CN109182506A - One kind serum/plasma excretion body miRNA marker relevant to atrial septum type congenital heart disease and its application - Google Patents

One kind serum/plasma excretion body miRNA marker relevant to atrial septum type congenital heart disease and its application Download PDF

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CN109182506A
CN109182506A CN201811167563.4A CN201811167563A CN109182506A CN 109182506 A CN109182506 A CN 109182506A CN 201811167563 A CN201811167563 A CN 201811167563A CN 109182506 A CN109182506 A CN 109182506A
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mirna
heart disease
serum
excretion
atrial septum
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CN109182506B (en
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莫绪明
徐诚
马思雨
束亚琴
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Nanjing Childrens Hospital of Nanjing Medical University
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Nanjing Childrens Hospital of Nanjing Medical University
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    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/178Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The present invention discloses a kind of serum/plasma excretion body miRNA marker relevant to atrial septum type congenital heart disease, excretion body correlation miRNA molecule marker and its primer of the present invention are preparing the application in the product for diagnosing atrial septum type congenital heart disease, can alleviate the technical issues of specific miRNA research relevant to atrial septum type congenital heart disease existing in the prior art still has some deficits.

Description

A kind of serum/plasma excretion body miRNA relevant to atrial septum type congenital heart disease Marker and its application
Technical field
The present invention relates to biotechnologys and diagnostic field, more particularly, to a kind of and atrial septum type congenital heart disease Relevant serum/plasma excretion body miRNA marker and its application.
Background technique
Atrial septum type congenital heart disease is common one of congenital heart malformation, is original atrial septum in embryonic development Occur exception in the process, causes to leave hole between left and right atrium.Atrial septal defect can individually occur, can also with it is other kinds of Cardiovascular malformation is simultaneously deposited.Congenital heart disease accounts for about the 0.8-1.2% of birth defect.Currently, there are about the 2000000 congenital hearts in China Popular name for patient, overall incidence 8 ‰ or so, there are about the births of 120,000~200,000 children with congenital heart disease every year.In the U.S., often About 8-10 children with congenital heart disease in 1000 live-born infants, every year about 40,000 children with congenital heart disease.With regard to the whole world From the point of view of range, atrial septal defect can account for the 10% of congenital heart disease total amount, be relatively common Congenital Heart vitium One of.It is shunted since atrium level exists, leads to corresponding abnormal hemodynamics, easily cause repeated respiratory infections, development It is slow, and it is possible to there are the complication such as congestive heart failure, pulmonary edema, subacute bacterial endocarditis, therefore should Early diagnosis, makes a definite diagnosis rear real-time monitoring, timely operative treatment.
Childhood in view of most patients with atrial septal defect, can be living without other symptoms in addition to easily catching a cold and waiting respiratory tract infection Moving can also be unrestricted;Patient one's mid-10s to 20s do not occur or only slight labour after shortness of breath or palpitaition, but mostly The number serious adults of atrial septal defect will appear it is tired, take exercise do not tolerate, palpitaition, shortness of breath, the symptoms such as edema;General 40 years old with Most humans exacerbation of symptoms afterwards, and often there is the performance of the arrhythmia cordis such as auricular fibrillation, auricular flutter and congestive heart failure. Operation stitching atrial septal defect effectively inhibits disease nature process, but untreated big ostium secundum defect clearly shortens existence Phase.Research report, 20 years Annual average mortality rates are lower before patients with atrial septal defect (first 10 years is 0.6%/year, second 10 Year is 0.7%/year), the 40th annual death rate increases to 4.5/, and the 60th annual death rate reaches for 7.5%/year.Therefore, early find, Early diagnosis, early treatment are the best modes for improving prognosis.
Currently, congenital atrial septal defect infant is found by cardiac auscultation mostly, rabat, ECG examination prompt are different Normal just further row echocardiography is clarified a diagnosis, but rabat and ECG examination are only when anatomical variations occurs in heart It can be seen that change.Echocardiogram is the preferred detection methods of Diagnosis of Atrial Septal Defect, can check defect, size and blood flow Dynamics changes.Two dimensional color Doppler ultrasound inspection can describe defect, size and blood flow direction, although accuracy rate Height, can not be as the method for extensive screening but costly.In addition, MRI can describe atrial septal defect and quantitatively divide Analyse hemodynamic index.Steady-state free precession imaging is the goldstandard of current measurement bulk of left ventricle and function, and phase velocity can With accurate quantification pulmonary circulation/body circulation blood flow ratio, but MRI is usually not applied to simple secondary openings or primary hole defect, unless Defect is unknown;High-resolution comparison CT can describe atrial septal defect in anatomical structure, but the electricity that the inspection generates Increase risk of cancer from radiation, thus limit the use of comparison CT, unless the spy undesirable for other Examined effect effects Determine patient.Cardiac catheterization is not used in Diagnosis of Atrial Septal Defect substantially, and Most patients row cardiac catheterization is scarce in order to block Damage.With the further investigation in the multiple stages developed to atrial septum type congenital heart disease and to excretion body microRNA (miRNA) continuous understanding, discovery excretion body miRNA participates in the occurrence and development of congenital heart disease, and plays in diagnosis Extremely important effect.Serum excretion body miRNA holds promise as the marker of atrial septum type congenital heart disease diagnosis.
Excretion body is a kind of capsule balloon-shaped structure that diameter is 30-150nm, is widely present in blood, saliva, urine, brain ridge In the biological fluids such as liquid.It is mainly derived from intracellular lysosome particle and invaginates the more vesica bodies to be formed, through the external film of more vesicas It is discharged into extracellular matrix with after cell membrane fusion.The outer membrane of excretion body is lipid bilayer structure, is inside enclosed with special egg White, mRNA and non-coding RNA etc..Non-coding RNA in excretion body is based on miRNA, and type is abundant, can influence Stem cell differentiation, orga- nogenesis, hemoposieis, tumour occurs and many physiological activities such as metabolism.
MiRNA is the single-stranded microRNA of non-coding for being widely present in the intracorporal 19-25nt of biology, by with target base Said target mrna or inhibit its translation because the complementary pairing of mRNA is degraded.Studies have shown that miRNA plays important work in heart development process With miRNA probably becomes the new way of congenital heart disease treatment and diagnosis.The non-coding of miRNA and target gene mRNA There is one-to-many relationships for sequence (3 '-UTR or 5'-UTR).MiRNA groups of tables are frequently found in congenital heart disease case Up to imbalance the phenomenon that, and these miRNA expression change and congenital heart disease generation it is closely related.And excretion body miRNA Compared with recycling miRNA, can have so that stable form is present in the body fluid such as serum, blood plasma of the mankind and is not easy to be degraded Advantage.
However, up to the present, related miRNA research is dedicated to the general orientation of congenital heart disease mostly, including between room Every type congenital heart disease, atrial septum type congenital heart disease, patent ductus arteriosus and tetralogy of Fallot, atrial septum type is first The cardiopathic specificity miRNA research of nature is very few.Therefore, the relevant miRNA of congenital heart disease is first to atrial septum type at present The cardiopathic specificity of nature is lower, increases the false positive of diagnosis, and it is congenital to cannot function as specific diagnosis atrial septum type The cardiopathic marker of property.Excretion body correlation miRNA is as molecular marker in the diagnosis of atrial septum type congenital heart disease Application report almost without, existing atrial septum type congenital heart disease diagnostic marker without for excretion body in disease The characteristics of miRNA expression variation.But stability and convenience of the excretion body miRNA in blood circulation, be it is a kind of minimally invasive and Effective atrial septum type congenital heart disease marker.
In view of this, the present invention is specifically proposed.
Summary of the invention
Primary and foremost purpose of the invention is in view of the above technical problems, to propose a kind of related to atrial septum type congenital heart disease Serum/plasma excretion body miRNA marker.
A second object of the present invention is to provide the primers of above-mentioned serum/plasma excretion body miRNA marker.
Third object of the present invention is to provide above-mentioned serum/plasma excretion body miRNA markers and its primer to prepare Application in the diagnosis of atrial septum type congenital heart disease or auxiliary diagnostic box.
Excretion body correlation miRNA molecule marker and its primer provided by the invention are first for diagnosing atrial septum type in preparation Application in the cardiopathic product of nature can be alleviated existing in the prior art relevant to atrial septum type congenital heart disease The technical issues of specific miRNA research still has some deficits.
Fourth object of the present invention is to provide the kit of the diagnosis of atrial septum type congenital heart disease or auxiliary diagnosis.With It is special to alleviate the detection existing in the prior art to atrial septum type congenital heart disease excretion body correlation miRNA molecule marker Not strong enough the technical problem of property.
The purpose of the present invention is be achieved through the following technical solutions:
The present invention provides a kind of serum/plasma excretion body miRNA molecule markers, can prepare for diagnosing or assisting Diagnose the product of atrial septum type congenital heart disease.
Excretion body miRNA molecule marker of the present invention includes increment in the type congenital heart disease excretion body of atrial septum One kind of the miRNA of expression.
The source of the excretion body includes body fluid and/or cell;The body fluid include but is not limited to serum, blood plasma, saliva, One of sputum, gastric juice or urine are a variety of.Preferably, body fluid used is serum or blood plasma.
Excretion body miRNA molecule marker of the present invention is miR-451a.
The present invention also provides the primers of the excretion body miRNA molecule marker.
The present invention provides a kind of primer of excretion body miRNA molecule marker, is SEQ ID NO.1 and SEQ ID NO.2.
The present invention also provides the excretion body miRNA molecule marker in preparation for diagnosis or auxiliary diagnosis room Application in type congenital heart disease product.
The present invention also provides the primers of the excretion body miRNA molecule marker to examine in preparation for diagnosing or assisting Application in disconnected atrial septum type congenital heart disease product.
Product of the present invention is reagent or kit.
In addition, the present invention also provides a kind of for diagnosing the kit of atrial septum type congenital heart disease, the reagent Box is for detecting excretion body miRNA molecule marker of the present invention.
The kit, the PCR upstream and downstream primer including detecting excretion body miRNA molecule marker can also include Excretion body miRNA molecule marker standard items and miRNA two-step method detection architecture.
Further, the kit, the kit are used for detection molecules marker miR-451a.
Further, the kit contains the primer of miR-451a;The upstream primer of the miR-451a has such as SEQ Nucleotide sequence shown in ID NO.1, downstream primer have the nucleotide sequence as shown in SEQ ID NO.2.
The kit can also include that PCR reacts common enzyme and reagent, such as reverse transcriptase, buffer, dNTPs, MgCl2, DEPC water and Taq enzyme etc.;Standard items and/or reference substance can also be contained.
The concentration of the standard items of the excretion body miRNA molecule marker can be 1013copy/μL。
Preferably, the standard items of the excretion body miRNA molecule marker are diluted to gradient standard items when in use.
Detection sample of the present invention for diagnosing the product of atrial septum type congenital heart disease is serum/plasma.
Beneficial effects of the present invention:
Excretion body miRNA molecule marker provided by the invention is found through experiments that with atrial septum type Congenital Heart Expression quantity is significant in the excretion body of disease, therefore excretion body correlation miRNA molecule marker provided in the present invention can be used as mark Risk of the will object for atrial septum type congenital heart disease is estimated and is detected, and has high sensitivity, and the strong advantage of accuracy is room Cabinet-type congenital heart disease diagnosis aspect provide certain application value, be atrial septum type congenital heart disease prevention and Diagnosis provides advantageous technical support, has good help to the screening and treatment of China atrial septum type congenital heart disease.Separately Outside, the present invention also provides the kits for diagnosing atrial septum type congenital heart disease, including detection excretion body miRNA molecule PCR upstream and downstream primer, excretion body miRNA molecule marker standard items and the miRNA two-step method detection architecture of marker.It is reversed Record high-efficient, primer specificity is strong, high sensitivity, detection comprehensively, quickly, it is accurate, be widely used, testing result can be used for clinic Diagnosing and treating.
Detailed description of the invention
Fig. 1 is the electrophoretogram that miRNA-451a is extracted and purified;
The amplification curve diagram of Fig. 2 miRNA-451a PCR product;
The solubility curve figure of Fig. 3 miRNA-451a primer;
The atrial septum Fig. 4 type congenital heart disease serum excretion body miR-451a detects box-shaped figure;
Fig. 5 serum excretion body miR-451a is detected to atrial septum type congenital heart disease risk evaluation result ROC curve.
Specific embodiment
The technical solution of the patent is explained in further detail With reference to embodiment.Described implementation Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill Personnel's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
Embodiment 1
One, experimental material and method
1.1 case selection
1.1.1 diagnostic criteria
The diagnosis of atrial septum type congenital heart disease is diagnosed as pure atrial septum type according to echocardiography in institute The infant of congenital heart disease.
1.2 case-data
The atrial septum type congenital heart disease for meeting the standard of being included in is collected in this research from December, -2017 in December, 2015 altogether Patient 30, all cases derive from Nanjing Children's Hospital cardiothoracic surgery, make a definite diagnosis through pathologic finding.Patient age 1-13 Year, the median age 2.5 years old.Normal group (" normal " refers to non-cardiac developmental deformity in this research) 29, the age 1-11 years old, in The position age 4 years old, both from attached Nanjing Children's Hospital children's health care section of university of Nanjing Medical University healthy premenopausal volunteers.The research side Case meets human trial ethical standard, and obtains the approval of Ethics Committee, subject it is tested it is preceding known, and obtain book Agree in face.
1.3 packet design
Atrial septum type congenital heart disease group VS Normal group, to reduce experimental error, atrial septum type Congenital Heart Disease group and Normal group respectively carry out 2 groups of technologies and repeat.
1.4 key instruments and reagent consumptive material
1.4.1 key instrument
Key instrument:
Full temperature oscillator Changzhou phenanthrene is general
Electronic balance Shanghai balance equipment factory
Pure water system Beijing epoch Electronic Instrument, Limited
Automatic dual pure water distiller Shanghai Yarong Biochemical Instrument Plant
SANYO company of ultra low temperature freezer Japan
Small desk centrifuge Sigma Co., USA
10 μ L, 20 μ L, 200 μ L, 1000 Eppendorf companies of μ L pipettor Germany
Bio-Rad company of the gel electrophoresis power supply U.S.
4 DEG C of refrigerated centrifuge Thermo Fisher Scientific companies
QuantStudio3 real-time fluorescence quantitative PCR instrument Thermo Fisher Scientific company
Biohazard Safety Equipment Haier, Qingdao Haier special type electric appliance
Standard PCR instrument A100, Hangzhou Lang Ji scientific instrument Co., Ltd
1.4.2 main agents and consumptive material
Main agents and consumptive material:
The synthesis of miRNA standard items powder Shanghai JiMa pharmacy Technology Co., Ltd
T4DNA ligase NEB (article number: M0202S includes 10 × T4DNA Ligase Buffer)
RNase inhibitor Fermentas (article number: K1622)
Transcriptase Shanghai Invitrogen Bioisystech Co., Ltd (article number: K1622, comprising RNase inhibitor, dNTPs、nuclease-free water)
T4 polynueleotide kinase NEB (article number: M0201S)
Nuclease free pure water Shanghai Invitrogen biology Co., Ltd (article number: K1622)
PCR reaction mixture ABI (article number: 4398965)
Upstream primer liquid (F primer) Shanghai JiMa pharmacy Technology Co., Ltd
General reverse primer liquid (R primer) Shanghai JiMa pharmacy Technology Co., Ltd
Uracil-DNA glycosylase NEB (article number: M0280S)
ExoRNeasy Serum/Plasma Midi Kit kit Qiagen company (article number 77044)
1.5 experimental methods and technology
1.5.1 materials
Clinical observation record is carried out according to the dedicated clinical observation table of this project.Atrial septum type children with congenital heart disease is admitted to hospital Afterwards, rear from morning to draw materials on an empty stomach.By trained seminar special messenger blood sampling, 4-5ml stands 1h under room temperature, 3,000rpm from Heart 10min collects upper serum, goes to -80 DEG C of refrigerators preservations for use after being placed in cryopreservation tube Liquid nitrogen storage.Sample is taken out when experiment This, thaws on ice, all equal 1 freeze thawing of detection blood sample.
1.5.2 serum excretion body miRNA-451a expression detects
1.5.2.1 experiment flow
(1) extraction and purifying of Sample serum excretion body miRNA-451a;(2) miRNA reverse transcription;(3) PCR amplification;(4) The configuration of miRNA two-step method molecular marker standard items;(5) calculating of miRNA-451a relative expression quantity.
1.5.2.2 the extraction and purifying of Sample serum excretion body miRNA-451a
(1) serum for suggesting only using pre- filtration, does not include the particle or Milipore Millex- of 0.8pm or more AA;
The XBP of 1 unit volume is added in the sample of (2) 1 unit volumes, gently overturns pipe 5 times to mix;
(3) mixture in (2) is poured into column spinner, is centrifuged 1 minute at 500xg.Liquid is abandoned, pillar is put back into original In collecting pipe;
(4) 3.5ml XWP buffer is added, is centrifuged 5 minutes at 5000xg, clean pillar and removes remaining buffer. Collecting pipe abandons together with liquid in pipe;
(5) column spinner is transferred in new collecting pipe;
(6) 700 μ L QIAzol are added into column, are centrifuged 5 minutes at 5000xg, collect lysate and are transferred to 2ml's In pipe;
(7) it is slightly vortexed and fills the pipe of lysate, and in (15 DEG C -25 DEG C) incubation 5min of room temperature;
(8) 3.5 μ L miRNeasy Serum/Plasma Spike-In Control (1.6x selectively actuatable: are added 108copies/ μ L working solution);
(9) 90 μ L chloroforms are added in pipe, covers tightly lid, acutely rocks 15s;
(10) (15 DEG C -25 DEG C) incubation 2-3min of room temperature;
(11) it is centrifuged 15min under 4 DEG C, 12000xg, centrifuge temperature is adjusted to 15-25 DEG C for upper strata aqueous phase after centrifugation It moves in new collecting pipe.The dehydrated alcohol of 2 times of volumes is added by 1:2, is mixed well for several times with pipettor piping and druming.Immediate operation In next step;
(12) 700 μ L samples are pipetted to RNeasy MinElute column spinner, column spinner is placed in 2ml collecting pipe.It closes the lid Son is centrifuged 15s at room temperature >=8000xg (>=10,000rpm), abandons liquid;
(13) remaining sample repeats previous step;
(14) 700 μ LRWT buffers are added in RNeasy MinElute column spinner, close the lid, in >=8000xg It is centrifuged 15s under (>=10,000rpm), abandons liquid;
(15) 500 μ L RPE buffers are added in column, close the lid, are centrifuged at >=8000xg (>=10,000rpm) 15s abandons liquid;
(16) 500 μ L RPE buffers are added in column, close the lid, are centrifuged at >=8000xg (>=10,000rpm) 2min abandons collecting pipe with liquid in pipe together;
(17) column spinner is moved in new 2ml collecting pipe, opens the lid of column spinner, be centrifuged 5min at full speed and dry film, Collecting pipe is abandoned together with liquid in pipe;
(18) column spinner is moved in new 1.5ml collecting pipe, 14 μ L RNase-free is added at column spinner center water.It gently closes the lid, stands 1min, be centrifuged 1min at full speed with eluted rna;
(19) RNA for collecting elution is used for reverse transcription.
It is the Sample serum excretion body of electrophoresis detection atrial septum type Children with Congenital Heart Disease and normal child such as Fig. 1 MiRNA-451a extract and purifying as a result, display atrial septum type congenital type heart disease infant and normal child serum excretion body In there is miRNA-451a.
1.5.2.3 miRNA-451a reverse transcription
1. in ice bath, be added in 100 μ L EP according to table 1 reaction system (in table 1 total system be 20 μ L, with specific reference to It need to be scaled addO-on therapy);
2. PCR instrument is arranged according to 1 parameter of table;
3. the lid for the EP pipe for having added system is covered, it is put into PCR instrument after centrifugation and carries out reverse transcription.
RNA reverse transcription reaction system:
The general reverse transcription reaction system component table of table 1
The general RNA reverse transcription condition of table 2
It is saved for subsequent PCR amplification for 4 DEG C after 10 times of cDNA dilutions.
1.5.2.4 PCR amplification
1. reaction system is added in 96 orifice plates according to table 3, and (total system is 20 μ L in table 3, with specific reference to need in ice bath It will addO-on therapy in proportion);
Wherein upstream primer and downstream primer sequence such as SEQ ID NO.1 and SEQ ID NO.2.
2. PCR instrument is arranged according to 4 parameter of table;
3. 96 orifice plates of system will have been added to be sealed with sealer, it is put into PCR instrument after centrifugation and is expanded.
PCR reaction system after the optimization of table 3
4 PCR thermal cycle conditions of table
As a result such as Fig. 2 and Fig. 3, the blood of based on PCR platform progress atrial septum type congenital type heart disease children and normal child The amplification curve diagram and solubility curve figure of clear excretion body miRNA-451a, as shown in Figure 2: miRNA-451a amplification curve is in " S " Type, amplification are normal;Fig. 3 is visible: miRNA-451a solubility curve is in unimodal, and the amplification of miRNA-451a is single, primer specificity Good Fig. 2 and Fig. 3 result prompts the detection method high reliablity.
1.5.2.5 the calculating of the relative expression quantity of miRNA-451a
The expression quantity Ct value of detection marker uses relative quantification formula value (2 according to Ct value-ΔΔCt), calculate miRNA phase To expression quantity.
1.5.3 the effect of the miRNA-451a two-step method detection congenital type heart disease auxiliary diagnosis detection kit of atrial septum type Fruit evaluation
1.5.3.1 experimental material
Serum sample comes from:
Normal group: normal children 29 of the same age (" normal " refers to non-cardiac developmental deformity in this research);
The congenital type heart disease group of atrial septum type: pure atrial septum type children with congenital heart disease 30.
Wherein, children of the children serum of the same age from the normal physical examination of children's health care section are normally organized;The congenital type heart disease of atrial septum type Group serum is diagnosed as the congenital type heart disease infant of pure atrial septum type in the court inpatient.
1.5.3.2 experimental method
The relative expression levels of miRNA-451a in above-mentioned serum sample are detected, specific step is as follows, such as nothing It illustrates, the same 1.5.2 of actual conditions parameter:
1. the extraction and purifying of serum excretion body miRNA-451a;
2. miRNA-451a reverse transcription;
3. PCR amplification;
4. miRNA two-step method molecular marker standard items configure;
5. the calculating of the relative expression quantity of miRNA-451a;
6. with 17.0 software of SPSS in normal children's group and pure atrial septal defect group serum excretion body of the same age MiRNA-451a relative expression levels carry out statistical analysis.
1.5.3.3 in the congenital type heart disease of atrial septum type miRNA-451a testing result and interpretation of result
PCR inspection of the miRNA-451a expression in normal group, the serum sample of the congenital type heart disease group of atrial septum type Survey result:
It is 0.52 ± 0.29 that the relative expression levels of normal group miRNA-451a, which are mean+SD, and atrial septum type is first Its type heart disease group is 1.07 ± 0.46.Statistical analysis is carried out to the relative expression levels of miRNA-451a in two groups of samples (Mann-Whitney U test), discovery serum excretion body miRNA-451a is in normal group and the congenital type heart disease of atrial septum type Group has significant difference (p < 0.001), illustrates excretion body miRNA marker and the significant phase of atrial septum type Congenital Heart disease forecasting It closes (Fig. 4).
Using normal group as control group, type congenital type heart disease group in atrial septum is disease group, to miRNA- in serum excretion body The relative expression levels of 451a carry out ROC curve analysis, AUC=0.8259, diagnostic sensitivity 70.00%, specificity 82.76%.With significant advantage (Fig. 5).
From the above results, serum excretion body miRNA-451a can be used as normal child and the congenital type heart of atrial septum type The potential marker of sufferer youngster progress Distinguishing diagnosis.The differentiation for carrying out normal child and the congenital type heart disease infant of atrial septum type is examined When disconnected, if the relative expression levels of miRNA-451a are greater than or equal to 0.76 in test serum excretion body, this it is to be measured artificial or Candidate is the congenital type cardiac of atrial septum type.
It is shown by serum excretion body miRNA marker diagnosis effect, compared with Color Sonography diagnosis effect, serum excretion Body miRNA has minimally invasive, the significant advantage of diagnosis effect.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations;To the greatest extent Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into Row equivalent replacement;And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution The range of scheme.
Sequence table
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Claims (10)

1. a kind of serum/plasma excretion body miRNA marker relevant to atrial septum type congenital heart disease, it is characterised in that should Marker is miR-451a.
2. the primer of serum/plasma excretion body miRNA marker described in claim 1.
3. primer according to claim 2, which is characterized in that the primer sequence are as follows: SEQ ID NO.1 and SEQ ID NO.2。
4. outside serum/plasma excretion body miRNA marker described in claim 1, serum/plasma described in claim 2 or 3 Secrete application of the primer of body miRNA marker in the diagnosis of preparation atrial septum type congenital heart disease or auxiliary diagnostic box.
5. a kind of atrial septum type congenital heart disease diagnosis or auxiliary diagnostic box, it is characterised in that the kit is for detecting Excretion body miR-451a in serum/plasma.
6. diagnostic kit according to claim 5, it is characterised in that the kit contains serum/plasma excretion body The primer of miR-451a in miRNA.
7. diagnostic kit according to claim 5, it is characterised in that the kit contains blood as claimed in claim 3 Clearly/blood plasma excretion body miRNA marker primer.
8. diagnostic kit described according to claim 6 or 7, it is characterised in that the kit can also include that PCR reaction is common Enzyme and reagent.
9. diagnostic kit described according to claim 6 or 7, it is characterised in that it is serum or blood that kit, which detects sample used, Slurry.
10. the described in any item kits of claim 5~7 are in the diagnosis of atrial septum type congenital heart disease or auxiliary diagnosis Using.
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