CN105420347A - Gene diagnosis reagent kit for systemic lupus erythematosus - Google Patents
Gene diagnosis reagent kit for systemic lupus erythematosus Download PDFInfo
- Publication number
- CN105420347A CN105420347A CN201410417499.6A CN201410417499A CN105420347A CN 105420347 A CN105420347 A CN 105420347A CN 201410417499 A CN201410417499 A CN 201410417499A CN 105420347 A CN105420347 A CN 105420347A
- Authority
- CN
- China
- Prior art keywords
- gene
- systemic lupus
- ly6e
- oasl
- interferon
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention relates to a gene diagnosis reagent kit for systemic lupus erythematosus. The gene diagnosis reagent kit comprises primers for amplification of the OASL gene, the IGS15 gene and the LY6E gene, and further comprises a primer for the house-keeping gene RPLPO and an amplification reagent. The gene diagnosis reagent kit overcomes the defects that an I type interferon induction gene lacks of specificity, the number of detection genes is large, much time and labor are consumed, and consumption is increased. A real-time fluorescence quantification PCR method is adopted, and the gene diagnosis reagent kit has the advantages of being fast, sensitive, accurate and the like; the expression of the LY6E gene, the ISG15 gene and the OASL gene is detected through a real-time fluorescence quantification PCR, improved interferon indexes are worked out, early diagnosis of an SLE patient is facilitated, an important basis and reference value can be provided for clinical disease confirmation, and thus early prevention and treatment of the disease are facilitated; the gene diagnosis reagent kit has application and popularization value.
Description
Technical field
The present invention relates to a kind of gene diagnosis kit of autoimmune disease, be a kind of gene diagnosis kit for systemic lupus erythematous in particular, belong to technical field of molecular biology.
Background technology
Autoimmune is that a class is uncared-for, the common lethal chronic disease disabled.Systemic lupus erythematous (systemicIupuserythematosus, SLE) is considered to the prototype disease of autoimmune disease, can involve any organ of health.Diagnosis current Main Basis Americanism diseases caused by dampness association's nineteen eighty-two of SLE formulates and the criteria for classification of revision in 1997,2009 systemic lupus erythematous international co-operation group (SLICC) again it is upgraded.But these SLE criteria for classifications all SLE is isolated as main purpose from autoimmune disorder disease spectrum, are unfavorable for differentiating or finding light-duty SLE and early stage SLE.And for SLE, delay treatment causes dead independent hazard factor, prompting early diagnosis is most important to patient's prognosis.
Currently existing a lot of attempt the research that applying biological mark helps diagnose SLE, but due to the height heterogeneity of disease, majority all fails.Nearly ten years, along with molecular biological progress, it is found that I type Interferon, rabbit level in SLE patient increases, this pathway gene pleomorphism site also with disease-related, and in human and animal, I type interferon therapy can be induced and be produced lupoid acne performance, illustrates that interferon-induced gene plays an important role in disease occurs.Before the present invention makes, interferon-induced gene is applied to clinical by existing trial, but nearly all research is all devoted to carry out the judgement of SLE Disease Activity, comprises studying also display interference element inducible gene expression level in early days and really showing relevant to disease activity score and some specific clinical.But long term follow-up afterwards observes display, interferon-induced gene expression dose does not change with Disease Activity change, in prompting SLE body the exception of I type Interferon, rabbit may be disease intrinsic, thus more helpful to diagnosis.
The interferon-induced gene of I type has reported relevant to SLE to have tens at least, and some of them are also relevant with other autoimmune diseases (as rheumatoid arthritis, sjogren syndrome, inflammatory diseases etc.), lack specificity.We first sift out 5 representational genes of most by factorial analysis early stage from 14 oligogenes, then contrasts with normal people and other immunological diseases patient, analyze its value in SLE diagnoses.Thereafter attempt detection 5 interferon-induced genetic expressions, find there is certain diagnostic value to systemic lupus erythematous, but it is many to detect gene number on the one hand, wastes time and energy and increases consumption; Analyze through Receiver operating curve on the other hand, wherein two to having diagnosed larger effect.
Summary of the invention
Object of the present invention is just to overcome above-mentioned defect, develops a kind of gene diagnosis kit for systemic lupus erythematous.
Technical scheme of the present invention is:
A gene diagnosis kit for systemic lupus erythematous, its technical characteristics is, includes the primer for these 3 genes of OASL, IGS15 and LY6E that increase, also comprises the primer of house-keeping gene RPLPO, and amplifing reagent.
Described each gene primer sequence is respectively:
OASL-F：5’-CCATCACGGTCACCATTGTG-3’
OASL-R：5’-ACCGCAGGCCTTGATCAG-3’
ISG15-F：5’-GGTGGACAAATGCGACGAA-3’
ISG15-R：5’-CAGCCCGCTCACTTGCT-3’
LY6E-F：5’-ACTGCGTGACTGTGTCTGCTA-3’
LY6E-R：5’-AGGCTGTGGCCAAATGTC-3’
RPLPO-F：5’-GTTTCATTGTGGGAGCAGACA-3’
RPLPO-R：5’-CATGGTGTTCTTGCCCATCA-3’
Described amplifing reagent comprises the hot start Taq polymerase, ROX inertia reference dye and the ultrapure water that are mixed with PCR damping fluid and dNTPs in advance.
Described amplifing reagent is made up of the component with lower volume: cDNA1.0 μ l, PremixExTaqTM (2 ×) 10 μ l, ROX inertia reference dye (50 ×) 0.4 μ l, upstream primer 0.4 μ l, downstream primer 0.4 μ l, ultrapure water 7.8 μ l.
The described expression being detected LY6E, IGS15, OASL by Real-time quantitative PCR, the mean number getting 3 detected values calculates the cycle number experienced when fluorescent signal arrives setting threshold value, cycling numerical value when deducting house-keeping gene RPLPO arrival threshold value calculates difference value, then by 2
-difference valuecalculate each gene Relative Expression values.
Describedly carry out result judgement by calculating improvement Interferon, rabbit index, with testing sample gene Relative Expression values-normal group average gene Relative Expression values, again divided by normal group genetic expression standard deviation, calculate individual gene Interferon, rabbit index, three values corresponding to LY6E, ISG15 and OASL are added, are improvement Interferon, rabbit index.
Described when improvement Interferon, rabbit index is 2.37, Sensitivity and Specificity of its diagnosis reaches best dividing value, is respectively 70% and 80%.
Present invention employs real time fluorescence quantifying PCR method, there is the features such as quick, sensitive, accurate, detect LY6E, ISG15 and OASL genetic expression by real-time fluorescence quantitative PCR and calculate improvement Interferon, rabbit index, contribute to the early diagnosis of SLE patient, can be clinical definite disease and important foundation and reference value are provided, thus be beneficial to the early prevention of this disease is treated, there is universal and application value.
Accompanying drawing explanation
Fig. 1---test kit provided by the invention carries out gene level detected result schematic diagram.
Fig. 2---the relation schematic diagram of gene expression detection level provided by the invention and infection.
Fig. 3---ROC area under a curve (AUC) schematic diagram of improvement Interferon, rabbit index.
Fig. 4---improvement Interferon, rabbit index compares schematic diagram with anti-dsDNA antibody to SLE diagnostic value.
Embodiment
Below by embodiment, the present invention is described in further detail.But it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.RNA can also be comprised in test kit and extract test kit and/or Reverse Transcription box.
Embodiment 1:
1. the determination of gene and primer optimization
Determine to diagnose the relevant interferon-induced gene of I type to be LY6E, ISG15 and OASL to SLE according to early-stage Study, by the lot of experiments moved in circles, and be optimized in conjunction with the Tm value of masterplate amount, primer, determine the increase primer of above 3 genes and the primer of amplification house-keeping gene RPLPO.
Concrete primer sequence is shown in sequence table (OASL:SEQIDNO.1-2; ISG15:SEQIDNO.3-4; LY6E:SEQIDNO.5-6; RPLPO:SEQIDNO.7-8).
2. testing sample process
Early morning gathers venous whole 2-3ml with EDTA vein vacuum disposable blood conventional blood collection pipe (U.S. BDBiosciences) under empty stomach quiescent condition, apply Trizol (American I nvitrogen) immediately and extract RNA, then with SuperscriptII Reverse Transcriptase kit (American I nvitrogen) reverse transcription for cDNA.Be stored in-70 DEG C for subsequent use.
3. real-time fluorescence quantitative PCR
96 orifice plates will be added for inspection cDNA, 1ul/ hole, add LY6E, ISG15, OASL and RPLPO primer (sequence is in table 1) respectively, add SYBRGreen luciferase assay reagent (the precious biotechnology company limited in Dalian) again, 20ul, inserts the quick real-time fluorescent quantitative PCR detection system of ABI7500 (U.S. AppliedBiosystems) and carries out PCR reaction altogether.Each Gene response repeats 3 times, and amplification condition is sex change first 95 DEG C 10 seconds, afterwards 95 DEG C 5 seconds-60 DEG C within 34 seconds, maintain 40 circulations.
4. data analysis
Pass through StepOne
tMsoftware (U.S. AppliedBiosystems) obtains the Ct value of each genetic expression, calculates the mean value repeated for 3 times.The Ct value deducting house-keeping gene RPLPO with goal gene Ct value calculates Δ Ct value, then by 2
-Δ Ctcalculate gene Relative Expression values.
Described amplifing reagent comprises the hot start Taq polymerase, ROX inertia reference dye and the ultrapure water that are mixed with PCR damping fluid and dNTPs in advance; Be made up of the component with lower volume: cDNA1.0 μ l, PremixExTaqTM (2 ×) 10 μ l, ROX inertia reference dye (50 ×) 0.4 μ l, upstream primer 0.4 μ l, downstream primer 0.4 μ l, ultrapure water 7.8 μ l.The expression of LY6E, IGS15, OASL is detected by Real-time quantitative PCR, the mean number getting 3 detected values calculates the cycle number experienced when fluorescent signal arrives setting threshold value, cycling numerical value when deducting house-keeping gene RPLPO arrival threshold value calculates difference value, then by 2
-difference valuecalculate each gene Relative Expression values.
Describedly carrying out result judgement by calculating improvement Interferon, rabbit index, with testing sample gene Relative Expression values-normal group average gene Relative Expression values, then divided by normal group genetic expression standard deviation, calculating individual gene Interferon, rabbit index.Three values corresponding to LY6E, ISG15 and OASL are added, are improvement Interferon, rabbit index.When improvement Interferon, rabbit index is 2.37, the Sensitivity and Specificity of its diagnositc system lupus erythematosus reaches best dividing value, is respectively 70% and 80%.
Embodiment 2:
1. compare the change that SLE patient and normal population and other Serum of Patients With Autoimmune Diseases improve Interferon, rabbit index
Gather normal people, SLE patient and other Serum of Patients With Autoimmune Diseases blood samples respectively, obtain each gene Relative Expression values by embodiment 1, respectively organize LY6E, ISG15, OASL gene expression dose.With (testing sample gene Relative Expression values-normal group average gene Relative Expression values)/normal group genetic expression standard deviation, mark is carried out to each gene level, again three values corresponding to LY6E, ISG15 and OASL are added, obtain improvement Interferon, rabbit index, relatively more each group improvement Interferon, rabbit index level difference.Compare with other Serum of Patients With Autoimmune Diseases with normal people, SLE patient LY6E, ISG15, OASL expression level and improvement Interferon, rabbit index all obviously raise (Figure 1A-D).In different autoimmune disease, improvement Interferon, rabbit index level removes scleroderma (SSc) patient levels and slightly increases, and other and normal phase ratio are without significant difference (Fig. 1 E).
2. observe the impact infected improvement Interferon, rabbit index
SLE patient is divided into concurrent infection group and non-concurrent infection group, respectively organizes LY6E, ISG15, OASL gene expression dose, find no group difference.Detect anti-Epstein-Barr virus antibody level in patient body simultaneously, also do not find and improve Interferon, rabbit index and there is dependency.(Fig. 2)
3. improve the research of Interferon, rabbit index Sensitivity and Specificity
Compare with other Serum of Patients With Autoimmune Diseases with normal people, when improvement Interferon, rabbit index is for diagnosing SLE, its ROC area under a curve (AUC) reaches 0.812 (95% fiducial limit 0.738-0.881).When Interferon, rabbit index is 2.37, the susceptibility of diagnosis SLE is 70%, and specificity is 80% (Fig. 3).Compared with anti-dsDNA antibody, the susceptibility higher (Fig. 4) of improvement Interferon, rabbit exponent pair SLE diagnosis.Detect 3 genetic expressions (comprising LY6E, ISG15 and OASL) and calculate improvement Interferon, rabbit index value after data being done stdn conversion and judge, more can increase the accuracy of diagnosis; And compared with expressing (as LY6E) with detection individual gene, this method decreases fluctuation, thus makes result more stable, is easy to repetition.
Claims (7)
1. a gene diagnosis kit for systemic lupus erythematous, is characterized in that, includes the primer for these 3 genes of OASL, IGS15 and LY6E that increase, also comprises the primer of house-keeping gene RPLPO, and amplifing reagent.
2. the gene diagnosis kit of a kind of systemic lupus erythematous according to claim 1, is characterized in that, each gene primer sequence is respectively:
OASL-F：5’-CCATCACGGTCACCATTGTG-3’
OASL-R：5’-ACCGCAGGCCTTGATCAG-3’
ISG15-F：5’-GGTGGACAAATGCGACGAA-3’
ISG15-R：5’-CAGCCCGCTCACTTGCT-3’
LY6E-F：5’-ACTGCGTGACTGTGTCTGCTA-3’
LY6E-R：5’-AGGCTGTGGCCAAATGTC-3’
RPLPO-F：5’-GTTTCATTGTGGGAGCAGACA-3’
RPLPO-R：5’-CATGGTGTTCTTGCCCATCA-3’。
3. the gene diagnosis kit of a kind of systemic lupus erythematous according to claim 1, is characterized in that described amplifing reagent comprises hot start Taq polymerase, ROX inertia reference dye and the ultrapure water being mixed with PCR damping fluid and dNTPs in advance.
4. the gene diagnosis kit of a kind of systemic lupus erythematous according to claim 3, it is characterized in that, amplifing reagent is made up of the component with lower volume: cDNA1.0 μ l, PremixExTaqTM (2 ×) 10 μ l, ROX inertia reference dye (50 ×) 0.4 μ l, upstream primer 0.4 μ l, downstream primer 0.4 μ l, ultrapure water 7.8 μ l.
5. the gene diagnosis kit of a kind of systemic lupus erythematous according to claim 4, it is characterized in that, the expression of LY6E, IGS15, OASL is detected by Real-time quantitative PCR, the mean number getting 3 detected values calculates the cycle number experienced when fluorescent signal arrives setting threshold value, cycling numerical value when deducting house-keeping gene RPLPO arrival threshold value calculates difference value, then by 2
-difference valuecalculate each gene Relative Expression values.
6. the gene diagnosis kit of a kind of systemic lupus erythematous according to claim 1, it is characterized in that, result judgement is carried out by calculating improvement Interferon, rabbit index, with testing sample gene Relative Expression values-normal group average gene Relative Expression values, again divided by normal group genetic expression standard deviation, calculate individual gene Interferon, rabbit index, three values corresponding to LY6E, ISG15 and OASL are added, be improvement Interferon, rabbit index.
7. the gene diagnosis kit of a kind of systemic lupus erythematous according to claim 6, is characterized in that, when improvement Interferon, rabbit index is 2.37, Sensitivity and Specificity of its diagnosis reaches best dividing value, is respectively 70% and 80%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410417499.6A CN105420347A (en) | 2014-08-21 | 2014-08-21 | Gene diagnosis reagent kit for systemic lupus erythematosus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410417499.6A CN105420347A (en) | 2014-08-21 | 2014-08-21 | Gene diagnosis reagent kit for systemic lupus erythematosus |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105420347A true CN105420347A (en) | 2016-03-23 |
Family
ID=55498882
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410417499.6A Pending CN105420347A (en) | 2014-08-21 | 2014-08-21 | Gene diagnosis reagent kit for systemic lupus erythematosus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105420347A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841951A (en) * | 2018-07-19 | 2018-11-20 | 南京鼓楼医院 | A kind of RNASE2 gene primer pair and its kit for systemic lupus erythematosus diagnosis and detection disease activity |
| CN109266734A (en) * | 2018-09-25 | 2019-01-25 | 深圳市人民医院 | Autoimmune disease diagnostic kit and application |
| CN114622017A (en) * | 2020-12-14 | 2022-06-14 | 中山大学孙逸仙纪念医院 | Application of ISG15 and ISG15 induced macrophages and secretion thereof in tumor treatment |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008070137A2 (en) * | 2006-12-06 | 2008-06-12 | Medimmune, Llc | Interferon alpha-induced pharmacodynamic markers |
| CN101473045A (en) * | 2006-04-24 | 2009-07-01 | 健泰科生物技术公司 | Methods and compositions for detecting autoimmune disorders |
-
2014
- 2014-08-21 CN CN201410417499.6A patent/CN105420347A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101473045A (en) * | 2006-04-24 | 2009-07-01 | 健泰科生物技术公司 | Methods and compositions for detecting autoimmune disorders |
| WO2008070137A2 (en) * | 2006-12-06 | 2008-06-12 | Medimmune, Llc | Interferon alpha-induced pharmacodynamic markers |
Non-Patent Citations (2)
| Title |
|---|
| XUEBING FENG ET AL: "Association of Increased Interferon-Inducible Gene Expression With Disease Activity and Lupus Nephritis in Patients With Systemic Lupus Erythematosus", 《ARTHRITIS & RHEUMATISM》 * |
| 黄静等: "系统性红斑狼疮患者I型干扰素诱导基因的表达", 《中华内科杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841951A (en) * | 2018-07-19 | 2018-11-20 | 南京鼓楼医院 | A kind of RNASE2 gene primer pair and its kit for systemic lupus erythematosus diagnosis and detection disease activity |
| CN109266734A (en) * | 2018-09-25 | 2019-01-25 | 深圳市人民医院 | Autoimmune disease diagnostic kit and application |
| CN114622017A (en) * | 2020-12-14 | 2022-06-14 | 中山大学孙逸仙纪念医院 | Application of ISG15 and ISG15 induced macrophages and secretion thereof in tumor treatment |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6931125B2 (en) | Assessment of JAK-STAT1 / 2 cell signaling pathway activity using mathematical modeling of target gene expression | |
| EP3461916A1 (en) | Assessment of jak-stat3 cellular signaling pathway activity using mathematical modelling of target gene expression | |
| CN105506115A (en) | DNA library for detection and diagnosis of hereditary cardiomyopathy causing genes and application thereof | |
| CN103725798B (en) | For detecting primer, test kit, the detection method of hemorrhagic fever with renal syndrome virus with RT-LAMP method | |
| CN108559776A (en) | A kind of biomarker and its application for sudden weak smart auxiliary diagnosis | |
| CN103805696A (en) | Micro RNA (Ribonucleic Acid) molecular marker for diagnosing rheumatoid arthritis and detection kit thereof | |
| CN104651513A (en) | Gout serum miRNAs biomarkers and method for detecting expression quantity thereof | |
| CN107699617B (en) | A kind of early diagnosis septicopyemia causes acute kidney injury molecular marked compound miR-452, kit and application | |
| CN108949979A (en) | A method of judging that Lung neoplasm is good pernicious by blood sample | |
| CN105420347A (en) | Gene diagnosis reagent kit for systemic lupus erythematosus | |
| CN103725800B (en) | Human adenovirus detection kit | |
| Addissouky | Detecting liver fibrosis by recent reliable biomarkers in viral hepatitis patients | |
| US20170145501A1 (en) | Apparatus and methods of using of biomarkers for predicting tnf-inhibitor response | |
| CN112730858A (en) | Diagnosis marker for rheumatic arthritis | |
| CN108300788A (en) | A kind of micro RNA combination and its application for detecting light-duty brain trauma | |
| CN104087672A (en) | Kit for quickly detecting number of human chromosomes 21 by multiplex real-time fluorescence quantitative PCR (polymerase chain reaction) technique | |
| CN108220416A (en) | A kind of kit and its application for being used to detect deficiency of Yin excessive internal heat constitution serum specificity miRNA | |
| CN108977533A (en) | It is a kind of for predicting the miRNA combination object of chronic hepatitis B inflammation damnification | |
| TWI598444B (en) | Method and gene marker for assessing risk of suffering breast cancer | |
| JP2013110969A (en) | Method for evaluating stress, stress-evaluation marker, method for creating stressed model animal, and stressed model animal | |
| CN105177130B (en) | It is used for assessing the mark of aids patient generation immune reconstitution inflammatory syndrome | |
| CN104984363B (en) | Applications of the ZMYM1 in Parkinson's diagnosis and treatment reagent is prepared | |
| CN104087671A (en) | Kit used for detecting number of human chromosomes 21 | |
| CN105671188A (en) | Molecular marker and primer set for diagnosing Mycobacterium tuberculosis infection, and application thereof | |
| US20220349888A1 (en) | Method for detecting brain tumor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20160323 |
|
| RJ01 | Rejection of invention patent application after publication |