CN106434907A - Application of miR-495 as marker for detecting ankylosing spondylitis - Google Patents
Application of miR-495 as marker for detecting ankylosing spondylitis Download PDFInfo
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Abstract
The invention discloses an application of miR-495 as a marker for detecting ankylosing spondylitis (AS). The sequence of the miR-495 is AAACAAACAUGGUGCACUUCUU. Micro RNA-495 (miR-495) is generally subjected to down-regulated expression in an HLA-B27 positive sample (AS patient) but high expression in an HLA-B27 negative sample (non-AS patient); and a potential marker is provided for AS diagnosis. A detection method of miR-495 is simple and easy to implement and mainly needs a quantitative PCR instrument such as a real-time fluorescent quantitative PCR instrument and can be implemented in a conventional laboratory, thereby greatly improving the detection practicability. Moreover, the detection of the miR-495 expression level is relatively accurate, and the minimum level of detection can realize single copy, thereby remarkably improving the detection accuracy of AS disease.
Description
Technical field
The invention belongs to field of biomedicine technology.More particularly, to Microrna -495 (miR-495) as inspection
Application in terms of the mark of survey ankylosing spondylitises.
Background technology
Ankylosing spondylitises (ankylosing spondylitis, AS) are that one kind mainly involves sacroiliac joint, axial bone
Chronic auto-immune diseases associated with inflammation.At present, reported according to available data, the sickness rate about 3 ‰ of China AS patient, real
Border quantity is likely larger than this ratio.AS is in high genetic, and it is main group of people at high risk that man is between twenty and fifty, and age of onset is usually 26
Year (male：Women=2:1), and AS early diagnosiss delay time at stop be 8-11.Because of its definite cause of disease and pathogenesis still not
Clear, lack specific short, and the features such as age of onset is early, the course of disease is long, it has also become a worldwide treatment difficult problem.This is to AS in early stage
Diagnosis, Disease Activity assessment and the aspect such as prediction and treatment propose great challenge.
The expression of HLA-B27 antigen has high correlation with ankylosing spondylitiss.Epidemiological study shows, AS and
There is certain ratio in SpA (another form of AS) and HLA-B27.In AS patient, the U.S., European and Chinese HLA-B27 sun
The ratio about 85-95% of property.In AS, HLA-B27 and c reactive protein (CRP：C-reactive protein) it is the most frequently used
Biomarker.But, have research to show, in a population, only 5% HLA-B27 positive individuals develop into AS or
SpA.HLA-B27 while more than 90%AS patient is the positive, but in general population only 5-10% for the positive, with total people
Mouth is compared, and the crowd of the HLA-B27 positive is 6% in the U.S.；Europe is 4-14%, higher with the ratio in Northern Europe；And in China be
8%；And AS is difficult to make a definite diagnosis as its symptom is similar to numerous disease.Secondly, HLA-B27 is in other seronegativity osteoarthrosis
Sick (such as Reiter ' s syndrome, psoriasis arthropathica, uveitis etc.) also have obvious expression, and this to a certain extent will drop
Its specificity low.And AS pathogenesis are unclear, age of onset is early, the course of disease is long, causes it will be by early diagnosiss
It is delayed significantly.And, the testing result of existing AS is mainly by HLA- in flow cytomery detection object peripheral blood
The expression of B27 is being judged, the instrument of needs is flow cytometer, and the instrument and equipment price is very high；And HLA-B27 is in streaming
The detection of cellular level, is difficult to judge (the marginal value of flow cytomery HLA-B27 for some results in marginal value
For 147), greatly limit the detection of of AS disease itself.
Although recent years, people were gradually increased to the understanding of AS, the pathogenesis of such as AS, inflammatory signals path, new bone
Formed and loss of cartilage, but clinically useful biomarker remains and is difficult to determine.Therefore, in assessment and the pipe of the disease
The aspects such as reason need the discovery of new mark, it is found that the early stage for AS is examined by the biomarker related with new AS is searched out
Disconnected, prevent and treatment provides new reference and reference.
Microrna (microRNA, miRNA) is class endogenouss, the non-coding RNA of long 20-25 nucleotide.Their master
Want function to interact by specificity to act on some mRNA, so as to inducing its degraded or suppressing its translation, be that a class is being given birth to
RNA molecule in life activity with critical function.There is the RNA molecule of critical function as a class, miRNA wide participation life
Each process of life activity：The differentiation of cell and development, propagation and apoptosis, different physiology and pathology, Ia disease
Deng.Although existing many researchs show：MiRNAs is with very important effect in the development of AS, but which miRNA can
Using the biomarker as AS diagnosis and prognosis, up to the present also relatively clearly come to a conclusion, even not same grinds
It is contrary to study carefully the conclusion that group obtains.Therefore, also needed to further by the use of miRNA as the diagnosis of AS or clinical practice
Research and exploration.
Content of the invention
The technical problem to be solved in the present invention is to overcome existing ankylosing spondylitises (AS) checkout and diagnosis mark and correlation
The defect and deficiency of technology, provides a kind of application of miRNA in detection ankylosing spondylitises.
It is an object of the invention to provide Microrna -495 (miR-495) is in the mark as detection ankylosing spondylitises
The application of aspect.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
Application of the miR-495 in terms of the mark as detection ankylosing spondylitises (AS), it is characterised in that described
The sequence of miR-495 (hsa-miR-495) is：AAACAAACAUGGUGCACUUCUU.
We have discovered that, under Microrna -495 (miR-495) is commonly in HLA-B27 positive sample (AS patient)
Mileometer adjustment reaches, and is high expression in the ' negative ' specimens (non-AS patient) of HLA-B27；One kind is provided potentially for the diagnosis of AS
Mark.
On instrument, the testing result of existing AS is mainly by flow cytomery detection object peripheral blood
The expression of HLA-B27 is being judged, the instrument of needs is flow cytometer, and the instrument and equipment price is very high, greatly limit
The detection of of AS disease itself.And the detection of miR-495 of the present invention is mainly real-time fluorescence quantitative PCR, basic in Routine Test Lab
Can buy, which greatly enhances the practicality of detection.
In addition, detection of the HLA-B27 in fluidic cell level, is difficult to judge (stream for some results in marginal value
The marginal value of formula cell instrument detection HLA-B27 is 147).And the detection of miR-495 expression of the present invention can be compared accurately,
The floor level of its detection can reach single copy, which greatly enhances the accuracy of detection.
Further, HLA-B27 comprehensive descision is combined using miR-495, diagnostic accuracy can be more substantially improved.
Therefore, miR-495 combines application of the HLA-B27 in terms of the mark as detection ankylosing spondylitises, also exists
Within protection scope of the present invention.
Judge, comprehensive analysis diagnose AS, can show by detecting that the expression of miR-495 combines the yin and yang attribute of HLA-B27
Write lifting detection accuracy and susceptiveness.
In addition, the application of the material of detection miR-495 or reagent in terms of the reagent of detection ankylosing spondylitises is prepared,
Within protection scope of the present invention.
Specifically, as a kind of preferred embodiment, the material of the detection miR-495 or reagent are PCR primer
Right, sequence is as follows：
Forward primer：5’-GGGCAAACAAACATGGTGCA-3’；
Downstream primer：5’-CAGTGCGTGTCGTGGAGT-3’.
It is highly preferred that the material of the detection miR-495 or reagent also include reverse transcriptase primer：
5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACG ACAAGAAGTG-3’.
Real-time fluorescence quantitative PCR is carried out using the primer, can sensitively detect very much the miR-495 expression water of sample
Flat.
A kind of test kit of detection ankylosing spondylitises, includes the PCR primer pair of detection miR-495.
Preferably, the sequence of the PCR primer pair is as follows：
Forward primer：5’-GGGCAAACAAACATGGTGCA-3’；
Downstream primer：5’-CAGTGCGTGTCGTGGAGT-3’.
It is highly preferred that the test kit also includes reverse transcriptase primer：
5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACG ACAAGAAGTG-3’.
In addition, within the skill of the art, can realize sample miR-495's by various means and experimental technique
Detection, can all reach the purpose of diagnosis AS.
The invention has the advantages that：
The invention provides miR-495 as detection ankylosing spondylitises mark in terms of application, be the detection of AS
There is provided a kind of new detection mark.
The detection of miR-495 is simple, and conventional molecular biosciences laboratory can be completed.Instrument is needed to be mainly quantitative PCR
Instrument, more preferably real-time fluorescence quantitative PCR instrument, Routine Test Lab can be bought substantially, substantially increase the practicality of detection.
In addition, the detection of miR-495 expression can be compared accurately, the floor level of its detection can reach single
Copy, which greatly enhances the detection accuracy to AS disease.
Description of the drawings
Fig. 1 is the method flow for diagnosing AS with miR-495 as detection object.
Fig. 2 is the solubility curve of MiR-495 quantitative fluorescent PCR.
Fig. 3 is electrophoresis detection PCR primer result.
Fig. 4 is the sensitivity results of quantitative fluorescent PCR.
Fig. 5 is pattern detection result.
Fig. 6 is clinical case monitoring result.
Specific embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the reagent of employing, method and apparatus are the art conventional reagent, method
And equipment.
Unless stated otherwise, following examples agents useful for same and material are commercial.
Embodiment 1
1st, sample
The HLA-B27 positive (HLA-B27+) sample number is 25 (having been determined as AS patient)；
HLA-B27 feminine gender (HLA-B27-) is 15 (having determined that non-AS patient).
2nd, method is as shown in Figure 1
Obtain the fresh blood 3-5ml of detection object first, then monokaryon therein is obtained by density gradient centrifugation thin
Born of the same parents, then extract the total serum IgE of corresponding cell, finally by real-time fluorescence quantitative PCR to the miR- in sample by Trizol method
495 genes are detected (with U6 as reference gene).
Specifically, the method for the real-time fluorescence quantitative PCR is as follows：
(1) reverse transcription obtains cDNA
A () reverse transcriptase primer sequence is as follows：
5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAAGAAGTG-3’
B () is according to system (10.0 μ l) as shown in table 1.Reacted with the program shown in table 2.
Table 1
| Reagent | Usage amount |
| 5×gDNA Eraser Buffer | 2.0μl |
| gDNA Eraser | 1.0μl |
| Total RNA | 500ng |
| RNase Free dH2O | 10 μ l of polishing |
Table 2
| Stage 1 | 42℃ | 2min |
| Stage 2 | 4℃ | 1hr |
C () is reacted with the program shown in table 4 according to the system shown in table 3.
Table 3
| Reagent | Usage amount |
| The reactant liquor of (b) system | 10.0μl |
| PrimeScript RT Enzyme Mix I | 1.0μl |
| RT primer | 1.0μl(0.5pmol) |
| 5×PrimeScript Buffer 2(for Real Time) | 4.0μl |
| RNase Free dH2O | 4.0μl |
Table 4
| Stage 1 | 42℃ | 30min |
| Stage2 | 85℃ | 5sec |
| Stage 3 | 4℃ | 1hr |
Note：3 system U6 gene of reference gene table is random primer, and concentration is 50pmol.And the stage1 in 4 program of table
In be 37 DEG C.
(2) PCR primer and condition
With the cDNA of above-mentioned acquisition as template, enter performing PCR amplification.
A () PCR primer is：
5 '-GGGCAAACAAACATGGTGCA-3 ' and 5 '-CAGTGCGTGTCGTGGAGT-3 '.
B () PCR system is as shown in table 5：
Table 5
| Reagent | Usage amount | Final concentration |
| SYBR Premix Ex Taq II(Tli RNaseH Plus)(2×) | 12.5μl | 1× |
| PCR Forward Primer(10μM) | 1.0μl | 0.4μM |
| PCR Reverse Primer(10μM) | 1.0μl | 0.4μM |
| CDNA solution | 4μl | 5 times of dilution |
| dH2O (sterile purified water) | 6.5μl |
C () PCR program is as shown in table 6：
Table 6
3rd, as shown in Figure 2, the solubility curve of MiR-495 quantitative fluorescent PCR is single for result, shows PCR primer specificity
Very well.
As shown in Figure 3, further electrophoresis detection PCR primer is single product, to demonstrate the spy of PCR primer further
The opposite sex.
4th, the result of such as accompanying drawing 4 shows, the sensitivity of quantitative fluorescent PCR is very high, can detect low-down template amount,
Ct value=39 during 1 template copy.
Further, show through substantial amounts of research and experimental verification, using real-time fluorescence quantitative PCR detection sample
MiR-495 gene, when ct value is more than 34, shows that miR-495 gene expression is relatively low, and sample is that the risk of AS patient is higher.
5th, such as 5 result of accompanying drawing shows, compared with non-AS patient, Microrna -495 (miR-495) expression of AS patient is universal
Notable downward, is but high expression in non-AS clinical samples.
The prison detection of 2 clinical case of embodiment
With a clinical definite as the sample of AS disease, the detection of MiR-495 is carried out.Method is with embodiment 1.
As a result as shown in Figure 6, Ct=34.8, shows that miR-495 gene expression is relatively low, sample be the risk of AS patient relatively
High.Testing result is coincide with actual.
Claims (9)
- Application of the 1.miR-495 in terms of the mark as detection ankylosing spondylitises, it is characterised in that the miR-495 Sequence be：AAACAAACAUGGUGCACUUCUU.
- 2.miR-495 combines application of the HLA-B27 in terms of the mark as detection ankylosing spondylitises.
- Application of the 3.miR-495 in terms of the reagent of detection ankylosing spondylitises is prepared.
- 4. the application of the material of detection miR-495 or reagent in terms of the reagent of detection ankylosing spondylitises is prepared.
- 5. application according to claim 4, it is characterised in that the material of the detection miR-495 or reagent are PCR primer To and/or reverse transcriptase primer；The PCR primer is to for forward primer：5 '-GGGCAAACAAACATGGTGCA-3 ', and downstream draws Thing：5’-CAGTGCGTGTCGTGGAGT-3’；The reverse transcriptase primer sequence is：5’- GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAAGAAGTG-3’.
- 6. a kind of detection ankylosing spondylitises test kit, it is characterised in that include detection miR-495 reagent.
- 7. test kit according to claim 6, it is characterised in that the reagent be.
- 8. test kit according to claim 7, it is characterised in that the sequence of the primer pair is as follows：Forward primer：5’-GGGCAAACAAACATGGTGCA-3’；Downstream primer：5’-CAGTGCGTGTCGTGGAGT-3’.
- 9. test kit according to claim 7, it is characterised in that also include reverse transcriptase primer, sequence is：5’- GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACAAGAAGTG-3’.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484613A (en) * | 2019-07-05 | 2019-11-22 | 新乡医学院第一附属医院 | A kind of ankylosing spondylitis early diagnosis marker |
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| CN104152452A (en) * | 2014-08-19 | 2014-11-19 | 中国人民解放军总医院第一附属医院 | Blood miRNA marker related to liver cancer and application thereof |
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Application publication date: 20170222 |