CN112143797A - HIST1H2BC applied to differential diagnosis of active tuberculosis - Google Patents

HIST1H2BC applied to differential diagnosis of active tuberculosis Download PDF

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CN112143797A
CN112143797A CN202011058666.4A CN202011058666A CN112143797A CN 112143797 A CN112143797 A CN 112143797A CN 202011058666 A CN202011058666 A CN 202011058666A CN 112143797 A CN112143797 A CN 112143797A
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hist1h2bc
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金奇
张笑冰
刘立国
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Institute of Pathogen Biology of CAMS
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Abstract

The invention belongs to the field of biological medicines, and relates to an application of a molecular marker HIST1H2BC in diagnosis of tuberculosis. The invention provides application of a reagent for detecting the expression level of a molecular marker HIST1H2BC in preparing a kit for diagnosing tuberculosis.

Description

HIST1H2BC applied to differential diagnosis of active tuberculosis
Technical Field
The invention belongs to the field of biological medicines, and relates to an application of a molecular marker HIST1H2BC 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 positive nodule rate of the bacteria only accounts for 30-40% of clinical tuberculosis cases at present, 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 current tuberculosis virus diagnosis methods are as follows: (I) etiology examination: the sputum smear bacteria examination and the sputum tubercle bacillus examination are simple and easy to implement, the accuracy is higher, and the tuberculosis can be accurately diagnosed by examining the tubercle bacillus in the sputum. Generally, three sputum specimens, i.e., night sputum, early morning sputum and immediate sputum, should be examined for the first diagnosis. Although it is a 'gold index' for diagnosing pulmonary tuberculosis, the diagnosis rate is low and the culture period is long. The result of the tubercle bacillus culture is high in reliability, and the tubercle bacillus drug sensitivity test can be carried out, but the application is limited after 6-8 weeks. (II) X-ray inspection: the chest X-line examination 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 the deficiency of the general X-ray examination. But is easily confused with other pulmonary diseases and requires the confirmation of a professional physician. (III) immunological diagnosis of pulmonary tuberculosis: 1. the tuberculin Pure Protein Derivative (PPD) test is commonly used, positive test is one of evidences of tubercle bacillus infection, but the false positive rate is high, and misdiagnosis is easy. 2. The positive detection of the antibody of tuberculosis in blood and sputum also helps diagnosis and is easy to have false positive rate. The BACTEC method for detecting the metabolites of Mycobacterium tuberculosis can isolate the mycobacteria generally within two weeks, but the amount of the bacteria can affect the days of positive result. 5. Polymerase Chain Reaction (PCR) has the advantage of 98-100% sensitivity and the disadvantage of poor specificity. (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.
HIST1H2BC, also known as the H2BC4 gene, encodes histone H2B1 type I-C/E/F/G/I clusterin (H2B clusterized histone 4), which is the core component of the nucleosome. The gene is a known gene, and the sequence is shown in a reference (Taylor, T.D.et al.human chromosome 11DNA sequence and analysis cloning gene identification.2006, Nature 440, 497-. DNA assembly is also accomplished by post-translocation modification of histones. The gene participates in the processes of antibacterial humoral immune response of organisms, natural humoral response of mucous membranes and the like. This gene mutation has been found in patients with extramammary Paget's disease, but has not been shown to correlate with this disease; serum deficiency results in high expression of this gene in fibroblast cultures; no research report related to tuberculosis is found at present.
Disclosure of Invention
The invention aims to provide application of a molecular marker HIST1H2BC 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.
The molecular marker HIST1H2BC is a known gene and can be extracted by the conventional method.
The invention is described in more detail below:
the invention aims to provide application of a reagent for detecting the expression level of a molecular marker HIST1H2BC in preparing a kit for diagnosing tuberculosis.
Application of detecting the change of the protein level of HIST1H2BC in preparing a kit for diagnosing tuberculosis.
The method for detecting the expression level of the molecular marker HIST1H2BC is a fluorescent quantitative PCR method.
Application of a reagent for detecting the expression level of a molecular marker HIST1H2BC in preparing a kit for diagnosing active tuberculosis.
Wherein, the application comprises the application of distinguishing patients with active tuberculosis, latent tuberculosis infection and non-latent inactive tuberculosis infection.
Wherein the tuberculosis is pulmonary tuberculosis or extrapulmonary tuberculosis.
The invention aims to provide application of HIST1H2BC in preparation of a tuberculosis diagnosis marker product.
The invention aims to provide application of HIST1H2BC in preparing a medical instrument for diagnosing tuberculosis, wherein the medical instrument for diagnosing tuberculosis takes HIST1H2BC as a diagnostic molecular marker.
Specifically, the kit comprises the following components:
CD15+ cell-specific antibody screening magnetic beads (Invitrogen);
RNeasy Plus Mini Kit(Qiangen.)
SuperScriptTM IV Reverse Transcriptase(Invitrogen.)
TaqMan DNA polymerase
TaqMan probe and gene amplification specific primer
Another object of the present invention is to provide a detection method, comprising the steps of:
(1) whole blood sample processing: taking whole blood of human or animals for incubation;
(2) magnetic separation: separating and lysing neutrophils;
(3) total RNA extraction: extracting total RNA of the neutrophils;
(4) synthesis of cDNA: performing reverse transcription on the RNA to obtain sample cDNA,
(5) real-time fluorescent quantitative PCR: and (3) carrying out real-time fluorescent quantitative PCR detection by using cDNA as a template and using HIST1H2BC specific primers and internal reference primers, and calculating to obtain the content of the HIST1H2BC gene in the sample.
And (4) conclusion:
the relative expression quantity of the gene in the host body is obtained by the research method, and statistical analysis shows that the expression quantity of the gene in the active tuberculosis combined with the latent infection and non-tuberculosis infection hosts has obvious change, so that the gene can be used for molecular identification of active tuberculosis and non-tuberculosis infected persons and tuberculosis latent infection and non-tuberculosis infected persons.
Preferably, the detection method of the present invention comprises the following steps:
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 and rapidly adding a portion of the beads to the diluted blood, tightly covering the lid, properly placing the flow tube on a Hula Mixer, incubating for 20min at 4 ℃ and 8rpm with rotation,
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 of protein low adsorption tube, standing on a magnetic frame for 2min, sucking off the supernatant,
taking the third product off the magnetic frame, adding 1.6ml of separating medium, blowing gently, standing on the magnetic frame for 2min, sucking off the supernatant, repeating 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 from cells
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 mul of 70% ethanol into the fluid penetrating liquid, mixing uniformly, transferring 700 mul to an RNeasy Mini column at 12,000rpm, centrifuging for 15s, discarding the fluid penetrating liquid,
adding 700 mul Buffer RW1 at 12,000rpm, centrifuging for 15s, discarding the flow-through liquid,
fourthly, 500 mul Buffer RPE is added and centrifuged at 12,000rpm for 15s, the flow-through liquid is discarded,
fifthly, adding 500 mul 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 SuperScriptTM IV VILOTMPerforming reverse transcription by the Master Mix to obtain cell sample cDNA,
5) detection of expression level of HIST1H2BC Gene
And (2) detecting the real expression condition of the target gene in the host body by utilizing fluorescence quantitative PCR reaction, adding a specific primer pair or an internal reference primer pair into the cDNA prepared in the steps as a template to perform real-time quantitative PCR, obtaining the amplification constants of the HIST1H2BC gene and the internal reference gene in each sample source template, and calculating the relative expression quantity of the target gene.
The method for detecting the expression level of the HIST1H2BC gene comprises the following steps:
(1) preparation of reaction System 1 and reaction System 2
Reaction system 1 (target gene): 20 μ L of polymerase was reacted by qPCR
Figure BDA0002711630280000051
Fast Advanced Master Mix, target Gene
Figure BDA0002711630280000053
Assay primer, and sample cDNA and nuclease-free water. Reaction system 2 (reference gene): 20 μ L of a mixture of
Figure BDA0002711630280000054
Fast Advanced Master Mix，
Figure BDA0002711630280000052
Assay primer, and sample cDNA and nuclease-free water.
Among them, HIST1H2BC and reference gene specific primers were commercial probes (Applied Biosystems, US), and 1 ul/well was added to each of the pre-dispensed qPCR reaction solutions. And (4) after fully mixing, loading the mixture on a machine, and collecting data.
(2) Real-time quantitative PCR detection
Putting each reaction system prepared in the step (1) in QuantStaudioTMReal-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 CARD16 gene in each template was 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 7.
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 could not be detected. The HIST1H2BC gene can distinguish active tuberculosis patients from latent tuberculosis infection, active tuberculosis patients from non-tuberculosis infection, and active tuberculosis patients from other lung infections, and can be used as a candidate biomarker for diagnosing the active tuberculosis.
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
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
Buffer RW 1: RW1 buffer solution
Buffer RPE: RPE buffer
SuperScriptTM IV VILOTM Master Mix:SuperScriptTMIV VILOTMMixed solution
cDNA: inverted DNA
Figure BDA0002711630280000061
Fast Advanced Master Mix：
Figure BDA0002711630280000062
Quick mixing liquid
Drawings
FIG. 1 shows the detection of relative expression level of HIST1H2BC in different groups of samples.
In the figure: HC: a normal control group of non-tuberculosis infection; LTBI: tuberculosis latent infection group; ATB: active tuberculosis patient group.
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,
Detecting the expression level of peripheral blood immune cells of active tuberculosis patients and tuberculosis mycobacteria infected patients.
Study object and method
1. Study object
1.1 study Subjects and inclusion criteria
In the study, 152 host samples were collected and divided into three groups, including Active Tuberculosis (ATB, 51 cases), Tuberculosis Infection (LTBI, 54 cases) and normal Control (HC, Health Control,102 cases). Wherein, the active tuberculosis sample is a confirmed tuberculosis inpatient from Shenzhen third people hospital, the diagnosis standard is according to the standard of the national health and industry Standard of people's republic of China (WS 288-2017) tuberculosis diagnosis, and the pathogenic detection of the sputum sample 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; tuberculosis infection samples refer to samples with clinical contact history, no clinical symptoms and positive IGRA) and normal control group samples (normal physical examination related indexes and negative 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 detailed information of the members to be enrolled is shown in Table 1.
TABLE 1 sample demographic data
Figure BDA0002711630280000071
1.2 sources of specimens
2.5mL of peripheral blood of the above-mentioned subjects (all with informed consent) 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 the peripheral blood), to obtain peripheral blood specimens.
2. Research method
The magnetic bead sorting is based on the combination of cell surface antigen and specific monoclonal antibody connected with magnetic bead, and the cell connected with magnetic bead via antibody in the external magnetic field is adsorbed and retained in the sorting column, while 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 stay 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, ODF3B sequence amplification specific primers and real-time fluorescent quantitative PCR related reagents.
2.1 enrichment of peripheral blood neutrophils
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.
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 to a corresponding 2ml of 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 slightly and uniformly blowing. Standing on a magnetic frame for 2min, and sucking off the supernatant. Repeat for 1 time.
350. mu.l of Buffer RLT (Qiagen, Germany) was added to lyse the cells, vortexed for 1min, and left at 4 ℃ until use.
2.2 Total RNA extraction from cells
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 mul 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 of 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.
2.3 Synthesis of cDNA
Taking total RNA of cells, and adopting SuperScriptTM IV VILOTMThe Master Mix (Invitrogen, US) was subjected to reverse transcription to obtain cell sample cDNA.
Detection of expression level of HIST1H2BC Gene
The real expression of the target gene in the host is detected by performing a fluorescent quantitative PCR reaction (TaqMan system). And (3) taking the cDNA prepared in the step 2.3 as a template, adding a specific primer pair or an internal reference primer pair to perform real-time quantitative PCR, obtaining the amplification constants of the HIST1H2BC gene and the internal reference gene in each sample source template, and calculating the relative expression quantity of the target gene. The method comprises the following specific steps:
(1) preparation of reaction System 1 and reaction System 2
Reaction system 1 (target gene): 20 μ L of polymerase was reacted by qPCR
Figure BDA0002711630280000093
Fast Advanced Master Mix, target Gene
Figure BDA0002711630280000094
Assay primer, and sample cDNA and nuclease-free water. Reaction system 2 (reference gene): 20 μ L of a mixture of
Figure BDA0002711630280000091
Fast Advanced Master Mix，
Figure BDA0002711630280000092
Assay primer, and sample cDNA and nuclease-free water.
(2) Real-time quantitative PCR detection (2) real-time quantitative PCR detection
Putting each reaction system prepared in the step (1) in QuantStaudioTMReal-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 CARD16 gene in each template was 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 7.
3. Results of the study
After processing the quantitative PCR results, the data were analyzed using GraphPad Prism 7, and the results are shown in figure 1. As can be seen from the figure, compared with the non-latent infection group and the latent infection group of the inactive tuberculosis, the relative expression level of the HIST1H2BC gene in the neutrophils in the active tuberculosis group is remarkably different from that in the healthy control group by P <0.0001, and is remarkably increased by P <0.0001 compared with that in the latent infection group.
4. And (4) conclusion: as can be seen from the figure, the HIST1H2BC gene can distinguish active tuberculosis patients, tuberculosis latent infection and inactive tuberculosis non-latent infection, and can be used as a candidate biomarker for diagnosing active tuberculosis.
Example 2 kit
The kit comprises the following components:
1. specific cell-enriched antibody-labeled magnetic beads, Dynabeads CD15(Invitrogen, US)
2. Total RNA extraction Kit from cells, RNeasy Plus Mini Kit (Qiagen, Germany)
cDNA reverse transcriptase, SuperScriptTM IV VILOTM Master Mix(Invitrogen，US)
qPCR fluorescent quantitative PCR detection system, comprising
Figure BDA0002711630280000101
PCR polymerase, buffer system, and
Figure BDA0002711630280000102
the primer and the probe for detecting the specificity of the exon region of Assay HIST1H2 BC.
The invention reports the detection based on the change of the expression level of the HIST1H2BC gene of specific cells contained in human peripheral blood for the first time, and compared with the detection of the change of the expression of the whole blood related gene of the peripheral blood, the detection is more sensitive and specific. The gene has the possibility and the practicability as a tuberculosis molecular diagnosis marker through the combination of serial commercialized reagents and the optimization of the process.
Reference documents:
1.Takuya Takeichi,Yusuke Okuno,Takaaki Matsumoto.et al.equent FOXA1-Activating Mutations in Extramammary Paget's Disease.Cancers 2020,12,820.
2.Shawn M.Lyons,1Clark H.Cunningham,1Joshua D.Welch，et al.A subset of replication-dependent histone mRNAs are expressed as polyadenylated RNAs in terminally differentiated tissues.Nucleic Acids Res.2016Nov 2；44(19):9190–9205。

Claims (10)

1. the application of the reagent for detecting the expression level of the molecular marker HIST1H2BC in the preparation of a kit for diagnosing tuberculosis.
2. Application of detecting the change of the protein level of HIST1H2BC in preparing a kit for diagnosing tuberculosis.
3. The use according to claim 1, wherein the method for detecting the expression level of the molecular marker HIST1H2BC is a fluorescent quantitative PCR method.
4. The use according to claim 1, characterized by the use of a reagent for detecting the expression level of the molecular marker HIST1H2BC 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:
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;
HIST1H2BC sequence amplification specific primer;
real-time fluorescence quantitative PCR related reagent.
The application of HIST1H2BC in preparing tuberculosis diagnosis marker products.
The application of HIST1H2BC in the preparation of a medical instrument for diagnosing tuberculosis, wherein HIST1H2BC 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
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 are rapidly orientedAdding a portion of magnetic beads into the diluted blood, tightly covering the cover, properly placing the flow tube on a Hula Mixer, incubating for 20min at 4 ℃ and 8rpm in a rotating manner,
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 of protein low adsorption tube, standing on a magnetic frame for 2min, sucking off the supernatant,
taking the third product off the magnetic frame, adding 1.6ml of separating medium, blowing gently, standing on the magnetic frame for 2min, sucking off the supernatant, repeating 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 from cells
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 mul of 70% ethanol into the fluid penetrating liquid, mixing uniformly, transferring 700 mul to an RNeasy Mini column at 12,000rpm, centrifuging for 15s, discarding the fluid penetrating liquid,
adding 700 mul Buffer RW1 at 12,000rpm, centrifuging for 15s, discarding the flow-through liquid,
fourthly, 500 mul Buffer RPE is added and centrifuged at 12,000rpm for 15s, the flow-through liquid is discarded,
fifthly, adding 500 mul 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 SuperScriptTMIV VILOTMPerforming reverse transcription by the Master Mix to obtain cell sample cDNA,
5) detection of expression level of HIST1H2BC Gene
And (2) detecting the real expression condition of the target gene in the host body by utilizing fluorescence quantitative PCR reaction, adding a specific primer pair or an internal reference primer pair into the cDNA prepared in the steps as a template to perform real-time quantitative PCR, obtaining the amplification constants of the HIST1H2BC gene and the internal reference gene in each sample source template, and calculating the relative expression quantity of the target gene.
CN202011058666.4A 2020-09-30 2020-09-30 HIST1H2BC applied to differential diagnosis of active tuberculosis Pending CN112143797A (en)

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