Method for rapidly detecting intracellular mycobacterium tuberculosis

Abstract

The invention discloses a method for rapidly detecting intracellular mycobacterium tuberculosis, which comprises the following steps: marking mycobacterium tuberculosis by using mScarlet, marking host cells by CFSE, rinsing part of extracellular bacteria which do not enter macrophages by PBS and sterilizing by gentamicin, detecting the survival amount of the intracellular mycobacterium tuberculosis by using a flow cytometer, and completing sample analysis within several hours. The experimental period is remarkably shortened, the research efficiency is improved, and the method is suitable for large-scale sample analysis.

Description

Technical Field

[0001] The invention relates to a detection method, in particular to a method for rapidly detecting intracellular tuberculosis bacilli. Background Art

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis) that poses a serious threat to global public health. According to statistics from the World Health Organization (WHO), more than 2 million people die from tuberculosis each year. Mycobacterium tuberculosis is a typical intracellular pathogen that can survive for a long time in host cells and evade clearance by the immune system. Studying the interaction between Mycobacterium tuberculosis and host cells, especially its survival and replication mechanism in host cells, is of great significance for the development of new therapeutic drugs and vaccines.

[0003] Currently, the main method for evaluating the survival of Mycobacterium tuberculosis in host cells is through colony-forming unit (CFU) counting. However, this method requires the infected cells to be lysed and inoculated in 7H10 medium, and the results can only be obtained after a culture period of up to one month, which seriously affects the experimental efficiency. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for rapidly and accurately detecting intracellular Mycobacterium tuberculosis, which solves the problems existing in the prior art of counting the number of intracellular Mycobacterium tuberculosis by colony forming units (CFU), which is time-consuming, has a large workload, and involves subjective judgment.

[0005] Technical solution: The method for rapid detection of intracellular tuberculosis bacilli of the present invention comprises labeling Mycobacterium tuberculosis with mScarlet, labeling host cells with carboxyfluorescein succinimidyl ester (CFSE), rinsing some extracellular bacteria that have not entered macrophages with PBS and sterilizing with gentamicin, and detecting the survival amount of intracellular tuberculosis bacilli by flow cytometry.

[0006] Preferably, the method specifically comprises the following steps:

[0007] (1) Bacterial resuscitation: Resuscitate Mycobacterium tuberculosis carrying the pJV53-mScarlet plasmid;

[0008] (2) Prepare inactivated tuberculosis bacteria as a negative control;

[0009] (3) Cell infection: THP-1 cells were first labeled with CFSE and then induced to differentiate into macrophages using phorbol methyl ester (PMA); inactivated and non-inactivated Mycobacterium tuberculosis carrying the pJV53-mScarlet plasmid were infected at the same infection dose;

[0010] (4) Removal of extracellular bacteria;

[0011] (5) Gentamycin for bactericidal effect;

[0012] (6) Flow cytometry: Scrape the infected cells and analyze them using a flow cytometer. Use the FITC channel to detect CFSE-labeled macrophages, and use the PE-Texas Red channel to detect Mycobacterium tuberculosis signals. + Circle PE-Texas Red in the cells + tuberculosis, and finally the survival amount of intracellular tuberculosis bacilli was quantified.

[0013] Preferably, the Mycobacterium tuberculosis in step (1) is leucine auxotrophic Mycobacterium tuberculosis (H37RvΔleuD).

[0014] Preferably, the resuscitation is carried out in a water bath at 37°C.

[0015] Preferably, the resuscitation time is 2 hours.

[0016] Preferably, in step (2), the Mycobacterium tuberculosis (H37RvΔleuD-mScarlet) carrying the pJV53-mScarlet plasmid revived in step (1) is added with ethanol, placed at room temperature, centrifuged to remove the supernatant, resuspended with complete medium, and centrifuged again to remove the supernatant, so as to completely remove the ethanol remaining in the tuberculosis bacteria.

[0017] Preferably, in step (2), 50 μl of the Mycobacterium tuberculosis (H37RvΔleuD-mScarlet) carrying the pJV53-mScarlet plasmid revived in step (1) is taken and 200 μl of 95% ethanol is added.

[0018] Preferably, the room temperature placement time is 30 minutes.

[0019] Preferably, the centrifugal speed is 8000-10000 rpm, and the centrifugal time is 5-10 min.

[0020] Preferably, the dosage of CFSE in step (3) is 5 μM; the concentration of phorbol ester (PMA) is 10-20 ng / ml; and the differentiation induction time is 24-36 hours.

[0021] Preferably, the concentration of phorbol ester (PMA) is 10 ng / ml.

[0022] Preferably, the differentiation induction time is 24 hours.

[0023] Preferably, the CFSE product number is 565082, BD.

[0024] Preferably, in step (3), inactivated and non-inactivated Mycobacterium tuberculosis (H37RvΔleuD-mScarlet) carrying the pJV53-mScarlet plasmid infect macrophages differentiated by PMA, and the infection dose is MOI=1.

[0025] Preferably, the infection time is 4 hours.

[0026] Preferably, in step (4), sterile phosphate buffered saline (PBS) is used for repeated rinsing to remove extracellular bacteria that have not entered the macrophages.

[0027] Preferably, the step (5) comprises:

[0028] (5-1) Replace the culture medium: add serum-containing culture medium containing gentamicin and continue culturing;

[0029] (5-2) Replace the medium with serum containing low concentration of gentamicin and continue culturing.

[0030] Preferably, the concentration of gentamicin in the serum-containing culture medium in step (5-1) is 200 μg / ml; and the concentration of gentamicin in the serum-containing culture medium in step (5-2) is 20 μg / ml.

[0031] Preferably, the culturing is carried out in a cell culture incubator.

[0032] Preferably, the serum-containing culture medium is RPMI-1640 culture medium containing 10% fetal bovine serum (FBS).

[0033] Preferably, the cell culture incubator conditions are 37° C. and 5% CO 2 .

[0034] Preferably, the continued culture time in step (5-1) is 2 hours.

[0035] Preferably, before replacing the low concentration of gentamicin in step (5-2), the cells are gently rinsed three times with sterile PBS.

[0036] Preferably, the continued culture time in step (5-2) is 24 hours.

[0037] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention uses mScarlet to label Mycobacterium tuberculosis, the host cells are labeled with CFSE, some extracellular bacteria that have not entered the macrophages are rinsed with PBS and sterilized with gentamicin, and the survival of intracellular bacteria is detected by flow cytometry. Sample analysis can be completed within a few hours, which significantly shortens the experimental cycle, improves research efficiency, and is suitable for large-scale sample analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a graph showing the intracellular bacterial content detected by flow cytometry after inactivation of H37RvΔleuD-mScarlet in Example 2 of the present invention in THP-1 cells;

[0039] Figure 2 This is a graph showing the intracellular bacterial content detected by flow cytometry after THP-1 cells were infected with live H37RvΔleuD-mScarlet in Example 2 of the present invention. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0041] Example 1

[0042] (1) Bacterial resuscitation: resuscitating Mycobacterium tuberculosis carrying the mScarlet plasmid;

[0043] (2) Prepare inactivated tuberculosis bacteria as a negative control;

[0044] (3) Cell infection: THP-1 cells were first labeled with CFSE and then induced to differentiate using phorbol esterase (PMA); inactivated and non-inactivated Mycobacterium tuberculosis were infected at the same infection dose;

[0045] (4) Removal of extracellular bacteria;

[0046] (5) Gentamycin kills extracellular bacteria;

[0047] (6) Flow cytometry: Scrape the infected cells and analyze them using a flow cytometer. Use the FITC channel to detect CFSE-labeled macrophages, and use the PE-Texas Red channel to detect Mycobacterium tuberculosis signals. + Circle PE-Texas Red in the cells + tuberculosis, and finally the survival amount of intracellular tuberculosis bacilli was quantified.

[0048] Example 2

[0049] (1) Bacterial resuscitation: The leucine auxotrophic Mycobacterium tuberculosis (H37RvΔleuD) carrying the pJV53-mScarlet plasmid was revived in a 37°C water bath in advance;

[0050] (2) Prepare inactivated tuberculosis bacteria as a negative control: take 50 μl H37RvΔleuD-mScarlet, add 200 μl 95% ethanol, place at room temperature for 30 min, centrifuge at 10,000 rpm for 5 min, remove the supernatant, resuspend H37RvΔleuD in complete medium, and centrifuge again to remove the supernatant to completely remove the residual ethanol in the tuberculosis bacteria for subsequent infection;

[0051] (3) Cell infection: THP-1 cells were first labeled with carboxyfluorescein succinimidyl ester (CFSE, 5 μM, Catalog No. 565082, BD) and then induced to differentiate with phorbol ester (PMA, 10 ng / ml) for 24 hours. Inactivated and non-inactivated Mycobacterium tuberculosis (H37RvΔleuD-mScarlet) were infected with the same infection dose (MOI = 1) for 4 hours;

[0052] (4) Removal of extracellular bacteria: Repeated rinsing with sterile phosphate-buffered saline (PBS) to remove extracellular bacteria that have not entered the macrophages;

[0053] (5) Replace the culture medium: add RPMI-1640 culture medium containing 200 μg / ml gentamicin and 10% fetal bovine serum (FBS), and continue to culture in a 37°C, 5% CO2 incubator for 2 h;

[0054] (6) Replace low-concentration gentamicin: Gently rinse the cells three times with sterile PBS, add RPMI-1640 medium containing 20 μg / ml gentamicin and 10% fetal bovine serum (FBS), and continue to culture in a 37°C, 5% CO2 incubator for 24 h;

[0055] (7) Flow cytometry: Infected cells were scraped and analyzed by flow cytometry. CFSE-labeled macrophages were detected using the FITC channel, and H37RvΔleuD-mScarlet signals were detected using the PE-Texas Red channel. + Circle PE-Texas Red in the cells + tuberculosis, and finally the survival of intracellular bacteria was quantified.

[0056] Figure 1The figure shows the intracellular bacterial content detected by flow cytometry after inactivation of H37RvΔleuD-mScarlet in THP-1 cells in Example 2. (A) The flow cytometric analysis software (CytExpert) circles all living cells; (B) The flow cytometric analysis software (CytExpert) circles out the adherent cells; (C) The flow cytometric analysis software (CytExpert) circles out the FITC + (D) Flow cytometry analysis software (CytExpert) circled the percentage of intracellular bacteria.

[0057] Figure 2 The following is a graph showing the intracellular bacterial content detected by flow cytometry after live H37RvΔleuD-mScarlet infected THP-1 cells in Example 2. (A) The flow cytometric analysis software (CytExpert) circles all live cells; (B) The flow cytometric analysis software (CytExpert) circles out the adherent cells; (C) The flow cytometric analysis software (CytExpert) circles out the FITC + (D) Flow cytometry analysis software (CytExpert) circled the percentage of intracellular bacteria.

[0058] From the above results, it can be seen that after the inactivated H37RvΔleuD-mScarlet infected THP-1 cells, the flow cytometer only detected 2.86% of the bacteria, while after the non-inactivated H37RvΔleuD-mScarlet infected THP-1 cells, the flow cytometer detected 17.15% of the bacteria. This shows that this method can quickly detect intracellular tuberculosis bacteria in macrophages.

[0059] The experimental method for rapid detection of intracellular tuberculosis bacteria proposed in this patent combines the advantages of fluorescent protein labeling and flow cytometry, and can efficiently and accurately quantify the survival of Mycobacterium tuberculosis in host cells. This method overcomes the shortcomings of the traditional CFU counting method, such as long cycle, low efficiency, and subjective judgment, and provides practical technical support for host-pathogen interaction research and drug screening for tuberculosis. In the future, this method is expected to play an important role in the fields of tuberculosis diagnosis, vaccine evaluation, and drug screening.

Claims

1. A method for rapid detection of intracellular tuberculosis bacilli, characterized in that: The method includes using mScarlet to label Mycobacterium tuberculosis, labeling host cells with carboxyfluorescein succinimidyl ester (CFSE), rinsing some extracellular bacteria that have not entered macrophages with PBS and sterilizing them with gentamicin, and detecting the survival amount of intracellular Mycobacterium tuberculosis by flow cytometry.

2. The method for rapid detection of intracellular tuberculosis bacilli according to claim 1, characterized in that: The specific steps include: (1) Bacterial resuscitation: Resuscitate Mycobacterium tuberculosis carrying the pJV53-mScarlet plasmid; (2) Prepare inactivated tuberculosis bacteria as a negative control; (3) Cell infection: THP-1 cells were first labeled with CFSE and then induced to differentiate into macrophages using phorbol methyl ester (PMA); inactivated and non-inactivated Mycobacterium tuberculosis carrying the pJV53-mScarlet plasmid were infected at the same infection dose; (4) Removal of extracellular bacteria; (5) Gentamycin for bactericidal effect; (6) Flow cytometry: Scrape the infected cells and analyze them using a flow cytometer. Use the FITC channel to detect CFSE-labeled macrophages, and use the PE-Texas Red channel to detect Mycobacterium tuberculosis signals. + Circle PE-TexasRed in the cells + tuberculosis, and finally the survival amount of intracellular tuberculosis bacilli was quantified.

3. The method for rapid detection of intracellular tuberculosis bacilli according to claim 2, characterized in that: The Mycobacterium tuberculosis described in step (1) is a leucine auxotrophic Mycobacterium tuberculosis (H37RvΔleuD).

4. The method for rapid detection of intracellular tuberculosis bacilli according to claim 2, characterized in that: In the step (2), the Mycobacterium tuberculosis (H37RvΔleuD-mScarlet) carrying the pJV53-mScarlet plasmid revived in step (1) is added with ethanol, placed at room temperature, centrifuged to remove the supernatant, resuspended with complete medium, and centrifuged again to remove the supernatant, thereby completely removing the ethanol remaining in the tuberculosis bacteria.

5. The method for rapid detection of intracellular tuberculosis bacilli according to claim 4, characterized in that: Take 50 μl of the Mycobacterium tuberculosis (H37RvΔleuD-mScarlet) carrying the pJV53-mScarlet plasmid revived in step (1) and add 200 μl of 95% ethanol.

6. The method for rapid detection of intracellular tuberculosis bacilli according to claim 2, characterized in that: The dosage of CFSE in step (3) is 5 μM; the concentration of phorbol ester (PMA) is 10-20 ng / ml; and the differentiation induction time is 24-36 hours.

7. The method for rapid detection of intracellular tuberculosis bacilli according to claim 2, characterized in that: In the step (3), inactivated and non-inactivated Mycobacterium tuberculosis (H37RvΔleuD-mScarlet) carrying the pJV53-mScarlet plasmid infect macrophages differentiated by PMA, and the infection dose is MOI=1.

8. The method for rapid detection of intracellular tuberculosis bacilli according to claim 2, characterized in that: In step (4), sterile phosphate buffered saline (PBS) is used for repeated rinsing to remove extracellular bacteria that have not entered the macrophages.

9. The method for rapid detection of intracellular tuberculosis bacilli according to claim 2, characterized in that: The step (5) comprises: (5-1) Replace the culture medium: add serum-containing culture medium containing gentamicin and continue culturing; (5-2) Replace the medium with serum containing low concentration of gentamicin and continue culturing.

10. The method for rapid detection of intracellular tuberculosis bacilli according to claim 9, characterized in that: The concentration of gentamicin in the serum-containing culture medium in step (5-1) is 200 μg / ml; the concentration of gentamicin in the serum-containing culture medium in step (5-2) is 20 μg / ml.

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