CN106435004B - Application of the serum excretion body miRNAs marker in endemic arsenic poisoning early diagnosis - Google Patents
Application of the serum excretion body miRNAs marker in endemic arsenic poisoning early diagnosis Download PDFInfo
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Abstract
The invention belongs to genetic engineerings and clinical medicine domain, and in particular to serum excretion body miRNAs marker relevant to endemic arsenic poisoning early diagnosis and its application, the marker are the combination of has-miR-155, has-miR-191.The marker and its primer can be used for preparing endemic arsenic poisoning early diagnosis kit.
Description
Technical field
The invention belongs to because of engineering and technical field of clinical medicine, and in particular to endemic arsenic poisoning early stage phase occurs for the mankind
Serum excretion body miRNA (microRNAs, the miRNAs) marker of pass and its application.
Background technique
Endemic arsenic poisoning referred to as arsenic disease, is a kind of biogeochemical disease, is to live in specific geographic environment
Under the conditions of resident, it is long-term by caused by drinking-water, air or the excessive inorganic arsenic of food intake with skin pigment depigmentation or/
With the systemic slow poisoning based on excessive calm, palm plantar angling and canceration.Ground arsenic disease is a kind of serious harm human health
Endemic disease.In addition to causing skin change, inorganic arsenic be International Cancer Research Center confirmation human carcinogen, can cause cutaneum carcinoma,
Lung cancer, and it is high-incidence with other visceral cancers.In grave illness area, after cutting off arsenic source or lesion is left, by still there is ground arsenic disease for many years
Generation, show that as caused by arsenic, murder by poisoning is sustainable and exist for a long time, and gradually show late injury-skin change, dislike
Property tumour and other diseases etc..Clinically, arsenic disease in ground is mostly arsenicalism performance.In different lesiones, due to taking arsenic medium not
Same and intake difference, clinical manifestation are not quite similar.Light ward patient often only have light cutaneous lesions and without apparent
Clinical symptoms.In grave illness area, physical signs of patient is obvious, is often accompanied by different degrees of clinical symptoms, while cardiovascular disease, hepatopathy, tumour
Etc. concurrent also more common.
Endemic arsenic poisoning is popular in many countries in the whole world, is just threaten more than the 5000 of at least 22 countries and regions at present
Ten thousand populations, it has also become worldwide one of the public health problem for seriously endangering human health, including drawn by the high arsenic water of long-term drinking
The drinking-water type endemic arsenism risen causes pollution that caused by coal burning type caused by room air or food pollution local with arsenic coal is used
Property arsenic poisoning, the latter one are the distinctive endemic arsenic poisoning types in China, are only popular in Guizhou (1976 confirm) and Shaanxi
(confirming for 2005) two provinces.
The Chinese government pays much attention to endemic arsenic poisoning prevention and control problem, has persistently been classified as state key since " 15 " and prevented
Disease and " health China by the year 2020 action plan " are controlled, obtains achievement of attracting attention in terms of environment intervention, crowd behaviour at present.
But since arsenic intoxication patients caused by coal influence factor is complicated, low arsenic pollution situation still has presence, and health hazard has cumulative bad and difficulty can
Inverse property, the state of an illness still sustainable development, especially mechanism are unknown and sick without specific treatment medicine or the pathogenic people of scheme after stopping contact
Feelings are not obviously controlled.Therefore, main target of the present invention is exactly that important life is explored in the early diagnosis of arsenic intoxication patients caused by coal
Object marker provides reliable basis for early diagnosis, controls arsenic poisoning effectively in early days.
Epigenetics refers to that DNA sequence dna does not change, but heritable the phenomenon that changing, packet has occurred in gene expression
Include DNA methylation (DNA methylation), histone modification (histone modification), non-coding RNA
(noncoding RNA, ncRNA) and chromatin remodeling etc. have many characteristics, such as heritable and reversible；Also it has been reported that table
Seeing science of heredity change may change earlier than hereditary information, and more universal compared with science of heredity change.Environment in recent years chemicals cause
The change of epigenetic mode is related with disease to be concerned, and furthers investigate epigenetic regulation mechanism in endemic arsenic poisoning
Effect and its process in caused body injury and tumour will become the emphasis of the invention studied.
MiRNA (microRNAs, i.e. miRNAs) is the research hotspot having just emerged in recent years, it is that one kind is about
The single strand RNA molecule of 19-23 nucleotide, multidigit in highly conserved on Genome noncoding regions, evolution, by with target gene
After the 3 ' areas UTR of mRNAs combine, target gene mRNA is sheared or inhibits to translate and down-regulation protein expression.MiRNAs can be adjusted
Physiology and the pathologic processes such as various biological process, including cell cycle, apoptosis, differentiation, development and metabolism are controlled, while
There is closely contact for generation and development with many diseases.From the lin-4 and let-7 quilt for participating in regulation nematode timing development
Since it was found that, miRNAs has been increasingly becoming the research hotspot of regulation mRNA stability and protein translation, respectively in 2002 and
Two degrees in 2003 are selected in the annual ten big technological breakthroughs of Science magazine.Now forecast miRNAs can at least regulate and control the thousands of mankind
Gene accounts for 30% or more of all genes.With going deep into for research, more and more miRNAs are found.Currently, miRNAs with
The relationship of tumour has become the emphasis of research, it has been found that expression and chronic lymphocytic of several miRNAs by negative regulator gene
Property leukaemia, liver cancer, lung cancer, breast cancer, colon cancer it is highly relevant.However, but serum miRNAs and endemic arsenic poisoning at present
Deng correlation report it is very few.
Excretion body is a kind of nanoscale lipid encapsulation body structure that diameter is 30-100nm, and nineteen eighty-three knits in sheep net for the first time
It is found in red blood cell, and 1987 are named as " exosome " by Johnstone.Wrapped up inside it albumen, mRNA and
The substances such as microRNA.Almost all kinds of cell including tumour cell can generate and discharge excretion body.Outside
It secretes body to be released by cell secretion, be propagated in the body fluid such as blood, it is last to be swallowed again by other cells, it is cell-cell communication
Important medium.More and more evidences show that the excretion body of host cell or tumor cell secretion takes part in tumour generation, life
Long, invasion and transfer.It is millions of to also demonstrate that tumour releases in a paper being published on " nature " (Nature) magazine
Carry they protein and genetic contents vesica.These vesicas are as " despatch vessel " or " scouting warship ", they are really
It has protected receiving organ and has been ready reception tumour cell.Particularly, it is anti-to trigger necessary molecule in receiving organ for excretion body
Answer --- tumour cell is welcome in inflammation, vascularization etc., is allowed and is proliferated when tumour cell reaches.However, blood
There is not been reported for the correlation of clear excretion body miRNAs and endemic arsenic poisoning etc..
Many evidences show that miRNAs can be stable in the presence of in body fluid, including saliva, urine, breast milk and blood.Cell
Outer miRNAs can be loaded into excretion body or microcapsule bubble, moreover it is possible to be loaded on high-density lipoprotein HDL, these can be protected
It protects them not to be degraded, is stabilized them.Transportation function with vesica is increasingly excavated, the miRNAs's in excretion body
Effect also receives more and more attention.They transmit information by circulating vesica, this is considered as what intercellular signal exchanged
The third approach weighs as the signal transduction that cell contact relies on and conduction this two approach mediated by shla molecule
It wants.There are excretion body and carrying miRNAs in newest research achievement discovery serum, so that miRNAs property is stable, content is rich
Richness is easy to quantitative detection, and there are significant disease specifics, it has been confirmed that serum excretion body miRNAs in lung cancer, colon cancer
Express spectra can be used as the potential source biomolecule marker of early diagnosis.This discovery is exciting, serum excretion body miRNAs conduct
The microRNA of a kind of non-coding modulability is possible to the biomarker for replacing traditional differential protein to be representative, developing
The frontier of biomarker.However application of the excretion body miRNAs in endemic arsenic poisoning early diagnosis monitoring is gone back in serum
It is not paid close attention to accordingly, if can find, the stable early stage to endemic arsenic poisoning falls ill relevant specific serum excretion body
MiRNAs researches and develops the diagnosis of corresponding disease, monitoring reagent box as biomarker, is not only in leading in the world in the field
Status can create the economic benefit to attract people's attention, and the prevention and treatment to China's endemic arsenic poisoning also will be primary strong promotion.
Summary of the invention
In view of the above technical problems, the present invention proposes a kind of serum excretion body relevant to endemic arsenic poisoning early diagnosis
MiRNA marker and its application.
A second object of the present invention is to provide the specific primers of above-mentioned miRNAs marker.
It is a still further object of the present invention to provide above-mentioned serum excretion body microRNA markers and its specific primer to exist
Prepare the application in endemic arsenic poisoning early diagnosis kit.
A further object of the present invention is to provide the kit of the early diagnosis of mankind's endemic arsenic poisoning or monitoring.
The purpose of the present invention is what is realized by following technical proposal:
A kind of serum excretion body miRNA marker relevant to endemic arsenic poisoning early diagnosis, the marker are has-
The combination of miR-155, has-miR-191.
The sequence of hsa-miR-155 is UUAAUGCUAAUCGUGAUAGGGGU (SEQ ID No.1),
The sequence of hsa-miR-191 is CAACGGAAUCCCAAAAGCAGCUG (SEQ ID No.2).
The specificity amplification primer of the miRNA marker, which is characterized in that the primer are as follows:
The upstream primer sequence of has-miR-155 is SEQ ID No.3, and downstream primer sequence is SEQ ID No.4；
The upstream primer sequence of has-miR-191 is SEQ ID No.5, and downstream primer sequence is SEQ ID No.6.
The miRNA marker is preparing the application in endemic arsenic poisoning early diagnosis kit.
The specificity amplification primer of the miRNA marker is in preparing endemic arsenic poisoning early diagnosis kit
Using.
A kind of endemic arsenic poisoning early diagnosis kit, the kit is for detecting has- in serum excretion body miRNAs
MiR-155 and has-miR-191.
The diagnostic kit contains the specificity amplification primer of the miRNA marker in the kit.
The diagnostic kit further includes the common reagent of round pcr in the kit.The reagent is can to measure
The reagent of these serum excretion body microRNA marker expression quantity in serum excretion body.
Another object of the present invention be to provide the object of the present invention is to provide extract serum excretion body method and mirror
It is fixed.
The present invention is described in detail as follows: the present inventor acquires standard compliant blood sample with S.O.P. (SOP),
System collects complete crowd's basic information and clinical data, and use RT-PCR method, TaqMan miRNA Array,
The one or more of Real-time PCR (dye method) method are detected.The experimental method specifically studied mainly includes
Following components:
One, research object selection and group basis:
A group: healthy control group (n=80,20 people's cDNA microarrays, one phase of 30 people are verified, 30 people independence crowds verifying), without it
His systemic major disease.
B group: severe eclampsia early stage group (n=80,20 people's cDNA microarrays, one phase of 30 people are verified, 30 people independence crowds verifying),
Without other systemic major diseases.
Two, blood serum separation and pre-treatment:
(1) fresh heparin anti-coagulating 5ml is centrifuged 5min in centrifuge 3000rpm, and the every 250 μ l of supernatant is taken to dispense to cleaning
In 1.5ml EP pipe.
(2) 63ulExoQuick reagent (4:1) is added in 250ul serum, 4 degree of incubation 30min-1h, after incubation, 1500g-
10000g is centrifuged 30min-1h.
(3) supernatant is sucked, 1500g is centrifuged 5min, and careful 1/10 deionization for exhausting liquid original sample volume is water-soluble
Solution precipitating, as excretion body.
(4) a part is detected for transmission electron microscope, is first fixed with glutaraldehyde.
(5) 1ml Trizol is added in a part of extracted excretion body precipitating, mixes and (blows and beats or be vortexed repeatedly), 4 DEG C
Place 10min.
(6) chloroform 200ul is added in (1ml Trizol:200ul chloroform) in proportion, acutely vibrates, 4 DEG C of placement 15min.
(7) 4 DEG C of 12000 × g are centrifuged 15min.It draws upper strata aqueous phase and manages (350ml) to new EP.
(8) isopropanol 500ul is added in (1ml Trizol:500ul isopropanol) in proportion, mixes, 4 DEG C of placement 10min.
(9) 4 DEG C of 12000 × g are centrifuged 10min, abandon supernatant, and RNA is sunken to tube bottom.
(10) 75% ethyl alcohol 1ml is added in (75% ethyl alcohol of 1ml Trizol:1ml) in proportion, mild to vibrate.
(11) 4 DEG C of 12000g are centrifuged 5min, as far as possible abandoning supernatant (it is primary to repeat 75% ethanol washing).
(12) room temperature is dried, and adds appropriate DEPC water dissolution precipitating RNA.
(13) -70 DEG C save treated sample.
Excretion body used in present invention experiment extracts reagent and is all from ExoQuickTM Exosome Precipitation
This kit of Solution (article No. EXOQ20A-1), similarly hereinafter.
Three, serum excretion body is identified.
The excretion body that serum extracts carries out transmission electron microscope detection after being fixed with the glutaraldehyde of appropriate concentration.
Four, Real-time PCR method measures serum excretion body miRNAs expression quantity.
1. the serum excretion body RNA for pre-treatment of learning from else's experience, obtains cDNA sample by RNA reverse transcription reaction.
Reverse transcription system is prepared shown according to the form below:
2. doing brief centrifugation after PCR pipe is mixed by inversion 3 times repeatedly, place 5 minutes on ice.
3. PCR pipe, which is put into PCR instrument, carries out reverse transcription, reaction condition is as follows:
Reverse transcription product is stored in 4 DEG C of refrigerators with the pre- amplification for next step.
4. the cDNA according to the form below reaction system after reverse transcription, which is prepared, carries out pre-expansion increasing:
The reaction condition expanded in advance is as shown in the table:
After being down to 4 DEG C, pre- amplified production is stored in 4 DEG C of refrigerators to be used for the Real-time PCR of next step reaction.
5. after brief centrifugation, 0.1TE (Ph8.0) 75 μ l being added, does brief centrifugation after being mixed by inversion again.Pre- amplified production can
To be directly used in following Real-time PCR.
Pre- amplified production carries out preparing reaction system shown in Real-time PCR according to the form below:
* loss of prime and high-volume 12.5% are considered.
6. detecting and miRNAs expression quantity in serum excretion body sample being organized in healthier control, endemic arsenic poisoning early stage
Difference.The normal healthy controls for having differences expression and endemic arsenic poisoning early stage patient serum excretion body miRNAs detected include
hsa-miR-155,hsa-miR-191.Copy number of these miRNAs in endemic arsenic poisoning early stage patient is significantly lower than strong
Health control group, and these miRNAs are expressed in serum excretion body with stability.
Five, Real-time PCR method verifies serum excretion body miRNAs expression quantity
1. designing the primer of 2 target miRNAs: using Stem-loop PCR method design primer.
2. fluorescent dye, which is added, carries out Real-time PCR reaction.
3. select independent crowd (control and each 30 people of case) to carry out Real-time PCR detection, it is as a result consistent to have 3
MiRNAs, specifically are as follows: hsa-miR-155, hsa-miR-191.
Therefore, it is finally confirmed as having differences the normal healthy controls of expression and endemic arsenic poisoning early stage blood serum excretion body
MiRNAs includes hsa-miR-155, hsa-miR-191.Their copy numbers in endemic arsenic poisoning early stage blood serum excretion body
Substantially lower than healthy control group, and these miRNAs are expressed in serum with stability.
Five, diagnostic reagent box preparation method:
According to above-mentioned a series of experiments as a result, the present inventor be also prepared for it is a kind of can be used for endemic arsenic poisoning early stage dynamic
The diagnostic kit of monitoring, the diagnostic kit include measurement experimenter's serum excretion body in be stabilized and it is detectable at
The primer and tool of ripe hsa-miR-155, hsa-miR-191.Diagnostic kit includes a collection of serum excretion body miRNAs primer,
It can also include the mixture of the reagents such as Taq enzyme, triphosphoric acid base deoxynucleotide.
The utility model has the advantages that the present invention uses serum excretion body miRNAs the marker of endemic arsenic poisoning EARLY STAGE EVALUATION
It is advantageous in that:
(1) serum excretion body is easy to extract, and needs serum amount few, reduces tester's serum extraction amount.
(2) serum excretion body miRNAs is a kind of new biomarkers, is different from traditional biological marker, not only surely
It is fixed, minimally invasive, be easy to detect, and it is quantitative accurate, the sensibility and specificity of endemic arsenic poisoning early diagnosis, phase will be greatly improved
Than in traditional biomarker (such as mRNA and protein), excretion body miRNAs is more stable and is easy to screen and accurately
Quantitative analysis, to become the biomarker of ideal new generation early diagnosis and prognosis evaluation.
(3) serum excretion body miRNA marker provided by the invention can be used as endemic arsenic poisoning early diagnosis marker,
It can avoid invasive diagnosis, and auxiliary diagnosis can be carried out in early stage, so that foundation is provided for the further testing in depth testing of clinician,
Quick and precisely to grasp the morbid state of patient and coincident with severity degree of condition, taking the control prece of more personalized to provide branch in time
It holds, delays and prevent progression of disease.
(4) present invention is verified using the sample for meeting endemic arsenic poisoning early stage and normal healthy controls crowd, it was demonstrated that this
Several marker expression amounts are there are significant difference and have stability, to illustrate that the marker has specificity, can be used as mark
Will object uses.
(5) present invention uses tight, multistage verifying and appraisement system, and initial stage screens a variety of blood by preliminary experiment
MiRNAs carries out secondary verifying using the methods of Real-time PCR and independent crowd verifies, ensure that the serum excretion body
The reliability of miRNA biomarker.
Detailed description of the invention
Fig. 1 is the experiment effect figure of the embodiment of the present invention 2.
Fig. 2 is the experiment effect figure of the embodiment of the present invention 5.
Specific embodiment
The present invention is further described below by embodiment.
Embodiment 1: to research object selection and group basis
Satisfactory endemic arsenic poisoning early stage patient was collected from cooperation unit Guiyang Medical College in December, 2013
And healthy control group blood sample has therefrom selected satisfactory 80 normal healthy controls, 80 by the arrangement to sample data
Experimental subjects of the example endemic arsenic poisoning early stage patient as Real-time PCR detection miRNAs expression.Specific sample is returned
Class standard is as follows:
A group: healthy control group (n=80,20 people's cDNA microarrays, one phase of 30 people are verified, 30 people independence crowds verifying), without it
His systemic major disease.
B group: endemic arsenic poisoning early stage group (verify, and 30 people independence crowds test by n=80,20 people's cDNA microarrays, one phase of 30 people
Card), it is endemic arsenic poisoning early stage through clinical diagnosis, without other systemic major diseases.
Embodiment 2: the extraction identification of serum excretion body
It prepares serum excretion body sample: a) taking 250ul serum；B) 63ulExoQuick reagent (4:1) is added, gently mixes
Even, 4 degree are incubated for -1 hour 30 minutes, and after incubation, 1500g-10000g is centrifuged -1 hour 30 minutes, abandon upper layer waste liquid；C) it sucks
It after supernatant, then is centrifuged 5 minutes with 1500g, carefully exhausts supernatant liquid, and with 1/10 deionized water dissolving of original sample volume
Precipitating；D) it after 4 degree of refrigerators of appropriate 2.5% glutaraldehyde of addition are 2-3 hours fixed, send to pattern detection room and carries out transmission electron microscope detection
Pre-treatment, it is final to identify excretion body, as shown in Figure 1.
Embodiment 3: the extraction of excretion body RNA
Prepare excretion body RNA:a) extract excretion body precipitating in be added 1ml Trizol, mix (repeatedly piping and druming or whirlpool
Rotation), 4 DEG C of placement 10min；B) chloroform 200ul is added in (1ml Trizol:200ul chloroform) in proportion, acutely vibrates, 4 DEG C of placements
15min；C) 4 DEG C of 12000g are centrifuged 15min, and absorption upper strata aqueous phase to new EP manages (350ml)；D) in proportion (1ml Trizol:
500ul isopropanol) isopropanol 500ul is added, it mixes, 4 DEG C of placement 10min；E) 4 DEG C of 12000g are centrifuged 10min, abandon supernatant, RNA
It is sunken to tube bottom；F) 75% ethyl alcohol 1ml is added in (75% ethyl alcohol of 1ml Trizol:1ml) in proportion, mild to vibrate；g)4℃
12000g is centrifuged 5min, as far as possible abandoning supernatant (it is primary to repeat 75% ethanol washing)；H) room temperature is dried, and adds appropriate DEPC water dissolution heavy
Shallow lake RNA；I) treated sample is saved for -70 DEG C.
Embodiment 4:TaqMan miRNA array screening
Preparation cDNA sample: cDNA is obtained by RNA reverse transcription reaction.The reaction system of reverse transcription includes 0.5 μ lmiRNA
Specific primer RT buffer, 2 μ l, 5 reverse transcription mixed liquor (Vazyme company) and nuclease-free water.Reaction step is 50 DEG C of incubations
15min, 85 DEG C of incubation 2min.(being carried out using corresponding miRNART primer by above-mentioned steps if for different miRNA)
According to the form below reaction system after reverse transcription, which is prepared, carries out pre-expansion increasing:
The reaction condition expanded in advance is as shown in the table:
After pre- amplified production brief centrifugation, 0.1 × TE (pH8.0) 75 μ l is added, does brief centrifugation after being mixed by inversion again.
Pre- amplified production is used directly for following real-time fluorescence quantitative PCR (qPCR).
Pre- amplified production carries out preparing reaction system shown in qPCR according to the form below:
* loss of prime and high-volume 12.5% are considered.
The difference of miRNAs express spectra, sieve in detection and healthier control, endemic arsenic poisoning early stage blood serum excretion body
Select the miRNAs for having 3 times of difference or more.Through bioinformatic analysis and results of animal, it is candidate to select wherein 2 conducts
One step of traveling of going forward side by side is demonstrate,proved, specifically: hsa-miR-155, hsa-miR-191.
The expression quantity of embodiment 5:Real-time PCR method measurement serum excretion body miRNAs
Design primer respectively carries out the serum excretion body of 60 normal healthy controls, 60 Endemic Arsenism Patients each
The quantitative Real-time PCR of miRNAs is detected.
(1) prepare cDNA sample: the excretion body RNA reverse transcription reaction of extraction obtains cDNA.The reaction system packet of reverse transcription
Include 0.5 μ lmiRNA specific primer buffer RT (20 μM), 2 μ 5 × reverse transcription of l mixed liquors (Vazyme company) and nuclease free
Water.Reaction step is 50 DEG C of incubations 15min, 85 DEG C of incubation 2min.
(2) Real-time PCR: dye method: taking 1 μ l cDNA template, and 5 μ l SYBR Green dye mixtures are added
Vazyme company, 0.2 μ l ROX mixed liquor, the corresponding forward primer of the above-mentioned single miRNA of 0.5 20 μM of μ l, on 0.5 20 μM of μ l
The corresponding reverse primer of single miRNA, 2.8 μ l nuclease-free waters are stated, 10 μ l systems carry out quantitative fluorescent PCR.The instrument used
It is ABI Prism7300 fluorescence quantitative PCR instrument, the reaction condition of PCR is: 95 DEG C, 20 seconds progress 1 circulation → 95 DEG C, 10 seconds,
60 DEG C, 20 seconds, 70 DEG C, 30 seconds carry out 40 circulations.Detect healthier control, endemic arsenic poisoning early stage patient serum excretion
The expression quantity ratio of the variation of miRNAs expression quantity in body sample, each group Sample serum excretion body miRNAs can use equation 2-(ΔΔCt)
It indicates, wherein Δ Ct=Ct (group1)-Ct (group2).In order to guarantee the comparativity between testing every time, we are on every plate
It is all provided with U6, calculation expression amount is adjusted using its expression quantity as internal reference.
It is obtained from interpretation of result, this 2 miRNAs of hsa-miR-155, hsa-miR-191 have significance difference between each group
Not, as shown in Fig. 2, non-parametric Trend analysis also shows identical difference.
The production for the miRNA diagnostic kit that embodiment 6 is early diagnosed and monitored for endemic arsenic poisoning:
Normal person and endemic arsenic poisoning early stage patient are determined by the method and Real-time PCR method that are sequenced first
The miRNA for thering is more than one to copy in serum excretion body.Then pass through the technology screenings such as quantitative PCR and endemic arsenic poisoning early stage
Relevant one kind human serum excretion body miRNA, as the index for predicting whether to suffer from endemic arsenic poisoning early stage and diagnose.Most
The quantity for filtering out corresponding serum miRNA afterwards is controlled at several, this is the essence for the optimization made on the basis of preliminary experiment
Letter.This kit includes a collection of serum miRNA primer, wherein the primer of miRNA include hsa-miR-155, hsa-miR-191,
Forward and reverse primer (being shown in Table 1) of U6.Can also have the related common reagent of round pcr, as Taq enzyme, PCR buffer, MgCl2,
Corresponding commercial product can also be used in reagents, these reagents such as triphosphoric acid base deoxynucleotide mixed liquor, dyestuff, can also purchase
Buy the mixed liquor of required reagent.The value of such kit is only to need a small amount of (1~2ml) blood of primary extraction, Ji Kejian
The variation tendency of serum excretion body miRNA marker is surveyed, then occurring by the trend endemic arsenic poisoning early stage can
Energy property or diagnosis endemic arsenic poisoning early stage disease, and be easy to carry out dynamic monitoring and observe therapeutic effect.
Specific kit forms are as follows:
Two pairs of primers below: SEQ ID NO.3 and SEQ ID NO.4, SEQID NO.5 and SE Q ID NO.6, each 20 μM
0.5μl。
5 reverse transcription mixed liquors (Vazyme company) and nuclease-free water can also be contained in kit.
It can also forward and reverse primer a pair (table 1) containing internal reference U6 in kit.
Or also containing the 0.5 general reverse primer of μ l20 μM in kit in addition to forward primer, 10 μ l TaqMan are general
PCR mixed liquor, 6.6 μ l H2O。
Reagent of the component in addition to primer in kit can be using in the prior art for the corresponding of miRNA content detection
Reagent.On the basis of a series of above-mentioned results of study, inventors demonstrated that using hsa-miR-155, hsa-miR-191 energy
It is enough well to separate endemic arsenic poisoning early stage patient and normal healthy controls.
Table 1
| Primer | Corresponding miRNA primer sequence |
| hsa-miR-155-F | ACACTCCAGCTGGGTTAATGCTAATCGTGAT |
| hsa-miR-155-R | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAG ACCCCTAT |
| hsa-miR-191-F | ACACTCCAGCTGGGCAACGGAATCCCAAAAG |
| hsa-miR-191-R | CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGCTGCT |
| URP | TGGTGTCGTGGAGTCG |
| U6-F | CGCTTCGGCAGCACATATACTAAAATTGGAAC |
| U6-R | GCTTCACGAATTTGCGTGTCATCCTTGC |
F: upstream primer, R: downstream primer.
Sequence table
<110>Nanjing Medical University
<120>application of the serum excretion body miRNAs marker in endemic arsenic poisoning early diagnosis
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> RNA
<213> hsa-miR-155
<400> 1
uuaaugcuaaucgugauaggggu 23
<210> 2
<211> 23
<212> RNA
<213> hsa-miR-191
<400> 2
caacggaaucccaaaagcagcug 23
<210> 3
<211> 31
<212> RNA
<213>hsa-miR-155 upstream primer
<400> 3
acactccagctgggttaatgctaatcgtgat 31
<210> 4
<211> 44
<212> RNA
<213>hsa-miR-155 downstream primer
<400> 4
ctcaactggtgtcgtggagtcggcaattcagttgagacccctat 44
<210> 5
<211> 31
<212> RNA
<213>hsa-miR-191 upstream primer
<400> 5
acactccagctgggcaacggaatcccaaaag 31
<210> 6
<211> 44
<212> RNA
<213>hsa-miR-191 downstream primer
<400> 6
ctcaactggtgtcgtggagtcggcaattcagttgagcagctgct 44
Claims (2)
1. a kind of serum excretion body miRNAs marker is preparing the application in endemic arsenic poisoning early diagnosis kit, the mark
Will object is the combination of has-miR-155, has-miR-191.
The specificity amplification primer of 2.miRNAs marker is preparing the application in endemic arsenic poisoning early diagnosis kit, mark
Will object is the combination of has-miR-155, has-miR-191, the primer are as follows:
The upstream primer sequence of has-miR-155 is SEQ ID No.3, and downstream primer sequence is SEQ ID No.4；
The upstream primer sequence of has-miR-191 is SEQ ID No.5, and downstream primer sequence is SEQ ID No.6.
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| CN201611183390.6A CN106435004B (en) | 2016-12-20 | 2016-12-20 | Application of the serum excretion body miRNAs marker in endemic arsenic poisoning early diagnosis |
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| CN107988368B (en) * | 2017-12-28 | 2020-06-30 | 中南大学湘雅医院 | Glioma diagnosis marker circ 9:33948374|33948587 and application thereof |
| CN111218507A (en) * | 2020-03-18 | 2020-06-02 | 昆明医科大学 | Application of XPO1 gene product in preparation of inorganic arsenic exposure diagnosis product |
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| CN101798594A (en) * | 2009-11-13 | 2010-08-11 | 北京命码生科科技有限公司 | Marker, detection method, biological chip and test box for detecting milk quality |
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| CN101798594A (en) * | 2009-11-13 | 2010-08-11 | 北京命码生科科技有限公司 | Marker, detection method, biological chip and test box for detecting milk quality |
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| Title |
|---|
| Role of MicroRNA-214–Targeting Phosphatase and Tensin Homolog in Advanced Glycation End Product-Induced Apoptosis Delay in Monocytes;Li-Min Li et al.;《The Journal of Immunology》;20110112;Supplemental Legends中的Supplemental Table 2 |
| 尿中卟啉类物质作为地砷病暴露标志物可行性探讨;郑玉建等;《新疆医科大学学报》;20090331;第32卷(第3期);第241-243页 |
| 生物标志物在地方性砷中毒研究中的应用及展望;张爱华;《中国地方病学杂志》;20050320;第24卷(第2期);第119-120页 |
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