CN1995386A - BCR-ABL gene fluorescence quantitative RT-PCR primer and probe and reagent kit - Google Patents

Abstract

The invention discloses a quantitative RT-PCR primer and probe and agent box of BCR-ABL fusing gene mRNA fluorescence with BCR-ABL fusing gene primer and probe sequence as SEQ ID NO1-4 and internal reference gene primer and probe sequence as SEQ ID NO5-7, wherein the agent box contains cell cracking liquid, water, RT-PCR reacting liquid, internal reference TBP RT-PCR reacting liquid, BCR-ABL fusing gene detecting probe, TBP internal reference gene testing probe, composite enzyme, standard material and comparing material; the box can test the expressive level of mRNA of P210BCR/ABL and P190BCR/ABL in the specimen, which provides the reference to diagnose, recurrent and treat chronic granulocytic leukemia and acute lymphocyte leukemia.

Description

BCR-ABL gene fluorescence quantitative RT-PCR primer and probe and test kit

Technical field

The invention belongs to biological technical field, particularly relate to the mRNA expression that quantitative reverse transcription PCR detects gene.

Background technology

ABL is a proto-oncogene, is positioned at karyomit(e) q34 No. 9, and gene product is a kind of non-receptor type tyrosine protein kinase, but does not have kinase activity usually.The BCR gene is positioned at karyomit(e) q11 No. 22, and normal her gene product is the kytoplasm phosphoric acid albumen of 160kD.Because t (9; 22) (q34; Q11) transposition, cause being positioned at No. 9 ABL proto-oncogenes on the karyomit(e) and be positioned at No. 22 BCR genes on the karyomit(e) arranged side by side, form new BCR-ABL melt stylobate because of, in this transposition, ABL proto-oncogene breaking point on No. 9 karyomit(e) is more constant, BCR gene break point on No. 22 is non-constant, and most breaking points are positioned at the exon upstream and downstream No. 14.Mainly contain b2a2, b3a2 and three kinds of hypotypes of e1a2 according to the different fusion genes that form of breaking point, wherein 99% CML patient belongs to preceding two kinds of hypotypes, an and only relevant exon (75bp) on these two kinds of hypotype molecular structures, but because this exon does not have expression product, therefore the two all is translated as a kind of fusion rotein----p210 albumen, and the expressed product of e1a2 hypotype is a p190 albumen, and this albumen is acute lymphoblastic leukemia (ALL) patient characteristic expression product.Because of p210 and the proteic expression of p190 have activated tyrosine protein kinase, changed the protein-tyrosine level and the Actin muscle binding ability of cell, upset normal signal transduction path, suppress the generation of apoptosis.

The BCR-ABL fusion gene causes chronic myelocytic leukemia (CML) and acute lymphoblastic leukemia (ALL) to detect common method at present has: 1. morphological examination, because leukemia cell's height heterogeneity and polymorphism, poor repeatability, be subject to the influence of subjective factor, its diagnosis consistently puts down about 60%～80%; 2. immunological detection method has two kinds: cell smear method and FCM.Cell smear method required equipment is simple, can clearly judge the position of cell antigen and antibodies at microscopically, more easily get rid of the nonspecific reaction that brings because of the membrane permeability processing when especially the pair cell endoantigen detects, but range estimation is difficult for a large amount of cells are counted, so susceptibility is lower, be generally 10-2, be mainly used in the detection of intracellular antigen at present; And FCM can detect the antigen more than 2 in the same cell simultaneously, and to the capable real-time analysis of a large amount of cells, accurately objective, simple and convenient.Experimental results show that to its flow system carry out repeatedly and long-time the cleaning after, detecting the obtainable high sensitive of MRD is 10-6, but that generally reach in the actually operating is 10-4.3. cytogenetics detection method, conventional direct method or the Short-term Culture method used carried out karyomit(e) RHG and shown band, but leukemia cell's di is low, and chromosome morphology is short and small, and normal showing is with unclearly, makes some complex structures or trickle change be difficult to accurate identification.4. fluorescence in situ hybridization (FISH), FISH sensitivity height, draw materials conveniently, marrow or peripheral blood all can be used as research object, instrument is not to cell metaphase, and all can detect the interphasic nucleus cell, also can carry out check and analysis equally to conceal type, anomaly, be diagnostic method directly perceived, responsive, quick, special.But, when double-colored list commonly used merges probe in detecting bcr/abl fusion gene, because stochastic distribution or optical superposition on bcr and the abl gene signal space can cause 4%～10% false positive rate.Too high false positive rate not only has influence on sensitivity and specificity, also can influence the explanation of investigator to experimental result to a certain extent.5. molecular Biological Detection mainly contains two kinds of PCR method: a kind ofly be direct in-situ RT-PCR, and the more direct and easy enforcement of this scheme, direct in-situ RT-PCR detects a slow grain bcr/abl mRNA, and to have specificity good, the susceptibility height, and need not extract advantage such as RNA.It is reported that bcr/abl fusion gene male CML patient prognosis is better, but ALL patient antithesis, CML+ALL often presents high-risk symptom, and its complete remission rate is low, remission time and lifetime short, early stage resistance, and alleviates the back recurrence rate and reach 100%.But, be easy to generate non-specific result because primer mispairing and non-specific annealing take place.Another kind is the real time fluorescent quantitative detection of not reporting as yet and using.

So-called real-time fluorescence quantitative PCR technology is meant in the PCR reaction system to add fluorophor, utilizes the fluorescent signal accumulation whole PCR process of monitoring in real time, the method for by typical curve unknown template being carried out quantitative analysis at last.In fluorescent quantitative PCR technique, common preceding 15 the round-robin fluorescent signals that react with PCR are as the fluorescence background signal, the default setting of fluorescence threshold is 10 times of standard deviation of 6-15 round-robin fluorescent signal, and the cycle number that is experienced when the fluorescent signal in each reaction tubes arrived preset threshold is made as the Ct value.Studies show that there is linear relationship in the logarithm of the Ct value of each template and the initial copy number of this template, initial copy number is many more, and the Ct value is more little.Utilize known initial copying to see that several standard substance can make typical curve, wherein X-coordinate is represented the logarithm of initial copy number, and mark is represented the Ct value in length and breadth.Therefore, as long as obtain the Ct value of unknown sample, can calculate the initial copy number of this sample from typical curve.

Also add a special fluorescent probe when adding a pair of primer in the real-time fluorescence quantitative PCR amplification procedure, commonly used is the Taqman fluorescent probe at present, this probe is an oligonucleotide, and two ends are mark report fluorophor and a cancellation fluorophor respectively.When probe was complete, the fluorescent signal that reporter group is sent out was absorbed by quenching group, so detect less than fluorescence; In the pcr amplification process, 5 '-3 ' end 5 prime excision enzyme activity of Taq enzyme is cut degraded with the probe enzyme, the report fluorophor is separated with quenching group, thereby the fluorescence monitoring system can receive fluorescent signal.Each working cycle detection of end first order fluorescence intensity, reaction just can get a working curve after finishing.

Because quantitative fluorescent PCR has good reproducibility, highly sensitive, quantitative result is characteristics accurately and reliably, so the medical science context of detection, the problem that some conventional sense methods cann't be solved has obtained solution.For example, the morphocytology inspection can directly observe tumour cell, but because the number that tumour drops in the circulation of blood is small, and the susceptibility of this inspection and specificity are all relatively poor, positive rate is low.The application that immunohistochemical method improves tumour cell painted specificity, particularly monoclonal antibody in circulation of blood makes painted specificity and susceptibility that bigger improvement arranged, and can find 10 ⁴-10 ⁵A tumour cell in the individual karyocyte.But this technology then needs specific antibody, the production relative complex.Simultaneously, false positive has limited this broad application.Compare with these technology, fluorescent quantitative PCR technique has tangible advantage, because the adjusting of most of genes betides transfer level, some tumour can be transcribed specific mrna, but it is synthetic not have corresponding protein, so the quantitative fluorescent PCR range of application is wider; PCR is simple and easy to do, as long as know the testing gene sequence, can design synthetic primer and carry out reverse transcription and amplification; This method has higher sensitivity and repeatability, has also guaranteed the accuracy of medical science detected result.So this The Application of Technology will be found small transfer to the tumour cell in the circulation of blood of early stage searching tumour patient, for guiding clinical treatment, improve patient's prognosis and produce very important effect.

Summary of the invention

Technical problem to be solved

Technical problem to be solved by this invention provides a kind of BCR-ABL fusion gene (P190 ^BCR/ABLAnd P210 ^BCR/ABL) mRNA fluorescence quantitative RT-PCR primer and probe and test kit, prior art is low to detection by quantitative poor accuracy, the sensitivity of BCR/ABL fusion gene mRNA expression level in chronic myelocytic leukemia (CML) and acute lymphoblastic leukemia (ALL) the patient cell, detection speed is slow to overcome, and is unfavorable for the defective of clinical classification, disease observation and the recurrence monitoring of chronic myelocytic leukemia (CML) and acute lymphoblastic leukemia (ALL).

Technical scheme

One of technical scheme of the present invention provides one group of BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR and detects primer and probe, comprises following sequence:

BCR-ABL fusion gene upstream and downstream primer:

P210 ^BCR/ABLUpstream primer SEQ ID NO1:5 '-CCG CTG ACC ATC AATAAG G-3 '

P190 ^BCR/ABLUpstream primer SEQ ID NO2:5 '-ACT GCC CGG TTG TCGTGT-3 '

P190 ^BCR/ABL, P210 ^BCR/ABLDownstream primer SEQ ID NO3:5 '-GTT CCA ACGAGC GGC TTC A-3 ';

BCR-ABL fusion gene detection probes is SEQ ID NO4:

5’-FAM-TCT TCC CAG CCC ACC ATC CC-TAMRA-3’

Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence to 5 ';

Perhaps with above-mentioned sequence homology greater than 85% sequence;

The perhaps complementary base sequences thereof of above-mentioned sequence;

Perhaps use any one or more than one the combination in the above-mentioned all sequences.

The optimal technical scheme that one group of above-mentioned BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detects primer and probe is that its sequence set also comprises the primer and the probe of internal control gene TBP gene:

TBP internal control gene upstream and downstream primer:

Upstream primer SEQ ID NO5:5 '-GTG CCC GAA ACG CCG AAT A-3 ',

Downstream primer SEQ ID NO6:5 '-CTG GAC TGT TCT TCA CTC TT-3 '

TBP internal control gene detection probes is SEQ ID NO7:

5’-FAM-AAT CCC AAG CGG TTT GCT GCG G-TAMRA-3’；

Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence to 5 ';

Perhaps with above-mentioned sequence homology greater than 85% sequence;

The perhaps complementary base sequences thereof of above-mentioned sequence;

Perhaps use any one or more than one the combination in the above-mentioned all sequences.

Two of technical scheme of the present invention provides a kind of BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit that contains above-mentioned primer and probe sequence, and it consists of: cell pyrolysis liquid, water, the BCR-ABL RT-PCR reaction solution that contains primer sequence SEQ ID NO1-3, confidential reference items TBP RT-PCR reaction solution, the BCR-ABL gene probe that contains probe sequence SEQ ID NO4, TBP gene probe, reversed transcriptive enzyme, archaeal dna polymerase, standard substance, negative control, positive control.

Said BCR-ABL RT-PCR reaction solution consists of: RT-PCR damping fluid, MgCl ₂, goal gene (BCR-ABL fusion gene) upstream and downstream primer, dNTPs, no RNA enzyme water.

One of optimal technical scheme of above-mentioned BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit is, primer sequence in the said confidential reference items TBP RT-PCR reaction solution is SEQ IDNO5 and 6, and said TBP gene probe sequence is SEQ ID NO7.

Said TBP RT-PCR reaction solution consists of: RT-PCR damping fluid, MgCl ₂, house-keeping gene (TBP) upstream and downstream primer, dNTPs, no RNA enzyme water.

Two of the optimal technical scheme of above-mentioned BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit is that said standard substance are following dna sequence dna SEQ ID NO8:

5’-TGTGAAACTC CAGACTGTCC ACAGCATTCC GCTGACCATC

AATAAGGAAG ATGATGAGTC TCCGGGGCTC TATGGGTTTC

TGAATGTCAT CGTCCACTCA GCCACTGGAT TTAAGCAGAG

TTCAAAAGCC CTTCAGCGGC CAGTAGCATC TGACTTTGAG

CCTCAGGGTC TGAGTGAAGC CGCTCGTTGG AACTCCAAGG

AAAACCTTCT CGCTGGACCC AGTGAAA-3’。

As special case, standard substance are to contain the T carrier that inserts the BCR-ABL specific and conserved sequence, and this carrier can be bred in escherichia coli DH5a.

Three of the optimal technical scheme of above-mentioned BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit is that said water is the water of no RNA enzyme.

Generally, the water preparation method of no RNA enzyme is: add DEPC (diethylpyrocarbonate) to deionized water, to final concentration be 0.01% (V/V), room temperature (22-25 ℃) was placed after 10-12 hour, 121 ℃, 20 minutes high pressure, room temperature is placed standby;

Four of the optimal technical scheme of above-mentioned BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit is that said negative control is the normal cell sample of no BCR-ABL fusion gene.

Five of the optimal technical scheme of above-mentioned BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit is that said positive control is the leukemia cell's sample that contains the BCR-ABL fusion gene.

Six of the optimal technical scheme of above-mentioned BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit is that said cell pyrolysis liquid is made up of cell pyrolysis liquid I and cell pyrolysis liquid II.

As special case, cell pyrolysis liquid I consists of: 320mM sucrose, 5mM MgCl2,1%TritonX-100,10mM Tris-HCl[pH7.5], water;

As special case, cell pyrolysis liquid II consists of: 4M guanidine thiocyanate, 0.75M Trisodium Citrate (pH7.0) and 10% sodium N-lauroyl sarcosinate (Sarcosyl 1g/10mL), 14.3M beta-mercaptoethanol, 2M sodium-acetate (pH4.0) and water-saturated phenol;

Said reversed transcriptive enzyme and the form of archaeal dna polymerase in test kit can be for mixing the mixed solution of two kinds of enzymes in above-mentioned each scheme.Generally speaking, archaeal dna polymerase is selected heat-resisting Taq archaeal dna polymerase for use.

Six of the optimal technical scheme of above-mentioned BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit is that the amount of each component is in every box: 1 bottle of 2mL of water of 1 bottle of 24mL of cell pyrolysis liquid I, 1 bottle of 6mL of cell pyrolysis liquid II, no RNA enzyme, BCR-ABL RT-PCR reaction solution 1 pipe 210 μ L, TBP RT-PCR reaction solution 1 pipe 210 μ L, BCR-ABL gene probe 1 pipe 30 μ L, TBP internal control gene probe 30 μ L, standard substance 1 pipe 10 μ L and negative control product 1 pipe 10 μ L.

Cell pyrolysis liquid in this test kit (being that RNA always extracts reagent) should be in 4 ℃ of preservations, and RT-PCR reagent is stored in-20 ℃, reduces multigelation as far as possible.

The nucleotide position in gene of primer that the present invention is designed and probe sequence, Tm value and product length are as follows:

PCR standard substance primer sequence

Primer	Nucleotide position	Base sequence (5 '-3 ')	Tm (℃)	Product (bp)
					The upstream	249 (HSA13146 6)	TGT GAA ACT CCA GAC TGT CC	60	227
The upstream	475 (HSA13146 6)	ACG AAA AGG TTG GGG TCA TT	58

The BCR/ABL fusion gene detects primer, probe sequence

Primer	Nucleotide position	Base sequence (5 '-3 ')	Tm (℃)	Product (bp)
					SEQ ID NO1	3282 (NM_00432 7)	CCG CTG ACC ATC AAT AAG G	58	90 or 165
SEQ ID NO2	1777 (NM_00432 7)	ACT GCC CGG TTG TCG TGT	58	167
					SEQ ID NO3	132 (NM_00515 7)	GTT CCA ACG AGC GGC TTC A	60	--
SEQ ID NO4	86 (NM_00515 7)	TCT TCC CAG CCC ACC ATC CC	72

Confidential reference items primer, probe sequence

Primer	Nucleotide position	Base sequence (5 '-3 ')	Tm (℃)	Product (bp)
					SEQ ID NO5	905 (NM_0031 94)	GTG CCC GAA ACG CCG AAT A	60	136
SEQ ID NO6	789 (NM_0031 94)	CTG GAC TGT TCT TCA CTC TT	58
					809 (NM_0031 94)	AAT CCC AAG CGG TTT GCT GCG G	70	-

The standard substance sequence

Standard substance	Nucleotide position	Base sequence (5 '-3 ')	Tm (℃)	Product (bp)
					SEQ ID NO8	249～494 (HSA13146 6)	TGTGAAACTC CAGACTGTCC ACAGCATTCC GCTGACCATC AATAAGGAAG ATGATGAGTC TCCGGGGCTC TATGGGTTTC TGAATGTCAT CGTCCACTCA GCCACTGGAT TTAAGCAGAG TTCAAAAGCC CTTCAGCGGC CAGTAGCATC TGACTTTGAG CCTCAGGGTC TGAGTGAAGC CGCTCGTTGG AACTCCAAGG AAAACCTTCT CGCTGGACCC AGTGAAA		227

The present invention also provides the using method of said test kit as follows:

Testing goal gene (BCR-ABL fusion gene) and house-keeping gene (TBP:TATA box binding protein) all should be set up positive control and negative control at first, at every turn.

[sample requirement]

The fresh peripheral blood or the sample of bone marrow of 1～2ml anti-freezing.

Gather venous blood or marrow 1～2ml in the aseptic centrifuge tube that contains antithrombotics, or in aseptic centrifuge tube, use EDTA to make antithrombotics (1.44mg/ml whole blood).Should use immediately after the sample collection, if can not use immediately, can 4 ℃ preserve 1-2 hour, but the time should not be after length, otherwise will influence detected result.Whole blood after treatment, the karyocyte that obtains can be preserved the several months in-80 ℃ or liquid nitrogen, can not influence detected result.

[operation steps]

One, total RNA extracts

1. fresh anticoagulated whole blood or marrow 1～1.5ml are placed 1.5ml Beckman centrifuge tube, 4 ℃ of centrifugal 10min of following 3000g abandon supernatant liquor.

2. down mending full cell pyrolysis liquid I in the confluent monolayer cells, mixing, ice bath 10min be mixing twice once more therebetween, and solution becomes shows red corpuscle cracking clearly.

3.4 ℃ following 3000g, centrifugal 10min precipitation karyocyte discards the supernatant liquor that contains lysing cell fully.

4. with (if still have red corpuscle in the precipitation, this operates 1～2 time to need repetition) after the cell pyrolysis liquid I repetition cracking 1 time, the supernatant that inclines blots the mouth of pipe.

5. adding 50 μ L in above-mentioned precipitation karyocyte does not have RNA enzyme water, breaks up with the suction of rifle head and forms limpid not sticking solution, adds 450 μ L cell pyrolysis liquid II again, blows and beats mixing repeatedly with pipettor, and room temperature was placed 5 minutes.

6. add 200 μ L trichloromethanes, with vibrate back and forth mixing 15 seconds of hand, room temperature was placed 5 minutes, and 4 ℃ 15, centrifugal 10 minutes of 000rpm moves into new centrifuge tube with supernatant.

7. add the equal-volume isopropyl alcohol, vortex vibration 5 seconds, precipitation at room temperature 10 minutes, 4 ℃ 15, centrifugal 5 minutes of 000rpm abandons supernatant.

8. 75% washing with alcohol that adds 500 μ L precoolings turns upside down 10 times, and 4 ℃ 10, centrifugal 5 minutes of 000rpm blots supernatant, precipitation drying at room temperature 10-20 minute, and adding 20 μ L does not have RNA enzyme water dilution mixing, uses immediately or-70 ℃ of preservations.

Two, RT-PCR

The centrifuge tube that BCR-ABL RT-PCR reaction solution, confidential reference items TBP (TATA box binding protein) RT-PCR reaction solution, BCR-ABL fusion gene probe, TBP house-keeping gene probe, mixed enzyme, standard substance, reference substance will be housed takes out from-20 ℃, place treat on the ice chest that it slowly melts after, low-speed centrifugal, with pipettor mentioned reagent is prepared goal gene (BCR-ABL fusion gene) and house-keeping gene (TBP:TATA box binding protein) RT-PCR reaction solution respectively, mixing, low-speed centrifugal places on the ice chest to have.

Prepare RT-PCR reaction solution (30 μ L system) by following composition:

Genes of interest RT-PCR reaction system 21 μ BCR-ABL RT-PCR reactant liquor L BCR-ABL fusion probes 3 μ L mixed enzyme 2 μ L RNA templates 4 μ L

House-keeping gene RT-PCR reaction system TBP RT-PCR reactant liquor 21 μ L TBP house-keeping gene probes 3 μ L mixed enzyme 2 μ L RNA templates 4 μ L

The RT-PCR reaction conditions: 37 ℃ 30 minutes; 95 ℃ 5 minutes; (95 ℃ 10 seconds, 60 ℃ 45 seconds, 40 circulations).

Three, the making of typical curve

With standard substance stock solution (6*10 ⁷Copies/ μ L) gradient dilution is 6*10 ⁶, 6*10 ⁵, 6*10 ⁴, 6*10 ³, 6*10 ²Copies/ μ L, getting 4 μ L is template, together carries out RT-PCR amplification, the quantitative curve of production standard with testing sample.

[result's judgement]

1. use the accompanying software of real-time fluorescence quantitative PCR instrument to carry out the setting of file preservation and threshold value.

2. choose the different concns standard substance, use corresponding software and analyze, draw typical curve and relevant information.

3. choose sample institute to detect the hole, use corresponding software to analyze, draw the initial copy number (i.e. [Copy] of testing sample goal gene and house-keeping gene goal gene and house-keeping gene should be arranged _Sample[Copy] _IPC).

4. according to the initial copy number that detects sample goal gene and house-keeping gene, calculate the genetic expression rate, i.e. Expression Ratio=[Copy] _Sample/ [Copy] _IPC

Beneficial effect

Test kit of the present invention is by extracting total RNA to fresh anticoagulation cirumferential blood karyocyte, in order to get rid of different specimens and to cause when extracting the difference that may exist on output, quality and the reverse transcription efficient at RNA to obtain the specific expressed real difference of target gene, selection is expressed with medium abundance, the lower TBP of variation proofreaies and correct and stdn as the internal reference gene between cell specimen, in conjunction with real time fluorescent quantitative Rt-PCR detection technique, can accurately detect P210 in the sample simultaneously again ^BCR/ABLAnd P190 ^BCR/ABLThe expression level of mRNA, for the early diagnosis of chronic myelocytic leukemia and acute lymphoblastic leukemia, recurrence, treatment plan determine and the judgement of prognosis provides important reference frame.

The present invention has set up the method for utilizing TaqMan technology for detection BCR-ABL fusion gene mRNA to express, and through chronic myelocytic leukemia (CML) and acute lymphoblastic leukemia (ALL) patient sample are detected, shows that this method is practical.Because present method has adopted quantitative fluorescent PCR, the detection sensitivity and the specificity that make the BCR-ABL fusion gene mRNA express improve greatly, reduce the false positive rate of conventional pcr amplification, thereby made the present invention can in the sample of minute quantity, obtain enough information.Simultaneously in order to get rid of different specimens and to cause when extracting the difference that may exist on output, quality and the reverse transcription efficient at RNA to obtain the specific expressed real difference of target gene, determine to express with medium abundance by screening, the lower TBP (being the TATA box binding protein) of variation proofreaies and correct and stdn as internal reference gene (also claiming house-keeping gene) between cell specimen.

In this test item, the present invention has adopted the most advanced at present real-time fluorescence quantitative PCR detection technique, under the prerequisite that keeps hypersensitivity, reduces false-positive interference as far as possible.Before the actual quantities fluorescence quantitative PCR detection technique occurs, people quantitatively will pass through PCR product electrophoresis to pcr template, again with electrophoresis result machine picture processing as calculated, determine what of final PCR product amount according to the height of electrophoretic band, or the PCR product of the tape label mode with ELISA detected, infer the amount that starting template more thus, but these methods in fact all have the sxemiquantitative of belonging to level, even because PCR condition optimization, the unstable of the operation of electrophoresis and subsequent step brings influence still can for result's analysis, thereby influences quantitative this purpose.Along with real-time fluorescence quantitative PCR The Application of Technology of the present invention, can accomplish accurate quantification veritably to pcr template, this high quick intelligence that has quantitatively not only kept conventional PCR, and because specificity fluorescent probe hybridization The Application of Technology, the specificity that makes quilt detect gene improves greatly.

Primer of the present invention, probe and test kit adopt fluorescent quantitative RT-PCR method, this method sensitivity, special, good reproducibility, and the result represents with copy number, accurately and reliably, is beneficial to unified standard.The method sensitivity, the recombinant plasmid quantitative templates by 10 times of gradient dilutions, can detect 10 copies, and make typical curve thus, and linearity range is wide by (0～10 ⁷Copy), the stability of method, repeatability, dependency are good, and relation conefficient is 0.997.Fluorescent quantitative RT-PCR method has wide practical use at aspects such as dynamic observing of CML, ALL diagnosis, curative effect and prognosis judgement and MRD.Because nearly all CML patient can both find the bcr/abl fusion gene, detect the diagnosis that the bcr/abl fusion gene helps CML, ALL, and cloudy commentaries on classics of bcr/abl fusion gene become the important indicator that CML, ALL curative effect and prognosis are judged.

Description of drawings

Fig. 1 is sample standard detection figure.

Fig. 2 is the sample standard graphic representation.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment instrument to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, as the molecular cloning operational manual, or the condition of advising according to manufacturer.All inorganic chemical reagents and organic solvent are available from Shanghai chemical reagent factory, and Trizol reagent is given birth to worker company available from Shanghai, and restriction enzyme is available from Shen, Shanghai energy lottery industry biotech firm, MMLV reversed transcriptive enzyme, Taq archaeal dna polymerase, Oligo (dT _{) 15-18}All available from U.S. Promega company, primer and probe are given birth to worker company by Shanghai and are synthesized.Eppendorf gradient type pcr amplification instrument is available from German Eppendorf company, and iCycle fluorescent PCR instrument is a BIO-RAD company product.

Embodiment 1

The real-time fluorescence quantitative RT-PCR method detects the expression and the application of people BCR-ABL fusion gene mRNA

One, primer and probe design and synthetic:

(GenBank accession number HSA131466) is template with wherein a kind of b3a2 hypotype of BCR-ABL fusion gene full length cDNA sequence, use the site of Oligo software analysis TaqMan primer and probe, and, therefrom select best of breed according to the situation of considering BCR and ABL two genomic dna sequences simultaneously.

Standard substance PCR upstream primer sequence is: 5 '-TGT GAA ACT CCA GAC TGT CC-3 '; The downstream primer sequence is: 5 '-ACG AAA AGG TTG GGG TCA TT-3 ';

Detect and be: 5 '-CCG CTG ACC ATC AAT AAG G-3 ' and 5 '-ACT GCC CGG TTG TCG TGT-3 ' with PCR upstream primer sequence; The downstream primer sequence is: 5 '-GTT CCA ACGAGC GGC TTC A-3 ';

The fluorescent probe sequence is: 5 '-FAM-TCT TCC CAG CCC ACC ATCCC-TAMRA-3 ';

Two, examination criteria product preparation:

Get the fresh peripheral blood of patient, separate the peripheral blood karyocyte, after the PBS washing, centrifugal collecting cell extracts total RNA with Trizol reagent, gets 1 μ gRNA, uses Oligo (dT in 25 μ L total reaction systems _{) 15-18}For after primer carries out reverse transcription to it, increase at the enterprising performing PCR of Eppendorf gradient type pcr amplification instrument with standard substance upstream and downstream primer, reaction conditions is 95 ℃ of sex change 5 minutes, carried out 35 cyclic amplifications in 45 seconds by 95 ℃ 20 seconds, 58 ℃ 20 seconds, 72 ℃ again, extend in 72 ℃ at last and be placed on 4 ℃ in 7 minutes.

Pcr amplification product is served the sea and is dodged brilliant biotech firm and clone (plasmid is provided by the company of answering) after electrophoresis detection, and with positive colony through sequence verification (by the order-checking of last sea base Kanggong department).Extract the positive colony plasmid, be standard substance, measure its concentration and be converted into (copy number/volume).

Three, sample detects:

Get leukaemic and 2 routine normal people's peripheral blood sample 1.5ml that 5 examples are determined through pathology, separate the outer karyocyte of periphery, after the PBS washing, centrifugal collecting cell, extract total RNA with Trizol reagent, get 4 μ L RNA extracting solutions, in 30 μ L total reaction systems, use with detection, on downstream primer and the house-keeping gene, downstream primer is simultaneously in the enterprising performing PCR amplification of iCycle fluorescent PCR instrument, reaction conditions is 37 ℃ of insulations 30 minutes, 95 ℃ of sex change 5 minutes, again by 95 ℃ 20 seconds, 58 ℃ 20 seconds, 72 ℃ were carried out 40 cyclic amplifications in 45 seconds, add the standard substance detection simultaneously and make typical curve, measurement result is handled according to typical curve through instrument and is calculated the BCR-ABL fusion gene of detection sample and the copy number of TBP, and the copy number according to both calculates BCR-ABL fusion gene expression rate again.

Five, result:

(1) preparation of standard substance

Through order-checking, the standard substance of above-mentioned design conform to expection fully, and the standard substance fragment sequence of recovery is as follows:

5’-TGTGAAACTC CAGACTGTCC ACAGCATTCC GCTGACCATC

AATAAGGAAG ATGATGAGTC TCCGGGGCTC TATGGGTTTC

TGAATGTCAT CGTCCACTCA GCCACTGGAT TTAAGCAGAG

TTCAAAAGCC CTTCAGCGGC CAGTAGCATC TGACTTTGAG

CCTCAGGGTC TGAGTGAAGC CGCTCGTTGG AACTCCAAGG

AAAACCTTCT CGCTGGACCC AGTGAAA-3’

(2) sample detection

The standard substance detected result is seen Fig. 1, and typical curve is seen Fig. 2.

5 routine patients and 2 routine normal people's peripheral blood sample fluorescence quantitative RT-RCR detected results are as follows:

Patient's sex year sample fusion gene TBP expression rate (Ratio)

Numbering age (copies/mL) is (Copy (copies/mL) _PML-RARa/

Copy _TBP)

1 male 43 peripheral blood 1.192*10 ²2.185*10 ¹5.46

2 women 51 peripheral blood 5.866*10 ⁰5.894*10 ⁰1.00

3 women 62 peripheral blood 1.164*10 ²1.099*10 ¹10.60

4 male 68 peripheral blood 3.254*10 ¹2.032*10 ¹1.60

5 women 36 peripheral blood 9.240*10 ¹1.324*10 ¹6.98

6 women 38 peripheral bloods---

7 male 57 peripheral bloods---

Adopting similar detection PCR upstream and downstream primer and 7 same routine samples to carry out original position direct in-situ RT-PCR detects.

Experimental program is as follows:

Reverse transcription reaction: reaction system 20 μ L. contain 200 μ mol/L dNTPs.1 μ moI/L downstream primers, RNA enzyme inhibitors 40U and Mo-MLV 20U, reaction solution is through a gene frame (GeneFrame, purchase company in Advanced Biotechnologies) be sealed in above-mentioned slide center with the zone of cell, slide is PCR instrument (Genius in position, Britain Techne company) 42 ℃ of reaction 30min finish the back and remove the gene frame in, get rid of and abandon reaction solution.

Original position PCR: reaction system 25 μ L contain 200 μ mol/L dNTPs, 1 μ mol/L upstream and downstream primer, 4.5mmol/L MgCl ₂, the dUTP of 10 μ mol/L digoxigenin labeleds and Taq enzyme 5U.Reaction solution is sealed in above-mentioned position equally, and after reaction conditions was 95 ℃ of sex change 5min, with 94 ℃ of sex change, 58 ℃ of annealing and 72 ℃ of each 1min of extension on the PCR instrument, carried out 25 circulations in position altogether.

Amplified production detects: the 2 * SSC that 1. develops a film develops a film 3 times, contains 0.The Buffer I of 1% tween 20 (0.1mol/L Tris-HCI pH7.5,0.15M MgCl ₂) wash each 5min 2 times; Contain the Buffer II incubated at room 30min of 5% bovine serum albumin (BSA) and 3% sheep serum, drip sheet with anti digoxin antibody again, hatch 2h for 37 ℃ in the wet box; 2. develop a film: the Buffer I that contains 0.1% Ye Wen-20 washes 3 times, and Buffer II washes 1 time again, Buffer III (0.1mol/L Tris-HCl pH9.5,0.1mol/L NaCl, 0.05mol/L MgCl ₂) wash each 5min 1 time; The about 30min of NBT/BCIP lucifuge dyeing, light microscopic is observed down, and the appropriate after washing of waiting to develop the color stops covered, glycogelatin mounting; 3. the result judges that to occur the blue-purple granule material in the cytoplasm positive, no blue-purple granule in the negative patient endochylema.

5 routine patients and 2 routine normal people's peripheral blood sample original position RT-PCR detected results are as follows:

Numbering sex age detection result of specimen

1 male 43 peripheries and the positive

The 2 women 51 peripheral blood positives

The 3 women 62 peripheral blood positives

The 4 male 68 peripheral blood positives

The 5 women 36 peripheral blood positives

6 women 38 peripheral blood feminine genders

7 male 57 peripheral blood feminine genders

Though, original position RT-PCR detected result is consistent with the fluorescence quantitative RT-RCR detected result, but its operating process excess enthalpy complexity, the result judges the influence that is subjected to subjective factor, and can only make qualitative detection, can not provide important reference frame for clinical treatment provides the judgement definite and prognosis of CML, ALL early diagnosis, recurrence, treatment plan.

The nucleotides sequence tabulation

SEQUENCE LISTING

＜110〉Shanghai Fosun Pharmaceutical (Group) Co., Ltd.

Fuxing Medical Science-Technology Development Co., Ltd., Shanghai

＜120〉BCR-ABL gene fluorescence quantitative RT-PCR primer and probe and test kit

<130>060802

<140>CN

<141>2006-08-10

<160>8

<170>PatentIn version 3.3

<210>1

<211>19

<212>DNA

<213>Homo sapiens

<220>

＜221〉BCR-ABL fusion gene P210 upstream primer

<222>(1)..(19)

<400>1

ccgctgacca tcaataagg 19

<210>2

<211>18

<212>DNA

<213>Homo sapiens

<220>

＜221〉BCR-ABL fusion gene P190 upstream primer

<222>(1)..(18)

<400>2

actgcccggt tgtcgtgt 18

<210>3

<211>19

<212>DNA

<213>Homo sapiens

<220>

＜221〉BCR-ABL fusion gene P210/P190 downstream primer

<222>(1)..(19)

<400>3

gttccaacga gcggcttca 19

<210>4

<211>22

<212>DNA

<213>Homo sapiens

<220>

＜221〉BCR-ABL fusion gene detection probes

<222>(1)..(20)

<223>n＝FAM，y＝TAMRA

<220>

<221>misc_feature

<222>(1)..(1)

<400>4

ntcttcccag cccaccatcc cy 22

<210>5

<211>19

<212>DNA

<213>Homo sapiens

<220>

＜221〉TATA box binding protein internal control gene upstream primer

<222>(1)..(19)

<400>5

gtgcccgaaa cgccgaata 19

<210>6

<211>20

<212>DNA

<213>Homo sapiens

<220>

＜221〉TATA box binding protein internal control gene downstream primer

<222>(1)..(20)

<400>6

ctggactgtt cttcactctt 20

<210>7

<211>24

<212>DNA

<213>Homo sapiens

<220>

＜221〉TATA box binding protein internal control gene detection probes

<222>(1)..(22)

<223>n＝FAM，y＝TAMRA

<220>

<221>misc_feature

<222>(1)..(1)

<400>7

naatcccaag cggtttgctg cggy 24

<210>8

<211>227

<212>DNA

<213>Homo sapiens

<220>

＜221〉standard substance dna sequence dna

<222>(1)..(227)

<400>8

tgtgaaactc cagactgtcc acagcattcc gctgaccatc aataaggaag atgatgagtc 60

tccggggctc tatgggtttc tgaatgtcat cgtccactca gccactggat ttaagcagag 120

ttcaaaagcc cttcagcggc cagtagcatc tgactttgag cctcagggtc tgagtgaagc 180

cgctcgttgg aactccaagg aaaaccttct cgctggaccc agtgaaa 227

Claims (10)

1. one group of BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detects primer and probe, comprises following sequence:

BCR-ABL fusion gene upstream and downstream primer:

P210 ^BCR/ABLUpstream primer SEQ ID NO1:5 '-CCG CTG ACC ATC AATAAG G-3 '

P190 ^BCR/ABLUpstream primer SEQ ID NO2:5 '-ACT GCC CGG TTG TCGTGT-3 '

P190 ^BCR/ABL, P210 ^BCR/ABLDownstream primer SEQ ID NO3:5 '-GTT CCA ACGAGC GGC TTC A-3 '

BCR-ABL fusion gene detection probes is SEQ ID NO4:

5’-FAM-TCT TCC CAG CCC ACC ATC CC-TAM RA-3’

Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence to 5 ';

Perhaps with above-mentioned sequence homology greater than 85% sequence;

The perhaps complementary base sequences thereof of above-mentioned sequence;

Perhaps use any one or more than one the combination in the above-mentioned all sequences.

2. detect primer and probe according to the described one group of BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR of claim, its sequence set also comprises the primer and the probe of internal control gene TBP gene:

TBP internal control gene upstream and downstream primer:

Upstream primer SEQ ID NO5:5 '-GTG CCC GAAACG CCG AATA-3 ',

Downstream primer SEQ ID NO6:5 '-CTG GAC TGT TCT TCA CTC TT-3 '

TBP internal control gene detection probes is SEQ ID NO7:

5’-FAM-AAT CCC AAG CGG TTT GCT GCG G-TAMRA-3’

Perhaps include holding or/and the sequence that 3 ' end prolongs of above-mentioned sequence to 5 ';

Perhaps with above-mentioned sequence homology greater than 85% sequence;

The perhaps complementary base sequences thereof of above-mentioned sequence;

Perhaps use any one or more than one the combination in the above-mentioned all sequences.

3. BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit that contains the described sequence of claim 1, it consists of: cell pyrolysis liquid, water, the BCR-ABL RT-PCR reaction solution that contains the described primer sequence SEQ of claim 1 ID NO1-3, confidential reference items TBP RT-PCR reaction solution, contain BCR-ABL fusion gene detection probes, TBP gene probe, reversed transcriptive enzyme, archaeal dna polymerase, standard substance, negative control, the positive control of the described probe sequence SEQ of claim 1 ID NO4.

4. according to claim 2 or 3 described BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit, it is characterized in that, primer sequence in the said confidential reference items TBP RT-PCR reaction solution is SEQ ID NO5 and 6, and said TBP gene probe sequence is SEQ ID NO7.

5. BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit according to claim 3 is characterized in that, said standard substance are following dna sequence dna SEQ ID NO8:

5’-TGTGAAACTC CAGACTGTCC ACAGCATTCC GCTGACCATC

AATAAGGAAG ATGATGAGTC TCCGGGGCTC TATGGGTTTC

TGAATGTCAT CGTCCACTCA GCCACTGGAT TTAAGCAGAG

TTCAAAAGCC CTTCAGCGGC CAGTAGCATC TGACTTTGAG

CCTCAGGGTC TGAGTGAAGC CGCTCGTTGG AACTCCAAGG

AAAACCTTCT CGCTGGACCC AGTGAAA-3’。

6. BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit according to claim 3 is characterized in that, said water is the water of no RNA enzyme.

7. BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit according to claim 3 is characterized in that, said negative control is the normal cell sample of no BCR-ABL fusion gene.

8. BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit according to claim 3 is characterized in that said positive control is the leukemia cell's sample that contains the BCR-ABL fusion gene.

9. BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit according to claim 3 is characterized in that said cell pyrolysis liquid is made up of cell pyrolysis liquid I and cell pyrolysis liquid II.

10. BCR-ABL fusion gene mRNA fluorescence quantitative RT-PCR detection kit according to claim 3, it is characterized in that the amount of each component is in every box: 1 bottle of 2mL of water of cell pyrolysis liquid I1 bottle 24mL, cell pyrolysis liquid II1 bottle 6mL, no RNA enzyme, BCR-ABL RT-PCR reaction solution 1 pipe 210 μ L, TBP RT-PCR reaction solution 1 pipe 210 μ L, BCR-ABL gene probe 1 pipe 30 μ L, TBP internal control gene probe 30 μ L, standard substance 1 pipe 1 μ L and negative control product 1 pipe 1 μ L.

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