CN112481370A - Application of BST1 as tuberculosis diagnosis molecular marker - Google Patents

Abstract

The invention belongs to the field of biological medicines, and relates to an application of a molecular marker BST1 in diagnosis of tuberculosis. The invention aims to provide application of a molecular marker BST1 in diagnosis of tuberculosis. The marker of the invention can be used as a marker for diagnosing tuberculosis or detecting mycobacterium tuberculosis infection, and has good sensitivity and specificity.

Description

Application of BST1 as tuberculosis diagnosis molecular marker

Technical Field

The invention belongs to the field of biological medicines, and relates to an application of a molecular marker BST1 in diagnosis of tuberculosis.

Background

Tuberculosis (TB) is a chronic infectious disease that seriously threatens human health caused by infection with Mycobacterium Tuberculosis (m.tb), and nearly one fourth of people worldwide are infected with Mycobacterium Tuberculosis and are chronically in a latent infection state, of which 5-10% of all life may develop active Tuberculosis. Because the biological characteristics of the mycobacterium tuberculosis such as thicker cell wall, higher fatty acid content, intracellular parasitism and the like, the sensitivity and the detection rate of the early tuberculosis and the rapid diagnosis are lower, and the breakthrough progress is not made. The diagnosis of active tuberculosis is mainly based on etiology detection and patient imaging diagnosis at present. The etiology detection is considered as the 'gold standard' for tuberculosis diagnosis, comprises a sputum smear of a patient, a sputum culture method and a molecular biology detection method, and mainly aims at detecting viable mycobacterium tuberculosis and gene components thereof existing in a host specimen; however, the existing data show that the positive nodule proportion of the bacteria only accounts for 30-40% of clinical tuberculosis cases, and the majority of cases can not be diagnosed by applying pathogenic results. On the other hand, although the imaging diagnosis is fast and sensitive, the imaging diagnosis has the defects of high false positive and incapability of distinguishing the tuberculosis from other lung infections. However, the detection of host-specific immune responses, such as Tuberculin Skin Test (TST) and interferon gamma release test (IGRA), can only determine whether tuberculosis infection is active or not, and cannot distinguish tuberculosis from non-tuberculous mycobacterial infection. Therefore, a new diagnosis method is urgently needed to realize early and rapid diagnosis of tuberculosis so as to achieve the aims of effectively treating individuals and controlling and eliminating tuberculosis transmission.

The tuberculosis diagnosis and auxiliary diagnosis which are clinically applied at present are mainly methods established from etiology and host, and certain defects and limitations still exist in the application. (I) etiology examination: the sputum smear examination of the patient is simple and easy, but the detection rate is low; sputum culture methods, such as roche slant culture and MGIT960, have high accuracy, but have long culture period and complex operation process, and need to be performed in a standard reference laboratory; molecular biological tests, such as GeneXpert, are directed to DNA molecules of tubercle bacillus in a sample, and cannot determine whether the tubercle bacillus is viable. The positive pathogen detection is the 'gold standard' for diagnosing active tuberculosis, however, the positive pathogen tuberculosis ratio of the clinical bacteria only accounts for 30-40% of tuberculosis cases at present, and the pathogenic results cannot be applied to diagnosis in more than half of cases. (II) imaging examination: the X-ray examination of the chest can discover tuberculosis at an early stage, can determine the position, the property and the range of a focus, can know the morbidity and can be used for judging the treatment effect, and is convenient to develop and easy to accept by patients; the CT of the chest can find small or hidden lesions and can make up for the deficiency of general X-ray examination. But is easily confused with other pulmonary diseases and requires the confirmation of a professional physician. Third, the methods for detecting the immune response of the host, such as Tuberculin Skin Test (TST) and interferon gamma release test (IGRA), can only determine whether tuberculosis infection is active tuberculosis, and cannot distinguish tuberculosis from non-tuberculous mycobacterial infection. Therefore, a new diagnosis method is urgently needed to realize early and rapid diagnosis of tuberculosis so as to achieve the aims of effectively treating individuals and controlling and eliminating tuberculosis transmission.

BST1(Bone Marrow Stromal Cell Antigen 1), is a Bone Marrow Stromal Cell Antigen 1. BST1 is located on chromosome 14q32.3 and codes for ADP-ribosyl/cyclic ADP-ribose hydroslase 2. The gene is a known gene, encodes 293 amino acids, and the sequence reference is described in (Nature 409(6822),860-921(2001) the molecule is related to B cell precursor cell maturation^[1]. The product amino acid composition was 33% similar to CD 38. BST1 is expressed in the bone stromal cell line of rheumatoid arthritis patients. In severe rheumatoid arthritis patients, overexpression of BST1 in the stromal cell population is associated with B cell polyclonal malformations to some extent. The change of the expression level of the BST1 gene can also be used for the prognosis evaluation of ovarian cancer treatment^[3]. In addition, the gene can promote the differentiation of mesenchymal cells; exon analysis suggests that nonsense mutations and polymorphisms of this gene may be associated with parkinson's disease. At present, no literature report about BST1 and tuberculosis of human is found.

Disclosure of Invention

The invention aims to provide application of a molecular marker BST1 in diagnosis of tuberculosis.

Our data show that the expression level of BST1 gene in Active Tuberculosis (ATB) sample is obviously increased compared with that in non-tuberculosis infection Control group (HC). At the same time, the expression level of BST1 in active Tuberculosis is also obviously higher than that in the Tuberculosis Latent Infection group (LTBI) and the bacterial pneumonia group (PN). Therefore, the gene can be used as a molecular diagnostic marker of TB and a differential diagnostic marker of TB and LTBI, TB and other lung infections.

The molecular marker BST1(Bone Marrow Stromal Cell Antigen 1) is Bone Marrow Stromal Cell Antigen 1. BST1 is located on chromosome 14q32.3 and codes for ADP-ribosyl/cyclic ADP-ribose hydroslase 2. The gene is a known gene and codes 293 amino acids, and the sequence reference is described in the article (Nature 409(6822),860-921 (2001).

The invention is described in more detail below:

the reagent for detecting the expression level of the molecular marker BST1 is applied to the preparation of a kit for diagnosing tuberculosis.

The invention relates to application of detecting the change of the protein level of BST1 in preparing a kit for diagnosing tuberculosis.

The method for detecting the expression level of the molecular marker BST1 is a fluorescent quantitative PCR method.

The invention relates to application of a reagent for detecting the expression level of a molecular marker BST1 in preparing a kit for diagnosing active tuberculosis.

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 invention relates to application of BST1 in preparing tuberculosis diagnosis marker products.

The invention discloses application of BST1 in preparing a medical instrument for diagnosing tuberculosis, wherein BST1 is used as a diagnostic molecular marker of the medical instrument for diagnosing tuberculosis.

Specifically, the kit comprises the following components:

specific cell-enriched antibody-labeled magnetic beads, Dynabeads CD15(Invitrogen, US); cell total RNA extraction Kit, RNeasy Plus Mini Kit (Qiagen, Germany); cDNA reverse transcriptase, SuperScript^TM IV VILO^TMMaster Mix (Invitrogen, US); a qPCR fluorescent quantitative PCR detection system comprises TaqMan Fsat Advanced Master Mix (Thermo Fishen Scientific, US),

specificity of the exon region of Assay BST1Detecting primer and probe.

Another object of the present invention is to provide a detection method, comprising the steps of:

1) whole blood sample processing

Aspirate 0.8ml of mixed whole blood into a 5ml flow tube, 1: 2, adding 1.6mL of 4 ℃ precooled separation liquid into the flow tube, and uniformly mixing by blowing and sucking; adding CD15⁺Magnetic beads (Invitrogen, US) and a aliquot of the beads was added quickly to the diluted blood, the lid was closed, the flow tube was properly placed on a Hula Mixer (Invitrogen, US), incubated at 4 ℃ for 20min with rotation at 8rpm,

2) magnetic separation

Taking out the incubated cells, performing instantaneous centrifugation, standing for 2min on a magnetic frame, and carefully sucking off the supernatant;

adding 1.6mL of separation liquid, gently blowing uniformly, transferring to a corresponding 2mL protein low adsorption tube, standing on a magnetic frame for 2min, sucking off the supernatant,

the third step of taking the third step of the magnetic frame down, adding 1.6mL of the separation solution, gently blowing the solution uniformly, standing the solution on the magnetic frame for 2min, sucking the supernatant, repeating the steps for 1 time,

fourthly, 350 mu L of Buffer RLT is added to the cells for cell lysis, the cells are vortexed and shaken for 1min and placed at 4 ℃ for standby,

3) total RNA extraction

The extraction of total RNA of the magnetic bead sorted neutrophils is carried out by adopting RNeasy Plus Mini Kit, and the specific operation is as follows:

preparation reaction system 1 and reaction system 2

Standing the cell lysate on a magnetic frame for 2min, sucking the cell lysate, transferring the cell lysate to a gDNA removal column, and centrifuging at 12,000rpm for 30 s;

adding 350 mu L of 70% ethanol into the fluid penetrating agent, mixing uniformly, transferring 700 mu L of the mixture into an RNeasy Mini column at 12,000rpm, centrifuging for 15s, discarding the fluid penetrating agent,

adding 700 mu L of Buffer RW1 at 12,000rpm, centrifuging for 15s, discarding the flow-through liquid,

fourthly, 500 muL of Buffer RPE at 12,000rpm was added, centrifugation was carried out for 15s, the flow-through liquid was discarded,

fifthly, adding 500 mu L Buffer RPE at 12,000rpm, centrifuging for 2min, replacing a new collecting pipe, uncapping and centrifuging for 1min at full speed,

sixthly, putting an RNeasy Mini column into a numbered 1.5mL EP tube, adding 30 mu L of water into the center of a column membrane, standing for 1min at 12,000rpm, centrifuging for 1min, placing the obtained RNA ice for later use,

4) synthesis of cDNA

Taking total RNA of cells, and adopting SuperScript^TM IV VILO^TMPerforming reverse transcription by the Master Mix to obtain cell sample cDNA,

5) real-time fluorescent quantitative PCR detection of relative expression quantity of BST1 gene

And (3) carrying out real-time quantitative PCR on the BST1 specific primer pair or the internal reference primer pair by using the cDNA prepared in the step as a template so as to obtain the relative expression quantity of the BST1 gene in each sample template.

The method comprises the following specific steps of detecting the relative expression of the BST1 gene by real-time fluorescent quantitative PCR:

(1) preparation of reaction System 1 and reaction System 2

Reaction System 1 (target Gene) was 20. mu.L, and polymerase was reacted by qPCR

Fast Advanced Master Mix, target Gene

Assay primer, and sample cDNA and nuclease-free water. The reaction system 2 (reference gene) was 20. mu.L, consisting of

Fast Advanced Master Mix，

Assay primer, and sample cDNA and nuclease-free water.

(2) Real-time quantitative PCR detection

Putting each reaction system prepared in the step (1) in QuantStaudio^TMReal-time quantitative PCR detection was performed on a 6and 7Flex real-time fluorescent quantitative PCR instrument (Applied Biosystems, US). Make itBy 2^-ΔΔCtThe relative expression level of the BST1 gene in each template is calculated.

Reaction conditions are as follows: 2min at 50 ℃; pre-denaturation at 5 ℃ for 3 min; at 95 ℃ for 1s, 60 ℃ for 20sec, 40 cycles, the fluorescence signal was collected during the extension phase.

(3) Statistical analysis the results of step (2) were statistically analyzed using GraphPad Prism 6.

Compared with the prior art, the invention has the beneficial effects that:

the existing tuberculosis diagnosis method is mainly based on etiology diagnosis, and the detection rate of etiologically positive patients in the population is about 30-50%; the remaining pathogenic negative patients cannot be detected; differential diagnosis of tuberculosis patients and latent infection patients and pneumonia patients also presents difficulties. Therefore, the detection of the change of the expression level of the BST1 gene in the peripheral blood of the host can be used for distinguishing (1) active tuberculosis patients from tuberculosis latent infection patients; (2) diagnosing active tuberculosis; (3) latent infected persons and general pneumonia. Therefore, the BST1 gene can be used as a candidate biomarker for diagnosis and differential diagnosis of the above conditions.

For the terms appearing in the description, the corresponding explanations and explanations are given here:

TB: tuberculosis, Tuberculosis

M.tb: mycobacterium tuberculosis. Mycobacterium tuberculosis

TST: tuboculin skin test, tuberculin test

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

PN: pneumoconia, pulmonary infection

CD 15: leukocyte differentiation antigen 15

Hula Mixer: hula mixer

Buffer RLT: RLT buffer solution

RNeasy Plus Mini Kit: RNA extraction kit

gDNA: genomic DNA

RNeasy Mini: RNA extraction kit

Buffer RW 1: RW1 buffer solution

Buffer RPE: RPE buffer

SuperScript^TM IV VILO^TM Master Mix:SuperScript^TMIV VILO^TMMixed solution

cDNA: inverted DNA

Fast Advanced Master Mix：

Quick mixing liquid

Drawings

FIG. 1 real-time quantitative PCR results.

ATB: active tuberculosis patients; LTBI: those with latent infection; HC: non-latent infected persons with inactive tuberculosis; PN: patients with pneumonia.

Detailed Description

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

Examples 1,

Technical scheme

1. Study object and method

1.1. Study object

Study subjects and inclusion criteria

The subjects included ATB, LTB, PN and HC. The ATB group determines the inpatients by hospitals, the diagnosis standard is according to the standard of the national people's republic of China sanitary industry standard (WS 288-2017) tuberculosis diagnosis, and the pathogeny detection is positive, namely: at least 1 of the sputum smear, the culture and the nucleic acid detection is positive, the past tuberculosis history (no old tuberculosis focus in inquiry and X-ray chest examination) is absent, the primary anti-tuberculosis treatment is carried out, and the medicine consumption is less than 7 days; the LTBI group has a history of clinical exposure, no clinical symptoms and positive IGRA; PN group, clinically confirmed lung infection (excluding viral pneumonia) to exclude ATB; HC group, normal for physical examination related indicators, negative for IGRA. All subjects were under 70 years of age or over 18 years of age, women without pregnancy or lactation, and combined with other severe chronic diseases and immunodeficiency disorders. The ethical standard customized by the etiology and biology institute of Chinese medical science/Beijing cooperative medical institute and Shenzhen third people hospital ethical committee is followed, and a notice is signed.

Sample information

265 host samples were collected in this study and divided into 4 groups including ATB, 51; LTBI, 54 cases; PN 58 cases and HC 102 cases. The detailed information of age, sex, etc. is shown in Table 1.

TABLE 1 sample demographic data

Origin of specimen

2.5mL of peripheral blood of the above-mentioned subject was collected, placed in an anticoagulant blood collection tube (BD Biosciences, US) containing lithium heparin, and turned upside down 5 to 6 times (for the purpose of uniformly mixing the anticoagulant and peripheral blood), to obtain a peripheral blood sample.

1.2 methods of investigation

Magnetic bead sorting is based on the combination of cell surface antigen and specific monoclonal antibody connected with magnetic bead, and in the external magnetic field, the cell connected with magnetic bead via antibody is adsorbed and retained in the sorting column, and the cell without the surface antigen has no magnetism because of being unable to combine with the specific monoclonal antibody connected with magnetic bead, and does not retain in the sorting column, so that the cell can be separated.

The kit comprises the following components: CD15+ cell enrichment magnetic beads and related reagents, a cell total RNA extraction separation column and related reagents, cDNA synthesis reverse transcriptase and related reagents, BST1 sequence amplification specific primers and real-time fluorescent quantitative PCR related reagents.

1.2.1.1 Whole blood sample processing

Following the commercial reagent protocol, 0.8ml of pooled whole blood was aspirated into a 5ml flow tube, 1: 2, adding 1.6mL of 4 ℃ precooled separation liquid into the flow tube, and uniformly mixing by blowing and sucking; adding CD15⁺Magnetic beads (Invitrogen, US) and a aliquot of the beads was added quickly to the diluted blood, the lid was closed, and the flow tube was properly placed on a Hula Mixer (Invitrogen, US) and incubated at 4 ℃ for 20min with 8rpm rotation.

1.2.1.2 magnetic separation

Taking out the incubated cells, performing instantaneous centrifugation, standing for 2min on a magnetic frame, and carefully sucking off the supernatant;

and adding 1.6mL of separation liquid, and slightly and uniformly blowing the separation liquid and transferring the separation liquid to a corresponding 2mL protein low adsorption tube. Standing on a magnetic frame for 2min, and sucking off the supernatant.

And taking the third step down from the magnetic frame, adding 1.6mL of separation liquid, and blowing gently and uniformly. Standing on a magnetic frame for 2min, and sucking off the supernatant. Repeat for 1 time.

350 μ L of Buffer RLT (Qiagen, Germany) was added to lyse the cells, vortexed for 1min, and left at 4 ℃ until needed.

1.2.2 Total RNA extraction

Total RNA from magnetic bead sorted neutrophils was extracted using the RNeasy Plus Mini Kit (Qiagen, Germany) as follows:

preparation reaction system 1 and reaction system 2

Standing the cell lysate on a magnetic frame for 2min, sucking the cell lysate, transferring the cell lysate to a gDNA removal column, and centrifuging at 12,000rpm for 30 s;

adding 350 mu L of 70% ethanol into the fluid for fluid penetration, and mixing uniformly. Transfer 700. mu.L to RNeasy Mini column, centrifuge at 12,000rpm for 15s, and discard the flow-through.

This was done by adding 700. mu.L Buffer RW1 at 12,000rpm, centrifuging for 15s, and discarding the flow-through.

Then, 500. mu.L of Buffer RPE was added thereto at 12,000rpm, and the mixture was centrifuged for 15 seconds to discard the flow-through solution.

Fifthly, adding 500 mu L Buffer RPE at 12,000rpm, centrifuging for 2min, replacing a new collecting pipe, and uncapping and centrifuging for 1min at full speed.

Sixthly, putting an RNeasy Mini column into a numbered 1.5mL EP tube, adding 30 mu L of water into the center of a column membrane, and standing for 1 min. The mixture was centrifuged at 12,000rpm for 1min, and the RNA was then placed on ice until use.

1.2.3 Synthesis of cDNA

Taking total RNA of cells, and adopting SuperScript^TM IV VILO^TMThe Master Mix (Invitrogen, US) was subjected to reverse transcription to obtain cell sample cDNA.

1.2.4 detection of relative expression quantity of BST1 Gene by real-time fluorescent quantitative PCR

And (3) carrying out real-time quantitative PCR on the BST1 specific primer pair or the internal reference primer pair by using the cDNA prepared in the step 1.2.3 as a template so as to obtain the relative expression quantity of the BST1 gene in each sample template. The method comprises the following specific steps:

(1) preparation of reaction System 1 and reaction System 2

Reaction System 1 (target Gene) was 20. mu.L, and polymerase was reacted by qPCR

Fast Advanced Master Mix, target Gene

Assay primer, and sample cDNA and nuclease-free water. The reaction system 2 (reference gene) was 20. mu.L, consisting of

Fast Advanced Master Mix，

Assay primer, and sample cDNA and nuclease-free water.

(2) Real-time quantitative PCR detection

Putting each reaction system prepared in the step (1) in QuantStaudio^TMReal-time quantitative PCR detection was performed on a 6and 7Flex real-time fluorescent quantitative PCR instrument (Applied Biosystems, US). Use 2^-ΔΔCtThe relative expression level of the BST1 gene in each template is calculated.

Reaction conditions are as follows: 2min at 50 ℃; pre-denaturation at 5 ℃ for 3 min; at 95 ℃ for 1s, 60 ℃ for 20sec, 40 cycles, the fluorescence signal was collected during the extension phase.

(3) Statistical analysis the results of step (2) were statistically analyzed using GraphPad Prism 6.

2. Results of the study

After processing the quantitative PCR results, t-tests were performed on the data using GraphPad Prism 6, and the results are shown in fig. 1. as can be seen from the figure, the expression level of BST1 was significantly different in TB versus HC, LTBI, and PN group samples, and the expression level of TB group genes was significantly higher than that in HC and LTBI groups (. about.p <0.001 and. about.p < 0.01); the expression level of genes in the PN group is obviously higher than that in the HC group and the LTBI group (P < 0.0001). Therefore, the gene can be used as a molecular marker for diagnosing tuberculosis patients; and differential diagnosis markers for distinguishing active tuberculosis from latent infection and tuberculosis infection from bacterial pneumonia.

Example 2 kit

The kit comprises the following components:

specific cell-enriched antibody-labeled magnetic beads, Dynabeads CD15(Invitrogen, US); cell total RNA extraction Kit, RNeasy Plus Mini Kit (Qiagen, Germany); cDNA reverse transcriptase, SuperScript^TM IV VILO^TMMaster Mix (Invitrogen, US); the qPCR fluorescent quantitative PCR detection system comprises TaqMan^TM Fsat Advanced Master Mix(Thermo Fishen Scientific,US)，

The detection primers and probes are specific to the Assay BST1 exon region.

Reference documents:

[1]Kaisho T,Ishikawa J,Oritani K,Inazawa J,et al.BST-1,a surface molecule of bone marrow stromal cell lines that facilitates pre-B-cell growth.Proc.Natl.Acad.Sci.U.S.A.1994Jun；91(12)5325-5329

[2]Ishikawa J,Kaisho T,Tomizawa H,et al.Molecular cloning and chromosomal mapping of a bone marrow stromal cell surface gene,BST2,that may be involved in pre-B-cell growth.Genomics.1995Apr 10；26(3):527-34.doi:10.1016/0888-7543(95)80171-h.

[3]Ortolan E,Arisio R,Morone S,Bovino P,Lo-Buono N,Nacci G,Parrotta R,Katsaros D,Rapa I,Migliaretti G,Ferrero E,Volante M,Funaro A.Functional role and prognostic significance of CD157 in ovarian carcinoma.J Natl Cancer Inst.2010Aug 4；102(15):1160-77.doi:10.1093/jnci/djq256.Epub 2010Jul 16.PMID:20639476.

[4]Wang C,Feng X,Xie S,Gu Z,Chan P.Wang C,et al.Exonic sequencing revealed no causative mutation in the BST1 gene in patients with Parkinson's disease.Neurobiol Aging.2013Nov；34(11):2695.e9-2695.e10.doi:10.1016/j.neurobiolaging.2013.05.024.Epub 2013Jul 2.

[5]Wang S,Xu YF,Ding XY,Liu ZR,Ding Y,Jin B,Wang S,Ding MP.Association between bone marrow stromal cell antigen 1gene polymorphisms and the susceptibility to Parkinson's disease:a meta-analysis.Neurosci Lett.2015Jul 10；599:120-4.doi:10.1016/j.neulet.2015.05.026.Epub 2015May 15.PMID:25986899.

[6]Ceroni F,Sagar A,Simpson NH,Gawthrope AJ,Newbury DF,Pinto D,Francis SM,Tessman DC,Cook EH,Monaco AP,Maestrini E,Pagnamenta AT,Jacob S.A deletion involving CD38 and BST1 results in a fusion transcript in a patient with autism and asthma.Autism Res.2014Apr；7(2):254-63.doi:10.1002/aur.1365.Epub 2014Mar 13.PMID:24634087；PMCID:PMC4309371.

[7]Satake W,Nakabayashi Y,Mizuta I,Hirota Y,Ito C,Kubo M,Kawaguchi T,Tsunoda T,Watanabe M,Takeda A,Tomiyama H,Nakashima K,Hasegawa K,Obata F,Yoshikawa T,Kawakami H,Sakoda S,Yamamoto M,Hattori N,Murata M,Nakamura Y,Toda T.Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson's disease.Nat Genet.2009Dec；41(12):1303-7.doi:10.1038/ng.485.Epub 2009 Nov 15.PMID:19915576.

[8]Mo W,Liu J,Zhang Z,Yu H,Yang A,Qu F,Hu P,Liu Z,Hu F.Astudy of single nucleotide polymorphisms in CD157,AIM2 and JARID2 genes in Han Chinese children with autism spectrum disorder.Nord J Psychiatry.2018 Apr；72(3):179-183.doi:10.1080/08039488.2017.1410570.Epub 2017 Dec 7.PMID:29216786.

Claims (10)

1. the application of the reagent for detecting the expression level of the molecular marker BST1 in preparing a kit for diagnosing tuberculosis.

2. Application of detecting change of BST1 protein level in preparing kit for diagnosing tuberculosis.

3. The use according to claim 1, wherein the method for detecting the expression level of the molecular marker BST1 is a fluorescent quantitative PCR method.

4. The use according to claim 1, characterized by the use of an agent for detecting the expression level of the molecular marker BST1 for the preparation of a kit for the diagnosis of active tuberculosis.

5. Use according to claim 1, characterized in that it comprises a method for differentiating between patients with active tuberculosis, latent tuberculosis infection and non-latent inactive tuberculosis infection.

6. Use according to claim 1, wherein the tuberculosis is pulmonary tuberculosis or extrapulmonary tuberculosis.

7. The use according to claim 1, wherein the kit further comprises the following components:

specific cell-enriched antibody-labeled magnetic beads, Dynabeads CD15(Invitrogen, US); cell total RNA extraction Kit, RNeasy Plus Mini Kit (Qiagen, Germany); cDNA reverse transcriptase, SuperScript^TM IV VILO^TMMaster Mix (Invitrogen, US); a qPCR fluorescent quantitative PCR detection system comprises TaqMan Fsat Advanced Master Mix (Thermo Fishen Scientific, US),

the detection primers and probes are specific to the Assay BST1 exon region.

The application of BST1 in preparing tuberculosis diagnosis marker products.

The application of BST1 in preparing a medical apparatus for diagnosing tuberculosis, wherein BST1 is used as a diagnostic molecular marker.

10. The use according to claim 1, wherein the detection method of the kit comprises the following steps:

1) whole blood sample processing

Aspirate 0.8ml of mixed whole blood into a 5ml flow tube, 1: 2, adding 1.6mL of 4 ℃ precooled separation liquid into the flow tube, and uniformly mixing by blowing and sucking; adding CD15⁺Magnetic beads (Invitrogen, US) and a aliquot of the beads was added quickly to the diluted blood, the lid was closed, the flow tube was properly placed on a Hula Mixer (Invitrogen, US), incubated at 4 ℃ for 20min with rotation at 8rpm,

2) magnetic separation

Taking out the incubated cells, performing instantaneous centrifugation, standing for 2min on a magnetic frame, and carefully sucking off the supernatant;

adding 1.6mL of separation liquid, gently blowing uniformly, transferring to a corresponding 2mL protein low adsorption tube, standing on a magnetic frame for 2min, sucking off the supernatant,

the third step of taking the third step of the magnetic frame down, adding 1.6mL of the separation solution, gently blowing the solution uniformly, standing the solution on the magnetic frame for 2min, sucking the supernatant, repeating the steps for 1 time,

fourthly, 350 mu L of Buffer RLT is added to the cells for cell lysis, the cells are vortexed and shaken for 1min and placed at 4 ℃ for standby,

3) total RNA extraction

The extraction of total RNA of the magnetic bead sorted neutrophils is carried out by adopting RNeasy Plus Mini Kit, and the specific operation is as follows:

preparation reaction system 1 and reaction system 2

Standing the cell lysate on a magnetic frame for 2min, sucking the cell lysate, transferring the cell lysate to a gDNA removal column, and centrifuging at 12,000rpm for 30 s;

adding 350 mu L of 70% ethanol into the fluid penetrating agent, mixing uniformly, transferring 700 mu L of the mixture into an RNeasy Mini column at 12,000rpm, centrifuging for 15s, discarding the fluid penetrating agent,

adding 700 mu L of Buffer RW1 at 12,000rpm, centrifuging for 15s, discarding the flow-through liquid,

fourthly, 500 muL of Buffer RPE at 12,000rpm was added, centrifugation was carried out for 15s, the flow-through liquid was discarded,

fifthly, adding 500 mu L Buffer RPE at 12,000rpm, centrifuging for 2min, replacing a new collecting pipe, uncapping and centrifuging for 1min at full speed,

sixthly, putting an RNeasy Mini column into a numbered 1.5mL EP tube, adding 30 mu L of water into the center of a column membrane, standing for 1min at 12,000rpm, centrifuging for 1min, placing the obtained RNA ice for later use,

4) synthesis of cDNA

Taking total RNA of cells, and adopting SuperScript^TM IV VILO^TMPerforming reverse transcription by the Master Mix to obtain cell sample cDNA,

5) real-time fluorescent quantitative PCR detection of relative expression quantity of BST1 gene

And (3) carrying out real-time quantitative PCR on the BST1 specific primer pair or the internal reference primer pair by using the cDNA prepared in the step as a template so as to obtain the relative expression quantity of the BST1 gene in each sample template.

Images