CN108441552A - A kind of and the relevant serum/plasma miRNA marker of intrahepatic cholestasis of pregnancy auxiliary diagnosis and its application - Google Patents
A kind of and the relevant serum/plasma miRNA marker of intrahepatic cholestasis of pregnancy auxiliary diagnosis and its application Download PDFInfo
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Abstract
The invention discloses a kind of and the relevant serum/plasma miRNA marker of intrahepatic cholestasis of pregnancy auxiliary diagnosis and its applications.A kind of and relevant serum/plasma miRNA marker of ICP auxiliary diagnosis is made of miR 371a 5p, miR 6865 5p and miR 1182.The primer of the serum/plasma miRNA marker.Application of the primer in preparing ICP auxiliary diagnostic boxes.Inventor by detach and study primiparity, single pregnancy ICP cases and with the miRNAs in healthy pregnant women control serum/blood plasma of its age-matched, one group is found to fall ill highly relevant high specific and the miRNAs of sensibility with ICP, and the ICP auxiliary diagnostic boxes that can be convenient for clinical application are developed, provide laboratory support for the screening and diagnoses and treatment of ICP.
Description
Invention field
The invention belongs to genetic engineering and reproductive medicine fields, are related to one kind and are examined with intrahepatic cholestasis of pregnancy auxiliary
Disconnected relevant serum/plasma miRNA marker and its application.
Background technology
Intrahepatic cholestasis of pregnancy (intrahepatic cholestasis of pregnancy, ICP) is a kind of
Pregnant related syndromes characterized by itch, Liver enzyme and bile acid increase are the gestational period to seriously endanger baby's health
Complication.Its incidence may be up to 12.0%, can cause the Averse pregnancy outcomes such as fetal distress in uterus, spontaneous pre-term, stillborn foetus,
Increase Perinatal mortality and morbidity.ICP to gestation it is maximum harm be occur it is difficult to predict fetus die by visitation of God, enclose production
The death rate of youngster is about 6-10 times of normal pregnant.It, may although having been reported that the several genes of ICP and albumen can change
The generation of ICP is participated in by approach such as Apoptosis, oxidative stress, lipid-metabolism, cell growth and immune responses, but is so far
Only, the exact cause of ICP is still indefinite.
ICP crowd is early diagnosed, early intervention and rational therapy can effectively reduce the disease onset risk and
Maternal complications substantially reduce disease pain and economic pressures that ICP is brought to mother and baby and its family.However at present clinically
The monitoring means of ICP is very limited, relies only on the lower bile acid screening of susceptibility.Therefore the sensitivity that research ICP occurs
Molecular events, and then screen early diagnosis and implementation that susceptible biomarker (susceptible biomarker) can be ICP
Intervene and effective means is provided, to promoting mankind's baby's health that there is great scientific meaning.
MicroRNAs (i.e. miRNAs) is a hot spot in molecular biology research field in recent years, its maturity state
It is a kind of small non-coding single strand RNA molecule for being about 18-24 nucleotide, has in evolution well-conserved.MiRNA's is main
Function be adjust in organism gene related with body growth, development, disease generating process expression.Since participation is adjusted
Since the lin-4 and let-7 of control nematode sequential development are found, miRNA is selected in 2002 and 2003 two degrees respectively
The annual ten big technological breakthroughs of Science magazines.Prediction miRNAs can at least regulate and control 5300 human genes, i.e., all bases within 2005
The 30% of cause.With going deep into for research, more and more miRNAs are constantly found.MiRNA under spotlight is gradually put
The cover for having taken off DNA radiance becomes " leading role " from " supporting role ", and new challenge is proposed to the Central Position of DNA.In recent years
Come, the relationship of miRNA and disease has become the focus and emphasis of research, and miRNA has been found that gestational diabetes mellitus can be used as
Diagnosis with pre-eclampsia and prognostic marker.
Research has confirmed that there are hundreds of kinds of miRNAs, these microRNAs s properties stabilization, contents in serum/plasma
It enriches, be easy to quantitative detection, and there are significant disease specifics.The technology of existing maturation, including qualitative and quantitative miRNA
The technology of molecule shows using serum miRNAs the method for Molecular biomarkers than traditional differential protein molecular labeling
Method will be more efficient, and frontier has been opened up for biomarker.
However, there is presently no the reports of the relatively stable biomarker for ICP auxiliary diagnosis, if can filter out
ICP is specifically or the serum/plasma miRNA serum s of unconventionality expression is as biomarker, and develops corresponding auxiliary diagnostic box,
By the very big diagnosis present situation for improving China's ICP.
Invention content
The primary and foremost purpose of the present invention is in view of the above technical problems, to propose a kind of and relevant serum/blood of ICP auxiliary diagnosis
Starch miRNA marker.
Second object of the present invention is to provide the primer of above-mentioned serum/plasma miRNA marker.
Third object of the present invention is to provide above-mentioned serum/plasma miRNA markers and its primer to prepare ICP auxiliary
Application in diagnostic kit.
4th purpose of the invention is to provide the kit for ICP auxiliary diagnosis.
The purpose of the present invention is what is realized by following technical proposal：
It is a kind of with the relevant serum/plasma miRNA marker of ICP auxiliary diagnosis, the marker by miR-371a-5p,
MiR-6865-5p and miR-1182 compositions.
The serum/plasma miRNA marker is as detection target spot answering in preparing ICP auxiliary diagnostic boxes
With.
The primer of the serum/plasma miRNA marker, these primers are preferred：The primer of miR-371a-5p is SEQ
ID No.1 and SEQ ID No.2；The primer of miR-6865-5p is SEQ ID No.3 and SEQ ID No.4；MiR-1182's draws
Object is SEQ ID No.5 and SEQ ID No.6.
Application of the serum/plasma miRNA marker in preparing ICP auxiliary diagnostic boxes.
Application of the primer of the serum/plasma miRNA marker in preparing ICP auxiliary diagnostic boxes.
A kind of ICP auxiliary diagnostics box, the kit are used to detect in serum/plasma miR-371a-5p in miRNA,
MiR-6865-5p and miR-1182.
The diagnostic kit, the kit contain miR-371a-5p, miR-6865-5p and miR- in serum/plasma
1182 primer.
The primer of the diagnostic kit, the serum/plasma miRNA marker which contains is preferred：miR-
The primer of 371a-5p is SEQ ID No.1 and SEQ ID No.2；The primer of miR-6865-5p is SEQ ID No.3 and SEQ
ID No.4；The primer of miR-1182 is SEQ ID No.5 and SEQ ID No.6.
The diagnostic kit can also include that PCR reacts common enzyme and reagent, such as reverse transcriptase, buffer solution,
DNTPs, MgCl2, DEPC water and Taq enzyme etc.；Standard items and/or reference substance can also be contained.
It is obtained specifically, serum/plasma miRNA marker of the present invention screens by the following method：(1) unified mark is established
Accurate sample storehouse and database：Standard compliant blood sample is acquired with S.O.P. (SOP), system collects complete people
Mouth learns data and clinical data.(2) serum/plasma miRNA serum differential expression spectrum analysis：Select ICP cases and ICP case ages
The control of matched healthy women, detects ICP cases and control serum/blood plasma miRNA express spectra and content, analysis ICP cases and
The general character and characteristic of serum/plasma miRNA serum, screen differential expression miRNAs, carry out the further multistage between healthy women control
Verification.(3) screening disease specific serum/blood plasma miRNA s：To the serum/plasma differential expression miRNAs that has screened in large sample
Quantitative analysis is carried out in crowd, determines ICP specific serums/blood plasma miRNA s.
Serum/plasma miRNA serum screening and the development of auxiliary diagnostic box of the present invention are according to ICP cases and normal healthy controls
Specific serum/blood plasma miRNA develop miRNAs diagnostic kits.
The present inventor acquires standard compliant blood sample with S.O.P. (SOP), and system collects complete population
Data, clinical data etc. (these data can be used for judging progression of disease, influence of the factors such as patient age for morbidity), and
Use RT-PCR, Real-time PCR method, the detection of Agilent miRNA chips etc..
The experimental method specifically studied includes mainly following components：
1. studying the selection of sample
(1) it is included in case group：For ICP diagnostic criteria with reference to ICP patient's practice guidelines (first edition), specific standards are as follows：1)
There is pruitus in Medium and late pregnancy, or with different degrees of jaundice；2) laboratory examination：Serum tolal bile acid (TBA) increases
High (>40 μm of ol/L), or increased with transaminase (ALT and AST) mild to moderate, it can be increased with bilirubin；3) gestation is to cause skin
Skin itch and biochemical abnormal sole cause；4) patient's ordinary circumstance is good, without obviously vomit, be off one's feed, weakness and other
Disease symptoms；5) above-mentioned symptom, sign, Biochemical Indices In Serum restore rapidly normal after giving a birth.Collect the ICP of complete clinical data
Patient 44.
(2) it is included in Normal group：Without complications of pregnancy and complication, cesarean section indication is breech presentation, anomaly of pelvis and society
Meeting factor etc., collects the normal pregnancies 44 of complete clinical data.
(3) two groups of exclusion criterias：1) suffer from other diseases in the liver and gallbladder；2) there is other complications of pregnancy such as hypertensive disorder in pregnancy
Or there are the blood, urine or biochemistry exception that cannot use that ICP is explained；3) there are general disease such as diabetes, hypertension, spirit and neurological disease
Deng；4) heredity or immunity disease are suffered from；5) there are blood transfusion, transplanting or immunization therapy history person；6) there is oral contraceptive history person.
This research is studied using 88 standard compliant samples altogether.
2.Trizol reagents (Invitrogen, Carlsbad, CA) and miRNeasy Mini Kit (QIAGEN companies) are carried
Serum/plasma total serum IgE is taken, is operated according to a conventional method.Usually lead to~5 μ g RNA/50ml serum or blood plasma.
3.Agilent miRNA (Capitalbio Technology companies) chip detects
(1) total serum IgE obtains cDNA samples by reverse transcription reaction
(2) Agilent miRNA chips detect, and obtain the express spectra of miRNA
(3) data analysis and processing
4.Real-time RT-PCR (qRT-PCR) method
(1) the serum/plasma total serum IgE for taking subject obtains cDNA samples by RNA reverse transcription reactions；
(2) design primer；
(3) Sybrgreen fluorescent dye determinations carry out the quantitative detection of miRNA；
(4) detect and compare the variation of ICP cases and the amount of miRNA in normal healthy controls serum/plasma sample.
5. diagnostic reagent box preparation method
Agilent miRNA chip detecting methods synthesis determines in ICP cases and normal healthy controls there is differential expression
MiRNA, by qRT-PCR technology screenings in ICP cases and normal healthy controls one group of big serum/blood of expression quantity and difference degree
MiRNA is starched, the index as ICP diagnosis.The related serum/plasma miRNA serum composition diagnosis examination with ICP morbidities finally filtered out
Agent box (miR-371a-5p, miR-6865-5p and miR-1182).Diagnostic kit includes the combination of these serum/plasma miRNA serums
The reagents such as primer, probe, Taq enzyme and dNTP.
6. statistical analysis technique
Compare demographic characteristics with student t inspection, TAB (μm ol/L), ALT (IU/L), AST (IU/L) it is horizontal and
The difference that miRNA Average expression levels are distributed between research object group.
We use Agilent miRNA chips in exploratory sample population (4 ICP cases and 4 normal healthy controls)
Result of study preliminary screening find that 16 miRNA up-regulation, 3 miRNA are lowered.Verify four kinds of miRNAs of differential expression
The relevance of (miR-371a-5p, miR-6865-5p, miR-1182, miR-6803-5p) and ICP incidences.Individual
The different expressions of miRNAs detections are with 2–△△CtIt indicates, wherein Δ Ct=CT samples–CT internal references, with hsa-miR-16 (primer SEQ
ID No.7 and SEQ ID No.8) it is reference gene, calculate relative expression quantity.To there is the miRNAs of statistically-significant difference another
It is further verified using qRT-PCR methods in outer 40 cases and 40 controls.
Statistical analysis applies SPSS16.0 statistical analysis softwares to complete.The horizontal P values of significance,statistical are set as
0.05, all statistical tests are two-sided test.
It is further instruction of the present invention below：
In above-mentioned 4 qualified ICP cases and 4 normal healthy controls, two groups of ages are by the accurate matching of individual.We
This two groups of crowds are detected as exploratory sample through Agilent miRNA chips and obtain correlated results.
It is detected according to Agilent miRNA chips, the present inventor detects in " intrahepatic cholestasis of pregnancy case "
Expression is had differences (relative to control group in the serum of group and " healthy women control " group>2 times up-regulation or<0.5 times of downward)
MiRNA includes：hsa-miR-514b-5p、hsa-miR-4656、hsa-miR-3614-5p、hsa-miR-6829-
5p、hsa-miR-6861-5p、hsa-miR-6806-5p、hsa-miR-371a-5p、hsa-miR-3202、hsa-miR-3156-
5p、hsa-miR-7109-5p、hsa-miR-6865-5p、hsa-miR-8060、hsa-miR-3138、hsa-miR-1182、
hsa-miR-4695-5p、hsa-miR-1185-2-3p、hsa-miR-6803-5p、hsa-miR-6068、hsa-miR-518e-
5p。
Select the CT values of two groups of research objects in Agilent miRNA chips no more than 35 and between each group individual of sample
The relatively uniform miRNAs of expression signal is further verified with qRT-PCR methods, to improve detection efficiency.
The miRNAs for meeting above-mentioned condition includes：MiR-371a-5p, miR-6865-5p, miR-1182, miR-6803-
5p。
Sybrgreen fluorescent dye determinations qRT-PCR has 3 kinds as a result, it has been found that in 40 ICP cases and 40 normal healthy controls
Expression feelings of the miRNAs (miR-371a-5p, miR-6865-5p, miR-1182) in " ICP cases " group and " normal healthy controls " group
There are significant differences for condition.
Logistic Regression Analysis the result shows that, miR-371a-5p, miR-6865-5p, miR-1182 with
There is notable association, the combination of this 3 kinds of miRNAs is more efficient as the biomarker of ICP for the morbidity of ICP.
According to above-mentioned experimental result, the present inventor is prepared for a kind of kit that can be used for ICP auxiliary diagnosis, including measuring
It is stabilized in experimenter's serum/blood plasma and detectable maturation miR-371a-5p, miR-6865-5p and miR-1182 draws
Object and other detection reagents.
Specifically, the dependent diagnostic examination that the combination of the primer of the combination of this 3 kinds of miRNAs or this 3 kinds of miRNAs is constituted
Agent box contributes to the early diagnosis of ICP, is clinician Accurate Diagnosis ICP, and control prece is taken to provide support in time, to most
Reducing to limits ICP leads to the risk of Averse pregnancy outcomes.
Beneficial effects of the present invention：
Serum/plasma miRNA (microRNAs/miRNAs) marker provided by the invention is diagnosed as ICP
Marker be advantageous in that：
Inventor by detach and study primiparity, single pregnancy ICP cases and with the healthy pregnant women pair of its age-matched
According to the miRNAs in serum/plasma, finds one group and fall ill highly relevant high specific and the miRNAs of sensibility with ICP, and
The ICP auxiliary diagnostic boxes of clinical application can be convenient for by developing, and laboratory support is provided for the screening and diagnoses and treatment of ICP.
Initial stage of the invention uses Agilent miRNA chips detection acquisition disease special and the serum/plasma of unconventionality expression
MiRNAs express spectras, and the method for application qRT-PCR is verified in large sample by fluorescent dye determination；Above method and
The application acceleration of strategy and the application that ensure that serum/plasma miRNA serum s biomarkers and diagnostic kit, are also other diseases
Reference in the development providing method and strategy of sick biomarker.
The present invention is diagnosed by controlling the influence factor to disease development such as age, research serum/plasma miRNA serum in ICP
Application prospect, illustrate the influence that the miRNAs of unconventionality expression is in progress for ICP, disclose its diagnostic value to ICP.Therefore,
Present invention obtains ICP morbidity specific serum/blood plasma miRNA s expression databases and Specific markers；Pass through serum/blood
The development and application for starching miRNAs biomarkers and diagnostic kit, may make the diagnosis of ICP more convenient and easy, for clinic
Doctor quick and precisely diagnoses ICP and remedy measures is taken to lay the foundation, and to find novel small point with potential treatment value
Sub- drug targets provide help.
Description of the drawings
Diagnostic values of Fig. 1 serum miRNA to ICP
(A) miR-371a-5p, (B) miR-6865-5p, (C) miR-1182, (D) use multiple regression analysis, 3 kinds
The ROC curve of the combination of miRNA.The combination of three kinds of miRNA (miR-371a-5p, miR-6865-5p and miR-1182) is in ROC
Maximum area is generated under curve (AUC).
Specific implementation mode
The arrangement of the collection and sample data of 1 sample of embodiment
Inventor has collected greatly in October, 2016 in March, 2017 from attached Wuxi healthcare hospital for women ＆ children of Nanjing Medical University
The peripheral blood sample of amount ICP patient and normal healthy controls pregnant woman (for the same period collect, and samples, dispenses, preserves item by the sample for research
Part is uniform), by the arrangement to sample data, inventor therefrom selected 88 samples for meeting following standard as
Agilent miRNA chips detect and a series of laboratory sample of follow-up qRT-PCR verifications：
1, the studies above object existsMedium and late pregnancyConfirm (with reference to ICP patient's practice guidelines (first edition)) when ICP screenings
It is defined as case for the pregnant woman of ICP.
2, the studies above object existsMedium and late pregnancyICP does not occur when pregnant week ICP screenings, with case group age, pregnant week
The healthy pregnant women matched is defined as compareing.
And situations such as system acquisition demographic data of these samples, clinical data.
The Agilent miRNA chips detection of miRNA in 2 serum/plasma of embodiment
Above-mentioned qualified 4 ICP cases and 4 normal healthy controls are detected through Agilent miRNA chips and obtain phase
Close result.The specific steps are：
1, the serum of " intrahepatic cholestasis of pregnancy case " group and " healthy women control " group patient is taken respectively
The Trizol reagents of 3 times of volumes are added in 600ul；
2, it is separated：37 DEG C of 15min are positioned over after oscillation 1min, in static preceding addition controlling in inter-sample difference
Join gene hsa-mir-16 (primer is SEQ ID No.7 and SEQ ID No.8), it is 10 to make its ultimate density-4pmol/μl.
Isometric chloroform is added in turbid solution, shakes 50s, room temperature 15min.4 DEG C of centrifugations 14,000rpm after static, centrifugation
15min；
3, RNA precipitate：Water phase is transferred to the centrifuge tube of new 15ml, the absolute ethyl alcohol of 1.5 times of water phase volumes is added, fills
Divide mixing；
4, with Qiagen miRNeasy Mini kit (article No.s:217004) total serum IgE is extracted：700ul samples are drawn every time
Into centrifugal column, 14,000rpm centrifugation 15s discard filtrate in collecting pipe, repeat to cross column to sample standard deviation；700ul washing lotions 1 are added,
14,000rpm centrifugation 15s, abandon filtrate in collecting pipe；500ul washing lotions 2,14,000rpm are added and centrifuge 15s, abandons in collecting pipe and filters
Liquid；It adds 500ul washing lotions 2,14,000rpm and centrifuges 2min, abandon filtrate in collecting pipe；Centrifugal column is put back to empty collecting pipe
In, 14,000rpm centrifugation 2min are to dry centrifuge tube；Centrifuge tube is put into new 1.5ml pipes, 50ul nuclease-free waters are added
(60 DEG C of preheatings), 10,000rpm centrifugation 1min；Liquid in pipe is refunded in centrifugal column, 14,000rpm centrifugation 1min, abandon from
Stem；Liquid in pipe is the total serum IgE sample extracted.It is quantitative with spectrophotometer.
5, the dephosphorylation of total serum IgE and label reaction：Taking 200ng, total serum IgE is tested after purification.Use Agilent public affairs
The miRNA Complete Labeling and Hyb Kit of department, key step include：1) dephosphorylation, using in kit
Alkaline phosphatase CIP under the action of remove RNA5 ' end phosphate group.2) label reaction, uses the T4RNA in kit
Cyanine3-pCp is connected to the ends RNA3 ', this step reaction efficiency ＞ 90% by ligase.3) concentration of label reaction product is taken out
It is dry.4) overnight hybridization in Agilent companies hybrid heater.During entire array experiment, the label of Agilent companies is added
External standard (Labeling spike-in) RNA and hybridization Quality Controls of external standard (Hyb spike-in) RNA as whole experiment process.
It is planted using Human miRNA Microarray, the Release 21.0,8*60K chips detection personage of Agilent companies
MiRNA express spectras.
6, chip cleaning and scanning
After hybridization, 0.2%SDS is first contained at 42 DEG C or so, the washing lotion of 2 × SSC | in wash 5min, then 0.2 ×
5min is washed in the washing lotion II of SSC, can be used to scan after slide drying.Using Agilent chip scanners (G2565CA) to clear
Chip after washing is scanned, and obtains hybridization picture.
7, data extraction and analysis
First, hybridization picture is analyzed and is carried using Agilent Feature Extraction (v10.7) softwares
Access evidence；Then data are normalized using Agilent GeneSpring softwares, and using GeneSpring softwares into
Variance analysis between row group, such as T-test methods carry out two groups of difference expression gene analyses；ANOVA methods carry out multigroup difference
Expressing gene is analyzed.
We tentatively sieve in 4 ICP cases and 4 normal healthy controls with the result of study of Agilent miRNA chips
Existing 16 miRNA up-regulations are published, 3 miRNA are lowered：：hsa-miR-514b-5p、hsa-miR-4656、hsa-miR-3614-
5p、hsa-miR-6829-5p、hsa-miR-6861-5p、hsa-miR-6806-5p、hsa-miR-371a-5p、hsa-miR-
3202、hsa-miR-3156-5p、hsa-miR-7109-5p、hsa-miR-6865-5p、hsa-miR-8060、hsa-miR-
3138、hsa-miR-1182、hsa-miR-4695-5p、hsa-miR-1185-2-3p、hsa-miR-6803-5p、hsa-miR-
6068、hsa-miR-518e-5p。
The qRT-PCR experiments of miRNA in 3 serum/plasma of embodiment
Select the CT values of two groups of research objects in Agilent miRNA chips no more than 35 and between each group individual of sample
The relatively uniform miRNAs of expression signal is further verified with qRT-PCR methods, to improve detection efficiency.
The miRNAs for meeting above-mentioned condition includes：MiR-371a-5p, miR-6865-5p, miR-1182, miR-6803-
5p。
According to above-mentioned Agilent miRNA as a result, selection miR-371a-5p, miR-6865-5p, miR-1182, miR-
6803-5p designs the primer of reverse transcription and qRT-PCR.To the single individual of the serum of " ICP cases " group and " normal healthy controls " group into
The qRT-PCR of row miRNA is detected, and is shown in Table 1.
Table 1:The differential expression of ICP groups and control group miR array detections miRNA
ICP (P) is more than the miRNAs. of 2.0 times of up-regulations or downward compared with healthy pregnant women (C)
Student's t test.
Implement stringent Quality Control in entire research process.Each sample continuously detects three times.All detections are all made of
Blind is completed in the case where not knowing sample background to avoid bias.MiRNA, which is quantitatively detected, uses Sybrgreen fluorescence
Dye method.
1, with Qiagen miRNeasy Mini kit (article No.s:217004) extract total serum IgE, then use spectrophotometer or
Qubit methods are quantitative, its integrality is detected with the methods of agarose gel electrophoresis or Agilent2100.
2, total serum IgE reverse transcription reaction：Using the Megaplex of neck ring structureTMRNA reverse transcriptase primer mixtures carry out.It is inverse
The preparation of responsive transcription system see the table below:
Reverse transcription reaction：In PCR instrument, 16 DEG C, 10min；37℃,30min；65℃,5min.
3, quantitative PCR reacts：
(1) miRNA RealTime PCR reaction systems preparation see the table below：
| Component | Standard Amount |
| Power SYBR Green PCR Master Mix(2×) | 10μL |
| miRNA Cdna sample | 1μL |
| miRNA Universal Sense Primer(10μM) | 0.5μL |
| miRNA Specific Anti-Sense Primer(10μM) | 0.5μL |
| Nuclease-Free Water | 8μL |
| Total Volume | 20μL |
(2) quantitative PCR reaction is carried out.It is as follows that program is arranged in PCR instrument:
4, data process＆analysis
MiRNA quantitative PCR detection data calculate gene relative expression's fold differences using CT (△ △ CT) method is compared.Number
Include mainly according to processing procedure：
The expression quantity ratio of two groups of Sample serum miRNAs can use equation 2–△CtIt indicates, wherein △ Ct=CT samples–CT internal references,
MiR-16 calculates relative expression quantity (miR-16 as internal reference:SEQ ID No.7 and SEQ ID No.8).
Sybrgreen fluorescent dye determination qRT-PCR results are shown in 80 samples, there is 3 kinds of miRNAs (miR-371a-
5p, miR-6865-5p, miR-1182) there are significant differences for expression between two groups, it the results are shown in Table 1, table 2.
Table 2:Verify sample expression of results
Student's t test
Making of the embodiment 4 for the miRNA kits of ICP auxiliary early diagnosis
The making of miRNA kits and operating process are detected based on Agilent miRNA chips, RT-PCR and real-
The technologies such as time PCR.Kit includes that (including the primer of following primer miR-371a-5p is serum/plasma miRNA serum primer
SEQ ID No.1 and SEQ ID No.2；The primer of miR-6865-5p is SEQ ID No.3 and SEQ ID No.4；miR-1182
Primer be SEQ ID No.5 and SEQ ID No.6.), can also there are the common enzyme and/or reagent needed for corresponding PCR reactions,
Such as：Reverse transcriptase, buffer solution, dNTPs, MgCl2 remove nuclease water, fluorescent dye or probe, Taq enzyme, general reverse primer
(URP：GTGCAGGGTCCGAGGT (SEQ ID NO.9)) etc., it can be selected according to the experimental method specifically used, these common enzymes
And/or reagent is well known to those skilled in the art, in addition it can have standard items and control (such as the nematode miR- of quantitative markization
39 samples etc.) and normal reference value.The value of this kit is only to need serum/plasma without other tissue samples,
The variation tendency of miRNA is detected by the fluorescence or sonde method most simplified, then early diagnosis ICP is assisted by the trend, not only
Stablize, it is easy to detect and quantitative accurate, the sensibility and specificity of medical diagnosis on disease is greatly improved, therefore this kit is put into
Practice can help that clinic is instructed accurately to make diagnosis.
3 invention of table is related to primer sequence
Sequence table
<110>Wuxi City healthcare hospital for women ＆ children
<120>It is a kind of with the relevant serum/plasma miRNA marker of intrahepatic cholestasis of pregnancy auxiliary diagnosis and its to answer
With
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacagtgcc 50
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
tgactcaaac tgtgggggc 19
<210> 3
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgactgaagt 50
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
taggtggcag aggagggact t 21
<210> 5
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacgtcaca 50
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gagggtcttg ggagggatgt 20
<210> 7
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgaccgccaa 50
<210> 8
<211> 24
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
gcttgtagca gcacgtaaat attg 24
<210> 9
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
gtgcagggtc cgaggt 16
<210> 10
<211> 50
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacacgccc 50
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
aaatctgggg gtggggggct 20
Claims (8)
1. a kind of and relevant serum/plasma miRNA marker of intrahepatic cholestasis of pregnancy auxiliary diagnosis, it is characterised in that
The marker is made of miR-371a-5p, miR-6865-5p and miR-1182.
2. the primer of serum/plasma miRNA marker described in claim 1.
3. primer according to claim 2, it is characterised in that the primer sets become：The primer of miR-371a-5p is such as
Shown in SEQ ID No.1 and SEQ ID No.2；The primer of miR-6865-5p is as shown in SEQ ID No.3 and SEQ ID No.4；
The primer of miR-1182 is as shown in SEQ ID No.5 and SEQ ID No.6.
4. serum/plasma miRNA marker described in claim 1 is preparing gestational period intrahepatic cholestasis as detection target
Application in disease auxiliary diagnostic box.
5. application of the primer according to claim 2 or 3 in preparing intrahepatic cholestasis of pregnancy auxiliary diagnostic box.
6. a kind of intrahepatic cholestasis of pregnancy auxiliary diagnostic box, it is characterised in that the kit is wanted for test right
Seek the serum/plasma miRNA marker described in 1.
7. diagnostic kit according to claim 6, it is characterised in that the kit contains according to claim 2 or 3
Primer.
8. diagnostic kit according to claim 6, it is characterised in that the kit can also include that PCR reacts common enzyme
And reagent.
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| CN112903851A (en) * | 2021-01-22 | 2021-06-04 | 无锡市妇幼保健院 | Serum/plasma metabolic molecular marker related to auxiliary diagnosis of intrahepatic cholestasis in pregnancy and application thereof |
| CN114085906A (en) * | 2020-08-24 | 2022-02-25 | 沈阳康为医学检验实验室有限公司 | Serum miRNA breast cancer diagnosis marker combination and detection kit thereof |
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| CN109666739A (en) * | 2019-02-22 | 2019-04-23 | 无锡市妇幼保健院 | One kind serum/plasma LncRNA marker relevant with intrahepatic cholestasis of pregnancy auxiliary diagnosis is combined and its is applied |
| CN109666739B (en) * | 2019-02-22 | 2019-07-30 | 无锡市妇幼保健院 | One kind serum/plasma LncRNA marker relevant with intrahepatic cholestasis of pregnancy auxiliary diagnosis is combined and its is applied |
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| CN110257497B (en) * | 2019-02-22 | 2020-11-24 | 无锡市妇幼保健院 | Serum/plasma LncRNA marker ENST00000449605.1 and application thereof |
| CN114085906A (en) * | 2020-08-24 | 2022-02-25 | 沈阳康为医学检验实验室有限公司 | Serum miRNA breast cancer diagnosis marker combination and detection kit thereof |
| CN112410414A (en) * | 2020-10-09 | 2021-02-26 | 嘉兴市妇幼保健院 | Application of pregnant woman serum exosome miRNA marker in preparation of early diagnosis product of fetal congenital heart disease and kit |
| CN112410414B (en) * | 2020-10-09 | 2022-07-08 | 嘉兴市妇幼保健院 | Application of pregnant woman serum exosome miRNA marker in preparation of early diagnosis product of fetal congenital heart disease and kit |
| CN112697895A (en) * | 2020-12-02 | 2021-04-23 | 无锡市妇幼保健院 | Application of palmitoyl carnitine as detection target in preparation of ICP (inductively coupled plasma) auxiliary diagnostic kit |
| CN112903851A (en) * | 2021-01-22 | 2021-06-04 | 无锡市妇幼保健院 | Serum/plasma metabolic molecular marker related to auxiliary diagnosis of intrahepatic cholestasis in pregnancy and application thereof |
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