 Assign the minimum value K min , that is, K min =K t,imax+1 , i=i max+1 ; if K t,imax+1 <0.98 but K t+0.25,imax+1 ≥K t,imax+1 , then t=t+0.25, return to step 2 and analyze the next time point;

[0117] Get K min Then terminate the data collection.

[0118] Step 5,

[0119] Analysis K min Corresponding i value, if i=2~n, report MIC value = [drug concentration corresponding to well i]; if i=1, report MIC value ≤ [drug concentration corresponding to well i=1]; if i=n+1, report MIC value>[drug concentration corresponding to well i=n].

[0120] Methods for determining sensitivity

[0121] Compare the MIC value with the breakpoints of sensitive, intermediate, and resistant in the CLSI standard and output a report.

[0122] (4) Perform drug susceptibility tests on the same drugs and bacteria according to the standard methods of CLSI, and compare the results interpreted according to the standard methods with the results of step (4).

[0123] Example 2

[0124] The differences between this embodiment and Embodiment 1 are as follows, and the parts not mentioned are the same as those in Embodiment 1.

[0125] In step (1), the bacteria to be tested are Salmonella and Enterococcus faecium;

[0126] In step (2), the drug is ceftazidime;

[0127] In step (2), i=1 is 0.5 μg / mL, i=2 is 1 μg / mL, i=3 is 2 μg / mL, i=4 is 4 μg / mL, i=5 is 8 μg / mL, i=6 is 16 μg / mL, i=7 is 32 μg / mL, i=8 is 64 μg / mL, i=9 is 128 μg / mL, and i=10 is 256 μg / mL.

[0128] Example 3

[0129] The differences between this embodiment and Embodiment 1 are as follows, and the parts not mentioned are the same as those in Embodiment 1.

[0130] In step (1), the bacteria to be tested are Staphylococcus aureus ATCC29213 and Staphylococcus sciuri ATCC29061;

[0131] In step (2), the drug is ceftazidime;

[0132] In step (2), i=1 is 0.5 μg / mL, i=2 is 1 μg / mL, i=3 is 2 μg / mL, i=4 is 4 μg / mL, i=5 is 8 μg / mL, i=6 is 16 μg / mL, i=7 is 32 μg / mL, i=8 is 64 μg / mL, i=9 is 128 μg / mL, and i=10 is 256 μg / mL.

[0133] Example 4

[0134] The differences between this embodiment and Embodiment 1 are as follows, and the parts not mentioned are the same as those in Embodiment 1.

[0135] In step (1), the bacteria to be tested are Pseudomonas aeruginosa ATCC 27853 and clinically isolated Klebsiella pneumoniae;

[0136] In step (2), the drugs are ceftazidime and levofloxacin respectively;

[0137] In step (2), i=1 corresponding to ceftazidime is 0.5 μg / mL, i=2 is 1 μg / mL, i=3 is 2 μg / mL, i=4 is 4 μg / mL, i=5 is 8 μg / mL, i=6 is 16 μg / mL, i=7 is 32 μg / mL, i=8 is 64 μg / mL, i=9 is 128 μg / mL, and i=10 is 256 μg / mL; i=1 corresponding to levofloxacin is 0.25 μg / mL, i=2 is 0.5 μg / mL, i=3 is 1 μg / mL, i=4 is 2 μg / mL, i=5 is 4 μg / mL, i=6 is 8 μg / mL, i=7 is 16 μg / mL, i=8 is 32 μg / mL, i=9 is 64 μg / mL, and i=10 is 128 μg / mL.

[0138] Example Results Analysis

[0139] The data analysis process of the present invention can be performed through a program to directly obtain the final result; in order to more clearly illustrate the technical solution and effect of the present invention, the results are analyzed in combination with the process data below.

[0140] (1) The data processing results of the drug sensitivity test of Staphylococcus aureus ATCC 29213 to levofloxacin in Example 1 are as follows: Figure 2a ; The data processing results of the drug sensitivity test of Escherichia coli ATCC 25922 to levofloxacin are as follows Figure 2b .

[0141] from Figure 2a It can be seen that when t is less than 5, K t,i Compared with 0.85, N=0; continue to monitor at the next time point, when t=5, K t,i Compared with 0.85, N=1, K less than or equal to 0.85 t,i Corresponding to K at 5h 5,1=0.81791 is the minimum value Kmin, corresponding to i=1, the bacteria grow before the lowest drug concentration of 0.25μg / mL in the gradient, and the output MIC value is ≤ [drug concentration corresponding to i=1], that is, MIC≤0.25μg / mL. Compared with the corresponding CLSI standard, the output judgment is sensitive. It only takes 5 hours to get the drug sensitivity test results.

[0142] from Figure 2b It can be seen that when t is less than 2.75, K t,i Compared with 0.85, N=0; continue to monitor at the next time point, when t=2.75, K t,i Compared with 0.85, N=1, at this time K less than or equal to 0.85 t,i Corresponding to K at 2.75h 2.75,1 =0.71137 is the minimum value K min , the bacteria that have grown before the lowest drug concentration of 0.25 μg / mL in the gradient corresponding to i=1 have output MIC value ≤ [drug concentration corresponding to i=1], that is, MIC≤0.25μg / mL, compared with the corresponding CLSI standard, and the output judgment is sensitive; it only takes 2.75h ​​to obtain the drug sensitivity test results.

[0143] The above results are consistent with the MIC results obtained by the standard method in Table 3. Table 3 shows the MIC values ​​of Staphylococcus aureus ATCC 29213 in the levofloxacin susceptibility test implemented by the standard method and the MIC values ​​of Escherichia coli ATCC 25922 in the levofloxacin susceptibility test.

[0144] Table 3

[0145]

[0146] (2) The data processing results of the Salmonella susceptibility test to ceftazidime in Example 2 are as follows: Figure 3a ; The data processing results of the drug sensitivity test of Enterococcus faecium to ceftazidime are as follows Figure 3b .

[0147] from Figure 3a It can be seen that when t is less than 2.75, K t,i Compared with 0.85, N=0; continue to monitor at the next time point, when t=2.75, K t,i Compared with 0.85, N=1, at this time K less than or equal to 0.85 t,i Corresponding to K at 2.75h 2.75,n+1 =K 2.75,11 =0.75058 is the minimum value K min, corresponding to the growth of bacteria at a drug concentration of 256 μg / mL before the gradient of i=11, the output MIC value is > [drug concentration corresponding to i=10], that is, MIC>256μg / mL, compared with the corresponding CLSI standard, and the output judgment is drug-resistant; it only takes 2.75h ​​to obtain the drug sensitivity test results.

[0148] from Figure 3b It can be seen that when t is less than 4, K t,i Compared with 0.85, N=0; continue to monitor at the next time point, when t=4, K t,i Compared with 0.85, N=1, at this time K less than or equal to 0.85 t,i Corresponding to 4h K 4,n+1 =K 4,11 =0.81025 is the minimum value K min , corresponding to the growth of bacteria at a drug concentration of 256 μg / mL before the gradient of i=11, the output MIC value is > [drug concentration corresponding to i=10], that is, MIC>256 μg / mL, compared with the corresponding CLSI standard, and the output judgment is drug resistance; it only takes 4 hours to obtain the drug sensitivity test results.

[0149] The above results are consistent with the MIC results obtained by the standard method in Table 4. Table 4 shows the MIC values ​​of clinically isolated Salmonella in the ceftazidime susceptibility test implemented according to the standard method and the MIC values ​​of clinically isolated Enterococcus faecium in the ceftazidime susceptibility test.

[0150] Table 4

[0151]

[0152] (3) The data processing results of the drug sensitivity test of Staphylococcus aureus ATCC 29213 to ceftazidime in Example 3 are as follows: Figure 4a ; The data processing results of the drug sensitivity test of Staphylococcus aureus ATCC 29061 to ceftazidime are as follows Figure 4b .

[0153] from Figure 4a It can be seen that when t is less than 6, K t,i Compared with 0.85, N=0; continue to monitor at the next time point, when t=6, K t,i Compared with 0.85, N=1, at this time K less than or equal to 0.85 t,i Corresponding to 6h K 6,5 =0.81639 is the minimum value K min, corresponding to i=5 gradient drug concentration before 8μg / mL, the bacteria grow, and the output MIC value = [drug concentration corresponding to i], that is, MIC=8μg / mL. CLSI has no corresponding standard, and the MIC value is directly output; it only takes 6 hours to get the drug sensitivity test results.

[0154] from Figure 4b It can be seen that when t is less than 4, K t,i Compared with 0.85, N=0; continue to monitor at the next time point, when t=4, K t,i Compared with 0.85, N=1, at this time K less than or equal to 0.85 t,i Corresponding to 4h K 4,6 =0.81982 is the minimum value K min , corresponding to i=6 gradient drug concentration before 16 μg / mL, the bacteria grew, and the output MIC value = [drug concentration corresponding to i], that is, MIC=16μg / mL. CLSI has no corresponding standard, and the MIC value is directly output; it only takes 4 hours to get the drug sensitivity test results.

[0155] The above results are consistent with the MIC results obtained by the standard method in Table 5. Table 5 shows the MIC values ​​of Staphylococcus aureus ATCC 29213 in the ceftazidime susceptibility test implemented according to the standard method and the MIC values ​​of Staphylococcus aureus ATCC 29061 in the ceftazidime susceptibility test.

[0156] Table 5

[0157]

[0158] (4) The data processing results of the ceftazidime susceptibility test of Pseudomonas aeruginosa ATCC 27853 in Example 4 are as follows: Figure 5a ; The data processing results of the drug sensitivity test of Klebsiella pneumoniae to levofloxacin are as follows Figure 5b .

[0159] from Figure 5a It can be seen that when t is less than 4.75, K t,i Compared with 0.85, N=0; continue to monitor at the next time point, when t=4.75, K t,i Compared with 0.85, N=1, at this time K less than or equal to 0.85 t,i Corresponding to K at 4.75h 4.75,3 =0.80807 is the minimum value K min , corresponding to i=3 gradient drug concentration before 2μg / mL, the bacteria grew, and the output MIC value = [drug concentration corresponding to i], that is, MIC=2μg / mL, compared with the corresponding CLSI standard, and the output judgment was sensitive; it only takes 4.75h to get the drug sensitivity test results.

[0160] from Figure 5b It can be seen that when t is less than 3.25, K t,i Compared with 0.85, N=0; continue to monitor at the next time point, when t=3.25, K t,i Compared with 0.85, N=2, K less than or equal to 0.85 t,i Corresponding to K at 3.25h 3.25,8 =0.79620 and K 3.25,9 =0.80195, where i=8 corresponds to 32μg / mL, i=9 corresponds to 64μg / mL, and the K value of the high concentration of 64μg / mL and the concentration gradient of the high concentration is taken. t,imax+1 K 3.25,10 Compared with 0.98, K 3.25,10 =0.93044<0.98 and K 3.50,10 =0.89401<K 3.25,10 =0.93044, then take K 3.25,10 is the minimum value K min , corresponding to i=10 gradient drug concentration before 128μg / mL, the bacteria grew, and the output MIC value = [drug concentration corresponding to i], that is, MIC=128μg / mL, compared with the corresponding CLSI standard, and the output judgment was drug resistance; it only takes 3.5h to get the drug sensitivity test results.

[0161] The above results are consistent with the MIC results obtained by the standard method in Table 6.

[0162] Table 6

[0163]

[0164] Table 6 shows the MIC values ​​of Pseudomonas aeruginosa ATCC 27853 in the ceftazidime susceptibility test and the MIC values ​​of Klebsiella pneumoniae in the levofloxacin susceptibility test implemented according to the standard method.

Claims

1. A drug sensitivity detection method, characterized in that: The following steps are involved: (1) Setting up the microplate: The microplate is equipped with positive control wells, negative control wells, and experimental wells, which are numbered with the natural number i. Positive control well number i=0; The experimental wells are numbered sequentially with positive integers from 1 to n, i=1, i=2, ..., i=n; Negative control well number i=n+1; Wherein, n is a positive integer greater than 2; The positive control well, negative control well and experimental well all contain the same concentration of fluorescent dye. The positive control well and the experimental well also contain the same concentration of bacteria. The experimental well also contains drugs. The drug concentrations of the experimental wells increase in a gradient order from number i=1, i=2, ..., i=n. (2) Incubate the microplate, collect fluorescence signals regularly and analyze the data; The steps to analyze the data are as follows: Step 1, Let the microplate reaction time t = 0h; Step 2, The fluorescence value F of each well at time t is measured t,i ; The relative growth fluorescence value of each well at time t is K t,i ; Among them, the relative growth fluorescence value of the experimental well at time t is K t,i =F t,i / F t,i-1 ,i=1,i=2,…,i=n, The relative growth fluorescence value of the positive control well at time t is K t,0 =F t,0 / F t,0 =1, The relative growth fluorescence value of the negative control well at time t is K t,n+1 =F t,n+1 / F t,n ; Step 3, The relative growth fluorescence value K of each well i at time t t,i Compared with 0.85, K appears at the same time t,i The number of micropores ≤0.85 is N; Step 4, When N=0, let t=t+(0.2~0.5)h, return to step 2, and analyze the next time point; When N=1, take K less than or equal to 0.85 t,i Assign the minimum value K min , that is, K min =K t,i ; When N ≥ 2, take all K values ​​less than or equal to 0.85 t,i Compare i value, i max is the maximum value among them; if i max =n+1, then K min =K t,n+1 ; if i max ≠n+1, then take K t,imax+1 Compared with 0.98; if K t,imax+1 ≥0.98, then K t,imax Assign the minimum value K min , that is, K min =K t,imax ; if K t,imax+1 <0.98 and K t+0.25,imax+1 <K t,imax+1 , then K t,imax+1 Assign the minimum value K min , that is, K min =K t,imax+1 ; if K t,imax+1 <0.98 and K t+0.25,imax+1 ≥K t,imax+1 , then let t = t + (0.2~0.5) h, return to step 2, and analyze the next time point; Get K min Then terminate the data collection; Step 5, Analysis K min Corresponding i value, if i=2~n, report MIC value = [drug concentration corresponding to well i]; if i=1, report MIC value ≤ [drug concentration corresponding to well i=1]; if i=n+1, report MIC value>[drug concentration corresponding to well i=n]; The fluorescent dye is an AIE fluorescent dye; the AIE fluorescent dye includes an AIE-1 molecule, and the AIE-1 molecule is selected from at least one of Formula 1; Formula 1 In Formula 1, R is each independently selected from hydrogen or methoxy; R1 is selected from , Z is an anion; R2 is selected from or , n1 is 1 or 2.

2. The drug sensitivity detection method according to claim 1, characterized in that: The AIE-1 molecule is selected from , At least one of .

3. The drug sensitivity detection method according to claim 1, characterized in that: The AIE fluorescent dye further includes an AIE-2 molecule; the AIE-2 molecule is selected from at least one of Formula 2; the molar ratio of the AIE-1 molecule to the AIE-2 molecule is 1:(5-10); Formula 2 In Formula 2, R1 and R2 are each independently selected from methyl or ethyl; R3, R4, and R5 are selected from methyl; n2 is 2 or 3; X - It is an anion.

4. The drug sensitivity detection method according to claim 3, characterized in that: The AIE-2 molecule is selected from , At least one of .

5. The drug sensitivity detection method according to claim 1, characterized in that: The bacteria are Staphylococcus aureus, Staphylococcus sciuri, Escherichia coli, Salmonella, Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, MRSA, Enterococcus faecium, Streptococcus agalactiae, Streptococcus pyogenes, Serratia marcescens or Stenotrophomonas maltophilia; the drugs are levofloxacin, ceftazidime, ampicillin, imipenem, cefuroxime, gentamicin, tetracycline, piperacillin / tazobactam or co-trimoxazole.

6. The drug sensitivity detection method according to claim 1, characterized in that: The final concentration of the fluorescent dye is 5~12μM; the final concentration of bacteria is any concentration between 0.5 McFarland turbidity and 0.5 McFarland turbidity diluted 1000 times; the concentration gradient of the drug is set according to the CLSI standard.

7. The drug sensitivity detection method according to claim 1, characterized in that: n is a positive integer ranging from 3 to 20.

8. The drug sensitivity detection method according to claim 1, characterized in that: The culture temperature is 28~37℃; the excitation wavelength of the fluorescence signal is 460nm, and the emission wavelength is 560nm.

9. The drug sensitivity detection method according to claim 1, characterized in that: Compare the MIC value with the breakpoints of sensitive, intermediate, and resistant in the CLSI standard and output a report.

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