CN107345244A - Detect method, primer and the kit of leukaemia TEL AML1 fusions - Google Patents
Detect method, primer and the kit of leukaemia TEL AML1 fusions Download PDFInfo
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- CN107345244A CN107345244A CN201610910280.9A CN201610910280A CN107345244A CN 107345244 A CN107345244 A CN 107345244A CN 201610910280 A CN201610910280 A CN 201610910280A CN 107345244 A CN107345244 A CN 107345244A
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q2600/16—Primer sets for multiplex assays
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Abstract
The invention provides primer pair and probe combinations, and present invention also offers the application of described primer pair and probe combinations in the product for preparing detection or auxiliary detection ALL；The present invention additionally provides kit and application containing the primer pair and probe combinations, and the system that the screening based on primer pair and probe combinations or kit is susceptible to suffer from the biological sample of ALL simultaneously.It the experiment proved that, primer pair and probe combinations specificity of the invention is good, high sensitivity, detection efficiency are high, and the TEL AML1 fusions and the double fluorescent PCR method of GAPDH reference genes established based on the primer pair and probe combinations are reproducible, high flux, sensitive, accurately and quickly, auxiliary diagnosis and the judgement of successive treatment effect for leukaemia provide more effective way.
Description
Technical field
The present invention relates to field of biological detection, the more particularly to method of detection leukaemia TEL-AML1 fusions, primer
And kit.
Background technology
Leukaemia is a kind of candidate stem cell malignant clone disease, because the leukaemia of clone is in marrow and its
A large amount of propagation accumulations, are suppressed normal hematopoiesis function, while infiltrate other tissues and organ in its hematopoietic tissue.White blood
Sick main hyperplasia and the infiltration for being presented with leukaemia.Non-specific lesion then for bleeding and histotrophic nutrition it is bad and downright bad,
Scabies secondary infection etc..The hyperplasia of leukaemia and infiltration are occurred mainly in marrow and other hematopoietic tissues, may also appear in complete
In the other tissues of body, normal erythroid cells, Megakaryocyte is caused to substantially reduce.Can be because of some leukaemias in marrow
Obvious proliferation or hyperactive, and it is in bois de rose or yellow green.Lymphoid tissue also can be by leukemiacell infiltration, later stage
Then enlargement of lymph nodes, part leukaemic have obvious central nervous system leukemia to change, or strongly fragrant for intravascular leucocyte
Stagnant, blood vessel peripheral white blood cells hyperplasia.The internal organs that leukemic infiltration most easily occurs are kidney, lung, heart and thymus gland, testis etc., clinical
Show as anaemia, bleeding, infection and liver, spleen, enlargement of lymph nodes and skeleton pain.
The examined person's subjectivity composition influence of diagnostic accordance rate and accuracy of the past cytomorphology parting is larger, and nearly two
The year research of leukemia molecule feature achieves obvious progress, especially forms fusion to chromosome translocation, has had some
As the molecular biology specificity marker of diagnosis different type leukaemia and unique foundation of determination diagnosis, pass through dialogue blood
Sick correlation fusion gene is detected, and can not only be provided for leukemia diagnosis, parting, clinical treatment and Index for diagnosis important
Foundation, while be also the detection basis of Minimal Residual Disease of Leukemia.Diagnostic significance weight of the detection of fusion to leukaemia
Greatly, acute degree, the clone's characteristic and parting of leukaemia can be evaluated, makes the diagnosis typing of leukaemia more scientific and specification
Change；1 × 10 can be detected6A leukaemia in individual karyocyte, there is other sides in terms of the early diagnosis of leukaemia
The unrivaled specificity of method and sensitiveness.The Prognostic significance of cytogenetics parting and disease is close, therefore fusion
Detect for instructing the selection of clinical personalized therapy program and judging prognosis tool to be of great significance, can refer to when just controlling
Scientific and reasonable selection long-term treatment regimen is led, avoids unnecessary insufficient therapy or over-treatment.
TEL-AML1t(12；21) it is most common chromosomal structural abnormality in ALL, by No. 12
TEL genes on chromosome form with the AML1 Gene Fusions on No. 21 chromosomes, are formed in TEL-AML1 fusions same
When often with the missing of another allele, transposition and missing complicate the leukemogenic mechanism of TEL/AML1.TEL
The CBRa that fusion with AML1 encodes AML1 loses and DNA binding abilities or is unable to normal transcription and plays activation target gene
Effect, i.e. AML1 is transformed into transcription inhibitory factor from activating transcription factor.TEL/AML1 fusions are in children acute lymph
Relatively conventional in cellular type leukaemia, especially B cell ALL, its positive expression are that prognosis is preferable
One of mark.
It is former that the conventional method of fusion detection mainly includes leukaemia's chromosome karyotype analysis, chromosome fluorescence
Position hybridization technique and PCR (PCR) technology.In practical operation, chromosome karyotype analysis is frequently subjected to detect
The influence of technical conditions and manual operation factor, chromosome fluorescence in-situ hybridization technical result is more directly perceived, but tests process
It is excessively cumbersome, it is necessary to reagent type it is various, waste time and energy, and result of the test needs veteran expert to carry out interpretation, as a result
Interpretation limits the application of these methods to a certain extent there is also larger subjectivity.And PCR
(PCR) real time fluorescence quantifying PCR method common in technology has a SYBR GreenI dye methods, double probe hybrid methods and
Taqman technologies etc..The SYBR GreenI wherein used in SYBR GreenI dye methods are due to being non-saturable dye, specificity
Not as double probe hybrid methods and Taqman methods, it is necessary to judge its specificity by observing solubility curve；And two probe method is miscellaneous
Friendship method cost again costly.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of reproducible, high flux, sensitive, accurately and quickly sieve
Choosing be susceptible to suffer from ALL biological sample system, present invention also offers for the system it is reproducible,
Specific good, high sensitivity, detection efficiency high and applied widely primer pair and probe combinations, and contain the primer pair
Kit and its application with probe combinations.
One aspect of the present invention provides primer pair and probe combinations, including two groups of primer pairs and two probes, described two groups
Primer pair includes the first primer pair and the second primer pair, and two probes include the first probe and the second probe, and described first
Probe is corresponding with the first primer pair, and second probe is corresponding with the second primer pair, and first primer pair includes SEQ
ID NO：1 and SEQ ID NO：Nucleotide sequence shown in 2, first probe include SEQ ID NO：Nucleotides shown in 3
Sequence；Second primer pair includes SEQ ID NO：4 and SEQ ID NO：Nucleotide sequence shown in 5, described second visits
Pin includes SEQ ID NO：Nucleotide sequence shown in 6.
Second aspect of the present invention provides described primer pair and probe combinations and is preparing detection or the acute leaching of auxiliary detection
Application in the product of bar chronic myeloid leukemia.
Third aspect present invention provides a kind of kit, including described primer pair and probe combinations.
In a preference, in addition to fluorescent quantitative PCR reagent.
In a preference, the fluorescent quantitative PCR reagent is # including the article No. from Roche companies
Reagent FastStart in 6402682001 FastStart Essential Probes Master kits
Essential Probes Master Mix(2×)。
In a preference, in addition to total RNA extraction reagent.
In a preference, the total RNA extraction reagent includes erythrocyte cracked liquid, TRIZOL, chloroform, isopropyl
At least one of alcohol and ethanol.
In a preference, in addition to reverse transcription reagents.
In a preference, the reverse transcription reagents include the article No. from Beijing Quanshijin Biotechnology Co., Ltd
To carry AU311-03 TransScript-Uni One-Step gDNA Removal and cDNA Synthesis
Reagent included in SuperMix Kit.
In a preference, in addition to positive control, negative control and blank control.
In a preference, the positive control is molten containing TEL-AML1 fusions and GAPDH reference genes
Liquid；The negative control is without TEL-AML1 fusions but contains the solution of GAPDH reference genes；The blank control
For physiological saline or deionized water.
Fourth aspect present invention provides described kit and is preparing detection or the auxiliary detection white blood of acute lymphoblastic
Application in the product of disease.
Fifth aspect present invention provides a kind of system for the biological sample for screening and being susceptible to suffer from ALL,
Including：
Nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus are used to extract the sample of nucleic acid in the biological sample；
Quantitative fluorescent PCR device, the quantitative fluorescent PCR device are connected with the nucleic acid-extracting apparatus, suitable for using
Primer pair and probe combinations described in claim 1 carry out quantitative fluorescent PCR reaction to the sample of nucleic acid；And
Judgment means, the judgment means are connected with the quantitative fluorescent PCR device, so as to anti-based on quantitative fluorescent PCR
The result answered, judges whether the biological sample is susceptible to suffer from ALL.
In a preference, the nucleic acid-extracting apparatus further comprises：
RNA extraction units, the RNA extraction units are used to extract RNA samples from biological sample；And
Reverse transcription unit, the reverse transcription unit are connected with the RNA extraction units, for being carried out to the RNA samples
Reverse transcription reaction, to obtain cDNA samples, the cDNA samples form the sample of nucleic acid.
In a preference, the reaction system of the quantitative fluorescent PCR reaction is as follows：
FastStart Essential Probes Master Mix (2 ×) 12.5 μ l, primer SEQ ID NO：1 He
SEQ ID NO：2 final concentration is 0.8 μM, primer SEQ ID NO：4 and SEQ ID NO：5 final concentration is 0.8 μM, is visited
Pin SEQ ID NO：3 and SEQ ID NO：6 final concentration is 0.4 μM, cDNA template 2ng, adds ultra-pure water to supply system to 25
μl。
In a preference, the quantitative fluorescent PCR response procedures are as follows：95 DEG C of 10min, 1 circulation；95 DEG C of 10s,
60 DEG C of 30s (collection fluorescence), 45 circulations；40 DEG C of 10s, 1 circulation.
The present invention " first probe is corresponding with the first primer pair, and second probe is relative with the second primer pair
Should " in " corresponding " refer to that the first probe can be supported the use individually with the first primer pair, similarly, the second probe and the
Two primer pairs can also be supported the use individually.
The biological sample of the present invention is blood, the cell or tissue for including DNA and/or RNA, or is plasmid, or is
DNA, cDNA, mRNA or cDNA fragment, or be the reagent containing DNA, cDNA, mRNA or cDNA fragment.When biological sample is
DNA, cDNA or cDNA fragment, or be the reagent containing DNA, cDNA or cDNA fragment, or when being plasmid, by its directly as
Sample of nucleic acid is with the primer and probe to carrying out real-time fluorescence PCR reaction；When biological sample is to contain DNA's and/or RNA
Blood, cell or tissue, or be mRNA, or for reagent containing mRNA when, be translated into DNA, cDNA or cDNA fragment
Sample of nucleic acid carries out real-time fluorescence PCR reaction with the primer and probe again.
Beneficial effects of the present invention include：
(1) present invention a plurality of multiple draws for what leukaemia TEL-AML1 fusions and GAPDH reference genes designed
Thing, probe have carried out a large amount of screenings, its comprehensive specificity, sensitivity, match composite amplification influence each other and difference is drawn
The suitability of thing probe combinations and fluorescent PCR amplification kit, finally filter out good specificity, high sensitivity, it is reproducible,
Applicability is wide and can detect the primer pair and probe combinations of leukaemia TEL-AML1 fusions and GAPDH reference genes simultaneously.
(2) present invention on the basis of foregoing primer pair and probe combinations for establishing leukaemia TEL-AML1 fusions
The double fluorescent PCR detection method of gene and GAPDH reference genes, can be simultaneously to TEL-AML1 fusions and GAPDH internal references
Gene carries out qualitative and quantitative detection, and reproducible, high flux, sensitive, accurately and quickly, for the auxiliary diagnosis of leukaemia
And successive treatment effect judges to provide more effective way.
Embodiment
Unless specifically indicated, term used herein has the general sense in art of the present invention.
Below with reference to specific embodiment, the present invention will be described, it is necessary to which explanation, these embodiments are only to say
Bright property, and be not considered as limiting the invention.Unreceipted particular technique or condition in embodiment, according to routine
Experiment condition, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW, Molecular
cloning：A laboratory manual, 2001), or the condition according to manufacturer's specification suggestion.Agents useful for same or
The unreceipted production firm person of instrument, being can be by the conventional products of acquisition purchased in market.
Embodiment 1
Present embodiments provide primer pair and probe combinations and its application.
The screening technique of primer pair and probe combinations is：For leukaemia TEL-AML1 fusions and GAPDH internal reference bases
Cause, design different primer and probes and combine and carried out optimal screening, its comprehensive stability, specificity, sensitivity, pairing
Composite amplification influence each other and the suitability of different primers probe combinations and fluorescent PCR amplification kit, final screening
Go out that specificity is good, reproducible and high sensitivity following can be detected in leukaemia TEL-AML1 fusions and GAPDH simultaneously
Join the double fluorescent PCR of gene primer pair and probe combinations.
The TEL-AML1 fusions fragment is as follows：
TGAACCACATCATGGTCTCTGTCTCCCCGCCTGAAGAGCACGCC ATGCCCATTGGGAGAATAGCAGGA
ATGCATACTTGGAATGAATCCTTC TAGAGACGTCCACGA(SEQ ID NO：7).
The GAPDH reference genes fragment is as follows：
TGGTATCGTGGAAGGACTCATGACCACAGTCCATGCCATCACTG CCACCCAGAAGACTGTGGATGGCC
CCTCCGGGAAACTGTGGCGTGAT GGCCGCGGGGCTCTCCAGAACATCATC(SEQ ID NO：8)
Described primer pair and the nucleotide sequence of probe combinations are as follows：
TEL-AML1-F：：5’-TGAACCACATCATGGTCTCTG-3’(SEQ ID NO：1)；
TEL-AML1-R：5’-TCGTGGACGTCTCTAGAAGGA-3’(SEQ ID NO：2)；
TEL-AML1 probe sequence is：5’-FAM-CTCCCCGCCTGAAGAGCACG -TAMRA-3’(SEQ ID NO：
3)
GAPDH-F：5’-TGGTATCGTGGAAGGACTCA-3’(SEQ ID NO： 4)；
GAPDH-R：5’-GATGATGTTCTGGAGAGCCC-3’(SEQ ID NO： 5)；
GAPDH probe sequence is：5’-HEX-CCATGCCATCACTGCCACCC-T AMRA-3’(SEQ ID NO：6)
Wherein HEX, FAM are fluorophor, and TAMRA is quenching group.
Leukaemia TEL-AML1 fusions and GAPDH reference genes can be directed to simultaneously using above-mentioned primer pair and probe
Carry out double fluorescent PCR amplifications.
Upper in application, above-mentioned primer pair and probe combinations can be applied to detect or aid in detection leukaemia TEL-AML1 to melt
Close on gene and GAPDH reference genes；It can also be used to prepare detection or the production of auxiliary detection ALL
Product.
Embodiment 2
A kind of kit and its application are present embodiments provided, the kit includes：
(1)SEQ ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：4、SEQ ID NO：5 and SEQ
ID NO：6 (coming from embodiment 1)；
(2) total RNA extraction reagent；
(3) reverse transcription reagents；
(4) fluorescent quantitative PCR reagent；
(5) positive control, negative control and blank control.
The total RNA extraction reagent includes erythrocyte cracked liquid, TRIZOL, chloroform, isopropanol and ethanol.
The reverse transcription reagents are to carry AU311-03's including the article No. from Beijing Quanshijin Biotechnology Co., Ltd
Included in TransScript-Uni One-Step gDNA Removal and cDNA Synthesis SuperMix Kit
Reagent.
It is #6402682001's that the fluorescent quantitative PCR reagent, which includes the article No. from Roche companies,
Reagent FastStart Essential Probes in FastStart Essential Probes Master kits
Master Mix(2×)。
The positive control is the solution containing TEL-AML1 fusions and GAPDH reference genes；The negative control
Without TEL-AML1 fusions but to contain the solution of GAPDH reference genes；The blank control be physiological saline or go from
Sub- water.
It is demonstrated experimentally that above-mentioned total RNA extraction reagent, reverse transcription reagents, fluorescent quantitative PCR reagent and and SEQ
ID NO：1、SEQ ID NO：2、SEQ ID NO：3、SEQ I D NO：4、SEQ ID NO：5 and SEQ ID NO：6 combine makes
With effect is more superior, is in particular in high specificity, reproducible and high sensitivity.
Application it is upper, mentioned reagent box can be applied to detect or aid in detect leukaemia TEL-AML1 fusions with
On GAPDH reference genes；It can also be used to the product for preparing detection or auxiliary detection ALL.
Embodiment 3
Present embodiments provide a kind of detection or auxiliary detection leukaemia TEL-AML1 fusions and GAPDH internal reference bases
The method of cause, such as judge biological sample whether containing leukaemia TEL-AML1 fusions according to the double fluorescent PCR results expanded
Gene, it the method use the primer pair and the kit of probe combinations or embodiment 2 of embodiment 1.
The above method comprises the following steps：
Step 1：The extraction of total serum IgE
5ml erythrocyte cracked liquids are added in 15ml centrifuge tubes, take EDTA-Na2 anticoagulations (sample to be tested) 2ml to add
In centrifuge tube, gently mix, be stored at room temperature 10 minutes, 600 × g centrifuges 5 min, removes supernatant, adds 2ml erythrocyte splittings
Liquid, precipitation is gently resuspended, is stored at room temperature 5 min, 600 × g centrifugation 5min, removes supernatant, 1ml TRIzol are added into pipe, are blown
Beat and mix, be completely dissolved precipitation, after room temperature places 5min, add 0.2ml chloroforms, vibration is mixed, and solution is all shifted
Into 1.5ml centrifuge tubes, 4 DEG C of 13000 × g centrifuge 10min, draw supernatant and are transferred in new centrifuge tube, add isometric
After isopropanol, fully vibration, 10m in are stored at room temperature, 4 DEG C of 13000 × g centrifuge 10min, remove supernatant, gently add 1ml 75%
Ethanol, 4 DEG C of 1 3000 × g centrifuge 10min, carefully remove supernatant, drying at room temperature 10-15min, add 20 μ l RN ase-free
Water, 10min dissolving precipitations are placed on ice, obtain total serum IgE.
Step 2：CDNA synthesis
The TransScript- Uni One- that article No. using Beijing Quanshijin Biotechnology Co., Ltd is AU311-03
Step gDNA Removal and cDNA Synthesis SuperMix Kit kit simultaneously will be total according to its specification
RNA reverse transcriptions remove genomic DNA into cDNA.
Step 3：Double fluorescent PCR is expanded
Using cDNA as template, double fluorescent PCR amplifications are carried out using primer pair and probe, while positive control, the moon are set
Property control and blank control.The positive control is the solution containing TEL-AML1 fusions and GAPDH reference genes；Institute
It is without TEL-AML1 fusions but containing the solution of GAPDH reference genes to state negative control；The blank control is physiology
Salt solution or deionized water.
Double fluorescent pcr amplification reaction system：FastStart Essential Probes Master Mix (2×)
12.5 μ l, primer SEQ ID NO：1 and SEQ ID NO：2 final concentration is respectively 0.8 μM, primer SEQ ID NO：4 and SEQ ID
NO：5 final concentration is respectively 0.8 μM, probe SEQ ID NO：3 and SEQ ID NO：6 final concentration is respectively 0.4 μM, cD NA moulds
The μ l of plate 2 (concentration is 1ng/ μ l), add ultra-pure water to supply system to 25 μ l.Positive control, negative control and the template of blank control
Dosage be 2 μ l.
Double fluorescent pcr amplification reaction condition：95 DEG C of 10min, 1 circulation；95 DEG C of 10s, 60 DEG C of 30s (collection fluorescence),
45 circulations；40 DEG C of 10s, 1 circulation.
Double fluorescent pcr amplification reaction is in Roche companiesOn 96 real-time fluorescence quantitative PCR instrument
Carry out, probe in detecting pattern is that FAM (483-533) and HEX (523-568) is combined.
Step 4：The fluoroscopic examination of double fluorescent pcr amplification product
FAM and HEX fluorescence signal is collected after terminating in 58 DEG C of 30s stages of each circulation.
The standard that the result of the double fluorescent PCR amplifications judges is as follows：
Threshold line is adjusted to more than background signal and negative amplification line, the reference gene GAPDH only when sample to be tested
As a result when positive, testing result just thinks effective.
Positive findings judges：FAM passages and HEX passages have amplification curve, and Ct values are respectively less than or equal to 38.0.
Negative findings judges：HEX passages have amplification curve and Ct value≤38.0, FAM channel C t values ＞ 38.0.
If HEX passages need to re-start detection without amplification curve, or Ct values ＞ 38.0.
The result of the positive control, negative control and the double fluorescent of blank control PCR amplifications should be with shown in table 1 one
Cause, further to prove the confidence level of the testing result of testing sample.
Table 1
Embodiment 4
The detection or auxiliary that the present embodiment is established to embodiment 3 are detected in leukaemia TEL-AML1 fusions and GAPDH
The method of ginseng gene has carried out specificity and repeatability checking, and material to be tested used is as follows：Examined through golden domain white
80 samples of blood patient anticoagulation.
To Leukemia Patients 80 samples of anticoagulation carry out the extraction of total serum IgE, cDNA synthesis, double fluorescent PCR respectively
Amplification and the fluoroscopic examination of double fluorescent pcr amplification product, each step are carried out according to the method for embodiment 3, each sample
3 repetitions are done, experiment every time includes a positive control, a negative control and a blank control.
Interpretation of result：
The detection established using embodiment 3 or auxiliary detection leukaemia TEL-AML1 fusions and GAPDH reference genes
Method detection experimental result with the report result that golden domain is examined compared with, sample yin and yang attribute coincidence rate is 100%, and smart
Density test shows：Batch in and batch between reproducible, the coefficient of variation ＜ 10% of testing result Ct values, therefore the present invention foundation
Detection or auxiliary detection leukaemia TEL-AML1 fusions and GAPDH reference genes method energy effective detection TEL-
AML1 fusions and GAPDH reference genes, it is specific and reproducible, available for leukaemia auxiliary diagnosis and subsequently control
Therapeutic effect judges, molecule diagnosis basis is provided for clinical diagnosis and treatment.
Embodiment 5
The detection or auxiliary that the present embodiment is established to embodiment 3 are detected in leukaemia TEL-AML1 fusions and GAPDH
The method of ginseng gene has carried out sensitivity checking.Positive cell precursor B cell leukemic cell line Reh (TEL- are extracted respectively
AML1 is positive) (ATCC sources) and negative cells HeLa (TEL-AML1 feminine genders) (ATCC sources) RNA, by positive cell Reh
RNA carry out 10 times of gradient dilutions with the RNA of negative cells HeLa cells since 1 μ g, it is minimum to be diluted to 10-6.Reverse transcription
Double fluorescent quantitative PCR amplification is carried out afterwards, and with 3,3 repetitions of embodiment, experiment every time includes a positive right experimental procedure
According to a negative control and a blank control.Experimental result shows that the detection sensitivity of Reh cells is 10-4, i.e., 104It is individual thin
Have 1 in born of the same parents just can detect it with cell positive TEL-AML1 by primer, kit and the method for the present invention
Fusion.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to claimed model without departing from theon the basis of the spirit of the present invention
Enclose.
Claims (10)
1. primer pair and probe combinations, it is characterised in that including two groups of primer pairs and two probes；
Two groups of primer pairs include the first primer pair and the second primer pair；
Two probes include the first probe and the second probe, and first probe is corresponding with the first primer pair, and described
Two probes are corresponding with the second primer pair；
First primer pair includes SEQ ID NO：1 and SEQ ID NO：Nucleotide sequence shown in 2, the first probe bag
The NO of ID containing SEQ：Nucleotide sequence shown in 3；
Second primer pair includes SEQ ID NO：4 and SEQ ID NO：Nucleotide sequence shown in 5, the second probe bag
The NO of ID containing SEQ：Nucleotide sequence shown in 6.
2. primer pair according to claim 1 and probe combinations are preparing detection or the auxiliary detection white blood of acute lymphoblastic
Application in the product of disease.
3. a kind of kit, it is characterised in that including the primer pair and probe combinations described in claim 1.
4. kit according to claim 3, it is characterised in that also including fluorescent quantitative PCR reagent；
Optional, it is #6402682001's that the fluorescent quantitative PCR reagent, which includes the article No. from Roche companies,
Reagent FastStart Essential Probes in FastStart Essential Probes Master kits
Master Mix(2×)。
5. kit according to claim 4, it is characterised in that also including total RNA extraction reagent；
Optional, the total RNA extraction reagent is included in erythrocyte cracked liquid, TRIZOL, chloroform, isopropanol and ethanol at least
It is a kind of；
Optional, in addition to reverse transcription reagents；
Optional, it is AU311-03's that the reverse transcription reagents, which include the article No. from Beijing Quanshijin Biotechnology Co., Ltd,
Included in TransScript-Uni One-Step gDNA Removal and cDNA Synthesis SuperMix Kit
Reagent.
6. kit according to claim 5, it is characterised in that also including positive control, negative control and blank control；
Optional, the positive control is the solution containing TEL-AML1 fusions and GAPDH reference genes；The feminine gender is right
According to without TEL-AML1 fusions but to contain the solution of GAPDH reference genes；The blank control is physiological saline or gone
Ionized water.
7. the kit any one of claim 3 to 6 is preparing detection or auxiliary detection ALL
Product in application.
A kind of 8. system for screening the biological sample for being susceptible to suffer from ALL, it is characterised in that including：
Nucleic acid-extracting apparatus, the nucleic acid-extracting apparatus are used to extract the sample of nucleic acid in the biological sample；
Quantitative fluorescent PCR device, the quantitative fluorescent PCR device is connected with the nucleic acid-extracting apparatus, suitable for using right
It is required that the primer pair and probe combinations described in 1 carry out quantitative fluorescent PCR reaction to the sample of nucleic acid；And
Judgment means, the judgment means are connected with the quantitative fluorescent PCR device, so as to what is reacted based on quantitative fluorescent PCR
As a result, judge whether the biological sample is susceptible to suffer from ALL.
9. system according to claim 8, it is characterised in that the nucleic acid-extracting apparatus further comprises：
RNA extraction units, the RNA extraction units are used to extract RNA samples from biological sample；And
Reverse transcription unit, the reverse transcription unit are connected with the RNA extraction units, for being reversed to the RNA samples
Record reaction, to obtain cDNA samples, the cDNA samples form the sample of nucleic acid.
10. system according to claim 9, it is characterised in that：
The reaction system of the quantitative fluorescent PCR reaction is as follows：
The FastStart Essential Probes Master Mix (2 ×) are that the article No. of Roche companies is #
Reagent in 6402682001 FastStart Essential Probes Master kits.
Optional, the program of the quantitative fluorescent PCR reaction is as follows：95 DEG C of 10min, 1 circulation；95 DEG C of 10s, 60 DEG C of 30s (are received
Collect fluorescence), 45 circulations；40 DEG C of 10s, 1 circulation.
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Application publication date: 20171114 |