CN108828235A - Application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis - Google Patents
Application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis Download PDFInfo
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Abstract
Application the invention discloses PGLYRP1 albumen as marker in diagnostic activities tuberculosis.Compared with Healthy People and tuberculosis latent infection person, the expression quantity of PGLYRP1 gene is dramatically increased in the PBMCs of active tuberculosis patient；The expression quantity of PGLYRP1 gene can be used for distinguishing active tuberculosis patient and tuberculosis latent infection person；The expression quantity of PGLYRP1 gene can be used for distinguishing active tuberculosis patient and Healthy People.Therefore, PGLYRP1 albumen and/or PGLYRP1 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 PGLYRP1 albumen is being diagnosed as marker
Application in active 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.
Peptide glycan is the pathogen-associated molecular pattern of gram-positive bacteria, and peptidoglycan recognition protein is a kind of important mode
Identification receptor plays an important role during resisting pathogen invasion.Peptidoglycan recognition protein 1
(peptidoglycan recognition protein 1, PGLYRP1) is also known as PGRP-S or Tag7, can be with immunity receptor
TREM-1 is incorporated into onthe surface of monocytes, activates the secretory cell factor with antibacterial action.The study found that PGLYRP1 can
To form compound in conjunction with HSP70, there is direct killing effect to control the growth of tumour to tumour cell.
Summary of the invention
The purpose of the present invention is diagnostic activities tuberculosis.
The present invention protects the substance for detecting PGLYRP1 albumen preparing the application in product first；The product
Function can be following a1) at least one of to a3)：A1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is activity
Property tuberculosis patient；A3) prevention and control tuberculosis.
The present invention also protects substance for detecting PGLYRP1 albumen and records judgment criteria first and/or judgment criteria
Third carrier is preparing the application in product；The function of the product can be following a1) at least one of to a3)：A1 it) diagnoses
Active tuberculosis；A2) whether diagnosis person under test is active tuberculosis patient；A3) prevention and control tuberculosis；
The judgment criteria first can be：If the expression quantity of PGLYRP1 albumen is higher than control periphery in person under test's peripheral blood
The expression quantity of PGLYRP1 albumen in blood, 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 PGLYRP1 albumen is lower than the expression quantity of PGLYRP1 albumen in control peripheral blood, then person under test is not or doubtful is not
Active tuberculosis patient；
The judgment criteria third can be：If the concentration of PGLYRP1 albumen is higher than control peripheral blood in person under test's peripheral blood
The concentration of middle PGLYRP1 albumen, then person under test be or it is doubtful be active tuberculosis patient；If in person under test's peripheral blood
The concentration of PGLYRP1 albumen lower than control peripheral blood in PGLYRP1 albumen concentration, 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 PGLYRP1 albumen concretely PGLYRP1 from the PBMCs separated in peripheral blood in the peripheral blood
In the expression quantity of albumen, serum in the expression quantity or blood plasma of PGLYRP1 albumen PGLYRP1 albumen expression quantity.
The present invention also protects the substance for detecting PGLYRP1 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
Tuberculosis patient；A3) prevention and control tuberculosis.
The present invention also protects the substance for detecting PGLYRP1 gene to produce with the carrier for recording judgment criteria second in preparation
Application in product；The function of the product can be following a1) at least one of to a3)：A1) diagnostic activities tuberculosis；a2)
Diagnose whether person under test is active tuberculosis patient；A3) prevention and control tuberculosis；
The judgment criteria second can be：If the expression quantity of PGLYRP1 gene is higher than control periphery in person under test's peripheral blood
The expression quantity of PGLYRP1 gene in blood, 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 PGLYRP1 gene is lower than the expression quantity of PGLYRP1 gene in control peripheral blood, then person under test is not or doubtful is not
Active tuberculosis patient；The control peripheral blood can be tuberculosis latent infection person or the peripheral blood of Healthy People.
The expression quantity of the PGLYRP1 gene concretely PGLYRP1 from the PBMCs separated in peripheral blood in the peripheral blood
The expression quantity of gene.
The present invention also protects a kind of kit, it may include for detecting the substance of PGLYRP1 albumen and/or for detecting
The substance of PGLYRP1 gene；The kit can have the function of following a1) at least one of to a3)：A1) diagnostic activities knot
Core 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 PGLYRP1 albumen " can be the expression for detecting PGLYRP1 albumen
(the corresponding judgement is marked for the substance (the corresponding judgment criteria first) of amount and/or the substance for detecting PGLYRP1 protein concentration
Standard is the third).
Any of the above-described " for the detecting the substance of PGLYRP1 gene " can be the expression for detecting PGLYRP1 gene
The substance of amount.
The expression quantity of any of the above-described PGLYRP1 albumen can be the relative expression of PGLYRP1 albumen reference internal reference albumen
Amount.
The expression quantity of any of the above-described PGLYRP1 gene can be the relative expression of PGLYRP1 gene reference reference gene
Amount.
The expression quantity of any of the above-described detection PGLYRP1 albumen specifically can be used Western Blot experiment and carry out.
Any of the above-described detection PGLYRP1 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 PGLYRP1 gene
The substance of " relative expression quantity of PGLYRP1 gene reference reference gene " includes that primer pair and internal control primer draw composition
Object is to combination；
The primer pair can be made of primer PGLYRP1-F and primer PGLYRP1-R；The target of the primer pair
Sequence 6 of the gene containing ordered list DNA fragmentation shown in the 626th to 786 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 PGLYRP1-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 PGLYRP1-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.
Using the expression quantity or detection PGLYRP1 gene ginseng of any of the above-described primer pair detection PGLYRP1 gene
Relative expression quantity than reference gene also belongs to protection scope of the present invention.
Using the expression quantity or detection PGLYRP1 gene ginseng of any of the above-described primer pair combine detection PGLYRP1 gene
Relative expression quantity than reference gene also belongs to protection scope of the present invention.
Above, using PGLYRP1 gene reference internal reference in any of the above-described primer pair combine detection person under test cDNA
The method of the relative expression quantity of gene 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 PGLYRP1 albumen as marker in exploitation diagnostic activities reagent lungy；
Y2) application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis；
Y3) application of the PGLYRP1 gene as marker in exploitation diagnostic activities reagent lungy；
Y4) application of the PGLYRP1 gene as marker in diagnostic activities tuberculosis.
(No. GeneID is any of the above-described PGLYRP1 albumen：NP_005082.1 in amino acid sequence) such as sequence table
Shown in sequence 5.(No. Genebank is any of the above-described PGLYRP1 gene：NM_005091.2 such as sequence of nucleotide sequence)
In table shown in sequence 6.
It is demonstrated experimentally that compared with Healthy People and tuberculosis latent infection person, in the PBMCs of active tuberculosis patient
The expression quantity of PGLYRP1 gene dramatically increases；The expression quantity of PGLYRP1 gene can be used for distinguishing active tuberculosis patient and
Tuberculosis latent infection person；The expression quantity of PGLYRP1 gene can be used for distinguishing active tuberculosis patient and Healthy People.Therefore,
The expression quantity of PGLYRP1 gene 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 PGLYRP1 gene in PBMCs.
Fig. 2 is PGLYRP1 gene in ROC curve analytic activity tuberculosis patient and the PBMCs of tuberculosis latent infection person
Relative expression quantity.
Fig. 3 is the opposite table of PGLYRP1 gene in the PBMCs of ROC curve analytic activity tuberculosis patient and Healthy People
Up to 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 gDNA Eraser 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 the U.S.
The product of Ambion company.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, PGLYRP1 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 (Cellular is used
Technology Ltd, USA) image and spot count are carried out, then make the following judgment：The number of spots of negative control hole is small
When 6, if the number of spots that the number of spots of detection hole subtracts negative control hole be 6 or more, if detection hole be positive, if
The number of spots that the number of spots of detection hole subtracts negative control hole is less than 6, then detection hole is negative；The spot of negative control hole
Point number be 6 or more when, if the number of spots of detection hole be negative control hole number of spots 2 times or more, if detect
Hole be the 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.Inspection
Gaging hole is the positive, then peripheral blood to be measured provides (i.e. the periphery blood specimen of tuberculosis latent infection person) by tuberculosis latent infection person；Inspection
Gaging hole is feminine gender, 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 for extracting 40 Healthy Peoples (equal informed consent) 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 PGLYRP1 gene in diagnostic activities tuberculosis
(No. GeneID is PGLYRP1 albumen：NP_005082.1 amino acid sequence) is as shown in sequence 5 in sequence table.It compiles
(abbreviation PGLYRP1 gene, No. Genebank is the gene of code PGLYRP1 albumen：NM_005091.2 such as sequence of nucleotide sequence)
In list 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：Take the thallus of Mycobacterium tuberculosis H37Rv, first Co 60 spoke
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 Kit
With 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 PGLYRP1 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 PGLYRP1 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 PGLYRP1 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, and by 10 μ L2 × Green Master Mix, 0.4 μ LPGLYRP1-F aqueous solution, (concentration is
10 μM), 0.4 μ L PGLYRP1-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 fluorescence quantitative PCR instrument real
When quantitative PCR detection.Use 2-ΔCtMethod calculates the relative expression quantity of PGLYRP1 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, PGLYRP1 gene in the PBMCs of active tuberculosis group
Relative expression quantity dramatically increase.
(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 PGLYRP1 gene in the PBMCs of active tuberculosis group and tuberculosis latent infection group, make
Receiver operating curve's analysis is carried out with GraphPad Prism 5.As a result see Fig. 2.The result shows that PGLYRP1 gene
Relative expression quantity can be used for distinguishing active tuberculosis patient and tuberculosis latent infection person.
According to the relative expression quantity of PGLYRP1 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 the phase of PGLYRP1 gene
It can be used for distinguishing active tuberculosis patient and Healthy People to expression quantity.
It is important that the above results show that the relative expression quantity of PGLYRP1 gene has in terms of diagnostic activities tuberculosis
Application value.
<110>No.309 Hospital of PLA
<120>Application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis
<160> 6
<170> PatentIn version 3.5
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Ser Ser Cys Asn Thr Pro Ala Ser Cys Gln Gln Gln Ala Arg Asn Val
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Gln His Tyr His Met Lys Thr Leu Gly Trp Cys Asp Val Gly Tyr Asn
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ctcctagcgg tctcccggac cctgccgccc tgccactatg tcccgccgct ctatgctgct 300
tgcctgggct ctccccagcc tccttcgact cggagcggct caggagacag aagacccggc 360
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gcacctgagc ctgcccttac gctatgtggt ggtatcgcac acggcgggca gcagctgcaa 480
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Claims (10)
1. the substance for detecting PGLYRP1 albumen is preparing the application in product；The function of the product is following a1) extremely
At least one of a3)：A1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is active tuberculosis patient；A3) prevent
Control tuberculosis.
2. substance for detecting PGLYRP1 albumen and recording judgment criteria first and/or prepared by the carrier of judgment criteria third
Application in product；The function of the product is at least one of following a1) to a3)：A1) diagnostic activities tuberculosis；a2)
Diagnose whether person under test is active tuberculosis patient；A3) prevention and control tuberculosis；
The judgment criteria first is：If the expression quantity of PGLYRP1 albumen is higher than in control peripheral blood in person under test's peripheral blood
The expression quantity of PGLYRP1 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 PGLYRP1 albumen is lower than the expression quantity of PGLYRP1 albumen in control peripheral blood, then person under test is not or doubtful is not
Active tuberculosis patient；
The judgment criteria third is：If the concentration of PGLYRP1 albumen is higher than in control peripheral blood in person under test's peripheral blood
The concentration of PGLYRP1 albumen, then person under test be or it is doubtful be active tuberculosis patient；If in person under test's peripheral blood
The concentration of PGLYRP1 albumen lower than control peripheral blood in PGLYRP1 albumen concentration, then person under test be not or it is doubtful be not activity
Property tuberculosis patient；
The control peripheral blood is the peripheral blood of tuberculosis latent infection person or Healthy People.
3. the substance for detecting PGLYRP1 gene is preparing the application in product；The function of the product is following a1) extremely
At least one of a3)：A1) diagnostic activities tuberculosis；A2) whether diagnosis person under test is active tuberculosis patient；A3) prevent
Control tuberculosis.
4. the substance for detecting PGLYRP1 gene is preparing the application in product with the carrier for recording judgment criteria second；Institute
The function of product is stated as at least one of following a1) to a3)：A1) diagnostic activities tuberculosis；A2) whether diagnosis person under test
For active tuberculosis patient；A3) prevention and control tuberculosis；
The judgment criteria second is：If the expression quantity of PGLYRP1 gene is higher than in control peripheral blood in person under test's peripheral blood
The expression quantity of PGLYRP1 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 PGLYRP1 gene is lower than the expression quantity of PGLYRP1 gene in control peripheral blood, then person under test is not or doubtful is not
Active tuberculosis patient；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 PGLYRP1 albumen and/or the substance for detecting PGLYRP1 gene；
The kit has the function of following a1) at least one of to a3)：A1) diagnostic activities tuberculosis；A2) diagnosis person under test is
No is 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 PGLYRP1 albumen " is the substance and/or use for detecting the expression quantity of PGLYRP1 albumen
In the substance of detection PGLYRP1 protein concentration；
" for the detecting the substance of PGLYRP1 gene " is the substance for detecting the expression quantity of PGLYRP1 gene.
7. application as claimed in claim 6 or kit, it is characterised in that：
The expression quantity of the PGLYRP1 albumen is the relative expression quantity of PGLYRP1 albumen reference internal reference albumen；
The expression quantity of the PGLYRP1 gene is the relative expression quantity of PGLYRP1 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 " PGLYRP1 gene reference internal reference base for detecting the expression quantity of PGLYRP1 gene
The substance of the relative expression quantity of cause " includes that primer pair and internal control primer combine the primer pair of composition；
The primer pair is made of primer PGLYRP1-F and primer PGLYRP1-R；The target gene of the primer pair contains
The sequence 6 of ordered list DNA fragmentation shown in the 626th to 786 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 PGLYRP1 albumen as marker in exploitation diagnostic activities reagent lungy；
Y2) application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis；
Y3) application of the PGLYRP1 gene as marker in exploitation diagnostic activities reagent lungy；
Y4) application of the PGLYRP1 gene as marker in diagnostic activities tuberculosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810965685.1A CN108828235A (en) | 2018-08-23 | 2018-08-23 | Application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810965685.1A CN108828235A (en) | 2018-08-23 | 2018-08-23 | Application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108828235A true CN108828235A (en) | 2018-11-16 |
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ID=64150540
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810965685.1A Pending CN108828235A (en) | 2018-08-23 | 2018-08-23 | Application of the PGLYRP1 albumen as marker in diagnostic activities tuberculosis |
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| Country | Link |
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| CN (1) | CN108828235A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061191A (en) * | 2018-08-23 | 2018-12-21 | 中国人民解放军第三〇九医院 | Application of the S100P albumen as marker in diagnostic activities tuberculosis |
| CN110283905A (en) * | 2019-08-05 | 2019-09-27 | 中国人民解放军总医院第八医学中心 | Based on ABCA2 quantitative fluorescent PCR diagnostic activities kit lungy |
| 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 |
| CN102732601A (en) * | 2011-04-13 | 2012-10-17 | 中国人民解放军第三〇九医院 | Isothermal nucleic acid amplification technique based kit for diagnosis of tuberculosis |
| CN102844444A (en) * | 2009-11-30 | 2012-12-26 | 贝勒研究院 | Blood transcriptional signature of active versus latent mycobacterium tuberculosis infection |
-
2018
- 2018-08-23 CN CN201810965685.1A patent/CN108828235A/en active Pending
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 |
| CN102732601A (en) * | 2011-04-13 | 2012-10-17 | 中国人民解放军第三〇九医院 | Isothermal nucleic acid amplification technique based kit for diagnosis of tuberculosis |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109061191A (en) * | 2018-08-23 | 2018-12-21 | 中国人民解放军第三〇九医院 | Application of the S100P albumen as marker in diagnostic activities tuberculosis |
| CN109061191B (en) * | 2018-08-23 | 2021-08-24 | 中国人民解放军第三〇九医院 | Application of S100P protein as marker in diagnosis of active tuberculosis |
| 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 |
| CN110283905A (en) * | 2019-08-05 | 2019-09-27 | 中国人民解放军总医院第八医学中心 | Based on ABCA2 quantitative fluorescent PCR diagnostic activities kit lungy |
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