CN106544435A - Needed for detection miR 206, reverse transcriptase primer is to the application in prediction test kit is prepared - Google Patents
Needed for detection miR 206, reverse transcriptase primer is to the application in prediction test kit is prepared Download PDFInfo
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Abstract
The invention discloses reverse transcriptase primer is to the application in prediction test kit is prepared needed for detection miR 206.The invention provides it is a kind of for predict it is that mild cognitive impairment is lapsed to Alzheimer, be present in serum or blood plasma in miRNAs marks miR 206, using the reverse transcriptase primer of specific designs to carrying out reverse transcription to 206 marks of miR, gained cDNA carries out quantitative fluorescent PCR again and can predict that mild cognitive impairment is lapsed to Alzheimer afterwards, and the reverse transcriptase primer is to can be applicable to predict the preparation of test kit.Gained test kit can be stablized, noinvasive, it is convenient and swift and it is quantitative accurately, prediction mild cognitive impairment the lapsing to Alzheimer that sensitivity is high and specificity is good.
Description
Technical field
The present invention relates to a kind of biotechnology and neuro-cognitive technical field, more particularly to a kind of detection be present in serum or
Reverse transcriptase primer needed for miR-206 in blood plasma is to preparing the reagent that prediction mild cognitive impairment is lapsed to Alzheimer
Application in box.
Background technology
Alzheimer (Alzheimer ' s disease, referred to as AD) is a kind of serious god for threatening human health
Jing degenerative disease, and mild cognitive impairment (Mild cognitive impairment, referred to as MCI) is between A Erci
Sea it is silent it is sick and normal aging between a kind of transitive state.According to the difference of infringement cognitive territory, mild cognitive impairment symptom can
With point forgetting type MCI (referred to as aMCI) and non-forgetting type MCI.Forgetting type MCI patient easily develops into AD, belongs to high-risk people
Group.Research prompting has 23% forgetting type MCI patient that AD was progressed in 2 years, and 50% patient progressed to AD in 4 years.Therefore,
Finding sensitive molecule mark is used to predict aMCI lapsing to AD, is the key issue for preventing and treating AD early.
Include to the detection method that AD is lapsed to about predicting aMCI at present:Applied Neuropsychology is tested (to general cognitive
Function is estimated), neuroimaging (26S Proteasome Structure and Function), cerebrospinal fluid Biomarkers etc., above method be used alone or
It is combined with each other and is studied, potentially contributes to predict aMCI lapsing to AD, but these methods have certain lacking in prediction
Fall into.Neuropsychological test is affected larger by tester and subjects subjective's factor, test result often have it is larger not
Definitiveness and limitation.As aMCI is the preclinical phase of AD, may not find in neuroimaging inspection structure and
The change of function is artificially lacked experience etc. and can all cause mistaken diagnosis or fail to pinpoint a disease in diagnosis.Although it have been found that what some aMCI were lapsed to AD
Cerebrospinal fluid mark (such as A β 1-42, T-tau, P-tau etc.) (Alexopoulos et al., 2013), but due to cerebrospinal fluid mark
This collection has certain traumatic, therefore, have to strictly grasp its indication and contraindication in clinical practice, additionally, brain ridge
Fluid inspection is limited to the Sensitivity and Specificity that mild cognitive impairment is diagnosed.Therefore, new specificity height and sensitivity are found
Good prediction aMCI is increasingly subject to attention both domestic and external to the mark that AD is lapsed to.
MicroRNAs (miRNAs) is non-coding tiny RNA, and length is 19-24bp.Ripe miRNAs is mainly by identification
3 '-UTR of said target mrna are simultaneously in combination, suppress the translation of said target mrna or cause target mrna degradation adjust after genetic transcription so as to play
Section function.The links of the growth of miRNAs regulating cells, development, differentiation and apoptosis, and the change with physiological statuss and disease
The generation of disease, development are closely related.In recent years, miRNA has become grinding for the fields such as molecular biology, hereditism and clinical medicine
Study carefully focus.MiRNAs is small molecule in addition, it is easy to by blood brain barrier, than detection in cerebrospinal fluid, cerebral tissue with more feasibility,
So the miRNAs of unconventionality expression can be used as prediction index biology.
Research finds that serum/plasma miRNA serum is highly stable, multigelation, different acid or alkali environments, high temperature, long-term preservation etc.
Under the conditions of, serum/plasma miRNA serum changes of contents is little, and now most of RNA can degrade.This is just serum miRNA conducts
Biomarker carries out detection there is provided possible.Serum/plasma miRNA serum detection sensitivity is high, traumatic little, favorable repeatability,
Expense is low, simple to operate, detects that the change of specific miRNA express spectras will be helpful to the early diagnosiss of various diseases.
However, there is presently no to be used to predict the report that aMCI is lapsed to AD with regard to serum/plasma miRNA serum s, if can screen
Go out serum/plasma miRNA serum s of unconventionality expression during aMCI is lapsed to AD as biomarker, and develop corresponding disease
Evolution prediction test kit, the preventing and treating to AD will be once strong promotion.
The information for being disclosed in the background section is merely intended to increase the understanding of the general background to the present invention, and should not
When the prior art for being considered to recognize or imply the information structure in any form well known to persons skilled in the art.
The content of the invention
For above-mentioned technical problem, The present invention gives a kind of for predicting that aMCI is present in serum or blood to what AD was lapsed to
MiRNAs mark miR-206 in slurry.For the miRNAs marks, it is an object of the invention to provide a kind of detection is present
In reverse transcriptase primer needed for the miR-206 marks in serum or blood plasma to preparing prediction mild cognitive impairment to alzheimer '
Application in the test kit that silent disease is lapsed to.The test kit can be stablized, noinvasive, it is convenient and swift and it is quantitative accurately, sensitivity it is high and
Good prediction mild cognitive impairment the lapsing to Alzheimer of specificity.
The purpose of the present invention is realized by following technical proposal：
Reverse transcriptase primer needed for a kind of miR-206 detected in being present in serum or blood plasma is to preparing prediction mild cognitive
Application in the test kit that obstacle is lapsed to Alzheimer.
In another embodiment, the test kit also includes that Taq enzyme and/or miR-206 are reversed for above-mentioned application
Reverse transcriptase, dNTPs, MgCl needed for record2, buffer, one or more in DEPC water.
It is above-mentioned to apply in another embodiment, also including miR-206 standard substance and/or reference substance.
It is a kind of slightly to recognize comprising the prediction of reverse transcriptase primer pair needed for miR-206 in being present in serum or blood plasma for detection
Know the test kit that obstacle is lapsed to Alzheimer.
Mentioned reagent box carries out inverse needed for reverse transcription in another embodiment, also including Taq enzyme and/or miR-206
Transcriptase, dNTPs, MgCl2, buffer, one or more in DEPC water.The concentration of main matter is as follows：
Reverse transcriptase：100U/ml；DNTPs mixed liquors:100mM；Taq enzyme：100U/ml；MgCl2：6mM.
Mentioned reagent box in another embodiment, also including miR-206 standard substance and/or reference substance.
In another embodiment, the primer pair includes thering is such as SEQ ID NO for above-mentioned application or test kit:1 institute
Show sequence and SEQ ID NO:The primer pair of sequence shown in 2.
In another embodiment, the mild cognitive impairment hinders for forgetting type mild cognitive for above-mentioned application or test kit
Hinder.
A kind of reverse transcription reaction system, using the miScript II RT kit test kits of German Qiagen, by with the following group
It is grouped into：
Shown conversely reverse record primer
In another embodiment, its reaction condition is above-mentioned reverse transcription reaction system：37 DEG C, 60min；Afterwards 95
DEG C, 5min.
Significant advantage is the present invention compared with prior art：
1st, the present invention by research baseline when and follow-up in 5 years after aMCI patients serums/blood plasma miRNA s, search out with
AMCI lapses to the serum/plasma miRNA serum s predicting marker miR-206 of the high specific and sensitivity of height correlation to AD, uses
Reverse transcriptase primer needed for the detection of the specific designs miR-206 obtains cDNA to carrying out reverse transcription, and gained cDNA enters performing PCR again
As a result can predict that mild cognitive impairment is lapsed to Alzheimer, and miR-206 specific reverse transcriptase primers can be used for
The development and application of prediction test kit so that more convenient and easy is become to the prediction that lapses to of AD to aMCI, clinician reviews
With the morbid state and the order of severity of prediction patient, more efficiently individuation control prece is taken to provide support in time, so as to
The occurrence and development of prevention AD.
2nd, specific reverse transcriptase primer pair miR-206 of the application design carries out the cDNA of reverse transcription acquisition to carry out fluorescence fixed
During amount PCR, amplification efficiency is higher, and specificity is more preferable, without miscellaneous peak during the miR-206 reverse transcriptase primers designed using the application.
3rd, serum/plasma miRNA serum s is a kind of new biological markers, advantage with its stable, noinvasive, detection side
Just it is fast, and quantitative accurate, substantially increase the Sensitivity and Specificity of disease forecasting, the successful exploitation of the biological markers
Contribute to the prediction of AD, be that the development of other predicting markers is offered reference.
4th, this invention is first by AD model mouses (SAMP8) hippocampal tissue miRNA chip express spectra and serum/plasma
The expression conjunctive use of miRNA, has more cognitive impairment specificity with the miRNA marker that this filters out.
5th, detect baseline when and follow-up in 5 years after aMCI patients serums/blood plasma miR-206 expressions change, first will
Serum/plasma miR-206 is used to predict aMCI lapsing to AD.
Description of the drawings
Fig. 1 is the melting song for carrying out quantitative fluorescent PCR using different primers to cDNA obtained by reverse transcription is carried out to miR-206
Line chart；Left figure is the use of the miR-206 reverse transcriptase primers of the application design, and right figure is the use of existing miR-206 reverse transcriptions
Primer；
Fig. 2 be serum/plasma miR-206 in baseline and after follow-up in 5 years when expression；
Fig. 3 be serum/plasma miR-132 after baseline and follow-up in 5 years when expression；
When Fig. 4 is baseline after serum/plasma miR-206 level and follow-up in 5 years patient's MMSE scores correlation analysiss；
When Fig. 5 is baseline after serum/plasma miR-206 level and follow-up in 5 years patient's MoCA scores correlation analysiss；
When Fig. 6 is baseline after serum/plasma miR-132 level and follow-up in 5 years patient's MMSE scores correlation analysiss；
When Fig. 7 is baseline after serum/plasma miR-132 level and follow-up in 5 years patient's MoCA scores correlation analysiss；
Diagnostic value of the serum/plasma miR-206 level to forgetting type mild cognitive impairment when Fig. 8 is baseline；
Diagnostic value of the serum/plasma miR-132 level to forgetting type mild cognitive impairment when Fig. 9 is baseline；
Figure 10 is the survival Analysis that serum/plasma miR-206 prediction aMCI is lapsed to AD；
Figure 11 is the survival Analysis that serum/plasma miR-132 prediction aMCI is lapsed to AD.
Specific embodiment
Below in conjunction with the accompanying drawings, the specific embodiment of the present invention is described in detail, it is to be understood that the guarantor of the present invention
Shield scope is not limited by specific embodiment.
Embodiment 1 sets up the method that prediction mild cognitive impairment is lapsed to Alzheimer
It is provided herein it is a kind of set up the method that prediction mild cognitive impairment is lapsed to Alzheimer, according to
Lower step is carried out：
(1) demographic data storehouse, clinical data storehouse and clinical samples storehouse are set up
With demographic data when S.O.P. collection baseline and follow-up, clinical data and blood sample：
The collection of baseline：Including to cognitive normal person and to more than 60 years old aMCI trouble in mild cognitive impairment baseline visit storehouse
The demographic data of person, clinical data and blood sample collection；
The collection of follow-up investigation object：With the aMCI patient of more than 60 years old to grind in mild cognitive impairment baseline visit storehouse
Studying carefully object carries out demographic data, clinical data and blood sample collection, and all object of study are rich by least more than 2 experiences
Rich neurosurgeon carries out clinical diagnosises and assessment, diagnosis basis Petersen diagnostic criterias (the Petersen RC of aMCI
et al.Arch Neurol.1999)；The follow-up of 5 years is carried out to object of study, is divided into two groups according to the difference of final result：aMCI-
AMCI (not lapsing to as AD groups) and aMCI-AD (lapsing to as AD groups).The diagnostic criteria of AD is according to NINCDS-ADRDA (US Nationals
Neuropathy, communication obstacle and apoplexy institute-alzheimer disease and relevant disease association) diagnostic criteria；Wherein do not lapse to
Group 330, lapses to group 128, and its basic condition is as shown in table 1.
The collection of blood sample：Jing after the informed consent of baseline and all object of study, 5ml venous blood is extracted on an empty stomach,
EDTA anticoagulants, after centrifugation, hemocyte are preserved in -80 DEG C for Total RNAs extraction afterwards, so as to the blood of Erecting and improving
Liquid Sample Storehouse and corresponding clinical data storehouse.
(2) with Senescence-accelerated mice P8 (SAMP8 Mus) as animal pattern, using Affymetrix miR-96 gene express spectra cores
Total serum IgE in piece GeneChip miRNA 2.0Array and SAMP8 Mus hippocampal neurons is hybridized, from SAMP8 Mus Hippocampus groups
In the miRNA express spectras knitted, the detection screening miRNAs related to aMCI, that is, select in H2O2Compare normal condition under incentive condition
7 miRNAs that miRNAs differential expressions are raised in lower hippocampal neuron, respectively miR-206, miR-132, miR-193b,
MiR-130b, miR-20a, miR-296 and miR-329.
(3) adopt that SYBR Green dye methods are carried out real-time fluorescence quantitative PCR and filtered out with detecting step (2) 7
MiRNAs is in baseline and during follow-up in 5 years in object of study (do not lapse to as AD groups and lapse to as AD groups) serum/plasma table in vivo
Change up to horizontal dynamic, the dynamic for analyzing serum/plasma miR expression changes the effect lapsed to AD in prediction aMCI,
The related miR-206 lapsed to AD to prediction aMCI is filtered out finally.
(4) miRNAs prediction test kits are developed according to the testing result of step (3), realizes the dynamic monitoring to AD and predict
There is the purpose of development in which, diagnostic kit is included for detecting the primer and reverse transcriptase of miR-206, buffer,
DNTPs, MgCl2, the reagent such as DEPC water and Taq enzyme.
Following examples will be described in more detail to each step in embodiment 1.
Embodiment 2 is detected related to mild cognitive impairment to screen to SAMP8 Mus hippocampal tissue miRNA express spectras
miRNA
Test method：SAMP8 Mus hippocampal neurons are randomly divided into into 2 groups, respectively：(1) Normal group；(2) peroxide
Change hydrogen (H2O2) stimulation group：Stimulation group adopts 200 μM of H2O2Stimulate hippocampal neuron 6h.Using total RNA extraction reagent box
(MiRNeasy Mini Kit, Qiagen, Germany) is respectively from Normal group and hydrogen peroxide (H2O2) sea is extracted in stimulation group
Total serum IgE in horse neuron.Using Affymetrix miR-96 gene chip of expression spectrum GeneChip miRNA 2.0Array with
The total serum IgE of said extracted is hybridized.The experimental data that Partek Genomic Suite software processes are obtained, detection Hippocampus god
The express spectra of miRNA during Jing is first.Filter out H2O2Stimulation group is than on miRNA differential expressions in the hippocampal neuron of Normal group
7 miRNA for adjusting, respectively miR-206, miR-132, miR-193b, miR-130b, miR-20a, miR-296 and miR-
329, detected in the serum/plasma of Patients with Mild Cognitive Impairment and cognitive normal person, cognitive normal person only carries out baseline
Detection, do not carry out follow-up.
The extraction of the serum/plasma total serum IgE of 3 Patients with Mild Cognitive Impairment of embodiment and cognitive normal person
The collection of blood sample：Jing after the informed consent of baseline and all object of study, 5ml venous blood is extracted on an empty stomach,
EDTA anticoagulants, after centrifugation, hemocyte are preserved in -80 DEG C, the blood sample storehouse of Erecting and improving and corresponding clinical data
Storehouse.
The extraction of serum/plasma total serum IgE：Using serum/plasma total RNA extraction reagent box (miRNeasy Serum/
Plasma test kits, Qiagen, Germany), total serum IgE is extracted from serum/plasma；Take 100 μ L serum/plasmas samples and add 500 μ
L QIAzol Lysis Reagent lysates, RNA are extracted with reference to kit specification, with miR-39 as exogenous internal reference
(miRNeasy Serum/Plasma Spike-In Control test kits, Qiagen, Germany).
The expression of miRNAs in the detection serum/plasma of embodiment 4
The total serum IgE that serum/plasma is extracted is carried out into reverse transcription：Using RNA Reverse Transcriptase kits (miScript II RT
Kit, Qiagen, Germany), reaction system is 22 μ l altogether, composed of the following components：5×miScript HiSpec buffer
(4μl),10×miScript Nucleics mix(2μl),miScript Reverse transcription mix(2μl),
RNase-free water (2 μ l), it is final concentration of in the plasma/serum total serum IgE template (10 μ l) extracted in embodiment 3 and reaction system
1 μM of positive reverse transcriptase primer, each 1 μ l of conversely reverse record primer；Reaction condition is：37 DEG C, 60min；95 DEG C afterwards, 5min,
Obtain cDNA templates.Finally miR-206 is only have selected as predicting marker in the application, below with miR-206 and miR-132
Illustrate.It is used for detecting the reverse primer sequences such as SEQ ID NO of miR-206 in serum/plasma in reverse transcription reaction system:
1 is shown, the forward primer sequence such as SEQ ID NO of miR-206:2 is shown, the reverse primer sequences such as SEQ ID of miR-132
NO:3 is shown, the forward primer sequence such as SEQ ID NO of miR-132:Shown in 4, each end primer sequence is as follows：
SEQ ID NO:1：
CGTGGAATGTAAGGAAGTGTGTGG；
SEQ ID NO:2：
CAGTGCGTGTCGTGGAGT；
SEQ ID NO:3：
GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCG
ACCA；
SEQ ID NO:4：
TGGAATGTAAGGAAGTGTGTGG。
Specific reverse transcriptase primer pair miR-206 of the application design carries out the cDNA of reverse transcription acquisition and carries out fluorescent quantitation
During PCR, amplification efficiency is higher, and more preferably, melting curve difference is as shown in figure 1, inverse using the miR-206 of the application design for specificity
Without miscellaneous peak during transcription primers.
The application is using in Fluorescent quantitative PCR (Real-time PCR) detection serum/plasma
The expression of miRNAs, Real-Time Fluorescent Quantitative PCR Technique are in PCR reaction systems to add fluorophor, are believed using fluorescence
Number accumulation realizes the whole PCR processes of real-time monitoring, the method for carrying out quantitative analyses to starting template.Ct values refer to each reaction
Fluorescence signal in pipe reaches the period experienced during the threshold value of setting.Purpose miRNA starting copy number is more, and Ct values are got over
It is little, on the contrary it is bigger.Can directly obtain in the case of purpose miRNA and internal reference amplification efficiency identical purpose miRNA relative to
The quantitative △ Ct=Ct miRNA-Ct internal references miRNA to be measured of internal reference, with miR-39 as internal reference miR, i.e., △ Ct=Ct purposes-
Ctcel-miR-39.Statistical analysiss are analyzed using SPSS16.0 softwares.Carried out using SYBR Green dye methods glimmering in real time
Fluorescent Quantitative PCR, reaction system are 25 μ l：2×QuantiTect SYBR Green PCR Master mix(12.5μl),10×
miScript Universal primer(2.5μl),10×miScript Primer Assay(2.5μl),RNase-free
Water (5.5 μ l), cDNA templates (2 μ l).Above by primer is included in the reagent of commercial channel purchase in the PCR system, often
Individual miRNA test experience enforcement 3 is parallel, with cel-miR-39 as internal reference.Using ABI 7500Fast type quantitative real time PCR Instruments
(ABI, the U.S.), Real-time PCR reaction conditions are：40 circulations, each circulation include 94 DEG C of reaction 15s, 55 DEG C of reactions
30s, 70 DEG C of reaction 34s, adds melting curve, obtains Δ Ct values, according to lg2–ΔCtFormula carry out the calculating of relative expression quantity.
Inventor is also evaluated to MMSE scores and MoCA scores, and wherein MMSE must be divided in simple intelligent spirit shape
State checks the scoring event in scale, and MoCA must be divided into the scoring situation in the Congnitive scale of Montreal.Above-mentioned acquired results
As shown in table 1 and Fig. 2-Fig. 7.
The essential information of 1. aMCI-aMCI and aMCI-AD group forgetting type Patients with Mild Cognitive Impairment of table
aRelative expression quantity lg2–ΔCt.
After Fig. 2 display follow-ups, serum/plasma miR-206 is expressed in aMCI-AD groups and is significantly higher than aMCI-aMCI groups, P<
0.001, and its changes of contents and time are in reciprocal action P<0.001.After Fig. 3 shows follow-up, serum/plasma miR-132 exists
Expression change no difference of science of statistics in aMCI-AD groups and aMCI-aMCI groups.The relative expression quantity of miR-206 in Fig. 2 and Fig. 3
Less, the actual expression of miR-206 is higher, steeper slopes represent gather way it is faster.Each scatterplot in Fig. 4 represents each follow-up
The situation of object of study, straight line represent the average case of follow-up investigation object, serum/plasma miR-206 water when Fig. 4 shows baseline
Put down and there is dependency, r=0.185, P with patient's MMSE scores after follow-up in 5 years<0.05.Serum/plasma when Fig. 5 shows baseline
After miR-206 levels and follow-up in 5 years there is dependency, r=0.164, P in patient's MoCA scores<0.05.Serum when Fig. 6 is baseline/
After blood plasma miR-132 levels and follow-up in 5 years there is no dependency, P in patient MMSE scores>0.05.Serum/blood when Fig. 7 is baseline
After slurry miR-132 levels and follow-up in 5 years there is no dependency, P in patient MoCA scores>0.05；
Embodiment 5 analyzes the effect that serum/plasma miR-206 and miR-132 are lapsed to AD in prediction aMCI
Excessive risk value is lapsed to miR-206 and miR-132 using SurvivalROC modules in R softwares and low-risk is lapsed to
The marginal value of value is predicted.The sensitivity that miR-206 and miR-132 diagnoses aMCI respectively is evaluated using area under ROC curve
And specificity：Under ROC curve, the span of area is 0.5～1, when area under ROC curve<When 0.5, represent that diagnosis is not intended to
Justice, when under ROC curve, area is between 0.5～0.7, diagnostic value is relatively low, and when between 0.7～0.9, diagnostic value is medium,
When more than 0.9, diagnostic value is higher.As shown in figure 8, area (AUC) is 0.95 under the ROC curve of miR-206, marginal value
(Cutoff) it is -2.01, specificity is 77.8%, sensitivity is 95.3%.As shown in Figure 10, below the ROC curve of miR-132
Product (AUC) is 0.83, and marginal value (Cutoff) is -1.985, and specificity is 85.2%, and sensitivity is 76.6%.
The result of SurvivalROC shows that miR-206 is higher to the diagnostic in baseline and follow-up of aMCI.
Using COX proportional hazard models assessment serum/plasma miR-206 and miR-132 in prediction aMCI is lapsed to AD
Effect.As a result as shown in table 2, Fig. 9 and Figure 11,
Table 2. analyzes the predictor during aMCI is lapsed to AD using COX proportional hazard models
Table 2 and Fig. 9 show that serum/plasma miR-206 is the predictor that aMCI is lapsed to AD, when in serum/plasma
During relative expression quantity≤- 2.01 of miR-206 (value is by SurvivalROC modules prediction in R softwares), aMCI is to AD
The risk for lapsing to is miR-206>4.22 times of (HR risk=4.22, P when -2.01<0.001), its 95% credibility interval
For 2.07-7.03.Figure 11 shows that serum/plasma miR-132 is not predictor that aMCI is lapsed to AD, P>0.05.As a result table
Bright serum/plasma miR-206 can predict forgetting type Patients with Mild Cognitive Impairment to alzheimer ' as independent predictor
Silent disease lapses to.It is i.e. glimmering with real-time according to the reverse transcription in the plasma/serum method for extracting total RNA and embodiment 4 in embodiment 3
Fluorescent Quantitative PCR method, detects to the miR-206 in sample plasma/serum, is fused as obtained by real-time fluorescence quantitative PCR
Curve, obtains Δ Ct values, calculates relative expression quantity lg2-ΔCt, when value≤- 2.01, aMCI to the risk that AD is lapsed to is
miR-206>4.22 times when -2.01.
In the application, classified variable adopts X 2 test, continuous variable to check using t.
Embodiment 6 is used for the making of the miRNAs test kits for predicting that aMCI is lapsed to AD
Test kit includes the reverse transcriptase primer of serum/plasma miR-206 to (sequence such as SEQ ID NO:1 and SEQ ID
NO:Shown in 2), the conventional reaction enzymes and/or reagent needed for can also having corresponding reverse transcription reaction and PCR reactions, such as reverse transcription
Enzyme, buffer, dNTPs, MgCl2, DEPC water and Taq enzyme etc..The main matter concentration that the test kit is included is reverse transcriptase：
100U/ml；DNTPs mixed liquors:100mM；Taq enzyme：100U/ml；MgCl2：6mM.Can be entered according to the concrete experimental technique for adopting
Row is selected, and these conventional enzymes and/or reagent are well known to those skilled in the art, can also have in addition standard substance and reference substance and
Normal reference value.The value of this test kit is to only need to serum/plasma without other tissue samples, and sample is conveniently obtained
Take, miRNAs detected in serum/plasma expression by the method for fluorescence or probe, then by trend aMCI to
AD's lapses to, the method is stable, noinvasive, it is easy to detect it is quick, and it is quantitative accurately, substantially increase disease forecasting sensitivity and
Specificity, therefore by this test kit input practice, the dynamic detection of AD is can aid in, it is clinician reviews and prediction patient
Morbid state and the order of severity, take more efficiently individuation control prece to provide support in time, so as to prevent sending out for AD
Raw and development.
It is aforementioned to the present invention specific illustrative embodiment description be in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to above-mentioned teaching, much can be changed
And change.The purpose of selecting and describing the exemplary embodiment is that explaining that the certain principles and its reality of the present invention should
With so that those skilled in the art can realize and using the present invention a variety of exemplaries and
A variety of selections and change.The scope of the present invention is intended to be limited by claims and its equivalents.
SEQUENCE LISTING
<110>The First Hospital of Hebei Medical University
<120>Needed for detection miR-206, reverse transcriptase primer is to the application in prediction test kit is prepared
<130> 162202
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 24
<212> DNA
<213>Synthetic primer
<400> SEQ ID No. 1
cgtggaatgt aaggaagtgt gtgg 24
<210> 2
<211> 18
<212> DNA
<213>Synthetic primer
<400> SEQ ID No. 2
cagtgcgtgt cgtggagt 18
<210> 3
<211> 50
<212> DNA
<213>Synthetic primer
<400> SEQ ID No. 3
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccgacca 50
<210> 4
<211> 50
<212> DNA
<213>Synthetic primer
<400> SEQ ID No. 4
tggaatgtaa ggaagtgtgt gg 22
Claims (10)
1. reverse transcriptase primer needed for the miR-206 during a kind of detection is present in serum or blood plasma is to preparing prediction mild cognitive barrier
Hinder to Alzheimer the application in the test kit for lapsing to.
It is 2. as claimed in claim 1 to apply, it is characterised in that：The test kit is also carried out needed for reverse transcription including miR-206
Reverse transcriptase, dNTPs, MgCl2, buffer, one or more in DEPC water.
It is 3. as claimed in claim 1 to apply, it is characterised in that：The test kit also includes miR-206 standard substance and/or control
Product.
4. it is a kind of comprising for detection be present in serum or blood plasma in reverse transcriptase primer pair needed for miR-206 prediction mild cognitive
The test kit that obstacle is lapsed to Alzheimer.
5. test kit as claimed in claim 4, it is characterised in that：Also including miR-206 carry out reverse transcriptase needed for reverse transcription,
dNTPs、MgCl2, buffer, one or more in DEPC water.
6. test kit as claimed in claim 4, it is characterised in that：Also include miR-206 standard substance and/or reference substance.
7. apply as claimed in claim 1 or test kit described in claim 4, its feature exists：The primer pair includes having such as
SEQ ID NO:Sequence shown in 1 and SEQ ID NO:The primer pair of sequence shown in 2.
8. apply as claimed in claim 1 or test kit described in claim 4, its feature exists：The mild cognitive impairment is something lost
Forget type mild cognitive impairment.
9. a kind of reverse transcription reaction system, using the miScript II RT kit test kits of German Qiagen, by following components
Composition：
10. reverse transcription reaction system as claimed in claim 9, it is characterised in that：The reaction bar of the reverse transcription reaction system
Part is：37 DEG C, 60min；95 DEG C afterwards, 5min.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610871775 | 2016-09-30 | ||
| CN2016108717755 | 2016-09-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106544435A true CN106544435A (en) | 2017-03-29 |
Family
ID=58395230
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611021546.0A Pending CN106544435A (en) | 2016-09-30 | 2016-11-15 | Needed for detection miR 206, reverse transcriptase primer is to the application in prediction test kit is prepared |
Country Status (1)
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| CN (1) | CN106544435A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114438195A (en) * | 2022-02-28 | 2022-05-06 | 暨南大学 | Alzheimer's disease detection kit, storage medium and electronic device |
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| CN104480106A (en) * | 2014-10-08 | 2015-04-01 | 河北医科大学第一医院 | Serum/plasma micro-RNA marker for detecting patients with mild cognitive impairment and application thereof |
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| CN104480106A (en) * | 2014-10-08 | 2015-04-01 | 河北医科大学第一医院 | Serum/plasma micro-RNA marker for detecting patients with mild cognitive impairment and application thereof |
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