CN112391441B - Method for identifying mycobacterium tuberculosis rifampicin heterogeneous drug resistance based on fluorescein flow cytometry - Google Patents
Method for identifying mycobacterium tuberculosis rifampicin heterogeneous drug resistance based on fluorescein flow cytometry Download PDFInfo
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Abstract
The invention relates to the field of drug sensitivity detection, in particular to a method for identifying the rifampicin heterogeneous drug resistance of mycobacterium tuberculosis by using a fluorescein-based flow cytometry, which comprises the following steps: s1, preparing working solution; s2, treating the analysis bacterial liquid for 10 days or 14 days by using the substitution liquid and the rifampicin working solution respectively; s3: respectively dyeing the treated bacterial liquids; s4: and (3) respectively detecting the dyed bacteria liquid, recording the average fluorescence intensity value of each bacteria liquid, calculating the sensitivity index of each bacteria liquid, and judging the sensitivity of each bacteria liquid to rifampicin according to the sensitivity index: when the sensitivity index is larger than a critical value, judging that the detected mycobacterium tuberculosis is heterogeneous drug-resistant mycobacterium tuberculosis or drug-resistant mycobacterium tuberculosis; judging the sensitive bacteria when the sensitivity index is lower than a critical value; the critical value ranges from 0.48 or 0.37. The method has high accuracy, can quickly obtain the detection result of the mycobacterium tuberculosis rifampicin heterogeneous drug resistance within 10 days, and has high accuracy, sensitivity and specificity.
Description
Technical Field
The invention relates to the field of drug sensitivity detection, in particular to a method for identifying rifampicin heterogeneous drug resistance of mycobacterium tuberculosis based on fluorescein flow cytometry.
Background
Flow cytometry has the advantages of high throughput, multiple parameters, high efficiency and the like, and is increasingly applied to microbial drug sensitive detection. The essence of detecting bacterial drug sensitivity is to monitor the viability of bacteria after drug treatment, and flow cytometry is to reflect the viability of bacteria by monitoring the difference in fluorescence values of bacteria under different conditions after interaction with fluorochromes. The sensitive bacteria have reduced activity after being acted by antibiotics, the activity of the drug-resistant bacteria is unchanged, if microorganisms with different activities or functional states are combined with specific fluorescent dye to emit fluorescence with different intensities, the drug sensitive information of the bacteria can be judged by detecting the change of the fluorescence intensity. Fluorescein diacetate is a hydrophobic fluorescein derivative with cell membrane permeability, can be catalyzed and hydrolyzed by esterase in cells to generate fluorescein emitting green fluorescence after entering the cells, dead cells without complete cell membranes or with weak metabolic activity cannot hydrolyze the fluorescein diacetate due to inactivation of the esterase, and therefore, the fluorescein diacetate is often used for detecting the cell activity. At present, flow cytometry based on the dye gradually becomes one of the methods for rapidly detecting the sensitivity of microbial drugs such as candida albicans, mycobacterium tuberculosis, staphylococcus aureus and the like.
Mycobacterium Tuberculosis (MTB), the causative agent of tuberculosis, is still one of the most major public health problems worldwide. In recent years, MTB rapidly shows drug resistance and tends to be enhanced due to unreasonable administration, side effects of other drugs, food safety, environmental factors, and the like, which decrease the immunity of human populations. Under the condition that no new medicine is available, accurate drug resistance detection becomes an important auxiliary link for treating tuberculosis caused by MTB. There are also many scholars doing this.
Heterogeneous resistance is a particular phenomenon of resistance. Although the drug resistance detection technology is mature at present, for example, the traditional ratio method DST is a method for detecting drug resistance with strong reliability and high sensitivity, the current technology must rely on genetic detection technology to detect and obtain the heterogeneous drug resistance of pathogenic bacteria. However, the genetic detection has the defects that only the drug resistance gene mutation clearly related to the phenotype can be detected, and the undetermined drug resistance gene cannot be identified, so that the detection accuracy is influenced, and the clinical reference value of the detection result is reduced.
At present, no other more accurate and convenient technical means are available for effectively applying to the heterogeneous drug resistance detection of mycobacterium tuberculosis.
Disclosure of Invention
Based on the above, the invention aims to provide a method for identifying rifampicin heterogeneous resistance of mycobacterium tuberculosis by using fluorescein-based flow cytometry, wherein the rifampicin heterogeneous resistance of the mycobacterium tuberculosis is detected by using fluorescein diacetate-based flow cytometry, the principle is based on phenotype, but the detection time is greatly shortened, and the method is faster and more efficient than a proportional method and an absolute concentration method and is more accurate than a genotype method. And because the gene is based on phenotype and does not depend on whether the drug resistance gene is clear or not, the application of the gene to the heterogeneous drug resistance detection of other drugs of the mycobacterium tuberculosis can be expanded besides rifampicin.
The technical scheme of the invention is as follows:
a method for identifying mycobacterium tuberculosis rifampicin heterogeneous resistance by fluorescein-based flow cytometry, comprising the steps of:
s1, preparation of working solution: respectively preparing fluorescein diacetate staining solution, rifampin working solution and mycobacterium tuberculosis analysis bacterial solution;
s2, carrying out drug treatment on the analysis bacterial liquid: dividing the analytical bacteria liquid into a control group and a test group, and respectively treating the analytical bacteria liquid of the control group and the test group for 10-14 days by using a substitution liquid and a rifampicin working solution;
s3: respectively staining the mycobacterium tuberculosis bacterial liquid treated in the step S2;
s4: respectively detecting, recording the average fluorescence intensity value of each bacterial liquid, calculating the sensitivity index of each bacterial liquid, and judging the sensitivity of each bacterial liquid to rifampicin according to the sensitivity index;
the calculation method of the sensitivity index comprises the following steps: sensitivity index = mean fluorescence intensity of experimental group/mean fluorescence intensity of control group;
the evaluation criteria for the sensitivity of rifampicin were: when the sensitivity index is larger than a critical value, judging that the detected mycobacterium tuberculosis is heterogeneous drug-resistant mycobacterium tuberculosis; judging the sensitive bacteria when the sensitivity index is lower than a critical value;
the critical value range is 0.48-0.37; the critical value is in inverse proportion to the number of treatment days;
the substitute liquid is a liquid without any anti-mycobacterium tuberculosis substance.
Preferably, when the processing time is 10 days in step S2, the critical value in step S4 is 0.48; when the processing time is 14 days in step S2, the threshold value in step S4 is 0.37.
Further, the preparation of the diacetate working solution in S1 includes the specific operations: and taking out the fluorescein diacetate fluorescent dye stock solution, unfreezing the fluorescein diacetate fluorescent dye stock solution in the dark condition, and diluting the fluorescein diacetate fluorescent dye stock solution by using PBS to prepare the fluorescein diacetate dyeing solution with the concentration of 0.5 mu g/mL.
Further, the preparation of the rifampicin working solution in S1 specifically comprises the following steps: taking out the rifampicin stock solution from the refrigerator, thawing at room temperature, and diluting with normal saline to obtain rifampicin working solution with concentration of 500 μ g/mL for use. The concentration of the rifampicin working solution can be any concentration theoretically, the concentration of the rifampicin working solution is set to be 500 mug/mL, the optimal rifampicin working solution concentration is selected by taking account of controllability of final drug concentration and My turbidity in subsequent operations, and on the premise of maintaining the final drug concentration, the volume of the added drug working solution is reduced as much as possible, so that the My turbidity of the bacteria solution is controlled more easily and accurately.
Further, the preparation of the mycobacterium tuberculosis analysis bacterial liquid described in S1 specifically includes: dipping the culture solution to grind the mycobacterium tuberculosis thallus into a bacterium solution homogenate, adding a proper amount of the culture solution, mixing uniformly, filtering by using a filter screen, and preparing into a 1 McLeod turbidity bacterium solution for later use.
Further, the culture solution comprises 7H9 culture solution; the growth speeds of the mycobacterium tuberculosis in different culture solutions are different, and the corresponding critical values of the sensitivity indexes are also different, and the critical value of the sensitivity index is suitable for a culture medium of a 7H9 culture solution and a culture medium with the same component as that of a 7H9 culture solution, or the culture medium with the same culture effect as that of a 7H9 culture solution; in theory, the method is still applicable to other culture solutions, but the sensitivity index critical value of the mycobacterium tuberculosis is determined according to the growth rate of the mycobacterium tuberculosis.
Further, the specific operation method in step S2 includes: setting a control group and an experimental group; the treatment method of the experimental group comprises the following steps: sufficiently and uniformly mixing the bacteria liquid, transferring a certain amount of bacteria liquid into a sterile test tube, adding rifampicin working solution to prepare bacteria liquid with final concentration of rifampicin of 50 mu g/mL and McLeod turbidity of 0.9, and incubating at constant temperature;
the control group treatment method comprises the following steps: and (3) fully and uniformly mixing the bacteria liquid, transferring the bacteria liquid of the same sample with the volume same as that of the bacteria liquid of the experimental group into a sterile test tube, adding a substitute liquid to supplement the volume of the bacteria liquid to be the same as that of the bacteria liquid of the experimental group, fully and uniformly mixing, and incubating at a constant temperature.
The incubation temperature, incubation time and other incubation environments and incubation conditions of the experimental group and the control group are completely the same.
Preferably, the substitution liquid includes physiological saline, sterile water, and the like.
Further, the incubation temperature of the incubation operation in the step S2 is 35-37 ℃, and the incubation time is 10-14 days; incubation at 37 ℃ for 10 days is preferred.
Further, the staining treatment of the mycobacterium tuberculosis bacterial solution in the step S3 includes: respectively transferring bacteria liquid in an experimental tube and a control tube, respectively performing centrifugal treatment, removing supernatant, adding normal saline into the precipitate, dipping the bacteria liquid by using a sterile glass rod, grinding the bacteria into bacteria liquid homogenate, vibrating and resuspending to obtain suspension, dispersing by using a bacteria ultrasonic dispersion instrument, filtering by using a filter screen, transferring the obtained bacteria liquid into a flow type sample loading tube, adding diacetate working solution with the same volume as the bacteria liquid, uniformly mixing, and dyeing in a dark place for 30 min.
The normal saline is adopted to dilute and precipitate and the bacteria are ground manually, so that the bacteria dispersion degree is better, and the problems that the fluorescence intensity value is inaccurate and the calculation of the sensitivity index is influenced finally due to incomplete or uneven dyeing caused by accumulation or mutual adhesion and superposition of the bacteria are avoided. After manual grinding and dispersion, the bacteria liquid is dispersed by an ultrasonic disperser, so that the dispersion degree of the bacteria liquid can be further ensured, and the operation efficiency and the result accuracy of the whole method are improved.
Further, the centrifugation condition in the step S3 is 13000 rpm centrifugation for 10 min.
Further, in step S3, physiological saline is added to the sediment in an amount equal to the volume of the removed bacterial fluid.
Further, the dispersion operation method of the ultrasonic disperser in step S3 includes: the two procedures of 5 seconds of ultrasound and 5 seconds of pause are carried out alternately for 30 seconds.
Further, the screens used in step S1 and step S3 are 300 mesh screens.
The invention has the beneficial effects that:
heterogeneous drug resistance is the main reason for the inconsistency between the DST and the detection result of genetic drug resistance, and the concentration and titer of the required drug in the culture medium and the skill of laboratory operators can determine the accuracy of the drug sensitivity test result. The method optimizes the experimental operation standards (such as culture solution selection, pretreatment operation before dyeing and the like), is convenient for experimenters to carry out uniformly, and simultaneously provides the optimal numerical relationship, thereby avoiding the experimental error accumulation (such as the accumulation of errors during reagent concentration conversion, errors of Mycoplasma turbidimetry and the like) in the operation process and reflecting the greater influence on the final evaluation result. The method of the invention adopts the final concentration of rifampicin of 50 mug/mL to treat the mycobacterium tuberculosis bacteria liquid to be detected for 10 days, the flow cytometry based on fluorescein diacetate can effectively distinguish rifampicin all-sensitive mycobacterium tuberculosis and a heterogeneous bacteria liquid sample containing 25% rifampicin resistant mycobacterium tuberculosis, and the detection sensitivity and specificity are optimal and are respectively 96.2% and 96.7%. The method can quickly obtain the detection result of the mycobacterium tuberculosis rifampicin heterogeneous drug resistance in 10 days, has higher accuracy (93.33 percent), is faster (4-6 weeks) than a proportional method and is more accurate (66.67 percent) than a genotype method.
Drawings
FIG. 1 is a comparison of the distribution of sensitivity indexes obtained when a bacteria liquid sample containing 25% (i.e., 75% of sensitive bacteria) and 50% (i.e., 50% of sensitive bacteria) of rifampicin-resistant Mycobacterium tuberculosis and a bacteria liquid sample containing 0% (i.e., 100% of sensitive bacteria) of rifampicin-resistant Mycobacterium tuberculosis are treated for 10 days by the detection method of the present invention. Wherein 0%, 25% and 50% respectively represent the proportion of rifampicin resistant strains in the bacterial liquid sample, and 0.48 is the threshold value for detecting rifampicin heterogeneous drug resistant mycobacterium tuberculosis strains and rifampicin sensitive mycobacterium tuberculosis strains by the method.
FIG. 2 is a comparison of the distribution of sensitivity indexes obtained when a sample of a bacterial liquid containing rifampicin-resistant M.tuberculosis 75% (i.e., containing 25% of sensitive bacteria) and 100% (i.e., containing 0% of sensitive bacteria) is treated for 10 days by the detection method of the present invention with a sample of a bacterial liquid containing rifampicin-resistant M.tuberculosis 0% (i.e., containing 100% of sensitive bacteria). Wherein 0%, 75% and 100% respectively represent the proportion of rifampicin resistant strains in the bacterial liquid sample, and 0.48 is the threshold value for detecting rifampicin heterogeneous drug resistant mycobacterium tuberculosis strains and rifampicin sensitive mycobacterium tuberculosis strains by the method.
FIG. 3 is a comparison of the distribution of sensitivity indexes obtained when a bacteria liquid sample containing 25% (i.e., 75% of sensitive bacteria) and 50% (i.e., 50% of sensitive bacteria) of rifampicin-resistant Mycobacterium tuberculosis is treated for 14 days by the detection method of the present invention, and a bacteria liquid sample containing 0% (i.e., 100% of sensitive bacteria) of rifampicin-resistant Mycobacterium tuberculosis is obtained. Wherein 0%, 25% and 50% respectively represent the proportion of rifampicin resistant strains in the bacterial liquid sample, and 0.37 is the cut-off value for detecting rifampicin heterogeneous drug resistant mycobacterium tuberculosis strains and rifampicin sensitive mycobacterium tuberculosis strains by the method.
FIG. 4 is a comparison of the distribution of sensitivity indexes obtained when a sample of a bacterial liquid containing rifampicin-resistant M.tuberculosis 75% (i.e., containing 25% of sensitive bacteria) and 100% (i.e., containing 0% of sensitive bacteria) is treated for 14 days by the detection method of the present invention with a sample of a bacterial liquid containing rifampicin-resistant M.tuberculosis 0% (i.e., containing 100% of sensitive bacteria). Wherein 0%, 75% and 100% respectively represent the proportion of rifampicin resistant strains in the bacterial liquid sample, and 0.37 is the cut-off value for detecting rifampicin heterogeneous drug resistant mycobacterium tuberculosis strains and rifampicin sensitive mycobacterium tuberculosis strains by the method.
Detailed Description
The implementations described in the exemplary embodiments below are not intended to represent all implementations consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.
The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.
The specific techniques or conditions not indicated in the examples of this application are performed according to the techniques or conditions described in the literature in the field or according to the product description. The reagents or instruments used in the examples of the present application are not indicated by manufacturers, and are all conventional products available from commercial sources and the like.
Example A method of the invention for the identification of rifampicin-heterogeneous resistance of Mycobacterium tuberculosis by fluorescein-based flow cytometry
1.1 apparatus and materials
The flow cytometer FACSAria II is manufactured by BD corporation in the United states, and the bacterial ultrasonic disperser BACSpreadERTM 1100 is manufactured by Bikang Biotech, Inc., Guangdong body; 300 mesh stainless steel cell filters were purchased from Saint Biotechnology, Inc., Middlebrook 7H10 solid medium and Middlebrook 7H9 liquid medium from BD, Inc., USA, fluorescein diacetate from LIFE, PBS (pH 7.4. + -. 0.1) from Gino biomedical technologies, Inc., Hangzhou.
1.2 identification method
S1, preparation of working solution:
fluorescein diacetate fluorescent dye: and (3) taking out the fluorescein diacetate fluorescent dye stock solution, unfreezing the stock solution in the dark condition, and diluting the stock solution by using PBS to prepare 0.5 mu g/mL working solution.
Preparing a rifampicin working solution: taking out from refrigerator at-80 deg.C to obtain a concentration of 2 × 10 4Mu g/mL rifampicin stock solution is thawed at room temperature, and after being completely dissolved, the rifampicin stock solution is diluted into rifampicin working solution with the concentration of 500 mu g/mL by using physiological saline for standby.
Preparation of an analytical bacterial liquid: dipping 7H9 culture solution by using a sterile glass rod, grinding the mycobacterium tuberculosis thallus into a bacterial liquid homogenate, adding a proper amount of 7H9 culture solution, uniformly mixing, and filtering by using a 300-mesh sterile stainless steel filter screen to obtain a bacterial liquid; carrying out turbidimetry by using a bacterial ultrasonic dispersion counter to prepare a 1 McLeptobacterium suspension for later use.
S2, analyzing the drug treatment of the bacterial liquid:
and (3) sufficiently and uniformly mixing the bacterial liquid of the S1, transferring 1800 mu L of bacterial liquid into a sterile test tube, adding 200 mu L of normal saline, tightly covering a cover, sufficiently and uniformly mixing, and placing the mixture in a thermostat with 37 ℃ (the temperature can be replaced by any temperature between 35 ℃ and 37 ℃) for incubation for 10 days (the temperature can be replaced by any number of days between 10 and 14), thus obtaining the control tube. And (3) sufficiently and uniformly mixing the bacterial liquid of S1, transferring 1800 mu L of bacterial liquid into a sterile test tube, adding 200 mu L of rifampicin working solution, preparing bacterial liquid with final rifampicin concentration of 50 mu g/mL, and placing the bacterial liquid in a thermostat with 37 ℃ (the temperature can be changed to any temperature between 35 ℃ and 37 ℃) for incubation for 10 days (the time can be changed to any day between 10 days and 14 days).
S3, staining of a mycobacterium tuberculosis suspension sample: taking out the experiment tube and the control tube from the thermostat, respectively transferring 1000 mu L of bacterial liquid into a high-temperature sterilization EP tube, centrifuging at 13000 rpm for 10 min, then removing supernatant, leaving precipitate, adding 1000 mu L of physiological saline into the precipitate, dipping the bacteria into sterile physiological saline by using a sterile glass rod, grinding the bacteria into bacterial liquid homogenate, shaking and resuspending to obtain suspension, dispersing by using a bacterial ultrasonic disperser (the two procedures of 5 seconds of ultrasound and 5 seconds of pause are alternately carried out for 30 seconds), filtering by using a 300-mesh stainless steel cell filter screen, transferring 300 mu L of bacterial liquid into a flow-type sample feeding tube, adding 300 mu L of fluorescein diacetate fluorescent dye working solution, uniformly mixing, and dyeing in a dark place for 30 min.
S4, detecting the bacterial liquid dyed in the S3 by using a flow cytometer, recording the average fluorescence intensity value of the bacterial liquid, and calculating the sensitivity index of the bacterial liquid; judging the sensitivity of the rifampicin to the rifampicin according to the sensitivity index; wherein the content of the first and second substances,
sensitivity index = mean fluorescence intensity of experimental tubes/mean fluorescence intensity of control tubes;
sensitive drug resistance threshold: when the sensitivity index is larger than the critical value, the mycobacterium tuberculosis to be detected is judged to be the heterogeneous drug-resistant mycobacterium tuberculosis, and when the sensitivity index is lower than the critical value, the mycobacterium tuberculosis is judged to be the rifampicin sensitive bacteria.
The selection range of the critical value is 0.48-0.37, and the critical value is in inverse proportion to the treatment days. When the treatment time is between 10 and 14 days (including 10 days and 14 days), any critical value in the range of 0.48 to 0.37 is theoretically applicable, with only differences in detection accuracy, sensitivity and specificity. The preferred scheme of the invention is as follows: when the processing time is 10 days in step S2, the threshold value in step S4 is 0.48; when the processing time is 14 days in step S2, the threshold value in step S4 is 0.37.
EXAMPLE two detection of Rifampicin heteroresistance of Mycobacterium tuberculosis Using the method of the present invention
Taking 30 strains of mycobacterium tuberculosis and 5 bacteria liquid samples which are determined to be rifampicin-heterogeneous drug-resistant mycobacterium tuberculosis, wherein each drug-resistant strain is respectively provided with 5 bacteria liquid samples, and the 5 bacteria liquid samples are respectively bacteria liquid samples containing rifampicin-resistant mycobacterium tuberculosis with the ratio of 0% (namely containing 100% of sensitive bacteria), 25% (namely containing 75% of sensitive bacteria), 50% (namely containing 50% of sensitive bacteria), 75% (namely containing 25% of sensitive bacteria) and 100% (namely containing 0% of sensitive bacteria).
According to the operation method of the embodiment 1, 150 prepared bacterial liquid samples are respectively detected according to the method of the embodiment one, when the drug (final concentration of rifampicin is 50 μ g/mL) is treated on the 10 th day and the 14 th day, the samples are respectively sampled, dyed and detected, the respective sensitivity indexes are calculated, and distribution graphs are made (see the picture 1 to the picture 4), wherein the picture 1 shows the sensitivity index distribution of each sample when bacterial liquid samples of concentration groups containing rifampicin-resistant mycobacterium tuberculosis accounting for 0%, 25% and 50% (each concentration group contains 30 samples of rifampicin-heterogeneously resistant mycobacterium tuberculosis) are treated on the 10 th day; FIG. 2 is a graph showing the sensitivity index distribution of each sample on day 10 of the treatment of the bacterial liquid sample containing concentration groups of rifampicin-resistant M.tuberculosis at ratios of 0%, 75% and 100%; FIG. 3 is a graph showing the sensitivity index distribution of samples on day 14 after treatment of the bacterial suspension samples containing concentration groups of 0%, 25% and 50% rifampicin-resistant Mycobacterium tuberculosis; FIG. 4 is a graph showing the sensitivity index distribution of each sample on day 14 of the treatment of the bacterial suspension sample containing rifampicin-resistant Mycobacterium tuberculosis at concentrations of 0%, 75% and 100%. The ROC curve analysis was performed again and the results are shown in Table 1.
Table 1: ROC curve analysis result of treated bacteria liquid fluorescein diacetate dyeing sensitivity index
Description of the drawings: "0% vs 25%" refers to the comparison between the bacteria liquid with rifampicin-resistant mycobacterium tuberculosis 0% and 25% in the mycobacterium tuberculosis heterogeneous bacteria liquid sample; the same holds true.
It can be seen that: when the sample is treated for 10 days and 14 days, the flow cytometry based on fluorescein diacetate can distinguish bacterial liquid samples containing different proportions of drug-resistant bacteria, wherein the identification effect on heterogeneous bacterial liquid samples containing 0 percent and 25 percent of rifampicin-resistant mycobacterium tuberculosis is the best, and the detection performance difference between samples in other proportions is not obvious.
The method can quickly obtain the detection result of rifampicin heterogeneous drug resistance of the mycobacterium tuberculosis within 10 days, distinguishes the rifampicin full-sensitive mycobacterium tuberculosis and a heterogeneous bacterium liquid sample containing 25% rifampicin drug-resistant mycobacterium tuberculosis by using a critical value standard of 0.48, has the best detection effect, has the sensitivity and specificity of 96.2% and 96.7% respectively, and has the higher accuracy of 93.33%. In 14 days, a 0.37 cutoff standard is used for identifying rifampicin full-sensitive mycobacterium tuberculosis and a heterogeneous bacterium liquid sample containing 25% rifampicin resistant mycobacterium tuberculosis, the detection effect is good, and the sensitivity and the specificity are respectively 96.7% and 93.3%.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (7)
1. A method for fluorescein-based flow cytometry for identifying rifampin-heterogeneous resistance of mycobacterium tuberculosis, the method comprising the steps of:
s1, preparation of working solution: preparing rifampicin working solution and mycobacterium tuberculosis analysis bacterial solution;
s2, setting a control group and an experimental group;
the treatment method of the experimental group comprises the following steps: transferring a certain amount of the bacterial liquid prepared in S1, adding a rifampicin working solution to prepare a bacterial liquid containing rifampicin with the final concentration of 50 mu g/mL and the McLeod turbidity of 0.9, and incubating at the constant temperature of 35-37 ℃ for 10 days or 14 days;
the treatment method of the control group comprises the following steps: transferring the bacterial liquid prepared in S1 with the same volume as that of the bacterial liquid of the experimental group, adding substitute liquid normal saline to supplement the volume of the bacterial liquid to be the same as that of the bacterial liquid of the experimental group, and incubating at the constant temperature of 35-37 ℃ for 10 days or 14 days;
s3: respectively transferring the bacterial liquid in the experimental group and the control group which are treated in S2, performing centrifugal treatment, removing supernatant, adding physiological saline into the precipitate, dipping sterile physiological saline by using a sterile glass rod, grinding the bacteria into bacterial liquid homogenate, vibrating, resuspending, redispersing, filtering by using a filter screen, transferring the obtained bacterial liquid, adding fluorescein diacetate staining solution, and performing dark staining for 30 min;
s4: respectively detecting the stained bacteria liquid in the S3, recording the average fluorescence intensity value of each bacteria liquid, calculating a sensitivity index, and judging the sensitivity of the detected mycobacterium tuberculosis to rifampicin according to the sensitivity index;
the calculation method of the sensitivity index comprises the following steps: sensitivity index = mean fluorescence intensity of experimental group/mean fluorescence intensity of control group;
the evaluation criteria for the sensitivity of rifampicin were: when the sensitivity index is larger than a critical value, judging that the detected mycobacterium tuberculosis is heterogeneous drug-resistant mycobacterium tuberculosis; when the sensitivity index is lower than the critical value, the detected mycobacterium tuberculosis is judged to be sensitive bacteria;
the critical value is 0.48 or 0.37; the method specifically comprises the following steps: when the processing time is 10 days in the step S2, the critical value in the step S4 is 0.48; when the processing time in the step S2 is 14 days, the critical value in the step S4 is 0.37;
the substitute liquid is a liquid which does not contain any substance against mycobacterium tuberculosis.
2. The method for the identification of rifampicin-heterogeneous resistance of mycobacterium tuberculosis by fluorescein-based flow cytometry as claimed in claim 1, wherein the rifampicin working solution in S1 is prepared by the following steps: prepare the rifampicin working solution with the concentration of 500 mug/mL.
3. The method for identifying rifampicin-heteroplasmy resistance of mycobacterium tuberculosis according to claim 1, wherein the preparation of the mycobacterium tuberculosis-analyzed bacterial liquid in S1 comprises the following steps: dipping the culture solution to grind the mycobacterium tuberculosis thallus into a bacterium solution homogenate, adding a proper amount of the culture solution, mixing uniformly, filtering by using a filter screen, and preparing into a bacterium solution with 1 McLeod turbidity.
4. The method for fluorescein-based flow cytometry for the identification of rifampicin-heterogeneous resistance of mycobacterium tuberculosis as claimed in claim 3, wherein the culture broth comprises 7H9 culture broth.
5. The method for the identification of rifampin-heterogeneous resistance of mycobacterium tuberculosis by fluorescein-based flow cytometry as claimed in claim 1, wherein the centrifugation condition in step S3 is 13000 rpm centrifugation for 10 min.
6. The method for the identification of rifampin-heterogeneous resistance of mycobacterium tuberculosis by fluorescein-based flow cytometry as claimed in claim 1, wherein the redispersion procedure in step S3 comprises: the two procedures of 5 seconds of ultrasound and 5 seconds of pause are alternately carried out by an ultrasonic disperser for 30 seconds.
7. The method for fluorescein-based flow cytometry for identifying rifampicin heterogeneous resistance of mycobacterium tuberculosis as claimed in claim 1 or claim 3, wherein the sieve is 300 mesh sieve.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1338518A (en) * | 2001-09-17 | 2002-03-06 | 复旦大学 | Secreted substance of medicines-resistant branched tubercle bacillus and its application |
| WO2007010641A1 (en) * | 2005-07-21 | 2007-01-25 | Kyushu University | Method for detection of microorganism and kit for detection of microorganism |
| WO2012085652A2 (en) * | 2010-12-23 | 2012-06-28 | Centre Hospitalier Universitaire Vaudois | Methods for differentiating between disease states |
| CN105358982A (en) * | 2013-07-04 | 2016-02-24 | Abm科技公司 | Method for the rapid determination of susceptibility or resistance of bacteria to antibiotics |
-
2021
- 2021-01-13 CN CN202110039850.2A patent/CN112391441B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1338518A (en) * | 2001-09-17 | 2002-03-06 | 复旦大学 | Secreted substance of medicines-resistant branched tubercle bacillus and its application |
| WO2007010641A1 (en) * | 2005-07-21 | 2007-01-25 | Kyushu University | Method for detection of microorganism and kit for detection of microorganism |
| WO2012085652A2 (en) * | 2010-12-23 | 2012-06-28 | Centre Hospitalier Universitaire Vaudois | Methods for differentiating between disease states |
| CN105358982A (en) * | 2013-07-04 | 2016-02-24 | Abm科技公司 | Method for the rapid determination of susceptibility or resistance of bacteria to antibiotics |
Non-Patent Citations (5)
| Title |
|---|
| Flow cytometric testing of susceptibilities of Mycobacterium tuberculosis isolates to ethambutol, isoniazid, and rifampin in 24 hours;S M Kirk 等;《J Clin Microbiol.》;19980630;第36卷(第6期);第1568–1573页 * |
| 应用流式细胞术鉴定死活结核分枝杆菌的实验研究;王伟亮 等;《中山大学学报（医学版）》;20190531;第40卷(第3期);第372-378页 * |
| 应用流式细胞术鉴定结核分枝杆菌利福平敏感性的研究;谢贝 等;《国际医药卫生导报》;20190601;第25卷(第11期);摘要，第1693页右栏第1段至第1696页左栏第2段，图1，表1 * |
| 流式细胞术在医学检验中的研究进展;陈丽;《右江民族医学院学报》;20160630;第３８卷(第３期);第335-336页 * |
| 目录 应用流式细胞术检测结核分枝杆菌对异烟肼的敏感性;龙会;《万方数据知识服务平台》;20150817;摘要，第1-42页 * |
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