CN108004316A - For predicting the kit of acute myocardial infarction AMI risk - Google Patents
For predicting the kit of acute myocardial infarction AMI risk Download PDFInfo
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- CN108004316A CN108004316A CN201810019374.6A CN201810019374A CN108004316A CN 108004316 A CN108004316 A CN 108004316A CN 201810019374 A CN201810019374 A CN 201810019374A CN 108004316 A CN108004316 A CN 108004316A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Abstract
The present invention relates to area of medical diagnostics, and in particular to a kind of kit for being used to predict acute myocardial infarction AMI risk, it includes the detection agent of microRNA 22 5p and/or microRNA 375.The invention further relates to microRNA 22 5p and/or microRNA 375 application in being used to predict the diagnosticum of acute myocardial infarction AMI risk is being prepared as molecular marked compound.Kit provided by the present invention has AMI diagnosis and prediction efficiency well, can predict the onset risk of AMI, instruct the prevention and treatment of AMI, mitigate the threat of AMI.
Description
Technical field
The present invention relates to area of medical diagnostics, in particular to a kind of examination for being used to predict acute myocardial infarction AMI risk
Agent box.
Background technology
(1) introduction
Angiocardiopathy, also known as circulation system disease, including acute rheumatic fever, chronic rheumatic heart disease, hypertension
Property disease, ischemic heart disease, cor pulmonale, that is, pulmonary heart disease and disease of pulmonary circulation, cranial vascular disease, and other hearts
With the circulation system disease such as blood vessel.World Health Organization's statistics of 2015 points out that angiocardiopathy is the No.1 of the whole world
The cause of the death, had 17,500,000 people to die of angiocardiopathy in 2012, accounted for the 31% of global dead sum.China cardiovascular patient patient
Number is 2.9 hundred million, its incidence, the death rate are occupied first of various diseases.The risk assessment of angiocardiopathy at present is mainly blood pressure, blood
The detection of the indexs such as sugar, blood fat, and in prevention and treatment, alleviation disease is generally taken according to medical history and imageological examination result
Cause, reduce risk factor and make the life better mode the methods of reduce new heart damage and occur, early prevention can not be reached
With the effect of quick diagnosis.
Acute myocardial infarction AMI (Acute myocardial infarction, AMI) is as high incidence in worldwide
It is always the research hotspot of angiocardiopathy one of with the angiocardiopathy of the death rate.AMI is that coronary artery is acute, continuation
Myocardial necrosis caused by hypoxic-ischemic.It is clinically to have violent and lasting retrosternal pain, rest and nitrate esters medicine more
Complete incidence graph is unable to, is increased with serum enzyme activities and progressive ECG Change, can complicated by arrhythmia, shock or the heart
Force failure, or even threat to life.In China, incidence is in obvious ascendant trend to AMI in recent years, is newly sent to few 500,000 every year, now suffers from
At least 2,000,000.AMI early, is fast and accurately diagnosed, is the effective behave for reducing the death rate.
The sensitivity and specificity of existing iconography and biomarker inspection result have certain limitation, it is impossible to reach
The effect of early prevention and quick diagnosis.Therefore, prevention and quick diagnosis acute myocardial infarction AMI are so as to successive treatment, it has also become doctor
Learn one of significant task with biological study.
(2)MicroRNAs
MicroRNAs (miRNAs) is the raw, tiny RNA of about 20-24 nucleotide of length, these generally existings in one kind
Small molecule eukaryotic gene expression regulation and control in have extensive effect.In nematode, drosophila, has sent out in the species such as mouse and people
Existing hundreds of miRNAs, there were significant differences for the expression in different tissues, different developmental phases.Several miRNAs also may be used
To adjust same gene, or can be by the combination of several miRNAs come the expression of some gene of finely regulating.MiRNAs tables
Expression patterns have the characteristics such as position phasic property and the timing of differentiation, prompt its molecule possible as controlling gene expression is participated in,
Thus have important practical significance to medical diagnosis on disease.
In nucleus, miRNAs is transcribed by DNA.The precursor (pre-miRNA) of miRNAs is by RNase III digestions
Formed after cutting.Then it is transported in cytoplasm, the ripe miRNA for being further cracked into 19 to 23 length of nucleotides is double
Chain nucleotide.Wherein a chain is degraded or is discharged from cell, and another chain is loaded on RNA induction silencing complexes
(RNA-RISC), target mRNA can be prevented to be converted into protein.
It is complementary with target gene transcription sequence that effects of the MicroRNA-RISC to target gene mRNA depends primarily upon it always
Degree, mainly have three kinds of modes.The first is turned off the mRNA molecules of target gene --- miRNA and target gene complete complementary knot
Close, the mode of action and function are closely similar with siRNA, finally cut said target mrna.Second is the translation for suppressing target gene ---
During effect with the not fully complementary combination of target gene, and then check translation without influence mRNA stability, this miRNA is current
It was found that most species (such as nematode lin-4).The third is to combine to suppress --- there is both the above binding mode：When with target base
When being combined because of complementation, cutting mRNA is directly targeted；When being not fully integrated with target gene, play a part of to adjust gene expression.
MiRNAs plays a great role during cell differentiation, biological development and disease development, has caused and has ground
Study carefully the extensive concern of personnel.As the further further investigation for the miRNAs mechanisms of action, and utilization are newest for example
The high-throughout technological means such as miRNAs chips is studied for the relation between miRNA and disease, and people are for high true
The network of core biological gene expression regulation, which understands, to reach a new high.This will also make miRNA be likely to become disease
The new biological marker of diagnosis, it is also possible to so that this molecule becomes medicine target, or simulates this molecule and carries out new drug development,
Treatment to human diseases provides a kind of new means.
(3) advantages of the miRNAs biomarker
Biomarker, for establishing the diagnosis of AMI patient, is initially started in the orientation analysis of myocardium protein, into
For the more and more important index of AMI diagnosis.Creatine kinase isozyme (CK-MB) and troponin (T or I) rise are that diagnosis is anxious
The important indicator of property myocardial infarction, and it is widely used to clinical diagnosis.Wherein, cardiac troponin (cTnI) is to generally acknowledge at present
" goldstandard " AMI diagnosis.However, the requirement with the Sensitivity and Specificity of early diagnosis AMI is continuously improved, new life
The exploration of thing marker also never stops.
Preferable biomarker has following characteristic：It can be obtained by the method for non-damage；Diagnostic result has
Higher sensitivity and specificity；It can be detected early stage disease, and specific change spy is presented with the development of disease
Sign, can reflect the physiopathologic specific variations of histoorgan；With fast and convenient detection technique.Compared to traditional AMI
Examination criteria, miRNAs meets all of the above feature, and specific inspection can be carried out by gene order specific amplification technology
Survey, than protein biomarker in terms of medical diagnosis on disease it is more effective.
In recent years, the morbidity and mortality of AMI remain high, and the people in every country and area are enjoyed it to disturb.
" it is early find, early diagnosis, early treatment " survival rate and cure rate of patient can be effectively improved so that mitigate patient spirit and
Financial burden.Therefore, low cost, timely and effectively AMI early diagnose means still have it is to be developed.MiRNAs is highly stable to be present in
In blood circulation, it is verified that playing an important role in being adjusted after the transcription and transcription of gene expression.And some researches show that,
MiRNAs is also detected in the urine of animal.The research of urinary biomarkers thing, will assign the mankind in disease prevention, examine
The possibility that all many-sided realizations such as disconnected, treatment and Prognosis scoveillance more improve.
MiRNAs can be as the biology in blood and urine to be shown to AMI specificity miRNAs results of study in blood
Marker, early diagnosis and Rapid identification for AMI, may be than traditional since it more early reaches the characteristic of expression peak value
Standard of perfection cTnI is more sensitive.However, existing study the population analysis for being based on small sample, and known miRNAs quantity is very
It is few, the correlation of miRNAs and AMI clinical characteristcs and potential prognostic value can not be assessed, its diagnostic value is needed into one
Walk larger range of screening and identification research.
(4) AMI specificity miRNAs biomarkers research has important theory and practice meaning
Detection miRNAs has certain clinical value, its biomarker that can be early diagnosed as AMI.Close at present
The research for occurring the biomarker of early stage as AMI in peripheral blood in circulation miRNAs it is less and current it is not yet found that
MiRNAs can be as the relevant report of biomarker in the AMI Urine in Patients samples of pass.It was found that more miRNAs, filter out
Specific expressed index in blood and urine specimen, sensitivity and specificity to improving diagnosis AMI, has important theory
And realistic meaning.
In view of this, it is special to propose the present invention.
The content of the invention
It is an object of the invention to find and acute myocardial infarction AMI (Acute myocardial infarction, AMI) phase
The blood plasma new bio marker of pass, for the early diagnosis and early warning of AMI patient, instructs the prevention and diagnosis of AMI, mitigates
The threat of AMI.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
Prepared the present invention relates to microRNA-22-5p and/or microRNA-375 as molecular marked compound for predicting
Application in the kit of acute myocardial infarction AMI risk.
Wherein, the Acession number of microRNA-22-5p and microRNA-375 are respectively MIMAT0004495
And MIMAT0000728, its nucleotide sequence is respectively such as SEQ ID NO:5 and SEQ ID NO:Shown in 6.
Present invention applicant by the blood plasma microRNA-22-5p to 32 AMI patients and 20 normal healthy controls,
The expression of microRNA-375 is compared, and studies the correlation of microRNA-22-5p, microRNA-375 and AMI,
Find the susceptible biological people's group marks of believable AMI.Step gathers AMI patient and healthy population using EDTA-Na anticoagulant tubes
Upper plasma is extracted after the centrifugation ten minutes of 5ML, 3000 × g, 25 DEG C of peripheral blood, blood plasma RNA is extracted using TRIZOL methods, is then adopted
With real time fluorescence quantifying PCR method expand microRNA-22-5p, microRNA-375 and outer ginseng cel-miR-39
Increase；Expressed in the normalization of microRNA-22-5p, microRNA-375 with respect to cel-miR-39 for calculating every sample respectively
Level, as a result tests by SPSS 16.0, with P ＜ 0.05 for significance test standard, finds in AMI patient and health
There are significant difference for expression in crowd.
Be used to predicting the kit of acute myocardial infarction AMI the invention further relates to a kind of, including microRNA-22-5p and/or
The detection agent of microRNA-375.
Compared with prior art, kit provided by the present invention has AMI diagnosis and prediction efficiency well, can be pre-
The onset risk of AMI is surveyed, the prevention and treatment of AMI is instructed, mitigates the threat of AMI.
Brief description of the drawings
, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution of the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in describing below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
Put, other attached drawings can also be obtained according to these attached drawings.
Fig. 1 be AMI patient and healthy population blood plasma in microRNA-22-5p expressions (i.e. with cel-miR-39 phases
Normalization than microRNA-22-5p is horizontal)；
Fig. 2 be AMI patient and healthy population blood plasma in microRNA-375 expressions (i.e. with cel-miR-39 phases
Normalization than microRNA-22-5p is horizontal)；
Wherein *：P ＜ 0.05, that is, have notable significant difference；
Fig. 3 for microRNA-136-3p and microRNA-4791 in AMI patient and healthy population blood plasma individually and the two
The performance curve (receiver operatingcharacteristic curve, abbreviation ROC curve) of conjunctive use, should
ROC curve, area under the curve (area under the curve, AUC), sensitivity (sensitivity), spy are shown in figure
Different in nature (specificity), wherein AUC reflects prediction efficiency, and (AUC=0.5, is not previously predicted efficiency；0.5 ＜ AUC ＜ 0.7 are very
Small predictive value；0.7 ＜ AUC ＜, 0.9 fairly accurate predictive values；0.9 ＜ AUC ＜ 1, very accurate predictive value).
Embodiment
Prepared the present invention relates to microRNA-22-5p and/or microRNA-375 as molecular marked compound for predicting
Application in the kit of acute myocardial infarction AMI risk.
Specifically, the detection agent the present invention relates to microRNA-22-5p and/or microRNA-375 is being prepared for pre-
Survey the application in the kit of acute myocardial infarction AMI risk.
The detection agent be used for by measure the given the test agent from subject in microRNA-22-5p and/or
The expression of microRNA-375 diagnoses whether the subject has acute myocardial infarction AMI risk；Wherein relative to health
Corresponding microRNA-22-5p and/or microRNA-375 expressions in human sample can raise, to indicate that subject has
Acute myocardial infarction AMI risk size.
The invention further relates to a kind of kit for being used to predict acute myocardial infarction AMI risk, including microRNA-22-5p
And/or the detection agent of microRNA-375.
Preferably, application or kit, the detection agent include one kind or more in following nucleotide sequences as described above
Kind：
A carrying for), can hybridizing with the microRNA-22-5p and/or microRNA-375 shows signal strength
The probe A of indicator；
B), the cDNA that can be obtained to microRNA-22-5p the and/or microRNA-375 reverse transcriptions is expanded
Primer pair；
C) with B) described in cDNA combined with display signal strength indicator probe B.
Specifically, the corresponding detection method of detection agent can be, in situ hybridization (particularly fluorescence in situ hybridization, FISH),
Northern Blot, qRT-PCR (using Taqman probes or fluorescent dye) and other can be used for RNA quantitative analyses
Method.
Preferably, application as described above or kit, showing the indicator of signal strength includes fluorescent material, biology
Any of element, radio isotope, digoxin.
Preferably, as described above application or kit, the fluorescent material include Alexa 350, Alexa 405,
Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminacrine, BODIPY 630/650, BODIPY
650/665th, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5- carboxyl -4 ', 5 '-two chloro- 2 ', 7 '-two
Methoxyl group fluorescein, 5- carboxyls -2 ', 4 ', 5 ', 7 '-tetrachlorofluorescein, 5-carboxyfluorescein, 5- carboxyrhodamines, 6- carboxyl sieve
Red bright, 6- carboxyls tetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7,6-FAM, dansyl Cl, fluorescein,
HEX, 6-JOE, NBD (7- nitro benzo -2- oxa-s -1,3- diazole), Oregon Green 488, Oregon Green 500,
The solid purple, cresyl blue of Oregon Green514, Pacific Blue, phthalic acid, terephthalic acid (TPA), M-phthalic acid, cresols
Purple, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluoresceins, rare earth gold
Belong to cryptate, three pairs of pyridine radicals diamines europiums, europium cryptate or chelate, diamines, dicyanin, the blue dyes of La Jolla
Material, allophycocyanin, allococyanin B, phycocyanin C, phycocyanin R, thiamines, algae red green white, phycoerythrin R,
REG, rhodamine are green, rhodamine isothiocyanates, rhodamine are red, ROX, TAMRA, TET, TRIT (different sulphur of tetramethylrhodamine
Alcohol), the one or more in tetramethylrhodamine and texas Red；
Preferably, the radio isotope includes110In、111In、177Lu、18F、52Fe、62Cu、64Cu、67Cu、67Ga、68Ga、86Y、90Y、89Zr、94mTc、94Tc、99mTc、120I、123I、124I、125I、131I、154-158Gd、32P、11C、13N、15O、186Re
、188Re、51Mn、52mMn、55Co、72As、75Br、76Br、82MRb and83One or more in Sr.
Preferably, application as described above or kit, in B) in, the microRNA-22-5p reverse transcriptions can be obtained
To the nucleotide sequences of the upstream and downstream primer of primer pair that are expanded of cDNA respectively such as SEQ ID NO:1 and SEQ ID NO:
Shown in 2；
The upstream and downstream primer for the primer pair that the cDNA that can be obtained to the microRNA-375 reverse transcriptions is expanded
Nucleotide sequence is respectively such as SEQ ID NO:3 and SEQ ID NO:Shown in 2.
Preferably, kit as described above, the kit is further included to be obtained for expanding outer ginseng microRNA reverse transcriptions
The primer pair of the cDNA arrived.
Preferably, kit as described above, the outer ginseng microRNA is cel-miR-39.
Preferably, kit as described above, the outer ginseng microRNA are the cDNA that cel-miR-39 reverse transcriptions obtain
Primer pair upstream and downstream primer nucleotide sequence respectively such as SEQ ID NO:4 and SEQ ID NO:Shown in 2.
Preferably, kit as described above, the kit further include RNA extracts reagents, reverse transcriptase, fluorescent quantitation
PCR reaction buffers, for carrying out quantitative indicator, sample treatment liquid, seedless sour water, archaeal dna polymerase, the moon to amplified production
Property control and positive control in one or more；
Preferably, the archaeal dna polymerase be selected from Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne,
Any in Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase, Klenow fragments
Kind；
Preferably, it is described amplified production is carried out quantitative indicator be selected from SYBR Green I, EvaGreen,
Any of PicoGreen, Peko Green, propidium iodide, calcein or hydroxynaphthol blue.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer, is
Can be with conventional products that are commercially available.
Embodiment
First, material and sample, which are collected, describes
AMI patient adds up to 32.The plasma specimen of normal population adds up to 19.The diagnosis of AMI is by generally acknowledging at present
The testing result of " goldstandard " --- cardiac troponin (cTnI) confirms.Each sample is added up to EDTA-Na anticoagulant tubes
Collect 5ml blood.
2nd, sample treatment and RNA extractions
After intravenous blood collection in 10min, after being centrifuged 10 minutes at 3000 × g, 25 DEG C, with no RNase and without thin
The suction nozzle of bacterium takes upper plasma to be saved backup for -80 DEG C in no RNase and abacterial EP pipes.RNA in blood plasma passes through
TRIZOL methods are extracted and purified.
3rd, real-time fluorescence quantitative PCR (SYBR dye methods)
With microRNA real-time fluorescence quantitative PCRs kit (the SYBR Premix of the precious biological Co., Ltd of DaLian, China
Ex Taq II, article No.：RR820A) microRNA-22-5p, microRNA-375 and outer ginseng cel-miR-39 are expanded,
Ct values (cycle threshold) are respectively obtained, pass through formula：(wherein：△ △ Ct=△ Ct (test specimen)-△
Ct (authentic specimen) △ Ct (test specimen)=Ct (test specimen, target gene)-Ct (test specimen, reference gene) △ Ct
(authentic specimen)=Ct (authentic specimen, target gene)-Ct (authentic specimen, reference gene)).Every sample is being calculated respectively
MicroRNA-22-5p, microRNA-375 expression, specific operation process is shown in specification.Each sample is in triplicate
Experiment.MicroRNA-22-5p, microRNA-375 and outer ginseng cel-miR-39 primer sequences are shown in Table 3.
4th, statistical analysis technique.
Counted with statistics software SPSS 16.0.Test of normality uses Shapiro-Wilk methods, and conspicuousness is poor
It is different to be examined (Mann-Whitney Test) with Mann-Whitney, performance curve (receiver operating
Characteristic curve, abbreviation ROC curve) and line under area (area under the curve, AUC) be used for evaluate
The single prediction efficiency of microRNA-22-5p, microRNA-375.Think there is significant difference as p ＜ 0.05.
Returned with binary logistic, establish the dependent equation of microRNA-22-5p, microRNA-375, obtain pre-
Survey probability P, and using prediction probability P for test variable carry out ROC curve analysis, then evaluate microRNA-22-5p with
The prediction efficiency of microRNA-375 conjunctive uses.Think there is significant difference as p ＜ 0.05.
5th, interpretation of result
1.AMI patient compares with the expression quantity of healthy population blood plasma microRNA-22-5p, p ＜ 0.05, there is extremely notable system
Meter learns difference.
2.AMI patient compares with the expression quantity of healthy population blood plasma microRNA-375, p ＜ 0.05, there is extremely notable statistics
Learn difference.
3. blood plasma microRNA-22-5p is to the prediction efficiency of AMI by ROC curve it is recognised that microRNA-22-5p
Prediction efficiency to AMI is good, and AUC is 0.847 (95%CI, 0.723-0.930).
4. blood plasma microRNA-375 is to the prediction efficiency of AMI by ROC curve it is recognised that microRNA-375 pairs
The prediction efficiency of AMI is good, and AUC is 0.788 (95%CI, 0.657-0.887).
5. conjunctive use blood plasma microRNA-22-5p and microRNA-375 passes through ROC curve to the diagnostic of AMI
It is recognised that conjunctive use blood plasma microRNA-136-3p and microRNA-4791 is good to the prediction efficiency of AMI, it is excellent
Individually used in it, AUC is 0.854 (95%CI, 0.732-0.935).
6th, conclusion
MicroRNA-22-5p and microRNA-375 is good to the prediction efficiency of AMI in blood plasma, can predict AMI
Onset risk, and the prediction efficiency of the conjunctive use of the two is better than individually using.
The expression of table 1AMI patient and microRNA in healthy population blood plasma
* represent AMI patient and organize VS. healthy populations：P<0.05.
The essential information of 2 microRNA of table
3 miR-22-5p, miR-375, cel-miR-39 primer information of table
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 is described in detail the present invention with reference to foregoing embodiments, but it will be understood by those of ordinary skill in the art that：Its
It can still modify to the technical solution described in foregoing embodiments, either to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
SEQUENCE LISTING
<110>University Of Qingdao
<120>For predicting the kit of acute myocardial infarction AMI risk
<160> 6
<170> PatentIn version 3.3
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<213>Artificial sequence
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tggtgtcgtg gagtcg 16
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<213>Artificial sequence
<400> 3
tttgttcgtt cggctcgcgt ga 22
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<213>Artificial sequence
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tcaccgggtg taaatcagct tg 22
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<213> Homo sapiens
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aguucuucag uggcaagcuu ua 22
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Claims (10)
1.microRNA-22-5p and/or microRNA-375 is being prepared for predicting acute myocardial infarction AMI as molecular marked compound
Application in the diagnosticum of risk.
2. a kind of be used to predict the kit of acute myocardial infarction AMI risk, it is characterised in that including microRNA-22-5p and/or
The detection agent of microRNA-375.
3. kit according to claim 2, it is characterised in that the detection agent includes one kind in following nucleotide sequences
It is or a variety of：
A), the instruction with display signal strength that can hybridize with the microRNA-22-5p and/or microRNA-375
The probe A of agent；
What B), the cDNA that can be obtained to microRNA-22-5p the and/or microRNA-375 reverse transcriptions was expanded draws
Thing pair；
C) with B) described in cDNA combined with display signal strength indicator probe B.
4. kit according to claim 3, it is characterised in that show signal strength indicator include fluorescent material,
Any of biotin, radio isotope, digoxin.
5. kit according to claim 4, it is characterised in that the fluorescent material includes Alexa 350, Alexa
405th, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, aminacrine, BODIPY 630/650,
BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, 5- carboxyl -4 ', 5 '-two is chloro-
2 ', 7 '-dimethoxyfluorescein, 5- carboxyls -2 ', 4 ', 5 ', 7 '-tetrachlorofluorescein, 5-carboxyfluorescein, 5- carboxyrhodamines,
6- carboxyrhodamines, 6- carboxyls tetramethylrhodamine, Cascade Blue, Cy2, Cy3, Cy5, Cy7,6-FAM, dansyl Cl,
Fluorescein, HEX, 6-JOE, NBD, Oregon Green 488, Oregon Green 500, Oregon Green514,
Pacific Blue, phthalic acid, terephthalic acid (TPA), M-phthalic acid, cresols consolidate purple, cresols royal purple, brilliant cresyl blue, to ammonia
Yl benzoic acid, erythrosine, phthalocyanine, azomethine, cyanine, xanthine, succinylfluoresceins, rare earth metal cryptate, three
Double pyridine radicals diamines europium, europium cryptate or chelates, diamines, dicyanin, La Jolla indigo plants dyestuff, allophycocyanin,
Allococyanin B, phycocyanin C, phycocyanin R, thiamines, algae red green white, phycoerythrin R, REG, rhodamine are green, sieve
Red bright isothiocyanates, rhodamine are red, ROX, TAMRA, TET, TRIT, tetramethylrhodamine and one kind in texas Red or
It is a variety of；
Preferably, the radio isotope includes110In、111In、177Lu、18F、52Fe、62Cu、64Cu、67Cu、67Ga、68Ga、86Y、90Y、89Zr、94mTc、94Tc、99mTc、120I、123I、124I、125I、131I、154-158Gd、32P、11C、13N、15O、186Re、188Re
、51Mn、52mMn、55Co、72As、75Br、76Br、82MRb and83One or more in Sr.
6. kit according to claim 3, it is characterised in that in B) in, can be anti-to the microRNA-22-5p
The nucleotide sequence of the upstream and downstream primer for the primer pair that obtained cDNA is expanded is transcribed respectively such as SEQ ID NO:1 and SEQ
ID NO:Shown in 2；
The nucleosides of the upstream and downstream primer for the primer pair that the cDNA that can be obtained to the microRNA-375 reverse transcriptions is expanded
Acid sequence is respectively such as SEQ ID NO:3 and SEQ ID NO:Shown in 2.
7. kit according to claim 6, it is characterised in that the kit is further included for expanding outer ginseng
The primer pair for the cDNA that microRNA reverse transcriptions obtain.
8. kit according to claim 7, it is characterised in that the outer ginseng microRNA is cel-miR-39.
9. kit according to claim 8, it is characterised in that the outer ginseng microRNA is cel-miR-39 reverse transcriptions
The nucleotide sequence of the upstream and downstream primer of the primer pair of obtained cDNA is respectively such as SEQ ID NO:4 and SEQ ID NO:Shown in 2.
10. according to claim 6-9 any one of them kits, it is characterised in that the kit further includes RNA extraction examinations
Agent, reverse transcriptase, quantitative fluorescent PCR reaction buffer, for amplified production is carried out quantitative indicator, sample treatment liquid,
One or more in seedless sour water, archaeal dna polymerase, negative control and positive control；
Preferably, the archaeal dna polymerase be selected from Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma,
Any of Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4DNA polymerase, Klenow fragments；
Preferably, it is described amplified production is carried out quantitative indicator be selected from SYBR Green I, EvaGreen, PicoGreen,
Any of Peko Green, propidium iodide, calcein or hydroxynaphthol blue.
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| US11718880B2 (en) | 2018-07-03 | 2023-08-08 | Beijing Medintell Biomed Co., Ltd | Marker and diagnosis method for noninvasive diagnosis of myocardial infarction |
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