CN105779606A - Markers for diagnosing and predicating focal segmental glomerulosclerosis - Google Patents
Markers for diagnosing and predicating focal segmental glomerulosclerosis Download PDFInfo
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- CN105779606A CN105779606A CN201610222290.3A CN201610222290A CN105779606A CN 105779606 A CN105779606 A CN 105779606A CN 201610222290 A CN201610222290 A CN 201610222290A CN 105779606 A CN105779606 A CN 105779606A
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Abstract
The invention discloses application of reagent of markers for detecting focal segmental glomerulosclerosis (FSGS) to preparation of a kit for diagnosing and predicating focal segmental glomerulosclerosis, the focal segmental glomerulosclerosis markers are one or more of miR-17, miR-451, miR-106a and miR-19B, By means of the markers for diagnosing and predicating focal segmental glomerulosclerosis, new blood markers are provided for clinical diagnosis of focal segmental glomerulosclerosis, peripheral blood testing can be directly conducted, and the markers have the advantages of being good in specificity and high in sensitivity, have good clinical diagnosis value and lay a foundation for implementation of hematology diagnosis of FSGS.
Description
Technical field
The invention belongs to detection field, the reagent being particularly used for detecting FSGS mark is examined in preparation
Application in the disconnected and test kit of indication FSGS, FSGS mark is
At least one in miR-17, miR-451, miR-106a and miR-19b.
Background technology
FSGS (focal segmental glomerulosclerosis, FSGS) is a kind of common constitutional
Renal glomerular disease, sickness rate in recent years is gradually increased, and in adult with primary nephrotic syndrome patient, about 30%～40% is FSGS.
Clinical manifestation atypism, great majority are starting with the nephrotic syndrome of onset concealment, as diagnosed not in time and treating, will development
For end stagerenaldisease.At present, the main bases of FSGS is Renal biospy, lacks special Blood diagnosis mark.Therefore,
Urgently need the specificity Blood diagnosis mark of one or more FSGS.
Microrna (microRNAs, miRNAs) is the endogenous non-coding RNA molecule that a class length is about 20～25nt,
Its not coded protein, but be combined with messenger RNA (mRNAs) 3 ' the untranslated region complementation of target gene, induce said target mrna
Degraded or the translation of suppression said target mrna, thus realize post-transcriptional gene regulation effect.Research confirms, miRNAs can regulate and control
The function of podocyte, the morbidity at FSGS plays pivotal role during progression of disease.Plasma circulation miRNAs refers to blood
It is free on extracellular miRNAs present in slurry, there is feature stable, sensitive and easy to detect, be that a class is emerging
Diagnosis molecular marker, the potential early diagnosis being applied to various disease and prognosis pass judgment on, such as: tumor, cardiovascular disease,
Hepatic disease, organ transplantation etc..Therefore, blood plasma miRNA s also gets a good chance of becoming FSGS early diagnosis and prognosis passes judgment on
Mark.
Summary of the invention
In view of this, it is an object of the invention to provide for the reagent detecting FSGS mark in preparation
Application in the test kit of diagnosis and indication FSGS, has filled up current without FSGS
The blank of mark, and there is the advantage that specificity is good, highly sensitive.
For achieving the above object, the present invention provides following technical scheme:
For detecting the reagent of FSGS mark at preparation diagnosis and indication FSGS
Test kit in application, it is characterised in that: described FSGS mark is miR-17, miR-451,
At least one in miR-106a and miR-19b.
Preferably, described FSGS mark is miR-17, miR-451, miR-106a or miR-19b.
It is furthermore preferred that described FSGS mark is miR-17, miR-451, miR-106a and
The combination of miR-19b.
For easy to detect, it is test kit by the agent combination of FSGS mark, it is preferred that described reagent
Box is ultraviolet spectrophotometry detection kit, fluorescent dye determination detection kit, quantitative real-time PCR detection kit
Or digital pcr method detection kit.
It is furthermore preferred that described test kit is micro-array chip reagent kit.
In the present invention, the detection sample of described diagnosis and indication FSGS is peripheral blood.
The beneficial effects of the present invention is: present invention firstly discloses the blood serum designated object of FSGS, it is possible to
Using peripheral blood as pattern detection, specificity is good, highly sensitive, and sampling is convenient, and solving prior art needs to be lived by kidney
Inspection could diagnose the defect of FSGS；And testing cost is low, it is not necessary to use special detection equipment, can
To use the detection such as existing fluorescence quantitative PCR method or micro-array chip, there is good market prospect.
Accompanying drawing explanation
In order to make the purpose of the present invention, technical scheme and beneficial effect clearer, the present invention provides drawings described below:
Fig. 1 is that in 40 example specimen, FSGS blood plasma expresses significantly reduced miRNAs.
Fig. 2 is 60 example specimen checking miR-17, and miR-451, miR-106a, miR-19b express significantly in FSGS blood plasma
Reduce.
Fig. 3 is miR-17, ROC curve (A:miR-17 when miR-451, miR-106a, miR-19b detect alone or in combination
ROC curve；B:miR-451ROC curve；C:miR-106aROC curve；D:miR-19b ROC curve；E: on
State 4 united ROC curve of miRNAs).
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.Unreceipted actual conditions in embodiment
Experimental technique, generally according to normal condition, such as institute in Molecular Cloning: A Laboratory guide (third edition, J. Pehanorm Brooker etc. writes)
The condition stated, or according to the condition proposed by manufacturer.
Embodiment 1, Taqman real-time quantitative PCR detection blood plasma miRNA s express spectra
Taking 20 example clinical diagnosises is FSGS patients blood plasma, takes 20 example Healthy Human Serums as comparison simultaneously, then extracts blood plasma
RNA, extracting method is with reference to mirVanaTMPARIS test kit operates, and specifically comprises the following steps that
1. in 400 μ L blood plasma, add isopyknic 2 × Denaturing Solution, whirlpool 30 seconds, fully mix, add
10 μ L cel-mir-39 (10nM), are immediately placed on 5 minutes on ice；
2. the phenol chloroform of 800 μ L, whirlpool 30-60 second are added；
3. in 12000 × g, under the conditions of 4 DEG C centrifugal 15 minutes, separate supernatant in clean centrifuge tube；
4. add and be equivalent to 1.25 times of volume room temperature 100% ethanol of supernatant in separating in supernatant, fully mix；
5. the liquid after mixing is inhaled in centrifugal column, if liquid volume is more than 700 μ L, repeatedly upper props, centrifugal 30 seconds until
Liquid crosses post completely；
6. adding 700 μ LmiRNA Wash Solution I in centrifugal column, 12000 × g is centrifuged 15 seconds, removes waste liquid, will
Centrifugal column is put back in collecting pipe；
7. adding 500 μ L Wash Solution 2/3 in centrifugal column, 12000 × g is centrifuged 15 seconds, removes waste liquid, by centrifugal column
Put back in collecting pipe, be repeated once；
8., in transfer centrifugal column and new collecting pipe, 50 μ L 95 DEG C of nuclease-free water of preheating are added to centrifugal column center,
Centrifugal 30 seconds, collect RNA.
Then with extract blood plasma RNA as template, by Taqman real-time quantitative PCR detection blood plasma miRNA s expression
Level, the primer that quantitative PCR uses is purchased from Thermo Fisher company, and article No. is as follows: miR-451 (Cat.#4427975,
ID:001141), miR-106a (Cat.#4427975, ID:002169), miR-19b (Cat.#4427975, ID:000396),
MiR-17 (Cat.#4427975, ID:002308).
Taqman real time quantitative PCR method is as follows, first carries out reverse transcription reaction and synthesizes the first chain cDNA (total system is 5 μ L):
Then at 16 DEG C, 30min, 42 DEG C, 30min, 85 DEG C, 5min reaction, 4 DEG C of preservations.
By above cDNA product ddH2O is template after diluting 2 times, and it is anti-that test kit provides primer to carry out Real-time PCR
Should.System is as follows: 5 μ LTaqman PCR MIX (TAKARA company), 0.25 μ Lprimers/probe, 2 μ LcDNA produce
Thing, 2.75 μ LddH2O, cumulative volume is 10 μ L.Then 2min, 95 DEG C, 15s, 60 DEG C, 30s reaction are reacted at 95 DEG C
40 circulations.Result identifies 4 miRNAs (miR-17, miR-451, miR-106a, miR-19b, nucleotide sequences
As shown in SEQ ID NO.1～SEQ ID NO.4) in FSGS blood plasma, express notable downward (P < 0.01), become FSGS and examine
Disconnected candidate markers, the most as shown in Figure 1.
The candidate markers checking of embodiment 2, FSGS diagnosis
Can 4 candidate markers that obtain for checking embodiment 1 screening be used for FSGS early diagnosis and prognosis judge, this reality
Execute example enlarged sample scale.Take 60 example plasma samples (30 example FSGS patients blood plasma and 30 example human normal plasmas), according to enforcement
The method of example 1 carries out real-time quantitative PCR, and result is as shown in Figure 2.Result shows, miR-17, miR-451 in 60 example specimen,
MiR-106a and miR-19b expresses in FSGS patients blood plasma and significantly reduces, and primarily determines that miR-17, miR-451, miR-106a
The mark can passed judgment on as FSGS early diagnosis and prognosis with miR-19b.
Then draw ROC curve according to testing result, pass judgment on miR-17, miR-451 with the size of area under curve (AUC),
The diagnostic sensitivity of miR-106a and miR-19b and specificity (AUC is closer to 1, represents sensitivity and specificity is the highest),
Result is as shown in Figure 3.Result shows, area under miR-17, miR-451, miR-106a and the independent detection curve of miR-19b
(AUC) it is respectively 0.82,0.77,0.82,0.86, shows miR-17, miR-451, miR-106a and miR-19b conduct
Mark diagnosis FSGS has higher specificity and sensitivity.Then by miR-17, miR-451, miR-106a and miR-19b
ROC curve is drawn after joint-detection, result display miR-17, miR-451, miR-106a and miR-19b joint-detection
AUC area is 0.92, shows that miR-17, miR-451, miR-106a and miR-19b joint-detection more individually detects and has more
High specificity and sensitivity.Therefore, miR-17, miR-451, miR-106a and miR-19b individually or miR-17, miR-451,
MiR-106a and miR-19b combination all can be as FSGS diagnosis and the new mark of indication.
The clinical practice in diagnosis FSGS of embodiment 3, blood plasma miRNA s-tag thing
In order to assess miR-17, miR-451, miR-106a and miR-19b as mark effect in FSGS clinical diagnosis
Really, collect the FSGS plasma specimen that 30 examples are made a definite diagnosis through renal tissue pathology, collect 30 example human normal plasmas as comparison simultaneously,
Carry out quantitative PCR according to the method for embodiment 1, detect the table of miR-17, miR-451, miR-106a and miR-19b respectively
Reach situation, and utilize 4 miRNAs marks to predict the probability that FSGS falls ill alone or in combination.Result shows: miR-17
When individually detecting, can be diagnosed to be 23 examples in the FSGS patient that 30 examples are made a definite diagnosis is FSGS, and its sensitivity is 76.7%,
In 30 example normal plasmas, it is possible to it is normal for being diagnosed to be 24 examples, and its specificity is 80.0%；When miR-451 individually detects, 30
Can be diagnosed to be 25 examples in the FSGS patient that example is made a definite diagnosis is FSGS, and its sensitivity is 83.3%, in 30 example normal plasmas,
It is normal for can being diagnosed to be 21 examples, and its specificity is 70.0%；When miR-106a individually detects, at the FSGS that 30 examples are made a definite diagnosis
Can be diagnosed to be 22 examples in patient is FSGS, and its sensitivity is 73.3%, in 30 example normal plasmas, it is possible to be diagnosed to be 25
Example is normal, and its specificity is 83.3%；When miR-19b individually detects, can diagnose in the FSGS patient that 30 examples are made a definite diagnosis
Going out 21 examples is FSGS, and its sensitivity is 70%；In 30 example normal plasmas, it is possible to it is normal for being diagnosed to be 29 examples, and it is special
Property is 96.7%；Use miR-17, miR-451, miR-106a and miR-19b joint-detection, at the FSGS that 30 examples are made a definite diagnosis
In patient, being diagnosed to be 24 examples is FSGS, and its sensitivity is 80%；In 30 example normal plasmas, miRNAs composite marker thing
Can be diagnosed to be 28 examples is normal control, and its specificity is 93.3%.Result above shows, miR-17, miR-451, miR-106a
Individually or a combination thereof is used equally to diagnose FSGS with miR-19b, and FSGS early diagnosis is had important clinical meaning.
For easy to detect, it is test kit by the agent combination of FSGS mark, such as ultraviolet spectrophotometry
Detection kit, fluorescent dye determination detection kit, quantitative real-time PCR detection kit or digital pcr method；For
Can batch detection, test kit can be made micro-array chip form.
Finally illustrating, preferred embodiment above is only in order to illustrate technical scheme and unrestricted, although by above-mentioned
The present invention is described in detail by preferred embodiment, it is to be understood by those skilled in the art that can in form and
In details, it is made various change, without departing from claims of the present invention limited range.
Claims (6)
1. for detecting the reagent of FSGS mark at preparation diagnosis and indication FSGS
Test kit in application, it is characterised in that: described FSGS mark is miR-17, miR-451,
At least one in miR-106a and miR-19b.
Application the most according to claim 1, it is characterised in that: described FSGS mark is miR-17,
MiR-451, miR-106a or miR-19b.
Application the most according to claim 1, it is characterised in that: described FSGS mark is miR-17,
The combination of miR-451, miR-106a and miR-19b.
Application the most according to claim 1, it is characterised in that: described test kit be ultraviolet spectrophotometry detection kit,
Fluorescent dye determination detection kit, quantitative real-time PCR detection kit or digital pcr method detection kit.
Application the most according to claim 4, it is characterised in that: described test kit is micro-array chip reagent kit.
Application the most according to claim 1, it is characterised in that: described diagnosis and the inspection of indication FSGS
Test sample is originally peripheral blood.
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| CN201610222290.3A CN105779606B (en) | 2016-04-11 | 2016-04-11 | The marker of diagnosis and indication focal segmental glomerulosclerosis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107561175A (en) * | 2017-08-10 | 2018-01-09 | 武汉大学 | A kind of evaluation method of glomerular sclerosis rat model |
| CN107586836A (en) * | 2017-09-22 | 2018-01-16 | 中国人民解放军南京军区南京总医院 | Detect LRRC55mRNA kit and its application in Focal segmental glomerulosclerosis disease diagnoses |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016036949A1 (en) * | 2014-09-03 | 2016-03-10 | The Regents Of The University Of California | Methods to determine the distribution profiles of circulating rnas |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016036949A1 (en) * | 2014-09-03 | 2016-03-10 | The Regents Of The University Of California | Methods to determine the distribution profiles of circulating rnas |
Non-Patent Citations (2)
| Title |
|---|
| MATTHIAS HACKL等: "miR-17，miR-19b，miR-20a，and miR-106a are down-regulated in human aging", 《AGING CELL》 * |
| 蔡晓懿: "血清microRNA在局灶节段性肾小球硬化和微小病变性肾病中表达研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107561175A (en) * | 2017-08-10 | 2018-01-09 | 武汉大学 | A kind of evaluation method of glomerular sclerosis rat model |
| CN107586836A (en) * | 2017-09-22 | 2018-01-16 | 中国人民解放军南京军区南京总医院 | Detect LRRC55mRNA kit and its application in Focal segmental glomerulosclerosis disease diagnoses |
| CN107586836B (en) * | 2017-09-22 | 2020-04-07 | 中国人民解放军南京军区南京总医院 | Kit for detecting LRRC55mRNA and application thereof in diagnosis of focal-segment glomerulosclerosis |
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Granted publication date: 20190402 |