CN107541564A - Molecular marked compound TCONS_00016233, kit and application - Google Patents
Molecular marked compound TCONS_00016233, kit and application Download PDFInfo
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- CN107541564A CN107541564A CN201710915173.XA CN201710915173A CN107541564A CN 107541564 A CN107541564 A CN 107541564A CN 201710915173 A CN201710915173 A CN 201710915173A CN 107541564 A CN107541564 A CN 107541564A
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- septicopyemia
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention discloses a kind of molecular marked compound TCONS_00016233, kit and application.The molecular marked compound is used to early diagnose, predict septicopyemia complicated with acute injury of kidney.TCONS_00016233 in septicopyemia and acute injury of kidney blood samples of patients expresses in high, by the expression for detecting TCONS_00016233 in blood samples of patients, it can be determined that whether patient suffers from septicopyemia acute injury of kidney or suffer from the probability of septicopyemia acute injury of kidney.Septicopyemia and acute injury of kidney early diagnosis, the means of prediction specifically, are sensitively realized the invention provides a kind of.
Description
Technical field
The present invention relates to molecular diagnostic techniques field, and in particular to one kind is used to early diagnose, predict that septicopyemia is concurrent
Molecular marked compound, kit and the application of acute injury of kidney.
Background technology
Septicopyemia is one of critical heavy patient's common disease factor and complication, and its case fatality rate is up to more than 50%.Meanwhile septicopyemia
Mass formed by blood stasis is also the common cause that acute injury of kidney occurs for patient with severe symptoms, once patients with sepsis complicated with acute injury of kidney, its disease
Dead rate may be up to 70%.Therefore, early diagnosis and therapy is the key for reducing the septicopyemia correlation AKI incidences of disease and case fatality rate.
The diagnosis of acute injury of kidney at present relies primarily on serum creatinine and urine volume, but serum creatinine and urine volume are affected by many factors, it is impossible to and
When reflect that renal function changes exactly, enough Sensitivity and Specificity is lacked to AKI diagnosis.It is existing to be lost, it is known that working as renal function
It can just be changed up to creatine concentration when 50%, and creatinine reaches the time that stable state needs several days, therefore can not reflect in time
Renal function.In addition, creatinine is by age, sex, race, body capacity condition, muscle catabolism, protein uptake, stomach
Many kidney other factors such as intestinal bleeding and medicine influence.It can be seen that serum creatinine rise often lags behind the deterioration of renal function, and can not
Accurately reflect the change of renal function.Therefore, hypersensitivity and specific biological markers are found, are examined for AKI early stage
Disconnected and prognosis evaluation is most important.
Long-chain non-coding RNA is the non-coding RNA that length is more than 200 nucleotides, and research shows that it is in a variety of diseases
Occur, unconventionality expression occur in evolution, can react advancing of disease and prognosis, and its express in blood it is relatively steady
It is fixed, as molecular marked compound have it is special, sensitive, quick, conveniently, it is with strong points the advantages of.
The content of the invention
An object of the present invention is to provide the molecular marked compound of a kind of energy early detection septicopyemia and acute injury of kidney,
To early diagnosing, predicting septicopyemia, simultaneously acute injury of kidney is significant.
A kind of molecular marked compound TCONS_ for being used to early diagnosing, predicting septicopyemia complicated with acute injury of kidney
00016233, sequence such as SEQ ID NO：Shown in 1.
The second object of the present invention is to provide above-mentioned molecular marked compound TCONS_00016233 application.Detect TCONS_
Application of the product of 00016233 expression in early diagnosis, prediction septicopyemia complicated with acute injury of kidney instrument is prepared.
The product includes：The system of TCONS_00016233 expressions is detected by RT-PCR or real-time quantitative PCR
Agent.
With the specific amplification TCONS_00016233 of RT-PCR detection TCONS_00016233 expressions primer sequence
It is classified as：
F：5’-AACGCTCTGACATCTTCCGT-3’
R：5’-TTCCGAATCCACAGATGGCG-3’.
The third object of the present invention is to provide a kind of examination for being used to early diagnosing, predicting septicopyemia complicated with acute injury of kidney
Agent box, include the reagent that TCONS_00016233 expressions are detected by RT-PCR or real-time quantitative PCR.
The described kit for being used to early diagnose, predicting septicopyemia complicated with acute injury of kidney, passes through RT- comprising a pair
PCR specific amplifications TCONS_00016233 primer, its primer sequence are：
F：5’-AACGCTCTGACATCTTCCGT-3’
R：5’-TTCCGAATCCACAGATGGCG-3’.
It is found by the applicant that TCONS_00016233 up-regulated expressions (Fig. 2) in septicopyemia and acute injury of kidney patient, are carried
It is the molecular marked compound of diagnosis, prediction septicopyemia and acute injury of kidney to show TCONS_00016233.The present invention is septicopyemia
Acute injury of kidney diagnosis, prediction provide strong molecular biology mechanism, have far-reaching clinical meaning and generalization.
Brief description of the drawings
The chip expression that Fig. 1 is LncRNA composes analysis result.
A. thermal map；B. the LncRNA, C. of pyemia AKI middle up-regulations compared with control group with control group and the non-AKI of pyemia
The pyemia AKI upper LncRNA lowered, D pyemias AKI and control group and non-AKI of pyemia of reconciling compared with the non-AKI of pyemia
The LncRNA of middle downward compared with control group；E, the non-AKI of pyemia are with control group up-regulation while in pyemia AKI and control group
The LncRNA that the LncRNA of downward, the non-AKI of pyemia are lowered in pyemia AKI and control group simultaneously with control group up-regulation；F generations
Table TCONS_00016233 raises in pyemia AKI compared with control group, is lowered in the non-AKI of pyemia compared with control group.
Fig. 2 is that RT-PCR demonstrates expression feelings of the TCONS_00016233 in control, pyemia AKI, the non-AKI of pyemia
Condition, A.TCONS_00016233 and internal reference GAPDH electrophoresis patterns；B is gray analysis.Difference has statistical significance.
Embodiment
The present invention is intended to further illustrate with reference to embodiments, is not intended to limit the present invention.
Embodiment 1：The screening molecular marked compound related to septicopyemia complicated with acute injury of kidney
1. sample is collected
Collect Healthy People, the non-acute injury of kidney of septicopyemia, septicopyemia complicated with acute impaired renal patient's blood preparation.
The preparation and quality analysis of 2.RNA samples
Total serum IgE is extracted for the Trizol of ShiJi Co., Ltd using health, concrete operation step is as follows：
1) take 2ml to collect the whole blood in the test tube that sodium citrate treats, be put into no enzyme centrifuge tube；
2) blood plasma is collected：3000rpm centrifuges 10min, and carefully siphoning away supernatant (blood plasma) at the top of sample is put into another nothing
In enzyme centrifuge tube；
3) 250 μ L plasma lipids are taken, are gone in 1.5ml centrifuge tube, 750 μ L TRIzol reagents, acutely vibration manually
Body extremely mixes.
4) sample is incubated 5 minutes in 15-30 DEG C after being homogenized.
5) 0.2ml chloroform is added in the sample of homogenate, covers tightly lid.Acutely vibration body is after 15 seconds manually, 15-30
It is incubated 2 to 3 minutes.12,000rpm is centrifuged 15 minutes at 4 DEG C.
6) mixing liquid is classified into the red phenol chloroform phase of lower floor, the colourless aqueous phase on intermediate layer core upper strata after centrifuging.RNA
All it is distributed in aqueous phase.500 μ L aqueous phases are drawn to be transferred in new centrifuge tube.
7) 500 μ L isopropanols are added in new centrifuge tube, mixed to precipitate RNA therein.15-30 DEG C is incubated 10 points after mixing
Zhong Hou, centrifuged 10 minutes in 4 DEG C of 12,000rpm.
8) supernatant is removed, adds at least 1ml 75% ethanol, cleans RNA precipitate.4 DEG C of 7,500 rpm centrifugation after vibration
5 minutes.
9) ethanol solution, air drying RNA precipitate 5-10 minutes are removed.
10) add without RNase water 20ul, blown and beaten repeatedly several times with pipettor.Then the centrifuge tube for filling RNA is covered, is deposited
In -80 DEG C of refrigerators.
11) RNA quality analyses：UseND-1000 is determined the RNA concentration and purity of said extracted.
3. high flux transcript profile is sequenced
1) RNA-seq reads position
2) transcript abundance is assessed
3) difference expression gene detects
4. result
RNA-seq results are as shown in figure 1, applicant's preliminary experiment have studied from 5 health using LncRNA chip means
Control, 15 pyemias are not with the blood plasma of AKI patient and 15 septic AKI patients.We are by analyzing microarray
Result learn：
(1) Healthy People is compared, there are 1084 lncRNA up-regulated expressions in the blood sample of pyemia AKI patient, 914
LncRNA expression is lowered；(2) Healthy People is compared, there are 538 lncRNA up-regulated expressions in blood plasma, 522 lncRNA expression are lowered；
(3) pyemia AKI has 1056 lncRNA up-regulated expressions compared with the non-AKI of pyemia, and 824 lncRNA expression are lowered；(4) purulence
The non-AKI of toxication has 207 with control group and pyemia the AKI lncRNA raised compared with control group, what expression was all lowered
LncRNA has 254；(5) under being expressed in the non-AKI of pyemia and up-regulated expression in control group up-regulation, pyemia AKI and control group
The lncRNA of tune has 110；Express and lower in the non-AKI of pyemia and control group, pyemia AKI and up-regulated expression in control group
LncRNA have 87.(6) expression quantity of the TCONS_00016233 in septicopyemia and acute injury of kidney blood is significantly higher than
The expression quantity of the non-acute injury of kidney of septicopyemia and Healthy People (Fig. 1 F).
Embodiment 2:RT-PCR verifies differential expression
1. chosen according to the testing result of high-flux sequence and carry out RT-PCR checkings.According to sample collection side in embodiment 1
Formula selection Healthy People, septicopyemia complicated with acute injury of kidney, the non-acute injury of kidney blood preparation of septicopyemia.
2.RNA extraction steps are the same as embodiment 1
3. reverse transcription：Operated, comprised the following steps that using the Reverse Transcriptase kit of ThermoFish companies：
1) agents useful for same is added, and is stored on ice, according to lower table row related experiment.
2) -20 DEG C store for future use.
4.RT-PCR is expanded
1) design of primers
The primer of PCR amplifications is designed according to TCONS_00016233 and Gapdh gene coded sequence, is given birth to as scheduled by Guangzhou
Thing Technology Co., Ltd. synthesizes.Specific primer sequence is as follows：
TCONS_00016233：
F：5 '-AACGCTCTGACATCTTCCGT-3 ' are (see SEQ ID NO：2)
R：5 '-TTCCGAATCCACAGATGGCG-3 ' are (see SEQ ID NO：3)
Gapdh genes：
F：5 '-CAAGGTCATCCATGACAACTTTG-3 ' are (see SEQ ID NO:4)
R：5 '-GTCCACCACCCTGTTGCTGTAG-3 ' are (see SEQ ID NO:5)
2) PCR reaction systems are prepared according to following table (it is ShiJi Co., Ltd that wherein Tap MasterMix premix systems, which are purchased to health)：
PCR response procedures
Denaturation and annealing repeat 35-40 circulation.
Agarose gel electrophoresis：Take 10 μ L PCR primers electrophoresis detection on 2% Ago-Gel, the fine jade of RT-PCR results
Lipolysaccharide electrophoresis photographs carry out gray scale scanning with image analysis system, and gray scale scanning value (densitometric scan value) is IA, and it represents solidifying
The brightness of purpose band on glue, that is, react the amount of purpose band.
The IA/ internal references GAPDH of relative quantity (IA ratios)=purpose fragment of PCR primer IA is calculated by appealing formula
Go out cDNA fragments healthy control group, pyemia complicated with acute injury of kidney group, the non-acute injury of kidney of septicopyemia IA ratios, should
Ratio is the relative quantity of RT-PCR products.
6. result：As a result as shown in Fig. 2 compared with Healthy People, TCONS_00016233 damages in pyemia complicated with acute kidney
Hinder the up-regulated expression in patient's blood；Compared with Healthy People, TCONS_00016233's non-acute impaired renal patient of septicopyemia exists
Expression in the non-acute impaired renal patient's blood of pyemia is lowered, consistent with RNA-seq results.
The explanation of above-described embodiment is at least used for the method and its core concept for understanding the present invention.Original of the invention is not being departed from
On the premise of reason, some improvement and modification are carried out to the present invention, these are improved and modification will also fall into the claims in the present invention
In protection domain.
Sequence table
<110>Xiangye No. 2 Hospital of Central South University
<120>Molecular marked compound TCONS_00016233, kit and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 835
<212> DNA
<213>Homo sapiens (Homo sapiens)
<400> 1
attaattctt taaaattatc aaattataca taacataatt tgccattttt tccatttttt 60
tccagacaga agtaacttct aaaacattgc tagggaaacg ctctgacatc ttccgtagag 120
gtgaaaaagc acaggtctga ctcaggtagc tgcgagttca agttcagccg caggctcttc 180
cccaaggtgc tggtcttcgc aggaacccct tgcaggcagc tgcaaagagc agcacggcgc 240
gcacacacag ggtccggctg gcgtctggcc agagcaaggg aacagtaagt gtgtcctgga 300
ggcgctcgcc atgcactcac acgccatctg tggattcgga agtgacctcc ccctcggttg 360
atgagggttc tctgcgtctg gagaacacag gacgtccaca ggaaagttct ccctcctgca 420
ctcccggctg gctctgaaaa ttatgaactc tcttttcctt aaaataaagg ctctaaaatt 480
gtgtgattcc acctccctga ggctaagcga aagagccgcg tctgcccctg tccttcccgg 540
gggctggcag ggagtgggtg cccggcggtg actcagccct gcagcctccc agcccttccc 600
cgtatgcggg caggtgatgc ggacgcctcc gaccctcgtg gggtttgtgg agagcgttga 660
tcagcgctgc cgtcagaaca ggacctgtgc gaggcctcgc agatgccggg agccgggcgg 720
tgctctcggt gcttctcatc aaatccactg gctcttcgag aggaagcaca tgtcacagcc 780
cagctcaaac aagaagcaag aaaatcactc ttcattttgg aattgtaaaa taaag 835
<210> 2
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 2
aacgctctga catcttccgt 20
<210> 3
<211> 20
<212> DNA
<213>Unknown (Unknown)
<400> 3
ttccgaatcc acagatggcg 20
<210> 4
<211> 23
<212> DNA
<213>Unknown (Unknown)
<400> 4
caaggtcatc catgacaact ttg 23
<210> 5
<211> 22
<212> DNA
<213>Unknown (Unknown)
<400> 5
gtccaccacc ctgttgctgt ag 22
Claims (6)
1. a kind of molecular marked compound TCONS_00016233, sequence such as SEQ ID NO：Shown in 1.
2. the product for detecting TCONS_00016233 expressions is preparing early diagnosis, prediction septicopyemia complicated with acute kidney damage
Hinder the application in instrument.
3. application according to claim 2, it is characterised in that the product includes：Pass through RT-PCR or real-time quantitative
PCR detects the preparation of TCONS_00016233 expressions.
4. application according to claim 3, it is characterised in that detect TCONS_00016233 expressions with RT-PCR
Specific amplification TCONS_00016233 primer sequence is：
F：5’-AACGCTCTGACATCTTCCGT-3’
R：5’-TTCCGAATCCACAGATGGCG-3’.
5. a kind of be used to early diagnosing, predicting the kit of septicopyemia complicated with acute injury of kidney, it is characterised in that comprising passing through
RT-PCR or real-time quantitative PCR detection TCONS_00016233 expressions reagent.
6. the kit according to claim 5 for being used to early diagnosing, predicting septicopyemia complicated with acute injury of kidney, it is special
Sign is that, comprising the primer a pair by RT-PCR specific amplifications TCONS_00016233, its primer sequence is：
F：5’-AACGCTCTGACATCTTCCGT-3’
R：5’-TTCCGAATCCACAGATGGCG-3’.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710915173.XA CN107541564B (en) | 2017-09-30 | 2017-09-30 | Molecular marked compound TCONS_00016233, kit and application |
| PCT/CN2018/102669 WO2019062429A1 (en) | 2017-09-30 | 2018-08-28 | Molecular marker tcons_00016233, kit using same, and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710915173.XA CN107541564B (en) | 2017-09-30 | 2017-09-30 | Molecular marked compound TCONS_00016233, kit and application |
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| Publication Number | Publication Date |
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| CN107541564A true CN107541564A (en) | 2018-01-05 |
| CN107541564B CN107541564B (en) | 2018-06-01 |
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| CN201710915173.XA Active CN107541564B (en) | 2017-09-30 | 2017-09-30 | Molecular marked compound TCONS_00016233, kit and application |
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| WO (1) | WO2019062429A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019062429A1 (en) * | 2017-09-30 | 2019-04-04 | 中南大学湘雅二医院 | Molecular marker tcons_00016233, kit using same, and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104278100A (en) * | 2014-10-16 | 2015-01-14 | 上海交通大学医学院附属仁济医院 | Applications of miRNA-188 in preparation of diagnosis reagent as labelled molecule |
| US20170240969A1 (en) * | 2014-08-14 | 2017-08-24 | Medizinische Hochschule Hannover | A circulating non-coding rna as predictor of mortality in patients with acute kidney injury |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107541564B (en) * | 2017-09-30 | 2018-06-01 | 中南大学湘雅二医院 | Molecular marked compound TCONS_00016233, kit and application |
-
2017
- 2017-09-30 CN CN201710915173.XA patent/CN107541564B/en active Active
-
2018
- 2018-08-28 WO PCT/CN2018/102669 patent/WO2019062429A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170240969A1 (en) * | 2014-08-14 | 2017-08-24 | Medizinische Hochschule Hannover | A circulating non-coding rna as predictor of mortality in patients with acute kidney injury |
| CN104278100A (en) * | 2014-10-16 | 2015-01-14 | 上海交通大学医学院附属仁济医院 | Applications of miRNA-188 in preparation of diagnosis reagent as labelled molecule |
Non-Patent Citations (3)
| Title |
|---|
| BU D ET AL.: "NONHSAT055523.2", 《NONCODE》 * |
| VOLDERS PJ ET AL.: "URS00008B7A11", 《NONCODE》 * |
| 李萍珠、徐炜新: "脓毒血症并发急性肾损伤早期诊断标志物的研究", 《中华检验医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019062429A1 (en) * | 2017-09-30 | 2019-04-04 | 中南大学湘雅二医院 | Molecular marker tcons_00016233, kit using same, and application thereof |
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| Publication number | Publication date |
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| WO2019062429A1 (en) | 2019-04-04 |
| CN107541564B (en) | 2018-06-01 |
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