Method and culture medium for detecting drug resistance of living cells in vitro

Abstract

The present invention relates to the field of rapid detection of cell drug resistance, and discloses a method for rapid, sensitive and accurate detection of drug resistance of living cells, comprising the following specific steps: incubating the living cells to be detected before detecting them, and continuously detecting the concentration of markers at different time points after incubation to achieve the above detection; adding a detection reagent mixture that can be used by the living cells to be detected and a test compound for drug resistance detection before incubation; the incubation refers to adding the living cells to be detected to a culture solution of a determined concentration for cultivation. The advantages of the present invention are that it can quickly and sensitively detect the growth of living cells, including the detection of their cell drug resistance, discover "hidden" drug resistance that cannot be detected by current methods, prevent super bacteria and drug-resistant tumors, and develop efficient new drugs, etc., which has high application value.

Description

Technical Field

[0001] The present invention relates to the field of rapid and highly sensitive detection of cell drug resistance, and in particular to a method for detecting partial cell drug resistance and corresponding detection compounds. Background Art

[0002] The emergence of a large number of super bacteria and tumor resistance is a public health problem that urgently needs to be solved. In addition to new drugs, a method that can detect drug resistance early may be able to solve the emergence of super bacteria and tumor resistance from the source.

[0003] Currently, commonly used laboratory testing methods are divided into three main categories: bacteriology, immunology, and molecular biology. Modern antimicrobial susceptibility testing in clinical microbiology in my country currently has some challenges, such as long testing times, inaccurate results that affect diagnostic outcomes, and the high cost of certain identification equipment, which makes it unaffordable for some small and medium-sized hospitals.

[0004] Currently, the main methods for bacterial drug sensitivity testing include the qualitative determination paper diffusion method (KB method), the quantitative determination dilution method, the E method and the fully automatic drug sensitivity instrument method, which generally take one day to produce results.

[0005] At present, there are many methods that can be used to detect tuberculosis in the laboratory and clinically. The most commonly used ones are sputum smear microscopy, Roche solid culture method, BACTEC MGIT960 system culture method, real-time fluorescence quantitative polymerase chain reaction (PCR), etc.

[0006] Existing methods for drug susceptibility testing of Mycobacterium tuberculosis have the following problems: sputum smears have high requirements for preparation, and microscopic examination needs to be completed manually, which greatly increases the workload of clinical laboratory personnel and is prone to omissions; due to the characteristics of the growth of Mycobacterium tuberculosis itself, the solid culture method is time-consuming; the use of mycobacterium rapid culture instruments and real-time fluorescent quantitative polymerase chain reaction (PCR) can shorten the culture time and is convenient to operate, but the bacteria are not viable, the accuracy is low, and the price is high, which is not conducive to promotion.

[0007] The problems existing in the above-mentioned existing technologies are partly due to the limitations of the detection methods themselves. However, in the process of cell resistance detection, the characteristics of the markers that need to be cultured to obtain the required concentration lead to the disadvantages of the above-mentioned detection methods such as long time required and low accuracy.

[0008] Therefore, it is necessary to develop better detection methods based on existing technologies and combined with the characteristics of cell-based drug sensitivity testing. Summary of the Invention

[0009] The present invention addresses the shortcoming of the prior art in that drug sensitivity testing of cells takes a long time, and provides a method for rapidly, sensitively and accurately detecting drug resistance of living cells.

[0010] During the experiment, we found that the existing detection methods seemed unable to detect "some" drug resistance of cells. Cells that tested negative (no drug resistance or low drug resistance) after being tested by existing detection methods were still found to have drug resistance in subsequent applications. Although the current theory cannot accurately explain the mechanism of the existence of this type of cell resistance, by changing the existing detection methods, we found that some new rapid detection methods seem to be able to achieve the effect of detecting the above-mentioned "undetectable" cell resistance by raising the "lower limit" of the cell resistance detection index. Through these new rapid detection methods, cells that tested negative using existing detection methods can be detected with more obvious drug resistance. At the same time, these new rapid detection methods have the advantages of fast, sensitive and accurate detection compared to existing detection methods.

[0011] In addition, it has been found in actual applications that the above-mentioned rapid detection method also has high detection accuracy and can perform quantitative detection. It can reliably detect "hidden" drug resistance (10-30%) that cannot be detected by various current endpoint methods or turbidity methods. It can prevent the emergence of super bacteria and tumor resistance, and improve the standards for new drug evaluation, which is conducive to the development of truly effective drugs.

[0012] To achieve the above purpose, the present invention can adopt the following technical solutions:

[0013] A method for rapidly, sensitively, and accurately detecting drug resistance in living cells comprises the following specific steps: incubating the living cells before testing, and detecting the concentration of a marker after incubation to achieve the aforementioned detection; adding a detection reagent mixture that can be utilized by the living cells and a test compound for drug resistance detection before incubation; incubating the living cells in a culture medium of a predetermined concentration; drug resistance refers to the inability of the test compound to completely inhibit the growth of the living cells. Alternatively, the marker concentration can be continuously detected at predetermined time intervals during the detection of the marker concentration after incubation.

[0014] The method also includes the following steps: under optimized conditions, the detection reagent mixture can use a rate method to track the growth of cells after administration and detect cell resistance; the rate method refers to continuously detecting the change of the concentration signal of the marker over time at predetermined time intervals; cell resistance includes resistance of cell growth higher than 20% that can be detected by conventional methods, and resistance of cell growth lower than 20% that cannot be detected by conventional methods.

[0015] It should be noted that the present application refers to: drug resistance (R resistance: the cell growth rate is more than 20% of the non-drug-treated cells), sensitivity (S sensitivity: the cell growth rate is less than 2% of the non-drug-treated cells), and partial drug resistance I (PR; the cell growth rate is 2-20% of the non-drug-treated cells) with a cell growth rate of less than 20% that cannot be detected by conventional methods.

[0016] It also includes a concentration adjustment step: adjusting the concentration of the cell culture medium used for incubation before incubation (including achieving the purpose of changing the number of cells per unit volume in the culture medium) so that the living cells to be detected can roughly achieve a more optimal growth rate. After the concentration adjustment step is completed, the concentration of the cells in the culture medium is diluted (including reducing the number of cells per unit volume in the culture medium), and the dilution range is 2-4000 times of McFarland 0.5 dilution. In the case where the in vitro living cells are fast-growing bacteria, a 40-fold dilution is used. It also includes an accelerated culture step: after the culture medium concentration that can achieve the optimal growth rate of the living cells to be detected is obtained, the concentration of the culture medium is increased during the incubation process.

[0017] The term "utilization" refers to the ability of the detection reagent mixture to enter the living cells to be detected. The term "utilization" refers to the ability of the detection reagent mixture to react with at least a portion of the components of the cells to be detected. The marker is a product having a color. The detection reagent mixture includes a compound that can be oxidized or reduced to a product that develops a color within the living cells to be detected. The detection reagent mixture can be selected from EZMTT and an electron coupling agent.

[0018] The method further includes an inhibition rate detection step: while incubating the living cells to be detected, adding an equal amount of the living cells to be detected to a culture medium of the same concentration to prepare a positive control group, and using a culture medium of the same concentration without the living cells to be detected to prepare a negative control group; further comparing the concentration of the detection marker after incubation with the positive and negative control groups to obtain the inhibition rate of the test compound on the living cells to be detected;

[0019] The inhibition rate is calculated as 1-(compound-negative) / (positive-negative). A step for identifying cell resistance is also included: specifically, detecting changes in cell growth rate after drug administration and comparing them with corresponding wild-type cells (compared under drug administration (0% growth) and no drug administration (100% growth) conditions).

[0020] By continuously detecting the concentration of the marker, the inhibitory effect of the antibacterial and bactericidal agents on the cells is detected. The living cells include fungi, bacteria, actinomycetes, chlamydia, mycoplasma, rickettsia, spirochetes, infectious organisms, various drug-resistant and drug-intolerant cells, and animal and human cells that can produce NAD(P)H.

[0021] The culture medium of slow-growing cells of Mycobacterium tuberculosis also includes an antibacterial agent; slow-growing cells refer to cells with a doubling time of more than 12 hours.

[0022] In addition, the application also discloses a method for incubating living cells to be detected, including a culture medium and a detection reagent mixture; the detection reagent mixture includes a compound having the following structural formula

[0023]

[0024] in,

[0025] A1 is and connected aromatic or heterocyclic groups;

[0026] A2 is and connected aromatic or heterocyclic groups;

[0027] A3 is and The connected phenyl groups;

[0028] R1 is H or at least one R1 substituent connected to A1, wherein the R1 substituents are independently selected from -NO2, -SO3H, -SO2NH2, halogen elements, and heterocyclic groups and alkyl groups fused to A1;

[0029] R2 is H or at least one R2 substituent connected to A2, wherein the R2 substituents are independently selected from NO2, methoxy, alkyl, phenyl and its derivatives connected through an azide group, halogen atoms, sulfonic acid groups and their salts, and phenyl or heterocyclic groups fused to A2;

[0030] R3 is an R3 substituent connected to A3; the R3 substituents are independently selected from sulfonic acid groups, carboxylic acid groups, substituted sulfonic acid groups, and substituted carboxylic acid groups;

[0031] The heterocyclic group includes 1-2 heteroatoms, and the heteroatoms are independently selected from Se, N, S or O.

[0032] The living cells that can be used include fungi, bacteria, actinomycetes, chlamydia, mycoplasma, rickettsia, spirochetes, infectious organisms, various drug-resistant and non-drug-resistant cells, and animal and human cells, etc., but dead cells cannot be detected under the steps recorded in this method because the number of dead living cells is zero and the detection reagent mixture cannot be used; in addition, although the detection instruments used in the embodiments of the present application include instruments that can detect the absorbance of the solution or similar instruments that can detect the color of the solution, these instruments can be existing instruments, or instruments with the detection function required by this application. The incubation refers to the cultivation of the living cells to be detected by culture medium. The general result of the cultivation is that the number of the living cells to be detected increases, but the technical solution recorded in this application does not require that the living cells that survive at the time of adding the detection reagent mixture before incubation continue to survive after the incubation is completed, nor does it require that the number of living cells after incubation absolutely increase. Generally, the technical solution of the present application only requires that the detection reagent mixture can be utilized by living cells, and the purpose of rapid detection can be achieved by detecting the product after the cells are utilized. The culture medium used in the incubation process can be any mixed solution that can be used for the growth of living cells to be detected, such as MH broth for bacteria, RPMI for fungi, and 7H9 culture medium for tuberculosis. The detection reagent mixture used in the embodiments of the present application is EZMTT, and other water-soluble tetrazolium disulfonate salts WST series, WST8, WST1, XTT, MTS, etc. can also be selected. Therefore, although EZMTT is non-toxic or basically non-toxic to cells, those compounds that have certain toxicity to cells can still be used as detection reagent mixtures. However, it is obvious that due to the toxicity to cell growth, the growth is inhibited but not to the extent that it cannot grow. The method described in this application can still be used, and although the results obtained are biased, it can also provide a certain IC. 50 And growth rate value. The test compound used in this application is a compound that needs to be tested for drug sensitivity (drug resistance) of specific cells. In the examples of this application, ampicillin, streptomycin, isoniazid, rifampicin, ethambutol, etc. are used as test compounds, and the living cells used are Escherichia coli, Mycobacterium tuberculosis, Gram-negative bacteria Pseudomonas aeruginosa, Candida albicans, mammalian cells, Gram-positive bacteria, fungal cells, tumor cells, blood cells, especially including various drug-resistant and non-drug-resistant cells. Those skilled in the art can determine the above-mentioned living cells and test compounds according to the needs of cells and compounds that need to be tested for drug sensitivity.

[0033] Furthermore, the detection method described in this application is mainly used when the markers are products generated after being utilized by cells. In other words, after the detection reagent mixture is utilized by living cells, it continuously generates detectable markers and the generation rate of the marker is related to the cell growth rate or the inhibition rate of the detection reagent mixture on the cells; therefore, if a detection reagent mixture can directly react with existing compounds in the cells or on the cell membrane, but has no or little relationship with the growth of living cells, there is no need to use the steps described in this application.

[0034] As an optional solution, embodiments of the present application further include an incubation concentration adjustment step: prior to incubation, the concentrations of the various components in the culture medium used for incubation are adjusted so that the living cells to be detected achieve approximately an optimized growth rate, where the growth rate refers to the growth rate over the entire incubation period. The optimized growth rate refers to the culture medium concentration at which, under other given incubation conditions, the living cells to be detected have a maximum or near-maximum growth rate within a certain incubation period.

[0035] Further as an optional scheme, in an embodiment of the present application, a pre-culture step is also included: before incubating the living cells to be detected, the living cells to be detected are cultured and the density of the optimal cells of the living cells to be detected in the culture medium is determined. The culture medium used in this step is the same as the culture medium used in the above step. This step is generally used to determine the concentration of the culture medium that can make the living cells to be detected roughly reach the optimized growth rate. There is no need to add a detection reagent mixture in this step. In addition, the role of this step is to select the concentration of bacteria in the experiment (relative to the culture medium concentration) based on the specificity of the cells.

[0036] As an optional solution, embodiments of the present application also include an accelerated culture step: after achieving a culture fluid concentration that optimizes the growth rate of the live cells to be detected, the concentration of a cell growth-accelerating component in the culture fluid is increased during the incubation process. Compared to the pre-culture step, this step increases the concentration of the culture fluid to enhance the cell growth rate within a short period of time, shortening the required detection time and improving the detection speed.

[0037] Further as an optional solution, in the embodiments of the present application, the utilization refers to the ability of the detection reagent mixture to enter the living cells to be detected, generally, and also includes reacting with components located on the cell membrane. In addition, whether the product after utilization can continue to stay in the living cells or be released by the cells in vitro or released into the culture medium due to cell death during incubation does not affect the use of the method described in the present application. Further as an optional solution, in the embodiments of the present application, the utilization refers to the ability of the detection reagent mixture to react with at least a portion of the components of the cells to be detected.

[0038] Further as an optional solution, in the embodiments of the present application, the marker is a product with color, and the color here refers to a color with a certain wavelength range on the spectrum, so white is not the color described in the present application.

[0039] As an alternative, in the embodiments of the present application, the detection reagent mixture is a compound that can be oxidized or reduced to a product that develops a color within the living cells to be detected, and the color varies depending on the detection reagent mixture used. In the case of EZMTT, the product is an orange-yellow formazan.

[0040] Further as an optional solution, in an embodiment of the present application, the detection reagent mixture is a compound that can react with NAD(P)H in the living cells to be detected and be reduced to a product that can show color in the presence of an electron coupling agent.

[0041] Further as an optional scheme, in an embodiment of the present application, an inhibition rate detection step is also included: while incubating the living cells to be detected, an equal amount of living cells to be detected is added to the culture medium of the same concentration to prepare a control group, before incubating the control group, a control compound of the same amount and concentration as the compound to be detected is added, and after incubating the control group, the concentration of the marker is detected and compared, and the inhibition rate of the test compound on the living cells to be detected is obtained by using test compounds of different toxicity.

[0042] In addition, the present invention also discloses a culture medium that can be used for the above-mentioned living cell incubation, comprising a culture medium and a detection reagent mixture; the detection reagent mixture has the following general structural formula:

[0043]

[0044] in,

[0045] A1 is and connected aromatic or heterocyclic groups;

[0046] A2 is and connected aromatic or heterocyclic groups;

[0047] A3 is and The connected phenyl groups;

[0048] R1 is H or at least one R1 substituent connected to A1, wherein the R1 substituents are independently selected from -NO2, -SO3H, -SO2NH2, halogen elements, and heterocyclic groups and alkyl groups fused to A1; especially the R1 substituent located at the para position of A1;

[0049] R2 is H or at least one R2 substituent connected to A2, and the R2 substituents are independently selected from NO2, methoxy, alkyl, phenyl and its derivatives connected through an azide group, halogen atoms, sulfonic acid groups and their salts, and phenyl or heterocyclic groups fused to A1; especially R2 substituents located at the ortho or para position of A2.

[0050] R3 is an R3 substituent connected to A3; the R3 substituents are independently selected from sulfonic acid, carboxylic acid, substituted sulfonic acid, substituted carboxylic acid; especially selected from Especially the R3 substituent is located in an ortho position relative to A3.

[0051] The heterocyclic group includes 1-2 heteroatoms, and the heteroatoms are independently selected from Se, N, S or O.

[0052] The above aromatic or heterocyclic groups are each independently selected from

[0053] In addition, additional sensitizers may be added to the above culture medium, such as PMS (commonly known as phenazine methosulfate) derivatives, especially methyl or ethyl derivatives thereof, or other compounds having similar electron coupling agent effects may be selected.

[0054] The culture medium can be prepared using the following media: LB and MH broth for Gram-negative and Gram-positive bacteria; (0.5-2x) Sauton medium, Middlebrook 7H9, Middlebrook 7H10, Middlebrook 7H11, Middlebrook 7H12, Kuang's medium, Proskauer's medium, sodium pyruvate cytochrome C medium, BACTEC MGIT 960 medium for Mycobacterium tuberculosis; RPMI medium for fungi; Basel medium Eagle, RPMI, DMEM, DMEM / F12, RPMI-HEPES, MEM, IMDM, HamF10, M199, L15, McCoy5A for animal cell culture, etc. These media are usually diluted 0.5-2x before use. For clarity, a considerable portion of the above names use the trade names of commercially available culture media.

[0055] In addition, growth accelerators, antimicrobial agents, and antibacterial synergists may be added to the above-mentioned culture medium; growth accelerators may be selected from various types of blood, hormones, oyster mushroom liquid, bean extract, OADC, etc., as well as trace amounts of malachite green, penicillin, nalidixic acid, polymyxin B, carbenicillin C, amphotericin B, trimethoprim, or mgit Pant antibiotics for inhibiting miscellaneous bacteria, which are used for addition to tuberculosis culture medium; serum-free culture media such as 1-20% BSA or B-27 are used for addition to mammalian cell culture medium;

[0056] The selected detection reagent mixture, EZMTT, is a newly synthesized monosulfonated tetrazolium salt. Its detection principle is based on the fact that NAD(P)H in living cells reduces EZMTT to a soluble orange-yellow formazan in the presence of an electron coupling agent. The formazan absorbance can be measured at 450 nm using a microplate reader, whereas dead cells do not. The absorbance measured by the microplate reader indirectly reflects bacterial viability. Within a certain range, the color change of EZMTT is proportional to the bacterial load. Furthermore, the assay is non-toxic, making it potentially applicable to the development of rapid drug susceptibility assays.

[0057] The present invention has the following significant technical effects:

[0058] The present invention can select the concentration of bacteria in drug sensitivity tests based on bacterial specificity. The present invention can have no lethal effect on bacteria, can perform continuous detection, and the data is reliable. The present invention can shorten the drug sensitivity test time (EZMTT for clinical fast-growing bacteria detection requires 3-4 hours; traditional methods require 1 day; EZMTT for tuberculosis requires 4-6 days; traditional methods require 4-10 days for liquids; and 1 month for solids), can detect partial inhibition (EZMTT obtains % inhibition rate; while traditional visual inspection methods can only qualitatively determine "presence / absence"), and quickly obtain accurate results. The present invention can detect multiple bacteria and multiple antibiotics in large quantities with high throughput and rapid detection. The present invention can provide the best combination of drugs for clinical use and achieve precise treatment. The detection reagent mixture of the present invention is inexpensive compared to others, is easy to promote, and reduces the economic burden. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The 24-hour endpoint method was used. In order to obtain the optimal detection conditions, different tetrazolium salts were used to detect the signals of live E. coli growth in vitro; all water-soluble tetrazolium salts had signals; LB culture medium was used as a negative control.

[0060] Figure 2 In order to obtain the optimal detection conditions, a seven-day endpoint method was used and different tetrazolium salts were used to track the in vitro growth signal of live Mycobacterium tuberculosis; among them, the EZMTT experimental result had the best signal.

[0061] Figure 3 In order to obtain the optimal detection conditions, different culture media were used to track the growth signal of live tuberculosis bacteria. The best signal was obtained after growing tuberculosis bacteria using the culture medium Middlebrook7H9.

[0062] Figure 4 The seven-day endpoint method was used to obtain the optimal detection conditions, and different additives were used to track the growth signal of Mycobacterium tuberculosis in vitro. The growth accelerator OADC produced the best signal.

[0063] Figure 5 .The 5-day continuous tracking rate method was used to obtain the optimal detection conditions. The results of 5-day tracking of H22 tumor cells cultured in vitro using different culture media were obtained.

[0064] Figure 6-1 、 6-2 . Using the 4-hour endpoint method, EZMTT was used to detect the drug-resistant E. coli ( Figure 6-1 ) and sensitive Escherichia coli ( Figure 6-2 ) in vitro culture to ampicillin resistance test; 4h test results showed that under conditions of higher bacterial concentrations, IC50 The MIC values ​​increased slightly (about 2 times), but did not affect the drug sensitivity test results.

[0065] Figure 7 Comparison of ampicillin resistance in clinically isolated Escherichia coli using a 4-hour endpoint assay using EZMTT as a detection indicator. Samples with known resistance (arranged from left to right, top to bottom): (1. Clinically identified as resistant) also showed resistance when tested with EZMTT as a detection reagent mixture; (2. Clinically identified as resistant) also showed resistance when tested with EZMTT as a detection reagent mixture; (3. Clinically identified as sensitive) also showed sensitivity when tested with EZMTT as a detection reagent mixture IC50. =13μg / ml; (4. Clinically identified as sensitive) The use of EZMTT as a detection reagent mixture also showed a sensitive IC50 of 14μg / ml, (5. Clinically identified as sensitive) However, the EZMTT method as a detection reagent mixture showed that it was not completely inhibited, but was in a partial inhibition state (R resistant: its cell growth rate was more than 20% of that of cells without drug administration, S sensitive: its cell growth rate was less than 2% of that of cells without drug administration, PR partial resistance (or HR hidden resistance): its cell growth rate was 2-20% of that of cells without drug administration).

[0066] Figure 8-1 、 8-2 The results of the growth curve of E. coli cells were compared: EZMTT was used as the detection indicator ( Figure 8-1 ) and the same sample was tested using the traditional turbidity method (24 hours) Figure 8-2 ), where under the same cell mass conditions, Figure 8-1 The OD450nm signal is 4, which is highly sensitive. Figure 8-2 The OD600nm signal was only 0.4, which was not sensitive enough, thus explaining why the turbidimetric method could not detect some of the inhibition of cell growth. The cell growth curve showed that the sensitivity of the EZMTT method was 10 times that of the turbidimetric method.

[0067] Figure 9-1 、 9-2 , 9-3 for comparison using EZMTT or other soluble tetrazolium salts (WST8 / CCK8) as detection indicators, wherein both reagents can detect completely resistant or completely sensitive bacteria, but WST8 has certain toxicity to partially resistant bacteria and cannot detect the drug resistance of bacteria E.

[0068] Figure 10To detect drug resistance of Mycobacterium tuberculosis using EZMTT as an indicator, the EZMTT assay was used to track cell growth daily under different growth conditions (different cell amounts (10- or 40-fold dilution), with or without nutrients (OADC), with or without drugs (S, I, R, E), and time effects of different detection times). The histogram shows that increasing the cell amount (10-fold dilution) and adding the nutrient OADC increased the signal; at the same time, EZMTT can be used for quantitative analysis and to detect the time effects of cell growth. The histogram represents the endpoint assay, and the curve represents the rate assay.

[0069] Figure 11 In vitro drug resistance testing of living Gram-positive bacteria: 4-hour drug sensitivity test results of Staphylococcus aureus (40-fold dilution of fresh bacterial solution of 0.5MCF), where A is IC50 = 129ng / ml after treatment with ampicillin; B is IC50 = 872ng / ml after treatment with kanamycin; C is IC50 = 87ng / ml after treatment with cefazolin.

[0070] Figure 12 In vitro drug resistance test of Gram-negative bacteria: Pseudomonas aeruginosa (0.5 MCF fresh bacterial solution diluted 40 times) 4 hours gentamicin susceptibility test (IC 50 =0.17 μg / ml) results.

[0071] Figure 13 To detect the drug resistance of living fungal cells in vitro: a 4-hour drug sensitivity test of Candida albicans (40-fold dilution of 0.5MCF fresh bacterial solution) showed that ampicillin was resistant to Candida albicans.

[0072] Figure 14-1 、 14-2 14-3 is a continuous detection rate method to detect drug resistance of living cells in vitro: EZMTT culture medium is used to detect drug resistance and partial drug resistance of tumor cells. The 5-day drug sensitivity test shows that the use of the patented new EZMTT culture medium and detection method (with and without drug addition, cell growth dose effect) can distinguish whether DOX can completely inhibit tumors ( Figure 14-2 ) and Paclitaxel partially inhibited tumor ( Figure 14-3 ); This explains the reported tumor resistance to Paclitaxel in vivo, and thus the present invention provides guidance for early prediction of tumor resistance.

[0073] Figure 15-1 、 15-215-3 is a continuous detection rate method to detect the drug resistance of living cells in vitro: the new EZMTT culture medium is used to detect the growth inhibition of HEK293t tumor cells after partial infection by lentivirus, among which the effect of viral infection on the growth of HEK293t cells: the middle Figure 15-2 This is a growth curve. The top line shows the growth of 2000 HEK293t cells. The bottom line shows the growth of 2000 HEK293t cells after virus transfection. Figure 15-1 2000 HEK293t cells. Figure 15-2 These are 2000 HEK293t transfected cells, and the fluorescence shows the cells transfected with viruses.

[0074] Figure 16-1 and 16-2 The EZMTT rate assay was used to detect the in vitro drug resistance of live cells of six E. coli strains (MG655 is a wild-type E. coli without mutations; the others are clinically isolated E. coli). Compared with the traditional turbidity assay, the EZMTT (OD450 nm) was able to detect cell growth due to drug resistance that was undetectable by the turbidity assay (OD600 nm) (details will be published in ACS Infecious Diseases, 2019, Detection of “Hidden” Antimicrobial Drug Resistance; corresponding author is Benfang Helen Ruan).

[0075] Figure 17 . The detected partial drug resistance (PR; showing 2-20% cell growth after drug administration) was verified, and the results showed that the increase in the signal of the detection marker was indeed consistent with cell growth (details will be published in ACS Infecious Diseases, 2019, Detection of "Hidden" Antimicrobial Drug Resistance; corresponding author Benfang Helen Ruan).

[0076] Figure 18The EZMTT rate assay was used to detect the drug resistance of 10 strains of Mycobacterium tuberculosis (H37Ra is a wild-type strain without mutations; the others are clinically isolated Mycobacterium tuberculosis). The assay was compared with traditional solid-state and liquid-based assays. The EZMTT had a 100% success rate, reduced the detection time for significant drug resistance (R) from 30 days to 3-5 days, and reduced the detection time for partial drug resistance (PR; growth in the presence of 2-20% drug) from 30 days to 5-7 days. These results were consistent with those of traditional solid-state or liquid-based assays (details will be published in ACS Infecious Diseases, 2019, "Detection of "Hidden" Antimicrobial Drug Resistance"; corresponding author is Benfang Helen Ruan).

[0077] Figure 19 The results of the in vitro drug resistance test of 6 E. coli strains (MG655 is a wild-type E. coli without mutations; the others are clinically isolated E. coli) to 12 total drugs were measured over a 24-hour period using multiple methods. The EZMTT assay (OD450 nm tracking detection) of the present invention can detect cell growth (2-20% growth) due to partial drug resistance that cannot be detected by the turbidity assay (OD600 nm), MTT endpoint assay, and EZMTT endpoint assay (details will be published in ACS Infecious Diseases, 2019, Detection of “Hidden” Antimicrobial Drug Resistance; corresponding author is Benfang Helen Ruan). DETAILED DESCRIPTION

[0078] The present invention is described in further detail below with reference to the embodiments.

[0079] Example 1

[0080] Perform an endpoint-based drug susceptibility test on E. coli: Prepare ampicillin solution (0-12.8 mg / ml; two-fold dilution gradient) and set aside. Use a dispenser to transfer 2 μl of the ampicillin solution to a 96-well plate. Adjust the freshly cultured E. coli to a concentration of 0.5 MCF in MH broth. Dilute the solution 20-640 times (preferably 40-fold) in MH broth containing a mixture of EZMTT (EZMTT can be replaced with various tetrazolium salts and PMS derivatives, including methyl PMS) detection reagents. Transfer 200 μl to a 96-well plate. Set up control wells, replacing 2 μl of the ampicillin solution with sterile water. Follow the same procedures as for the drug-treated wells. Incubate the 96-well plate in a 37°C incubator. After 4 hours, measure the OD450 and OD600 values ​​using the endpoint method.

[0081] Example 2

[0082] Use the rate method to conduct a drug sensitivity (drug resistance) test on Mycobacterium tuberculosis: prepare streptomycin, isoniazid, rifampicin, and ethambutol solutions for use. According to the conventional drug detection concentration, take the drug solution to be used into a screw-cap tube. Use 7H9 culture medium to adjust the concentration of freshly cultured Mycobacterium tuberculosis to 0.5MCF, and then use 7H9 culture medium containing EZMTT (which can also be replaced by other tetrazolium salts and PMS derivatives, including methyl PMS, the same as the following examples) to dilute 4-40 times (preferably 20 times) and add 4ml to the drug-containing tube. Set up control wells, replace the drug solution to be used with sterile water in the control wells, the one with bacteria is the positive control (signal), and the one without bacteria is the negative control (background). Place the tube in a 37°C incubator for culture. After 5 days, measure the OD450 and OD600 values ​​every day. When the signal / background is >2 times, calculate the inhibition rate and judge the drug resistance. After one day, the assay was terminated when the signal / background ratio was >4-fold.

[0083] Example 3

[0084] Use the endpoint method to conduct a drug sensitivity test on the Gram-negative bacteria Pseudomonas aeruginosa (40-fold dilution of fresh bacterial solution of 0.5MCF): prepare gentamicin solution (0-12.8mg / ml; two-fold dilution concentration gradient) for use. Use a spray gun to take 2μl of gentamicin solution to be used in a 96-well plate for use. Adjust the fresh culture of Pseudomonas aeruginosa to a concentration of 0.5MCF with MH broth culture medium, and then dilute it 20-640 times (preferably 40 times) with MH broth containing EZMTT detection reagent mixture, and take 200μl to a 96-well plate. Set up a control well, replace 2ul of gentamicin solution in the control well with sterile water, and the rest of the operation is the same as the dosing well. Place the 96-well plate in a 37°C incubator and culture it. After 4 hours, detect the OD450 and OD600 values.

[0085] Example 4

[0086] In vitro drug resistance testing of live Gram-positive bacteria: Staphylococcus aureus (40-fold dilution of a fresh 0.5 MCF culture) was used for drug susceptibility testing. Ampicillin, kanamycin, and cefazolin solutions (0-12.8 mg / ml; two-fold dilution gradient) were prepared and set aside. Using a dispenser, 2 μl of the solution was transferred to a 96-well plate. Freshly cultured Staphylococcus aureus was cultured to a concentration of 0.5 MCF using MH broth. The solution was then diluted 20-640-fold (preferably 40-fold) in MH broth containing the EZMTT assay reagent mixture, and 200 μl was transferred to a 96-well plate. Control wells were established, and 2 μl of the solution was replaced with sterile water. All other procedures were the same as for the drug wells. The 96-well plate was incubated at 37°C. After 4 hours, the OD450 and OD600 values ​​were measured.

[0087] Example 5

[0088] Use the fungus Candida albicans (40-fold dilution of fresh bacterial solution at 0.5 MCF) for drug sensitivity testing: Prepare ampicillin solution (0-12.8 mg / ml; two-fold dilution concentration gradient) for use. Use a spray gun to transfer 2 μl of the ampicillin solution to a 96-well plate for use. Adjust the freshly cultured E. coli strain to a concentration of 0.5 MCF with MH broth, then dilute it 20-640 times (preferably 40-fold) with MH broth containing an EZMTT detection reagent mixture, and transfer 200 μl to a 96-well plate. Set up control wells, replace 2 ul of the ampicillin solution with sterile water in the control wells, and follow the same procedures as for the dosing wells. Place the 96-well plate in a 37°C incubator and incubate for 4 hours. Detect the OD450 and OD600 values.

[0089] Example 6

[0090] Use the rate method to conduct a drug sensitivity test on tumor cells HCT116 (1000-8000): prepare the anti-tumor drug solution (two-fold dilution concentration gradient; or combination drug) for use. Use a spray gun to take 2μl of the drug solution to a 96-well plate for use. Dilute the freshly cultured HCT116 with RPMI or other culture medium containing serum of the EZMTT detection reagent mixture (1000 per well) and take 200μl to a 96-well plate. Set up a control well, replace the 2ul of the drug solution in the control well with sterile water, and the rest of the operation is the same as the dosing well. Place the 96-well plate in a 37°C incubator and culture it. Detect the OD450 and OD600 values ​​after 4 hours, 1 day, 2 days, 3 days, 4 days, and 5 days.

[0091] Example 7

[0092] Viral infection experiments were performed using the rate assay on HEK293T tumor cells (1000-8000 cells / well): Freshly cultured HEK293T cells were diluted with RPMI (e.g., serum) containing the EZMTT assay mixture (1000 μl per well) and 200 μl was transferred to a 96-well plate. A large amount of lentivirus was added. Control wells were prepared, with the virus replaced with sterile water. All other procedures were the same. The 96-well plate was incubated at 37°C and the OD450 and OD600 values ​​were measured after 4 hours, 1 day, 2 days, 3 days, 4 days, and 5 days.

[0093] Example 8

[0094] Based on Examples 1-6 above, the following compounds can also be used as a detection reagent mixture using a rate assay to replace the EZMTT component in Example 1. The detection reagent mixture used in this example can be used at a concentration equivalent to that of EZMTT in Example 1, with a rough concentration range of 0.1-50 mM being proposed. The detection reagent mixture used in this example comprises low-toxic, highly effective compounds. Given the requirement for low toxicity in living cells to be detected, some compounds are non-toxic or substantially non-toxic to living cells.

[0095] Further explanation of the accompanying drawings

[0096] Attachment Figure 1-18 The data obtained from combining different experimental results are compared based on the experimental steps described in Example 1 by replacing the compounds and experimental conditions described in the accompanying figure description with the corresponding contents described in Example 1. Therefore, the repeated data in the accompanying figure correspond to multiple experiments, which were obtained by partially replacing the contents described in Example 1. Figure 10 As shown, EZMTT was used as a detection indicator and the contents described in Example 1 were used to detect the drug resistance of clinical drug-resistant bacteria. To further compare with the above results, the traditional liquid fluorescence method was used as a reference. Table 1 below compares the results of the traditional endpoint method (solid method liquid fluorescence method) and the EZMTT rate method for detecting clinical tuberculosis-resistant bacteria. Table 2 below compares the results of the clinically used endpoint method (MIC and inhibition zone), the turbidity detection rate method and the EZMTT rate method for detecting clinical Escherichia coli-resistant bacteria.

[0097] Table 1 (Details will be published in ACS Infecious Diseases, 2019, Detection of “Hidden” Antimicrobial Drug Resistance; corresponding author: Benfang Helen Ruan). Figure 18Results analysis: Mycobacterium tuberculosis drug resistance detection; 30-day solid culture endpoint method, 14-day liquid oxygen consumption endpoint method, 3-7 day EZMTT rate method (rapid, accurate, quantitative)

[0098] c The growth rate of living cells

[0099] R: Resistant, S: Susceptible, PR: Partially resistant or "hidden" resistance

[0100]

[0101]

[0102] Table 2 (Details will be published in ACS Infecious Diseases, 2019, Detection of “Hidden” Antimicrobial Drug Resistance; corresponding author: Benfang Helen Ruan) and Figure 16 Results Analysis: Comparison of Escherichia coli Antimicrobial Resistance Detection Results: EZMTT Rate Method, Turbidimetric Rate Method, and Clinical Endpoint Methods (VITEK and KB)

[0103]

[0104]

[0105] a S sensitive (growth under <2% drug inhibition), R resistant (growth under >20% drug inhibition), I partially resistant (HR hidden resistance; growth under 2-20% drug inhibition), where MNO is a bacteriostatic agent and CFP is a bactericidal agent. Figure 19 The result is calculated.

[0106] In addition to the detection reagent mixture described in Example 1 above, any combination of groups described in A1-3 and R1-3 described in the technical solution of this application can also be used; some of the more important group combinations are listed one by one here:

[0107]

[0108]

[0109]

[0110]

[0111] The compound CPD1-51 described above was used as a detection reagent mixture using the steps described in Example 1, and a drug sensitivity test was performed using E. coli, wherein ampicillin was used as the test compound; the rest of the steps were the same as in Example 1. After performing each experiment, it was found that the results were similar to the turbidity 600nm detection results in Figure 8, but the detection time was greatly shortened and the sensitivity was higher.

[0112] In short, the above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the patent of the present invention.

Claims

1. A method for detecting drug resistance of living cells in vitro, It is characterized in that The method comprises the following specific steps: adjusting the concentration of the cell culture medium used for incubation so that the living cells to be detected can roughly reach a more optimal growth rate, adding a detection reagent mixture that can be used by the living cells to be detected and a test compound for drug resistance detection for incubation, and continuously detecting the concentration of the marker at a predetermined time interval after incubation to achieve the above detection; The incubation refers to adding the living cells to be tested into a culture solution of a determined concentration for culture; drug resistance refers to the inability of the tested compound to completely inhibit the growth of the living cells to be tested; After the concentration adjustment step is completed, the concentration of the cell culture fluid is diluted in a range of 2-4000 times of McFarland 0.5 dilution; or when the in vitro living cells are fast-growing bacteria, a 40-fold dilution is used; Under optimized conditions, the detection reagent mixture can use the rate method to track the growth of cells after administration and detect cell resistance, specifically including detecting the change in cell growth rate after administration and comparing it with the growth rate of corresponding wild-type cells; the rate method refers to continuously detecting the change in the concentration signal of the marker over time at a predetermined time interval; While incubating the living cells to be detected, an equal amount of the living cells to be detected are added to a culture medium of the same concentration to prepare a positive control group, and a culture medium of the same concentration without the living cells to be detected is used to prepare a negative control group; the concentration of the detection marker after incubation with the positive and negative control groups is further compared to obtain the inhibition rate of the living cells to be detected by the compound to be detected; The living cells include fungi, bacteria, animals and human cells that can produce NAD(P)H; The detection reagent mixture includes a compound having the following structural formula: Among them, A1 is and Aromatic or heterocyclic groups connected to each other; A2 is and Aromatic or heterocyclic groups connected to each other; A3 is and The attached phenyl groups; R1 is H or at least one R1 substituent connected to A1, wherein the R1 substituents are independently selected from -NO 2 、-SO 3 H, -SO 2 NH 2 , a halogen element, and a heterocyclic group or an alkyl group fused with the A1; R2 is H or at least one R2 substituent connected to A2, wherein the R2 substituents are independently selected from NO 2 , methoxy, alkyl, phenyl connected by an azide group, halogen atoms, sulfonic acid groups and salts thereof, and the phenyl or heterocyclic group fused to A2; R3 is an R3 substituent connected to A3; The R3 substituents are each independently selected from: The heterocyclic group includes 1-2 heteroatoms, and the heteroatoms are independently selected from Se, N, S or O; The detection reagent mixture is EZMTT and an electron coupling agent; The aromatic or heterocyclic groups of the detection reagent mixture are each independently selected from 2. The method for detecting drug resistance of living cells in vitro according to claim 1, It is characterized in that By continuously detecting the concentration of the marker, the inhibitory effect of the antibacterial and bactericidal compounds on the cells is detected.

3. The method for detecting drug resistance of living cells in vitro according to claim 1, It is characterized in that The culture solution also includes a sensitizer; the sensitizer includes PMS.

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