Rapid phenotypic drug susceptibility testing method for Mycobacterium tuberculosis infection samples

Abstract

This invention discloses a rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infected samples. The method utilizes the incorporation of deuterium into biomolecules synthesized by metabolically active Mycobacterium tuberculosis in a heavy water (D2O) environment, thereby generating C-D characteristic peaks in single-cell Raman spectroscopy. The inhibition of metabolic activity by anti-tuberculosis drugs leads to a decrease in the C-D signal. By calculating the metabolic change rate between drug-treated and untreated samples, the bacterial drug response can be quantified, and susceptibility / resistance determination and MIC value can be established. This invention provides a rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis based on single-cell Raman spectroscopy and heavy water labeling technology (Raman-DIP). This method detects the metabolic changes of individual bacteria under the action of anti-tuberculosis drugs to determine drug susceptibility and minimum inhibitory concentration (MIC), enabling a rapid detection process from sample to result.

Description

Technical Field

[0001] This invention relates to the field of microbiology and biochemical detection and analysis technology, and in particular to a method for rapid detection of Mycobacterium tuberculosis using single-cell Raman-DIP combined with heavy water labeling technology. Mycobacterium tuberculosis Phenotypic detection methods for MTB (Medium-to-Thinning) drug susceptibility. Background Technology

[0002] Mycobacterium tuberculosis is the main pathogen causing tuberculosis, and the increase in multidrug-resistant and extensively drug-resistant tuberculosis has seriously affected global prevention and control efforts. While traditional culture-based phenotypic drug susceptibility testing is considered the "gold standard," it is time-consuming and carries a high risk of contamination, often requiring 3-8 weeks to obtain results, which is detrimental to timely clinical adjustments of medication. Molecular diagnostic methods (such as Xpert MTB / RIF) can provide rapid results, but they only detect known resistance sites and cannot identify phenotypic resistance caused by novel or unknown mutations. Furthermore, they cannot distinguish between dead and live bacteria, leading to a high likelihood of false positives.

[0003] Patent CN202311653817 discloses a method for detecting drug resistance in drug-resistant Mycobacterium tuberculosis, but it has the following shortcomings: 1. Sample limitations: It is only applicable to pure bacterial cultures and cannot be directly detected in complex clinical samples (such as sputum), which limits its practical application; 2. Complex detection method: The detection of bacteria is carried out by fixing them on a filter plate or filter membrane. The operation steps are complicated and can easily cause uneven signal distribution, affecting repeatability. 3. Lack of standardized algorithms for result judgment: The proportion of CD peak intensity change is used as the judgment basis, but the threshold setting depends on experimental experience and lacks unified statistical or algorithmic standards, resulting in insufficient repeatability and comparability. The detection time is relatively long: sufficient bacterial cells need to be cultured before detection, the pretreatment is time-consuming, and the detection cycle still takes several days.

[0004] Patent CN202110178116.4 discloses a kit and method for rapid detection of drug resistance in Mycobacterium tuberculosis, but it has the following shortcomings: 1. Sample limitations: It is only applicable to pure bacterial cultures and cannot be directly detected in complex clinical samples (such as sputum), which limits its practical application; 2. Functional limitations: This patent can only distinguish between sensitive and resistant strains. It cannot further quantify the minimum inhibitory concentration (MIC), and therefore cannot provide a quantitative basis for personalized drug administration.

[0005] 3. Insufficient quantitative analysis: No unified normalization or statistical model has been established, and the judgment is still based on the difference in relative peak intensity, resulting in poor data comparability; The literature Rapid Mycobacterium abscessus The antimicrobial susceptibility testing based on antibiotic treatment response mapping via Raman Microspectroscopy has the following shortcomings: 1. Different target bacterial species: The research subjects are rapidly growing nontuberculous mycobacteria. Mycobacterium abscessus The feasibility of this study in slow-growing bacteria such as Mycobacterium tuberculosis (MTB) has not been verified. 2. Sample limitations: It is only applicable to pure bacterial cultures and cannot be directly detected in complex clinical samples (such as sputum), which limits its practical application; 3. Functional limitations: This patent can only distinguish between sensitive and resistant strains. It cannot further quantify the minimum inhibitory concentration (MIC), and therefore cannot provide a quantitative basis for personalized drug administration.

[0006] Therefore, there is an urgent need for a new method that maintains the accuracy of metabolic detection while taking into account biosafety, universality, and algorithm standardization, so as to achieve phenotypic determination of MTB drug sensitivity in a short period of time. Summary of the Invention

[0007] The technical problem this invention aims to solve is to address the shortcomings of the existing technology by providing a rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis (MTB) infected samples. This invention provides a rapid phenotypic drug susceptibility detection method for MTB based on single-cell Raman spectroscopy and heavy water labeling (Raman-DIP) technology. This method determines drug susceptibility and minimum inhibitory concentration (MIC) by detecting metabolic changes in a single bacterium under the action of anti-tuberculosis drugs, and can complete the detection process from sample to result in a short time.

[0008] The principle of this invention: This invention utilizes deuterium to incorporate into biomacromolecules synthesized by metabolically active Mycobacterium tuberculosis in a heavy water (D2O) environment, thereby generating CD characteristic peaks in single-cell Raman spectroscopy; the inhibition of metabolic activity by anti-tuberculosis drugs leads to a decrease in CD signal. By calculating the metabolic change rate between drug-treated and untreated samples, the drug response of bacteria can be quantified, and sensitivity / resistance can be determined, as well as the MIC value can be established.

[0009] The solution of the present invention has the following significant advantages: 1. Introduce standardized quantitative indicators MR and algorithm thresholds to replace empirical judgments, resulting in objective and repeatable results.

[0010] 2. Applicable to complex samples (such as sputum), direct detection can be achieved through optimized preprocessing and signal analysis.

[0011] 3. The testing cycle is significantly shortened (≤50 hours), while maintaining consistency with the gold standard test results.

[0012] 4. Scalable: It can be further developed into an automated instrument platform.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is: a rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples, comprising the following steps: S1. Provide a test sample, wherein the test sample is a Mycobacterium tuberculosis colony sample isolated from the test subject or a sputum sample containing Mycobacterium tuberculosis obtained from the test subject. S2, Effects with drugs: S2-1. For Mycobacterium tuberculosis colony samples, incubate them separately in drug treatment systems containing different concentrations of anti-tuberculosis drugs. S2-2. For sputum samples, they are added to a drug treatment system containing anti-tuberculosis drugs and incubated. The concentration of anti-tuberculosis drugs in the drug treatment system is set according to the resistance breakpoint concentration specified in the CLSI Antimicrobial Susceptibility Testing Standard. S3, Heavy water labeling: Add heavy water to the drug treatment system and incubate; S4. Sample inactivation: The product obtained in step S3 is inactivated to obtain an inactivated bacterial sample. S5. Raman spectroscopy acquisition: Acquire single-cell Raman spectral data of inactivated bacterial samples; S6. Data Processing and Indicator Calculation: S6-1. Preprocess the collected spectra; S6-2, 2040-2300cm -1 The band range is defined as the CD band, covering 2800-3100cm. -1 The band range is defined as the CH band, and the Raman spectral peak area within the CD band is obtained and denoted as S. C-D Then, the area of ​​the Raman spectral peak in the CH band is obtained and denoted as S. C-H ; S6-3. Calculate the metabolic index MI using the following formula: MI=S C-D / (S) C-D +S C-H ); S6-4. The metabolic ratio MR is calculated using the following formula to characterize the degree of cellular metabolic inhibition after drug treatment: ; Among them, MIdrug is the metabolic index of the drug treatment group, MIpositive is the positive control group, and MInegative is the negative control group. No anti-tuberculosis drugs were added to the positive control group, and no anti-tuberculosis drugs and heavy water were added to the negative control group. S7. Determine the test results: S7-1. For Mycobacterium tuberculosis colony samples, determine the minimum inhibitory concentration (MIC) of anti-tuberculosis drugs against Mycobacterium tuberculosis based on the following two conditions: (a) MR is less than 75%; (b) MR is significantly different from the control group, i.e., the significance p value is ≤0.05; The lowest drug concentration that meets the criteria is taken as the MIC value; S7-2. For sputum samples, compare the MR with the pre-obtained classification threshold T for each anti-tuberculosis drug. n For comparison, n represents the number of types of anti-tuberculosis drugs. If MR ≤ T n The MR was determined to be sensitive to the anti-tuberculosis drug; if MR > T n It was determined that the bacteria were resistant to the anti-tuberculosis drug for Mycobacterium tuberculosis.

[0014] Preferably, the anti-tuberculosis drugs include rifampin, isoniazid, streptomycin, and ethambutol.

[0015] Preferably, the concentration gradient of the anti-tuberculosis drug in the drug treatment system in step S2-1 is set according to the gold standard liquid drug susceptibility test to determine the minimum inhibitory concentration.

[0016] Preferably, in S2-2, the resistance breakpoint concentrations of the four anti-tuberculosis drugs are as follows: rifampin 2 μg / mL, isoniazid 0.25 μg / mL, streptomycin 4 μg / mL, and ethambutol 8 μg / mL.

[0017] Preferably, step S3 specifically involves adding heavy water to the drug treatment system to achieve a final volume concentration of 20-80%, and continuing incubation for 12-48 hours.

[0018] Preferably, step S3 specifically involves adding heavy water to the drug treatment system to achieve a final volume concentration of 50%, and continuing incubation for 24 hours.

[0019] Preferably, step S4 specifically involves heating the product obtained in step S3 in a water bath at 95°C for 10 minutes to completely inactivate the bacteria, thereby obtaining an inactivated bacterial sample.

[0020] Preferably, in step S7-2, the classification thresholds for the four anti-tuberculosis drugs are as follows: The classification thresholds for rifampin are T1=0.5612, for isoniazid T2=0.7392, for streptomycin T3=0.5961, and for ethambutol T4=0.7040.

[0021] Preferably, in step S7-2, the classification threshold of anti-tuberculosis drugs is obtained by the following method: the concentration of anti-tuberculosis drugs in the drug treatment system is set in advance according to the resistance breakpoint concentration specified in the CLSI Antimicrobial Susceptibility Testing Standard, and then the drug treatment system is used to treat Mycobacterium tuberculosis. The ROC curve of anti-tuberculosis drugs is plotted based on drug resistance and MR value, and the MR value corresponding to the maximum Yoden index is the classification threshold of anti-tuberculosis drugs.

[0022] Preferably, the rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples includes the following steps: S1. Provide a test sample, wherein the test sample is a pure culture of Mycobacterium tuberculosis or a colony sample obtained from the sputum, cerebrospinal fluid or bronchoalveolar lavage fluid of the test subject, or a sputum sample containing Mycobacterium tuberculosis obtained from the test subject. S2, Effects with drugs: S2-1. For Mycobacterium tuberculosis colony samples, they were added to drug treatment systems containing different concentrations of anti-tuberculosis drugs and incubated separately. The concentration gradient of the anti-tuberculosis drugs in the drug treatment system was set according to the gold standard liquid drug susceptibility test to determine the minimum inhibitory concentration. S2-2. For sputum samples, incubate them in a drug treatment system containing anti-tuberculosis drugs. The concentration of anti-tuberculosis drugs in the drug treatment system shall be set according to the resistance breakpoint concentration specified in the CLSI Antimicrobial Susceptibility Testing Standard. Anti-tuberculosis drugs include rifampin, isoniazid, streptomycin, and ethambutol. The resistance breakpoint concentrations of the four anti-tuberculosis drugs are as follows: rifampin 2 μg / mL, isoniazid 0.25 μg / mL, streptomycin 4 μg / mL, and ethambutol 8 μg / mL. S3, Heavy water labeling: Add heavy water to the drug treatment system to make the final volume concentration of heavy water 50%, and continue incubation for 24 hours; S4. Sample inactivation: The product obtained in step S3 is heated in a 95°C water bath for 10 min to completely inactivate the bacterial cells and obtain an inactivated bacterial cell sample. S5. Raman spectroscopy acquisition: Using a 532nm laser confocal Raman microscope system, more than 20 single-cell Raman spectral data were randomly collected from the inactivated bacterial samples. S6. Data Processing and Indicator Calculation: S6-1. Perform background subtraction and normalization on the acquired spectrum; S6-2, 2040-2300cm -1 The band range is defined as the CD band, covering 2800-3100cm. -1 The band range is defined as the CH band, and the Raman spectral peak area within the CD band is obtained and denoted as S. C-D Then, the area of ​​the Raman spectral peak in the CH band is obtained and denoted as S. C-H ; S6-3. Calculate the metabolic index MI using the following formula: MI=S C-D / (S) C-D +S C-H ); S6-4. The metabolic ratio MR is calculated using the following formula to characterize the degree of cellular metabolic inhibition after drug treatment: ; Among them, MIdrug is the metabolic index of the drug treatment group, MIpositive is the positive control group, and MInegative is the negative control group. No anti-tuberculosis drugs were added to the positive control group, and no anti-tuberculosis drugs and heavy water were added to the negative control group. S7. Determine the test results: S7-1. For Mycobacterium tuberculosis colony samples, determine the minimum inhibitory concentration (MIC) of anti-tuberculosis drugs against Mycobacterium tuberculosis based on the following two conditions: (a) MR is less than 75%; (b) MR is significantly different from the control group, i.e., the significance p value is ≤0.05; The lowest drug concentration that satisfies either condition (a) or (b) is taken as the MIC value; S7-2. For sputum samples, the MR is compared with the pre-obtained classification threshold Tn for anti-tuberculosis drugs, where n is the number of types of anti-tuberculosis drugs. If MR ≤ Tn, it is determined that Mycobacterium tuberculosis is sensitive to the anti-tuberculosis drug; if MR > Tn, it is determined that Mycobacterium tuberculosis is resistant to the anti-tuberculosis drug. The classification thresholds for the four anti-tuberculosis drugs are as follows: rifampin T1=0.5612, isoniazid T2=0.7392, streptomycin T3=0.5961, and ethambutol T4=0.7040.

[0023] The beneficial effects of this invention are: Compared with the prior art, the present invention has at least the following advantages: 1. The testing cycle is significantly shortened. This invention, based on single-cell Raman spectroscopy and heavy water labeling technology, directly assesses changes in bacterial metabolic activity under drug stress, allowing for phenotypic determination without waiting for visible growth. The overall detection cycle is 50 hours, significantly shorter than traditional drug susceptibility testing (3-8 weeks); with future integration of automated sampling and data processing modules, the total detection time can be further reduced to approximately 38 hours.

[0024] 2. The sample has broad applicability and strong clinical feasibility. This invention is applicable to both pure bacterial cultures and complex matrix samples (such as simulated sputum). By mixing bacterial strains into the sputum before pretreatment and performing full-process verification, it ensures that the detection conditions are consistent with real clinical samples, and has good versatility and scalability.

[0025] 3. The judgment criteria are objective and repeatable, and the results are highly consistent. A statistical threshold algorithm based on ROC curves and the maximum Yoden index was introduced to replace the traditional empirical judgment criteria, achieving standardization and reproducibility of classification thresholds. The sputum sample detection results achieved 100% classification consistency with the gold standard liquid susceptibility testing method for four first-line anti-tuberculosis drugs; the minimum inhibitory concentration (MIC) results for pure culture samples achieved 100% basic consistency with the gold standard method.

[0026] 4. Safe and easy to operate, with potential for automation and point-of-care testing (POCT). This invention employs a 95℃ 10min heat inactivation process to ensure biosafety; simultaneously, the process is modular and requires no DNA extraction or amplification. This system can be further integrated into closed detection chips or microfluidic systems to achieve point-of-care testing (POCT), possessing broad prospects for clinical translation.

[0027] 5. The test results showed strong phenotypic correlation. Raman-DIP technology reflects drug efficacy by directly detecting the degree of inhibition of bacterial metabolic activity. It can identify phenotypic drug resistance caused by unknown or atypical mutations, making up for the limitations of molecular detection methods and providing a more reliable basis for precision medicine in clinical practice. Attached Figure Description

[0028] Figure 1 This is a flowchart of the detection process of the method of the present invention; Figure 2 This is a schematic diagram of the peak areas of the CH4 and CH5 bands obtained from single-cell Raman spectroscopy. Figure 3 The MR values ​​and ROC curves for four anti-tuberculosis drugs are shown. Figure 4 Single-cell Raman spectra of simulated sputum samples with different drug resistance; Figure 5To simulate the drug resistance test results of sputum samples; Figure 6 The results show the MIC values ​​of pure bacterial samples. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0030] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. For examples where specific conditions are not specified, conventional conditions or conditions recommended by the manufacturer are followed. For reagents or instruments whose manufacturers are not specified, they are all commercially available products.

[0032] Example 1 A rapid phenotypic drug susceptibility testing method for Mycobacterium tuberculosis infection samples includes the following steps: S1. Provide test samples. In this embodiment, pure culture samples and simulated sputum samples are prepared using the MTB standard strain H37Rv for subsequent testing. Specifically, the test samples include: (1) Pure culture samples: MTB standard strain H37Rv colonies in Roche medium were inoculated into 7H9 liquid medium and cultured for 4-5 weeks before being used directly; this was used to simulate Mycobacterium tuberculosis colony samples isolated from the test subjects. (2) Simulated sputum sample: The bacterial suspension of MTB standard strain H37Rv was mixed with sputum from healthy individuals at a volume ratio of 1:4. The mixture was then digested with 4wt% NaOH, neutralized with PBS, and collected by centrifugation to obtain a simulated sputum sample, which was used to simulate sputum samples containing Mycobacterium tuberculosis obtained from the tested human body. S2, Effects with drugs: S2-1. For pure culture samples, they are added to drug treatment systems containing different concentrations of anti-tuberculosis drugs and incubated separately. The concentration gradient of anti-tuberculosis drugs in the drug treatment system is set according to the gold standard liquid drug susceptibility test to determine the minimum inhibitory concentration (MIC). S2-2. For simulated sputum samples, they are added to a drug treatment system containing anti-tuberculosis drugs for incubation. The concentration of anti-tuberculosis drugs in the drug treatment system is set according to the resistance breakpoint concentration specified in the CLSI Antimicrobial Susceptibility Testing Standard to distinguish between drug-sensitive and drug-resistant strains. Anti-tuberculosis drugs include rifampin, isoniazid, streptomycin, and ethambutol. The resistance breakpoint concentrations of the four anti-tuberculosis drugs are as follows: rifampin 2 μg / mL, isoniazid 0.25 μg / mL, streptomycin 4 μg / mL, and ethambutol 8 μg / mL. S3, Heavy water labeling: Heavy water is added to the drug treatment system to make the final volume concentration of heavy water 50%, and incubation continues for 24 hours; metabolically active bacteria incorporate deuterium into cellular components, thereby forming CD vibration signals in Raman spectroscopy; S4. Sample inactivation: The product obtained in step S3 is heated in a 95°C water bath for 10 min to completely inactivate the bacteria and obtain the inactivated bacterial sample. S5. Raman spectroscopy acquisition: Using a 532nm laser confocal Raman microscope system (50x objective lens, integration time 7s), more than 20 single-cell Raman spectral data were randomly collected from the bacterial cell samples after fire extinguishing. S6. Data Processing and Indicator Calculation: S6-1. Perform background subtraction and normalization on the acquired spectrum; S6-2, 2040-2300cm -1 The band range is defined as the CD band, covering 2800-3100cm. -1 The band range is defined as the CH band, and the Raman spectral peak area within the CD band is obtained and denoted as S. C-D Then, the area of ​​the Raman spectral peak in the CH band is obtained and denoted as S. C-H ; S6-3. Calculate the metabolic index MI using the following formula: MI=S C-D / (S) C-D +S C-H ); Single-cell Raman spectroscopy data were used to calculate the metabolic index (MI), defined as the combined intensity of the CD (2040 - 2300 cm⁻¹) band relative to the sum of the intensities of the CD and CH (2800 - 3100 cm⁻¹) bands. MI reflects the degree of D₂O binding and cellular anabolic metabolic activity. S6-4. The metabolic ratio MR is calculated using the following formula to characterize the degree of cellular metabolic inhibition after drug treatment: ; Among them, MIdrug is the metabolic index of the drug treatment group, MIpositive is the positive control group, and MInegative is the negative control group. No anti-tuberculosis drugs were added to the positive control group, and no anti-tuberculosis drugs and heavy water were added to the negative control group. S7. Determine the test results: S7-1. For pure culture samples, determine the minimum inhibitory concentration (MIC) of anti-tuberculosis drugs against Mycobacterium tuberculosis based on the following two conditions: (a) MR is less than 75%; (b) MR is significantly different from the control group, i.e., the significance p value is ≤0.05; The lowest drug concentration that satisfies either condition (a) or (b) is taken as the MIC value; the detection results obtained in the experiment are basically consistent with the gold standard MIC by 100%; S7-2. For simulated sputum samples, the MR (Mutual Detection Ratio) is compared with the pre-obtained classification threshold Tn for anti-tuberculosis drugs, where n is the number of anti-tuberculosis drug types. If MR ≤ Tn, Mycobacterium tuberculosis is determined to be sensitive to the anti-tuberculosis drug; if MR > Tn, it is determined to be resistant to the anti-tuberculosis drug. This strategy achieved 100% consistency with the gold standard classification in the validation experiment. The classification thresholds for the four anti-tuberculosis drugs are as follows: rifampin T1=0.5612, isoniazid T2=0.7392, streptomycin T3=0.5961, and ethambutol T4=0.7040.

[0033] The classification thresholds for anti-tuberculosis drugs were obtained as follows: Four pure-cultured bacterial strains were treated with the resistance breakpoint concentrations (rifampin 2 μg / mL, isoniazid 0.25 μg / mL, streptomycin 4 μg / mL, ethambutol 8 μg / mL) as specified in the CLSI Antimicrobial Susceptibility Testing Standards (4*4 sets of experiments). The resistance modulotropic index (MR) values ​​of the four strains under each drug treatment were calculated. Since the drug resistance of the strains was known, ROC curves were plotted for each of the four drugs based on their resistance and MR values. The MR value corresponding to the maximum Yoden index (maximum sum of sensitivity and specificity) was used as the threshold. The thresholds were then applied to simulated sputum samples for validation.

[0034] In this embodiment, all strains were derived from the Fifth People's Hospital of Suzhou. H37Rv is the standard strain of Mycobacterium tuberculosis; the others are clinical isolates of Mycobacterium tuberculosis, numbered 3569, 1075, and 7349, respectively. These strain numbers correspond to the patient numbers in the clinical reports.

[0035] Reference Figure 1 Here is a flowchart of the detection process of the method of the present invention; Reference Figure 2The figure shows a schematic diagram of the peak areas of the CH4 and CH5 bands obtained from single-cell Raman spectroscopy. The symbols in the figure are as follows: D represents the 50% heavy water labeled group, and H represents the control group without heavy water. UV 1h d represents the bacterial suspension dried on an aluminum sheet irradiated with UV light for 1 hour; UV 1h l represents the bacterial suspension irradiated with UV light for 1 hour; 95℃ for 10 min and 80℃ for 30 min are the parameters for inactivating the bacterial suspension in a metal bath, respectively.

[0036] Reference Figure 3 Figure 1 shows the calculated MR values ​​and ROC curves for four anti-tuberculosis drugs. Specifically, EH in the figure represents the calculated MR values ​​of rifampin, isoniazid, streptomycin, and ethambutol applied to pure bacterial samples, with blue indicating susceptibility and red indicating known resistance. IL represents the ROC curves for the four drugs, and the obtained thresholds are marked in EH.

[0037] Reference Figure 4 The figures show single-cell Raman spectra of simulated sputum samples with different drug resistance levels. A and D represent the spectra after treatment with four different drugs. R (resistance) and S (sensitivity) are the gold standard results.

[0038] Reference Figure 5 The figure shows the results of drug resistance testing on simulated sputum samples. In the figure, AD corresponds to four first-line anti-tuberculosis drugs: rifampin (RIF), isoniazid (INH), streptomycin (STR), and ethambutol (EMB). The figure illustrates the MR of four simulated sputum samples (H37Rv, 3569, 1075, 7349 strains + MTB-negative sputum) treated with the four first-line anti-tuberculosis drugs. The dashed line represents the MR classification threshold established in pure culture, and the bar color represents the gold standard result: blue indicates sensitivity, and red indicates resistance. This reflects 100% classification consistency. Figure 5 It is based on Figure 4 (Results obtained from spectral analysis).

[0039] Reference Figure 6 The figure shows the MIC values ​​of pure bacterial samples. It illustrates the MR values ​​of four MTB strains exposed to gradient concentrations of four first-line anti-tuberculosis drugs. Each cell represents the MR of the strain under a specific drug concentration. `neg` is the negative control (no drug, no heavy water labeling), and `0` is the positive control (no drug, but with heavy water labeling). The shaded squares correspond to the MIC values ​​obtained by the gold standard method (BMD), and the black squares correspond to the MIC values ​​obtained by this patented method (Raman-DIP). The last two columns show the drug resistance results obtained by the two methods: sensitive (S) / resistant (R), demonstrating that the new method is essentially 100% consistent with the gold standard method.

[0040] Essential agreement (EA): The MIC value of the test system differs from the MIC value of the reference method or comparison method by no more than one or two console dilutions (bacteria) or two console dilutions (yeast). EA is not calculated when the method being evaluated is the disc diffusion method.

[0041] The above is the general concept of the present invention. Based on this, detailed embodiments and comparative examples are provided below to further illustrate the present invention.

[0042] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infected samples, characterized in that, Includes the following steps: S1. Provide a test sample, wherein the test sample is a Mycobacterium tuberculosis colony sample isolated from the test subject or a sputum sample containing Mycobacterium tuberculosis obtained from the test subject. S2, Effects with drugs: S2-1. For Mycobacterium tuberculosis colony samples, incubate them separately in drug treatment systems containing different concentrations of anti-tuberculosis drugs. S2-2. For sputum samples, they are added to a drug treatment system containing anti-tuberculosis drugs and incubated. The concentration of anti-tuberculosis drugs in the drug treatment system is set according to the resistance breakpoint concentration specified in the CLSI Antimicrobial Susceptibility Testing Standard. S3, Heavy water labeling: Add heavy water to the drug treatment system and incubate; S4. Sample inactivation: The product obtained in step S3 is inactivated to obtain an inactivated bacterial sample. S5. Raman spectroscopy acquisition: Acquire single-cell Raman spectral data of inactivated bacterial samples; S6. Data Processing and Indicator Calculation: S6-1. Preprocess the collected spectra; S6-2, 2040-2300cm -1 The band range is defined as the CD band, covering 2800-3100cm. -1 The band range is defined as the CH band, and the Raman spectral peak area within the CD band is obtained and denoted as S. C-D Then, the area of ​​the Raman spectral peak in the CH band is obtained and denoted as S. C-H ; S6-3. Calculate the metabolic index MI using the following formula: MI=S C-D / (S C-D +S C-H ); S6-4. The metabolic ratio MR is calculated using the following formula to characterize the degree of cellular metabolic inhibition after drug treatment: ； Among them, MIdrug is the metabolic index of the drug treatment group, MIpositive is the positive control group, and MInegative is the negative control group. No anti-tuberculosis drugs were added to the positive control group, and no anti-tuberculosis drugs and heavy water were added to the negative control group. S7. Determine the test results: S7-1. For Mycobacterium tuberculosis colony samples, determine the minimum inhibitory concentration (MIC) of anti-tuberculosis drugs against Mycobacterium tuberculosis based on the following two conditions: (a) MR is less than 75%; (b) MR is significantly different from the control group, i.e., the significance p value is ≤0.05; The lowest drug concentration that meets the criteria is taken as the MIC value; S7-2. For sputum samples, compare the MR with the pre-obtained classification threshold T for each anti-tuberculosis drug. n For comparison, n represents the number of types of anti-tuberculosis drugs. If MR ≤ T n The MR was determined to be sensitive to the anti-tuberculosis drug; if MR > T n It was determined that the bacteria were resistant to the anti-tuberculosis drug for Mycobacterium tuberculosis.

2. The rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples according to claim 1, characterized in that, in, Anti-tuberculosis drugs include rifampin, isoniazid, streptomycin, and ethambutol.

3. The rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples according to claim 2, characterized in that, The concentration gradient of the anti-tuberculosis drug in the drug treatment system in step S2-1 is set according to the gold standard liquid drug susceptibility test and is used to determine the minimum inhibitory concentration.

4. The rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples according to claim 2, characterized in that, In S2-2, the resistance breakpoint concentrations of the four anti-tuberculosis drugs are as follows: rifampin 2 μg / mL, isoniazid 0.25 μg / mL, streptomycin 4 μg / mL, and ethambutol 8 μg / mL.

5. The rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples according to claim 1, characterized in that, Step S3 specifically involves adding heavy water to the drug treatment system to achieve a final volume concentration of 20-80%, and continuing incubation for 12-48 hours.

6. The rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples according to claim 5, characterized in that, Step S3 specifically involves adding heavy water to the drug treatment system to achieve a final volume concentration of 50%, and continuing incubation for 24 hours.

7. The rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples according to claim 1, characterized in that, Step S4 specifically involves heating the product obtained in step S3 in a 95°C water bath for 10 minutes to completely inactivate the bacteria, thereby obtaining an inactivated bacterial sample.

8. The rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples according to claim 1, characterized in that, In step S7-2, the classification thresholds for the four anti-tuberculosis drugs are as follows: The classification thresholds for rifampin are T1=0.5612, for isoniazid T2=0.7392, for streptomycin T3=0.5961, and for ethambutol T4=0.7040.

9. The rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples according to claim 8, characterized in that, In step S7-2, the classification threshold of anti-tuberculosis drugs is obtained by the following method: the concentration of anti-tuberculosis drugs in the drug treatment system is set in advance according to the resistance breakpoint concentration specified in the CLSI Antimicrobial Susceptibility Testing Standards. Then, the drug treatment system is used to treat Mycobacterium tuberculosis. The ROC curve of anti-tuberculosis drugs is plotted based on drug resistance and MR value. The MR value corresponding to the maximum Yoden index is the classification threshold of anti-tuberculosis drugs.

10. The rapid phenotypic drug susceptibility detection method for Mycobacterium tuberculosis infection samples according to claim 9, characterized in that, Includes the following steps: S1. Provide a test sample, wherein the test sample is a pure culture of Mycobacterium tuberculosis or a colony sample obtained from the sputum, cerebrospinal fluid or bronchoalveolar lavage fluid of the test subject, or a sputum sample containing Mycobacterium tuberculosis obtained from the test subject. S2, Effects with drugs: S2-1. For Mycobacterium tuberculosis colony samples, they were added to drug treatment systems containing different concentrations of anti-tuberculosis drugs and incubated separately. The concentration gradient of the anti-tuberculosis drugs in the drug treatment system was set according to the gold standard liquid drug susceptibility test to determine the minimum inhibitory concentration. S2-2. For sputum samples, incubate them in a drug treatment system containing anti-tuberculosis drugs. The concentration of anti-tuberculosis drugs in the drug treatment system shall be set according to the resistance breakpoint concentration specified in the CLSI Antimicrobial Susceptibility Testing Standard. Anti-tuberculosis drugs include rifampin, isoniazid, streptomycin, and ethambutol. The resistance breakpoint concentrations of the four anti-tuberculosis drugs are as follows: rifampin 2 μg / mL, isoniazid 0.25 μg / mL, streptomycin 4 μg / mL, and ethambutol 8 μg / mL. S3, Heavy water labeling: Add heavy water to the drug treatment system to make the final volume concentration of heavy water 50%, and continue incubation for 24 hours; S4. Sample inactivation: The product obtained in step S3 is heated in a 95°C water bath for 10 min to completely inactivate the bacterial cells and obtain an inactivated bacterial cell sample. S5. Raman spectroscopy acquisition: Using a 532nm laser confocal Raman microscope system, more than 20 single-cell Raman spectral data were randomly collected from the inactivated bacterial samples. S6. Data Processing and Indicator Calculation: S6-1. Perform background subtraction and normalization on the acquired spectrum; S6-2, 2040-2300cm -1 The band range is defined as the CD band, covering 2800-3100cm. -1 The band range is defined as the CH band, and the Raman spectral peak area within the CD band is obtained and denoted as S. C-D Then, the area of ​​the Raman spectral peak in the CH band is obtained and denoted as S. C-H ; S6-3. Calculate the metabolic index MI using the following formula: MI=S C-D / (S C-D +S C-H ); S6-4. The metabolic ratio MR is calculated using the following formula to characterize the degree of cellular metabolic inhibition after drug treatment: ； Among them, MIdrug is the metabolic index of the drug treatment group, MIpositive is the positive control group, and MInegative is the negative control group. No anti-tuberculosis drugs were added to the positive control group, and no anti-tuberculosis drugs and heavy water were added to the negative control group. S7. Determine the test results: S7-1. For Mycobacterium tuberculosis colony samples, determine the minimum inhibitory concentration (MIC) of anti-tuberculosis drugs against Mycobacterium tuberculosis based on the following two conditions: (a) MR is less than 75%; (b) MR is significantly different from the control group, i.e., the significance p value is ≤0.05; The lowest drug concentration that satisfies either condition (a) or (b) is taken as the MIC value; S7-2. For sputum samples, the MR is compared with the pre-obtained classification threshold Tn for anti-tuberculosis drugs, where n is the number of types of anti-tuberculosis drugs. If MR ≤ Tn, it is determined that Mycobacterium tuberculosis is sensitive to the anti-tuberculosis drug; if MR > Tn, it is determined that Mycobacterium tuberculosis is resistant to the anti-tuberculosis drug. The classification thresholds for the four anti-tuberculosis drugs are as follows: rifampin T1=0.5612, isoniazid T2=0.7392, streptomycin T3=0.5961, and ethambutol T4=0.7040.

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