CN102605063A - In-situ hybridization detection kit and in-situ hybridization detection method for mRNA (messenger RNA) level in prophase of human cardiac pathologic evolution, and application of in-situ hybridization detection method - Google Patents
In-situ hybridization detection kit and in-situ hybridization detection method for mRNA (messenger RNA) level in prophase of human cardiac pathologic evolution, and application of in-situ hybridization detection method Download PDFInfo
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Abstract
The invention discloses an in-situ hybridization detection kit which comprises a hybridization probe and a marker, and further discloses a method for using the kit to perform in-situ hybridization detection on mRNA (messenger RNA) of CTnT (cardiac troponin T) closely related to early myocardial damage pathologic evolution. The method includes the steps: firstly, contacting RNA to be detected in substrate with the hybridization probe to form a hybrid complex under the condition that the homologous complementary hybridization probe and a target sequence can form a stable hybrid complex; and secondly, detecting the hybrid complex. The kit and the method can be used for detecting gene expression quantity on RNA level, can attain indexes (protein detection indexes) earlier than existing clinical biochemical detection, and can truly screen the RNA level in the prophase of cardiomyopathy. In addition, the method is simple, convenient, low-cost and convenient for application to county and district hospitals.
Description
Technical field
The present invention relates to field of biological detection, more particularly, relate to manage the correlation detection technology that mRNA expresses change (pathology evolution process) in the differentiation with people's myocardosis.
Background technology
According to the data that domestic and international authoritative institution provides, the newly-increased number more than 300 ten thousand of the annual cardiovascular diseases of China, death toll nearly 2,500,000; The patient more than 900 ten thousand, the annual newly-increased cardiovascular patient more than 1,200 ten thousand in the whole world, and death toll is near more than 1,000 ten thousand; The patient has more than 100,000,000 ten thousand approximately; According to world authoritative institution prediction, to the year two thousand thirty above number will double, this is one group of fearful numeral.Cardiovascular diseases diagnosis and treatment cost is increasingly high; (poor area maybe be higher by patient's year medical expense 100,000; Developed regions possibly exceed 200,000 far away), more than 900 ten thousand patients, annual cost is 1.8 trillion Renminbi; Deduction cost 35% is about more than 6 hundred billion, has every year 1.2 trillion Renminbi to consume in vain approximately.And mortality is very high, and in all diseases, the mortality ratio of cardiovascular diseases remains first.Therefore, existing clinical cardiovascular diseases diagnosis and treatment pattern must change, and innovative point of the present invention is to accomplish preventative examination in advance, in time gets involved preventative regulation and control and prophylactic treatment then, accomplishes preventiveing treatment of disease of gene level cardiovascular diseases.
The inventor is in the middle discovery that studies for a long period of time, and the major reason that causes the cardiovascular death rate not fallen is to accomplish real early stage diagnosis and treatment.Diagnose according to biochemical (myocardial damage involved enzyme class must the detect) index in current experiments chamber, major part all rests on the observation of curative effect after cardiovascular diseases forms back detection and sick sending out.
Along with molecular biotechnology is perfect day by day, functional genomics, the deep expansion of research such as disease gene group, sick in order to seek more early stage examination cardiovascular diseases, treatment cardiovascular diseases and Prevention of Cardiovascular, make great progress.So far, we might do more accurate early screening and diagnosis on the one-level functional transcription product (mRNA level or microRNA) of gene, before cardiovascular pathological changes early stage or myocardial cell injury, just can accomplish early prediction and examination.The present invention adopts the nucleic acid hybridization in situ technology, selects many group clinical samples, and myocardial damage patient, high risk population (three high crowds), normal control carry out check and analysis to the early warning of CTnT gene mRNA and various myocardium pathologies.
The contriver is in long term studies; Drawn a kind of new concept, the clinical diagnosis and treatment pattern of cardiovascular diseases and other clinical major disease must change, and can not only stop present treating the disease affected (morbidity back diagnosis and treatment); Accomplish preventative diagnosis and treatment; Accomplish to preventive treatment of disease, only in this way could reduce the M & M of major disease, reduce social cost and medical treatment cost.Therefore, the contriver innovates theoretical and technical in the rna level kit for screening and medicine of exploitation and production major disease.Particularly screen clinical samples (normal population, high risk population, cardiovascular patient); Broken through healthy tissues and pathological tissues consistency research and development thinking relatively; Seek and develop the cardiovascular pathological changes rna level in early stage; Develop closely relatedly with cardiovascular diseases early gene physiopathology, and the extremely important target of clinical meaning is sent out the early prevention property diagnosis and treatment that diagnosis and treatment pattern in back becomes with cardiovascular diseases clinically; Strive for the time and the space of cardiovascular diseases diagnosis and treatment, reached the sick generation of Prevention of Cardiovascular.
Human cardiac troponin T (CTnT) is attached on the cardiac muscle and the tropomyosin of Skelettmuskel, in case during myocardial damage, be present in intracytoplasmic free type CTnT and get into very soon in the blood, can be comparatively fast, reflecting myocardium degree of injury earlier.As the high responsive and specificity marker thing of myocardial damage, troponin has the important clinical meaning to detecting the acute coronary syndrome patient's myocardial ischemia and the classification of risks.The specific to human cardiac muscle property troponin gene mRNA test kit of our research and development; Be used for monitoring free CTnT gene to human peripheral serum; The degree whether the early screening cardiac muscle damages and damage is for examination and preventative detection are done in the morbidity of diseases such as acute myocardial infarction, unstable angina pectoris, acute myocarditis, acute coronary syndrome, hypertension middle and advanced stage, hyperlipidemia, mellitus initial stage in earlier stage.Simultaneously, the troponin mrna concentration also can be used for passing judgment on the risk that functions in patients with unstable angina is suffered from myocardial infarction and cardiac death, can judge also whether patients with coronary heart disease should accept anticoagulant such as the treatment of platelet glycoprotein II b/ III a receptor antagonist.Dynamically detect the mRNA of CTnT gene, observe its positive reaction at any time, timely reliable basis is provided for pointing out prognosis mala and diagnoses and treatment Acute Myocardial Infarction (AMI) and acute ischemic coronary syndrome (AICS) etc.Analyze from the pathophysiological mechanism that myocardial damage takes place; The space-time that the cardiac troponin expression of gene is translated out than cardiac troponin wants Zao; Susceptibility and stability clearer and more definite (because; Sometimes mRNA and protein expression are asynchronous, sometimes have mRNA to express, and protein expression is not necessarily arranged).Therefore, the clinical meaning of the mRNA test kit of our this troponin gene of Application and Development is more important, mainly is to above-mentioned disease incidence examination in early stage, secondly is the degree judgement with myocardial damage in the above-mentioned disease pathology evolution process.
Heart is human recycle system's a power.Heart promotes blood flow, to organ, tissue ample amount of blood flow is provided, and with supply oxygen and various nutritive substance, and takes away metabolic end product (like carbonic acid gas, urea and uric acid etc.), makes cell keep normal metabolism and function.And myocardiumly can make when impaired the heart running obstacle, serious harm people's life and health occur.
Plasma lactic dehydrogenase (LDH), Tn (CK) and isozyme (CK-MB) thereof have been used to for many years clinical as myocardiac mark; Weak point is that the myocardium enzyme tissue specificity is not high; It is not high that enzymic activity detects tolerance range, and sensitivity is not enough to the auxiliary myocardial infarction of making a definite diagnosis.Along with the research that deepens continuously to the myocardial damage mark, find that gradually some susceptibilitys are high, specificity is mark preferably.Cardiac troponin (CTn) is established as the New Set of diagnosis myocardial damage with its excellent susceptibility and specificity.Regression diagnosis different (＞6 hours) with the enzyme mark; Cardiac troponin can detect myocardial damage rapidly; The whole process that real-time reflecting myocardium is impaired; Wherein CTnT all is superior to CK-MB and isozyme and becomes really have myocardium specific mark in diagnosis, monitoring and prognosis to myocardial damage, also is to be independent of the important serological index that can be used for judging infarct kitchen range range size and prognosis outside the electrocardiogram(ECG.Change of serum C TnT is the specificity and the sensitivity label thing of myocardial cell injury.CTnT is a kind of structural protein, is one of subunit of cardiac troponin mixture, participate in to regulate that cardiac muscle is thick, the gliding motility of thin filament, and specific heart is expressed, and IC is high, myocardiumly continue to discharge when impaired, in the blood transformation period short with the susceptibility height.And the mRNA detection method that adopts the CTnT gene is than with the ELISA method of cardiac troponin early response myocardial damage pathologic process more.
The test kit that we adopt nucleic acid hybridization in situ technology and immunohistochemical methods method to combine; MRNA to 80 routine healthy person control groups and 17 routine Acute Myocardial Infarction (AMI) patients, 23 routine unstable angina pectoris patients (UAP), 18 routine stable stenocardia (SA) and 15 routine hyperpietics' peripheral blood serum flesh calcium CTnT gene detects, and detects AMI group three indexs of patient's heart zymogram (LDH, CK, CK-MB) and myocardium myo calcium CTnT kit gene simultaneously.Data show that CTnT is 98.1% to the susceptibility of AMI, and specificity is 100%, and AMI group patient CTnT positive rate is apparently higher than the ELISA detected value of heart zymogram and cardiac troponin, and none routine CTnT concentration surpasses the upper limit concentration of normal health group.Therefore, this test kit of our independent development can be used for early screening and auxiliary diagnosis myocardial damage pathologic process and prognosis.
At present the high flux gene chip analytical technology is all adopted in the research of CTnT gene; And these methods are used for the scientific research aspect more, the incompatibility clinical application, and detect RNA than genetic analysis more science (the DNA analysis major part is on the presentation of susceptibility; MRNA is functional embodiment); Than analysis of protein more reliable (mRNA and albumen are transcribed sometimes asynchronous, and mRNA has expression, and albumen is not transcribed).Detection technique and test kit according to existing literature data CTnT gene level do not appear in the newspapers.
Hybridization in situ technique (in situ hybridization) combines molecular biology and cytochemistry technology, is probe with the nucleic acid molecule of mark, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is to make the nucleic acid strand (being probe) that contains distinguished sequence, process mark; Under optimum conditions with histocyte in the complementary nucleic acid strand be that target nucleic acid is hybridized; With radioautograph or immunocytochemistry label probe is surveyed again, thereby shown special DNA or RNA molecule at cell in-situ.
The probe of in situ hybridization is molecule or sequence the unknown but the known nucleic acid molecule of molecule (though indeterminate this molecule full sequence of known array; But what target molecule known its is directed against), the kind of probe can be divided into dna probe, cDNA probe, cRNA probe and synthetic oligonucleotide probe again by the properties of nucleic acids difference.For the ease of spike, probe must be used certain means mark in addition, is beneficial to later detection.Affinity tag commonly used comprises radionuclide and non-radioactive marker's two big classes.Isotopic label commonly used has
3H,
35S,
125I with
32P.Advantages such as susceptibility is high though isotopic label has, the back of the body end is comparatively clear because ri all can damage human and environment, have by the substituted trend of heterotope recently.At present the most frequently used in the heterotope affinity tag have three kinds of vitamin H, digoxin and resorcinolphthaleins.The method that detects these affinity tags all is a sensitive extremely.
According to used probe and to detect nucleic acid difference can be divided into DNA-DNA again, RNA-DNA, RNA-RNA hybridization.No matter but the hybridization of any form, all must be through five big processes, promptly histiocytic fixing, prehybridization, hybridization, flushing and demonstration.The present invention adopts the hybridization mode of RNA-RNA; Synthetic homologous probe (RNA) and the target RNA that detects are the principles that adopts base complementrity (hybridization reverse complemental); Simultaneously through long-time research with observe, if wash-out starts fully and termination place the result not influence of residue to detecting.
In view of the diagnosis of cardiovascular diseases clinically at present (the biochemical indicator thing all is the diagnosis after most of cardiovascular diseases takes place) is the diagnosis in late period, treatment also is a treatment of late stage, the treatment pattern that causes mortality ratio not fallen.Original intention of the present invention is to want to change at present the diagnosis and treatment pattern of major disease clinically, becomes preventative preventiveing treatment of disease from treating the disease affected, reaches preventative diagnosis and treatment.Adopt nucleic acid hybridization in situ technology and immunohistochemical methods method to combine to detect myocardial damage to become mRNA sick early stage and express and change, done the technological breakthrough of novelty, provide the horizontal examination of the early stage mRNA of myocardial damage pathology technological.Make the technology that a new real early screening of myocardial damage pathology mRNA level in early stage has been arranged clinically, for the diagnosis and treatment of clinical myocardial damage are raced against time and the space.
Summary of the invention
The object of the invention at first provides a kind of hybridization in situ detection kit, and it comprises in situ hybridization detection probes and affinity tag.
Secondly, the present invention also to provide the mentioned reagent box be used for various myocardial damage pathologies examinations in early stage and patient treatment after the relevant in situ hybridization detection method of early warning.
For realizing the object of the invention, technical scheme of the present invention is following: the present invention at first provides a kind of hybridization in situ detection kit, and it comprises hybridization probe and affinity tag; Wherein, Described hybridization probe is the complementary sequence of sequence shown in the sequence table SEQ ID NO.1, the sequence number of target gene: AY277394, and CTnT consecutive nucleotides sequence length is 1102bp; CDS:213 ... 893bp is positioned at karyomit(e) 1q32 " on.(probe sequence of the present invention is 680bp; Do not have problems in that probe mark is technical, repeat specification once, probe is a homologous probe; The sequence of probe is the hybridization complementary base sequence of CTnT gene mRNA, and the process of probe mark is exactly a base reverse complemental process).
A preferred version of test kit of the present invention is that described affinity tag is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, resorcinolphthalein, enzyme and nano material.
A preferred version of test kit of the present invention is also to comprise hybridization solution.
A preferred version of test kit of the present invention is also to comprise toughener.
A preferred version of test kit of the present invention is also to comprise developer.
Canceration CTnT kit for screening in early stage using value of the present invention is that to various myocardial damage pathology examinations in early stage, the especially examination in early stage of heart stalk, and myocardial damage disease is sent out back or course of disease judge of treatment back and early warning, further cooperates clinical treatment.
The present invention also provides a kind of detection method of CTnT in situ hybridization, may further comprise the steps:
(1) described in the above hybridization probe and target sequence can form under the condition of stablizing hybridization complex, and RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect said hybridization complex.
Detection method of the present invention, wherein preferably, the described condition of stablizing hybridization complex that forms is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour.
Detection method of the present invention, wherein preferably, described substrate is selected people's blood preparation for use.More preferably be that described blood preparation is from various myocardial damage patients in early stage, various cardiovascular diseases high risk population, healthy normal population.
Detection method of the present invention, wherein preferably, described myocardial damage high risk population, cardiovascular diseases build up a family fortune well family, various cardiovascular patient (particularly the heart obstructs early stage patient).
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine, and is detected object with CTnT, and synthesising probing needle is the complementary sequence of CTnT sequence, and the substrate of detection is the expression amount of mRNA in the blood of human body.The display packing of hybridization in situ technique can provide sxemiquantitative or the quantitative expression deciding degree of CTnT.Judge the expression amount of above mRNA according to the immunohistochemical methods colour developing of hybridization back; Normal people CTnT does not express; Promptly do not develop the color, CTnT expresses or high expression level at the myocardosis philtrum, and the high risk population has a certain amount of little 14-28%; With the normal control crowd apparent difference is arranged relatively, this expression of gene amount is all higher than normal people expression amount.
The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that the molecular biology insider all knows, and the concrete operations step is to carry out quantitative analysis, result's report under sample disposal, prehybridization, hybridization, immunohistochemical staining, the mirror, and wherein the concrete steps of hybridization comprise:
1). sample to be measured is put into reactive tank;
2). instrument discards liquid automatically, adds Digestive system automatically;
3). instrument discards liquid automatically, and the back is fixing automatically;
4). instrument discards liquid automatically, automatically prehybridization (42 ℃);
5). instrument discards liquid automatically, cleans automatically;
6). instrument discards liquid automatically, automatically hybridization (42 ℃);
7). instrument discards liquid automatically, cleans automatically;
8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);
9). instrument discards liquid automatically, cleans colour developing automatically;
10). take out the mounting microscopy.
The scheme of a preferred embodiment of the present invention is: with CTnT synthetic nucleic probe with digoxigenin labeled (cDNA of digoxigenin labeled, RNA and oligonucleotide probe; Not only probe has a biotin labeling advantage; Also having overcome biotin labeled probe is organized the endogenous vitamin H to do shortcomings such as sorrow in the crossover process in position); MRNA nucleic acid to be measured in this hybridization probe and the blood of human body is hybridized,, under light microscopic, observe existence and the location of mRNA again with the method colour developing of immunohistochemical methods; According to painted cell count, judge the expression amount of purpose mRNA.
The inventive method is a nucleic acid hybridization in situ technology commonly used at present; This method is through detecting the CTnT expression amount in the substrate cell; Be used for confirming that various myocardium pathology develop the mRNA variable quantity of early stage (particularly heart stalk is early stage), the various cardiovascular pathological changes of early warning (myocardial infarction) whether take place and the treatment of various cardiovascular patient after prognosis prediction.Because CTnT does not express in the normal people; If the CTNT expression amount increases, explain that the myocardial damage pathologic process begins, explain that myocardial damage takes place; Or the variation of myocardial damage patient myocardium pathomechanism after treating and curative effect judge, thereby the early diagnosis information of acquisition myocardial damage.A test kit can many person-portions use or person-portion use.
The present invention has following beneficial effect: clinical meaning of the present invention is to follow the tracks of the variation that detects CTnT expression amount in (special heart stalk) generation of various cardiovascular pathological changes and the pathology evolution process more in early days, and the various cardiovascular diseasess of early warning take place, development trend.Diagnostic kit of the present invention is with other detects and cardiovascular diseases mark clinically, has apparent different with other inspections clinically.The present invention can be at mRNA level detection CTnT unconventionality expression; Before cardiovascular diseases biochemical indicator (existing clinical indices) produces unusually; Also do not take place to accomplish the information acquisition of above abnormal gene expression early before the heart stalk, give real early warning of clinical myocardial damage patient.So just might implement early screening, early prevention, the early treatment of myocardial damage, might be from the lethality of the various cardiovascular diseasess of thorough radical cure on the source.
In addition, characteristics highly sensitive, high specificity that test kit provided by the invention has, simultaneously, detection method of the present invention is convenient and simple for operation, can be widely used and promoted in Municipal Hospitals.
Description of drawings
Fig. 1 is a CTnT hybridization in situ technique schema of the present invention.
Fig. 2 is myocardial damage patient CTnT expression an increasing picture in the embodiment of the invention.
Fig. 3 is that high risk population CTnT expresses picture in the embodiment of the invention.
Fig. 4 is that normal people CTnT expresses picture in the embodiment of the invention.
Embodiment
Below in conjunction with embodiment, content of the present invention is described more specifically.Should be appreciated that following embodiment is used for explanation and non-limiting content of the present invention, any pro forma change or accommodation will fall into protection scope of the present invention.
Prepare the in situ hybridization test kit of present embodiment according to ordinary method, this test kit comprises that wherein: the probe mark thing of present embodiment is selected digoxin for use with hybridization probe, affinity tag, the specification sheets of CTnT design.
The test kit hybridization solution is formed:
| Digestive system | 100 μ L/ |
1 pipe/box | Colourless transparent liquid |
| Protection liquid | 100 μ L/ |
1 pipe/box | Colourless transparent liquid |
| Prehybridization solution | 1300 μ L/ pipe | 2 pipe/boxes | Colourless transparent liquid |
| The justice hybridization solution | 10 μ L/ |
1 pipe/box | Colourless transparent liquid |
| The antisense hybridization solution | 10 μ L/ |
1 pipe/box | Colourless transparent liquid |
| Confining liquid | 1000 μ L/ |
1 pipe/box | Colourless transparent liquid |
| The alkaline |
1 μ L/ |
1 pipe/box | Colourless transparent liquid |
| Developer A | 175 μ L/ |
1 pipe/box | Yellow liquid |
| Developer B | 320 μ L/ |
1 pipe/box | Colourless transparent liquid |
| The damping fluid I | The 90mL/ |
1 bottle/box | Light yellow or colourless transparent liquid |
| The damping fluid II | The 80mL/ |
1 bottle/box | Light yellow or colourless transparent liquid |
| The damping fluid III | The 20mL/ bottle | 3 bottle/boxes | Light yellow or colourless transparent liquid |
| The damping fluid IV | The 90mL/ |
1 bottle/box | Light yellow or colourless transparent liquid |
| Stationary liquid | The 90mL/ |
1 bottle/box | Colourless transparent liquid |
| The positive control sample | 6/box | ? | ? |
The reagent preparation working concentration
1). 10 * damping fluid I is diluted to 1 * damping fluid I with tri-distilled water by 1:10;
2). 20 * damping fluid II is diluted to 2 * damping fluid II with tri-distilled water by 1:10;
Be diluted to 0.2 * damping fluid II by 1:100; Be diluted to 0.1 * damping fluid II by 1:200;
3). 10 * damping fluid III is diluted to 1 * damping fluid III with tri-distilled water by 1:10;
4) .10 * damping fluid IV with tri-distilled water by 1:10 be diluted to * the damping fluid IV (get 1#, 2#, each 10mL of 3#, add water to 100mL both can).
Embodiment 2
Use the implementation process of nucleic acid hybridization in situ detection method to each group blood preparation CTnT expression amount:
1). get two of samples to be measured;
2). in glass jar, add Digestive system (Digestive system 100 μ L add 1 * damping fluid I 99.9ml, are working concentration) 50 ml, 37 ℃ of water-bath preheating 10min put 16 slides into, handle 12 min, use 1 * damping fluid I to wash 5min again for 37 ℃;
3). (protection liquid 1ml adds 1 * damping fluid
to the protection liquid with 0.2%; 99ml is working concentration) wash 10min; Tri-distilled water is washed 5min (above process is all carried out at glass jar); Take out slide, let its seasoning;
4). slide is put into the box of preserving moisture, add prehybridization solution 25 μ L/ sheets (being added in the place of cell), covered, the lid box of tightly preserving moisture is placed in 42 ℃ of constant water bath box more than the 3h;
5). take out slide, discard deckglass, slide is put into glass jar, the ethanol with 70%, 90%, 95% is respectively washed 2min, takes out seasoning;
6). slide is put into the box of preserving moisture, and one adds just hybridization solution 25 μ L/ sheets, and another adds antisense hybridization solution 25 μ L/ sheets, and covered is covered the box of tightly preserving moisture, and is placed on 16-24h in 42 ℃ of constant water bath box;
7). take out slide, discard deckglass, slide is put into glass jar:
In 42 ℃ of constant water bath box, wash twice, each 15min with 2 * damping fluid II;
In 42 ℃ of constant water bath box, wash once each 15min with 0.2 * damping fluid II;
In 42 ℃ of constant water bath box, wash twice, each 15min with 0.1 * damping fluid II;
8). wash 30s with 1 * damping fluid III, take out slide, seasoning;
9). slide is put into the box of preserving moisture, add 0.5% confining liquid (the 1ml confining liquid adds 5ml 1 * damping fluid III), 100 μ L/ sheets, cover the box of tightly preserving moisture, at room temperature act on 30min.(this step need not add deckglass);
10). take out slide, wash 30s with 1 * damping fluid III, seasoning;
11). slide is put into the box of preserving moisture; Add X-AP antibody (getting a pipe alkaline phosphatase enzyme antibody) 100 μ L/ sheets, cover the box of tightly preserving moisture and at room temperature act on 30min to wherein adding 1.8ml 1 * damping fluid III; Time can not be long, otherwise can produce false positive (this step need not add deckglass);
12). take out slide, wash 3 times, each 15min with 1 * damping fluid III;
13). wash 2min with 1 * damping fluid IV, add developer (developer A73.3 μ L, developer B157.5 μ L is added in 30mL 1 * damping fluid IV, mixing), room temperature lucifuge 16h is to more than the 18h;
14). wash 5min with tri-distilled water, seasoning, (add with glycerine 10% 1 * damping fluid I mixing) mounting microscopy.
Nucleic acid hybridization in situ detection method of the present invention is used digoxigenin labeled with goal gene; Become the RNA nucleic probe; The RNA nucleic acid to be measured of probe and human leukocytes is hybridized,, therefore under light microscopic, observe existence and the location of RNA again with the method colour developing of immunohistochemical methods; According to painted cell count, judge the expression amount of purpose RNA.
Cardiovascular patient 20 examples, high risk population (three high crowds) 20 examples, 20 of normal control groups.Take out all people's to be checked peripheral blood 3-5 milliliter (separation white corpuscle) and do in situ hybridization.The result representes that all cardiovascular patient CTNT expression amounts are low, and cell dyeing is shallow; The high risk population expresses slightly and reduces, decimal dyeing; Normal control group CTNT expression amount is high, the dyeing of cell great majority, and concrete outcome is seen Fig. 2, Fig. 3, Fig. 4.
| The cardiovascular diseases number | Expression amount % | High-risk number | Expression amount % | Normal number | |
| 1 | ? 76 | 1 | ?12 | 1 | 0 |
| 2 | ? 72 | 2 | ?23 | 2 | 0 |
| 3 | ? 77 | 3 | ?16 | 3 | 0 |
| 4 | 86? | 4 | 10? | 4 | 0? |
| 5 | 70 | 5 | ?8 | 5 | 0 |
| 6 | 80 | 6 | 14? | 6 | 0 |
| 7 | ? 69 | 7 | ?8 | 7 | ? 2 |
| 8 | ? 67 | 8 | ?6 | ? 8 | 2 |
| 9 | ? 76 | 9 | ?8 | 9 | 0 |
| ? 10 | ? 68 | ? 10 | ?14 | ? 10 | 0 |
| ? 11 | ? 76 | ? 11 | ? 9 | ? 11 | 0 |
| ? 12 | 80 | ? 12 | ?15 | ? 12 | 0 |
| ? 13 | ? 76 | ? 13 | ?14 | ? 13 | 0 |
| ? 14 | ? 86 | ? 14 | ?12 | ? 14 | 0 |
| ? 15 | ? 82 | ? 15 | ?19 | ? 15 | 2 |
| ? 16 | ? 79 | ? 16 | ?16 | ? 16 | 0 |
| ? 17 | ? 74 | ? 17 | ?14 | ? 17 | 0 |
| ? 18 | ? 78 | ? 18 | ?10 | ? 18 | 0 |
| ? 19 | ? 82 | ? 19 | ?13 | ? 19 | 1 |
| 20 | 80 | 20 | 15 | 20 | 0 |
SEQUENCE?LISTING
< 110>Rui bends biotechnology (Shanghai) Co., Ltd.
< 120>people's myocardosis reason develops the horizontal hybridization in situ detection kit of mRNA and detection method and application in earlier stage
<130> 、
<160> 1
<170> PatentIn?version?3.5
<210> 1
<211> 1102
<212> DNA
<213> Homo?sapiens
<400> 1
gagcagacgc?ctccaggatc?tgtcggcagc?tgctgttctg?agggagagca?gagaccatgt 60
ctgacataga?agaggtggtg?gaagagtacg?aggaggagtg?agagcaggag?gaggcagcgg 120
aagaggatgc?tgaagcagag?gctgagaccg?aggagaccag?ggcagaagaa?gatgaagaag 180
aagaggaagc?aaaggaggct?gaagatggcc?caatggagga?gtccaaacca?aagcccaggt 240
cgttcatgcc?caacttggtg?cctcccaaga?tccccgatgg?agagagagtg?gactttgatg 300
acatccaccg?gaagcgcatg?gagaaggacc?tgaatgagtt?gcaggcgctg?atcgaggctc 360
actttgagaa?caggaagaaa?gaggaggagg?agctcgtttc?tctcaaagac?aggatcgaga 420
gacgtcgggc?agagcgggcc?gagcagcagc?gcatccggaa?tgagcgggag?aaggagcggc 480
agaaccgcct?ggctgaagag?agggctcgac?gagaggagga?ggagaacagg?aggaaggctg 540
aggatgaggc?ccggaagaag?aaggctttgt?ccaacatgat?gcattttggg?ggttacatcc 600
agaagacaga?gcggaaaagt?gggaagaggc?agactgagcg?ggaaaagaag?aagaagattc 660
tggctgagag?gaggaaggtg?ctggccattg?accacctgaa?tgaagatcag?ctgagggaga 720
aggccaagga?gctgtggcag?agcatctata?acttggaggc?agagaagttc?gacctgcagg 780
agaagttcaa?gcagcagaaa?tatgagatca?atgttctccg?aaacaggatc?aacgataacc 840
agaaagtctc?caagacccgc?gggaaggcta?aagtcaccgg?gcgctggaaa?tagagcctgg 900
cctccttcac?caaagatctg?ctcctcgctc?gcacctgcct?ccggcctgca?ctcccccagt 960
tcccgggccc?tcctgggcac?cccaggcagc?tcctgtttgg?aaatggggag?ctggcctagg 1020
tgggagccac?cactcctgcc?tgcccccaca?cccactccac?accagtaata?aaaagccacc 1080
acacacaaaa?aaaaaaaaaa?aa 1102
Claims (10)
1. a hybridization in situ detection kit comprises hybridization probe and affinity tag, it is characterized in that, described hybridization probe is the complementary sequence of sequence shown in the sequence table SEQ ID NO.1.
2. test kit as claimed in claim 1 is characterized in that, described affinity tag is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, resorcinolphthalein, enzyme and nano material.
3. test kit as claimed in claim 1 is characterized in that this test kit also comprises hybridization solution.
4. test kit as claimed in claim 1 is characterized in that this test kit also comprises synergistic agent.
5. test kit as claimed in claim 1 is characterized in that this test kit also comprises developer.
6. CTnT gene hybridization in situ detection method is characterized in that this method may further comprise the steps:
(1) can form under the condition of stablizing hybridization complex at described hybridization probe of claim 1 and target sequence, RNA to be measured in the substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect said hybridization complex.
7. detection method as claimed in claim 6 is characterized in that, the described condition of stablizing hybridization complex that forms is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour.
8. detection method as claimed in claim 6 is characterized in that, described substrate is selected the mRNA sample in the human blood for use.
9. detection method as claimed in claim 8 is characterized in that, described blood mRNA composition is selected from cardiovascular disorder, hypertension, hyperlipidemia, hyperglycemia and heart stalk high risk population, normal people's sample in earlier stage.
10.CTnT gene detects the application in early stage myocardial necrosis, damage and the various cardiovascular diseases hybridization in situ detection kit in preparation.
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| CN101535813A (en) * | 2006-09-18 | 2009-09-16 | 霍夫曼-拉罗奇有限公司 | Natriuretic peptides for diagnosing cardiac complications due to coronary catheterization |
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| CN101535813A (en) * | 2006-09-18 | 2009-09-16 | 霍夫曼-拉罗奇有限公司 | Natriuretic peptides for diagnosing cardiac complications due to coronary catheterization |
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| Title |
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| DU Y H等: "AY277394.1", 《GENBANK》 * |
| 金贺: "病毒性心肌炎心肌结构蛋白的损伤及其法医学意义", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
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