CN101624620A - Kit for quantitatively detecting AML1/ETO mRNA level - Google Patents
Kit for quantitatively detecting AML1/ETO mRNA level Download PDFInfo
- Publication number
- CN101624620A CN101624620A CN200810116600A CN200810116600A CN101624620A CN 101624620 A CN101624620 A CN 101624620A CN 200810116600 A CN200810116600 A CN 200810116600A CN 200810116600 A CN200810116600 A CN 200810116600A CN 101624620 A CN101624620 A CN 101624620A
- Authority
- CN
- China
- Prior art keywords
- aml1
- eto
- sequence
- real
- time quantitative
- 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit for quantitatively detecting an AML1/ETO mRNA level. The kit comprises a standard product which is used for manufacturing a standard curve, an inner reference gene real-time quantitative PCR system and an AML1/ETO real-time quantitative PCR system. The kit can accurately, quickly and quantitatively detect the AML1/ETO mRNA level, is used for diagnosing acute myelogenous leukemia and monitoring minimal residual diseases in a treatment process, and provides an important molecular basis for accurate diagnosis of clinical diseases, determination of a treatment proposal, curative effect evaluation and prognosis.
Description
Technical field
The present invention relates to a kind of test kit of detection by quantitative AML1/ETO mRNA level.
Background technology
Acute myeloid leukaemia (AML) chromosome translocation t (8; 21) (q22; Q22) on molecular level, show as AML1 gene and ETO gene and form fusion gene AML1/ETO, have approximately among the adult of 8-12% and the children's primary AML and contain this fusion gene, there is 40% M2 type AML patient to contain this fusion gene approximately, minority case report is also arranged among M0 type, M1 type and the M4 type AML patient in addition.The alpha subunit of AML1 genes encoding heterodimer transcription factor CBF, it is the key factor that hemopoietic system is grown, ETO albumen then is one and transcribes common supressor.Also not fully aware of about the mechanism of AML1/ETO initiation AML at present.AML1/ETO fusion gene type is single, and the breaking point on the AML1 is all on intron 5, and therefore the breaking point on the ETO adopts a cover primer can carry out pcr amplification AML1/ETO fusion gene all in the upstream of exon 2.Adopt round pcr to detect and have important use value in the monitoring of AML1/ETO fusion gene for minimal residual disease (MRD) in diagnosis, prognosis and the therapeutic process of AML.
Fa Ming real-time quantitative PCR technology (RQ-PCR) had realized the quantitative leap of PCR meaning from qualitative to real in recent years.Compare with regular-PCR, RQ-PCR also has the following advantages except can be quantitatively: (1) by adding probe or by making melting curve in the PCR reaction system, makes that specificity strengthens, sensitivity improves; (2) stopped pipe operation in the amplification procedure, the real-time collecting data have reduced opportunities for contamination, reduce false positive results; (3) need not electrophoresis, shortened the operating time; (4) estimate sample quality by quantitative internal control gene, reduced false negative result.RQ-PCR key problem in technology part based on the TaqMan probe is: also added the TaqMan probe in the PCR reaction system except the primer of upstream and downstream, 5 of this probe ' end and 3 ' end indicates fluorescence report group (as FAM or TET) and fluorescent quenching group (as TAMRA) respectively.Before the PCR reaction, probe is complete, and fluorescence report group fluorescent signal emitted is by the cancellation of fluorescent quenching group; Carrying out along with the PCR reaction, the circumscribed activity of Taq enzyme performance 5 ' → 3 ', make the fluorescence report group that scales off away from the fluorescent quenching group, produce fluorescent signal, fluorescence signal intensity is directly proportional with the amount of original template DNA, when fluorescence signal intensity during greater than threshold value (10 SD more than the baseline), can be by fluorescent probe monitoring in real time regularly, the CT value of each sample is (in the pcr amplification process, fluorescent signal begins to be entered by background the pairing cycle number of flex point of exponential growth phase) be directly proportional with the logarithmic value of starting template amount, can utilize typical curve to calculate the amount of goal gene and internal control gene respectively according to the CT value, the two ratio that is divided by be the level relatively of goal gene.
The amount that calculates starting template must be utilized typical curve, the making of typical curve is to adopt the standard substance of the known initial amount of process serial dilution to carry out RQ-PCR, standard substance can be the cDNA of known quality or the plasmid of known copy number, the former makes fairly simple, but the unknown sample result who obtains is not directly perceived, than indigestion; Although and the plasmid standard preparation is more loaded down with trivial details, utilize it can calculate the copy number of unknown sample, and visual result, be present most widely used standard substance type.
The purpose that internal control gene is selected is in order to eliminate the difference of aspects such as mRNA quality between the different samples, quantity and reverse transcription efficient, the selection of internal control gene has following three to require greatly: (1) is equal stably express in all karyocytes, have nothing to do with differentiation series and differential period, do not tested the influence of processing; (2) there is not pseudogene; (3) expression level and Degradation Level are consistent with goal gene.
Adopt the clinical value of RQ-PCR technology for detection AML1/ETO mRNA level to be mainly reflected in following three broad aspect: (1) diagnosis: AML1/ETO mRNA is peculiar by AML, especially M2 type AML patient, and acute lymphoblastic leukemia (ALL) patient does not express this fusion gene, therefore adopt RQ-PCR technology for detection AML1/ETO mRNA level to help making a definite diagnosis of AML, and seek out the responsive special molecular indexes that is fit to the MRD monitoring.In the up-to-date WHO Case definition with AML1/ETO (+) AML single-row be a kind of independent type.(2) therefore prognosis: AML1/ETO (+) patient's prognosis bona adopts RQ-PCR technology for detection AML1/ETO mRNA level patient's layering can be helped the selection of clinical treatment.(3) monitoring of MRD: the treatment to AML1/ETO (+) AML patient at present comprises chemotherapy and hematopoietic stem cell transplantation two big classes, they can make most of patient obtain genetics to alleviate fully, and the monitoring of MRD thereafter can only rely on the most responsive RQ-PCR technology of generally acknowledging at present.The MRD level determine be therapeutic evaluation, treatment plan selection (as change chemotherapy regimen, at transplant patient's clinical intervention measures such as donor lymphocyte infusion) important evidence, for the prevention clinical recurrence, improve patient's remission rate, curative ratio and survival rate and have great importance.
Summary of the invention
The test kit that the purpose of this invention is to provide a kind of detection by quantitative AML1/ETO mRNA level.
The test kit of detection by quantitative AML1/ETO mRNA level provided by the present invention comprises standard substance, internal control gene real-time quantitative PCR system and the AML1/ETO real-time quantitative PCR system that is used for the production standard curve;
Described AML1/ETO real-time quantitative PCR system comprises upstream primer, downstream primer and TaqMan probe, the nucleotide sequence of the upstream primer of described AML1/ETO real-time quantitative PCR system is shown in sequence in the sequence table 1, the nucleotide sequence of downstream primer is shown in sequence in the sequence table 2, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 3.
Described internal control gene is preferably abl gene, and during as internal control gene, described internal control gene real-time quantitative PCR system comprises upstream primer, downstream primer and TaqMan probe with abl gene; The nucleotide sequence of the upstream primer of described internal control gene real-time quantitative PCR system is shown in sequence in the sequence table 4, and the nucleotide sequence of downstream primer is shown in sequence in the sequence table 5, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 6;
The Master Mix that also comprises fluorescent PCR in above-mentioned internal control gene real-time quantitative PCR system, the AML1/ETO real-time quantitative PCR system.
Among the present invention, 3 of the TaqMan probe in above-mentioned AML1/ETO real-time quantitative PCR system and the internal control gene real-time quantitative PCR system ' end is connected with fluorescent quenching group TAMRA, and 5 ' end is connected with fluorescence report group FAM.
Standard substance in the test kit of above-mentioned detection by quantitative AML1/ETO mRNA level are the plasmid standard that contains abl gene, and the nucleotide sequence of described abl gene is shown in sequence in the sequence table 7.
Use for convenience, the test kit of above-mentioned detection by quantitative AML1/ETO mRNA level also comprises negative control and AML1/ETO positive control.
The test kit of detection by quantitative AML1/ETO mRNA level of the present invention has following characteristics:
1, susceptibility height: can repeat susceptibility is 5 copies.
2, cost is low: experimental system only is 10 μ l, and each composition consumption all reduces over half, thereby cost is obviously reduced.In addition, the shared typical curve of testing goal gene and internal control gene, not only easy but also reduce cost.
3, practical: this test kit comprises internal control gene real-time quantitative PCR system and plasmid standard simultaneously, can quantitatively go out simultaneously the copy number of AML1/ETO fusion gene and internal control gene ABL in the sample, finally calculates sample AML1/ETO mRNA level.
4, easy to use: the cDNA that only need add testing sample during use can begin the PCR reaction.
5, the shelf lives is long: can prolonged preservation (＞1.5 years) under-20 ℃ of conditions, and do not influence the detection effect.
Test kit of the present invention can be accurately, fast, quantitative assay AML1/ETO mRNA level, be used for the diagnosis of acute myeloid leukaemia and the monitoring of therapeutic process minimal residual disease, for the determining of the making a definite diagnosis of clinical disease, treatment plan, therapeutic evaluation and prognosis provide important molecule foundation.
Description of drawings
Fig. 1 is for utilizing internal control gene real-time quantitative PCR system amplification 1 * 10
6-1 * 10
2The typical curve of the ABL plasmid standard preparation of copy
Embodiment
If the pcr amplification efficient unanimity of two kinds of genes, it also is the same utilizing two typical curves of their plasmid standard making so respectively, and promptly the slope of typical curve and vertical intercept and gene type are irrelevant.Therefore the present invention is after the pcr amplification efficient of verifying goal gene AML1/ETO and internal control gene ABL is consistent, adopt the copy number of simultaneously quantitative goal gene of a typical curve and internal control gene, so not only easy economy, more can eliminate the difference that plasmid quantitatively brings, improve the accuracy of RQ-PCR.
The preparation of the test kit of embodiment 1, detection by quantitative AML1/ETO mRNA level
One, the design of primer and probe in the test kit of detection by quantitative AML1/ETO mRNA level
AML1/ETO real-time quantitative PCR system is as follows:
(1) upstream primer (being arranged in AML1 exon 5): 5 '-CACCTACCACAGAGCCATCAAA-3 ' (sequence table sequence 1), final concentration is 0.3 μ M;
(2) downstream primer (being arranged in the ETO exon 2): 5 '-ATCCACAGGTGAGTCTGGCATT-3 ' (sequence table sequence 2), final concentration is 0.3 μ M;
(3) TaqMan probe (being arranged in the ETO exon 2): 5 '-AACCTCGAAATCGTACTGAGAAGCACTCCA-3 ' (sequence table sequence 3), final concentration is 0.2 μ M;
(4) the Master Mix of fluorescent PCR (available from American AB I company).
Internal control gene real-time quantitative PCR system is as follows:
(1) upstream primer (being positioned at the ABL exon 2):
5 '-TGGAGATAACACTCTAAGCATAACTAAAGGT-3 ' (sequence 4 in the sequence table), final concentration are 0.3 μ M;
(2) downstream primer (being arranged in the ABL exon 3): 5 '-GATGTAGTTGCTTGGGACCCA-3 ' (sequence table sequence 5), final concentration is 0.3 μ M;
(3) TaqMan probe (being arranged in the ABL exon 3): 5 '-CCATTTTTGGTTTGGGCTTCACACCATT-3 ' (sequence table sequence 6), final concentration is 0.2 μ M;
(4) MasterMix of fluorescent PCR (available from American AB I company).
3 of TaqMan probe in above-mentioned AML1/ETO real-time quantitative PCR system and the internal control gene real-time quantitative PCR system ' end is connected with fluorescent quenching group TAMRA, and 5 ' end is connected with fluorescence report group FAM.
Two, the preparation of ABL plasmid standard
The ABL plasmid standard comprises 1 * 10
6Copy/μ L, 1 * 10
5Copy/μ L, 1 * 10
4Copy/μ L, 1 * 10
3Copy/μ L and 1 * 10
2The plasmid standard of five concentration of copy/μ L, its concrete preparation method is as follows:
Establishing criteria product sequence covers and greater than the primer of the principle design pcr amplification ABL plasmid standard of RQ-PCR amplified production, concrete sequence is: upstream primer 5 '-CCTTCAGCGGCCAGTAGC-3 ', downstream primer 5 '-GGACACAGGCCCATGGTAC-3 '.Extract total RNA of the stripped peripheral blood mononuclear cell of normal people, its reverse transcription become cDNA and be that template is carried out pcr amplification with this cDNA, pcr amplification product is carried out agarose gel electrophoresis detection, recovery, purifying, product behind the purifying is connected on the pGEM-T Easy carrier, and transfection TOP10 competent escherichia coli cell, utilize blue hickie screening positive clone.Extract positive colony and carry out plasmid and carry for a short time and check order, sequencing result shows that the deoxyribonucleotide sequence that is connected to the abl gene on the pGEM-T Easy carrier is shown in sequence in the sequence table 7.Wherein, from 5 ' the 142nd the-the 172nd of end is the upstream primer of the internal control gene real-time quantitative PCR system of above-mentioned steps one, from 5 ' the 210th the-the 237th of end is the TaqMan probe sequence of the internal control gene real-time quantitative PCR system of above-mentioned steps one, is the downstream primer of the internal control gene real-time quantitative PCR system of above-mentioned steps one from 5 ' the 245th the-the 265th of end.
The positive colony that above-mentioned sequencing result is correct carries out carrying in the plasmid, and measures plasmid concentration, calculates plasmid copy number, carries out 10 times of serial dilutions, is distributed into 1 * 10 at last
6Copy/μ L, 1 * 10
5Copy/μ L, 1 * 10
4Copy/μ L, 1 * 10
3Copy/μ L, 1 * 10
2Copy/μ L, 1 * 10
1Copy/μ L, put-80 ℃ frozen standby.
Three, the preparation of negative control plasmid-WT1 and AML1/ETO positive control
1, the preparation of negative control plasmid-WT1
Extract total RNA that 1 example has been diagnosed as acute promyelocytic leukemia patient BMNC, its reverse transcription is become cDNA, with this cDNA is template, is primer PCR amplification WT1 gene with 5 '-ccacagcacagggtacgaga-3 ' and 5 '-ctcagatgccgaccgtacaa-3 '.Pcr amplification product is carried out agarose gel electrophoresis detect, the product behind recovery, the purifying is connected on the pGEM-T Easy carrier, and transfection TOP10 competent escherichia coli cell, utilizes blue hickie screening positive clone.Extract positive colony and carry out plasmid and carry for a short time and check order, sequencing result shows that the deoxyribonucleotide sequence that is connected to the WT1 gene on the pGEM-T Easy carrier is shown in sequence in the sequence table 8.The correct plasmid of the sequencing result that obtains is negative control plasmid-WT1.
2, the preparation of AML1/ETO positive control
Separating 1 example is the stripped BMNC of AML1/ETO (+) patients with acute myeloid leukemia from the first visit of the People's Hospital, extracts its total RNA, and its reverse transcription is become cDNA, and this cDNA is the AML1/ETO positive control.
Four, using method
All testing samples internal control gene ABL that all need increase calculates the copy number of ABL and determines the quality of testing sample.The patient of testing sample qualified (sample of ABL copy number 〉=30000 thinks qualified, detects otherwise need extract sample again again), AML1/ETO fusion gene again increases.Concrete using method is as follows:
1, gets stripped patient's marrow to be detected or peripheral blood mononuclear cell, extract its total RNA, and its reverse transcription is become cDNA.
2, the cDNA that adds the testing sample of 1 μ L step 1 in the internal control gene real-time quantitative PCR system of 9 μ L above-mentioned steps one carries out the real-time quantitative PCR reaction, calculates the quality of copy number and the definite testing sample of ABL then.
Qualified (sample of ABL copy number 〉=30000 thinks qualified for testing sample, otherwise need extract sample again detects again) the patient, the cDNA that adds the testing sample of 1 μ L step 1 in the AML1/ETO real-time quantitative PCR system of 9 μ L above-mentioned steps one carries out the real-time quantitative PCR reaction.
The condition of above-mentioned real-time quantitative PCR reaction is: 1 circulation of 50 ℃ of 2min earlier; 1 circulation of 95 ℃ of 10min then; 95 ℃ of 15s again, 62 ℃ of 1min, 40 circulations.
3, the making of typical curve: the copy number that adds two preparations of 1 μ L above-mentioned steps in the internal control gene real-time quantitative PCR system of 9 μ L above-mentioned steps one respectively is 1 * 10
6, 1 * 10
5, 1 * 10
4, 1 * 10
3And 1 * 10
2The ABL plasmid standard of five concentration, 1 circulation of 50 ℃ of 2min earlier; 1 circulation of 95 ℃ of 10min then; 95 ℃ of 15s again, 62 ℃ of 1min, 40 circulations.The production standard curve, wherein 1 * 10
2The ABL plasmid standard of copy is made two pipe parallel pipes simultaneously, ABL plasmid standards of all the other each copy pipe that increases respectively.
4, every crowd of RQ-PCR reacts also need increase the simultaneously positive and negative control.The AML1/ETO positive control that in the AML1/ETO real-time quantitative PCR system of 9 μ L above-mentioned steps one, adds three preparations of 1 μ L above-mentioned steps; The negative control that adds three preparations of 1 μ L above-mentioned steps in the internal control gene real-time quantitative PCR system of 9 μ L above-mentioned steps one carries out the real-time quantitative PCR reaction.
5, the AML1/ETO mRNA level of testing sample is calculated according to following formula:
The condition of storage of the test kit of detection by quantitative AML1/ETO mRNA level of the present invention is: 4 ℃ of lucifuges store at least 2 months or-20 ℃ of lucifuges store 1.5 years.
Five, the susceptibility of the test kit of detection by quantitative AML1/ETO mRNA level of the present invention, specificity and stability are measured
1, susceptibility: can repeat susceptibility is 5 copies
Each 5 parts of the ABL plasmid standards of 5 copies that will prepare according to the method for above-mentioned steps two, utilize the internal control gene real-time quantitative PCR system of above-mentioned steps one to carry out repeated experiments 5 times respectively, in the each experiment of result amplification curve is arranged all, therefore the repeated susceptibility of internal control gene real-time quantitative PCR system of the present invention is 5 copies.
The AML1/ETO positive control of above-mentioned steps three preparations is carried out serial dilution, be template with the cDNA behind the serial dilution respectively, utilize the AML1/ETO real-time quantitative PCR system of above-mentioned steps one to carry out 5 batches of repetition RQ-PCR reaction, each ABL plasmid standard that all increases simultaneously that reacts, the production standard curve, be equivalent to 5 copies and greater than the AML1/ETO positive control after the dilution of 5 copies amplification curve arranged all in the each experiment of result, therefore the repeated susceptibility of AML1/ETO real-time quantitative PCR system of the present invention is 5 copies.
2, specificity
We utilize the AML1/ETO real-time quantitative PCR system of above-mentioned steps one to detect negative control plasmid-WT1, and amplification curve does not appear in the result, confirm that test kit of the present invention has specificity.
Because human nuclear hematopoietic cell all contains abl gene, utilize the internal control gene real-time quantitative PCR system of above-mentioned steps one to detect the WT1 plasmid that does not contain abl gene, amplification curve does not appear in the result, confirms that the ABL detection has specificity.
3, stability
Sample used in the following stability experiment is the stripped BMNC of voluntary donor all from the People's Hospital.
(1) difference analysis in the daytime
1. extract total RNA of sample G717, its reverse transcription is become cDNA, and be template with this cDNA, utilize the internal control gene real-time quantitative PCR system of above-mentioned steps one to carry out the RQ-PCR reaction, carry out 5 batches of experiments altogether, the result is as shown in the table, and as can be known from the table data, the poor in the daytime variation coefficient (CV) is 1.83%.
| Sample number | The CT value | Detection time |
| ??1 | ??22.44 | ??06-6-10AM |
| ??2 | ??22.91 | ??05-6-17PM |
| ??3 | ??22.84 | ??05-7-1PM |
| ??4 | ??23.35 | ??05-7-8AM |
| ??5 | ??22.3 | ??05-7-15AM |
2. extract total RNA of sample A352, its reverse transcription is become cDNA, and be template with this cDNA, utilize the AML1/ETO real-time quantitative PCR system of above-mentioned steps one to carry out the RQ-PCR reaction, carry out 5 batches of experiments altogether, the result is as shown in the table, and as can be known from the table data, the poor in the daytime variation coefficient (CV) is 0.61%.
| Sample number | The CT value | Detection time |
| ??1 | ??31.31 | ??06-3-20PM |
| ??2 | ??31.67 | ??06-3-24AM |
| ??3 | ??31.19 | ??06-4-14AM |
| ??4 | ??31.23 | ??06-9-21PM |
| ??5 | ??31.28 | ??06-9-22AM |
(2) day interior difference analysis
1. extract total RNA of sample H236, its reverse transcription is become cDNA, and be template with this cDNA, utilize the internal control gene real-time quantitative PCR system of above-mentioned steps one to carry out the RQ-PCR reaction, the same batch of RQ-PCR reaction of having carried out 5 parallel holes, the result is as shown in the table, and as can be known from the table data, a day interpolation variation coefficient (CV) is 1.35%.
| Sample number | The CT value | Detection time |
| ??1 | ??22.02 | ??05-9-30AM |
| ??2 | ??22.2 | ??05-9-30AM |
| ??3 | ??22.21 | ??05-9-30AM |
| ??4 | ??21.83 | ??05-9-30AM |
| ??5 | ??22.64 | ??05-9-30AM |
2. extract total RNA of sample A610, its reverse transcription is become cDNA, and be template with this cDNA, utilize the AML1/ETO real-time quantitative PCR system of above-mentioned steps one to carry out the RQ-PCR reaction, the same batch of RQ-PCR reaction of having carried out 5 parallel holes, the result is as shown in the table, and as can be known from the table data, a day interpolation variation coefficient (CV) is 1.30%.
| Sample number | The CT value | Detection time |
| ??1 | ??31.53 | ??06-9-27AM |
| ??2 | ??31.28 | ??06-9-27AM |
| ??3 | ??31.73 | ??06-9-27AM |
| ??4 | ??31.18 | ??06-9-27AM |
| ??5 | ??30.66 | ??06-9-27AM |
4, be the influence of the storing temp of example detection kit and storage time with the ABL plasmid standard of different copy numbers to experimental result
Utilize test kit of the present invention to detect 1 * 10
3And 1 * 10
7The ABL plasmid standard room temperature lucifuge of copy stored for 1 week, 4 ℃ of lucifuges store 3 weeks, 6 weeks, 9 weeks and 12 weeks, the detected result when-20 ℃ of lucifuges store 18 months, detected result CT value representation, concrete outcome is shown in following two tables, data as can be seen from table, variation along with storing temp and storage time, the no considerable change of CT value shows that test kit is still very stable.
10
3Different storing temps of the ABL plasmid standard of copy and the CT value under the time change
| Numbering | Storing temp (℃) | The CT value | Storage time |
| ??1 | Room temperature | ??31.39 | 1 week |
| ??2 | ??4 | ??31.3 | 3 weeks |
| ??3 | ??4 | ??30.92 | 6 weeks |
| ??4 | ??4 | ??31.18 | 9 weeks |
| ??5 | ??4 | ??30.34 | 12 weeks |
| ??6 | ??-20 | ??31.89 | 18 months |
10
7Different storing temps of the ABL plasmid standard of copy and the CT value under the time change
| Numbering | Storing temp (℃) | The CT value | Storage time |
| ??1 | Room temperature | ??17.41 | 1 week |
| ??2 | ??4 | ??17.66 | 3 weeks |
| ??3 | ??4 | ??16.87 | 6 weeks |
| ??4 | ??4 | ??16.65 | 9 weeks |
| ??5 | ??4 | ??16.24 | 12 weeks |
| ??6 | ??-20 | ??17.82 | 18 months |
Sequence table
<160>8
<210>1
<211>22
<212>DNA
＜213〉artificial sequence
<400>1
cacctaccac?agagccatca?aa????????????22
<210>2
<211>22
<212>DNA
＜213〉artificial sequence
<400>2
atccacaggt?gagtctggca?tt????????????22
<210>3
<211>30
<212>DNA
＜213〉artificial sequence
<400>3
aacctcgaaa?tcgtactgag?aagcactcca????30
<210>4
<211>31
<212>DNA
＜213〉artificial sequence
<400>4
tggagataac?actctaagca?taactaaagg?t????????????????????????????????????31
<210>5
<211>21
<212>DNA
＜213〉artificial sequence
<400>5
gatgtagttg?cttgggaccc?a???????????????????????????????????????????????21
<210>6
<211>28
<212>DNA
＜213〉artificial sequence
<400>6
ccatttttgg?tttgggcttc?acaccatt????????????????????????????????????????28
<210>7
<211>316
<212>DNA
＜213〉artificial sequence
<400>7
ccttcagcgg?ccagtagcat?ctgactttga?gcctcagggt?ctgagtgaag?ccgctcgttg?????60
gaactccaag?gaaaaccttc?tcgctggacc?cagtgaaaat?gaccccaacc?ttttcgttgc????120
actgtatgat?tttgtggcca?gtggagataa?cactctaagc?ataactaaag?gtgaaaagct????180
ccgggtctta?ggctataatc?acaatgggga?atggtgtgaa?gcccaaacca?aaaatggcca????240
aggctgggtc?ccaagcaact?acatcacgcc?agtcaacagt?ctggagaaac?actcctggta????300
ccatgggcct?gtgtcc????????????????????????????????????????????????????316
<210>8
<211>151
<212>DNA
＜213〉artificial sequence
<400>8
ccacagcaca?gggtacgaga?gcgataacca?cacaacgccc?atcctctgcg?gagcccaata????60
cagaatacac?acgcacggtg?tcttcagagg?cattcaggat?gtgcgacgtg?tgcctggagt????120
agccccgact?cttgtacggt?cggcatctga?g???????????????????????????????????151
Claims (8)
1, a kind of test kit of detection by quantitative AML1/ETO mRNA level comprises the standard substance, internal control gene real-time quantitative PCR system and the AML1/ETO real-time quantitative PCR system that are used for the production standard curve;
Described AML1/ETO real-time quantitative PCR system comprises upstream primer, downstream primer and TaqMan probe; The nucleotide sequence of described AML1/ETO real-time quantitative PCR system upstream primer is shown in sequence in the sequence table 1, and the nucleotide sequence of downstream primer is shown in sequence in the sequence table 2, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 3.
2, test kit according to claim 1 is characterized in that: when described internal control gene was abl gene, described internal control gene real-time quantitative PCR system comprised upstream primer, downstream primer and TaqMan probe; The nucleotide sequence of the upstream primer of described internal control gene real-time quantitative PCR system is shown in sequence in the sequence table 4, and the nucleotide sequence of downstream primer is shown in sequence in the sequence table 5, and the nucleotide sequence of TaqMan probe is shown in sequence in the sequence table 6;
3, test kit according to claim 2 is characterized in that: the Master Mix that also comprises fluorescent PCR in described internal control gene real-time quantitative PCR system, the AML1/ETO real-time quantitative PCR system.
4, test kit according to claim 3, it is characterized in that: 3 of the TaqMan probe in described AML1/ETO real-time quantitative PCR system and the internal control gene real-time quantitative PCR system ' end is connected with fluorescent quenching group TAMRA, and 5 ' end is connected with fluorescence report group FAM.
5, test kit according to claim 1 is characterized in that: described standard substance are the plasmid standard that contains abl gene.
6, test kit according to claim 5 is characterized in that: the nucleotide sequence of described abl gene is shown in sequence in the sequence table 7.
7, test kit according to claim 6 is characterized in that: described test kit also comprises the AML1/ETO positive control.
8, test kit according to claim 7 is characterized in that: described test kit also comprises negative control.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810116600A CN101624620A (en) | 2008-07-11 | 2008-07-11 | Kit for quantitatively detecting AML1/ETO mRNA level |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810116600A CN101624620A (en) | 2008-07-11 | 2008-07-11 | Kit for quantitatively detecting AML1/ETO mRNA level |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101624620A true CN101624620A (en) | 2010-01-13 |
Family
ID=41520612
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810116600A Pending CN101624620A (en) | 2008-07-11 | 2008-07-11 | Kit for quantitatively detecting AML1/ETO mRNA level |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101624620A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161990A (en) * | 2010-02-24 | 2011-08-24 | 北京雅康博生物科技有限公司 | Kit for quantificationally detecting BRAF (Block Repeat Active Flag) mutation |
| CN102925573A (en) * | 2012-09-29 | 2013-02-13 | 李艳 | Kit for detecting protein expression indexes of acute myelogenous leukemia1-eighttwentyone (AML1-ETO) fusion gene messenger ribonucleic acid (mRNA) |
| CN102925559A (en) * | 2012-09-29 | 2013-02-13 | 童永清 | Kit for quantitatively detecting W515 site mutation of MPL genes |
-
2008
- 2008-07-11 CN CN200810116600A patent/CN101624620A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161990A (en) * | 2010-02-24 | 2011-08-24 | 北京雅康博生物科技有限公司 | Kit for quantificationally detecting BRAF (Block Repeat Active Flag) mutation |
| CN102925573A (en) * | 2012-09-29 | 2013-02-13 | 李艳 | Kit for detecting protein expression indexes of acute myelogenous leukemia1-eighttwentyone (AML1-ETO) fusion gene messenger ribonucleic acid (mRNA) |
| CN102925559A (en) * | 2012-09-29 | 2013-02-13 | 童永清 | Kit for quantitatively detecting W515 site mutation of MPL genes |
| CN102925573B (en) * | 2012-09-29 | 2014-12-10 | 李艳 | Kit for detecting protein expression indexes of acute myelogenous leukemia1-eighttwentyone (AML1-ETO) fusion gene messenger ribonucleic acid (mRNA) |
| CN102925559B (en) * | 2012-09-29 | 2014-12-10 | 童永清 | Kit for quantitatively detecting W515 site mutation of MPL genes |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101624621B (en) | Kit for quantitatively detecting BCR/ABL mRNA level | |
| Drewry et al. | Differentially expressed microRNAs in the aqueous humor of patients with exfoliation glaucoma or primary open-angle glaucoma | |
| Lusardi et al. | MicroRNAs in human cerebrospinal fluid as biomarkers for Alzheimer’s disease | |
| JP6342329B2 (en) | Method for predicting responsiveness to treatment with an EGFR inhibitor | |
| CN105112522A (en) | Detection primer combination and kit for HER2 (human epidermal growth factor receptor 2) gene amplification | |
| CN103451268B (en) | A kind of standard substance of detection line plastochondria A3243G heterozygous mutant rate, test kit and detection method thereof | |
| CN108753984A (en) | The biomarker combinations and its application and kit of prediction or diagnosis Malignant cerebral gliomas postoperative recurrence | |
| CN104862310A (en) | Schizophrenia biomarker, screening method and kit | |
| CN103667453B (en) | Method, primers and probe for detecting relative expression quantity of 11q23/MLL fusion genes | |
| Margery‐Muir et al. | Establishment of Gene Copy Number–Specific Normal Ranges for Serum C4 and Its Utility for Interpretation in Patients With Chronically Low Serum C4 Concentrations | |
| CN101624620A (en) | Kit for quantitatively detecting AML1/ETO mRNA level | |
| CN101624622A (en) | Kit for quantitatively detecting PML/RARalpha mRNA level | |
| CN101624623A (en) | Kit for quantitatively detecting ABL mRNA level | |
| CN108130362A (en) | Kit and application for EGFR genetic mutation detection | |
| CN101298630A (en) | Method for identifying miRNA in blood serum of colon cancer patient by Solexa technology | |
| CN102827935B (en) | Reagent kid for quantitatively testing mRNA (messenger ribonucleic acid) level of FIP1L1-PDGFRA (feline infectious peritonitis 1 like 1-platelet-derived growth factor receptor alpha) fusion genes | |
| CN110257496A (en) | Clopidogrel pharmaceutical relevant gene CYP2C19 genetic polymorphism detection primer sets, kit and detection method | |
| CN112481379B (en) | Primer probe combination and kit for detecting Ph-like ALL fusion gene | |
| CN110066868A (en) | A kind of primer sets, kit and the detection method of aspirin pharmaceutical relevant gene genetic polymorphism detection | |
| CN107002124A (en) | For the method for the survival prognosis for determining the patient with cancer of pancreas | |
| EP4112745A1 (en) | Biomarkers for predicting a patient's response to bcg therapy, methods and uses based thereon | |
| CN108913774A (en) | C-KIT somatic mutation gene detection KIT and detection method thereof | |
| CN104561257B (en) | A kind of taking non-coding RNA Tmevpg1 as detecting or diagnose myasthenia gravis detection kit and the application of screening marker | |
| US20170159125A1 (en) | Micro-rna biomarkers for haemolysis and methods of using same | |
| KR101988578B1 (en) | A marker for the determination of Mibyeong |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20100113 |