CN107904300B - mRNA capable of predicting severe ischemia risk of human coronary atherosclerotic heart disease and application thereof - Google Patents
mRNA capable of predicting severe ischemia risk of human coronary atherosclerotic heart disease and application thereof Download PDFInfo
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Abstract
mRNA capable of predicting severe ischemia risk of human coronary atherosclerotic heart disease and application thereof. The sequence of the biomarker is shown in SEQ ID NO. 1. The expression level of the mRNA can be used as a target point and a basis for treating the serious ischemic heart disease caused by the human coronary atherosclerosis, and can also be used for screening treatment medicines for the disease and evaluating the effect; the primer can be used for preparing a quantitative PCR kit for clinical auxiliary diagnosis of the diseases.
Description
Technical Field
The invention belongs to the field of molecular biological diagnosis of coronary heart disease, and relates to biomarkers related to assessment, prediction and diagnosis of severe ischemia risk of coronary atherosclerotic heart disease and clinical application thereof.
Background
Cardiovascular diseases are the first killers threatening human health on a global scale, and death caused by the cardiovascular diseases exceeds tumors, and the death causes caused by various diseases jump the top. In China, the death caused by cardiovascular diseases accounts for about 25% of the total death causes, and the proportion tends to rise year by year, thereby bringing huge economic and social burden to China. Coronary atherosclerotic heart disease (CAD), short Coronary heart disease, is one of the most common heart diseases; mainly caused by the fact that the coronary artery has atherosclerotic lesions to cause the stenosis or obstruction of the blood vessel cavity, and the ischemia and anoxia and even necrosis of the myocardial cells. Recent studies have shown that chronic inflammatory responses mediated by immune cells in the circulating blood, such as monocytes, lymphocytes, are the basis for atherosclerosis and cardiovascular disease; along with the inflammatory reaction, the immune cells such as monocytes and lymphocytes in the blood regulate the gene expression level thereof, and participate in the process of the generation and development of atherosclerosis. Therefore, the search for markers of immune cell gene expression in blood as effective diagnostic and intervention targets would bring hope for the treatment of the disease.
At present, clinically, if coronary artery single-branch or multi-branch vessel stenosis is found to be more than 70% by cardiac coronary angiography, the myocardium is considered to have serious ischemia risk, and a stent needs to be implanted to rebuild blood circulation. In view of the high morbidity and mortality of CAD, early diagnosis, judgment of ischemic risk, prediction of myocardial necrosis risk are very important, and especially effective markers that can be used for early diagnosis and risk stratification of CAD will have important value. There are many methods for diagnosing coronary heart disease, such as imaging Emission Computed Tomography (ECT). ECT is a method of examination using a radionuclide, and although it has advantages such as high sensitivity, it is expensive; the conventional methods such as electrocardiogram and ultrasonic examination are not high in sensitivity and specificity; the current gold standard for CAD diagnosis is coronary angiography, but it is costly and traumatic. In laboratory diagnosis, although various blood biochemical markers such as troponin, myosin, creatine kinase isozyme and the like have been used for diagnosis of CAD, clinical specificity and detection sensitivity of these biochemical markers are low since various cells in the whole body can synthesize and secrete these substances into blood. Particularly, in the aspect of judging ischemia risk, when or what degree of ischemia of a coronary heart disease patient needs to be reestablished, methods such as invasive coronary angiography and a blood flow reserve evaluation technology (such as FFR) are needed for determining. However, currently, there is no reliable hematological diagnostic marker for coronary atherosclerotic ischemic heart disease. Therefore, a simple and noninvasive method for evaluating the ischemia degree of patients with coronary heart disease, judging the ischemia risk, predicting myocardial ischemia and even necrosis, evaluating the curative effect of the medicine and making a hematologic marker with high prediction specificity for long-term prognosis is urgently needed in clinic.
Disclosure of Invention
The technical problem to be solved is as follows: the invention provides mRNA capable of predicting severe ischemia risk of human coronary atherosclerotic heart disease and application thereof.
The technical scheme is as follows: an mRNA which can predict the serious ischemia risk of human coronary atherosclerotic heart disease and has a sequence shown in SEQ ID NO. 1.
The mRNA is applied to the preparation of a kit for predicting or diagnosing the severe ischemia risk of human coronary atherosclerotic heart disease.
The kit contains a sequence shown by SEQ ID NO. 1.
The kit contains a primer pair of a sequence shown by SEQ ID NO.1, wherein the primer pair is as follows:
5’-CCAGCAAGCCCACTGCACCC-3’；
5’-GGAAGTCCTCTGACTCTTGG-3’。
the application of the mRNA in screening drugs for improving myocardial blood supply aiming at human coronary atherosclerotic heart disease.
A kit for predicting or diagnosing the serious ischemic risk of human coronary atherosclerotic heart disease contains mRNA shown in SEQ ID NO. 1.
Has the advantages that: the invention provides an mRNA biomarker related to coronary atherosclerosis, which can be used as a target for treating severe ischemic heart disease caused by human coronary atherosclerosis and can also be used for evaluating the curative effect of a treatment medicament for the disease; the detection primer can be prepared into a kit and used for clinical auxiliary diagnosis of the disease.
Drawings
FIG. 1: an amplification curve chart of the biomarker shown in SEQ ID NO. 1;
FIG. 2: GAPDH amplification profile;
FIG. 3: a melting curve chart of the biomarker shown as SEQ ID NO. 1;
FIG. 4: GAPDH melting profile;
FIG. 5: gel electrophoresis picture of PCR product of the biomarker shown in SEQ ID NO. 1;
FIG. 6: the relative expression level of the biomarkers shown in SEQ ID NO.1 of patients (positive group) without coronary atherosclerosis or coronary artery stenosis degree less than 70% and patients (negative group) without severe ischemia indication with the coronary artery stenosis degree more than or equal to 70% confirmed by coronary angiography is detected.
Detailed Description
The invention is further illustrated by the following examples. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a process are given, but the scope of the present invention is not limited to the following embodiments. Conditions, methods and the like not shown in the following examples were carried out according to conventional or manufacturer-recommended conditions.
Example 1
The kit comprises the following components:
1. human peripheral blood lymphocyte separation (Tianjin sea object, cat # LST1077)
2. Primers for the biomarkers shown in SEQ ID NO.1, specific primers were designed using Primer5 software and synthesized by Invitrogen (Shanghai) Inc. (http:// www.thermofisher.com).
3. RNA extraction kit InvitrogenTMTRIzol Reagent (U.S. Invitrogen corporation, cat # 15596018)
4. Reverse transcription reagent PrimeScriptTMRT reagent Kit with gDNA Eraser (TaKARA, Dalianbao bio, cat # R047)
5. FastStart Universal SYBR Green Master (American Roche Co., Ltd., cat # 04913914001)
6. Agarose (Spain BIOWEST Co., Cat number: 111860)
7. Nucleic acid dye Gel-Red (Biyuntian, goods number D0139)
8. 6 XDNA sample buffer (Biyuntian, good number D0071)
The experimental steps are as follows:
1. patients with chest discomfort were confirmed by coronary angiography and divided into two groups: 1) coronary atherosclerosis leads to coronary stenosis of more than or equal to 70%, i.e. patients (positive group) with the indication of myocardial revascularization by stent placement; 2) patients with coronary atherosclerotic plaques or atherosclerotic plaques with a stenosis degree of < 70% (negative group). Taking 10mL of peripheral blood of a patient, and extracting Peripheral Blood Mononuclear Cells (PBMC) according to the separation step of the separation liquid of the peripheral blood lymphocytes of the Tianjin tall ocean organism;
2. according to InvitrogenTMExtracting PBMC total RNA from TRIzol Reagent operating instructions;
3. detecting the ratio of A260 to A280, and analyzing the purity of the total RNA;
4. according to TAKARA reverse transcription kit instructions, the first step of removing genomic DNA reaction;
incubate at 42 ℃ for 2 minutes (or room temperature for 5 minutes) for use.
5. Carrying out reverse transcription to form cDNA according to the instruction of a TAKARA reverse transcription kit, and adding the following components into an EP tube;
incubate PCR instrument for 15 min at 37 ℃. Heating at 85 deg.C for 5 min, and standing at 4 deg.C.
6. Real-time fluorescent quantitative PCR: by using Roche
Amplification was performed using a 480II PCR instrument with internal reference GAPDH, sequence 5'-GGATTTGGTCGTATTGGG-3' (forward), 5'-GGAAGATGGTGATGGGATT-3' (reverse).
Preparation of qPCR reaction system
qPCR reaction program set-up
7. Calculating the relative expression Rate
CtA is set as the Ct value of the biomarker shown in the sample SEQ ID NO.1, CtB is the Ct value of the internal reference GAPDH of the sample, and the delta Ct value is CtA-CtB. The average value M.DELTA.Ct of the negative group.DELTA.Ct was taken (negative group). According to 2-ΔΔC tThe relative expression of the target genes of the two groups of samples is calculated by the formula: when Δ Δ Ct is Δ Ct (positive group) -M Δ Ct (negative group) ═ X, the expression level of the biomarker shown in SEQ ID No.1 in the positive group sample is 2 in the negative group-XAnd (4) doubling. The mean was taken and the statistical significance was assessed by unpaired t-test.
8. Detection of product specificity by DNA agarose gel electrophoresis
1) Installing an electrophoresis tank: the gel bed of the BioRad electrophoresis apparatus was cleaned, air dried, the openings at both ends were sealed with tape, placed on a horizontal table, and a sample comb was inserted.
2) Preparation of agarose gel: agarose was weighed and dissolved in the electrophoresis buffer, 1% gel (1g agarose in 100mL distilled water) was added, the mixture was heated in a microwave oven or a boiling water bath until completely melted (not heated to boiling), 5. mu.L gel-red was added, and the mixture was shaken well.
3) Glue pouring: the agarose solution cooled to 60 ℃ was gently poured onto a horizontal plate of an electrophoresis tank.
4) After the agarose gel is solidified, adding an electrophoresis buffer solution into the electrophoresis tank, and then pulling out the comb.
5) Sample adding: and (3) uniformly mixing the DNA sample and the sample adding buffer solution according to the volume ratio of 5:1, adding the mixed solution into sample tanks by using a micropipette, adding 10-20 mu L of the mixed solution into each tank, and recording the sample application sequence and the sample adding amount of the sample.
6) Electrophoresis: installing electrode wire, connecting one end of sample application hole with cathode and the other end with anode, turning on power supply, adjusting voltage to 3-5V/cm, performing electrophoresis for 1-3hr, and stopping electrophoresis when bromophenol blue moves to 1-2cm from the front edge of gel.
7) And (4) observation: the gel was removed and observed under a 300nm UV lamp.
The results show that
1. The ratio of extracted RNA A260 to extracted RNA A280 is about 2.0, and the extraction purity meets the experimental requirements.
2. Real-time fluorescence quantitative PCR amplification is carried out on the biomarker shown in SEQ ID NO.1 and a GAPDH curve. FIG. 1 is an amplification curve of the biomarker shown in SEQ ID NO.1, and FIG. 2 is an amplification curve of GAPDH, which is a smooth amplification curve.
3. And (3) detecting the specificity of the amplification product: FIG. 3 is a melting curve of the biomarker shown in SEQ ID NO.1, and FIG. 4 is a GAPDH melting curve, both of which are single peaks. FIG. 5 is a gel electrophoresis diagram of PCR products of the biomarkers shown in SEQ ID NO.1, which shows a single band and corresponds to the expected size (159bp), and M represents a DNA marker. The results prove that the PCR kit of the biomarker shown in SEQ ID NO.1 has good specificity.
4. The application of the kit comprises: the relative expression quantity of the biomarker shown in SEQ ID No.1 of a patient with severe ischemic heart disease caused by coronary atherosclerosis in peripheral mononuclear leukocytes is detected, and the biomarker shown in the SEQ ID No.1 of the positive group is higher than that of the negative group, so that the biomarker can be used for predicting and assisting diagnosis of myocardial ischemia of a CAD patient (figure 6).
Sequence listing
<110> Nanjing university of medical science
<120> mRNA capable of predicting severe ischemia risk of human coronary atherosclerotic heart disease and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7852
<212> DNA
<213> myosin light chain kinase transcription variants (Homo sapiens)
<400> 1
ggcgctgagc gagctcggag cccgcgctgt gcgcctgcgg ccggggcgcc ccgccgagcg 60
ccggtgcccc ggctcccggg ccgccttcgc cgcgcgggaa ggattcttca aaattaacag 120
aaaccaattc gggccagctg aagagaaaaa ataaaggtgg ctcccggctg cctctgctgc 180
agttcagagc aacttcagga gcttcccagc cgagagcttc aggacgcctt tcctgtccca 240
ctggcccagt tgccacaaca aacaacagag aagacggtga ccatggggga tgtgaagctg 300
gttgcctcgt cacacatttc caaaacctcc ctcagtgtgg atccctcaag agttgactcc 360
atgcccctga cagaggcccc tgctttcatt ttgccccctc ggaacctctg catcaaagaa 420
ggagccaccg ccaagttcga agggcgggtc cggggttacc cagagcccca ggtgacatgg 480
cacagaaacg ggcaacccat caccagcggg ggccgcttcc tgctggattg cggcatccgg 540
gggactttca gccttgtgat tcatgctgtc catgaggagg acaggggaaa gtatacctgt 600
gaagccacca atggcagtgg tgctcgccag gtgacagtgg agttgacagt agaaggaagt 660
tttgcgaagc agcttggtca gcctgttgtt tccaaaacct taggggatag attttcagct 720
ccagcagtgg agacccgtcc tagcatctgg ggggagtgcc caccaaagtt tgctaccaag 780
ctgggccgag ttgtggtcaa agaaggacag atgggacgat tctcctgcaa gatcactggc 840
cggccccaac cgcaggtcac ctggctcaag ggaaatgttc cactgcagcc gagtgcccgt 900
gtgtctgtgt ctgagaagaa cggcatgcag gttctggaaa tccatggagt caaccaagat 960
gacgtgggag tgtacacgtg cctggtggtg aacgggtcgg ggaaggcctc gatgtcagct 1020
gaactttcca tccaaggttt ggacagtgcc aataggtcat ttgtgagaga aacaaaagcc 1080
accaattcag atgtcaggaa agaggtgacc aatgtaatct caaaggagtc gaagctggac 1140
agtctggagg ctgcagccaa aagcaagaac tgctccagcc cccagagagg tggctcccca 1200
ccctgggctg caaacagcca gcctcagccc ccaagggagt ccaagctgga gtcatgcaag 1260
gactcgccca gaacggcccc gcagaccccg gtccttcaga agacttccag ctccatcacc 1320
ctgcaggccg caagagttca gccggaacca agagcaccag gcctgggggt cctatcacct 1380
tctggagaag agaggaagag gccagctcct ccccgtccag ccaccttccc caccaggcag 1440
cctggcctgg ggagccaaga tgttgtgagc aaggctgcta acaggagaat ccccatggag 1500
ggccagaggg attcagcatt ccccaaattt gagagcaagc cccaaagcca ggaggtcaag 1560
gaaaatcaaa ctgtcaagtt cagatgtgaa gtttccggga ttccaaagcc tgaagtggcc 1620
tggttcctgg aaggcacccc cgtgaggaga caggaaggca gcattgaggt ttatgaagat 1680
gctggctccc attacctctg cctgctgaaa gcccggacca gggacagtgg gacatacagc 1740
tgcactgctt ccaacgccca aggccagctg tcctgtagct ggaccctcca agtggaaagg 1800
cttgccgtga tggaggtggc cccctccttc tccagtgtcc tgaaggactg cgctgttatt 1860
gagggccagg attttgtgct gcagtgctcc gtacggggga ccccagtgcc ccggatcact 1920
tggctgctga atgggcagcc catccagtac gctcgctcca cctgcgaggc cggcgtggct 1980
gagctccaca tccaggatgc cctgccggag gaccatggca cctacacctg cctagctgag 2040
aatgccttgg ggcaggtgtc ctgcagcgcc tgggtcaccg tccatgaaaa gaagagtagc 2100
aggaagagtg agtaccttct gcctgtggct cccagcaagc ccactgcacc catcttcctg 2160
cagggcctct ctgatctcaa agtcatggat ggaagccagg tcactatgac tgtccaagtg 2220
tcagggaatc caccccctga agtcatctgg ctgcacaatg ggaatgagat ccaagagtca 2280
gaggacttcc actttgaaca gagaggaact cagcacagcc tttgtatcca ggaagtgttc 2340
ccggaggaca cgggcacgta cacctgcgag gcctggaaca gcgctggaga ggtccgcacc 2400
caggccgtgc tcacggtaca agagcctcac gatggcaccc agccctggtt catcagtaag 2460
cctcgctcag tgacagcctc cctgggccag agtgtcctca tctcctgcgc catagctggt 2520
gacccctttc ctaccgtgca ctggctcaga gatggcaaag ccctctgcaa agacactggc 2580
cacttcgagg tgcttcagaa tgaggacgtg ttcaccctgg ttctaaagaa ggtgcagccc 2640
tggcatgccg gccagtatga gatcctgctc aagaaccggg ttggcgaatg cagttgccag 2700
gtgtcactga tgctacagaa cagctctgcc agagcccttc cacgggggag ggagcctgcc 2760
agctgcgagg acctctgtgg tggaggagtt ggtgctgatg gtggtggtag tgaccgctat 2820
gggtccctga ggcctggctg gccagcaaga gggcagggtt ggctagagga ggaagacggc 2880
gaggacgtgc gaggggtgct gaagaggcgc gtggagacga ggcagcacac tgaggaggcg 2940
atccgccagc aggaggtgga gcagctggac ttccgagacc tcctggggaa gaaggtgagt 3000
acaaagaccc tatcggaaga cgacctgaag gagatcccag ccgagcagat ggatttccgt 3060
gccaacctgc agcggcaagt gaagccaaag actgtgtctg aggaagagag gaaggtgcac 3120
agcccccagc aggtcgattt tcgctctgtc ctggccaaga aggggacttc caagaccccc 3180
gtgcctgaga aggtgccacc gccaaaacct gccaccccgg attttcgctc agtgctgggt 3240
ggcaagaaga aattaccagc agagaatggc agcagcagtg ccgagaccct gaatgccaag 3300
gcagtggaga gttccaagcc cctgagcaat gcacagcctt cagggccctt gaaacccgtg 3360
ggcaacgcca agcctgctga gaccctgaag ccaatgggca acgccaagcc tgccgagacc 3420
ctgaagccca tgggcaatgc caagcctgat gagaacctga aatccgctag caaagaagaa 3480
ctcaagaaag acgttaagaa tgatgtgaac tgcaagagag gccatgcagg gaccacagat 3540
aatgaaaaga gatcagagag ccaggggaca gccccagcct tcaagcagaa gctgcaagat 3600
gttcatgtgg cagagggcaa gaagctgctg ctccagtgcc aggtgtcttc tgacccccca 3660
gccaccatca tctggacgct gaacggaaag accctcaaga ccaccaagtt catcatcctc 3720
tcccaggaag gctcactctg ctccgtctcc atcgagaagg cactgcctga ggacagaggc 3780
ttatacaagt gtgtagccaa gaatgacgct ggccaggcgg agtgctcctg ccaagtcacc 3840
gtggatgatg ctccagccag tgagaacacc aaggccccag agatgaaatc ccggaggccc 3900
aagagctctc ttcctcccgt gctaggaact gagagtgatg cgactgtgaa aaagaaacct 3960
gcccccaaga cacctccgaa ggcagcaatg ccccctcaga tcatccagtt ccctgaggac 4020
cagaaggtac gcgcaggaga gtcagtggag ctgtttggca aagtgacagg cactcagccc 4080
atcacctgta cctggatgaa gttccgaaag cagatccagg aaagcgagca catgaaggtg 4140
gagaacagcg agaatggcag caagctcacc atcctggccg cgcgccagga gcactgcggc 4200
tgctacacac tgctggtgga gaacaagctg ggcagcaggc aggcccaggt caacctcact 4260
gtcgtggata agccagaccc cccagctggc acaccttgtg cctctgacat tcggagctcc 4320
tcactgaccc tgtcctggta tggctcctca tatgatgggg gcagtgctgt acagtcctac 4380
agcatcgaga tctgggactc agccaacaag acgtggaagg aactagccac atgccgcagc 4440
acctctttca acgtccagga cctgctgcct gaccacgaat ataagttccg tgtacgtgca 4500
atcaacgtgt atggaaccag tgagccaagc caggagtctg aactcacaac ggtaggagag 4560
aaacctgaag agccgaagga tgaagtggag gtgtcagatg atgatgagaa ggagcccgag 4620
gttgattacc ggacagtgac aatcaatact gaacaaaaag tatctgactt ctacgacatt 4680
gaggagagat taggatctgg gaaatttgga caggtctttc gacttgtaga aaagaaaact 4740
cgaaaagtct gggcagggaa gttcttcaag gcatattcag caaaagagaa agagaatatc 4800
cggcaggaga ttagcatcat gaactgcctc caccacccta agctggtcca gtgtgtggat 4860
gcctttgaag aaaaggccaa catcgtcatg gtcctggaga tcgtgtcagg aggggagctg 4920
tttgagcgca tcattgacga ggactttgag ctgacggagc gtgagtgcat caagtacatg 4980
cggcagatct cggagggagt ggagtacatc cacaagcagg gcatcgtgca cctggacctc 5040
aagccggaga acatcatgtg tgtcaacaag acgggcacca ggatcaagct catcgacttt 5100
ggtctggcca ggaggctgga gaatgcgggg tctctgaagg tcctctttgg caccccagaa 5160
tttgtggctc ctgaagtgat caactatgag cccatcggct acgccacaga catgtggagc 5220
atcggggtca tctgctacat cctagtcagt ggcctttccc ccttcatggg agacaacgat 5280
aacgaaacct tggccaacgt tacctcagcc acctgggact tcgacgacga ggcattcgat 5340
gagatctccg acgatgccaa ggatttcatc agcaatctgc tgaagaaaga tatgaaaaac 5400
cgcctggact gcacgcagtg ccttcagcat ccatggctaa tgaaagatac caagaacatg 5460
gaggccaaga aactctccaa ggaccggatg aagaagtaca tggcaagaag gaaatggcag 5520
aaaacgggca atgctgtgag agccattgga agactgtcct ctatggcaat gatctcaggg 5580
ctcagtggca ggaaatcctc aacagggtca ccaaccagcc cgctcaatgc agaaaaacta 5640
gaatctgaag aagatgtgtc ccaagctttc cttgaggctg ttgctgagga aaagcctcat 5700
gtaaaaccct atttctctaa gaccattcgc gatttagaag ttgtggaggg aagtgctgct 5760
agatttgact gcaagattga aggataccca gaccccgagg ttgtctggtt caaagatgac 5820
cagtcaatca gggagtcccg ccacttccag atagactacg atgaggacgg gaactgctct 5880
ttaattatta gtgatgtttg cggggatgac gatgccaagt acacctgcaa ggctgtcaac 5940
agtcttggag aagccacctg cacagcagag ctcattgtgg aaacgatgga ggaaggtgaa 6000
ggggaagggg aagaggaaga agagtgaaac aaagccagag aaaagcagtt tctaagtcat 6060
attaaaagga ctatttctct aaaactcaaa aaaaaaaaaa aaactcaaga tagtaaaagc 6120
acctagtgtg atagattatc ggttaggtca tttgtgggtt gattcttcag aaacagcagt 6180
tgatacctag cagcgttatt gatgggcatt aatctatgtt agttggcacc ttaagatact 6240
agtgcagcta gatttcattt agggaaatca ccagtaactt gactgaccaa ttgattttag 6300
agagaaagta accaaaccaa atatttatct gggcaaagtc ataaattctc cacttgaatg 6360
cgctcatgaa aaataaggcc aaaacaagag ttctgggcca cagctcagcc cagagggttc 6420
ctggggatgg gaggcctctc tctccccacc ccctgactct agagaactgg gttttctccc 6480
agtactccag caattcattt ctgaaagcag ttgagccact ttattccaaa gtacactgca 6540
gatgttcaaa ctctccattt ctctttcccc ttccacctgc cagttttgct gactctcaac 6600
ttgtcatgag tgtaagcatt aaggacatta tgcttcttcg attctgaaga caggtccctg 6660
ctcatggatg actctggctt ccttaggaaa atatttttct tccaaaatca gtaggaaatc 6720
taaacttatc ccctctttgc agatgtctag cagcttcaga catttggtta agaacccatg 6780
ggaaaaaaaa aatccttgct aatgtggttt cctttgtaaa ccaggattct tatttgtgct 6840
gttatagaat atcagctctg aacgtgtggt aaagattttt gtgtttgaat ataggagaaa 6900
tcagtttgct gaaaagttag tcttaattat ctattggcca cgatgaaaca gatttcaact 6960
gataaagagc tggagaactc catgtacttt ggaatctcct ccaagatagc cagagtttaa 7020
tacatcttca ttctcaacac tctccaaaga acttgaccta ccttatgggt tccatatttt 7080
tcttcttaaa tgtgcatcaa tcatgccttg cccccaacct ttaaatatat tcttagacct 7140
ggtaaatgca ctcagacttg cgtctttagg aatttttaac tttctttcac tacattggca 7200
cttaaatttt ttctttataa agctttttga aggtcataaa caaagaccat aattgatgat 7260
agacctaata catttcctct gtgtgtgtgt gtaacattcc aaatactttt tttttctttt 7320
ccactgtttg taaggtgcaa caatttaata tttttaaggg actttttaag agttccttaa 7380
gaaccaattt aaaattactt cagtgcaatc ctacacagta tcaacattag aattttgata 7440
ttagtcttat gttatcttcc attctatttt tatctgcttt ttgctgctag tttcaaactg 7500
ccagtatttt tccttttgct tttaaaatag ttacaatatt tttcatgata gccacagtat 7560
tgccacagtt tattataata aagggttttt atttgattta gcgcattcaa agcttttttc 7620
tatcactttt gtgttcagaa tataaccttt gtgtgcgtgt atgttgtgtg tgtgcatgtg 7680
tggcgtatat gtgtgttaca ggttaatgcc ttcttggaat tgtgttaatg ttctcttggt 7740
ttattatgcc atcagaatgg taaatgagaa cactacaact gtagtcagct cacaattttt 7800
aaataaagga taccacagtg catgctgttt gttcaaaaaa aaaaaaaaaa aa 7852
<210> 2
<211> 20
<212> DNA
<213> primer 1 (Artificial sequence)
<400> 2
ccagcaagcc cactgcaccc 20
<210> 3
<211> 20
<212> DNA
<213> primer 2 (Artificial sequence)
<400> 3
ggaagtcctc tgactcttgg 20
Claims (1)
1. The application of a primer pair for detecting mRNA shown in SEQ ID NO.1 in the preparation of a kit for diagnosing the severe ischemic risk of human coronary atherosclerotic heart disease, wherein the severe ischemic risk indicator of human coronary atherosclerotic heart disease is that the coronary stenosis degree is more than or equal to 70 percent, and the primer pair is as follows: 5'-CCAGCAAGCCCACTGCACCC-3', respectively; 5'-GGAAGTCCTCTGACTCTTGG-3' are provided.
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| CN107904300A (en) | 2018-04-13 |
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