CN103937891A - Multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes - Google Patents
Multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes Download PDFInfo
- Publication number
- CN103937891A CN103937891A CN201410153342.7A CN201410153342A CN103937891A CN 103937891 A CN103937891 A CN 103937891A CN 201410153342 A CN201410153342 A CN 201410153342A CN 103937891 A CN103937891 A CN 103937891A
- Authority
- CN
- China
- Prior art keywords
- primer
- leukemia fusion
- fusion gene
- kit
- pcr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6858—Allele-specific amplification
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of gene detection, and in particular relates to a multiplex PCR (polymerase chain reaction) kit for detecting leukemia fusion genes. The kit comprises a conventional multiplex PCR component, and chimeric primer and common primer pairs aiming at the common leukemia fusion genes. Chimeric primers are sequences of adding common primers to the 5' ends of specific primers of seven shear isomers of the common leukemia fusion genes BCR/ABL, PML/RARalpha, AML1/ETO and E2A/PBX1. By using the multiplex PCR kit for detecting the leukemia fusion genes, seven leukemia fusion genes including AML1/ETO, BCR/ABLe1a2, BCR/ABLe13a2, BCR/ABLe14a2, PML/RARalphaL, PML/RARalphaS and E2A/PBX11 with high incidence can be simultaneously detected, so that the consumption of reagents can be saved, the detection cost can be reduced, and meanwhile, the detection efficiency is improved. Moreover, the highest detection sensitivity of the kit can reach 10 copies per 25mu L, and the kit is simple in operation and high in clinical detection rate, so that the kit has clinical popularization and application values.
Description
Technical field
The invention belongs to gene test field, be specifically related to the multiple PCR reagent kit of four kinds of common leukemia fusion genes of a kind of detection.
Background technology
Multiplex PCR principle of work is to add multipair primer in same PCR reaction system, simultaneously the increase PCR reaction of multiple DNA fragmentations, as one of important deriving technology of PCR, the method has high efficiency, economical and convenient, can greatly save time and the many advantages such as reagent cost.This technology has been widely used in the fields such as biotechnology, quarantine and medical science detection at present.
In multi-PRC reaction, generally comprise following reagent: more than two couples primer pair, dNTP, MgCl
2, PCR damping fluid, template DNA, Taq DNA polysaccharase and other adjuvants (DMSO, glycerine, BSA), conventionally in order to obtain reasonable amplification, understand the usage quantity to reagent, the reaction conditions of pcr amplification, such as elongating temperature, extension time, annealing time, annealing temperature, PCR cycle number etc., be optimized.
Leukaemic's genetics characteristics complex is changeable, comprise chromosomal transposition, disappearance, sudden change and inversion etc., when these chromosome aberrations, in most of situation, can produce new fusion gene, these fusion genes can be used as the dissimilar leukemic molecular marker of diagnosis, in addition, the treatment plan of dissimilar leukemia chemotherapy and radiotherapy is also different.Therefore, the detection of fusion gene has important guiding value to leukemic diagnosis, prognosis, monitoring minimal residual disease and direction of medication usage etc., has become one of most important index of leukemia diagnosis standard.
BCR/ABL, PML/RAR α, AML1/ETO, E2A/PBX1 are four kinds of modal leukemia fusion genes.Wherein, approximately 95% patients with chronic myelocytic leukemia can detect BCR/ABL fusion gene; More than 85% acute promyelocytic leukemia relates to PML/RAR alpha fusion gene; And AML1/ETO fusion gene is one of modal chromosome abnormalities, in M2 hypotype acute myeloid leukemia, account for 30%-50%; E2A/PBX1 fusion gene sees the children of about 3-5% and 3% adult B cellular type acute lymphoblastic leukemia, is the important detection index in children acute lymphoblastic leukaemia diagnosis and treatment.Based on above reason, choose leukemia fusion gene that this four kinds of sickness rate the are high detection target as this research.
As one of important deriving technology of PCR, multiplex PCR has many advantages: (1) high efficiency, reduce operating process, and can detect multiple genes or fragment simultaneously; (2) economical and convenient, can save time and reagent cost greatly.It can, once detect multiple common leukemia fusion gene in experiment simultaneously, effectively make up the deficiency of chromosome karyotype analysis, has improved the recall rate of invisible translocation chromosome.At present, the mainly Multiplex RT-PCR detection method taking Poul Jorgensen as representative of clinical conventional leukemia molecule diagnostic method, these class methods are generally divided into reaction system eight pipes, every pipe detects dissimilar fusion gene and product clip size exists notable difference, judges fusion gene type according to reaction tubes position, amplified production place and clip size.Because multipair primer in same reaction system easily interacts, as form hairpin structure, dimeric structure etc., thereby affect amplification efficiency, this is to be divided into the major cause that eight pipes detect, but this has also caused such detection method complexity, loaded down with trivial details simultaneously, operator's relevant speciality is had relatively high expectations, be difficult to promote.
Summary of the invention
Goal of the invention of the present invention is to provide a kind of Multiplex RT-PCR detection kit of common four kinds of leukemia fusion genes, utilize it can detect 7 shearing isomer of BCR-ABL, PML/RAR α, AML1/ETO, these four kinds of common leukemia fusion genes of E2A/PBX1, specifically comprise: BCR/ABL e1a2, BCR/ABL e13a2, BCR/ABL e14a2, PML/RAR α L-type, PML/RAR α S type, E2A/PBX1 I type and AML1/ETO.
To achieve the above object of the invention, the technical solution used in the present invention is: a kind of multiple PCR reagent kit that detects leukemia fusion gene, described multiple PCR reagent kit comprises: conventional multiplex PCR assembly, also comprise chimeric primers and consensus primer pair for common leukemia fusion gene, described consensus primer is to comprising following nucleotide sequence:
SEQ ID NO.1: the sense primer 5 '-AGACATCGTAGGTAGTGACA-3 ' of consensus primer centering; SEQ ID NO.2: the antisense primer 5 '-GAACGGACGACAATACAGTG-3 ' of consensus primer centering;
The described chimeric primers for common leukemia fusion gene is many chimeric primers pair for common leukemia fusion gene BCR/ABL, PML/RAR α, AML1/ETO, E2A/PBX1, specifically as shown in following nucleotide sequence:
In technique scheme, described conventional multiplex PCR assembly comprises: positive control primer, Taq archaeal dna polymerase, dNTP, MgCl2, PCR damping fluid, deionized water.
Positive control can be used to detect cDNA template quality and reaction system amplification efficiency, and the present invention is preferably based on the positive control primer of GAPDH gene, and concrete sequence is: SEQ ID NO.15, AGGTCGGAGTCAACGGATTTG; SEQ ID NO.16, GTGATGGCATGGACTGTGGT.
In technique scheme, in the time carrying out PCR reaction, every concentration for the chimeric primers of common leukemia fusion gene is 1 μ M.
In technique scheme, in the time carrying out PCR reaction, the concentration of consensus primer is 10 μ M.
Two principle is followed in the consensus primer design of technique scheme: when (1) consensus primer and template to be measured increase, no matter how condition is optimized, all there is no specificity product; (2), in the time having amplified production with the multi-primers of consensus primer, necessarily can amplify specificity product by consensus primer.The sequence that adds consensus primer at the special primer 5 ' end for BCR/ABL, PML/RAR α, AML1/ETO, the conventional leukemia fusion gene of E2A/PBX1, is referred to as chimeric primers.
The multiple PCR reagent kit of above-mentioned detection leukemia fusion gene can be used for the detection of the common fusion gene of leukemia, specifically be divided into and first carry out two-wheeled PCR, when first round PCR, primer is many chimeric primers, the concentration of each chimeric primers is 1 μ M, and template is peripheral blood leucocyte cDNA template to be detected; The second primer while taking turns PCR is consensus primer, and the concentration of consensus primer is 10 μ M, and detecting template is the product of first round reaction.The fusion gene plasmid template for detection of system specificity and susceptibility having built has (BCR/ABLe1a2 type and e13a2, the L-type of e14a2 type, PML/RAR α and S type, AML1/ETO, E2A/PBX1 I type); Two-wheeled PCR adopts 25 μ L reaction systems, and other component of reaction system is: the Taq polymerase 0.15 μ L of 5U/ μ L, the MgCl of 25mM
22 μ L, the dNTP 0.5 μ L of 10mM, 10xTaqBuffer 2.5 μ L, deionized water.The reaction conditions of two-wheeled PCR is: first 95 DEG C of 5min; Secondly 95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, totally 35 circulations; Last 72 DEG C of 5min.Second takes turns PCR finishes laggard row agarose gel electrophoresis analysis, result shows BCR/ABL e1a2, the BCR/ABL e13a2 that the multiple PCR reagent kit of this detection leukemia fusion gene can the common leukemia fusion gene of specific detection, and e14a2, PML/RAR α L-type, PML/RAR α S type, E2A/PBX1 I type and AML1/ETO7 shear isomer; Utilize the plasmid template building, this detection system susceptibility reaches as high as 10 copy/25 μ L.
In multiple PCR products detects, positive control primer is for people GAPDH gene; Negative control is not add the same reaction system that detects template.
Because technique scheme is used, the present invention compared with prior art has the following advantages:
(1), the present invention discloses a kind of multiple PCR reagent kit that detects leukemia fusion gene first, can detect four kinds simultaneously and comprise 7 the highest several leukemia fusion genes of leukemia sickness rate of shearing isomer, not only can save using amount of reagent and reduce testing cost, having improved detection efficiency simultaneously.
(2), while utilizing test kit disclosed by the invention to detect, first round PCR reaction institute adds chimeric primer concentration and is only required to be every of 1 μ M/, second to take turns added consensus primer concentration be 10 μ M; Reducing chimeric primers concentration is conducive to add more multipair chimeric primers and improves multiplex PCR and detect flux.
(3), utilize multiple PCR reagent kit disclosed by the invention to carry out conventional leukemia fusion gene to detect and can avoid the multipair primer existing in prior art to interact, thereby affect the defect of detected result; Detection sensitivity of the present invention reaches as high as 10 copy/25 μ L, and simple to operate, carries out without being in charge of, and clinical recall rate is high, has clinic popularization and application values.
Brief description of the drawings
Fig. 1 is consensus primer and the genomic PCR reaction result of Healthy People electrophorogram in embodiment mono-;
Fig. 2 is the PCR reaction product electrophorogram of the plasmid template detection consensus primer susceptibility that in embodiment, two utilizations build;
Fig. 3 is the principle of work schematic diagram of consensus primer in embodiment tri-;
Fig. 4 utilizes the plasmid template having built to detect the PCR reaction product electrophorogram of body series susceptibility in embodiment tri-;
Fig. 5 utilizes the plasmid template having built to detect the specific PCR reaction product of body series electrophorogram in embodiment tetra-;
Fig. 6 is clinical detection result figure in embodiment five.
Embodiment
Below in conjunction with drawings and Examples, the invention will be further described:
Embodiment mono-:
The present embodiment carries AML1/ETO, BCR/ABL e1a2, BCR/ABL e13a2, BCR/ABL e14a2, PML/RAR α L-type, PML/RAR α S type, E2A/PBX1 I type fusion gene patient sample checking multi-primers in conjunction with the feasibility of the principle of work of consensus primer and the susceptibility of detection with clinical definite.
The multiple PCR reagent kit of the detection leukemia fusion gene that the present embodiment adopts, comprising: conventional multiplex PCR assembly, also comprises chimeric primers and consensus primer pair for common leukemia fusion gene.Consensus primer to and for the chimeric primers (with the multi-primers sequence of consensus primer) of common leukemia fusion gene and the fusion gene of corresponding detection in table 1.
Table 1
Above-mentioned conventional multiplex PCR assembly comprises: positive control primer, Taq archaeal dna polymerase, dNTP, MgCl
2, PCR damping fluid, deionization.The concrete sequence of positive control primer is: SEQ ID NO.15:AGGTCGGAGTCAACGGATTTG; SEQ ID NO.16:GTGATGGCATGGACTGTGGT.
Embodiment mono-, consensus primer specific detection
By consensus primer SEQ ID No.1 and the SEQ ID No.2 amplification Healthy People genomic dna of design, the specificity of checking consensus primer.Two principle is followed in the design of the present embodiment consensus primer: when (1) consensus primer and template to be measured increase, no matter how condition is optimized, all there is no specificity product; (2), in the time having amplified production with the multi-primers of consensus primer, necessarily can amplify specificity product by consensus primer.
25 μ L PCR reaction systems specifically comprise: two each 1 μ L of 10 μ M concentration consensus primer, the Taq polymerase 0.15 μ L of 5U/ μ L, the MgCl of 25mM
22 μ L, the dNTP 0.5 μ L of 10mM, 10xTaqBuffer 2.5 μ L, DNA profiling 1 μ L, deionized water 16.85 μ L.PCR reaction conditions is: 95 DEG C of sex change 5min; 95 DEG C of sex change 30s, 50-60 DEG C of annealing 30s, 72 DEG C are extended 30S, totally 35 circulations; Last 72 DEG C of 5min.Wherein, 1-6 tube reaction system annealing temperature is set as respectively 50 DEG C, 52 DEG C, 54 DEG C, 56 DEG C, 58 DEG C and 60 DEG C.Agarose gel electrophoresis detects 6 pipe amplified productions, accompanying drawing 1 is the reaction result electrophorogram of consensus primer and Healthy People genomic dna, 1-6 swimming lane is 50-60 DEG C of thermograde, the positive contrast of PC, M are DNA standard molecular weight, agarose gel electrophoresis result shows that consensus primer, under different annealing temperature condition, can not amplify specific band in human genome.
Embodiment bis-, consensus primer sensitivity Detection
Detect the plasmid containing consensus primer sequence of different concns by consensus primer SEQ ID No.1 and SEQ ID No.2.Dilute gradients with deionized water by 10 times by after extracting from intestinal bacteria containing the plasmid of consensus primer sequence, from 10
7copy 10 to
1copy dilution becomes the DNA cloning template of different concns plasmid template as PCR reaction system; Adopt 25 μ L PCR reaction systems: every 25 μ L PCR reaction systems comprise: the Taq polymerase 0.15 μ L of 5U/ μ L, the MgCl of 25mM
22 μ L, the dNTP 0.5 μ L of 10mM, 10xTaqBuffer 2.5 μ L, the each 1 μ L of consensus primer of two concentration 10 μ M, the plasmid template 1 μ L of different concns, deionized water 16.85 μ L; PCR reaction conditions is: 95 DEG C of sex change 5min; 95 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C are extended 30S, totally 35 circulations; Last 72 DEG C of 5min.Detect amplified production with agarose gel electrophoresis, accompanying drawing 2 be different concns plasmid template respectively with the reaction result electrophorogram of consensus primer, the 1-7 swimming lane respectively concentration of corresponding plasmid template is 10
1copy, 10
2copy is until 10
7copy, experimental result shows that this institute consensus primer susceptibility can reach 10
2copy.
Embodiment tri-, multiplex PCR system sensitivity Detection
Utilize the cDNA plasmid template of the L of the e1a2 of BCR/ABL and e14a2 type, PML/RAR α and S type, AML1/ETO, E2A/PBX1I type fusion gene, adopt multiple PCR technique, by specificity and the susceptibility of two-wheeled PCR reaction detection consensus primer and multi-primers, the primer is SEQ ID No.1-SEQ ID No.14.Its principle of work as shown in Figure 3, in first round PCR reaction, increase by chimeric primers, between the chimeric primers reduction primer of use low concentration, there is interactional probability, to reduce dimer and the non-specific amplification that may exist, obtain two ends with the product fragment of consensus primer sequence as the second template of taking turns amplification; Second to take turns PCR reaction be the product amplified fragments for the first round based on consensus primer, and object is to improve the susceptibility of whole system, can detect containing the less sample of template copy number for some.
Concrete detection method is as follows: by the plasmid template of the L of the e1a2 that contains BCR/ABL and e14a2 type, PML/RAR α and S type, AML1/ETO, E2A/PBX1I type fusion gene respectively by 10 times of dilution gradients from 10
8copy is diluted to 10
1copy.Adopt PCR reaction system identical with above-described embodiment three and reaction conditions, the plasmid template of every kind of different weaker concns is carried out to multiplex PCR system sensitivity Detection.Second takes turns pcr amplification product agarose gel electrophoresis the results are shown in accompanying drawing 4.A-f e1a2 and the e14a2 type carrier of corresponding BCR/ABL respectively in Fig. 4, the L of PML/RAR α and S type, AML1/ETO, E2A/PBX1I type, its corresponding detection sensitivity is respectively 10 copy/25 μ L, 10
2copy/25 μ L, 10
3copy/25 μ L, 10
4copy/25 μ L, 10
3copy/25 μ L, 10
4copy/25 μ L.Wherein in Fig. 4 a 1-7 swimming lane respectively the concentration of corresponding plasmid template be 10
1copy, 10
2copy is until 10
7copy, in Fig. 4 b-4f 1-8 swimming lane respectively the concentration of corresponding plasmid template be 10
8copy, 10
7copy is until 10
1copy, NC is the same reaction system that does not add template, M is DNA standard molecular weight.
Embodiment tetra-, multiplex PCR system specific detection
Concrete detection method is as follows:
When first round PCR, the primer is SEQ ID No.3-SEQ ID No.14, totally 12 chimeric primers, and 12 primer mixing are diluted to 1 μ M/ bar with deionized water.Adopt 25 μ L PCR reaction systems to comprise: 12 primer mixed diluting liquid 2 μ L of 1 μ M/ bar, the cDNA plasmid template 1 μ L of 65ng/ μ L fusion gene, the Taq polymerase 0.25 μ L of 5U/ μ L, the MgCl of 25mM
22 μ L, the dNTP 0.5 μ L of 10mM, 10xTaqBuffer 2.5 μ L, deionized water 16.75 μ L.Reaction conditions is: 95 DEG C of sex change 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C are extended 30S, totally 35 circulations; Last 72 DEG C of 5min; The second primer while taking turns PCR is consensus primer SEQ ID No.1 and SEQ ID No.2, and template used is first round PCR reaction product.Adopt 25 μ L PCR reaction systems, comprising: 1 μ L first round PCR reaction product, the Taq polymerase 0.25 μ L of 5U/ μ L, the MgCl of 25mM
22 μ L, the dNTP 0.5 μ L of 10mM, 10xTaqBuffer 2.5 μ L, a pair of 10 μ M consensus primers respectively add 1 μ L, deionized water 16.75 μ L.Reaction conditions is: 95 DEG C of sex change 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C are extended 30S, totally 35 circulations; Last 72 DEG C of 5min.The amplified production of PCR is taken turns in agarose gel electrophoresis detection second, detected result is shown in accompanying drawing 5, wherein 1-6 swimming lane is followed successively by BCR/ABL e1a2, BCR/ABL e14a2, E2A/PBX1 I type, PML/RAR α L-type, PML/RAR α S type, AML1/ETO, the negative contrast of NC, M is DNA standard molecular weight, product band size is respectively 434bp, 330bp, 397bp, 540bp, 615bp, 490bp as seen from the figure, utilizes this system amplification to differentiate and distinguishes various shearing isomer.
Embodiment five, clinical samples detect
Gather the clinical 7 kinds of leukemia isomer peripheral blood in patients that 25 examples detect containing this detection kit of having made a definite diagnosis, extract total RNA according to TRIzol single stage method and chloroform-isopropanol method.Reverse transcription reaction is totally 40 μ L, comprising: total RNA 4 μ L, and the 6 random primer 2 μ L of 100 μ M, 5xReaction Buffer 8 μ L, 10mM dNTP 2 μ L, the M-MuLV 1 μ L of 200 U/ μ L, the RNasin 1 μ L of 20U/ μ L, all the other are DEPC water.37 DEG C of 45min, 95 DEG C of 5min ,-20 DEG C of cDNA templates that preservation obtains, for subsequent use.
Utilize multiple PCR reagent kit disclosed by the invention to detect, be specifically divided into two-wheeled PCR, when first round PCR, primer is SEQ ID No.3-SEQ ID No.14, totally 12 chimeric primers, and 12 primer mixed dilutings become 1 μ M/ bar.
The first round 25 μ L PCR reaction system comprises: above-mentioned 12 primer mixed diluting liquid 2 μ L of 1 μ M/ bar, 1 μ L cDNA template, the Taq polymerase 0.25 μ L of 5U/ μ L, the MgCl of 25mM
22 μ L, the dNTP 0.5 μ L of 10mM, 10xTaqBuffer 2.5 μ L, deionized water 16.75 μ L; PCR reaction conditions is: 95 DEG C of sex change 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C are extended 30S, totally 35 circulations; Last 72 DEG C of 5min.
The second primer while taking turns PCR is consensus primer SEQ ID No.1 and SEQ ID No.2, and template used is first round PCR reaction product.25 μ L PCR reaction systems comprise: 1 μ L first round PCR reaction product, the Taq polymerase 0.25 μ L of 5U/ μ L, the MgCl of 25mM
22 μ L, the dNTP 0.5 μ L of 10mM, 10xTaqBuffer 2.5 μ L, a pair of 10 μ M consensus primers respectively add 1 μ L, deionized water 16.75 μ L; Reaction conditions is: 95 DEG C of sex change 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C are extended 30S, totally 35 circulations; Last 72 DEG C of 5min.
Detect the above-mentioned second amplified production of taking turns PCR with agarose gel electrophoresis, accompanying drawing 6 is 7 kinds of isomer electrophoresis result that this multiplex PCR detects.In figure, 1-5 swimming lane is followed successively by BCR/ABL e1a2, BCR/ABL e13/e14a2, PML/RAR α L-type, PML/RAR α S type, AML1/ETO, and 6 swimming lanes are E2A/PBX1I type, and 7 swimming lanes are BCR/ABL e14a2, the negative contrast of NC; M is standard molecular weight.Table 2 is the detected result comparison that clinical detection method and the present embodiment method detect clinical positive sample type and amount detection, clinical detection method is with reference to Niels Pallisgaard et al. Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Screening of 29 Translocations and Chromosomal Aberrations in Acute Leukemia. Blood, (1998) 92,574-588.Fig. 6 and table 2 result show, utilizes the multiple PCR reagent kit of detection leukemia fusion gene disclosed by the invention to detect specificity and the verification and measurement ratio of leukemia fusion gene consistent with the result of clinical detection, and the present invention can be used for clinical detection.
Above result shows that the present invention can detect four kinds simultaneously and comprise 7 the highest leukemia fusion genes of leukemia sickness rate of shearing isomer, for BCR/ABL e1a2, e13a2 and e14a2 type, PML/RAR α L-type and S type, E2A/PBX1 and AML1/ETO fusion gene, not only can save using amount of reagent and reduce testing cost, having improved detection efficiency simultaneously.In addition, detection sensitivity of the present invention reaches as high as 10 copy/25 μ L, and simple to operate, and clinical recall rate is high, has clinic popularization and application values.
Table 2 clinical detection method and the inventive method detect clinical positive sample type and amount detection
SEQUENCE LISTING
<110> University Of Suzhou
Mono-kind of <120> detects the multiple PCR reagent kit of leukemia fusion gene
<160> 16
<210> 1
<211> 20
<212> DNA
<213> synthetic
<400> 1
AGACATCGTAGGTAGTGACA 20
<210> 2
<211> 20
<212> DNA
<213> synthetic
<400> 2
GAACGGACGACAATACAGTG 20
<210> 3
<211> 40
<212> DNA
<213> synthetic
<400> 3
AGACATCGTAGGTAGTGACATCCCTCGCAGAACTCGCAAC 40
<210> 4
<211> 43
<212> DNA
<213> synthetic
<400> 4
GAACGGACGACAATACAGTGACACCATTCCCCATTGTGATTAT 43
<210> 5
<211> 42
<212> DNA
<213> synthetic
<400> 5
AGACATCGTAGGTAGTGACAAGATGCTGACCAACTCGTGTGT 42
<210> 6
<211> 43
<212> DNA
<213> synthetic
<400> 6
GAACGGACGACAATACAGTGACACCATTCCCCATTGTGATTAT 43
<210> 7
<211>39
<212> DNA
<213> synthetic
<400> 7
AGACATCGTAGGTAGTGACACAAGAAAGCCAGCCCAGAG 39
<210> 8
<211> 40
<212> DNA
<213> synthetic
<400> 8
GAACGGACGACAATACAGTGTGACCCCATAGTGGTAGCCT 40
<210> 9
<211> 36
<212> DNA
<213> synthetic
<400> 9
AGACATCGTAGGTAGTGACAGCAGGAGCAGGTAGTG 36
<210> 10
<211> 40
<212> DNA
<213> synthetic
<400> 10
GAACGGACGACAATACAGTGTGACCCCATAGTGGTAGCCT 40
<210> 11
<211> 40
<212> DNA
<213> synthetic
<400>11
AGACATCGTAGGTAGTGACATGGCTGGCAATGATGAAAAC 40
<210>12
<211> 38
<212> DNA
<213> synthetic
<400> 12
GAACGGACGACAATACAGTGAGGAGGAGGAAGAAGAGG 38
<210>13
<211> 39
<212> DNA
<213> synthetic
<400> 13
AGACATCGTAGGTAGTGACAGGCCTGCAGAGTAAGATAG 39
<210>14
<211> 41
<212> DNA
<213> synthetic
<400> 14
GAACGGACGACAATACAGTGCATGTTGTCCAGCCGCATCAG 41
<210>15
<211> 21
<212> DNA
<213> synthetic
<400> 15
AGGTCGGAGTCAACGGATTTG 21
<210>16
<211> 20
<212> DNA
<213> synthetic
<400> 16
GTGATGGCATGGACTGTGGT 20
Claims (4)
1. one kind is detected the multiple PCR reagent kit of leukemia fusion gene, described multiple PCR reagent kit comprises: conventional multiplex PCR assembly, it is characterized in that, also comprise chimeric primers and consensus primer pair for common leukemia fusion gene, described consensus primer is to comprising following nucleotide sequence:
SEQ ID NO.1: the sense primer 5 '-AGACATCGTAGGTAGTGACA-3 ' of consensus primer centering; SEQ IDNO.2: the antisense primer 5 '-GAACGGACGACAATACAGTG-3 ' of consensus primer centering;
The described chimeric primers for common leukemia fusion gene is many chimeric primers pair for conventional leukemia fusion gene BCR/ABL, PML/RAR α, AML1/ETO, E2A/PBX1 respectively, specifically as shown in following nucleotide sequence:
。
2. the multiple PCR reagent kit that detects according to claim 1 leukemia fusion gene, is characterized in that, described conventional multiplex PCR assembly comprises: positive control primer, Taq archaeal dna polymerase, dNTP, MgCl
2, PCR damping fluid, deionized water.
3. the multiple PCR reagent kit that detects according to claim 1 leukemia fusion gene, is characterized in that, while carrying out PCR reaction, every concentration for the chimeric primers of common leukemia fusion gene is 1 μ M.
4. the multiple PCR reagent kit that detects according to claim 1 leukemia fusion gene, is characterized in that, while carrying out PCR reaction, the concentration of consensus primer is 10 μ M.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410153342.7A CN103937891B (en) | 2014-04-16 | 2014-04-16 | A kind of multiple PCR reagent kit detecting leukemia fusion gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410153342.7A CN103937891B (en) | 2014-04-16 | 2014-04-16 | A kind of multiple PCR reagent kit detecting leukemia fusion gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103937891A true CN103937891A (en) | 2014-07-23 |
| CN103937891B CN103937891B (en) | 2015-11-04 |
Family
ID=51185758
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410153342.7A Expired - Fee Related CN103937891B (en) | 2014-04-16 | 2014-04-16 | A kind of multiple PCR reagent kit detecting leukemia fusion gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103937891B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293962A (en) * | 2014-10-20 | 2015-01-21 | 苏州大学 | Method for screening general primers |
| CN104561331A (en) * | 2015-01-21 | 2015-04-29 | 苏州云泰生物医药科技有限公司 | Primer and probe for detecting leukemia-related fusion gene and kit of primer |
| CN105483117A (en) * | 2015-12-11 | 2016-04-13 | 广州医科大学附属肿瘤医院 | PCR (polymerase chain reaction) specificity improving method |
| CN106544437A (en) * | 2016-11-25 | 2017-03-29 | 徐州医科大学 | A kind of multiple fluorescence PCR test kit and method of detection leukemia fusion gene |
| CN108456726A (en) * | 2018-04-19 | 2018-08-28 | 深圳会众生物技术有限公司 | Spinal muscular atrophy genetic test probe, primer and kit |
| WO2022007980A1 (en) * | 2020-07-10 | 2022-01-13 | 北京工业大学 | Combination waste light emitting diode packaging material pyrolysis treatment and method of recovery of rare earth elements from phosphor powder |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102352411A (en) * | 2011-09-29 | 2012-02-15 | 苏州大学 | Multiplex polymerase chain reaction (PCR) amplification method and reagent kit |
| CN102676638A (en) * | 2011-03-08 | 2012-09-19 | 苏州大学附属第一医院 | Method and kit for detecting drug-resistance mutation site of ABL kinase domain of BCR/ABL fusion gene |
-
2014
- 2014-04-16 CN CN201410153342.7A patent/CN103937891B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676638A (en) * | 2011-03-08 | 2012-09-19 | 苏州大学附属第一医院 | Method and kit for detecting drug-resistance mutation site of ABL kinase domain of BCR/ABL fusion gene |
| CN102352411A (en) * | 2011-09-29 | 2012-02-15 | 苏州大学 | Multiplex polymerase chain reaction (PCR) amplification method and reagent kit |
Non-Patent Citations (2)
| Title |
|---|
| HANY ARIFFIN ET AL: "Validation of a Multiplex RT-PCR Assay for Screening Significant oncogene fusion transcripts in children with Acute Lymphoblastic Leukaemia", 《SINGAPORE MED J》, vol. 44, no. 10, 31 December 2003 (2003-12-31) * |
| 李志刚等: "多重RT-PCR方法同时检测29种白血病融合基因", 《中华血液学杂志》, vol. 24, no. 5, 30 May 2003 (2003-05-30), pages 257 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293962A (en) * | 2014-10-20 | 2015-01-21 | 苏州大学 | Method for screening general primers |
| CN104293962B (en) * | 2014-10-20 | 2017-01-11 | 苏州大学 | Method for screening general primers |
| CN104561331A (en) * | 2015-01-21 | 2015-04-29 | 苏州云泰生物医药科技有限公司 | Primer and probe for detecting leukemia-related fusion gene and kit of primer |
| CN104561331B (en) * | 2015-01-21 | 2016-08-31 | 苏州云泰生物医药科技有限公司 | A kind of primer detecting leukaemia correlation fusion gene and probe and kit thereof |
| CN105483117A (en) * | 2015-12-11 | 2016-04-13 | 广州医科大学附属肿瘤医院 | PCR (polymerase chain reaction) specificity improving method |
| CN105483117B (en) * | 2015-12-11 | 2019-04-30 | 广州医科大学附属肿瘤医院 | A method of improving polymerase chain reaction specificity |
| CN106544437A (en) * | 2016-11-25 | 2017-03-29 | 徐州医科大学 | A kind of multiple fluorescence PCR test kit and method of detection leukemia fusion gene |
| CN106544437B (en) * | 2016-11-25 | 2019-11-19 | 徐州医科大学 | A kind of multiple fluorescence PCR kit detecting leukemia fusion gene |
| CN108456726A (en) * | 2018-04-19 | 2018-08-28 | 深圳会众生物技术有限公司 | Spinal muscular atrophy genetic test probe, primer and kit |
| WO2022007980A1 (en) * | 2020-07-10 | 2022-01-13 | 北京工业大学 | Combination waste light emitting diode packaging material pyrolysis treatment and method of recovery of rare earth elements from phosphor powder |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103937891B (en) | 2015-11-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103937891B (en) | A kind of multiple PCR reagent kit detecting leukemia fusion gene | |
| JP5750710B2 (en) | Cancer marker, cancer evaluation method and evaluation reagent using the same | |
| CN102352411B (en) | Multiplex polymerase chain reaction (PCR) amplification method and reagent kit | |
| Tesorero et al. | Novel regulatory small RNAs in Streptococcus pyogenes | |
| Yuhong et al. | Identification and characterization of immune-related microRNAs in blunt snout bream, Megalobrama amblycephala | |
| JP6970732B2 (en) | RNA detection kit and method | |
| CN110004234B (en) | Chicken anti-salmonella infection related miRNA and application thereof | |
| Liu et al. | The early response expression profiles of miRNA-mRNA in farmed yellow catfish (Pelteobagrus fulvidraco) challenged with Edwardsiella tarda infection | |
| CN102965427A (en) | Establishment of female genital cancer relevant SNP sites multiple detection method | |
| Supadmanaba et al. | Interrelationship between miRNA and splicing factors in pancreatic ductal adenocarcinoma | |
| CN107177676B (en) | Use of long-chain non-coding RNA NONHSAT113026 as molecular marker for diagnosing kidney cancer | |
| CN108315321A (en) | The detection of K-ras gene mutation sites in urine ctDNA | |
| US20150247202A1 (en) | Microrna based method for diagnosis of colorectal tumors and of metastasis | |
| CN103882140A (en) | Rapid diagnosis kit for mycobacterium tuberculosis | |
| CN116083579A (en) | circRNA-miRNA-mRNA regulation network for non-small cell lung cancer diagnosis and application thereof | |
| CN102936619B (en) | Method for quantitative determination of escherichia coli RNA, and specialized standard substance and application thereof | |
| CN108315322A (en) | The detection in EGFR genetic mutation site in urine ctDNA | |
| CN102925590B (en) | Detection primer and method for detecting bombyx mori cytoplasmic polyhedrosis virus (BmCPV) | |
| Aljawadi et al. | MicroRNAs (20a, 146a, 155, and 145) expressions in a sample of Iraqi patients with multiple sclerosis | |
| Kaboré et al. | Macrophage migrating inhibitory factor expression is associated with Trypanosoma brucei gambiense infection and is controlled by trans-acting expression quantitative trait loci in the Guinean population | |
| CN111471769A (en) | Method for amplifying circular RNA, specific amplification primer and application | |
| CN102747166A (en) | PCR-SNP (Polymerase Chain Reaction-Single Nucleotide Polymorphism) detection method of mycoplasma pneumonia drug resistant strain | |
| CN109337973A (en) | A kind of primer design method, primer, kit, method and system copying number variation for verifying specific DNA fragments | |
| Wu et al. | MicroRNA-301a inhibits the progression of osteosarcoma by regulating DEC2 | |
| CN103103274A (en) | Fluorescent quantitative polymerase chain reaction (PCR) detection method for HSP90 (heat shock protein 90) gene of miichthys miiuy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151104 Termination date: 20200416 |