CN102140452A - Single-chain non-encoding Micro RNA (Ribonucleic Acid) III and application thereof - Google Patents
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Abstract
The invention discloses a single-chain non-encoding Micro RNA (Ribonucleic Acid), wherein the sequence of the single-chain non-encoding Micro RNA200a is shown as the SEQ ID NO.1. The invention simultaneously discloses an application of the single-chain non-encoding Micro RNA in preparing reagent kit for diagnosing serious hepatitis B. The invention also discloses a reagent kit for diagnosing the serious hepatitis B, which comprises a PCR (Polymerase Chain Reaction) reagent, a probe, an upstream primer and a downstream primer, wherein the sequence of the probe is shown as the SEQ ID NO.5; the sequence of the upstream primer is shown as the SEQ ID NO.3; and the sequence of the downstream primer is shown as the SEQ ID NO.4.
Description
Technical field
The present invention relates to expression level and the effect in prediction and diagnosis of hepatitis b worsening thereof in the hepatitis B critically ill patient blood plasma of a kind of MicroRNA.
Background technology
At present the whole world estimates at 3.5 hundred million chronic viral hepatitis B virus (HBV) carrier, has 1/3rd population to infect HBV.In China, hepatitis B be the most seriously, one of transmissible disease the most widely, infection rate has 1.2 hundred million people to carry HBV up to 60% approximately.The sickness rate of hepatitis B occupies the transmissible disease first place, and death toll occupies the 3rd of transmissible disease.
The hepatitis B hepatitis gravis is that a class shows as the syndromes that a large amount of hepatic necrosis of short-term cause liver failure, and its mortality ratio is high, and development rapidly, and is dangerous and clinical prognosis is poor.And the healthy hepatopathy of serious threat people, therefore, early diagnosis is so that in time take therapy measure to be even more important.
MicroRNA is the non-coding single stranded RNA fragment of about 22nt of being produced by Dicer enzyme cutting back of the hair clip shape structure precursor (pre-MicroRNA) of about 70nt of being transcribed by the body native gene, high conservative during evolution, can be by matching with the specific base complementrity of target gene mRNA, cause the degraded of target gene mRNA or suppress its translation, widely the negative regulation target gene expression.MicroRNA intervenes instrument as a kind of new gene, has advantages such as efficient, special, quick, for the research of gene regulating and the development of gene therapy have brought new visual angle.MicroRNA is highly stable, and the research and development that are used for reagent for clinical diagnosis have advantages such as good reproducibility, easy operation.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of non-coding of strand MicroRNA200a that utilizes and prepares diagnostic kit with diagnosing myasthenia hepatitis.
In order to solve the problems of the technologies described above, the invention provides the non-coding of a kind of strand MicroRNA, the non-coding of strand MicroRNA200a is the sequence shown in the SEQ ID NO:1.
The present invention also provides the non-coding of above-mentioned strand MicroRNA the application in the test kit of the B-mode hepatitis gravis of preparation diagnosis simultaneously.
The present invention also provides a kind of test kit that is used to diagnose B-mode hepatitis gravis simultaneously, comprises PCR reagent, probe, upstream primer and downstream primer; Probe is the sequence shown in the SEQ ID NO:5, and upstream primer is the sequence shown in the SEQ ID NO:3, and downstream primer is the sequence shown in the SEQ ID NO:4.
As the improvement that is used to diagnose the test kit of B-mode hepatitis gravis of the present invention: PCR reagent is 1 * TaqManUniversal PCR MasteMix.
MicroRNA is the single stranded RNA (about 22～23 Nucleotide) of a class high conservative, bioinformatic analysis shows, each microRNA may regulate hundreds of target genes, prompting microRNA may participate in regulating signal transduction pathways numerous in the cell activities, plays a role in a series of processes such as cell proliferation, differentiation, apoptosis, immune response and vasculogenesis.In addition, the unconventionality expression of MicroRNA will cause the generation of physiological unusual and disease, and the generation of disease also will cause the change of the expression level of MicroRNA on the contrary, and people can utilize it to reflect the degree of disease and make corresponding treatment.
The MicroRNA sequencing is simple relatively, by the MicroRNA that exists in the human peripheral blood of genetic engineering technique separation and purification with comparalive ease, and it is checked order.Up to the present reported and surpassed 800 MicroRNA sequences, but because the complicacy of MicroRNA function, function and the purposes of most of MicroRNA still belong to the unknown, therefore, seek and develop that useful MicroRNA is used for clinical diagnosis and treatment is the current research focus.U.s.a. applied biosystem company has researched and developed MicroRNA chip (TaqMan MicroRNA Arrays) according to present present Research, it comprises the reverse transcription primer of most of known MicroRNA, utilizes this chip can detect these MicroRNA changes of expression level situations of known array.The present invention utilizes this chip that MicroRNA in the hepatitis B patient with severe symptoms blood plasma is detected, with normal artificial contrast, the patient is made a more comprehensive MicroRNA collection of illustrative plates of ratio, therefrom find out and the closely-related MicroRNA molecule of hepatitis B worsening change of illness state.Utilize this molecule can research and develop prediction and diagnosis worsening of hepatitis diagnostic reagent.
In the present invention, the contriver observes the change situation of MicroRNA under the hepatitis B severe state by detecting the expression level of MicroRNA 200a in the hepatitis B patient with severe symptoms blood plasma.The present invention chooses the normal people and has been diagnosed as hepatitis B patient with severe symptoms's blood plasma, extracts RNA from blood plasma, utilizes MicroRNA chip of expression spectrum (u.s.a. applied biosystem company
Human MicroRNA Array A (B)) MicroRNA is analyzed.Found that MicroRNA 200a significantly raises in hepatitis B patient with severe symptoms blood plasma, that is, this MicroRNA expresses in hepatitis B patient with severe symptoms blood plasma and obviously increases than the normal people.Prompting MicroRNA 200a plays important regulation in the hepatitis B worsening, this MicroRNA can be used as the diagnosis marker of hepatitis B worsening simultaneously.
Adopt method of the present invention to come early diagnosis hepatitis B worsening, have following advantage:
The present invention has confirmed the potential target spot of MicroRNA200a as diagnosis hepatitis B hepatitis gravis, can be used for rapid detection and diagnosis.
MicroRNA is highly stable in blood plasma and serum, and they are effectively protected in order to avoid contact RNases, still can keep stable under the condition of harsh and unforgiving environments.Therefore, to make that its actual value and the test value that obtains patient coincide on one's body fine for their stability.The detection technique of microRNA express spectra is ripe in the serum, the common technology of research microRNA express spectra mainly contains Northern hybridization, expression chip, real-time fluorescence quantitative PCR and Solexa order-checking etc. at present, method is quick, convenient, and tolerance range and sensitivity can be satisfied clinical demand.
Serum microRNA mainly contains following advantage as the mark of clinical diagnosis and prognosis: the damage of detection is little, good stability, highly sensitive, can be applicable to the early stage detection that changes of disease.Have not yet to see the microRNA that is used for the diagnosis of hepatitis b worsening, but microRNA is used to diagnose other diseases to obtain many encouraging effects: for example blood plasma miR-92 successfully is used as the molecular marker of colorectal cancer, sensitivity can reach 89%, specificity reaches 70%, and the expression amount of miR-92 significantly reduces before than art behind the tumor resection; Early diagnosis can improve survival rate greatly and improve prognosis.
Description of drawings
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Fig. 1 is a MicroRNA chip of expression spectrum detected result, and MicroRNA 200a expresses hardly among the normal people, the then tangible high expression level of hepatitis B patient with severe symptoms.
Embodiment
The extraction of total RNA in embodiment 1 blood plasma
Choose Zhejiang University Medical College The First Affiliated Hospital, hepatitis B patient with severe symptoms's 4 examples are chosen the normal people that 4 example ages, sexes are complementary simultaneously, extract the 4ml whole blood; Promptly extract hepatitis B patient with severe symptoms's whole blood, each 4 example of whole blood of normal people.As confidential reference items, relative expression's level of MicroRNA is with 2 with U6snRNA
-Δ CtExpression, detection method is identical.Real-time fluorescence quantitative PCR provides according to u.s.a. applied biosystem company
MicroRNA Arrays operates.
We mix (that is, 4 routine patients' blood plasma mixes, and 4 routine normal peoples mix) on the same group blood plasma not in order to reduce individual difference.Utilize mirVana
TMPARIS
TMKit comes to extract total RNA from plasma sample, extracting method is according to the specification sheets operation of this test kit, and process is as follows successively:
1. with plasma sample (maximum volume 625 μ L) (freeze thawing on ice) and isopyknic 2X Denaturing Solution room temperature mixing.If plasma sample is less than 100ul, add Cell Disruption Buffer earlier to 〉=100 μ L, add isopyknic 2X Denaturing Solution again, abundant at once mixing (vortex 30s) is hatched 5min on ice.
2. the acidifying phenol/chloroform that is provided in the adding test kit is to above-mentioned mixed solution.
3. vortex shakes the 30-60s mixing.
4. the centrifugal 5min of room temperature 10,000 * g isolates water and organic phase (water is on the upper strata).
5. shift the water that is positioned at the upper strata and manage to new EP, and the mark volume.
6. 100% ethanol (room temperature) that adds 1.25 times of volumes of water arrives water, fully mixing.
7. Filter column is placed Collection Tube (collection tube), draw lysate and alcoholic acid mixture (being step 6 gained) to Filter column.
8.10, the centrifugal 30s of 000 * g.
9. abandon filtrate, repeated centrifugation is till all lysate/alcohol mixtures all filter Filter column.Filter column is placed collection tube again.Add 700 μ L MicroRNA Wash Solution 1 to Filter column.The centrifugal 15s of 10,000 * g.
10. abandon filtrate, put Filter column to identical collection tube.
11. add 500 μ L Wash Solution 2/3 to Filter column.The centrifugal 15s of 10,000 * g abandons filtrate, puts Filter column to identical collection tube.
12. repeating step 11.
13.10 000 * g sky is from 1min.
14. shift Filter column to new collection tube.
15. add the elutriant of 100 μ L preheatings (95 ℃) or nuclease free water to Filter column central authorities, close lid, the centrifugal 30s of 10,000 * g abandons post, collects total RNA ,-20 ℃ preserve or more low temperature preserve.
Used reagent is conventional reagent in following examples, can obtain by commercial mode, for example, can be available from American AB I company.
Embodiment 2 increases in advance
1. carry out reverse transcription (RT)
(1) the following reagent of freeze thawing:
Megaplex?RT?Primers
MgCl
2
Reaction system:
3 μ L (1 to 350ng) total RNA (embodiment 1 gained)
4.5 μ L of RT reaction mix (as table 1)
(2) the following reagent of mixing (as described in Table 1) is little from pipe to 1.5ml.
Table 1
(3) inversion is managed 6 times with mixing, the centrifugal a little several seconds once.
(4) at 96-well
Optical Reaction Plate or
Among the 8-Tube Strip, draw 4.5 μ L RT reaction mixtures to each hole or pipe.
(5) add the total RNA of 3 μ L (1 to 350ng) to each hole or the pipe that contain the RT reaction mixture, obtain the mixed solution of 7.5ul volume.
(6) use
Clear Adhesive Film or
Optical Strip Caps seals, and then is inverted plate or manages 6 times with mixing, and is once centrifugal a little.
(7) ice bath 5min.
(8) following RT operational conditions (table 2) is set, reaction volume: 7.5 μ l.
Table 2
The cDNA that obtains can-15 to-25 ℃ preserves more than the week.
2. pre-amplification reaction
Pre-amplification reaction volume: 25 μ L
2.5 μ L RT product (being the cDNA of above-mentioned RT step gained)
22.5 μ L PreAmp reaction mix (as shown in table 3 below)
(1) freeze thawing Megaplex on ice
TMPreAmp Primers is inverted and manages 6 times with mixing.
(2) reverberate bottle with mixing
PreAmp Master Mix (2 *).
(3) the following reagent of mixing (table 3) is little from pipe to 1.5ml.
Table 3
(4) inversion is managed 6 times with mixing, and is once centrifugal a little.
(5) at 96-well
Optical Reaction Plate or
Among the 8-Tube Strips, add 2.5 μ L RT products to corresponding hole or pipe.
(6) add 22.5 μ L of PreAmp reaction mix again in 96-well plate or MicroAmp Optical 8-Tube.
(7) use
Clear Adhesive Film or
Optical Strip Caps seals, and is inverted plate or manages 6 times with mixing, and is once centrifugal a little.
(8) ice bath 5min.
(9) reaction volume (μ L): 25
(10) move according to following reaction conditions (table 4).
Table 4, reaction conditions
3. diluting reaction system
(1) takes out 96-well plate or 8-tube strips.
(2) once centrifugal a little.
(3) add 75 μ L of, 0.1 * TE pH 8.0 to each hole or pipe.
(4) plate or channel closure are inverted plate or are managed 6 times with mixing, and be once centrifugal a little.
(5) pre-expansion volume increase thing is kept at-15 to-25 ℃.Perhaps product is put and carried out follow-up real-time fluorescence quantitative PCR reaction on ice.
Embodiment 3, real-time fluorescence quantitative PCR
The preparation of PCR reaction solution:
1. the pre-expansion volume increase thing of the dilution of freeze thawing on ice preservation is inverted 6 times with mixing, and is once centrifugal a little.
2. reverberate bottle with mixing TaqMan Universal PCR Master Mix.
3. it is little from pipe following reagent (table 5) to be added to 1.5mL.
Table 5
Be inverted 6 times with mixing, then once centrifugal a little.
4. add 100 μ L PCR reaction mix in the passage separately of TaqMan MicroRNA Array.
5. centrifugal, then seal.
6. go up sample and move 384 well TaqMan Low Density Array default thermal-cycling conditions.
7. collecting the Δ Ct value of each MicroRNA after reaction is finished analyzes.Specifically as shown in table 6.
Table 6
MicroRNA chip of expression spectrum detected result, MicroRNA 200a expresses hardly among the normal people, and hepatitis B patient with severe symptoms is tangible high expression level then, can be used as the diagnosis marker of hepatitis B worsening.
Contain four kinds of compositions: A:1 * TaqMan Universal PCR MasteMix in the test kit, B: probe (SEQ ID NO:5), C: upstream primer (SEQ ID NO:3), D: downstream primer (SEQ ID NO:4).Wherein, A is a regular-PCR reagent, can obtain by commercial mode; B, C, D are the special probe and the primers of the 5uM concentration of the MicroRNA 200a sequences Design elite according to the present invention, need dilute during actual the use; Also can directly select 0.2uM probe (SEQ ID NO:5), 1.5uM upstream primer (SEQ ID NO:3) and 0.7uM downstream primer (SEQ ID NO:4) for use.
Using method is specific as follows:
1, extracting RNA:
Extract patient's to be checked 4ml whole blood, extract total RNA in the blood plasma according to embodiment 1 described method then;
2, pre-amplification:
(1), carries out reverse transcription (RT)
1) melt following reagent:
Stem-loop reverse transcriptase primer (SEQ ID NO:2)
MgCl
2
2) reaction system:
3 μ L (1 to 350ng) total RNA (above-mentioned steps 1) gained)
4.5 μ L of RT reaction mix (as table 7)
3) the following reagent of mixing (as described in Table 7) is little from pipe to 1.5ml.
Table 7
4) working method is with the reverse transcription (RT) of embodiment 2.
(2) pre-amplification reaction
1) reaction volume that increases in advance: 25 μ L
2.5 μ L RT product (being the cDNA of above-mentioned RT step gained)
22.5 μ L PreAmp reaction mix (as shown in table 8 below)
2) freeze thawing Megaplex on ice
TMPreAmp Primers is inverted and manages 6 times with mixing.
3) reverberate bottle with mixing
PreAmp Master Mix (2 *).
4) the following reagent of mixing (table 8) is little from pipe to 1.5ml.
Table 8
5) working method is with the pre-amplification reaction of embodiment 2.
(3) the diluting reaction system is with the diluting reaction system of embodiment 2.
3, real-time fluorescence quantitative PCR
(1) preparation of PCR reaction solution:
1) the pre-expansion volume increase thing of the dilution of freeze thawing on ice preservation is inverted 6 times with mixing, and is once centrifugal a little.
2) reverberate bottle with mixing 1 * TaqMan Universal PCR MasteMix;
3) it is little from pipe following reagent (table 9) to be added to 0.2mL.
Table 9
| The reagent composition | Volume/ |
| 1×TaqMan?Universal?PCR? |
1 |
| Pre-expansion volume increase thing | 0.67 |
| 0.2uM probe (SEQ ID NO:5) | 1.25 |
| 1.5uM upstream primer (SEQ ID NO:3) | 1.25 |
| 0.7uM downstream primer (SEQ ID NO:4) | 1.25 |
| Nuclease-free?water | 4.58 |
| Cumulative volume | 10ul |
4) be inverted 6 times with mixing, then once centrifugal a little, seal.
5) go up sample operation PCR reaction: the PCR response procedures is: 95 ℃ of 10min, move 60 ℃ of 1min of 40 95 ℃ of 15s of circulation of following program then.
6) the average Ct value defined of gained was Ct1 after reaction was finished.
4, judge:
Adopt same patient's cDNA to detect with quantitative fluorescent PCR at U6snRNA, method and program the same (its used probe and primer are universal standard product, can by
Company buys).The average Ct value defined of gained was Ct2 after reaction was finished.
ΔCt＝Ct1-Ct2。When Δ Ct value＜5, be judged to be the hepatitis B worsening.
4 people that choose 4 people that are known as healthy person in advance respectively and be known as the hepatitis B critically ill patient in advance use embodiment 4 described test kits (comprising using method) to detect respectively as experimental subjects, and the result is shown in table 10 and table 11;
Table 10, normal healthy controls group
Table 11, hepatitis B critically ill patient group
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
SEQ?ID?NO：1
auaacacugu?cugguaacga?ugu 23
SEQ?ID?NO：2
ctcaactggt?gtcgtggagt?cggcaattca?gttgagacat?cgtt 44
SEQ?ID?NO：3
tctcaactgg?tgtcgtggag?tc 22
SEQ?ID?NO：4
aggctgcact?gataactctg?tctg 24
SEQ?ID?NO：5
caattcagtt?gagacatcgt 20
Claims (4)
1. the non-coding of strand MicroRNA, it is characterized in that: the non-coding of strand MicroRNA 200a is the sequence shown in the SEQ ID NO:1.
2. the application of the non-coding of strand as claimed in claim 1 MicroRNA in the test kit of the B-mode hepatitis gravis of preparation diagnosis.
3. a test kit that is used to diagnose B-mode hepatitis gravis is characterized in that: comprise PCR reagent, probe, upstream primer and downstream primer; Described probe is the sequence shown in the SEQ ID NO:5, and upstream primer is the sequence shown in the SEQ ID NO:3, and downstream primer is the sequence shown in the SEQ ID NO:4.
4. the test kit that is used to diagnose B-mode hepatitis gravis according to claim 3 is characterized in that: described PCR reagent is 1 * TaqMan Universal PCR MasteMix.
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|---|---|---|---|---|
| US20100021914A1 (en) * | 2006-11-23 | 2010-01-28 | Querdenker Aps | Oligonucleotides for modulating target rna activity |
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| US20100021914A1 (en) * | 2006-11-23 | 2010-01-28 | Querdenker Aps | Oligonucleotides for modulating target rna activity |
Non-Patent Citations (2)
| Title |
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