CN102094074A - Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1 - Google Patents
Fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting leukemia fusion gene TEL-AML1 Download PDFInfo
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Abstract
The invention discloses a fluorescent reverse transcription-polymerase chain reaction (RT-PCR) kit for quantitatively detecting a leukemia translocation ETS leukemia acute myelocytic leukemia (TEL-AML)1 fusion gene and belongs to the field of in-vitro nucleic acid diagnosis. The kit comprises a quantitative reference substance, a negative reference substance, a positive reference substance, RT-PCR enzyme, PCREnhancer, diethypyrocarbonate (DEPC) water, fluorescent PCR reaction liquid I, fluorescent PCR reaction liquid II and ribose nucleic acid (RNA) extraction liquid. The kit comprises an RT-PCR system in which a fluorescent PCR technology is taken as basis, and forward and reverse primers and a fluorescent probe aiming at AMLI and TEL-AML1, can detect the RNA of AMLI and TEL-AML1 simultaneously under the same PCR condition, detect whether TEL-AML1 gene fusion occurs in a clinical sample conveniently and quickly, and provides important basis for leukemia diagnosis, typing, clinical treatment and prognosis diagnosis, and a new train of thought for clinical treatment.
Description
Technical field
The invention belongs to external detection of nucleic acids field, relate in particular to a kind of fluorescence RT-PCR (polymerase chain reaction) test kit that in clinical sample, detects leukaemic TEL-AML1 gene fusion.
Background technology
Leukemia is the malignant disease of hemopoietic system, is commonly called as " leukemia ", is one of domestic ten big malignant tumours occurred frequently.China leukaemic is about 3～4 people/100,000 populations, and is the highest with leukemic sickness rate in children's's the malignant tumour, annual increases with 30,000 to 40,000 speed at least.According to investigations, the sickness rate of China＜10 years old childhood lymphoblastic leukemia is 2.28/10 ten thousand, and any age all can fall ill, and the male sex's sickness rate is higher than the women.Oncogene active and unconventionality expression are relevant with leukemic generation, and chromosomal fracture can make the position of oncogene be moved and be activated with displacement.Chromosome translocation causes gene fusion, and the unconventionality expression of these fusion genes and genes involved thereof causes immoderate cell growth and vicious transformation, is the important mechanism of leukemia morbidity.
Acute lymphoblastic leukemia (ALL) accounts for 75% in leukemia of children, TEL-AML1t (12; 21) be modal chromosomal structural abnormality among the children ALL, form by the AML1 gene fusion on the TEL gene on No. 12 karyomit(e) and No. 21 karyomit(e), often follow another allelic disappearance when the TEL-AML1 fusion gene forms, transposition and disappearance make the leukemogenic mechanism of TEL/AML complicated.The fusion of TEL and AML1 loses and DNA binding ability or can not normal transcription and bring into play the effect of activation target gene the CBRa of AML1 coding, and promptly AML1 is transformed into transcription inhibition factor from activating transcription factor.In addition, the 12p disappearance is also involved the CD-KNIB gene in its downstream when causing TEL genetically deficient, because the plain dependent kinase arrestin of this gene energy code period P27, have and stop cell to enter the s phase from the G1 phase, suppress the effect of cell proliferation, quickened leukemia cell's hypertrophy so it is got involved.SHURTLE equals nineteen ninety-five and finds that there is TEL-AML1 fusion gene and relevant with prognosis in nearly 30 preceding B acute lymphoblastic leukemia children.
The positive ALL of TEL-AML1 fusion gene mostly occurred at 1～10 years old, and the person accounted for 76.2% in 1～5 years old, all t (12; 21) patient expresses B family cellular immunization phenotype, sees that so that CD9, the CD10 positive more dexamethasone is better to the positive leukemic result of treatment of TEL-AML1.Existing mainly is chromosome karyotype analysis to leukemia detection of fusion proteins method, but because 12p is very similar when routine shows band with the 21q end, therefore conventional cytogenetic methods recall rate only 0.05%, needs to use FISH or RT-PCR method and improves and detect positive rate.FISH method complicated operation, length consuming time also needs the diagnostic comments of large-scale instrument and equipment such as fluorescent microscope and pathologist, and is strict to the requirement of feeler mechanism, is unfavorable for detecting fast of clinical sample; RT-PCR result can not monitor in real time, and product needs detected through gel electrophoresis, causes false positive and false negative result easily.
The fluorescent PCR technology was taken the lead in succeeding in developing by U.S. PE company in nineteen ninety-five, and it has the high sensitivity of PCR, the specificity of DNA hybridization and the advantage of spectroscopic techniques accurate quantification concurrently, and visual result can directly be monitored the variation in the PCR process.Compare with regular-PCR, but its result's Real Time Observation, product does not need detected through gel electrophoresis, and stopped pipe is operated fully, has reduced the risk of PCR product pollution effectively.
The fluorescent probe of fluorescent PCR technology forms broad variety with development, as the Taqman probe, and FRET probe etc., what present method related to is the Taqman probe technique.Its principle of work is: it can be cut active the degraded by 5 ' → 3 ' enzyme of DNA synthetic enzyme (for example Taq enzyme), 5 ' end of probe has a fluorescence report group, 3 ' end has a fluorescent quenching group, when two groups are earlier close mutually, because the transmission ofenergy effect takes place, reporter group can not send fluorescence, but carrying out along with amplified reaction, the reporter group of 5 ' end splits away off along with the hydrolysis of probe, no longer with the effect of cancellation generation transmission ofenergy, thereby can send fluorescence, be caught by signal sensor.Often the group that combines with probe 5 ' end has FAM (6-Fluoresceincarboxylic acid), TET (tetrachloro-6-Fluoresceincarboxylic acid), JOE (2,7-dimethyl-4,5-two chloro-6-Fluoresceincarboxylic acids), HEX (chlordene-6-methyl fluorescein) or VIC etc., often the fluorescent quenching group that combines with 3 ' end often is TAMRA (6-carboxyl tetramethylrhodamin) or DABCYL (4-(4 '-oxane amino-benzene azo) phenylformic acid) etc.
Also do not utilize the fluorescence quantitative RT-RCR technology to carry out the product that leukemia antigen-4 fusion protein gene TEL-AML1 detects in the market.
Summary of the invention
For overcoming traditional method, the invention provides a specific specificity height, cost is low and can makes the testing product of detection by quantitative the shortcoming that leukemia fusion rotein TEL-AML1 detects.
The invention provides the fluorescence RT-PCR test kit of a kind of detection by quantitative leukemia fusion gene TEL-AML1, comprise quantitative reference material, negative reference material, positive reference substance, fluorescent PCR reaction solution I, fluorescent PCR reaction solution II; Described fluorescent PCR reaction solution I comprises AML1 gene primer, AML1 specificity fluorescent probe; Described fluorescent PCR reaction solution II comprises TEL-AML1 fusion gene primer, TEL-AML1 specificity fluorescent probe.
Described quantitative reference material, positive reference substance are to contain the PMD18-T vector plasmid that inserts AML1 gene and TEL-AML1 fusion gene distinguished sequence, and described insertion sequence is as follows respectively:
AML1 gene insertion sequence is
TCGGAGCGAAAACCAAGACAGGTCACTGTTTCAGCCTCACCCCTCTAGCCCTACATCTCTCTTTCTTCTCCCCTCTGCTGGATACCTCTGGGACTC 96bp；
TEL-AML1 fusion gene insertion sequence is
GCACGCCATGCCCATTGGGAGAATAGCAGAATGCATACTTGGAATGAATCCTTCTAGAGACGTCCACGATGCCAGCACGAGCCGCCGCTTCACGCCGCCTTCCACCG 107bp。
Described quantitative reference material is 1.0 * 10 for depositing concentration
10-5.0 * 10
10The recombinant plasmid of copies/ml.Can be used to do the reference of quantitative judgement by gradient dilution during use.
Described positive reference substance recombinant plasmid concentration is 1.0 * 10
6-5.0 * 10
6Copies/ml.
Described negative reference material is the negative serum of normal no TEL-AML1 amalgamation and expression.
Described AML1 gene primer sequence is as follows:
The forward primer TCGGAGCGAAAACCAAGACAG 21 of augmentation detection AML1 gene;
Augmentation detection AML1 gene reverse primer GAGTCCCAGAGGTATCCAGCAG 22.
Described AML specificity fluorescent probe sequence is as follows:
Detect the probe TCAGCCTCACCCCTCTAGCCCTAC 24 of AML1 gene.
Described TEL-AML1 fusion gene primer sequence is as follows:
The forward primer GCACGCCATGCCCATTGGGA 20 of augmentation detection TEL-AML1 fusion gene;
The reverse primer CGGTGGAAGGCGGCGTGAAG 20 of augmentation detection TEL-AML1 fusion gene.
Described TEL-AML1 specificity fluorescent probe sequence is as follows:
Detect the probe AGCAGAATGCATACTTGGAATGAATCCT 28 of TEL-AML1 fusion gene.
Described AML1 and TEL-AML1 specificity fluorescent probe are the Taqman probe, and what label probe 5 ' was held is a kind of fluorescence report group, can be FAM, TET, JOE, VIC, HEX, ROX, TAMRA, CY3, CY5; 3 ' end be a kind of fluorescent quenching group, can be TAMRA, DABCYL, NFQ.In reaction system of the present invention, can use one or more fluorescent probe, the fluorescence report group of institute's mark can be for a kind of or two kinds.
Described test kit also comprises RT-PCR enzyme, PCR Enhancer, DEPC water and RNA extracting solution.
The present invention compared with prior art has the following advantages and effect:
1. quantitatively accurately, have the high sensitivity of PCR, the specificity of DNA hybridization and the advantage of spectroscopic techniques accurate quantification concurrently, visual result can directly be monitored the variation in the PCR process.Compare with regular-PCR, but its result's Real Time Observation, product does not need detected through gel electrophoresis, and stopped pipe is operated fully, has reduced the risk of PCR product pollution effectively;
2. sensitivity and specificity height.Because take the double insurance design of specificity and primer and probe, sensitivity and specificity all improve a lot, can before occurring, clinical symptom detect whether producer merges;
3. detection speed is fast, adds that sample extraction only needs 2-3 hour altogether;
4. step is simple, and the result represents that with copy number quantitative result has not only shortened detection time accurately and reliably, has reduced the generation of polluting, and has improved the reliability of analytical data;
5. can carry out high-throughout pattern detection simultaneously.
Fluorescent PCR sensitivity height in a word, specificity is good, monitoring reaction process in real time, reaction times can be controlled in two and one-half-hours, and be the stopped pipe operation, need not subsequent disposal, can avoid reaction product to pollute to greatest extent, can replace that conventional cell genetics detects or common RT-PCR detects and carries out the TEL-AML1 gene fusion and cause leukemic diagnosis.Detected result not only can be judged for leukemia diagnosis, somatotype, clinical treatment and prognosis provides important evidence, also is simultaneously the basis that the leukemia minimal residual disease detects.The leukemic pathogenesis of decorrelation that helped dark people, and provide new thinking for clinical treatment.
Description of drawings:
Fig. 1 is the fluorescent PCR amplification curve of quantitative reference material: curve from left to right is respectively (5.0 * 10
7, 5.0 * 10
6, 5.0 * 10
5, 5.0 * 10
4Copies/ml);
Fig. 2 is the typical curve of quantitative reference material, and relation conefficient=0.999729 illustrates that linear relationship is very good, can be used for the quantitative analysis of RNA copy number;
The fluorescent PCR amplification curve of the negative reference material of Fig. 3, positive reference substance.
Fig. 4 is the AML1 and the TEL-AML1 fluorescence RT-PCR amplification curve of clinical positive sample.Measured positive sample AML1 amplification curve Ct value is between 24～30, and measured positive sample AML1 amplification curve Ct value is between 30～38, all within quantitative reference material linearity range.
Embodiment:
Be to be understood that, unaccounted normal condition and method among the following embodiment, usually according to the conventional employing method of affiliated field experimenter: as " molecular cloning experiment guide " third edition of Sa nurse Brooker and Russell chief editor, or the step and the condition of advising according to manufacturer.
Embodiment 1: the preparation of test kit
1. primer and probe design and synthetic
Use Primer Express 2.0, respectively in the non-integration region of AML1 gene and TEL and AML1 gene fusion zone, the design screening is in the probe of conserved regions, the upstream and downstream primer of design AML1 gene and TEL-AML1 fusion gene on the probe basis that chooses.Primer and probe all entrust specialized company's (giving birth to the worker) synthetic, and wherein primer is the PAGE purifying, and probe is the HPLC purifying, probe 5 ' end flag F AM fluorophor, 3 ' end mark TAMRA fluorophor.
Extension increasing sequence such as table 1:
Table 1. specific probe and primer sequence
| The sequence title | Oligonucleotide sequence (5 '-3 ') | Base length (bp) | |
| The AML1 probe | TCAGCCTCACCCCTCTAGCCCTAC | 24 | |
| The TEL-AML1 probe | AGCAGAATGCATACTTGGAATGAATCCT | 28 | |
| The AML1 forward | TCGGAGCGAAAACCAAGACAG | 21 | |
| The AML1 reverse primer | GAGTCCCAGAGGTATCCAGCAG | 22 | |
| The TEL-AML1 forward primer | GCACGCCATGCCCATTGGGA | 20 | |
| The TEL-AML1 reverse primer | CGGTGGAAGGCGGCGTGAAG | 20 |
2.AML1 the preparation of the quantitative reference material of plasmid positive template
The AML1 plasmid is used to prepare quantitative reference material as positive template in the present embodiment.
Use the non-integration region sequence of synthetic amplification AML1 primer amplified AML1 in the step 1, purified PCR product is connected on the PMD18-T carrier; Be converted into then in the bacillus coli DH 5 alpha competent cell; By blue hickie screening, make up the AML1 recombinant plasmid dna as the sensitivity reference material.The AML1 recombinant plasmid that makes up is identified through two-way dna sequencing.Extract plasmid, ultraviolet spectrophotometer is quantitative, is diluted to 5.0 * 10
10Copies/ml is in-20 ℃ of preservations.
3.TEL-AML1 the preparation of fusion gene positive reference substance
The TEL-AML1 plasmid is used to prepare positive reference substance as positive template in the present embodiment.
Use synthetic amplification TEL-AML1 primer amplified TEL-AML1 fusion gene distinguished sequence in the step 1, purified PCR product is connected on the PMD18-T carrier; Be converted into then in the bacillus coli DH 5 alpha competent cell; By blue hickie screening, make up the TEL-AML1 recombinant plasmid dna as positive reference substance.The TEL-AML1 recombinant plasmid that makes up is identified through two-way dna sequencing.Extract plasmid, ultraviolet spectrophotometer is quantitative, is diluted to 5.0 * 10
10Copies/ml is in-20 ℃ of preservations.
4. reference substance is selected
Use the negative contrast of normal human serum; TEL-AML1 recombinant plasmid (5 * 10
6Copies/ml) positive contrast.
5. fluorescent PCR reaction solution I forms
Table 2. fluorescent PCR reaction solution I forms
| Material name | Final concentration |
| PCR?Buffer | (0.8-2)× |
| MgCl 2 | 2-5mM |
| dNTP | 0.1-0.5mM |
| The AML1 primer | 0.1-0.50μM |
| The AML1 probe | 0.1-0.50μM |
| PCR?Enhancer | 0.4-2U |
| The RT-PCR enzyme | 0.5-3U |
| H 2O | In right amount |
| The DNA masterplate | 15μL |
| Cumulative volume | 50μL |
6. fluorescent PCR reaction solution II forms
Table 3. fluorescent PCR reaction solution II forms
| Material name | Final concentration |
| PCR?Buffer | (0.8-2)× |
| MgCl 2 | 2-5mM |
| dNTP | 0.1-0.5mM |
| The TEL-AML1 primer | 0.1-0.50μM |
| The TEL-AML1 probe | 0.1-0.50μM |
| PCR?Enhancer | 0.4-2U |
| The RT-PCR enzyme | 0.5-3U |
| H 2O | In right amount |
| The DNA masterplate | 15μL |
| Cumulative volume | 50μL |
Embodiment 2: the use of test kit
1. sample extraction
Use total RNA of RNA extracting solution extracting testing sample blood
Operation steps:
1) gets the 0.5ml centrifuge tube, add 150 μ LRNA extracting solutions, add 100 μ L samples to be tested (EDTA anticoagulation) then, fully mixing.
2) add 50 μ L chloroforms, 4 ℃, 13000rpm, centrifugal 15min.
3) with the upper strata after centrifugal to the new centrifuge tube, add 100 μ L Virahols, room temperature is placed 10min.
4) 4 ℃, 13000rpm, centrifugal 15min.
5) remove supernatant gently, place on the thieving paper, add 300 μ L, ice precooling 75% ethanol, 4 ℃, 13000rpm, centrifugal 10min.
6) remove supernatant gently, place on the thieving paper, briefly centrifugal, abandon supernatant with the suction that the rifle head is careful, drying at room temperature 1-5min.
7) add 30 μ L DEPC water, dissolving naturally places standby (in the 2h) on ice.
2. quantitatively reference material is prepared
The quantitative reference material of AML1 plasmid positive template is carried out gradient dilution with DEPC water, and concentration is respectively 5.0 * 10
7, 5.0 * 10
6, 5.0 * 10
5, 5.0 * 10
4Copies/ml.
3. positive reference substance is prepared
TEL-AML1 fusion gene positive reference substance is carried out gradient dilution with DEPC water, it is diluted to final concentration 5.0 * 10
6Copies/ml.
4. pattern detection
Get the nucleic acid that extracts in the step 1 respectively, the quantitative reference material of the gradient dilution that obtains in the step 2, as the RNA template, add RT-PCR enzyme, PCR Enhancer and contain the specific PCR primer and the fluorescent quantitation reaction solution I of specificity fluorescent probe in, form PCR reaction system I.Get in the step 1 positive reference substance and the negative control that obtain in the nucleic acid that extracts, the step 3 respectively, as the RNA template, add RT-PCR enzyme, PCR Enhancer and contain the specific PCR primer and the fluorescent quantitation reaction solution II of specificity fluorescent probe in, form PCR reaction system II.In PCR reaction system I, add respectively and diluted the good AML1 plasmid standard (5.0 * 10 of mixing
7, 5.0 * 10
6, 5.0 * 10
5, 5.0 * 10
4Copies/ml), the standard that quantitatively judges as sample.
Each main component of system is as follows:
5. response procedures
The fluorescence detection channel of collecting the FAM fluorescent signal is set, reaction tubes is put into fluorescent PCR instrument (ABI7500) begin amplification, response procedures is as follows:
Table 4.PCR response procedures
6. the result judges
The Ct value (cycle number) of baseline scope is selected automatically for 6-15 or by software, and setting threshold makes it to surpass the maximum of random amplification curve.Fluorescent PCR instrument difference, the Ct value of gained baseline scope can be different.
7. quality control standard
(1) all kinds of contrast quality control product judged results such as following table:
Table 5. quality control product standard testing result
| ? | Quality control product | The |
| 1 | Negative control | There is not |
| 3 | |
22＜Ct value≤25 |
The fluorescent PCR amplification curve of negative contrast of Fig. 3 and positive control, wherein negative control does not have amplification, positive control Ct=23.
(2) quantitative reference material Quality Control determination methods: the quantitative reference material plasmid of AML1 plasmid positive template of gradient dilution, make typical curve through amplification, when relation conefficient＞0.99, can be used for the quantitative analysis of AML1 and TEL-AML1RNA copy number; Otherwise to test again.Fig. 1 is the amplification curve that obtains after the quantitative reference material amplification, and the typical curve that obtains according to this curve is Fig. 2.
7. result's report:
Fig. 4 is the AML1 and the TEL-AML1 fluorescence RT-PCR amplification curve of clinical sample.Measured positive sample Ct value is between 20～38, and within quantitative reference material linearity range, and sample AML1 amplification curve Ct value is less than TEL-AML1 amplification curve Ct value.
The judging criterion of sample results is as follows:
Table 6. report pattern detection result
Sequence table
＜110〉Shanghai Yue Loong Clinical Laboratory center company limited
＜120〉detection by quantitative leukemia fusion gene TEL-AML1 fluorescence RT-PCR test kit
<160>8
<210>1
<211>96
<212>DNA
＜213〉Genus Homo (HOMO)
<400>1
tcggagcgaa?aaccaagaca?ggtcactgtt?tcagcctcac?ccctctagcc?ctacatctct 60
ctttcttctc?ccctctgctg?gatacctctg?ggactc 96
<210>2
<211>107
<212>DNA
＜213〉Genus Homo (HOMO)
<400>2
gcacgccatg?cccattggga?gaatagcaga?atgcatactt?ggaatgaatc?cttctagaga 60
cgtccacgat?gccagcacga?gccgccgctt?cacgccgcct?tccaccg 107
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, with forward primer as augmentation detection AML1 gene in the detection by quantitative leukemia fusion gene TEL-AML1 fluorescence RT-PCR test kit.
<400>3
tcggagcgaa?aaccaagaca?g 21
<210>4
<211>22
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, to use augmentation detection AML1 gene reverse primer in the detection by quantitative leukemia fusion gene TEL-AML1 fluorescence RT-PCR test kit.
<400>4
gagtcccaga?ggtatccagc?ag 22
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, with forward primer as augmentation detection TEL-AML1 fusion gene in the detection by quantitative leukemia fusion gene TEL-AML1 fluorescence RT-PCR test kit.
<400>5
gcacgccatg?cccattggga 20
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, with reverse primer as augmentation detection TEL-AML1 fusion gene in the detection by quantitative leukemia fusion gene TEL-AML1 fluorescence RT-PCR test kit.
<400>6
cggtggaagg?cggcgtgaag 20
<210>7
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, with probe as detection AML1 gene in the detection by quantitative leukemia fusion gene TEL-AML1 fluorescence RT-PCR test kit.
<400>7
tcagcctcac?ccctctagcc?ctac 24
<210>8
<211>28
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to nucleic acid base pairing mechanism and the design of gene chip hybridization condition, with probe as detection TEL-AML1 fusion gene in the detection by quantitative leukemia fusion gene TEL-AML1 fluorescence RT-PCR test kit.
<400>8
agcagaatgc?atacttggaa?tgaatcct 28
Claims (10)
1. the fluorescence RT-PCR test kit of a detection by quantitative leukemia fusion gene TEL-AML1 is characterized in that, comprises quantitative reference material, negative reference material, positive reference substance, fluorescent PCR reaction solution I, fluorescent PCR reaction solution II; Described fluorescent PCR reaction solution I comprises AML1 gene primer, AML1 specificity fluorescent probe; Described fluorescent PCR reaction solution II comprises TEL-AML1 fusion gene primer, TEL-AML1 specificity fluorescent probe.
2. test kit as claimed in claim 1, it is characterized in that, described quantitative reference material, positive reference substance are respectively and contain the PMD18-T vector plasmid that inserts AML1 gene and TEL-AML1 fusion gene distinguished sequence, and described insertion sequence is as follows respectively: AML1 gene insertion sequence is
TCGGAGCGAAAACCAAGACAGGTCACTGTTTCAGCCTCACCCCTCTAGCCCTACATCTCTCTTT
CTTCTCCCCTCTGCTGGATACCTCTGGGACTC 96bp；
TEL-AML1 fusion gene insertion sequence is
GCACGCCATGCCCATTGGGAGAATAGCAGAATGCATACTTGGAATGAATCCTTCTAGAGACGTC
CACGATGCCAGCACGAGCCGCCGCTTCACGCCGCCTTCCACCG 107bp。
3. test kit as claimed in claim 1 or 2 is characterized in that: described quantitative reference material is 1.0 * 10 for depositing concentration
10-5.0 * 10
10The recombinant plasmid of copies/ml.
4. test kit as claimed in claim 1 or 2 is characterized in that, described positive reference substance recombinant plasmid concentration is 1.0 * 10
6-5.0 * 10
6Copies/ml.
5. test kit as claimed in claim 1 is characterized in that: described negative reference material does not have the negative serum of TEL-AML1 amalgamation and expression for the normal people.
6. test kit as claimed in claim 1 is characterized in that, described AML1 gene primer sequence is as follows:
The forward primer TCGGAGCGAAAACCAAGACAG 21 of augmentation detection AML1 gene;
The reverse primer GAGTCCCAGAGGTATCCAGCAG 22 of augmentation detection AML1 gene.
7. test kit as claimed in claim 1 is characterized in that, described TEL-AML1 fusion gene primer sequence is as follows:
The forward primer GCACGCCATGCCCATTGGGA 20 of augmentation detection TEL-AML1 fusion gene;
The reverse primer CGGTGGAAGGCGGCGTGAAG 20 of augmentation detection TEL-AML1 fusion gene.
8. test kit as claimed in claim 1 is characterized in that, described specificity fluorescent probe sequence is as follows:
Detect the probe TCAGCCTCACCCCTCTAGCCCTAC 24 of AML1 gene;
Detect the probe AGCAGAATGCATACTTGGAATGAATCCT 28 of TEL-AML1 fusion gene.
9. as claim 1 or 8 described test kits, it is characterized in that, described specificity fluorescent probe is the Taqman probe, and what label probe 5 ' was held is a kind of fluorescence report group, can be FAM, TET, JOE, VIC, HEX, ROX, TAMRA, CY3, CY5; 3 ' end be a kind of fluorescent quenching group, can be TAMRA, DABCYL, NFQ.
10. test kit as claimed in claim 1 is characterized in that, also comprises RT-PCR enzyme, PCR Enhancer, DEPC water and RNA extracting solution.
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| 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) |
| CN107345244A (en) * | 2016-10-12 | 2017-11-14 | 深圳市儿童医院 | Detect method, primer and the kit of leukaemia TEL AML1 fusions |
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| 何军等: "儿童急性淋巴细胞白血病TEL/AML1融合基因的检测及其临床意义", 《临床检验杂志》 * |
| 李志刚等: "儿童急性淋巴细胞白血病TEL-AMLl融合基因的表达水平与临床特点、早期治疗反应的关系", 《中国实验血液学杂志》 * |
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| CN107345244A (en) * | 2016-10-12 | 2017-11-14 | 深圳市儿童医院 | Detect method, primer and the kit of leukaemia TEL AML1 fusions |
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| CN113106149A (en) * | 2021-04-26 | 2021-07-13 | 福建和瑞基因科技有限公司 | Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance |
| CN113106149B (en) * | 2021-04-26 | 2023-01-17 | 福建和瑞基因科技有限公司 | Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance |
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