WO2019056926A1 - 一种快速定量检测抗菌药物敏感性的试剂及其应用方法 - Google Patents

一种快速定量检测抗菌药物敏感性的试剂及其应用方法 Download PDF

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WO2019056926A1
WO2019056926A1 PCT/CN2018/102508 CN2018102508W WO2019056926A1 WO 2019056926 A1 WO2019056926 A1 WO 2019056926A1 CN 2018102508 W CN2018102508 W CN 2018102508W WO 2019056926 A1 WO2019056926 A1 WO 2019056926A1
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drug
reagent
detection
antibacterial
atp
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PCT/CN2018/102508
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French (fr)
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羌维兵
童明庆
张怡
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江苏中新医药有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G01N21/763—Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56938—Staphylococcus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9446—Antibacterials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
    • G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)

Definitions

  • the invention relates to a reagent for rapidly detecting sensitivity of a pathogenic microorganism to an antibacterial drug and an application method thereof.
  • the methods of the conventional antimicrobial Susceptibility Tests (AST) used in clinical microbiology laboratories mainly include a disk diffusion method, a dilution method, and an E-test method. These methods are judged by visual observation of the growth of microorganisms, and it is often necessary to culture for more than 16 hours to report the drug susceptibility results.
  • the Chinese patent "Rapid Drug Sensitivity Test Kit” (ZL201120469811) uses the dilution method for drug susceptibility test, and the color test method shortens the test time, but since the sensitivity of the color test method is low, it takes 9-11 hours to get the drug. Sensitive results.
  • Biomerieux's VITEK 2 automated instrument takes up to 4 hours to get a susceptibility result.
  • Other automated instruments for clinical testing include: BD's Phoenix-100 automated bacterial identification/drug sensitivity system, Beckman-Coulter's MicroScan microbial identification and drug susceptibility analysis system, and Thermo Scientific's Sensititre ARIS 2X ID/AST system. These instruments generally use the turbidimetric method to observe the kinetic changes of the number of bacteria, which is limited by the low sensitivity of the turbidimetric method. The concentration of the bacteria required for the susceptibility test is large, and it needs to be cultured for more than 4 hours.
  • Wu et al. established a rapid method for the detection of bacterial antibiotic susceptibility by adenosine triphosphate-bioluminescence method. Based on the micro-broth dilution method, ATP bioluminescence method was used to detect bacteria after culture for a period of time, and the calculation was compared with the blank. Luminescence rate to determine whether the bacteria are resistant or not. Because ATP bioluminescence detection technology has high sensitivity, it can shorten the culture time, and the results are interpreted within 4 hours, and no high concentration of bacteria liquid is needed (ATP bioluminescence method is used to detect the establishment and application of bacterial drug sensitivity technology, Zhejiang Clinical Medicine, October 2015 Volume 17, Number 10: 1680).
  • a microplate luminometer that is less equipped with primary care facilities must be used and an additional autosampler system is required.
  • this method requires the addition of two reagents: bacterial lysate and ATP detection reagent, the lysate and ATP detection reagent are injected through the auto-injection system of the microplate luminescence detector; at least two auto-injection systems are required.
  • the flashing luminescent reagent has a shorter luminescence duration, and the luminescence intensity decays faster with time. It is necessary to "detect the luminescence value immediately" after adding the ATP detection reagent, otherwise the detection result is affected.
  • the concentration gradient of the antibacterial drug set in this method is small, and the MIC (minimum inhibitory concentration) cannot be measured. Therefore, this method only qualitatively reports sensitivity and does not provide an exact MIC value. The quantitative results of MIC values are more important for guiding clinicians to take medication.
  • the object of the present invention is to develop a kit for rapidly detecting the sensitivity of an antibacterial drug, which can not only give a drug susceptibility result in a shorter time, but also provide an exact MIC (minimum inhibitory concentration) value, and has low hardware requirements. .
  • the invention provides a novel kit for rapidly detecting the sensitivity of an antibacterial drug, which is based on a micro-broth dilution method, comprising an antibacterial plate, a medium and a bacterial ATP detecting reagent.
  • the culture medium provided by the test kit is adjusted to a suitable concentration and added to the antibacterial drug plate.
  • the bacterial ATP detection reagent is added to the antibacterial drug plate, and then the antibacterial drug plate is added.
  • the intensity of the luminescence is measured in a multi-function plate reader, the MIC value is determined based on the relative luminescence intensity, and sensitivity, intermediate or resistance is reported.
  • the invention provides a kit for rapidly detecting the sensitivity of an antibacterial drug, characterized in that:
  • the reagent is a detection reagent containing a bacterial ATP releasing agent and a bioluminescent substance, and can perform bacterial lysis and ATP release and detection in one step.
  • the bioluminescent substance is a glow type bioluminescent substance
  • the ratio of the two substances is different, and the mass ratio of the two substances is 1:4 to 1:6, preferably 1:4.7. .
  • Preferred bacterial ATP detection reagents include bacterial ATP releasing agents, buffers, luciferase, D-luciferin, stabilizers and the like.
  • the bacterial ATP releasing agent is preferably a surfactant selected from one or more of cetyltrimethylammonium bromide, benzalkonium chloride, chlorhexidine and tritonone.
  • Preferred bacterial ATP detection reagents are chlorhexidine acetate (0.15%-1.20%), Triton X-100 (1.5%-2.5%), luciferase (0.02%-0.05%) and D-fluorescence. An aqueous solution of 0.1% to 0.2%. The percentage is the mass ratio.
  • the bacterial ATP detecting reagent used in one example of the present invention consists of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (150-250 mM), cetyltrimethyl bromide Ammonium (0.06%-0.10%), chlorhexidine acetate (0.15%-1.2%), Triton (1.5%-2.5%), Magnesium chloride (0.3%-0.5%), Ethylenediaminetetraacetic acid (0.7%- 1.0%), sodium fluoride (0.05%-0.1%), sodium pyrophosphate (0.01%-0.02%), luciferase (0.02%-0.05%), D-luciferin (0.1%-0.2%) And water.
  • the bacterial ATP detecting reagent of the present invention can realize the functions of the two reagents of the prior art bacterial lysate and ATP detecting reagent in one step. During the test, it is not necessary to inject the lysate first, and then inject the ATP detection reagent, which not only reduces the number of steps, but also eliminates the need to configure the autosampler system.
  • the antibacterial drug sheet of the present invention has a series of concentration gradient antibacterial drugs having a concentration of ten or more per drug.
  • concentration range includes the interpretation breakpoint listed in the CLSI file, and contains the value of the quality control bacteria to meet the needs of the MIC measurement.
  • the specific concentration gradient of antibacterial drugs varies greatly depending on the nature of the antibacterial drugs, such as piperacillin concentrations of 12800, 6400, 3200, 1600, 800, 400, 200, 100, 50, and 25 ⁇ g/mL, while cyclopropane
  • concentration of sand star is 200, 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78 and 0.39 ⁇ g/mL.
  • microplate luminescence detector used in the prior art can be used, but also a multi-function plate reader equipped with a primary medical institution (see the embodiment of the present invention), no additional equipment is required regardless of the instrument used. Autosampler system.
  • the bacterial ATP detecting reagent of the present invention is a glow type luminescent reagent
  • the luminescence intensity is relatively stable after the addition of the reagent and can be continued for a period of time, and is detected immediately after the reagent is not added, and the detection reagent is added to the antibacterial drug plate after use, and then By placing the antimicrobial plate into the instrument for testing, the automated sample introduction system is not required.
  • the multi-function plate reader is an instrument that is available in most primary care facilities and related clinical departments.
  • the microplate luminescence detector is a specialized instrument, and the configuration rate of the above mechanism is relatively low.
  • the comparison file method does not suggest that the multi-function plate reader can achieve the detection accuracy required by its method.
  • the antibacterial drug plate provided by the kit of the present invention may be provided with various antibacterial drugs according to actual needs; the types of antibacterial drugs vary according to the type of bacteria to be tested, each antibacterial drug includes multiple concentrations, and the form of the antibacterial drug is according to different drugs. Depending on the specific circumstances. When the conditions permit, the preferred drug form is a dry powder or granule that is fixed to the bottom of the well plate to facilitate storage and transportation of the kit.
  • the culture medium provided by the kit of the present invention refers to the CLSI (American Society for Clinical and Laboratory Standards) standards, and depending on the type of the test bacteria, Mueller-Hinton Broth (CAMHB) and RPMI- can be used to adjust the cation. 1640 broth medium or other media required for susceptibility testing.
  • CLSI American Society for Clinical and Laboratory Standards
  • the kit of the present invention is applied by adding the bacteria to be tested to the antibacterial plate for 2 hours, adding the bacterial ATP detecting reagent to the antibacterial plate, and then placing the whole antibacterial plate into the multi-function reading plate.
  • the luminous intensity was measured in the instrument, and the relative luminous intensity of the antibacterial drug well relative to the positive hole without the antibacterial drug was calculated.
  • the bacteria to be tested added to the antibacterial drug plate are adjusted to the desired concentration of the bacteria to be tested, and the required concentration is determined according to actual needs, such as determining the appropriate concentration by reference to the CLSI standard (non-hypertrophic bacteria)
  • the concentration is about 5 x 10 5 CFU/mL).
  • the relative luminous intensity is calculated as follows:
  • Relative luminous intensity (antibacterial drug hole luminescence intensity / positive hole luminescence intensity) ⁇ 100%
  • the MIC value (minimum inhibitory concentration) of the drug was obtained from the minimum drug concentration with a relative luminescence intensity ⁇ 80%, and the conclusion was obtained by referring to the CLSI standard for drug resistance.
  • CLSI vertices concentration provided by CLSI, when the MIC ⁇ sensitive vertices, the bacteria to be tested are sensitive to the antibacterial drug; when the MIC ⁇ drug resistance breakpoint, the test bacteria are resistant to the antibacterial drug; when the MIC is between two folds When the spots are between, the sensitivity of the test bacteria to the antibacterial drug is intermediate.
  • kits of the present invention to detect the sensitivity of Escherichia coli and Staphylococcus aureus to 30 different antibacterial agents, respectively, and simultaneously detecting 10 strains of Enterobacteriaceae to ampicillin and cefazolin, 10 strains of Staphylococcus Sensitivity to erythromycin and penicillin. All of the above tests can report the MIC value within 2 hours, and are within the tolerances allowed by the CLSI standard compared to the values reported by the standard methods of CLSI.
  • the kit of the present invention can be quantitatively detected as well as qualitatively.
  • the kit of the present invention is simpler to operate: only the negative hole of the antibacterial plate is used, Positive wells and antimicrobial drug breakpoints (including sensitive breakpoints and drug resistance breakpoints), the rest of the operation is consistent with the above quantitative test.
  • the specific operation method is: adding the bacteria to be tested to the antibacterial plate for 2 hours, adding the bacterial ATP detecting reagent to the antibacterial plate, and then placing the antibacterial plate into the multi-function plate reader to detect the corresponding hole.
  • Luminescence intensity the relative luminescence intensity of the antibiotic drug well relative to the positive hole without the antibacterial drug was calculated.
  • the antibacterial drug breakpoint concentration hole is determined by the CLSI standard, and there are large differences between different microorganisms and antibacterial drugs.
  • the relative luminous intensity is calculated as follows:
  • Relative luminous intensity (antibacterial drug hole luminescence intensity / positive hole luminescence intensity) ⁇ 100%
  • the microorganism to be tested When the relative luminescence intensity of the sensitive puncturing point and the resistance puncturing point concentration are both ⁇ 80%, the microorganism to be tested is sensitive to the antibacterial drug; when the relative luminescence intensity of the sensitive punctate concentration is >80%, and the relative concentration of the drug resistance vertices is relative If the luminous intensity is ⁇ 80%, the microorganism to be tested is sensitive to the sensitivity of the antibacterial drug; when the relative luminescence intensity of the sensitive puncturing point and the drug resistance puncturing point are both >80%, the microorganism to be tested is resistant to the antibacterial drug.
  • HEPES N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid
  • CTAB cetyltrimethylammonium bromide
  • Triton X-100 Triton X-100
  • magnesium chloride hexahydrate 0.86 g of disodium ethylenediaminetetraacetate dihydrate (EDTA)
  • EDTA disodium ethylenediaminetetraacetate dihydrate
  • fluorine Sodium hydride and 0.01 g sodium pyrophosphate were dissolved; after adjusting the pH of the solution to 7.5 with 1 M sodium hydroxide, the filter was sterilized by using a 0.22 ⁇ m filter; 35.8 mg of luciferase (Sigma) and 168 mg of D-luciferin were added. (Sigma), dissolved, stored at -20 °C.
  • Example 2 Bacterial ATP detection reagent for detecting bacteria
  • Escherichia coli ATCC 25922
  • Staphylococcus aureus ATCC 29213
  • the two bacteria were separately diluted into a series of concentrations with CAMHB broth, and a certain volume of the diluted bacterial solution was transferred to a blank multi-well plate, and a certain volume of the bacterial ATP detection reagent prepared in Example 1 was added to the multi-function plate reader. (PerkinElmer), the luminescence value of each well was examined, and three wells were repeated for each concentration.
  • Tables 1 and 2 show the luminescence values of different numbers of Escherichia coli and Staphylococcus aureus, respectively.
  • Figures 1 and 2 show the relationship between the detected luminescence intensity and the number of bacteria. It can be seen from the figure that the luminescence intensity is positively correlated with the number of bacteria in a large range. Considering the background and its standard deviation, the minimum detection limits for Escherichia coli and Staphylococcus aureus by luminescence method can reach 25 CFU and 20 CFU, respectively. This result indicates that the formulated bacterial ATP detection reagent can be applied to the detection of bacteria, and has a very high detection sensitivity, and can detect a very low number of bacteria.
  • Table 1 shows the luminescence values of different numbers of Escherichia coli
  • CFU Number of bacteria
  • RLU Luminous intensity
  • Table 2 shows the luminescence values of different amounts of Staphylococcus aureus
  • CFU Number of bacteria
  • RLU Luminous intensity
  • Escherichia coli ATCC 25922
  • Staphylococcus aureus ATCC 29213
  • the No. 1 column was used as the negative control
  • the No. 6 column was used as the positive control
  • the A line was cefazolin
  • the B and E lines were piperacillin
  • the C and G lines were netilmicin
  • the D line was levofloxacin.
  • Line F is minocycline.
  • Table 3 shows the relative luminescence intensity detected after 2 h of antibiotics at different concentrations. Due to the positive correlation between luminescence intensity and the number of bacteria, the change in relative luminescence intensity reflects the change in the number of bacteria. When the concentration of antibiotics increases and the relative luminescence intensity decreases, the number of bacteria can be considered to decrease. This result indicates that the change in the number of bacteria after 2 hours of antibiotic action can be detected by luminescence, and the bacterial ATP of the present invention is used. Detection reagents are expected to report drug susceptibility results within 2 hours.
  • Table 3 shows the relative luminescence intensity of bacteria after different concentrations of antibiotics
  • Preparation method In the 384-well plate, No. 1 is listed as a negative control, No. 24 is listed as a positive control, and No. 2-11 and No. 14-23 are antibiotics.
  • the cefazolin was formulated into a concentration of 3200, 1600, 800, 400, 200, 100, 50, 25, 12.5, 6.25 ⁇ g/mL, and added to the No. 2-11 hole of the A row, 0.5 ⁇ L per well.
  • the oxacillin was formulated into 1600, 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125 ⁇ g/mL and added to wells No. 14-23 of row A, 0.5 ⁇ L per well.
  • Other concentrations of antimicrobial agents were similarly added to the corresponding wells and finally dried under vacuum.
  • Example 5 The kit is applied to detect the MIC value of various antibacterial drugs
  • CMCC 44103 Escherichia coli
  • ATCC 25923 Staphylococcus aureus
  • Rapid detection of MIC values using the Rapid Sensitive Drug Sensitivity Test Kit Select several colonies from cultured agar plates to make 0.5 mL of turbidity suspension directly in CAMHB broth and further dilute with broth 300 times; in the antibacterial plate, 25 ⁇ L of broth was added to the negative column, and 25 ⁇ L of the diluted bacterial solution was added to the antibacterial and positive columns respectively; after incubating at 37 ° C for 2 h, 25 ⁇ L of bacterial ATP detection reagent was added to each well and placed. In the multi-function plate reader, the luminescence value of each hole is recorded; the data is processed, and the MIC value is determined by the minimum concentration of the relative illuminance ⁇ 80%. At the same time, the experiment was carried out by using a micro-broth dilution method to determine the standard MIC value.
  • Table 4 shows the comparison of the MIC values of the various antibiotics tested by the kit with the MIC detected by standard methods.
  • the results show that the MIC values of the kit detection method are mostly the same as those of the standard method.
  • the results of the standard method are one-fold ratio gradient, the methodological tolerance of the standard method is one-fold deviation from the gradient, so it can be said that the kit detection method
  • the results are consistent with the dilution method.
  • the kit of the present invention is applicable to the detection of MIC values of a variety of common antibiotics.
  • Example 6 The kit is applied to detect the MIC value of multiple strains of bacteria
  • the resuscitation test strain was cultured on TSA plate medium at 37 ° C for 18-24 h. Rapid detection of MIC values using the Rapid Sensitive Drug Sensitivity Test Kit: Select several colonies from cultured agar plates to make 0.5 mL of turbidity suspension directly in CAMHB broth and further dilute with broth 300 times; in the antibacterial plate, 25 ⁇ L of broth was added to the negative column, and 25 ⁇ L of the diluted bacterial solution was added to the antibacterial and positive columns respectively; after incubating at 37 ° C for 2 h, 25 ⁇ L of bacterial ATP detection reagent was added to each well and placed.
  • the illuminating value of each hole is recorded; the data is processed, and the MIC value is determined by determining the minimum concentration of the relative luminous intensity ⁇ 80%.
  • the experiment was carried out by using a micro-broth dilution method to determine the standard MIC value.
  • Tables 5 and 6 show the MIC values of Enterobacter and Staphylococcus using kits and comparison with standard methods, respectively.
  • the test results show that the MIC value of the kit detection method is mostly the same as that of the standard method.
  • the methodological tolerance of the standard method is a deviation ratio gradient, so it can be said that the kit detects
  • the result of the method is consistent with the dilution method.
  • the kit of the present invention can be applied to the determination of the MIC value of a plurality of strains.

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Abstract

一种快速定量检测抗菌药物敏感性的试剂盒，具有可直接检测细菌ATP的试剂；一次加入所述试剂后一步实现细菌的裂解和ATP的释放与检测，2小时便可得到药敏实验结果，可以使用任何微孔板式发光检测仪，而且不需要使用自动进样系统。一步加样操作，不需要分别注入裂解液和ATP检测试剂。可以确定MIC值，并同时报告耐药与否。

Description

一种快速定量检测抗菌药物敏感性的试剂及其应用方法 技术领域：
本发明涉及一种快速检测病原微生物对抗菌药物敏感性的试剂及其应用方法。
背景技术：
目前临床微生物实验室使用的常规的抗菌药物敏感性试验(Antimicrobial Susceptibility Tests，AST)的方法主要有纸片扩散法、稀释法、E-test法等。这些方法由于通过肉眼观察微生物生长情况进行结果评判，往往必须培养16个小时以上才能报告药敏结果。
中国专利《快速药敏检测试剂盒》(ZL201120469811)采用稀释法进行药敏试验，通过显色检测方法缩短了试验用时，但由于显色检测方法的灵敏度较低，需9-11小时才能得到药敏结果。
尽管自动化仪器极大地提高了药敏试验的速度，但仍需要至少4小时得到药敏结果。而且这些仪器成本均较高，基层医院和相关临床科室使用受限。
例如Biomerieux的VITEK 2自动化仪器，最快也需要4小时得到药敏结果。其它临床检测的自动化仪器还包括：BD的Phoenix-100全自动细菌鉴定/药敏系统、Beckman-Coulter的MicroScan微生物鉴定与药敏分析系统和Thermo Scientific的Sensititre ARIS 2X ID/AST系统等。这些仪器普遍采用比浊法观测细菌数量的动力学变化，受限于比浊法的低灵敏度，药敏试验所需的菌液浓度较大，且需要培养4h以上。
吴潇等采用三磷酸腺苷-生物发光法建立了一种快速检测细菌抗生素药敏的方法，以微量肉汤稀释法为基础，采用ATP生物发光法检测培养一段时间后的细菌，通过计算与空白比较的发光率来判断细菌的耐药与否。由于ATP生物发光检测技术灵敏度高，可以缩短培养用时，在4h内判读结果，且无需高浓度菌液(ATP生物发光法检测细菌药敏技术的建立及应用，《浙江临床医学》2015年10月第17卷第10期：1680)。
但上述方法仍有如下缺点：
一是对检测仪的硬件配置有较高要求：
必须使用基层医疗机构较少配备的微孔板式发光检测仪，而且需要额外配备自动进样系统。
二是操作较繁琐，因此该技术较难在基层医院使用：
1)需要通过自动进样系统先后分别注入两种试剂
因为该方法需要分别加入两种试剂：细菌裂解液和ATP检测试剂，通过微孔板发光检测仪的自动进样系统先后注入裂解液和ATP检测试剂；需要至少配备有两个自动进样系统。
2)采用的是闪光型发光试剂，必须在加入ATP检测试剂后立刻检测发光值
闪光型发光试剂的发光持续时间较短，发光强度随时间衰减比较快，必须在加入ATP检测试剂后“立刻检测发光值”，否则影响检测结果。
三是无法满足MIC(最低抑菌浓度)测定的需求：
该方法中设置的抗菌药物的浓度梯度少，无法测定MIC(最低抑菌浓度)。因此该方法只是定性报告敏感性，并没有提供确切的MIC值。而定量结果MIC值对于指导临床医生用药则更为重要。
因此，需要开发一种对设备要求较低，而且能够测定MIC(最低抑菌浓度)值的快速的抗菌药物敏感试验的方法和试剂盒，便于各级医院和相关临床科室及时检测和监测药敏结果，为抗菌治疗提供简单、快速的解决方案。
发明内容
本发明的目的是研制一种快速检测抗菌药物敏感性的试剂盒，不仅能够在更短的时间内给出药敏结果，并提供确切的MIC(最低抑菌浓度)值，而且对硬件要求低。
本发明提供了一种新的快速检测抗菌药物敏感性的试剂盒，该试剂盒以微量肉汤稀释法为基础，包括有抗菌药物板、培养基和细菌ATP检测试剂。应用该试剂盒时，将待测细菌用试剂盒提供的培养基调至合适的浓度加入到抗菌药物板中，培养2小时后，将细菌ATP检测试剂加入到抗菌药物板中，而后将抗菌药物板放入多功能读板仪中检测发光强度，根据相对发光强度确定MIC值，并报告敏感、中介或耐药。
本发明提供了一种快速检测抗菌药物敏感性的试剂盒，其特征是，：
1)具有可直接检测细菌ATP的试剂：
所述试剂是含有细菌ATP释放剂和生物发光物质的检测试剂，可以一步实现细菌的裂解和ATP的释放与检测。
所述生物发光物质是辉光型生物发光物质；
辉光型生物发光物质的主要成分虽然也是萤光素酶和D-萤光素，但二者的比例不同，这两种物质的质量比是1:4至1:6，优选为1:4.7。
优选的细菌ATP检测试剂含有细菌ATP释放剂、缓冲液、萤光素酶、D-萤光素和稳定剂等。
其中，所述细菌ATP释放剂优选为表面活性剂，选自十六烷基三甲基溴化铵、苯扎氯铵、氯己定和曲拉通中的一种或多种。
优选的细菌ATP检测试剂是含有醋酸氯己定(0.15％-1.20％)、曲拉通X-100(1.5％-2.5％)、萤光素酶(0.02％-0.05％)和D-萤光素(0.1％-0.2％)的水溶液。所述百分比是质量比。
本发明的一个实例中所用的细菌ATP检测试剂由以下成分组成：N-2-羟乙基哌嗪-N'-2-乙磺酸(150-250mM)、十六烷基三甲基溴化铵(0.06％-0.10％)、醋酸氯己定(0.15％-1.2％)、曲拉通(1.5％-2.5％)、氯化镁(0.3％-0.5％)、乙二胺四乙酸(0.7％-1.0％)、氟化钠(0.05％-0.1％)、焦磷酸钠(0.01％-0.02％)、萤光素酶(0.02％-0.05％)、D-萤光素(0.1％-0.2％)和水。
本发明的细菌ATP检测试剂可以一步实现现有技术中细菌裂解液和ATP检测试剂两种试剂的功能。检测时，不需要先注入裂解液，再注入ATP检测试剂，不仅可以减少操作步骤，也不需要配置自动进样系统。
2)具有系列浓度梯度的抗菌药物板
本发明的抗菌药物板中具有系列浓度梯度的抗菌药物，所述系列浓度梯度是每种药物具有十个或以上的浓度。
对于不同的检测药物的具体浓度值根据实际需要设定。比如，参考CLSI文件设定，浓度范围包括CLSI文件所列的解释折点，且包含质控菌的值，可以满足MIC测定的需求。
抗菌药物的具体浓度梯度因为抗菌药物的性质而存在比较大的差异，例如哌拉西林的浓度为12800、6400、3200、1600、800、400、200、100、50和25μg/mL，而环丙沙星的浓度为200、100、50、25、12.5、6.25、3.12、1.56、0.78和0.39μg/mL。
3)可使用任何具有发光检测功能的仪器进行检测
使用任何具有发光检测功能的仪器都可以进行检测，得到所需的结果。而且不需要额外配备自动进样装置。
如，不仅可以使用现有技术使用的微孔板式发光检测仪，还可以用基层医疗机构都有配备的多功能读板仪(见本发明实施例)，无论使用何种仪器都不需要额外配备自动进样系统。
由于本发明的细菌ATP检测试剂是辉光型发光试剂，加入试剂后发光强度相对稳定且能持续一段时间，不需要加入试剂后立即检测，在使用时该检测试剂加入到抗菌药物板后，再将抗菌药物板放入到仪器中检测，就可以不需要自动进样系统。
多功能读板仪是多数基层医疗机构和相关临床科室都有配备的仪器。而微孔板式发光检测仪是专用化仪器，在上述机构的配置率比较低。
而对比文件方法并没有提示使用多功能读板仪可以实现其方法所要求的检测精度。
本发明的试剂盒提供的抗菌药物板中可以根据实际需要设置多种抗菌药物；抗菌药物的种类根据待测菌的种类变化，每种抗菌药物包括有多个浓度，抗菌药物的形态根据不同药物的具体情况而定。条件允许的情况下，优选的药物形态是固着在孔板底部的干燥粉状或颗粒状，便于试剂盒的保管运输。
本发明的试剂盒提供的培养基是参照CLSI(美国临床和实验室标准协会)的标准，根据待测菌的种类变化而定，可选用调节阳离子的Mueller-Hinton肉汤(CAMHB)、RPMI-1640肉汤培养基或其它药敏试验所需培养基。
本发明的试剂盒的应用方法是：将待测细菌加入到抗菌药物板中培养2h后，将所述细菌ATP检测试剂加入到抗菌药物板中，然后将整个抗菌药物板放入多功能读板仪中检测发光强度，计算抗菌药物孔相对无抗菌药物的阳性孔的相对发光强度。
上述加入到抗菌药物板中的待测细菌是用培养基调至所需浓度的待测细菌，所述所需浓度，根据实际需要而定，如参照CLSI标准确定合适的浓度(非苛养菌的浓度为约5×10 5CFU/mL)。
相对发光强度的计算方法如下：
相对发光强度＝(抗菌药物孔发光强度/阳性孔发光强度)×100％
从相对发光强度≤80％的最小药物浓度得到该药物的MIC值(最低抑菌浓度)，同时参照CLSI标准获得耐药与否的结论。根据CLSI提供的折点浓度，当MIC≤敏感折点时，待测菌对该抗菌药物敏感；当MIC≥耐药折点时，待测菌对抗菌药物耐药；当MIC介于两个折点之间时，待测菌对该抗菌药物的敏感度为中介。
采用本发明的试剂盒分别检测了大肠埃希氏菌和金黄色葡萄球菌对30种不同的抗菌药物的敏感性；同时分别检测了10株肠杆菌对氨苄西林和头孢唑啉，10株葡萄球菌对红霉素和青霉素的敏感性。以上的检测都可以在2h内报告MIC值，且与CLSI的标准方法报告的值相比都在CLSI标准允许的误差范围内。
本发明的试剂盒不仅可以定量检测，也可以定性检测。
实际应用中，如果只需要快速地得知抗菌药物耐药与否的结论，而不需要获得MIC值的情况下，用本发明的试剂盒操作更为简便：只使用抗菌药物板的阴性孔、阳性孔和抗菌药物折点浓度孔(包括敏感折点和耐药折点)，其余的操作与上述定量检测时一致。
具体操作方法是：将待测细菌加入到抗菌药物板中培养2h后，将所述细菌ATP检测试剂加入到抗菌药物板中，然后将抗菌药物板放入多功能读板仪中检测对应孔的发光强度，计算抗菌药物孔相对无抗菌药物的阳性孔的相对发光强度。抗菌药物折点浓度孔参照 CLSI标准确定，不同的微生物以及抗菌药物存在比较大的差异。例如检测肠杆菌科对氨苄西林的敏感性时选取阴性孔、阳性孔、400μg/mL(敏感折点)和1600μg/mL(耐药折点)的孔；检测肠杆菌科对头孢唑啉的敏感性时选取阴性孔、阳性孔、100μg/mL(敏感折点)和400μg/mL(耐药折点)的孔。
相对发光强度的计算方法如下：
相对发光强度＝(抗菌药物孔发光强度/阳性孔发光强度)×100％
当敏感折点和耐药折点浓度的相对发光强度都≤80％，则待测微生物对该抗菌药物敏感；当敏感折点浓度的相对发光强度＞80％，而耐药折点浓度的相对发光强度≤80％，则待测微生物对该抗菌药物敏感程度中介；当敏感折点和耐药折点浓度的相对发光强度都＞80％，则待测微生物对该抗菌药物耐药。
本发明的优点是：
1、更快速得到药敏实验结果
与对比文件相比，时间节约了一半。2小时便可得到药敏实验结果。
2、硬件要求低，可满足基层医院和相关临床科室的需要
可以使用微孔板式发光检测仪或常用的具有发光检测功能的多功能读板仪进行监测，不需要使用自动进样系统。
3、操作简便
一步加样操作，不需要分别注入裂解液和ATP检测试剂。
4、可以确定MIC值，并同时报告耐药与否
并不是仅仅定性确定药敏结果，而是得到MIC值，可以帮助临床医生适时选择合适的药物，及早避免产生或加重细菌的耐药。
附图说明：
图1不同数量的大肠埃希氏菌检测的发光值；
图2不同数量的金黄色葡萄球菌检测的发光值。
具体实施方式
以下实例仅用于说明和解释本发明，并不能限定本发明。本领域人员根据本发明的原理和方法用其它不同的药物、细菌和ATP检测试剂均有可能达到本发明的效果。
实施例1 配制细菌ATP检测试剂
按照以下步骤配制细菌ATP检测试剂：
(1)使用新鲜制备的高纯水配制100mL 150-250mM的N-2-羟乙基哌嗪-N'-2-乙磺酸(HEPES)溶液；加入0.15-1.20g醋酸氯己定，1.5-2.5mL曲拉通X-100(Triton X-100)， 0.3-0.5g六水合氯化镁，0.7-1.0g二水合乙二胺四乙酸二钠盐(EDTA)；使用1M的氢氧化钠调节溶液pH为7.5后，使用0.22μm的滤膜过滤除菌；加入20-50mg萤光素酶(Sigma)和100-200mg D-萤光素(Sigma)，溶解，-20℃保存。
或(2)使用新鲜制备的高纯水配制100mL 150-250mM的N-2-羟乙基哌嗪-N'-2-乙磺酸(HEPES)溶液；加入0.06-0.10g十六烷基三甲基溴化铵(CTAB)，0.15-1.20g醋酸氯己定，1.5-2.5mL曲拉通X-100(Triton X-100)，0.3-0.5g六水合氯化镁，0.7-1.0g二水合乙二胺四乙酸二钠盐(EDTA)，0.05-0.10g氟化钠和0.01-0.02g焦磷酸钠溶解；使用1M的氢氧化钠调节溶液pH为7.5后，使用0.22μm的滤膜过滤除菌；加入20-50mg萤光素酶(Sigma)和100-200mg D-萤光素(Sigma)，溶解，-20℃保存。
又或(3)使用新鲜制备的高纯水配制100mL 200mM的N-2-羟乙基哌嗪-N'-2-乙磺酸(HEPES)溶液；加入0.08g十六烷基三甲基溴化铵(CTAB)，0.16g醋酸氯己定，1mL曲拉通X-100(Triton X-100)，0.4g六水合氯化镁，0.86g二水合乙二胺四乙酸二钠盐(EDTA)，0.08g氟化钠和0.01g焦磷酸钠溶解；使用1M的氢氧化钠调节溶液pH为7.5后，使用0.22μm的滤膜过滤除菌；加入35.8mg萤光素酶(Sigma)和168mg D-萤光素(Sigma)，溶解，-20℃保存。
实施例2 细菌ATP检测试剂检测细菌
为了考察细菌ATP检测试剂的性能，复苏大肠埃希氏菌(ATCC 25922)和金黄色葡萄球菌(ATCC 29213)于TSA平板培养基上，37℃培养18-24h。用CAMHB肉汤将两种菌分别稀释成一系列浓度，移取一定体积的稀释菌液到空白多孔板中，加入一定体积的实施例1中配制的细菌ATP检测试剂，放入多功能读板仪(PerkinElmer)中，检测各孔的发光值，每个浓度重复三个孔。
表1和表2分别是检测不同数量的大肠埃希氏菌和金黄色葡萄球菌的发光值，图1和图2所示的是检测的发光强度与细菌数量之间的关系。从图中可以看到，在很大的范围内发光强度与细菌数成正相关性。考虑到背景及其标准偏差，发光法检测大肠埃希氏菌和金黄色葡萄球菌的最低检测限分别可以达到25CFU和20CFU。该结果表明配制的细菌ATP检测试剂可以应用于细菌的检测，且具有非常高的检测灵敏度，可以检测非常低数量的细菌。
表1检测不同数量大肠埃希氏菌的发光值
细菌数(CFU) 发光强度(RLU)
0 149.0±15.4
25 677.3±100.0
250 2281.3±53.5
2500 20042.3±905.6
25000 168955.7±6059.5
250000 2034397.0±87456.9
表2检测不同数量金黄色葡萄球菌的发光值
细菌数(CFU) 发光强度(RLU)
0 95.3±20.8
20 464.7±56.1
200 1472.3±167.7
2000 13032.0±1926.1
20000 122109.3±2550.2
200000 1552810.0±78042.5
实施例3 检测抗生素作用后细菌数量
为了检测抗生素作用2h后细菌数量的变化，复苏大肠埃希氏菌(ATCC 25922)和金黄色葡萄球菌(ATCC 29213)于TSA平板培养基上，37℃培养18-24h。在96孔板中以1号列作为阴性对照，6号列作为阳性对照；A行是头孢唑啉，B和E行是哌拉西林，C和G行是奈替米星，D行是左氧氟沙星，F行是米诺环素。在孔板的1到6号列各孔均加入50μL的CAMHB肉汤，将抗生素使用CAMHB肉汤稀释到所需浓度(头孢唑啉4μg/mL，哌拉西林16μg/mL，奈替米星2μg/mL，左氧氟沙星0.125μg/mL，米诺环素1μg/mL)，在2号列加入50μL上述浓度的抗生素稀释液到对应的行中，采用逐级倍比稀释的方法稀释抗生素至3-5号列，5号列多余的50μL溶液去掉。从培养的琼脂平皿中挑选几个菌落直接在肉汤中制成0.5麦氏单位浊度的菌悬液，并进一步使用肉汤稀释150倍；A-D行加入大肠埃希氏菌稀释液，E-G行加入金黄色葡萄球菌稀释液，在1号列加入50μL肉汤，在2-6号列分别加入50μL的稀释菌液。37℃培养2h后，在各孔加入100μL的细菌ATP检测试剂，放入多功能读板仪中，记录各孔的发光值。实验重复3块多孔板，对数据进行处理。
表3是不同浓度抗生素作用2h后检测的相对发光强度，由于发光强度与细菌数量之间的正相关性，相对发光强度的变化反映了细菌数量的变化。抗生素浓度增大，相对发光强度降低，则可以认为细菌的数量的降低，这一结果表明，抗生素作用2h后的细菌数量的变化是可以通过发光法检测出来的，同时表明使用本发明的细菌ATP检测试剂有望在2 h内报告药敏结果。
表3检测不同浓度抗生素作用后细菌的相对发光强度
Figure PCTCN2018102508-appb-000001
实施例4抗菌药物敏感性快速检测试剂盒的制备
包含如下：
1)抗菌药物板：
制备方法：384孔板中，1号列为阴性对照，24号列为阳性对照，2-11号列和14-23号列是抗生素列。将头孢唑啉配成3200、1600、800、400、200、100、50、25、12.5、6.25μg/mL的浓度，分别加入到A行的2-11号孔，每孔0.5μL。将苯唑西林配成1600、800、400、200、100、50、25、12.5、6.25、3.125μg/mL的浓度，分别加入到A行的14-23号孔，每孔0.5μL。类似地将其它浓度的抗菌药物加入到对应的孔中，最后负压干燥。
2)10mL细菌ATP检测试剂一瓶(成分如前述)。
3)20mL的CAMHB肉汤(或其他肉汤，依据检测菌的不同而配备不同培养基)一瓶。
实施例5 试剂盒应用于检测多种抗菌药物的MIC值
为了考察试剂盒应用于检测多种抗菌药物的MIC值，复苏大肠埃希氏菌(CMCC 44103)和金黄色葡萄球菌(ATCC 25923)于TSA平板培养基上，37℃培养18-24h。使用抗菌药物敏感性快速检测试剂盒进行快速检测MIC值：从培养的琼脂平皿中挑选几个菌落直接在CAMHB肉汤中制成0.5麦氏单位浊度的菌悬液，并进一步使用肉汤稀释300倍；在抗菌药物板中，阴性列加入25μL肉汤，在抗菌药物列和阳性列分别加入25μL的稀释菌液；37℃培养2h后，在各孔加入25μL的细菌ATP检测试剂，放入多功能读板仪中，记录各孔的发光值；对数据进行处理，以相对发光强度≤80％的最小浓度确定为检测MIC值。同时采用微量肉汤稀释法进行实验，确定标准MIC值。
表4所示的是试剂盒检测多种抗生素对细菌MIC值与标准方法检测的MIC的比较。结果表明，试剂盒检测法与标准法的MIC值多数相同，虽然偶尔出现结果差一个倍比梯度的情况，但标准方法的方法学允许误差是偏差一个倍比梯度，因此可以说试剂盒检测法的结果与稀释法是一致的。本发明的试剂盒是可以应用于检测常见的多种抗生素的MIC值的。
表4试剂盒检测法与标准法测定的MIC结果(μg/mL)
Figure PCTCN2018102508-appb-000002
Figure PCTCN2018102508-appb-000003
实施例6 试剂盒应用于检测多株菌的MIC值
为了考察试剂盒应用于检测多株菌的情况，复苏实验菌株于TSA平板培养基上，37℃培养18-24h。使用抗菌药物敏感性快速检测试剂盒进行快速检测MIC值：从培养的琼脂平皿中挑选几个菌落直接在CAMHB肉汤中制成0.5麦氏单位浊度的菌悬液，并进一步使用肉汤稀释300倍；在抗菌药物板中，阴性列加入25μL肉汤，在抗菌药物列和阳性列分别加入25μL的稀释菌液；37℃培养2h后，在各孔加入25μL的细菌ATP检测试剂，放入多功能读板仪中，记录各孔的发光值；对数据进行处理，以相对发光强度≤80％的最小 浓度为确定检测MIC值。同时采用微量肉汤稀释法进行实验，确定标准MIC值。
表5和表6分别是采用试剂盒检测肠杆菌和葡萄球菌的MIC值及与标准方法的比较。检测结果表明，试剂盒检测法与标准法的MIC值多数相同，虽然偶尔出现结果差一个倍比梯度的情况，但标准方法的方法学允许误差是偏差一个倍比梯度，因此可以说试剂盒检测法的结果与稀释法是一致的。本发明的试剂盒可以适用于多株菌的MIC值的测定。
表5试剂盒检测法与标准法测定的MIC结果(μg/mL)
Figure PCTCN2018102508-appb-000004
表6试剂盒检测法与标准法测定的MIC结果(μg/mL)
Figure PCTCN2018102508-appb-000005
Figure PCTCN2018102508-appb-000006

Claims (10)

  1. 一种快速定量检测抗菌药物敏感性的试剂盒，其特征是，具有可直接检测细菌ATP的试剂；所述直接检测，是一次加入所述试剂后一步实现细菌的裂解和ATP的释放与检测。
  2. 权利要求1所述的试剂盒，其特征是，还含有具有系列浓度梯度的抗菌药物板，所述系列浓度梯度是每种药物具有十个或以上的浓度，且所述药物固着于孔板底部。
  3. 权利要求1所述的试剂盒，其特征是，可使用任何具有发光检测功能的仪器进行检测，且不需要配备自动进样装置。
  4. 权利要求1所述的试剂盒，所述试剂是含有细菌ATP释放剂和辉光型生物发光物质的检测试剂。
  5. 权利要求4所述的试剂盒，所述细菌ATP释放剂为表面活性剂，选自十六烷基三甲基溴化铵、苯扎氯铵、氯己定和曲拉通中的一种或多种；
    所述辉光型生物发光物质是含萤光素酶、D-萤光素的物质，特征是：萤光素酶与D-萤光素的质量比是1:4～6，
  6. 权利要求5所述的试剂盒，萤光素酶与D-萤光素的质量比是1:4.7。
  7. 权利要求1所述的试剂盒的使用方法，其特征是：将待测细菌加入到抗菌药物板中培养后，加入所述可直接检测细菌ATP的检测试剂，将整个抗菌药物板放入检测仪中检测各孔的发光强度，然后计算抗菌药物孔相对无抗菌药物的阳性孔的相对发光强度；
    其特征是：
    1)一步加样操作，不需要先注入裂解液；
    2)不要求加入试剂后立即检测；
    3)不需要自动进样设备；
    4)培养2小时后获得结果。
  8. 权利要求7所述的使用方法，所述检测仪是任何具有发光检测功能的仪器。
  9. 权利要求7所述的使用方法，当只需要定性获得抗菌药物耐药与否的结论时，所 用抗菌药物板不需要具有系列浓度梯度，只有抗菌药物折点浓度；对比阴性孔、阳性孔和抗菌药物折点浓度孔的发光强度即可，其余操作与权利要求7的方法一致；所述抗菌药物折点是敏感折点和耐药折点两个浓度。
  10. 一种快速定量检测抗菌药物敏感性的方法，其特征是，使用可直接检测细菌ATP的试剂；所述直接检测，是一次加入所述试剂后一步实现细菌的裂解和ATP的释放与检测。
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