CN203613173U - Detection kit taking miR-15a as aortic dissection clinical screening molecular marker - Google Patents

Abstract

The utility model discloses a detection kit taking miR-15a as an aortic dissection clinical screening molecular marker. The detection kit is characterized by comprising a box body, wherein a first fixture, a second fixture, a protective cover, a plurality of RNA (ribonucleic acid) extraction reagent containers, a plurality of RNA reverse transcription reagent containers and a plurality of real-time quantitative PCR (polymerase chain reaction) reagent containers are arranged in the box body; a plurality of holes or grooves for fixing the RNA extraction reagent containers are formed in the first fixture; a plurality of grooves for fixing the RNA reverse transcription reagent containers and the real-time quantitative PCR reagent containers are formed in the second fixture; the second fixture is filled with a refrigerant, and is thermally isolated from the first fixture and the box body; the protective cover is made of a thermal insulation material, is arranged above the second fixture, is in sealed detachable connection with the second fixture, and is used for insulating heat and preventing the loss of the RNA reverse transcription reagent containers and the real-time quantitative PCR reagent containers.

Description

MiR-15a is as the detection kit of dissection of aorta Clinical screening molecular marked compound

Technical field

The utility model relates to technical field of biological, and particularly a kind of miR-15a is as the detection kit of dissection of aorta Clinical screening molecular marked compound.

Background technology

Dissection of aorta refers to that in aorta, high speed and high pressure blood flow enters middle rete or membrane junction place of China and foreign countries by the inner membrance tearing port on aorta wall, thereby form true and false two chambeies of aorta, and expand along aorta long axis direction, cause aortoclasia to cause death or important organ (as: heart, brain, kidney, enteron aisle etc.) acute ischemia causes severe complication, the state of an illness is dangerous, is to involve one of the most serious disease of aorta; Can be divided into Aortic Dissection (acute aortic dissection, AAD), subacute dissection of aorta (subacute aortic dissection, SAD) and chronic dissection of aorta by the course of disease.We find in clinical practice, and a lot of dissection of aorta patients are obtaining before operative treatment or even before clarifying a diagnosis because pathology aortoclasia is dead.Have investigation to show, the mortality ratio of untreated Stanford A type patients with acute aortic dissection their early stage is up to 1%～2%/h.Survival through rational therapy can significantly improve, and especially the patient of underwent operative treatment, will realize in a short time survival rate and be increased to and be greater than 70% from high mortality risk.But in clinical position, the inspection of carrying out for patient's routine of violent pectoralgia is electrocardiogram(ECG and fluoroscopy of chest, these inspections all lack susceptibility and specificity to the diagnosis of Aortic Dissection.Thereby the patient of dissection of aorta often can not obtain diagnosis and treatment timely and effectively, in addition there is the subacute dissection of aorta patient of part not show the typical clinical performance of dissection of aorta, cause this rate of clinical misdiagnosis of disease (being reported for the first time by Morgagni in 1761) of being found and study more than 200 year by medical worker of dissection of aorta still up to 39%, some patients were only just finds to have suffered from dissection of aorta in the time of postmortem.Especially in the time that Stanford A type Aortic Dissection involves coronary artery, mistaken diagnosis is that the possibility of coronary heart disease, acute myocardial infarction (acute myocardial infarction, AMI) is very large; Patient is often bestowed anti-freezing, antiplatelet or even thromboembolism treatment, causes state of an illness malignant progression.Therefore, clarifying a diagnosis in time and take reasonably treatment is one of key for the treatment of dissection of aorta patient.

At present, the diagnosis of dissection of aorta mainly depends on imaging examination, for example: robot calculator x-ray tomography blood vessel imaging, through esophagus doppler ultrasound or nuclear magnetic resonance (NMR) vessel imaging, these inspections have very high susceptibility and specificity to Aortic Dissection.But these inspections are to examine doctor at head to think that this patient has and suffer from dissection of aorta and carry out possible in the situation that, and first the doctor that need to see and treat patients gets rid of and has the disease such as acute coronary syndrome, pulmonary infarction similar clinical manifestation, that sickness rate is higher; In addition the operation of these inspections and waiting process need the plenty of time, often can make patient lose precious getting up early diagnosis and treatment opportunity.Therefore, finding the clinical applicable early diagnosis biomarker of dissection of aorta is one of major issue at present urgently to be resolved hurrily, and the monitoring and the prognosis that disease condition development are lapsed to significant simultaneously.Academia is that the biomarker of finding dissection of aorta has carried out large quantity research, found such as: c reactive protein, DDi, unstriated muscle myoglobulin heavy chain, calcium conditioning albumen etc. may with the related a series of biomarkers of the generation of Aortic Dissection, but all fail to be used widely in the clinical diagnosis and treatment of Aortic Dissection owing to lacking specificity, the transformation period factor such as limitation short or that be subject to false chamber thrombosis.To this, Ranasinghe etc. have proposed the general characteristic that desirable early diagnosis index should possess: highly sensitive, high specificity, the course of disease that can reflect disease lapse to, quick, easy-to-use, cheap, there is good potential applicability in clinical practice and commercial supplier widely; For the research of dissection of aorta biomarker provides theoretical standard.

In clinic diagnosis process, many laboratory examination projects with its rapidly, can complete and disease is had to the higher feature such as susceptibility and specificity at bedside, be widely applied.The great discovery Microrna (mircoRNA, miRNA) of science is that a class extensively exists the also small molecules non-coding RNA of high conservative human and animal in recent years; MiRNA has played the part of very important role in cardiovascular organization cell development and differentiation and structure formation exception procedure; analysis detects for miRNA in dissection of aorta group and healthy group of (donator of cadaver) aorta sample early stage in this seminar; obtain the miRNA express spectra that interlayer aortic tunica media significantly changes, we have also found the miR-22 lowering in dissection of aorta (J vasc surg.2011 relevant to HSP27 and EC-SOD simultaneously; 53:1341-9).Recently study and show, miRNA, because having the characteristic of tissue specificity and high conservative, makes it have the possibility as disease biomarker.For example: Ai etc., by patients of acute myocardial infarction and control group comparison, express in conjunction with crossing of miR-1 in mouse model, confirm that miR-1 level significantly increases in patients of acute myocardial infarction circulating, fall ill and after two weeks, return to the variation tendency of basal level; Professors Jing Qing etc. have reported the miR-208a significantly increasing in the relative Healthy People circulating of patients of acute myocardial infarction, its blood plasma level lapses to conditions of patients and changes, and the feature that in peripheral blood, miR-208a occurs early than cardiac troponin, prompting miR-208a may be more responsive early sign thing of acute myocardial infarction.

Utility model content

The utility model is based on above-mentioned background technology, for the problem that solves current existence is carried out, that object is to provide is highly sensitive, high specificity, the course of disease that can reflect disease lapse to, quick, easy-to-use, cheap, there is good potential applicability in clinical practice and the dissection of aorta detection kit of commercial supplier widely.To achieve these goals, the utility model has adopted following technical scheme.

A kind of miR-15a is as the detection kit of dissection of aorta Clinical screening molecular marked compound, it is characterized in that, have: box body, in box body, be provided with the first fixture, the second fixture, a plurality of RNA extraction of protective cover reagent container, a plurality of RNA reverse transcription reagent container and a plurality of real-time quantitative PCR reagent container, wherein, the first fixture is provided with a plurality of hole or grooves that extract reagent container for fixing RNA; The second fixture is provided with a plurality of grooves for fixing RNA reverse transcription reagent container and real-time quantitative PCR reagent container, in the second fixture, is filled with cold-retaining agent, and isolates with the first fixture and box body heat; Protective cover is made up of lagging material, is arranged on the second fixture top, removably connects, for heat insulation and prevent that RNA reverse transcription reagent container and real-time quantitative PCR reagent container from losing with the second fixture is airtight.

In addition, this test kit also has consumptive material portion, is arranged in box body, loads RNA and extracts required adsorption column and centrifuge tube.

In addition, in this test kit, it is column bottle that RNA extracts reagent container; RNA reverse transcription reagent container and real-time quantitative PCR reagent container shape are different.

The beneficial effect of utility model

The utility model can guarantee in transportation, and reverse transcription reagent and real-time quantitative PCR reagent, in low-temperature condition, prevent its inactivation; Can prevent that similar reverse transcription reagent and real-time quantitative PCR reagent from obscuring mutually; Can also be fast and simple dissection of aorta is carried out to Clinical screening, use simple, cheap, highly sensitive, high specificity, meanwhile, the reagent using has commercial supplier widely.

Accompanying drawing explanation

Fig. 1 is the three-dimensional structure diagram of the utility model lid while opening;

Fig. 2 is the utility model cross-sectional view;

Fig. 3 is the utility model sectional view.

Embodiment

Below in conjunction with accompanying drawing, embodiment of the present utility model is described in detail.

Fig. 1 is the three-dimensional structure diagram of the utility model lid while opening; Fig. 2 is the utility model cross-sectional view.

As shown in Figure 1, 2, the miR-15a that the utility model provides extracts reagent container 4, RNA reverse transcription reagent container 5, real-time quantitative PCR reagent container 6, weighting material 7 and ice chest 8 as the detection kit of dissection of aorta Clinical screening molecular marked compound by box body 1, protective cover 2, consumptive material portion 3, RNA and forms.Box body 1 is rectangular parallelepiped, has openable lid.Consumptive material portion 3, for being arranged on two grid in box body 1, holding respectively and extracts RNA consumptive material used, comprises centrifuge tube and adsorption column.It is that four RNA extract reagent bottle that RNA extracts reagent container 4, is respectively used to hold the water (RNas/DNase-free water) that extracts RNA lysate, protein liquid removal, elutriant and nuclease free used; RNA reverse transcription reagent container 5 is 12 centrifuge tubes that are contained with RNA reverse transcription reagent, cross section is circular, be respectively used to hold the mixing solutions (dNTPs) of RNA enzyme inhibitors solution, reversed transcriptive enzyme solution, reversed transcriptive enzyme damping fluid, external source object of reference solution, external source object of reference reverse transcriptase primer solution, miR-15a reverse transcriptase primer solution and four kinds of deoxyribonucleotides, wherein RNA enzyme inhibitors, the each pipe of reversed transcriptive enzyme, each two pipes of other reagent; Real-time quantitative PCR reagent container 6 is 12 centrifuge tubes that are contained with real-time quantitative PCR reagent, cross section is regular hexagon, hold respectively real-time quantitative PCR mixed solution, external source object of reference special primer and the probe solution and miR-15a special primer and the probe solution that contain archaeal dna polymerase, the real-time quantitative PCR mixed solution that wherein contains archaeal dna polymerase is eight pipes, each two pipes of external source object of reference special primer and probe solution and miR-15a special primer and probe solution.It is identical with the miRNeasy Mini Kit of Qiagen company with consumptive material that above-mentioned RNA extracts reagent, RNA reverse transcription reagent and enforcement quantitative PCR reagent and ABI company

microRNA Assays(article No. 4427975),

microRNA Reverse Transcription Kit(article No. 4366597) and

universal PCR Master Mix II, no UNG(article No. 4440048) identical.Wherein,

microRNA Assays comprises two portions, and a part for reverse transcription is: 5 × RT primer(reverse transcriptase primer); A part, for pcr amplification, has comprised amplimer and specific probe, for:

small RNA Assay (20 ×).The miRNA assays of ABI company uses unified article No., and specifically certain miRNA has again its numbering, and the Assays ID that relates to the miR-15a of this project is 000389; The Assays ID of external source object of reference cel-miR-39-3p is 000200.In box body 1, being provided with weighting material 7, is a L shaped cardboard, and cardboard is provided with RNA and extracts the corresponding hole of reagent container 4, is fixed.In box body 1, be also provided with ice chest 8, ice chest 8 is a rectangular parallelepiped, it is made by lagging material with the face that weighting material 7 and box body 1 contact, in ice chest, be filled with cold-retaining agent, ice chest upper surface is processed with the groove corresponding with RNA reverse transcription reagent container 5, real-time quantitative PCR reagent container 6, will be fixing.

Fig. 3 is the utility model sectional view.

As shown in Figure 3, protective cover 2 covers ice chest 8 tops, can prevent in transportation, and centrifuge tube is because come off and lose from ice chest 8.

The miR-15a that use the utility model provides is as the detection kit of dissection of aorta Clinical screening molecular marked compound, first extract the RNA in reagent and consumptive material extraction detected object blood plasma with RNA, then reverse transcription becomes cDNA, then carry out the relative amount of the reverse transcription product of miR-15a in real-time quantitative PCR detection cDNA, according to the amount examination dissection of aorta patient of miR-15a in detected object blood plasma.Through recipient's operating characteristics (ROC) tracing analysis to detected result, detection specificity of the present utility model is better than existing method, convenient and swift.

The beneficial effect of embodiment

In embodiment of the present utility model, the fixing reverse transcription reagent container of ice chest and the real-time quantitative PCR reagent container of cold-retaining agent filled in employing, guaranteed in transportation, reverse transcription reagent and real-time quantitative PCR reagent is all the time in low-temperature condition, can be in transportation inactivation; In addition, adopt difform centrifuge tube to hold reverse transcription reagent and real-time quantitative PCR reagent, avoided obscuring reagent and caused the failure of an experiment; Meanwhile, on to the Clinical screening of dissection of aorta, in keeping hypersensitivity, improve specificity, had significant progress than prior art.

Above, be only a kind of embodiment of the present utility model, be not used for limiting practical range of the present utility model, i.e. all equivalences of doing according to the content of the utility model claim change and modify, and all should be technology category of the present utility model.

Claims (3)

1. miR-15a, as a detection kit for dissection of aorta Clinical screening molecular marked compound, is characterized in that, has:

Box body, is provided with the first fixture, the second fixture, a plurality of RNA extraction of protective cover reagent container, a plurality of RNA reverse transcription reagent container and a plurality of real-time quantitative PCR reagent container in described box body,

Wherein, described the first fixture is provided with a plurality of hole or grooves that extract reagent container for fixing described RNA;

Described the second fixture is provided with a plurality of grooves for fixing described RNA reverse transcription reagent container and real-time quantitative PCR reagent container, in described the second fixture, is filled with cold-retaining agent, and isolates with described the first fixture and described box body heat;

Described protective cover is made up of lagging material, is arranged on described the second fixture top, with airtight the removably connecting of described the second fixture, for heat insulation and prevent that RNA reverse transcription reagent container and described real-time quantitative PCR reagent container from losing.

2. miR-15a according to claim 1, as the detection kit of dissection of aorta Clinical screening molecular marked compound, is characterized in that, also has:

Consumptive material portion, is arranged in described box body, loads RNA and extracts required adsorption column and centrifuge tube.

3. miR-15a according to claim 1, as the detection kit of dissection of aorta Clinical screening molecular marked compound, is characterized in that:

Wherein, described RNA extraction reagent container is column bottle;

Described RNA reverse transcription reagent container and described real-time quantitative PCR reagent container shape are different.

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