CN106554990A - Can be used for the blood miR-3135b detection kits of coronary artery calcification diagnosis - Google Patents
Can be used for the blood miR-3135b detection kits of coronary artery calcification diagnosis Download PDFInfo
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Abstract
The present invention relates to a kind of blood miR-3135b detection kits that can be used for coronary artery calcification diagnosis, which is included for expanding the primer of miR-3135b.Moreover, it relates to be used to expand purposes of the primer of miR-3135b in the blood test reagent box for preparing the diagnosis that can be used for coronary artery calcification.
Description
Technical field
The invention belongs to molecular diagnosis field, more particularly to can be used for the diagnosis of coronary artery calcification
Blood test reagent box.Moreover, it relates to the primer for being used to expand miR-3135b can in preparation
For the purposes in the blood test reagent box of the diagnosis of coronary artery calcification.
Background technology
Coronary artery calcification is considered as a kind of benign, with age when which is originally found
And the aging phenomenon for occurring, but later with the proof of substantial amounts of research, it has been found that coronary blood
Pipe calcification is a critically important hazards of major cardiovascular events.Coronary artery calcification can be led
Cause the reduction of vascular compliance, abnormal vascular contractile response, or even the perfusion for hindering cardiac muscle.70
In crowd more than year, 90% male sex and 67% women have the generation of coronary artery calcification,
Several factors such as hypertension, diabetes, dyslipidemia, Overweight-obesity, smoking etc., are all proved to be hat
The hazards of shape arteries calcification.Coronary artery calcification itself does not clinically have characteristic
Performance, but patient find coronary artery calcification be had very serious cardiovascular event,
Therefore, the early diagnosis of coronary artery calcification just becomes particularly important.
The existing diagnostic method of osteoporosis
Coronary artery CT radiographies are the means that non-aggressiveness main at present diagnoses coronary artery calcification, and
The coronary blood based on coronary artery CT radiographies proposed in nineteen ninety first by Agararon et al.
Pipe Calcify integial, also become quantitative description angiosteosis degree so that atherosclerotic burden it is most normal
Standard.
But, above-mentioned inspection spends high, complex steps, therefore it is sensitiveer, special, easy to need discovery badly
In detection, clearly and effectively biomarker, coronary artery calcification is made accurately, timely
Diagnosis.
Present Research of the microRNA as biomarker
There are hundreds of kind of miRNAs, these small molecules in having confirmed serum/plasma in current research
RNAs is stable in properties, rich content, be easy to quantitative determination, and there is significant disease specific.It is existing
Some mature technologies, including qualitative, quantitative miRNA molecule detection technique shows using serum miRNAs
It is more more efficient than traditional differential protein molecular detecting method as the method for molecular marker, it is molecule mark
Will thing has opened up frontier.
Many microRNA molecules related to disease of cardiovascular system are had discovered that, the heart is participated in respectively
Myohypertrophia, the regulation of blood vessel inner skin cell function and activity, endothelial cell and smooth muscle cell function, but
It is to yet there are no reports of the microRNA as coronary artery calcification mark in serum/plasma.
If filtering out the serum/blood of the unconventionality expression in serum/plasma in coronary artery calcification generating process
Slurry miRNAs is used as biomarker, and develops corresponding diagnostic kit, to coronary artery calcium
The diagnosis present situation of change will be once strong promotion.
The content of the invention
The present inventor's long campaigns miRNA related research work, in in-depth study, Wo Mentong
Cross substantial amounts of experiment to find：
MiRNA-3135b (5 '-GGCUGGAGCGAGUGCAGUGGUG-3 ') in blood, be
The specific biomarkers of coronary artery calcification morbidity, so as to complete the present invention.
That is, the present invention includes：
1. a kind of blood test reagent box, which includes：For expanding the primer of miR-3135b, wherein,
The primer for expanding miR-3135b is the reverse transcription primer and miR-3135b of miR-3135b
CDNA amplimer.
2. the blood test reagent box according to item 1, which is used for the diagnosis of coronary artery calcification.
3. the blood test reagent box according to item 1 or 2, wherein, the miR-3135b's is anti-
The nucleotide sequence of transcription primers such as SEQ ID NO:Shown in 1.
SEQ ID NO:1：
5’-GAATCGAGCACCAGTTACGCAATGCCGAGGTCGACTTCCTAGATTT
TTTT TTTTTTTTTT-3’
4. the blood test reagent box according to any one of item 1～3, wherein, the miR-3135b
The amplimer of cDNA include：
Upstream primer, its nucleotide sequence such as SEQ ID NO:Shown in 2；And/or
Downstream primer, its nucleotide sequence such as SEQ ID NO:Shown in 3.
SEQ ID NO:2：
5’-GGCTGGAGCGAGTGCAGTGGTG-3’
SEQ ID NO:3：
5’-GAATCGAGCACCAGTTACGC-3’
5. the blood test reagent box according to any one of item 1～4, which also includes：For from blood
The reagent of middle purifying miR-3135b.
6. it is used for diagnosing the blood of coronary artery calcification in preparation for expanding the primer of miR-3135b
Purposes in liquid detection kit, the blood test reagent box include：For expanding drawing for miR-3135b
Thing, wherein, the primer for expanding miR-3135b be miR-3135b reverse transcription primer and
The amplimer of the cDNA of miR-3135b.
7. the purposes according to item 6, wherein, the nucleosides of the reverse transcription primer of the miR-3135b
Acid sequence such as SEQ ID NO:Shown in 1.
8. the purposes according to item 6 or 7, wherein, the amplification of the cDNA of the miR-3135b
Primer includes：
Upstream primer, its nucleotide sequence such as SEQ ID NO:Shown in 2；And/or
Downstream primer, its nucleotide sequence such as SEQ ID NO:Shown in 3.
9. the purposes according to any one of item 6～8, wherein, the blood test reagent box also includes：
For the reagent for purifying miR-3135b from blood.
It should be noted that in this manual, blood includes blood plasma, serum and whole blood, preferably blood
Slurry, serum.
The present inventor studies blood by controlling the influence factor to coronary artery calcification development such as age
Application prospect of the clearly/blood plasma miRNA in coronary artery calcification diagnosis, illustrates unconventionality expression
Impacts of the miRNAs to coronary disease disease progression, discloses the value of its examination and early diagnosis.Therefore, this
A person of good sense obtains the Specific marker of the serum/plasma miRNA serum s expression of Coronary Calcification morbidity specificity.
By the development to the serum/plasma biomarker and diagnostic kit and application, it is thus only necessary to patient's
Serum or blood plasma detect serum miRNA levels without any other tissue, can extend coronary artery calcium
The Testing index of change, improves the sensitivity of detection, can cause the diagnosis of Coronary Calcification and treat convenient
It is easy, it is that clinician quick and precisely grasps conditions of patients, is that the evaluation of clinical therapeutic efficacy lays the foundation.
Description of the drawings
Fig. 1 shows correlation of the miR-3135b expressions with coronary artery calcification score in serum.
Specific embodiment
In an aspect, the present invention provides a kind of (the blood testing examination of the present invention of blood test reagent box
Agent box), which includes：For expanding the primer of miR-3135b, wherein, it is described for expanding miR-3135b
Primer be miR-3135b reverse transcription primer and miR-3135b cDNA amplimer.
The blood test reagent box of the present invention can be used for the diagnosis of coronary artery calcification.In this specification,
" diagnosis " can be made a definite diagnosis, or to make a definite diagnosis offer reference information.
The nucleotides sequence of miR-3135b is classified as 5 '-GGCUGGAGCGAGUGCAGUGGUG-3 ',
Its amplimer is designed (including reverse transcription primer and cDNA for the miRNA of known nucleotide sequence
Amplimer) be it is well known to those skilled in the art, for example can be according to《Molecular Cloning:A Laboratory guide》
(J. Pehanorm Brookers (Sambrook.J.) etc. write, and Huang Peitang etc. is translated, the 3rd edition, and teaching 2005) comes
Be designed, or using the computer software Premier 6.0 of exploitation (such as Premier companies) come
It is designed.
Particularly, using SEQ ID NO:The reverse transcription primer of the miR-3135b shown in 1, SEQ ID
NO:The upstream primer and SEQ ID NO of the cDNA of amplification miR-3135b shown in 2:Shown in 3
During the downstream primer of the cDNA of amplification miR-3135b, the recall rate of the blood test reagent box of the present invention
(sensitivity) fully meets the requirement as diagnostic kit in medical apparatus statutes up to 95%.This theory
In bright book, recall rate is referred to：In positive sample with the confirmation of " goldstandard " method, determined by additive method
For the ratio of positive sample.The art confirms that " goldstandard " of coronary artery calcification is coronary artery
CT radiographies.
Therefore, the nucleotide sequence of the reverse transcription primer of the miR-3135b is preferably such as SEQ ID NO:1
It is shown.The amplimer of the cDNA of the miR-3135b is preferably such as SEQ ID NO:Shown in 2
Upstream primer and/or such as SEQ ID NO:Downstream primer shown in 3.
The blood test reagent box of the present invention can also be comprising for the purifying miR-3135b from blood
Reagent.For from blood purify miRNA reagent and method be it is well known by persons skilled in the art,
Qiagen serum microRNA detection kit product descriptions are referred to for example.As for from
The reagent that miRNA is purified in blood can be included for example：Chloroform, RNAase inhibitor, DEPC
Water, ethanol etc..
The blood test reagent box of the present invention is applicable to using real-time quantitative PCR in blood
The method detected by miR-3135b, therefore, which can also be used comprising real-time quantitative PCR reaction
Enzyme and/or reagent, for example：Reverse transcriptase, buffer solution, dNTPS, MgC12, go nuclease water,
Fluorescent dye or probe, Taq enzyme etc..These enzymes and/or reagent be it is well known to those skilled in the art, can
Suitably selected according to the concrete detection method for adopting.Additionally, the blood test reagent box of the present invention can be with
Comprising standard items and control (nematode miR-39 samples of such as quantitative markization etc.) and normal reference value (1-3*105
Copy/ul).Using the blood test reagent box of the present invention, it is only necessary to which blood is without other tissue samples
Product, detect miR-3135b by fluorescence or sonde method, easy to detect, and quantitative accurate, greatly improve
The Sensitivity and Specificity of medical diagnosis on disease.
In one aspect of the method, the present invention also provides above-mentioned for expanding the primer of miR-3135b in preparation
Purposes in blood test reagent box.Wherein, the blood test reagent box can be such as present invention
Blood test reagent box.
Embodiment
Embodiment 1
Confirm the expression of miRNA-3135b in serum with the coronary artery damage detected in patient
The obvious positive correlation of level.
The preparation of plasma sample
1). experimenter's blood, room temperature or 4 degree of preservations are gathered using EDTA anticoagulant tubes, enter in one hour
The process of row next step.
2) 4 degree of .1900g (3000rpm) is centrifuged 10 minutes
3). upper strata yellow liquid is taken for blood plasma
4) .16000g is centrifuged 10 minutes
5). supernatant is taken, -80 degree preserve stand-by.
The extraction of miRNA in blood plasma
Using QIAGEN companies of U.S. miRNeasy Serum/Plasma kit
(217184),miRNeasy Serum/Plasma Spike-in-control(219610).
Concrete grammar is as follows：
1). the serum that -80 degree are preserved is melted, 200ul is respectively taken.
2). the QIAzol Lysis Reagent (providing with kit) of 1ml are provided, are fully mixed.
3). place 5 minutes under room temperature (15-25 degree).
4). add 3.5ul miRNeays Serum/Plasma Spike-in Control
(1.6×108Copies/ul), fully mix.
5). 200ul chloroforms are added, is fully mixed 15 seconds.
6). place 2-3 minutes under room temperature (15-25 degree).
7) .4 degree 12000g is centrifuged 15 minutes.
8). supernatant is gone in new centrifuge tube, adds 100% ethanol of 900ul, is mixed.
9). draw 700ul and RNeasy MinElute spin (providing with kit), 8000g under room temperature are provided
Centrifugation 15 seconds.
10). say that remaining sample repeat step 9 is operated, remove the liquid being centrifuged.
11). 700ul reagent RWT (providing with kit) are provided in RNeasy MinElute Spin,
8000g is centrifuged 15 seconds, discards the liquid of centrifugation.
12). 500ul reagent RPE (providing with kit) are provided in RNeasy MinElute Spin,
8000g is centrifuged 15 seconds, discards the liquid of centrifugation.
13). the ethanol of 500ul 80%, 8000g centrifugations 2 are added in RNeasy MinElute Spin
Minute, discard liquid.
14) .RNeasy MinElute Spin are put in new centrifuge tube, and 13000g is centrifuged 5 minutes, with
Remove alcohol.
15) .RNeasy MinElute Spin are put in new centrifuge tube, add the water for removing RNase of 14ul,
Centrifuge maximum (top) speed is centrifuged 2 minutes, the RNA that collection is obtained in obtaining serum.
The tailing method reverse transcription reaction of miRNA-3135b in blood plasma
Using QIAGEN companies of U.S. miScript II RT kit (article No.s：218161).
Concrete grammar is as follows：
1). 10 × miSript Nucleics Mix and 5 × miScript Hispec buffer (are carried with kit
For) fully melt
2). reverse transcription reaction is as follows：
3). mix, 37 degree are reacted 60 minutes
4) .95 degree, 5 minutes, places on ice.
5). the cDNA after obtain 20 microlitres of reverse transcription reactions is diluted into 25 times, to 500 microlitres,
- 20 degree are preserved in case the detection of next step real-time quantitative PCR.
The expression of miRNA-3135b in real-time quantitative PCR detection blood plasma
Using QIAGEN companies of U.S. miScript SYBR Green PCR kit (article No. 218073)
Carry out real-time quantitative PCR detection.
Comprise the following steps that：
1) PCR reaction systems are built as follows：
2) mix, response procedures are as follows：
95 DEG C, 15min；(94 DEG C, 15sec, denaturation；55 DEG C, 30sec, annealing；70 DEG C, 30sec,
Extend) carry out 40 circulations；
3) in serum, the expression of miRNA-3135b is expressed as control with cel-miR-39, with 2Ct (cel-miR-39–miRNA-3135b)Numerical value be serum in miRNA-3135b expression.
The correlation analysis of the coronary artery damage that miRNA-3135b expression is detected with patient in serum
Entered with the coronary artery damage detected in patient using the expression of miRNA-3135b in serum
The expression of miRNA in line correlation analysis, wherein serum is represented with Log2, with GraphPad
Prism5 does statistical analysis.As a result show, the water of miR-3135b levels and coronary artery damage in serum
It is flat that obvious positive correlation (R=0.4642) is presented (Fig. 1).
2 recall rate of embodiment is verified
Subject recruitment standard：
Age is in the male sex of 50-70 year, healthy, height 162-175cm, without pharmacological dependence, nothing
Spirit, mental disease, hereditary disease and infectious disease medical history；Without severe allergy history；Without malnutrition.
Clinical detection index：
(1) coronary artery CT radiographies, obtain coronary artery damage
(2) in serum, the expression of miRNA-3135b is detected
In experimenter's serum, the extraction of miR-3135b and detection, interpretation of result are with embodiment 1.
Experimental result：
According to current clinical coronary calcification examination criteria, detect that CAS is more than by coronary artery CT
100 is Coronary Calcification patient.The serum of 200 experimenters is have collected in our current research, according to above-mentioned
Embodiment 1 is detected, with microRNA copy numbers more than 1*104Copy/ul is positive indication
(being defined as osteoporosis), has 95 people, while being carried out with the method for coronary artery CT to this crowd of people
Coronary Calcification detection, has 100 people's Calcify integials more than 100, comprising aforementioned 95 people in it is complete
Portion, therefore, recall rate is 95% (95/100).
Claims (9)
1. a kind of blood test reagent box, which includes：For expanding the primer of miR-3135b, wherein,
The primer for expanding miR-3135b is the reverse transcription primer and miR-3135b of miR-3135b
CDNA amplimer.
2. blood test reagent box according to claim 1, which is used for coronary artery calcification
Diagnosis.
3. blood test reagent box according to claim 1, wherein, the miR-3135b's
The nucleotide sequence of reverse transcription primer such as SEQ ID NO:Shown in 1.
4. blood test reagent box according to claim 1, wherein, the miR-3135b's
The amplimer of cDNA includes：
Upstream primer, its nucleotide sequence such as SEQ ID NO:Shown in 2；And/or
Downstream primer, its nucleotide sequence such as SEQ ID NO:Shown in 3.
5. blood test reagent box according to claim 1, which also includes：For pure from blood
Change the reagent of miR-3135b.
6. it is used for expanding the primer of miR-3135b in preparation for diagnosing the blood of coronary artery calcification
Purposes in liquid detection kit, the blood test reagent box include：For expanding drawing for miR-3135b
Thing, wherein, the primer for expanding miR-3135b be miR-3135b reverse transcription primer and
The amplimer of the cDNA of miR-3135b.
7. purposes according to claim 6, wherein, the reverse transcription primer of the miR-3135b
Nucleotide sequence such as SEQ ID NO:Shown in 1.
8. purposes according to claim 6, wherein, the expansion of the cDNA of the miR-3135b
Increasing primer includes：
Upstream primer, its nucleotide sequence such as SEQ ID NO:Shown in 2-5；And/or
Downstream primer, its nucleotide sequence such as SEQ ID NO:Shown in 6.
9. purposes according to claim 6, wherein, the blood test reagent box also includes：For
The reagent of miR-3135b is purified from blood.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152567A (en) * | 2014-08-19 | 2014-11-19 | 中国人民解放军总医院第一附属医院 | Application of miRNA-199a in preparation of diagnostic kit |
| CN104694639A (en) * | 2014-02-14 | 2015-06-10 | 中国航天员科研训练中心 | Blood detection kit for osteoporosis diagnosis |
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2015
- 2015-09-25 CN CN201510623077.9A patent/CN106554990A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694639A (en) * | 2014-02-14 | 2015-06-10 | 中国航天员科研训练中心 | Blood detection kit for osteoporosis diagnosis |
| CN104152567A (en) * | 2014-08-19 | 2014-11-19 | 中国人民解放军总医院第一附属医院 | Application of miRNA-199a in preparation of diagnostic kit |
Non-Patent Citations (1)
| Title |
|---|
| W. LIU等: "Circulating miRNAs as potential biomarkers of coronary artery calcification and their roles of predicting coronary artery disease", 《ESC CONGRESS 2015》 * |
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