CN109061191A - Application of the S100P albumen as marker in diagnostic activities tuberculosis - Google Patents
Application of the S100P albumen as marker in diagnostic activities tuberculosis Download PDFInfo
- Publication number
- CN109061191A CN109061191A CN201810965540.1A CN201810965540A CN109061191A CN 109061191 A CN109061191 A CN 109061191A CN 201810965540 A CN201810965540 A CN 201810965540A CN 109061191 A CN109061191 A CN 109061191A
- Authority
- CN
- China
- Prior art keywords
- gene
- tuberculosis
- albumen
- expression quantity
- under test
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/118—Prognosis of disease development
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Application the invention discloses S100P albumen as marker in diagnostic activities tuberculosis.Compared with Healthy People and tuberculosis latent infection person, the expression quantity of S100P gene is dramatically increased in the PBMCs of active tuberculosis patient；The expression quantity of S100P gene can be used for distinguishing active tuberculosis patient and tuberculosis latent infection person；The expression quantity of S100P gene can be used for distinguishing active tuberculosis patient and Healthy People.Therefore, S100P albumen and/or S100P gene can be used as marker diagnostic activities tuberculosis.The present invention has great application value.
Description
Technical field
The invention belongs to Medical Immunology diagnostic techniques fields, and in particular to S100P albumen is lived as marker in diagnosis
Application in dynamic property tuberculosis.
Background technique
Tuberculosis is infectious disease caused by mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), mainly
Pass through respiratory infectious.It may occur in which three kinds of different as a result, first is that immunity of organisms is preferable, MTB directly quilts after MTB infection human body
It removes；Second is that MTB is inhibited by immunity of organism, but can not be fully erased, develop as tuberculosis latent infection (latent
Tuberculosis infection, LTBI)；Third is that MTB is proliferated rapidly in body, active tuberculosis is developed into.Tuberculosis
Disease is the serious infectious diseases that China needs emphasis prevention and control.
Diagnosis of tuberculosis mainly has the methods of imaging diagnosis, tulase diagnosis and immunology diagnosis at present, but has one
Fixed disadvantage.Imaging diagnosis is difficult to differentiate between pulmonary tuberculosis and other pulmonary diseases.It is high that tulase diagnoses false negative.Immunology is examined
It is disconnected mainly divide antibody test and cellular immunity detect (such as tuberculin skin test (tuberculin skin test, TST) and
Interferon release test (interferon gamma release assays, IGRA)).TST and IGRA is to pass through detection
The immune main treating tuberculosis of body is cellullar immunologic response to evaluate tuberculosis infection situation.The country is existing mostly by TST as main inspection
Survey means, generally by purified protein derivative (PPD) (PPD) strong positive or in a short time from feminine gender switch to it is positive and without clinical tuberculosis evidence
Person is judged as tulase latent infection person.Due to TST feature be it is easy to operate, cheap, have become at present clinically most
A kind of common and the simplest tubercle bacillus affection diagnostic method.But PPD is the antigen mixture slightly mentioned from mycobacterium tuberculosis,
Comprising 200 multiple proteins, wherein being much the common antigen ingredient of non-tuberculous mycobacteria and BCG vaccine, therefore TST is determined
Poor specificity is detected, is also easy to produce false positive results in BCG vaccine (BCG) inoculation crowd and non-tuberculous mycobacteria infection population.
TST can only only have 70-80% according to the reaction power auxiliary diagnosis of skin, sensitivity.In addition TST there are when check fee, need
It wants subject to pay a return visit (72h), skin test operation and result and explains there is the disadvantages of subjective dependence.IGRA is inhaled using enzyme linked immunological
Adhesion test (ELISA) or Enzyme linked immunospot (ELISPOT) method quantitatively detect subject's whole blood or the single core of peripheral blood
IFN-γ detection release reaction of the cell to Specific Antigen of Mycobacterium Tuberculosis (ESAT6, CFP10 and TB7.7), for
The diagnosis of tubercle bacillus affection, but IGRA is difficult to differentiate between active tuberculosis and latent tuberculosis infection.Activity cannot be early diagnosed
Property tuberculosis, on the one hand lead to delay treatment, increase medical expense and the death rate；On the other hand it not can be effectively controlled the infection sources,
Cause diffusion lungy.Therefore, special, effective active tuberculosis diagnostic reagent is developed to tuberculosis prevention and treatment with important
Meaning.
S100 calbindin P (S100calcium binding protein P, S100P) is EF hand-type calbindin
One member of white S100 family has connection, protein phosphorylation, transduction calcium signal and transcriptional control between mediated cell
Etc. functions, participate in cell differentiation and mature, research finds that S100P is different in the expression of the tumours such as liver cancer, lung cancer, gastric cancer and cancer of pancreas
Often, can be used as tumour early diagnosis, by stages with assessment prognosis molecular marker.
Summary of the invention
The purpose of the present invention is diagnostic activities tuberculosis.
The present invention protects the substance for detecting S100P albumen preparing the application in product first；The function of the product
Can be following a1) at least one of to a3): a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is activity
Tuberculosis patient；A3) prevention and control tuberculosis.
The present invention also protects substance for detecting S100P albumen and records judgment criteria first and/or judgment criteria third
Carrier preparing the application in product；The function of the product can be following a1) at least one of to a3): a1) diagnosis is lived
Dynamic property tuberculosis；A2) whether diagnosis person under test is active tuberculosis patient；A3) prevention and control tuberculosis；
The judgment criteria first can are as follows: if the expression quantity of S100P albumen is higher than control peripheral blood in person under test's peripheral blood
The expression quantity of middle S100P albumen, then person under test be or it is doubtful be active tuberculosis patient；If in person under test's peripheral blood
The expression quantity of S100P albumen lower than control peripheral blood in S100P albumen expression quantity, then person under test be not or it is doubtful be not activity
Property tuberculosis patient；
The judgment criteria third can are as follows: if the concentration of S100P albumen is higher than in control peripheral blood in person under test's peripheral blood
The concentration of S100P albumen, then person under test be or it is doubtful be active tuberculosis patient；If S100P egg in person under test's peripheral blood
White concentration lower than control peripheral blood in S100P albumen concentration, then person under test be not or it is doubtful be not active tuberculosis sufferer
Person；
The control peripheral blood can be tuberculosis latent infection person or the peripheral blood of Healthy People.
The expression quantity of the S100P albumen concretely S100P albumen from the PBMCs separated in peripheral blood in the peripheral blood
Expression quantity, in serum in the expression quantity or blood plasma of S100P albumen S100P albumen expression quantity.
The present invention also protects the substance for detecting S100P gene preparing the application in product；The function of the product
Can be following a1) at least one of to a3): a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is activity knot
Core patient；A3) prevention and control tuberculosis.
The present invention also protects the substance for detecting S100P gene preparing product with the carrier for recording judgment criteria second
In application；The function of the product can be following a1) at least one of to a3): a1) diagnostic activities tuberculosis；A2 it) examines
Whether disconnected person under test is active tuberculosis patient；A3) prevention and control tuberculosis；
The judgment criteria second can are as follows: if the expression quantity of S100P gene is higher than control peripheral blood in person under test's peripheral blood
The expression quantity of middle S100P gene, then person under test be or it is doubtful be active tuberculosis patient；If in person under test's peripheral blood
The expression quantity of S100P gene lower than control peripheral blood in S100P gene expression quantity, then person under test be not or it is doubtful be not activity
Property tuberculosis patient；The control peripheral blood can be tuberculosis latent infection person or the peripheral blood of Healthy People.
The expression quantity of the S100P gene concretely S100P gene from the PBMCs separated in peripheral blood in the peripheral blood
Expression quantity.
The present invention also protects a kind of kit, it may include for detecting the substance of S100P albumen and/or for detecting
The substance of S100P gene；The kit can have the function of following a1) at least one of to a3): a1) diagnostic activities tuberculosis
Disease；A2) whether diagnosis person under test is active tuberculosis patient；A3) prevention and control tuberculosis.
Any of the above-described " for the detecting the substance of S100P albumen " can be the expression quantity for detecting S100P albumen
Substance (corresponding the judgment criteria first) and/or substance (the corresponding judgment criteria the third) for detecting S100P protein concentration.
Any of the above-described " for the detecting the substance of S100P gene " can be the expression quantity for detecting S100P gene
Substance.
The expression quantity of any of the above-described S100P albumen can be the relative expression quantity of S100P albumen reference internal reference albumen.
The expression quantity of any of the above-described S100P gene can be the relative expression quantity of S100P gene reference reference gene.
The expression quantity of any of the above-described detection S100P albumen specifically can be used Western Blot experiment and carry out.
Any of the above-described detection S100P protein concentration is particularly used in Elisa experiment and carries out.
Any of the above-described substance or any of the above-described described for detecting for detecting the expression quantity of S100P gene
The substance of " relative expression quantity of S100P gene reference reference gene " includes the primer of primer pair and internal control primer to composition
To combination；
The primer pair can be made of primer S100P-F and primer S100P-R；The target gene of the primer pair
Sequence 6 containing ordered list DNA fragmentation shown in the 194th to 328 from 5 ' ends；
The internal control primer primers F and primer R to can be made of；The target gene of the internal control primer pair can be people's internal reference base
The all or part of cause.
The primer S100P-F can be following a1) or a2):
A1) single strand dna shown in the sequence 1 of sequence table；
A2) there is phase by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1
The DNA molecular of congenerous.
The primer S100P-R can be following a3) or a4):
A3) single strand dna shown in the sequence 2 of sequence table；
A4) there is phase by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of congenerous.
The primers F can be following b1) or b2):
B1) single strand dna shown in the sequence 1 of sequence table；
B2) there is phase by sequence 1 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 1
The DNA molecular of congenerous.
The primer R can be following b3) or b4):
B3) single strand dna shown in the sequence 2 of sequence table；
B4) there is phase by sequence 2 by the substitution and/or deletion and/or addition of one or several nucleotide and with sequence 2
The DNA molecular of congenerous.
Any of the above-described primer pair also belongs to protection scope of the present invention.
In expression quantity or detection S100P gene reference using any of the above-described primer pair detection S100P gene
The relative expression quantity of ginseng gene also belongs to protection scope of the present invention.
Using in the expression quantity or detection S100P gene reference of any of the above-described primer pair combine detection S100P gene
The relative expression quantity of ginseng gene also belongs to protection scope of the present invention.
Above, using S100P gene reference internal reference base in any of the above-described primer pair combine detection person under test cDNA
The method of the relative expression quantity of cause is concretely: using person under test cDNA as template, using any of the above-described primer pair or
Then any of the above-described internal control primer uses 2 to real-time fluorescence quantitative PCR is carried out-ΔCtMethod, which calculates, to be obtained.The person under test
CDNA can be the cDNA of the PBMCs separated in person under test's peripheral blood.
Any of the above-described internal reference albumen can be GAPDH albumen.
Any of the above-described reference gene can be GAPDH gene.
The present invention also protects Y1) or Y2) or Y3) or Y4):
Y1) application of the S100P albumen as marker in exploitation diagnostic activities reagent lungy；
Y2) application of the S100P albumen as marker in diagnostic activities tuberculosis；
Y3) application of the S100P gene as marker in exploitation diagnostic activities reagent lungy；
Y4) application of the S100P gene as marker in diagnostic activities tuberculosis.
Sequence in the amino acid sequence of any of the above-described S100P albumen (No. GeneID are as follows: NP_005971.1) such as sequence table
Shown in column 5.In the nucleotide sequence of any of the above-described S100P gene (No. Genebank are as follows: NM_005980.2) such as sequence table
Shown in sequence 6.
It is demonstrated experimentally that compared with Healthy People and tuberculosis latent infection person, S100P in the PBMCs of active tuberculosis patient
The expression quantity of gene dramatically increases；The expression quantity of S100P gene can be used for distinguishing active tuberculosis patient and tuberculosis is latent
The infected；The expression quantity of S100P gene can be used for distinguishing active tuberculosis patient and Healthy People.Therefore, S100P gene
Expression quantity has important application value in terms of diagnostic activities tuberculosis.
Detailed description of the invention
Fig. 1 is that real-time fluorescence quantitative PCR detects active tuberculosis patient, tuberculosis latent infection person and Healthy People
The relative expression quantity of S100P gene in PBMCs.
Fig. 2 is S100P gene in ROC curve analytic activity tuberculosis patient and the PBMCs of tuberculosis latent infection person
Relative expression quantity.
Fig. 3 is the relative expression of S100P gene in the PBMCs of ROC curve analytic activity tuberculosis patient and Healthy People
Amount.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
Ficoll-Paque PLUS is the product of U.S. GE company.96 orifice plates are the product of Millipore company.AIM
VTMMedium serum free medium is the product of gibco company, catalog number 12055091.RPMI 1640 culture medium is
The product of Gibco company, catalog number 11875-093.IFN-γ ELISPOT detection kit is the production for being company up to section
Product.IFN-γ monoclonal captures antibody, IFN-γ detection antibody, tubercle bacillus differential mixed polypeptide A, tubercle bacillus differential mixing
Polypeptide B and phytohemagglutin phytolectin are the component in IFN-γ ELISPOT detection kit.TRIzolTMReagent is
The product of Invitrogen company.PrimeScriptTMRT reagent Kit with gDNAEraser is TaKaRa
The product of Biotechnology company.KAPATM Fast quantification PCR kit is the production of KapaBiosystems company
Product.2 × Green Master Mix is KAPATM Component in fast quantification PCR kit.Nuclease-free water is beauty
The product of Ambion company of state.Fluorescence quantitative PCR instrument is the product of Roche Holding Ag.
Cleaning solution: the PBS buffer solution of pH7.4,0.01M containing 0.05% (v/v) polysorbas20.
Application of the relative expression quantity in diagnostic activities tuberculosis of embodiment 1, S100P gene
One, the acquisition of periphery blood specimen
1, the periphery blood specimen of tuberculosis latent infection person and the periphery blood specimen of Healthy People are distinguished
The sign or symptom of tuberculosis latent infection person and health per capita without tuberculosis morbidity, are distinguished in accordance with the following steps:
A, it is coated with
(1) 96 orifice plates are taken, every hole is added 100 μ L IFN-γ monoclonals and captures antibody, and 4 DEG C of coatings are overnight.
(2) after completing step (1), 96 orifice plate is taken, abandons liquid phase, the PBS buffer solution washing two of pH7.4,0.01M is added
Secondary (each 1min), pats dry.
(3) after completing step (2), 96 orifice plate is taken, pH7.4,0.01M that 200 μ L contain 2% (v/v) BSA is added in every hole
PBS buffer solution, 37 DEG C of incubation 1h.
(4) after completing step (3), 96 orifice plate is taken, abandons liquid phase, it is primary that the rinse of RPMI 1640 culture medium is added.
B, the preparation of PBMCs suspension
(1) the RPMI 1640 culture medium of 2mL peripheral blood to be measured and 2mL is uniformly mixed；Then it is slowly added into being equipped with
In the sterile centrifugation tube of 3mL Ficoll-Paque PLUS, room temperature, 2000rcf are centrifuged 20min, are from top to bottom divided into three layers.
(2) it after completing step (1), draws middle layer and is transferred in the centrifuge tube of the RPMI 1640 culture medium equipped with 2mL.
(3) after completing step (2), the RPMI 1640 culture medium that 8mL is preheated to 37 DEG C is added into the centrifuge tube, uses
Dropper gently blows and beats mixing, and room temperature, 1400rpm are centrifuged 7min.
(4) after completing step (3), the centrifuge tube is taken, abandons supernatant, the culture of RPMI 1640 that 6mL is preheated to 37 DEG C is added
Liquid is resuspended, and room temperature, 1400rpm are centrifuged 7min.
(5) after completing step (4), the centrifuge tube is taken, abandons supernatant, addition is preheated to 37 DEG C of AIM VTMMedium is without blood
Clear culture medium is resuspended, and obtaining concentration is 2.5 × 106The PBMCs suspension of a/mL.
C, immunodotting detects
With reference to the specification of IFN-γ ELISPOT detection kit, immunodotting detection is carried out using the kit.Reagent
The equal by specification of dosage carries out.Specific step is as follows:
(1) take into 96 orifice plate of step a, every hole be added 100 μ L step b preparation PBMCs suspension (about 2.5 ×
105A PBMC).
(2) after completing step (1), tubercle bacillus differential mixed polypeptide A is added in each detection hole or tubercle bacillus differential is mixed
Close polypeptide B；Serum free medium is added in each negative control hole；Phytohemagglutin phytolectin is added in each Positive control wells.
(3) after completing step (2), 96 orifice plate is placed in incubator, 37 DEG C, 5%CO2Cultivate 20h.
(4) after completing step (3), 96 orifice plate is taken, abandons supernatant, the ice water of 200 μ L pre-cooling, 4 DEG C of placement 10min are added
(purpose is lytic cell).
(5) after completing step (4), 96 orifice plate is taken, abandons supernatant, 5 times is washed with cleaning solution and (200 μ L is added every time to wash
Liquid is washed, washs 1min every time), it pats dry.
(6) after completing step (5), 96 orifice plate is taken, every hole is added 100 μ L IFN-γ and detects antibody (Avidin mark
Note) dilution (being mixed by the PBS buffer solution of 99 parts by volume pH7.4,0.01M and 1 parts by volume IFN-γ detection antibody), 37
DEG C be incubated for 1h.
(7) after completing step (6), 96 orifice plate is taken, abandons supernatant, 5 times is washed with cleaning solution and (200 μ L is added every time to wash
Liquid is washed, washs 1min every time), it pats dry.
(8) after completing step (7), take 96 orifice plate, every hole be added the streptomysin of 100 μ LHRP label dilution (by
The streptomysin of the PBS buffer solution of 99 parts by volume pH7.4,0.01M and 1 parts by volume HRP label mixes), 37 DEG C of incubation 1h.
(9) after completing step (8), 96 orifice plate is taken, abandons supernatant, 5 times is washed with cleaning solution and (200 μ L is added every time to wash
Liquid is washed, washs 1min every time), it pats dry.
(10) after completing step (9), 96 orifice plate is taken, zymolyte is added in every hole, at room temperature black out colour developing 15-45min.
(11) after completing step (10), 96 orifice plate is taken, with distilled water flushing 3 times (purpose is stopped reaction), then
96 orifice plate is stored at room temperature, naturally dry.
(12) after completing step (11), 96 orifice plate is taken, immunodotting calculating instrument (CellularTechnology is used
Ltd, USA) image and spot count are carried out, then make the following judgment: when the number of spots of negative control hole is less than 6, if
The number of spots of detection hole subtract the number of spots of negative control hole be 6 or more, then detection hole be it is positive, if the spot of detection hole
The number of spots that point number subtracts negative control hole is less than 6, then detection hole is negative；The number of spots of negative control hole is 6
When a above, if the 2 times or more for the number of spots that the number of spots of detection hole is negative control hole, if detection hole be it is positive,
If the number of spots of detection hole than negative control hole number of spots less than 2 times, if detection hole be feminine gender.Detection hole is sun
Property, then peripheral blood to be measured provides (i.e. the periphery blood specimen of tuberculosis latent infection person) by tuberculosis latent infection person；Detection hole is yin
Property, then peripheral blood to be measured provides (i.e. the periphery blood specimen of Healthy People) by Healthy People.
2, the acquisition of periphery blood specimen
(1) active tuberculosis group: 25 periphery blood specimens.
25 periphery blood specimens: 25 patient's (all trouble for being clinically diagnosed as active tuberculosis disease are extracted respectively
The equal informed consent of person) peripheral blood 2-3mL, be placed in the pipe of anticoagulant blood-collecting containing EDTA, turn upside down 5-6 times (purpose be anti-freezing liquid and
Peripheral blood mixes), obtain 25 periphery blood specimens.
(2) tuberculosis latent infection group: 30 periphery blood specimens.
30 periphery blood specimens: 30 tuberculosis latent infection persons for being clinically diagnosed as tuberculosis latent infection are extracted respectively
The peripheral blood 2-3mL of (equal informed consent) is placed in the pipe of anticoagulant blood-collecting containing EDTA, and turning upside down 5-6 times, (purpose is for anti-freezing liquid and outside
All blood mixes), obtain 30 periphery blood specimens.
(3) healthy control group: 40 periphery blood specimens.
40 periphery blood specimens: the peripheral blood 2-3mL of 40 Healthy Peoples (equal informed consent) is extracted respectively, is placed in containing EDTA
Anticoagulant blood-collecting pipe turns upside down 5-6 times (purpose is that anti-freezing liquid and peripheral blood mix), obtains 40 periphery blood specimens.
95 periphery blood specimens need to be placed in room temperature (do not freeze or refrigerate), and standing time is less than 6h.
Two, application of the relative expression quantity of S100P gene in diagnostic activities tuberculosis
The amino acid sequence of S100P albumen (No. GeneID are as follows: NP_005971.1) is as shown in sequence 5 in sequence table.Coding
In the nucleotide sequence of the gene (abbreviation S100P gene, No. Genebank are as follows: NM_005980.2) of S100P albumen such as sequence table
Shown in sequence 6.
1, the acquisition of the cDNA of 95 periphery blood specimens
(1) preparation of PBMCs suspension
The peripheral blood to be measured of b in step 11 is replaced in step 12 95 periphery blood specimens, other steps respectively
It is constant, obtain the PBMCs suspension of 95 periphery blood specimens.
(2) RNA is extracted
The RNA of the PBMCs suspension of 95 periphery blood specimens is extracted respectively.Specific step is as follows:
1. taking 96 orifice plates, it is separately added into the PBMCs suspension of 95 periphery blood specimens.Every 150 μ L of hole.
2. after completing step 1., taking 96 orifice plate, the dilution of 50 μ L Mycobacterium tuberculosis H37Rv lysates is added in every hole
Liquid is (by AIM VTMMedium serum free medium dilutes Mycobacterium tuberculosis H37Rv lysate and obtains；Protein concentration is 10 μ g/
ML), mix.
The preparation method of Mycobacterium tuberculosis H37Rv lysate: the thallus of Mycobacterium tuberculosis H37Rv, first Co 60 spoke are taken
It is resuspended according to inactivation, then with PBS buffer solution, obtains re-suspension liquid；Re-suspension liquid is subjected to somatic cells using super-pressure biomixer
Broken, 4 DEG C, 12000rcf centrifugation 10min collect supernatant；Supernatant is taken, protein concentration is measured using BCA method.
3. taking 96 orifice plate, 37 DEG C, 5%CO after completing step 2.216h is cultivated, TRIzol is then usedTM
Reagent extracts RNA.
(3) synthesis of cDNA
The RNA for taking the PBMCs suspension of 95 periphery blood specimens respectively, using PrimeScriptTM RT reagent
Kitwith gDNA Eraser carries out reverse transcription, obtains the cDNA of 95 periphery blood specimens.
2, the preparation of primer pair combination
According to the nucleotide sequence of S100P gene, primer pair shown in table 1 is designed and synthesized.
According to the nucleotide sequence of GAPDH gene, internal control primer pair shown in table 1 is designed and synthesized.
Primer pair combination is by primer pair and internal control primer to forming.
Each primer (HPLC purifying) is synthesized by Shanghai Sheng Gong Biotechnology Co., Ltd.
Table 1
3, the relative expression quantity of real-time fluorescence quantitative PCR detection S100P gene
Respectively using the cDNA of 95 periphery blood specimens as template, the primer pair or internal control primer that are prepared using step 2
To progress real-time quantitative PCR, and then obtain the relative expression quantity of S100P gene in each template.Specific step is as follows:
(1) reaction system 1 and reaction system 2 are prepared
Reaction system 1 is 20 μ L, by 10 μ L2 × Green Master Mix, 0.4 μ LS100P-F aqueous solution (concentration 10
μM), 0.4 μ L S100P-R aqueous solution (concentration be 10 μM), 2 μ L templates (5-20ng) and 7.2 μ L nuclease-free waters form.
Reaction system 2 is 20 μ L, and by 10 μ L2 × Green Master Mix, 0.4 μ L GAPDH-F aqueous solution, (concentration is
10 μM), 0.4 μ L GAPDH-R aqueous solution (concentration be 10 μM), 2 μ L templates (5-20ng) and 7.2 μ L nuclease-free waters form.
(2) real-time quantitative PCR detects
Each reaction system that step (1) is prepared is existedIt is carried out on 480 II fluorescence quantitative PCR instruments real
When quantitative PCR detection.Use 2-ΔCtMethod calculates the relative expression quantity of S100P gene in each template.
Reaction condition: 95 DEG C of initial denaturation 3min；95 DEG C of 5s, 60 DEG C of 30sec, 40 circulations, fluorescence signal are extending the stage
Acquisition.
Experimental result is shown in Fig. 1 (TB is active tuberculosis group, and LI is tuberculosis latent infection group, and Nor is healthy control group).
The result shows that compared with healthy control group and tuberculosis latent infection group, S100P gene in the PBMCs of active tuberculosis group
Relative expression quantity dramatically increases.
(3) it statisticallys analyze
It is for statistical analysis using result of the GraphPad Prism 5 to step (2).
According to the relative expression quantity of S100P gene in the PBMCs of active tuberculosis group and tuberculosis latent infection group, use
GraphPad Prism 5 carries out Receiver operating curve's analysis.As a result see Fig. 2.The result shows that S100P gene is opposite
Expression quantity can be used for distinguishing active tuberculosis patient and tuberculosis latent infection person.
According to the relative expression quantity of S100P gene in the PBMCs of active tuberculosis group and healthy control group, use
GraphPad Prism 5 carries out Receiver operating curve's analysis.As a result see Fig. 3.The result shows that S100P gene is opposite
Expression quantity can be used for distinguishing active tuberculosis patient and Healthy People.
The above results show that the relative expression quantity of S100P gene has important answer in terms of diagnostic activities tuberculosis
With value.
<110>No.309 Hospital of PLA
<120>application of the S100P albumen as marker in diagnostic activities tuberculosis
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 1
aaggtgctga tggagaag 18
<210> 2
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 2
cacgaacacg atgaactc 18
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 3
tgttgccatc aatgacccct 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 4
tcgccccact tgattttgga 20
<210> 5
<211> 95
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 5
Met Thr Glu Leu Glu Thr Ala Met Gly Met Ile Ile Asp Val Phe Ser
1 5 10 15
Arg Tyr Ser Gly Ser Glu Gly Ser Thr Gln Thr Leu Thr Lys Gly Glu
20 25 30
Leu Lys Val Leu Met Glu Lys Glu Leu Pro Gly Phe Leu Gln Ser Gly
35 40 45
Lys Asp Lys Asp Ala Val Asp Lys Leu Leu Lys Asp Leu Asp Ala Asn
50 55 60
Gly Asp Ala Gln Val Asp Phe Ser Glu Phe Ile Val Phe Val Ala Ala
65 70 75 80
Ile Thr Ser Ala Cys His Lys Tyr Phe Glu Lys Ala Gly Leu Lys
85 90 95
<210> 6
<211> 510
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 6
tgaggctgcc ttataaagca ccaagaggct gccagtggga cattttctcg gccctgccag 60
cccccaggag gaaggtgggt ctgaatctag caccatgacg gaactagaga cagccatggg 120
catgatcata gacgtctttt cccgatattc gggcagcgag ggcagcacgc agaccctgac 180
caagggggag ctcaaggtgc tgatggagaa ggagctacca ggcttcctgc agagtggaaa 240
agacaaggat gccgtggata aattgctcaa ggacctggac gccaatggag atgcccaggt 300
ggacttcagt gagttcatcg tgttcgtggc tgcaatcacg tctgcctgtc acaagtactt 360
tgagaaggca ggactcaaat gatgccctgg agatgtcaca gattcctggc agagccatgg 420
tcccaggctt cccaaaagtg tttgttggca attattcccc taggctgagc ctgctcatgt 480
acctctgatt aataaatgct tatgaaatga 510
Claims (10)
1. the substance for detecting S100P albumen is preparing the application in product；The function of the product is following a1) to a3)
At least one of: a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is active tuberculosis patient；A3) prevention and control
Tuberculosis.
2. the substance for detecting S100P albumen is produced with the carrier for recording judgment criteria first and/or judgment criteria third in preparation
Application in product；The function of the product is at least one of following a1) to a3): a1) diagnostic activities tuberculosis；A2 it) examines
Whether disconnected person under test is active tuberculosis patient；A3) prevention and control tuberculosis；
The judgment criteria first are as follows: if the expression quantity of S100P albumen is higher than S100P in control peripheral blood in person under test's peripheral blood
The expression quantity of albumen, then person under test be or it is doubtful be active tuberculosis patient；If S100P albumen in person under test's peripheral blood
Expression quantity lower than control peripheral blood in S100P albumen expression quantity, then person under test be not or it is doubtful be not active tuberculosis sufferer
Person；
The judgment criteria third are as follows: if the concentration of S100P albumen is higher than S100P egg in control peripheral blood in person under test's peripheral blood
White concentration, then person under test be or it is doubtful be active tuberculosis patient；If the concentration of S100P albumen in person under test's peripheral blood
Lower than control peripheral blood in S100P albumen concentration, then person under test be not or it is doubtful be not active tuberculosis patient；
The control peripheral blood is the peripheral blood of tuberculosis latent infection person or Healthy People.
3. the substance for detecting S100P gene is preparing the application in product；The function of the product is following a1) to a3)
At least one of: a1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is active tuberculosis patient；A3) prevention and control
Tuberculosis.
4. the substance for detecting S100P gene is preparing the application in product with the carrier for recording judgment criteria second；It is described
The function of product is at least one of following a1) to a3): a1) diagnostic activities tuberculosis；A2) diagnosis person under test whether be
Active tuberculosis patient；A3) prevention and control tuberculosis；
The judgment criteria second are as follows: if the expression quantity of S100P gene is higher than S100P in control peripheral blood in person under test's peripheral blood
The expression quantity of gene, then person under test be or it is doubtful be active tuberculosis patient；If S100P gene in person under test's peripheral blood
Expression quantity lower than control peripheral blood in S100P gene expression quantity, then person under test be not or it is doubtful be not active tuberculosis sufferer
Person；The control peripheral blood is the peripheral blood of tuberculosis latent infection person or Healthy People.
5. a kind of kit, including the substance for detecting S100P albumen and/or the substance for detecting S100P gene；It is described
Kit has the function of following a1) at least one of to a3): a1) diagnostic activities tuberculosis；A2) diagnosis person under test whether be
Active tuberculosis patient；A3) prevention and control tuberculosis.
6. the application as described in Claims 1-4 is any or the kit as described in claim 5, it is characterised in that:
" for the detecting the substance of S100P albumen " is for detecting the substance of the expression quantity of S100P albumen and/or for examining
Survey the substance of S100P protein concentration；
" for the detecting the substance of S100P gene " is the substance for detecting the expression quantity of S100P gene.
7. application as claimed in claim 6 or kit, it is characterised in that:
The expression quantity of the S100P albumen is the relative expression quantity of S100P albumen reference internal reference albumen；
The expression quantity of the S100P gene is the relative expression quantity of S100P gene reference reference gene.
8. application or kit as claimed in claims 6 or 7, it is characterised in that:
The substance or described for detecting " S100P gene reference reference gene for detecting the expression quantity of S100P gene
The substance of relative expression quantity " includes that primer pair and internal control primer combine the primer pair of composition；
The primer pair is made of primer S100P-F and primer S100P-R；The target gene of the primer pair is containing orderly
The sequence 6 of list DNA fragmentation shown in the 194th to 328 from 5 ' ends；
The internal control primer is formed to by primers F and primer R；The target gene of the internal control primer pair is the whole of people's reference gene
Or part.
9. primer pair described in claim 8.
10.Y1) or Y2) Y3) or Y4):
Y1) application of the S100P albumen as marker in exploitation diagnostic activities reagent lungy；
Y2) application of the S100P albumen as marker in diagnostic activities tuberculosis；
Y3) application of the S100P gene as marker in exploitation diagnostic activities reagent lungy；
Y4) application of the S100P gene as marker in diagnostic activities tuberculosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810965540.1A CN109061191B (en) | 2018-08-23 | 2018-08-23 | Application of S100P protein as marker in diagnosis of active tuberculosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810965540.1A CN109061191B (en) | 2018-08-23 | 2018-08-23 | Application of S100P protein as marker in diagnosis of active tuberculosis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109061191A true CN109061191A (en) | 2018-12-21 |
| CN109061191B CN109061191B (en) | 2021-08-24 |
Family
ID=64756822
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810965540.1A Active CN109061191B (en) | 2018-08-23 | 2018-08-23 | Application of S100P protein as marker in diagnosis of active tuberculosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109061191B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825575A (en) * | 2019-04-08 | 2019-05-31 | 首都医科大学附属北京胸科医院 | Auxiliary diagnosis miRNA marker lungy and its application |
| CN110286231A (en) * | 2019-06-19 | 2019-09-27 | 中国人民解放军总医院第八医学中心 | Substance for detecting CD160 albumen is used for the application in diagnostic activities product lungy in preparation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102150043A (en) * | 2008-06-25 | 2011-08-10 | 贝勒研究院 | Blood transcriptional signature of mycobacterium tuberculosis infection |
| CN102844444A (en) * | 2009-11-30 | 2012-12-26 | 贝勒研究院 | Blood transcriptional signature of active versus latent mycobacterium tuberculosis infection |
| CN108828235A (en) * | 2018-08-23 | 2018-11-16 | 中国人民解放军第三〇九医院 | Application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis |
-
2018
- 2018-08-23 CN CN201810965540.1A patent/CN109061191B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102150043A (en) * | 2008-06-25 | 2011-08-10 | 贝勒研究院 | Blood transcriptional signature of mycobacterium tuberculosis infection |
| CN102844444A (en) * | 2009-11-30 | 2012-12-26 | 贝勒研究院 | Blood transcriptional signature of active versus latent mycobacterium tuberculosis infection |
| CN108828235A (en) * | 2018-08-23 | 2018-11-16 | 中国人民解放军第三〇九医院 | Application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825575A (en) * | 2019-04-08 | 2019-05-31 | 首都医科大学附属北京胸科医院 | Auxiliary diagnosis miRNA marker lungy and its application |
| CN110286231A (en) * | 2019-06-19 | 2019-09-27 | 中国人民解放军总医院第八医学中心 | Substance for detecting CD160 albumen is used for the application in diagnostic activities product lungy in preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109061191B (en) | 2021-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kala et al. | Diagnosis of scrub typhus: recent advancements and challenges | |
| CN110244048A (en) | Application of the SERPING1 albumen as marker in exploitation diagnostic activities reagent lungy | |
| JP2002532064A (en) | Tuberculosis diagnostic test | |
| CN108828235A (en) | Application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis | |
| CN107076744A (en) | For the composition for the mycobacterium tuberculosis for diagnosing latent infection | |
| CN109061191A (en) | Application of the S100P albumen as marker in diagnostic activities tuberculosis | |
| JP2001507130A (en) | Diagnosis of allergic bronchopulmonary aspergillosis | |
| CN109991417B (en) | Immune marker for tuberculosis and application | |
| EP1418936A2 (en) | Pharmaceutical compositions for preventing or treating th1 and th2 cell related diseases by modulating the th1/th2 ratio. | |
| CN109187987A (en) | Application of the MS4A3 albumen as marker in diagnostic activities tuberculosis | |
| CN110286231A (en) | Substance for detecting CD160 albumen is used for the application in diagnostic activities product lungy in preparation | |
| CN114560929B (en) | Monoclonal antibody aiming at coronavirus NP protein and application thereof | |
| RU2504784C1 (en) | Diagnostic technique for secondary immunodeficient disease in pulmonary tuberculosis | |
| KR20200040803A (en) | Compositions and methods for treatment and treatment choice of atopic dermatitis | |
| KR102132964B1 (en) | Method for Diagnosing Scrub Typhus Using Exosome derived from Orientia tsutsugamushi | |
| US20090208997A1 (en) | GPR18 as a Biomaker for TH1 Mediated Immune Response | |
| JP4269064B2 (en) | Test method for myasthenia gravis and test agent therefor | |
| Nasser et al. | B-cell monoclonal lymphocytosis in chronic hepatitis C virus infection | |
| Junker et al. | COVID-19 patient serum less potently inhibits ACE2-RBD binding for various SARS-CoV-2 RBD mutants | |
| KR101919403B1 (en) | Recombinant protein and use thereof | |
| JP5474802B2 (en) | A sensitive and rapid method of using chimeric receptors to identify autoimmune diseases | |
| WO2007069423A1 (en) | Diagnostic marker for allergy | |
| CN112114146A (en) | Kit for diagnosing active tuberculosis | |
| CN113631723A (en) | Application of tuberculosis marker in tuberculosis diagnosis and curative effect evaluation | |
| CN108387745B (en) | Application of CD4+ T lymphocyte characteristic protein in identification of latent tuberculosis infection and active tuberculosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |