CN109680058A - Application and kit of the miR-101 level detection in diabetic microvascular complication diagnostic reagent - Google Patents
Application and kit of the miR-101 level detection in diabetic microvascular complication diagnostic reagent Download PDFInfo
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Abstract
本发明公开了microRNA101水平检测在糖尿病微血管病变诊断试剂盒中的应用以及检测试剂和试剂盒。本发明研究发现在糖尿病微血管病变患者人群中，血浆microRNA101水平低于糖尿病非微血管病变患者，因此本发明以miRNA101作为分子标记，研发出糖尿病微血管病变的早期诊断试剂和试剂盒，其含有可检测血液中miRNA101的RNA表达水平的试剂，所述检测方法包括：1)获取糖尿病微血管病变患者血液标本；2)检测患者血浆中microRNA101水平，评估患者是否存在微血管病变，microRNA101水平可通过实时定量PCR方法检测。本发明的方法可以早期提示糖尿病微血管病变的高危患者，可帮助进行早期临床干预，及时正确地治疗以阻止糖尿病微血管病变的进展，具有较好的操作性和重现性。The invention discloses the application of microRNA101 level detection in a diagnostic kit for diabetic microangiopathy, as well as the detection reagent and the kit. The research of the present invention finds that in the population of diabetic microangiopathy patients, the level of plasma microRNA101 is lower than that of diabetic non-microangiopathy patients. Therefore, the present invention uses miRNA101 as a molecular marker to develop early diagnostic reagents and kits for diabetic microangiopathy, which contain blood detectable blood A reagent for the RNA expression level of miRNA101 in the patient, the detection method includes: 1) obtaining blood samples from patients with diabetic microangiopathy; 2) detecting the level of microRNA101 in the plasma of the patient to evaluate whether the patient has microvascular disease, and the level of microRNA101 can be detected by real-time quantitative PCR method . The method of the invention can prompt patients with high risk of diabetic microangiopathy at an early stage, can help early clinical intervention, and can timely and correctly treat to prevent the progress of diabetic microangiopathy, and has good operability and reproducibility.
Description
Technical field
The invention belongs to fields of biomedicine, detect more particularly, to miR-101 level and examine in diabetic microvascular complication
Application in disconnected, and be able to detect the detection reagent of miR-101 level and preparing diabetic microvascular complication diagnostic kit
In application, and corresponding diagnostic reagent and kit.
Background technique
International Diabetes Federation's report, whole world diabetic in 2013 have raised year by year up to 3.84 hundred million
Trend, diabetes, which have become, endangers one of major disease of public health[1].The disease incidence of the current maturity-onset diabetes in China exists
12% or so, seriously endanger people's health.Diabetic microvascular complication is one of most common complication of diabetes, can be induced
Microvascular lesion, diabetic nephropathy, diabetic retinopathy, diabetic neuropathy.Microvascular lesion directly adds
The heart ischemia of weight Coronary Heart Disease Patients, damages heart function, promotes the coronary heart disease even generation of heart infarction；Renal blood vessels lesion leads to kidney
Disease, kidney failure；Retinal microvascular disease finally can lead to blindness etc.[2].Therefore, diabetic microvascular complication has been
Through increasingly by the attention of researcher.Currently, because clinical intervention measure is unable to the development of reverting diabetes microangiopathies
Process[3], therefore high-risk patient can only be determined early in control diabetic microvascular complication by early warning, and is done
In advance, to prevent its occurrence and development.
The concealment of diabetic microvascular complication onset, in early days without apparent clinical symptoms, and does not have specific diagnosis index, only
Have and to a certain extent, organic impairment occurs, is just found: for example, clinical use microalbumin in the state of an illness
Urine is to detect the diabetic nephropathy as caused by microangiopathies[4]；It is can be found that using retina underwent eye-ground vascular fluorescence visualization technology
Diabetic retina microcirculation change etc., and all these means can not early diagnose the patient of diabetic microvascular complication.
Currently, there is some researchs specifically for the early diagnosis of diabetic microvascular complication, such as TGF-β 1, BMP-7 in the world[5],
Corpuscular protein precursor (Progranulin), brain natriuretic peptide N-terminal segment (NT-proBNP), 25-hydroxyvitamin D (25OH-D) etc..These
Marker is related to diabetic microvascular complication morbidity[6-8], but specificity is poor.Therefore, there are not specificity and sensitivity also at present
Property high biomarker be used for the early diagnosis of diabetic microvascular complication.Hypersensitivity high specific is developed as a result,
Diabetic microangiopathy kit has important clinical meaning and clinical value.
Microrna 101 (microRNA101, abbreviation miR-101) is an important tumor suppression of discovered in recent years
MiRNA has the function of dual: promoting cell stem cell and transfer.By inhibiting the expression of EZH2, miR-101 is not only
It can control the small proliferation of tumour, also can control its transfer and invasive ability.Current research suggests that it passes through in modulating vascular
Skin growth factor C inhibits the migration and invasion of liver cancer cells, but there is not yet is applied in terms of diabetic microangiopathy diagnosis
Report.
1.Turnbull,F.M.,et al.,Intensive glucose control and macrovascular
outcomes in type 2diabetes.Diabetologia,2009.52(11):p.2288-98.
2.Patel,A.,et al.,Intensive blood glucose control and vascular
outcomes in patients with type 2diabetes.N Engl J Med,2008.358(24):p.2560-72.
3.Huang,E.S.,et al.,Projecting the future diabetes population size
and related costs for the U.S.Diabetes Care,2009.32(12):p.2225-9.
4.Currie,G.,G.McKay,and C.Delles,Biomarkers in diabetic nephropathy:
Present and future.World J Diabetes,2014.5(6):p.763-76.
5.Wong,M.G.,et al.,Circulating bone morphogenetic protein-7and
transforming growth factor-beta1are better predictors of renal end points in
patients with type 2diabetes mellitus.Kidney Int,2013.83(2):p.278-84.
6.Xu,L.,et al.,Serum Levels of Progranulin Are Closely Associated
with Microvascular Complication in Type 2Diabetes.Dis Markers,2015.2015:
p.357279.
7.Keech,Serum 25-Hydroxyvitamin D:A Predictor of Macrovascular and
Microvascular Complications in Patients With Type 2Diabetes.diabetes care,
2015.
8.Hamano,K.,et al.,N-terminal fragment of probrain natriuretic
peptide is associated with diabetes microvascular complications in type
2diabetes.Vasc Health Risk Manag,2014.10:p.585-9.
Summary of the invention
The present inventor passes through the study found that in diabetic microvascular complication patients, blood plasma
MicroRNA101 level is lower than the non-microangiopathies patient of diabetes.On this basis, the invention proposes be able to detect blood plasma
The reagent of middle microRNA101 expression preparation diagnosis diabetic microvascular complication reagent or kit in application,
Corresponding diagnostic method and diagnostic kit.
The first aspect of the present invention provides the reagent for the microRNA101 level in blood plasma that is able to detect in preparation diabetes
The diagnostic reagent of microangiopathies or the purposes in diagnostic kit.
According to the present invention, the reagent for being able to detect microRNA101 level in blood plasma is detection microRNA101
The reagent of rna level.
According to the present invention, the diabetic microvascular complication include but is not limited to occur diabetic kidney,
The microangiopathies of the organ or tissues such as eye, heart, nerve, toes, can lead to diabetes with secondary nephrosis,
Diabetic retinopathy, diabetic cardiomyopathy, diabetic nerve system lesion, diabetes etc..
In an embodiment of the invention, the diabetic microvascular complication occurs in kidney of diabetic patients
Microangiopathies.
In an embodiment of the invention, the diabetic microvascular complication occurs in diabetic's eye
Microangiopathies.
In an embodiment of the invention, the diabetic microvascular complication occurs in diabetic's heart
Microangiopathies.
According to the present invention it is possible to be detected in blood plasma using the detection method of various detection rna levels known in the art
The rna level of microRNA101, such as real-time quantitative PCR, RNA trace, in situ hybridization, southern blotting technique, slot blot, nuclease
Protect analysis, RNA micro-array chip etc..
The second aspect of the present invention, provides a kind of diagnostic reagent of diabetic microvascular complication, and the diagnostic reagent includes
It is able to detect the detection reagent of the rna level of microRNA101.
As one embodiment of the present invention, the reagent can be detected described by the method for real-time quantitative PCR
The rna level of microRNA101.The reagent for being able to detect the rna level of microRNA101 in blood plasma is real-time quantitative
PCR reagent.
In one embodiment of the invention, the real-time quantitative PCR reagent includes microRNA101 amplimer, example
If the amplimer can be following sequence: 5 '-TACAGTACTGTGATAACTGAA-3 '.
In one embodiment of the invention, the real-time quantitative reagent further includes downstream primer.The downstream primer is
Universal primer, such as: 5 '-GCGTGCTACAGTACTGTGATAACTG-3 '.
Further, the real-time quantitative PCR reagent further include: archaeal dna polymerase and report molecule.For example, the report
Molecule can be fluorescent dye or reporter probe.The fluorescent dye is preferably SYBR Green I.The reporter probe is preferred
For TaqMan fluorescence probe.
Further, in the real-time quantitative PCR reagent further include: the buffer for PCR reaction.
Further, in the real-time quantitative PCR reagent further include: for extracting the reagent of RNA, such as phenol, chloroform
And isopropanol.
Further, in the real-time quantitative PCR reagent further include: for the reagent of reverse transcription RNA, such as reverse transcriptase
And reverse transcriptase primer.In one embodiment of the invention, the reverse transcriptase primer can be Oligo (dT), random primer or
The specific reverse transcriptase primer of person microRNA101；Such as the specific reverse transcriptase primer is 5 '-GTCGTATCCAGTGCA
GGGTCCGAGGTATTCGCACTGGATAC GACTTCACT-3’。
As another embodiment of the invention, the reagent can be detected by the method for RNA micro-array chip
The expression of the microRNA101.The reagent for being able to detect the rna level of microRNA101 in blood plasma is that RNA is micro-
Array chip detection reagent.
In one embodiment of the invention, the RNA micro-array chip detection reagent includes that miRNA chip and solid phase carry
Body.
In one embodiment of the invention, the RNA micro-array chip detection reagent includes capture probe.
In one embodiment of the invention, the RNA micro-array chip detection reagent further includes fluorescent marker.
Further, the RNA micro-array chip detection reagent further includes label buffer, hybridization buffer.
As another embodiment of the invention, the reagent can be by described in the detection of the method for RNA trace
The expression of microRNA101.The reagent for being able to detect the rna level of microRNA101 in blood plasma is the inspection of RNA trace
Test agent.
In one embodiment of the invention, the RNA blotting detection reagent includes that can hybridize instead with microRNA101
The probe answered.Preferably, the probe is the probe of mark molecule label.In one embodiment of the invention, institute
Stating mark molecule can be isotope, fluorescent molecule, nanogold, digoxin, biotin, lock nucleic acid.
In one embodiment of the invention, the RNA blotting detection reagent includes immobilon-p.Preferably, the solid phase
Film can be cellulose acetate film or nylon membrane.
In one embodiment of the invention, the RNA blotting detection reagent includes hybridization buffer and cleaning solution.
The third aspect of the present invention, also provides a kind of diagnostic kit of diabetic microvascular complication, and the kit contains
There is the detection reagent of the above-mentioned rna level for being able to detect microRNA101.
Further, the kit further includes the container for storing the detection reagent.
Further, the kit further includes using the teachings of the kit, such as product description, use
Schematic diagram etc..
The present invention also provides the application methods of the diagnostic reagent or diagnostic kit: making the detection reagent and to test sample
Product contact under conditions of microRNA101 in being suitable for the detection reagent detection sample to be tested, by reaction system
The measurement for the detection ingredient for being included is to realize the detection to microRNA101.
In one embodiment of the invention, described method includes following steps: obtaining the blood sample of subject
This, the rna expression for detecting microRNA101 in the blood sample is horizontal.
According to the present invention, the blood sample can be the peripheral blood of subject.
According to the present invention, the subject is diabetic.
According to the present invention, the method can be with are as follows: and the peripheral blood of subject is taken, mononuclearcell is separated, extracts RNA, it is inverse
Transcription RNA simultaneously carries out real-time quantitative PCR, and the rna expression for detecting microRNA101 in the peripheral blood sample is horizontal.
According to the present invention, the method can be with are as follows: and the peripheral blood of subject is taken, mononuclearcell is separated, extracts RNA, with
Probe carries out chip hybridization reaction, and the rna expression for detecting microRNA101 in the peripheral blood sample is horizontal.
According to the present invention, the method can be with are as follows: takes the peripheral blood of subject, separates mononuclearcell, extracts RNA, RNA
Gel electrophoresis separation is carried out, RNA is shifted and is fixed on immobilon-p, solid phase RNA hybridizes with the probe molecule of label, to special
In conjunction with the developed image of probe molecule detected.
According to the present invention, the method also includes judging that the rna expression of microRNA101 in the blood sample is horizontal high
Low step.
The judgement is carried out according to the threshold value of microRNA101 expression.The threshold value is microRNA101 given
Average expression level in crowd.The given crowd is the crowd for suffering from diabetes and microangiopathies not occurring.For example, working as
Given crowd is with diabetes and when the crowd of microangiopathies not occurring, if in blood plasma microRNA101 rna level
When giving horizontal 50% of crowd lower than this, then it is judged as positive, that is, for patient or the height that diabetic microangiopathies occur
Endanger patient；It is on the contrary then for feminine gender.Since there are certain false positive rate and false negative rates for the detection, can further tie
It closes clinical symptoms and other detection means makes diagnosis.For example, very low for the rna level of microRNA101 in blood plasma
Diabetic can carry out further microangiopathies detection to it, make a definite diagnosis and whether generate microangiopathies, and carry out corresponding
Treatment.For example, the patients of diabetic heart microangiopathies can occur for suspection, due to coronary angiography higher cost and
There are inspection risks, using microRNA101 level in blood plasma as reference index, may prompt as to whether that row coronary angiography is needed to make a definite diagnosis.
MicroRNA101 level belongs to composite target, and when microangiopathies occurs in diabetic, diabetic keratopathy either occurs
The decline of expression can all occur in nephrosis, diabetic ocular microangiopathies or diabetic heart microangiopathies etc..
Term definition and explanation:
Term " RNA " refers to ribonucleic acid, is to form long chain molecule through phosphodiester bond condensation by ribonucleotide.
Term " microRNA101 " is identical with " miR-101 ", " miRNA-101 " meaning, refers to Microrna -101, for one kind
Adjust the small-sized non-coding RNA of gene expression.
Term " subject " or " patient " are used interchangeably, and refer to diabetic herein.
Term " reverse transcription " is identical as " reverse transcription " meaning, refers to using RNA as template, by reverse transcriptase catalytic deoxidation nucleosides
The process of sour synthetic DNA.
Meaning is identical herein with " peripheral blood " for term " blood ", refers to as patient's blood obtained from in-vitro evaluation purpose
Liquid, including haemocyte and blood plasma；Term " blood plasma " is from the upper component obtained after the anticoagulant individual whole blood centrifugation of EDTA.
Beneficial effects of the present invention:
The present inventor passes through the study found that in diabetic microvascular complication patients, blood plasma
MicroRNA101 level is lower than the non-microangiopathies patient of diabetes.The present invention is using miRNA-101 as molecular labeling, research and development
The early diagnosis kit of diabetic microvascular complication out can be with auxiliary diagnosis by the detection of microRNA101 expression
To prompt the high-risk patient of diabetic microvascular complication in early days, further helps to carry out early clinic intervention, correctly control in time
The progress to prevent diabetic microvascular complication is treated, such as prevents the occurrence and development of diabetic nephropathy, and even can by treatment
So that the pathological change of diabetic nephropathy takes a turn for the worse, to quality of life in patients with diabetes is improved, saving patient vitals has weight
Want meaning.
The present invention detects in patients blood plasma using clinical diabetes microangiopathies blood samples of patients as test object
MicroRNA101 is horizontal, so establish it is a kind of can fast and simple measurement patient whether there is the methods of microangiopathies, overcome
The shortcomings that each microangiopathies detection method as known in the art, such as invasive and high risk etc..With the diabetes heart
For dirty microangiopathies, there are many detection method of coronary heart disease Coronary Artery Lesions, but there are experimental implementation complexity, sample for many methods
Difficulty is obtained, stenosis in coronary artery can not be embodied, have the shortcomings that invasive and high risk.Detection and diagnosis side of the invention
Method is that the clinical research of diabetic microvascular complication is provided convenience.
In clinical application, after being diagnosed as diabetes according to the sings and symptoms of patient, according to the diagnosis of the invention described
Reagent and kit with auxiliary diagnosis and can judge patient of diabetes by microRNA101 expression in measurement patients blood plasma
Person whether there is microangiopathies, and the convenient anticipation to conditions of patients is improved through other micro- blood of screening method complete detection
The specific aim of pipe lesion.
Specific embodiment
The present invention is described further with reference to embodiments.It should be noted that embodiment cannot function as to this hair
The limitation of bright protection scope, it will be understood by those skilled in the art that, any improvements introduced on the basis of the present invention and variation all exist
Within protection scope of the present invention.
Chemical reagent used in following embodiment is all conventional reagent, commercially available.
The division of endocrinology inpatient for inquiring Wuhan Union Hospital, collects in clinical laboratory
New diabetic's EDTA anticoagulant blood-collecting pipe (purple head tube) venous blood of being admitted to hospital, subject's Clinical symptoms basic condition are as shown in table 1.
Blood sample is the residual blood after blood routine detection, measures about 1-2mL.
This research has chosen Diabetic Nephropathy patients and patients with diabetic retinopathy is diabetic microvascular complication
Representative, judged with urine detection result whether the diabetic has occurred microangiopathies: for diabetes
Nephrosis early stage prompts urine protein positive with routine urinalysis；For diabetic nephropathy advanced stage, using decreased renal function as standard.Diabetes
Retinopathy is that foundation judges with patient eyeground testing result.Only diabetes, and exclude it and each micro- blood of internal organs occurs
Patient of the patient of pipe lesion as the non-microangiopathies of diabetes, that is, the diabetes B (T2DM) in following table 1 and 2 is suffered from
Person.
1 subject's Clinical symptoms of table
Sample process:
1, after collecting blood sample, 1500rpm is centrifuged 10min；
2, take upper plasma in new EP pipe, -80 DEG C save for use.
RNA is extracted:
Blood plasma total serum IgE RNAiso Plus*(Takara, 9108) is extracted, specific steps:
1, blood plasma takes out from -80 DEG C of refrigerators, inserts and slowly melts on ice, 2000rpm after thawing, 4 DEG C of centrifugation 10min take
Clearly；
2 ,+700 μ L RNAiso Plus of 300 μ L blood plasma, acutely concussion mixes, ice bath 5min；
3, add 200 μ L (1/5 volume) chloroform, acutely 12-15 times reverse, ice bath 10min；
4,4 DEG C, 12000rpm is centrifuged 15min, takes supernatant in new EP pipe；
5, plus with the isometric isopropanol of supernatant, turn upside down 12-15 times, -20 DEG C of standing 30min；
6,4 DEG C, 12000rpm is centrifuged 30min, abandons supernatant；
7, add 75% ethanol washing of 1mL Fresh, 4 DEG C, 7500rpm is centrifuged 5min；
8, plus 1mL dehydrated alcohol washs, and 4 DEG C, 7500rpm is centrifuged 5min；
9, supernatant is abandoned, residual ethanol is dried；
10、10μL RNA-free H2O dissolves RNA, and Quawell surveys RNA concentration.
Reverse transcription:
Use PrimeScriptTMRT reagent Kit (Takara, RR037A), standard step:
1,200ngRNA+0.4 μ L primer working solution, with RNA-free H2O complements to 6 μ L systems, 70 DEG C of 10min, and 4 DEG C
Reaction terminating；
2, above-mentioned system adds 2 μ 5 × PrimeScript of L Buffer, 0.5 μ L PrimeScript RT Enzyme Mix
I, RNA-free H2O complements to 10 μ L systems.42 DEG C of 60min, 70 DEG C of 10min, 4 DEG C of reaction terminatings.
Because blood plasma RNA content is extremely low, reverse transcription system replaces 200ng RNA total amount with 2.6 μ L RNA suspensions.
Real-time quantitative PCR (qRT-PCT):
With TB GreenTMPremix Ex TaqTM(Tli RNaseH Plus) (Takara, RR420A), specific steps:
1, reaction system (10 μ L):
2, real-time program:
95 DEG C of 20s, 1 circulation；
95 DEG C of 10s, 60 DEG C of 20s, 70 DEG C of 10s, 40 circulations
Experimental group:
Subject is divided into simple T2DM group according to its basic condition, T2DM merges microangiopathies patient (including DM kidney
Disease and DM retinopathy).
Real-time quantitative PCR interpretation of result: each PCR reaction takes fixed fluorescence threshold, measures miRNA101 in sample and expands
The CT value of increasing.
The expression relative level of the miRNA101 of blood sample is used 2-ΔΔCtMethod is calculated, using U6 gene as ginseng
According to gene.
Calculated result is analyzed using SPSS20.0 statistical software, applies Levene method of inspection and t method of inspection pair first
The measurement data of all statistics carries out homogeneity test of variance and significance test.
The relative level that table 2miRNA101 is expressed in subject's blood plasma
3 independent sample inspection result of table
According to table 2, table 3 data it is found that the non-microangiopathies of diabetes and diabetic microvascular complication patient
MicroRNA101 expresses relative level, and there are statistical differences, and in diabetic microvascular complication patients blood plasma
MicroRNA101 expresses relative level mean value and is significantly lower than the non-microangiopathies crowd of diabetes.The above results show in blood plasma
MicroRNA101 expression can be used as the reference index of microangiopathies early diagnosis, when it is significantly lower than given crowd
When the expression of (such as the non-microangiopathies crowd of diabetes), the probability that microangiopathies occur for subject is greatly increased.
More than, embodiments of the present invention are illustrated.But the present invention is not limited to above embodiment.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in guarantor of the invention
Within the scope of shield.
Sequence table
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Claims (10)
1. being able to detect the reagent of microRNA101 level in blood plasma in the diagnostic reagent for preparing diabetic microvascular complication or examining
Purposes in disconnected kit.
2. purposes according to claim 1, which is characterized in that described to be able to detect microRNA101 level in blood plasma
Reagent is the reagent for detecting the rna level of microRNA101.
3. a kind of diagnostic reagent of diabetic microvascular complication, which is characterized in that the RNA comprising being able to detect microRNA101
Horizontal detection reagent.
4. purposes according to claim 1 or 2 or diagnostic reagent as claimed in claim 3, which is characterized in that it is described can
The detection reagent of the rna level of detection microRNA101 can be detected described by the method for real-time quantitative PCR
The rna level of microRNA101；
Preferably, the detection reagent includes the amplimer of real-time quantitative PCR；Preferably, the amplimer is following sequence
Column: 5 '-TACAGTACTGTGATAACTGAA-3 '；
It is preferred that the detection reagent further includes universal primer sequence: 5 '-GCGTGCTACAGTACTGTGATAACTG-3 '；
Preferably, the detection reagent further include: archaeal dna polymerase and report molecule；It is preferred that the report molecule is fluorescent dye
Or reporter probe；The fluorescent dye is preferably SYBR Green I；The reporter probe is preferably TaqMan fluorescence probe；
Preferably, in the detection reagent further include: the buffer for PCR reaction；
Preferably, in the detection reagent further include: for extracting the reagent of RNA；
Preferably, in the detection reagent further include: reverse transcriptase and reverse transcriptase primer；
Preferably, the reverse transcriptase primer is that the specific reverse transcriptase of Oligo (dT), random primer or microRNA101 draw
Object；It is preferred that the specific reverse transcriptase primer is 5 '-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGA
CTTCACT-3’。
5. purposes according to claim 1 or 2 or diagnostic reagent as claimed in claim 3, which is characterized in that it is described can
The detection reagent of the rna level of detection microRNA101 can be detected described by the method for RNA micro-array chip
The expression of microRNA101；
It is preferred that the reagent for being able to detect the rna level of microRNA101 in blood plasma is RNA micro-array chip detection reagent；
Preferably, the detection reagent includes miRNA chip and solid phase carrier；
Preferably, the detection reagent includes capture probe；
Preferably, the detection reagent further includes fluorescent marker；
Preferably, the detection reagent further includes label buffer, hybridization buffer.
6. purposes according to claim 1 or 2 or diagnostic reagent as claimed in claim 3, which is characterized in that it is described can
The detection reagent for detecting the rna level of microRNA101 can detect the microRNA101's by the method for RNA trace
Expression；
It is preferred that the reagent for being able to detect the rna level of microRNA101 in blood plasma is RNA blotting detection reagent；
Preferably, the detection reagent include can be with the probe of microRNA101 hybridization reaction；Preferably, the probe is
The probe of mark molecule label；It is highly preferred that the mark molecule is selected from isotope, fluorescent molecule, nanogold, digoxin, life
Object element, lock nucleic acid；
Preferably, the detection reagent includes immobilon-p；
Preferably, the detection reagent includes hybridization buffer and cleaning solution.
7. a kind of diagnostic kit of diabetic microvascular complication, which is characterized in that the kit contains such as claim 3-6
Described in any item diagnostic reagents；
Preferably, the kit includes the container for storing the detection reagent；
Preferably, the kit includes such as product description, using schematic diagram using the teachings of the kit.
8. purposes according to claim 1 or 2 or the described in any item diagnostic reagents of claim 3-6 or claim 7
The diagnostic kit, which is characterized in that the diabetic microvascular complication be occur kidney of diabetic patients, eye and
Heart appoints the microangiopathies of one or more.
9. the application method of any one of the claim 3-6 diagnostic reagent, which is characterized in that make the detection reagent with it is to be measured
Sample contacts under conditions of microRNA101 in being suitable for the detection reagent detection sample to be tested, by reaction system
Included in detection ingredient measurement to realize the detection to microRNA101.
10. application method according to claim 9, which comprises the steps of: obtain the blood sample of subject
This, the rna expression for detecting microRNA101 in the blood sample is horizontal；
Preferably, the blood sample can be the peripheral blood of subject；
Preferably, the subject is diabetic；
Preferably, which comprises take the peripheral blood of subject, separate mononuclearcell, extract RNA, reverse transcription RNA goes forward side by side
Row real-time quantitative PCR, the rna expression for detecting microRNA101 in the peripheral blood sample are horizontal；
Preferably, which comprises take the peripheral blood of subject, separate mononuclearcell, extract RNA, carry out core with probe
Piece hybridization reaction, the rna expression for detecting microRNA101 in the peripheral blood sample are horizontal；
Preferably, which comprises take the peripheral blood of subject, separate mononuclearcell, extract RNA, RNA carries out gel electricity
Swimming separation, RNA is shifted and is fixed on immobilon-p, solid phase RNA hybridizes with the probe molecule of label, to the probe of specific bond
The developed image of molecule is detected；
Preferably, the method also includes judging the step of the rna expression level height of microRNA101 in the blood sample
Suddenly.
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| CN201910024714.9A CN109680058A (en) | 2019-01-10 | 2019-01-10 | Application and kit of the miR-101 level detection in diabetic microvascular complication diagnostic reagent |
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| CN201910024714.9A CN109680058A (en) | 2019-01-10 | 2019-01-10 | Application and kit of the miR-101 level detection in diabetic microvascular complication diagnostic reagent |
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