CN112630447A

CN112630447A - Glutathione used for identification and diagnosis of active tuberculosis

Info

Publication number: CN112630447A
Application number: CN202011512556.0A
Authority: CN
Inventors: 金奇; 郑建华; 刘立国; 邓佳珩
Original assignee: Institute of Pathogen Biology of CAMS
Current assignee: Institute of Pathogen Biology of CAMS
Priority date: 2020-12-20
Filing date: 2020-12-20
Publication date: 2021-04-09

Abstract

The invention relates to a method for diagnosing active tuberculosis by using glutathione as biomarker to detect the glutathione content in urine, and uses the specific content as diagnostic index, and if the content is lower than the index, the result is judged to be positive.

Description

Glutathione used for identification and diagnosis of active tuberculosis

Technical Field

The invention relates to a diagnostic reagent, in particular to application of Glutathione (Glutathione) to identification and diagnosis of active tuberculosis.

Background

Tuberculosis (TB) is a chronic infectious disease that seriously threatens human health and is caused by infection of Mycobacterium Tuberculosis (m.tb), one of ten causes of death worldwide and the main cause of death caused by a single infectious source. Nearly one quarter of the world's population is infected with mycobacterium tuberculosis and remains chronically in a latent infection state, with 5% to 10% of all cases having the potential to develop active tuberculosis throughout life. The progression from latent infection with mycobacterium tuberculosis to active disease constitutes a persistent threat to the spread of tuberculosis. Geographically, the majority of tuberculosis cases in 2018 were in southeast asian (44%), with china accounting for 9% of the worldwide population, second only to india (27%). Therefore, the rapid and accurate diagnosis of tuberculosis infection state is one of the important components for tuberculosis prevention and control. However, the biological characteristics of the mycobacterium tuberculosis such as thicker cell wall, higher fatty acid content, intracellular parasitism and the like are adopted, so that the sensitivity and the detection rate of the early tuberculosis and the rapid diagnosis are lower, and the breakthrough progress is not made. Especially in countries and regions with poor health management and poor medical care levels, nearly half of the patients with tuberculosis that are negative in etiology still cannot be diagnosed scientifically, reasonably, quickly and effectively, and even the conditions are delayed, resulting in drug resistance and spread. Therefore, how to realize the early and rapid diagnosis of tuberculosis patients and research and develop a new specific biomarker for the early diagnosis of tuberculosis becomes a key scientific problem to be solved urgently in China and the world in the important public health field.

The current tuberculosis virus diagnosis methods are as follows: (I) etiology examination: the sputum smear bacteria examination and the sputum tubercle bacillus examination are 'golden indexes' for diagnosing the pulmonary tuberculosis, are simple, convenient and easy to operate, have higher accuracy, and can confirm the diagnosis of the tuberculosis by checking the tubercle bacillus in the sputum. Generally, three sputum specimens, namely night sputum, early morning sputum and instant sputum, need to be examined for the first diagnosis, the result reliability is high, and tubercle bacillus can be cultured for susceptibility test. However, the diagnosis rate is low, the cultivation of tubercle bacillus takes 6-8 weeks, the period is too long, and the application is limited. (II) X-ray examination: the X-ray examination of chest can find tuberculosis at early stage, and can determine the position, property and range of the focus, understand the disease condition and judge the treatment effect, and the development is convenient, and the patient is willing to accept. The CT of the chest can find small or hidden lesions and can make up for the deficiency of general X-ray examination. But the examination is relatively expensive and easily confused with other lung diseases, requiring a specialized doctor to make a definitive diagnosis. (III) immunological diagnosis of pulmonary tuberculosis: 1. tuberculin Pure Protein Derivative (PPD) test, positive test is one of the common evidences of infecting tubercle bacillus, but the false positive rate is high, and misdiagnosis is easy. 2. The antibody detection of blood or sputum tuberculosis, the positive antibody in blood or sputum also helps diagnosis, but false positive rate is easy to appear, and latent infection and active tuberculosis cannot be distinguished. BACTEC, which is a diagnostic method by detecting metabolites of Mycobacterium tuberculosis, mycobacteria can be isolated typically for two weeks, but the amount of bacteria affects the time at which a positive result occurs. 4. Polymerase Chain Reaction (PCR) has the advantage of 98-100% sensitivity and the disadvantage of poor specificity. 5. The gamma interferon release assay (IGRA), although high in both sensitivity and specificity, is expensive and difficult to achieve for high throughput detection. (IV) other checks: can only be used as auxiliary diagnosis and can not be used as diagnosis basis. 1. The fiber bronchoscopy can directly or indirectly judge the pathological changes in the bronchus and the lung, has the functions of biopsy, lavage, video recording, picture taking in the trachea and the like, and is particularly useful for diagnosis and differential diagnosis. 2. Thoracoscopy and mediastinoscopy can be used to observe enlarged lymph nodes in the thoracic cavity and mediastinum, and biopsy can be taken to facilitate diagnosis and differential diagnosis. 3. Ultrasonic examination is mainly used for diagnosing and differentially diagnosing pleural effusion.

The molecular marker research of tuberculosis diagnosis at present mainly has two directions of mycobacterium tuberculosis and host, but the demand of tuberculosis diagnosis can not be met. The experiment carries out non-invasive metabonomics analysis on active tuberculosis patients and latent infection patients, searches related molecular markers for tuberculosis diagnosis on the metabonomic level, and provides a path for researching the molecular markers for the tuberculosis diagnosis.

Glutathione (glutathione) is a tripeptide composed of glutamic acid, cysteine and glycine, which participates in many important detoxification reactions and has a redox buffering effect to prevent oxidative damage and regulate redox signals in the body. It has been confirmed that glutathione can become a carrier molecule for nitric oxide by forming S-nitrosothione, thereby inhibiting the growth of mycobacterium tuberculosis, but it has not been studied as a biomarker in the diagnosis of tuberculosis.

The current commonly used tuberculosis diagnosis standard is generally bacteriology, namely positive sputum test for anti-nitrogen bacillus or positive sputum culture, or molecular biological test such as PCR or T-SPOT test, and if positive, the tuberculosis is diagnosed.

On the basis of the prior art, the invention adopts a biomarker method, and unexpectedly discovers a new biomarker, namely glutathione, because the glutathione is a very familiar substance, the detection method is simple and convenient, and the cost is low. Therefore, the invention provides a new tuberculosis diagnosis method, which adopts glutathione as a biomarker, detects the content of glutathione in urine, takes the value of specific content as a diagnosis index, and judges the urine to be positive when the value is lower than the diagnosis index.

Disclosure of Invention

The invention aims to provide application of glutathione as a biomarker in tuberculosis diagnosis. The invention discovers for the first time that the glutathione can be used as a biomarker for diagnosing active tuberculosis or latent tuberculosis infection, and further discovers that the glutathione has good sensitivity and specificity.

The more detailed technical scheme of the invention is described as follows:

the invention provides application of glutathione in preparation of a tuberculosis diagnosis reagent.

The invention further provides application of the glutathione in preparation of medical instruments for tuberculosis diagnosis.

The diagnosis of tuberculosis takes glutathione as a diagnosis molecular marker.

The invention provides a reagent combination, which can be used for directly or indirectly extracting and separating glutathione from urine, further detecting the content of the glutathione and using the content of the glutathione as a diagnostic index to determine whether a urine provider has tuberculosis, namely the reagent combination takes the glutathione as a detection target and takes the glutathione as a biomarker for diagnosing the tuberculosis.

Therefore, the invention further provides the application of the reagent combination in the preparation of a kit for diagnosing tuberculosis.

The biomarker glutathione of the invention, which is a known compound, can be extracted or detected according to any one of the existing methods, including any one of the methods for detecting glutathione, such as those reported in the prior documents: an enzyme cycling method, a colorimetric method, a fluorescence method, a chromatography method, a high performance capillary electrophoresis method, a flow cytometry method, a sodium nitrosoferricyanide method, a tetraoxypyrimidine method, a DTNB method and the like, preferably a chromatography method, more preferably a high performance liquid chromatography method, most preferably a method using a combination of ultra high performance liquid chromatography and mass spectrometry.

The application of the invention comprises the application of distinguishing active tuberculosis patients, tuberculosis latent infection patients and inactive tuberculosis non-latent infection patients.

The tuberculosis is pulmonary tuberculosis or extrapulmonary tuberculosis.

The body fluid is urine.

Specifically, the kit of the invention comprises any reagent which can be used for extracting and separating glutathione,

the kit of the present invention includes any one of the existing kits for detecting glutathione, such as: glutathione analytical kit Shanghai crystal antibiotic, Glutathione detection kit for Shanghai Jinning scientific research, Glutathione detection kit-Tongyu, merck Glutathione detection kit, Glutathione Assay kit (fluorometer), Glutathione kit gshe elisa kit and the like.

Preferably, the kit of the present invention comprises a glutathione standard control, and further comprises instructions for the detection method, reference values for the detection standard, organic or inorganic reagents, a preparation method of a solvent, and the like.

The technical scheme of the invention is obtained by research, and the research method comprises the following steps:

1. technical scheme

1.1 Subjects and methods

1.1.1 subjects

Study subjects and inclusion criteria

In the study subjects, the active tuberculosis comes from confirmed tuberculosis inpatients diagnosed in chest hospitals in Beijing, the diagnosis standard is in accordance with the standard of the national sanitation industry Standard (WS 288-2017) tuberculosis diagnosis of the people's republic of China, and the pathogenic detection is positive: at least 1 sputum smear or sputum culture is positive, no past tuberculosis history (no old tuberculosis focus in inquiry and X-ray chest examination) exists, primary antituberculosis treatment is carried out, and the administration time is less than 7 days; the latent infected person has tuberculosis exposure or contact history, the tuberculosis immunological examination is positive, the sputum smear or sputum culture is negative, and the chest X-ray examination is normal; the etiology and immunology of the non-latent infected persons (control group) with inactive tuberculosis were negative. The detailed information of age, sex and the like of 30 healthy control groups, 30 tuberculosis latent infection groups and 30 active tuberculosis groups are selected and shown in table 1. All subjects were under 70 years of age, women without pregnancy or lactation and combined with other severe chronic diseases and immunodeficiency disorders. The ethical standard of Chinese medical academy of sciences/Beijing cooperative medical academy pathogenic biology research institute and Beijing thoracic hospital ethical Committee is followed, and an informed opinion notice is signed.

TABLE 1 sample demographic data

Item	Active tuberculosis	Latent tuberculosis infection	Non-tuberculosis control group
				Total number of	30	30	30
Age (mean median)])	34.9[29.5]	58.7[58.5]	58.9[59]
				Sex
Men n (%)	16(53.33)	17(56.67)	13(43.33)
				Woman n (%)	14(46.67)	13(43.33)	17(56.67)

Origin of specimen

The midstream urine of morning urine from the study subjects was collected separately in 50ml centrifuge tubes and centrifuged at 4000 Xg for 10 minutes at room temperature, and the supernatant was separated and stored below-20 ℃ to obtain urine samples for analysis.

1.1.2 methods of investigation

Metabonomics is a research mode for identifying and analyzing metabolites and finding out the relative relation between the metabolites and physiological and pathological changes based on the combination of liquid mass spectrometry and chromatography. Since metabolites are mostly functional molecules, signal molecules, the measurement of metabolite concentrations is a more direct method to characterize the metabolic state of cells, tissues and organisms compared to other omics. Ultra Performance Liquid Chromatography (UPLC) has the advantages of high analysis flux, high speed, high sensitivity and high separation degree. The Mass spectrometry technology is an identification technology for measuring the Mass-to-charge ratio of ions, Time of Flight Mass Spectrometer (TOF-MS) is one of the commonly used Mass spectrometry types, a Mass analyzer is an ion drift tube (ion drift tube), the ion drift tube flies to an ion receiver at a constant speed after acceleration, and the arrival Time and the abundance of each ion are recorded to obtain the Mass-to-charge ratio of the ion, so that a Mass spectrogram is formed.

1.1.2.1 urine sample handling

After thawing the sample, 100. mu.l of the supernatant was added with 300. mu.l of methanol and 5. mu.l of DL-o-trichlorophenylalanine (2.8mg/mL, internal standard) and vortexed for 30 s. After storage at-40 ℃ for 1 hour, vortexed for 30s, centrifuged at 12000rpm at 4 ℃ for 15 minutes, 200. mu.l of the supernatant was transferred to a sample tube to prepare for loading.

1.1.2.2 UPLC-TOF-MS

Separation was performed using Ultimate 3000LC in combination with Q active MS (Thermo) and mass spectrometric identification analysis was performed with ESI-MS (targeted MS/MS mode). The liquid phase system was loaded with Thermo Hyper gold C18 (100X 2.1mm 1.9 μm). The mobile phase consists of a solvent A (0.1% formic acid-5% acetonitrile-water) and a solvent B (0.1% formic acid-acetonitrile), and gradient elution is carried out (0-1.0 min, 100-100% A, 1.0-6.0 min, 100-80% A, 6.0-8.0 min, 80-70% A, 8.0-12.0 min, 70-65% A, 12.0-13.0 min, 65-30% A, 13.0-15.0 min, 30-5% A, 15.0-16.0min, 5-5% A, 16.0-17.0min, 5-100% A, 17.0-18.0min and 100% A). The flow rate of the mobile phase was 0.3mL min-1. The column temperature was maintained at 40 ℃ and the sample manager temperature was set to 4 ℃. ESI-Mass Spectrometry parameters were as follows: heater temperature 300 ℃, sheath gas flow rate, 45 arb; secondary gas flow, 15 arb; scavenging flow, 1 arb; spraying voltage, 3.2 kV; capillary temperature, 350 ℃; S-Lens RF level, 60%.

1.1.3 data analysis

Raw data were collected and aligned using a Compound Discover (3.0, Thermo) based on the m/z value and retention time of the ion signal. The ion data was combined and imported into the SIMCA-P program (version 14.1) to normalize the data. Results were subjected to unpaired T-test statistical analysis using GraphPad Prism 7.

2. Results of the study

The data obtained by SIMAC-P is imported into GraphPad Prism 7, and the data is visually analyzed, and the result is shown in figure 1. As can be seen, the relative expression levels of glutathione in the active tuberculosis group were significantly different from those in the healthy control group (P <0.0001) and the latent infection group (P <0.0001) compared with those in the non-latent infection group and latent infection group of the inactive tuberculosis; the relative expression level of glutathione in the latently infected group was significantly different from that in the healthy control group (. about.P < 0.001). Can distinguish healthy people and tuberculosis patients, latent infection and tuberculosis patients, so the compound glutathione can be used as a molecular diagnosis marker of tuberculosis.

3. And (4) conclusion: glutathione can clearly distinguish active tuberculosis patients from tuberculosis latent infection and inactive tuberculosis non-latent infection, and can be used as a candidate biomarker for diagnosing active tuberculosis.

The invention, through the research, obtains the conclusion that glutathione can be used as a biomarker of tuberculosis, and a detection result is obtained through a related detection method, and the result can be used for diagnosing and evaluating the tuberculosis and the severity thereof. Or judging whether tubercle bacillus is infected or not according to the result, and whether the infection is active infection or not.

According to the research, the invention obtains a judgment standard, and preliminarily determines that the normal value of the glutathione content in the urine is 12.306-22.704 micrograms/ml, if the normal value is lower than the limit, the result is a positive result, and if the normal value is lower than 3 micrograms/ml, the possibility of active tuberculosis is considered to be higher. Within this normal range, negative results are obtained.

The following is a noun explanation of the invention:

TB: tuberculosis, Tuberculosis

M.tb: mycobacterium tuberculosis

PPD (p): tuberculin pure protein derivative experiment

IGRA: interferon gamma release assays, gamma interferon release assay

ATB: active Tuberculosis, Active Tuberculosis

LTBI: latent Tuberculosis Infection in late Tuberculosis

HC: health Control, non-tuberculosis infection Control group

And (3) UPLC: ultra Performance Liquid Chromatography

TOF-MS: time of Flight Mass Spectrometers, Time of Flight Mass Spectrometry

ESI-: electron Spray Ionization negative mode of electrospray ion source

Reference documents:

[1]World Health Organization.Global tuberculosis report 2019.Available from: http://www.who.int/tb/publications/global_report/en.

[2]Hur Y,Crampin A C,Chisambo C,et al.Identification of Immunological Biomarkers Which May Differentiate Latent Tuberculosis from Exposure to Environmental Nontuberculous Mycobacteria in Children.Clinical and Vaccine Immunology,2014,21(2):133-142.

[3]Nicholson J K,Connelly J,Lindon J C,et al.INNOVATIONMetabonomics:a platform for studying drug toxicity and gene function.Nat Rev Drug Discov,2002,1(2):153-161

[4]A.J.Cooper,J.T.Pinto,P.S.Callery,Reversible and irreversible protein glutathionylation: biological and clinical aspects,Expert Opin Drug Metab Toxicol 7(2011)891-910.

drawings

FIG. 1 shows the relative quantitation result of glutathione

Note: TB: active tuberculosis patients; LTBI: those with latent infection; HC: non-latent infected persons with inactive tuberculosis.

The specific implementation mode is as follows:

the invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.

Example 1

50mL of midstream urine of a Wangxian test subject was collected, centrifuged at 4000 Xg for 10 minutes at room temperature, the supernatant was separated, 100. mu.l of the supernatant was taken, and 300. mu.l of methanol and 5. mu.l of DL-o-trichlorophenylalanine (2.8mg/mL, internal standard) were added and vortexed for 30 s. Storing at-40 deg.C for 1 hr, vortexing for 30s, centrifuging at 12000rpm at 4 deg.C for 15 min, transferring 200 μ l of supernatant to sample tube, and detecting by the following liquid chromatography-mass spectrometry method:

The detection results are as follows:

after the ion data are introduced into SIMAC-P software for normalization and conversion, the content of glutathione in the sample is measured to be about 1.614 micrograms/microliter.

Normal values: 12.306-22.704 micrograms/ml, below which the test result is positive and has a greater probability of being an active infection.

Example 2

The midstream urine of the test subject, 50mL, was collected, centrifuged at 4000 Xg for 10 minutes at room temperature, the supernatant was separated, 100. mu.l of the supernatant was taken, and 300. mu.l of methanol and 5. mu.l of DL-o-trichlorophenylalanine (2.8mg/mL, internal standard) were added and vortexed for 30 s. Storing at-40 deg.C for 1 hr, vortexing for 30s, centrifuging at 12000rpm at 4 deg.C for 15 min, transferring 200 μ l of supernatant to sample tube, and detecting by the following liquid chromatography-mass spectrometry method:

The detection results are as follows:

after the ion data are introduced into SIMAC-P software for normalization and conversion, the content of glutathione in the sample is measured to be about 13.661 micrograms/microliter.

Normal values: 12.306-22.704 micrograms/ml, the detection result is within the limit range, and the result is negative.

Claims

1. Application of glutathione in preparing a reagent for diagnosing tuberculosis.

2. The application of glutathione in the preparation of medical instruments for tuberculosis diagnosis.

3. The use according to any one of claims 1 or 2, wherein the diagnosis of tuberculosis is using glutathione as a diagnostic molecular marker.

4. A reagent combination is used for directly or indirectly extracting and separating glutathione from body fluid, further detecting the content of the glutathione, and using the content of the glutathione as a diagnostic index to determine whether a body fluid provider has tuberculosis.

5. A reagent combination according to claim 4, wherein the bodily fluid is urine.

6. The reagent combination of claim 4, which is a kit comprising any reagent that can be used for extracting and separating glutathione.

7. The reagent combination of claim 6, wherein the kit comprises glutathione standard reference substances, and further comprises instructions for the detection method, reference values of the detection standard, organic or inorganic reagents, a preparation method of a solvent and the like.

8. Use of the combination of reagents according to claim 4 for the preparation of a kit for the diagnosis of tuberculosis.

9. Use according to any of claims 1 or 2, comprising a method for distinguishing between patients with active tuberculosis, latent tuberculosis infection and non-latent inactive tuberculosis infection.

10. The use according to any one of claims 1 or 2, wherein the tuberculosis is pulmonary tuberculosis or extrapulmonary tuberculosis.