CN101705302B - Kit for assisting to diagnose multiple myeloma - Google Patents
Kit for assisting to diagnose multiple myeloma Download PDFInfo
- Publication number
- CN101705302B CN101705302B CN2009102372296A CN200910237229A CN101705302B CN 101705302 B CN101705302 B CN 101705302B CN 2009102372296 A CN2009102372296 A CN 2009102372296A CN 200910237229 A CN200910237229 A CN 200910237229A CN 101705302 B CN101705302 B CN 101705302B
- Authority
- CN
- China
- Prior art keywords
- sequence
- magec1
- gene
- multiple myeloma
- plasmid
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for assisting to diagnose multiple myeloma. The kit comprises a specific primer pair A and a probe A aiming at MAGEC1/CT7 gene, wherein the specific primer pair A is a primer pair consisting of nucleotide sequences shown in a sequence 1 and a sequence 2 of a sequence table; and the nucleotide sequence of the probe A is shown in a sequence 3 of the sequence table. The kit not only can be applied to qualitatively diagnosing the expression of the MAGEC1/CT7 gene of the multiple myeloma, but also can diagnose the expression level of the MAGEC1/CT7 gene of the multiple myeloma by internal reference gene. The invention provides a fast, reliable and accurate new way for diagnosis of the multiple myeloma, provides basis for observing curative effect and dynamically observing minimal residual diseases and can play an important role in the field of medical detection.
Description
Technical field
The present invention relates to a kind of test kit of assisting to diagnose multiple myeloma.
Background technology
(multiple myeloma is plasmacytic malignant tumour MM) to multiple myeloma, it is characterized by abnormal plasmocyte malignant proliferation in the marrow; Excessive monoclonal immunoglobulin (IgG, IgA, IgD or IgE) or Bence Jones Bence Jones protein matter (free monoclonicity κ or γ light chain) appear in serum or the urine; Often with multiple molten bone property infringement, hypercalcemia, anaemia; Kidney damage is prone to cause lung, urinary system infection.Account for second of hematological system tumor at the sickness rate of China's multiple myeloma, median survival interval is 3～5 years.At present; The treatment of multiple myeloma mainly comprises conventional chemotherapy, stem cell transplantation and newtype drug treatment; Application like immunoregulation druge, proteasome inhibitor etc. though the state of an illness is effectively controlled, seldom has the patient to obtain and alleviates fully even cure.More and more evidences shows that the allelic heterogeneity of multiple myeloma cells has determined the different clinical consequence to a great extent.
The diagnosis of multiple myeloma now develops into cytobiology and clinical medicine bonded comprehensive diagnos pattern from the diagnosis of simple cell morphology.The routine diagnosis technology has clone's property rearrangement of various electrophoresis, immunohistochemical methods, flow cytometry, PCR detection heavy chain immunoglobulin or light chain gene etc.; But all the time; The molecular target that lacks the special diagnosis of multiple myeloma, prognosis molecule mark and treatment monitoring makes the curative effect to the treatment multiple myeloma be difficult to make the more assessment of science.
Cancer testis antigen (Cancer testis antigen; CT antigen) be a kind of immunogenic protein; Identified more than 40 family in the period of 15 in the past; Based on its specific expressed and immunogenicity in tumour patient, CT antigen has been considered to have the target of the human malignancies immunotherapy of application prospect, and might become important diagnostic and prognosis sign.Recently the MM patient's high frequency of discovering is expressed CT antigen, and can induce antibody-mediated and the cell-mediated immunoreation of T.The result of treatment part of allogeneic stem cell transplantation is the result by the immunological effect of the T cell demonstration in donor source; To the antigenic immunoreation of CT, prompting CT antigen possibly be the natural target of donor source alloimmunity or even spontaneous anti-myeloma immunne response.The obvious continuous expression CT of the MM patient of the residual malignant plasma cell of marrow antigen so CT antigen is the potential sign to minimal residual disease, can also be used for remaining in behind the targeted therapy myeloma cell of patient's marrow.Through detecting plasmacytic quantity of marrow and peripheral blood paraprotein level; Having found has strong correlation between CT antigenic expression and myelomatosis patient's clinical disease course; CT antigen possibly is not only independently tumor-marker, and is the important indicator that early stage recurrence and survival rate significantly descend.MAGEC1/CT7 (melanoma-associated antigen-C1/ cancer testis antigen 7) is modal a kind of CT antigen presentation among the MM; Detect positive rate and can reach 65%～82%; High expression level in tumour cell; In normal cell, do not express, the high frequency of the tumour-specific of MAGEC1/CT7 and lasting expression make it most possibly become effective MM diagnosis, prognosis, curative effect monitoring index and immunotherapy target.
At present, shortcoming such as being used to of having reported in the world detected the method that MAGEC1/CT7 expresses and be mainly qualitative PCR and immunohistochemical method, and these method ubiquity sensitivity are low, specificity is not high, waste time and energy makes to use to receive certain limitation.
Summary of the invention
The test kit that the purpose of this invention is to provide a kind of assisting to diagnose multiple myeloma.
The test kit of assisting to diagnose multiple myeloma provided by the invention comprises that the Auele Specific Primer that is directed against the MAGEC1/CT7 gene is to first and probe first; Said Auele Specific Primer is that the primer that nucleotide sequence is formed shown in the sequence 2 of sequence 1 and sequence table of sequence table is right to first; The nucleotide sequence of said probe first is shown in the sequence 3 of sequence table.
Said test kit also can comprise positive plasmid; Said positive plasmid is for containing MAGEC1/CT7 gene or its segmental recombinant plasmid.Said positive plasmid can be the recombinant plasmid of the MAGEC1/CT7 gene fragment that contains shown in the sequence 7.Said positive plasmid specifically can be the MAGEC1/CT7 gene fragment shown in the sequence 7 of pMD18-T carrier and sequence table is connected the recombinant plasmid that obtains.
Said test kit can comprise also that the Auele Specific Primer that is directed against internal control gene is to second and probe second; Said internal control gene is an abl gene.Said Auele Specific Primer specifically can be the primer that nucleotide sequence is formed shown in the sequence 5 of sequence 4 and sequence table of sequence table to second right; The nucleotide sequence of said probe second specifically can be shown in the sequence 6 of sequence table.
Said test kit also can comprise the confidential reference items plasmid; Said confidential reference items plasmid is for containing abl gene or its segmental recombinant plasmid.Said confidential reference items plasmid can be and contains the segmental recombinant plasmid of the abl gene shown in the sequence 8.Said confidential reference items plasmid specifically can be the abl gene fragment shown in the sequence 8 of pMD18-T carrier and sequence table is connected the recombinant plasmid that obtains.
The present invention also protects to the Auele Specific Primer of MAGEC1/CT7 gene first and the application of probe first in the test kit of preparation assisting to diagnose multiple myeloma; Said Auele Specific Primer is that the primer that nucleotide sequence is formed shown in the sequence 2 of sequence 1 and sequence table of sequence table is right to first; The nucleotide sequence of said probe first is shown in the sequence 3 of sequence table.
In the marrow or peripheral blood of multiple myeloma patients, MAGEC1/CT7 genetic expression, and in normal people's marrow or peripheral blood, the MAGEC1/CT7 gene is not expressed.And be in the marrow or peripheral blood of multiple myeloma patients of different I SS phase, MAGEC1/CT7 expression of gene level has otherness.Test kit of the present invention not only can be applied to the etiologic diagnosis multiple myeloma, can also pass through internal control gene, measures patient MAGEC1/CT7 expression of gene level.The present invention for the diagnosis of multiple myeloma provide one fast, reliably, new way accurately, for dynamic observing of observation of curative effect, minimal residual disease provides foundation.The present invention will play a significant role at the medical science detection range.
Description of drawings
Fig. 1 is the RQ-PCR fluorescence curve that positive control plasmid sensitivity detects among the embodiment 3.
Fig. 2 is the typical curve of positive control plasmid RQ-PCR amplification among the embodiment 5; Show between the different dilution samples of Ct value and be linear dependence (relation conefficient＞0.99) with the positive control plasmid.
Fig. 3 is the typical curve of the RQ-PCR amplification of confidential reference items control plasmid among the embodiment 5; Show between the different dilution samples of Ct value and be linear dependence (relation conefficient＞0.99) with the confidential reference items control plasmid.
Fig. 4 is among the embodiment 6, and 25 patients' marrow mesoplasmatocyte number accounts for the relation of the per-cent and the MAGEC1/CT7 gene expression dose of bone marrow nucleated cell.
Fig. 5 is among the embodiment 6, and the CD38+/CD138+ cell accounts for the relation of the per-cent and the MAGEC1/CT7 gene expression dose of bone marrow nucleated cell in 31 patients' the marrow.
Fig. 6 is among the embodiment 6; The per-cent that CD38+/CD138+ cell in 31 patients' the marrow is accounted for bone marrow nucleated cell by＜1% (1 group, n=9), 1%-10% (2 groups, n=14),＞10% (3 groups; When n=8) being divided into three groups, each organizes the MAGEC1/CT7 gene expression dose.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.
To the Auele Specific Primer of MAGEC1/CT7 gene to first (amplified production is 79bp) and probe first:
Upstream primer MAG-FP:5 '-TTGTCTTCTGGGAACCTTGACTC-3 ';
Downstream primer MAG-RP:5 '-TGAGGGACACATACATCCTAAAAGC-3 ';
Probe MAG-T-probe:FAM-ACTGCCTGGGCCTCCTCTGCTGT-BHQ (5 '-3 ').
To the Auele Specific Primer of abl gene (internal control gene) to second and probe second:
Upstream primer ENF1003:5 '-TGGAGATAACACTCTAAGCATAACTAAAGGT-3 ';
Downstream primer ENR1063:5 '-GATGTAGTTGCTTGGGACCCA-3 ';
Probe ENPr1043:FAM-CCATTTTTGGTTTGGGCTTCACACCATT-TAMRA (5 '-3 ').
Auele Specific Primer to abl gene is pressed synthetic (the Gabert J of reference to second and probe second; BeillardE; Van der Velden VH; Et al.Standardization and quality control studiesof ' real-time ' quantitative reverse transcriptase polymerase chain reactionof fusion gene transcripts for residual disease detection in leukemia-aEurope Against Cancer program.Leukemia.2003,17:2318-57.)
The preparation of embodiment 2, relevant plasmid
One, the preparation of positive control plasmid (plasmid that contains the MAGEC1/CT7 gene fragment)
CDNA with the marrow of MAGEC1/CT7 genetic expression male multiple myeloma patients (volunteer) is a template, carries out pcr amplification, and the nucleotide sequence of amplified production is shown in the sequence 7 (632bp) of sequence table; After amplified production is purified, be cloned into pMD18-T plasmid (pMD18-T carrier system, the white good company in Shanghai; Catalog number: D101A); With the recombinant plasmid transformed bacillus coli DH 5 alpha (day root biochemical corp, catalog number: CD101-02) competence, screening positive clone checks order after extracting plasmid and purifying; The result shows and has obtained positive control plasmid (skeleton plasmid is the pMD18-T plasmid, at cDNA shown in the insertion sequence 7 of ECOR V site).
The used primer of pcr amplification is to as follows:
CT7FP：5’-ACATCCTCACCCTCAGGAGGG-3’；
CT7RP：5’-GACGAGGATCGTCTCAGGTCAGC-3’。
Two, the preparation of confidential reference items control plasmid (containing the segmental plasmid of abl gene)
With K562 clone (the female willing biochemical industry in Shanghai ltd; China's cell bank classical collection cell centre cell catalogue: cDNA QK10269) is a template, carries out pcr amplification, and the nucleotide sequence of amplified production is shown in the sequence 8 of sequence table; After amplified production is purified; Be cloned into the pMD18-T plasmid, with recombinant plasmid transformed bacillus coli DH 5 alpha competence, screening positive clone checks order after extracting plasmid and purifying; The result shows and has obtained confidential reference items control plasmid (skeleton plasmid is the pMD18-T plasmid, at cDNA shown in the insertion sequence 8 of ECOR V site).
The used primer of pcr amplification is to as follows:
ABL-FP：5’-TGGAGATAACACTCTAAGCATAACTAAAGGT-3’；
ABL-RP：5’-GATGTAGTTGCTTGGGACCCA-3’。
Three, negative control plasmid
The pMD18-T plasmid.
The sensitivity of embodiment 3, test kit detects
One, the assembling of test kit
Test kit is made up of following assembly: the Auele Specific Primer that is directed against the MAGEC1/CT7 gene of embodiment 1 preparation is to first and probe first; The positive control plasmid of embodiment 2 preparations.
Two, the sensitivity of test kit detects
6 MAGEC1/CT7 male multiple myeloma patients (volunteer) cDNA sample and positive control plasmid are carried out the detection of MAGEC1/CT7 gene, and patient cDNA sample carries out 10 times of gradient dilutions to 10 respectively
-5, 10 times of gradient dilutions of positive control plasmid are for containing 10 respectively
5, 10
4, 10
3, 10
2, 10
1, 10
0The MAGEC1/CT7 gene fragment of individual copy (through measuring the copy number that absorbance calculates MAGEC1/CT7 gene fragment in the positive control plasmid).
Method is following:
1, RNA extracts
Under aseptic condition, extract the RNA (or the RNA of peripheral blood sample also can) of each patient's bone marrow prepare (The People's Hospital of Peking University) with TRIzol test kit (available from American I nvitrogen company) and reference reagent box specification sheets; Method is: use the EDTA anti-freezing; Isolate mononuclearcell with lymphocyte layering liquid (specific density 1.077g/L, Shanghai China smart High Seience Technology Co., Ltd.), with isolated mononuclearcell with saline water washing 2 times after; Add 1000 μ l TRIzol reagent; Blow and beat repeatedly with the application of sample rifle, make cytoclasis, RNA extracts according to the explanation carrying out of TRIzol test kit.After the RNA that extracts dries, add the deionized water dissolving RNA that an amount of DEPC handles, on the DNA/RNA ultraviolet spectrophotometer, survey RNA concentration and purity, A260/A280＞1.8 show that the RNA purity of being extracted is higher as a result.
2, cDNA is synthetic
The public system of rt (RT) (20 μ L):
5 * RT damping fluid, 4 μ L
0.1M?DTT 2μL
DNTPs (10mM, Promega company) 1 μ L
Random hexamer primer (0.1 μ g/ μ l, Invitrogen company) 1 μ L
MMLV reversed transcriptive enzyme (200U/ μ L, Invitrogen company) 1 μ L
RNaseOUT
TMRNase suppressor factor (40U/ μ L, Invitrogen company) 1 μ L
Public system packing 1 pipe of per 19 μ L RT, every pipe adds RNA 1 μ g (RNA is 65 ℃ of 5min at first, ice bath 5min).37 ℃ of reaction 65min, 95 ℃ of 5min deactivation reversed transcriptive enzymes, 4 ℃ of preservations are subsequent use.
3,6 MAGEC1/CT7 male multiple myeloma patients cDNA samples and positive control plasmid are carried out the sensitivity detection
Each sample that step 2 obtains carries out 10 times of gradient dilutions, on fluorescence real-time quantitative PCR appearance (7500-FAST of American AB I company type), carries out RQ-PCR.
PCR reaction system (10 μ l): the upstream primer 0.4 μ M shown in the sequence 1; Downstream primer 0.4 μ M shown in the sequence 2, the TaqMan probe 0.2 μ M shown in the sequence 3, the public system of 2 * TaqMan universal PC R 5 μ l (ABI companies; The U.S.), cDNA (or plasmid) 1 μ l; All the other are deionized water.PCR reaction conditions: 50 ℃ of 2min, 1 circulation; 95 ℃ of 10min, 1 circulation; 95 ℃ of 15s, 60 ℃ of 1min, 40 circulations.Analyze every crowd of RQ-PCR as a result the time threshold value all be fixed as 0.2.
The amplification curve characteristic of 10 times of serial dilutions of 6 MAGEC1/CT7 male multiple myeloma patients cDNA samples is seen table 1.
The amplification curve characteristic of 10 times of serial dilutions of table 1 multiple myeloma patients cDNA sample
| Patient's numbering | Highly diluted sensitivity | Undiluted sample mean Ct value | The average Ct value of highly diluted sensitivity | The dilution curve slope | Relation conefficient |
| 1 | -4 | 20.81 | 34.5 | -3.38 | 0.988 |
| 2 | -4 | 22.11 | 36.05 | -3.45 | 0.995 |
| 3 | -4 | 19.93 | 33.48 | -3.42 | 0.996 |
| 4 | -4 | 24.12 | 36.54 | -2.87 | 0.999 |
| 5 | -5 | 19.72 | 37.95 | -3.32 | 0.995 |
| 6 | -4 | 21.78 | 37.23 | -3.94 | 0.993 |
The result shows, uses the Auele Specific Primer to the MAGEC1/CT7 gene provided by the invention first and probe first are detected the multiple myeloma patients of 6 routine MAGEC1/CT7 gene masculines through RQ-PCR, and sensitivity can reach 10
-4Or 10
-5Extent of dilution.
The application of embodiment 4, test kit
One, the assembling of test kit
Test kit is made up of following assembly: the Auele Specific Primer that is directed against the MAGEC1/CT7 gene of embodiment 1 preparation is to first and probe first; The positive control plasmid and the negative control plasmid of embodiment 2 preparations.
Two, the application of test kit
Detect MAGEC1/CT7 expression of gene situation in the marrow of 125 multiple myeloma patients (wherein 51 are the multiple myeloma patients not controlling or recur), the normal donors (volunteer) of 22 examples, method is following:
1, RNA extracts
With 1 of the step 2 of embodiment 3.
2, cDNA is synthetic
With 2 of the step 2 of embodiment 3.
3, the detection of sample to be tested
Each sample that step 2 obtains carries out RQ-PCR on fluorescence real-time quantitative PCR appearance (7500-FAST of American AB I company type).
Every batch of RQ-PCR experiment all is provided with positive control (the positive control plasmid with isopyknic 10 times of gradient dilutions replaces sample), negative control (replacing sample with isopyknic negative control plasmid) and no template blank and (uses isopyknic H
2O replaces sample).PCR reaction system (10 μ l): the upstream primer 0.4 μ M shown in the sequence 1; Downstream primer 0.4 μ M shown in the sequence 2, the TaqMan probe 0.2 μ M shown in the sequence 3, the public system of 2 * TaqMan universal PC R 5 μ l (ABI companies; The U.S.), cDNA (or plasmid) 1 μ l; All the other are deionized water.PCR reaction conditions: 50 ℃ of 2min, 1 circulation; 95 ℃ of 10min, 1 circulation; 95 ℃ of 15s, 60 ℃ of 1min, 40 circulations.Analyze every crowd of RQ-PCR as a result the time threshold value all be fixed as 0.2.
The result of qualitative detection sees table 2.
Table 2 real-time quantitative polymerase chain reaction detects MAGEC1/CT7 genetic expression positive rate among the MM patient
| Group | Sum | Negative | Positive | Positive rate (%) |
| Patient's sample | 125 | 24 | 101 | 80.8 |
| Just control or recur the patient | 51 | 6 | 45 | 88.3 |
| Normal people's sample of |
22 | 22 | 0 | 0 |
The result shows that test kit of the present invention can be used for the detection of multiple myeloma.For the negative sample of detected result in the multiple myeloma patients, possibly be owing to other Expression of Related Genes of existence in this patient's the marrow, and not have the MAGEC1/CT7 expression of gene.
The expression amount of embodiment 5, MAGEC1/CT7 and multiple myeloma ISS dependency by stages
One, the assembling of test kit
Test kit is made up of following assembly: the Auele Specific Primer that is directed against the MAGEC1/CT7 gene of embodiment 1 preparation is to first and probe first; The Auele Specific Primer that is directed against abl gene of embodiment 1 preparation is to second and probe second; Positive control plasmid, negative control plasmid and the confidential reference items control plasmid of embodiment 2 preparations.
Two, the application of test kit
Detection is in the expression amount of MAGEC1/CT7 in the different I SS multiple myeloma patients (volunteer) by stages, and method is following:
1, RNA extracts
With 1 of the step 2 of embodiment 3.
2, cDNA is synthetic
With 2 of the step 2 of embodiment 3.
3, the detection of sample to be tested
Each sample that step 2 obtains carries out RQ-PCR on fluorescence real-time quantitative PCR appearance (7500-FAST of American AB I company type).
The setting of positive control, negative control and blank: (the positive control plasmid with isopyknic 10 times of gradient dilutions replaces sample to positive control; Contain 10 respectively
5, 10
4, 10
3, 10
2, 10
1, 10
0The MAGEC1/CT7 gene fragment of individual copy), negative control (replacing sample with isopyknic negative control plasmid) and no template blank (are used isopyknic H
2O replaces sample).PCR reaction system (10 μ l): the upstream primer 0.4 μ M shown in the sequence 1; Downstream primer 0.4 μ M shown in the sequence 2, the TaqMan probe 0.2 μ M shown in the sequence 3, the public system of 2 * TaqMan universal PC R 5 μ l (ABI companies; The U.S.), cDNA (or plasmid) 1 μ l; All the other are deionized water.PCR reaction conditions: 50 ℃ of 2min, 1 circulation; 95 ℃ of 10min, 1 circulation; 95 ℃ of 15s, 60 ℃ of 1min, 40 circulations.
The setting of confidential reference items contrast: the confidential reference items control plasmid with isopyknic 10 times of gradient dilutions replaces sample; Contain 10 respectively
6, 10
5, 10
4, 10
3, 10
2, 10
1The abl gene fragment of individual copy; PCR reaction system (10 μ l): the upstream primer 0.3 μ M shown in the sequence 4; Downstream primer 0.3 μ M shown in the sequence 5, the TaqMan probe 0.2 μ M shown in the sequence 6, the public system of 2 * TaqMan universal PC R 5 μ l (ABI companies; The U.S.), cDNA (or plasmid) 1 μ l; All the other are deionized water.PCR reaction conditions: 50 ℃ of 2min, 1 circulation; 95 ℃ of 10min, 1 circulation; 95 ℃ of 15s, 60 ℃ of 1min, 40 circulations.
Analyze every crowd of RQ-PCR as a result the time, the MAGEC1/CT7 threshold value all is fixed as 0.2, and the ABL threshold value all is fixed as 0.08.
The typical curve of positive control plasmid RQ-PCR amplification is seen Fig. 2, and the typical curve equation is logMAGEC1/CT7=(Ct-31.232)/-3.281 (r=0.995).The typical curve of confidential reference items control plasmid RQ-PCR amplification is seen Fig. 3, and the typical curve equation is log ABL=(Ct-38.465)/-3.221 (r=0.998).
SDS software utilizes the C of sample
TBe worth, calculate the copy number of each part sample ABL and goal gene MAGEC1/CT7 according to typical curve respectively.
The result shows that the MAGEC1/CT7 expression amount is relevant by stages with ISS, and the MAGEC1/CT7 expression amount increases with ISS by stages, sees table 3.
Table 3ISS by stages and the relation between the MAGEC1/CT7 gene expression dose
| ISS by stages | Sample number | Mean ± |
| 1 | 2 | 77±95 |
| 2 | 13 | 677±1484 |
| 3 | 9 | 2327±2329 |
The dependency of the expression amount of embodiment 6, MAGEC1/CT7 and marrow plasmocyte, CD38+/CD138+ cell count
One, the assembling of test kit
Test kit is made up of following assembly: the Auele Specific Primer that is directed against the MAGEC1/CT7 gene of embodiment 1 preparation is to first and probe first; The Auele Specific Primer that is directed against abl gene of embodiment 1 preparation is to second and probe second; Positive control plasmid, negative control plasmid and the confidential reference items control plasmid of embodiment 2 preparations.
Two, the dependency of MAGEC1/CT7 gene expression amount and marrow plasmocyte number
Detect the expression amount of MAGEC1/CT7 in 25 multiple myeloma patients (volunteer), method is following:
1, RNA extracts
With 1 of the step 2 of embodiment 3.
2, cDNA is synthetic
With 2 of the step 2 of embodiment 3.
3, the detection of sample to be tested
With 3 of the step 2 of embodiment 5.
Simultaneously, the sample of bone marrow smear is carried out morphological examination, detect the quantity of these 25 patients' marrow mesoplasmatocyte through microscope.25 patients' marrow mesoplasmatocyte accounts for the quantity per-cent and the MAGEC1/CT7 gene mRNA level of bone marrow nucleated cell, sees Fig. 4.Visible by Fig. 4, MAGEC1/CT7 expression amount and plasmocyte are counted positive correlation (r=0.435, P＜0.05).
Three, the dependency of MAGEC1/CT7 gene expression amount and CD38+/CD138+ cell count
Detect the expression amount of MAGEC1/CT7 in 31 multiple myeloma patients (volunteer), method is following:
1, RNA extracts
With 1 of the step 2 of embodiment 3.
2, cDNA is synthetic
With 2 of the step 2 of embodiment 3.
3, the detection of sample to be tested
With 3 of the step 2 of embodiment 5.
Simultaneously, use flow cytometer, detect the quantity of CD38+/CD138+ cell in this 31 patients' the marrow.The CD38+/CD138+ cell accounts for the quantity per-cent and the MAGEC1/CT7 gene mRNA level of bone marrow nucleated cell in 31 patients' the marrow, sees Fig. 5.Visible by Fig. 5, MAGEC1/CT7 expression level and CD38+/CD138+ positive cell percentage be proportionate (r=0.41, P＜0.05).
According to above detected result; The per-cent that CD38+/CD138+ cell in 31 patients' the marrow is accounted for bone marrow nucleated cell by＜1% (1 group, n=9), 1%-10% (2 groups, n=14),＞10% (3 groups; N=8) be divided into three groups; Each is organized the MAGEC1/CT7 gene expression dose and sees Fig. 6, and along with the increase that the CD38+/CD138+ cell accounts for the per-cent of bone marrow nucleated cell, the MAGEC1/CT7 gene expression dose also increases; Wherein the 1%-10% group is compared with＞10% group, and the increase of MAGEC1/CT7 gene expression dose has the difference (P＜0.05) of statistical significance.
Sequence table
< 110>The People's Hospital of Peking University
< 120>a kind of test kit of assisting to diagnose multiple myeloma
<130>CGGNARY92636
<160>8
<210>1
<211>23
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>1
ttgtcttctg?ggaaccttga?ctc 23
<210>2
<211>25
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>2
tgagggacac?atacatccta?aaagc 25
<210>3
<211>23
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>3
actgcctggg?cctcctctgc?tgt 23
<210>4
<211>31
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>4
tggagataac?actctaagca?taactaaagg?t 31
<210>5
<211>21
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>5
gatgtagttg?cttgggaccc?a 21
<210>6
<211>28
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>6
ccatttttgg?tttgggcttc?acaccatt 28
<210>7
<211>632
<212>DNA
< 213>Genus Homo people (Homo sapiens)
<400>7
gacgaggatc?gtctcaggtc?agcggaggga?ggagacttat?agacctatcc?agtcttcaag 60
gtgctccaga?aagcaggagt?tgaagacctg?ggtgtgaggg?acacatacat?cctaaaagca 120
ccacagcaga?ggaggcccag?gcagtgccag?gagtcaaggt?tcccagaaga?caaaccccct 180
aggaagacag?gcgacctgtg?aggccctaga?gcaccacctt?aagagaagaa?gagctgtaag 240
ccggcctttg?tcagagccat?catgggggac?aaggatatgc?ctactgctgg?gatgccgagt 300
cttctccaga?gttcctctga?gagtcctcag?agttgtcctg?agggggagga?ctcccagtct 360
cctctccaga?ttccccagag?ttctcctgag?agcgacgaca?ccctgtatcc?tctccagagt 420
cctcagagtc?gttctgaggg?ggaggactcc?tcggatcctc?tccagagacc?tcctgagggg 480
aaggactccc?agtctcctct?ccagattccc?cagagttctc?ctgagggcga?cgacacccag 540
tctcctctcc?agaattctca?gagttctcct?gaggggaagg?actccctgtc?tcctctagag 600
atttctcaga?gccctcctga?gggtgaggat?gt 632
<210>8
<211>124
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>8
tggagataac?actctaagca?taactaaagg?tgaaaagctc?cgggtcttag?gctataatca 60
caatggggaa?tggtgtgaag?cccaaaccaa?aaatggccaa?ggctgggtcc?caagcaacta 120
catc 124
Claims (4)
1. the test kit of an assisting to diagnose multiple myeloma comprises that the Auele Specific Primer that is directed against the MAGEC1/CT7 gene is to first and probe first; Said Auele Specific Primer is that the primer that nucleotide sequence is formed shown in the sequence 2 of sequence 1 and sequence table of sequence table is right to first; The nucleotide sequence of said probe first is shown in the sequence 3 of sequence table.
2. test kit as claimed in claim 1 is characterized in that: said test kit also comprises positive plasmid; Said positive plasmid is the recombinant plasmid that contains the MAGEC1/CT7 gene fragment shown in the sequence 7.
3. test kit as claimed in claim 2 is characterized in that: said positive plasmid is for being connected the recombinant plasmid that obtains with the MAGEC1/CT7 gene fragment shown in the sequence 7 of pMD18-T carrier and sequence table.
4. the Auele Specific Primer that is directed against the MAGEC1/CT7 gene is to first and the application of probe first in the test kit of preparation assisting to diagnose multiple myeloma; Said Auele Specific Primer is that the primer that nucleotide sequence is formed shown in the sequence 2 of sequence 1 and sequence table of sequence table is right to first; The nucleotide sequence of said probe first is shown in the sequence 3 of sequence table.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102372296A CN101705302B (en) | 2009-11-05 | 2009-11-05 | Kit for assisting to diagnose multiple myeloma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102372296A CN101705302B (en) | 2009-11-05 | 2009-11-05 | Kit for assisting to diagnose multiple myeloma |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101705302A CN101705302A (en) | 2010-05-12 |
| CN101705302B true CN101705302B (en) | 2012-07-11 |
Family
ID=42375554
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2009102372296A Expired - Fee Related CN101705302B (en) | 2009-11-05 | 2009-11-05 | Kit for assisting to diagnose multiple myeloma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101705302B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102336828B (en) * | 2010-07-26 | 2013-06-26 | 中国医学科学院基础医学研究所 | Multiple myeloma specific protein and its special detection kit |
| CN102242209B (en) * | 2011-07-05 | 2013-12-11 | 北京大学人民医院 | Quantitative detection kit based on multiple genes for assisting diagnosis of patients with multiple myeloma |
| CN102251034B (en) * | 2011-07-05 | 2012-11-21 | 北京大学人民医院 | Quantitative test kit for assisting diagnosing multiple myeloma patients on basis of MAGE-C2/CT10 gene |
| CN110218789B (en) * | 2019-04-22 | 2022-10-04 | 曾文炳 | Gene probe composition and kit for predicting overall survival rate of multiple myeloma patients |
| CN111476754B (en) * | 2020-02-28 | 2022-12-09 | 中国人民解放军陆军军医大学第二附属医院 | Bone marrow cell image artificial intelligence auxiliary grading diagnosis system and method |
| CN114480645B (en) * | 2022-01-13 | 2024-06-18 | 上海交通大学医学院附属仁济医院 | Multiple myeloma depletion NK cell subgroup, characteristic gene and application thereof |
-
2009
- 2009-11-05 CN CN2009102372296A patent/CN101705302B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN101705302A (en) | 2010-05-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Venner et al. | Molecular landscape of T cell–mediated rejection in human kidney transplants: prominence of CTLA4 and PD ligands | |
| Wisdom et al. | Single cell analysis reveals distinct immune landscapes in transplant and primary sarcomas that determine response or resistance to immunotherapy | |
| Bleakley et al. | Leukemia-associated minor histocompatibility antigen discovery using T-cell clones isolated by in vitro stimulation of naive CD8+ T cells | |
| CN101705302B (en) | Kit for assisting to diagnose multiple myeloma | |
| Altboum et al. | Digital cell quantification identifies global immune cell dynamics during influenza infection | |
| AU2019204503A1 (en) | Receptor gene for peptide cancer antigen-specific T cell | |
| Lin et al. | Circulating tumor cell as a biomarker for evaluating allogenic NK cell immunotherapy on stage IV non-small cell lung cancer | |
| CN109280704A (en) | RASD1 is preparing the application in B-ALL diagnosis and prognosis evaluation reagent kit as marker | |
| CN110106249A (en) | Application of the long-chain non-coding RNA NONHSAG039951.2 as diagnosing osteoporosis marker | |
| CN101974529B (en) | TGF-beta specific siRNA containing free triphosphoric acid group and application thereof | |
| CN110157794A (en) | Application of the long-chain non-coding RNA NONHSAT005760.2 as diagnosing osteoporosis marker | |
| CN105969892A (en) | Application of CSRP2 serving as marker for evaluating prognostic risk of adult B-ALL patients | |
| Schuhmacher et al. | A strong host response and lack of MYC expression are characteristic for diffuse large B cell lymphoma transformed from nodular lymphocyte predominant Hodgkin lymphoma | |
| Greco et al. | Gene expression analysis in SV-40 immortalized human corneal epithelial cells cultured with an air-liquid interface | |
| CN102251033B (en) | Quantitative detection kit for assistant diagnosis of multiple myeloma patient based on MAGE-A3 gene | |
| CN103451303B (en) | Kit for detecting expression level of human ERCC1 (excision repair cross complementation 1) through PCR (polymerase chain reaction) method | |
| Hallas et al. | Immunohistochemical distinction of ABC and GCB in extranodal DLBCL is not reflected in mutation patterns | |
| CN102242209B (en) | Quantitative detection kit based on multiple genes for assisting diagnosis of patients with multiple myeloma | |
| CN102251035A (en) | Quantitative detection kit for SSX-2 gene-based assisted diagnosis of multiple myeloma patients | |
| CN107029238B (en) | Applications of the LINC01094 in diagnosis and treatment cerebral arterial thrombosis | |
| Perco et al. | Molecular signature of mice T lymphocytes following tolerance induction by allogeneic BMT and CD40‐CD40L costimulation blockade | |
| Wisdom et al. | A single cell atlas reveals distinct immune landscapes in transplant and primary tumors that determine response or resistance to immunotherapy | |
| CN102643899B (en) | Kit for diagnosing acute myelocytic leukemia | |
| Arnaoaut et al. | CDX2 gene expression in acute lymphoblastic leukemia | |
| CN107619862B (en) | Sperm production disorder pathogenic gene NFATC1 for mild anemia men and detection method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120711 Termination date: 20171105 |