CN103571945B - Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types - Google Patents
Method, primer, probe and kit for screening and identifying BCR-ABL unusual fusion types Download PDFInfo
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Abstract
The invention discloses a method, primer, probe and kit for detecting six unusual fusion types of BCR-ABL fusion genes such as e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3. The method comprises the following steps: performing primary screening on blood samples by adopting two tubes of fluorescent quantitative polymerase chain reaction (PCR), and quantitatively indentifying specific types of the blood samples belonging to the a2 or a3 group through primary screening judgment. Therefore, the screening cost is reduced, and the detection throughput is also improved. According to the detection method, primer, probe and kit, high sensitivity, high specificity and high detection throughput can be realized, and the method is a rapid and accurate type screening and identifying method.
Description
Technical field
The invention belongs to life science and biological technical field, in particular to the non-common pattern of fusion method for distinguishing of a kind of Clinical screening BCR-ABL fusion gene, primer, probe and test kit, adopt Real-Time Fluorescent Quantitative PCR Technique, preliminary examination and type qualification are carried out to these the 6 kinds non-common types of BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3, e14a3 in human blood sample.
Background technology
BCR-ABL fusion gene is by No. 9 chromosome long arm transposition to 22 chromosome long arm, causes ABL proto-oncogene and BCR gene to occur to merge and produces.T (9; 22) (q34; Q11) No. 22 karyomit(e)s after transposition are also called Philadelphia chromosome (Ph karyomit(e)).Ph karyomit(e) and BCR-ABL fusion gene are the molecular basises that chronic myelocytic leukemia (Chronic Myeloid Leukemia, CML) produces.In addition, BCR-ABL fusion gene is also shown in acute lymphoblastic leukemia (AcuteLymphoblastic Leukemia, ALL), rare in acute myeloid leukemia (Acute Myeloblastic Leukemia, AML).
When No. 9 karyomit(e)s and No. 22 karyomit(e) generation transpositions, the fracture position on No. 9 karyomit(e)s is usually located on the 1st intron of abl gene, is secondly intron 2; Fracture position on No. 22 karyomit(e)s then can be positioned on multiple sections of BCR gene, therefore can form multiple fusion type.In various fusion type, have three kinds to be modal, be respectively: e13a2(is also called b2a2), e14a2(is also called b3a2) and e1a2; It is exactly the BCR-ABL P210 often said that first two merges type, and the latter is often called BCR-ABL P190.In addition, report that more non-common type then has: e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3.In CML patient, BCR-ABL e13a2 and e14a2 two kinds of pattern of fusion account for altogether ~ and 98%; But not common fusion type at least accounts for 1-2%, even more.In adult and children ALL patient, BCR-ABL positive rate is respectively ~ and 25% and ~ 3%; This wherein e13a2, e14a2 and e1a2 account for altogether ~ 99%; But not common fusion type also at least accounts for 1-2%.
At present, clinical in doing the examination of BCR-ABL fusion gene to doubtful CML, ALL and AML patient, usually only make detection for e13a2, e14a2 and e1a2 tri-kinds of types, other non-common types are then chance on by when chromosome karyotype analysis or Fluorescence in situ hybridization (FISH) technical inspection.There is following shortcoming in this examination mode judged by visual inspection: (1) examination to the non-common type of BCR-ABL fusion gene exists undetected situation.First, the prerequisite of chromosome karyotype analysis need be cultivated the karyocyte in sample, has mitosis figures; If cell cultures failure, without mitosis figures, then cannot analyze.Secondly, in cell cultures breeding, certain class cell meeting hypertrophy, if normal cell hypertrophy, then can " flood " by sick cell, cause the normal illusion of karyotype.And when cannot bone marrow extraction, extract peripheral blood do chromosome karyotype analysis, also have false negative occur.Moreover the chromosomal transposition of some case is extremely small, occur invisible Ph karyomit(e), this cannot find in the chromosome examination being undertaken analyzing by visual inspection.And during FISH method detection BCR-ABL fusion, although without culturing cell, also can detect invisible Ph karyomit(e), its inspection charge is too high, and most of patient can not select the method to detect, therefore does not have the effect of real examination.(2) the concrete type of BCR-ABL cannot be distinguished, thus just cannot carry out the monitoring of MRD (Minimal Residua Disease, MRD) targetedly.Chromosome karyotype analysis is identified chromosomal transposition by naked eyes; Equally, FISH is by fluorescent signal, and observe the fusion of coloured differently body, submicroscopic change cannot be observed.(3) power is detected as and sensitivity is low.
At present, even if identify the non-common type of BCR-ABL fusion gene targetedly, also be first by carrying out multiplex PCR (m μ ltiplex PCR) or nest-type PRC (nested PCR) method increases, then in conjunction with agarose gel electrophoresis or order-checking.The shortcoming that multiplex PCR carries out examination in conjunction with agarose gel electrophoresis or order-checking is as follows: (1) exists non-specific amplification, there will be many PCR primer bands, easily occur false positive during electrophoresis; (2) when being analyzed by electrophoresis, result judges to there is subjectivity, and particularly when the monitoring to MRD, Critical Result cannot carry out clear and definite analysis and judge, and can be increased to causing cost by order-checking, and the time cycle greatly extends; (3) sensitivity is lower than nest-type PRC and fluorescent PCR; (4) qualitative detection can only be carried out; (5) length consuming time, if need 5-6 hour by electroresis appraisal PCR primer; If by order-checking qualification, then need 10-12 hour.And nest-type PRC carries out examination in conjunction with agarose gel electrophoresis or order-checking also there is following shortcoming: (1) process is loaded down with trivial details, easily pollutes, and during electroresis appraisal, the judgement of Critical Result is more subjective; (2) PCR reaction system is many, and cost is high, is not suitable for high-throughout pattern detection; (3) qualitative detection can only be carried out; (4) length consuming time, if need 5-6 hour by electroresis appraisal PCR primer; If by order-checking qualification, then need 10-12 hour.Therefore be no matter first by carrying out multiplex PCR (m μ ltiplex PCR) or nest-type PRC (nested PCR) method increases, the non-common type of BCR-ABL fusion gene is identified in recycling agarose gel electrophoresis or order-checking, all can not meet highly sensitive and that specificity is good advantage simultaneously.
Summary of the invention
In view of the deficiency of the non-common type method of current examination BCR-ABL fusion gene, the present invention designs for detecting the non-common pattern of fusion method for distinguishing of BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 these 6 kinds, primer, probe and test kit, thus can in high sensitivity, with high specificity, high-throughput ground and examination rapidly and accurately and qualification BCR-ABL fusion gene non-common type.
The object of the present invention is to provide for non-other primer of common pattern of fusion of examination BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 these 6 kinds and probe, it is characterized in that, the nucleotide sequence of described primer and probe is as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
E1F：5’-CTCGCAGAACTCGCAACAG-3’
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
Further, described primer and probe also comprise for increasing as the primer of the house-keeping gene ABL of internal reference and probe, and its nucleotide sequence is as follows:
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
Present invention also offers for the identification of non-other primer of common pattern of fusion of BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 these 6 kinds and probe, it is characterized in that,
The primer of qualification e6a2 type and the nucleotide sequence of probe as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
The primer of qualification e8a2 type and the nucleotide sequence of probe as follows:
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
The primer of qualification e19a2 type and the nucleotide sequence of probe as follows:
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
The primer of qualification e1a3 type and the nucleotide sequence of probe as follows:
E1F：5’-CTCGCAGAACTCGCAACAG-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
The primer of qualification e13a3 type and the nucleotide sequence of probe as follows:
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
The primer of qualification e14a3 type and the nucleotide sequence of probe as follows:
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
Further, identify that the nucleotide sequence of primer and probe being selected from any one type in e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 type also comprises amplification as the primer of the house-keeping gene ABL of internal reference and probe, its nucleotide sequence is as follows:
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’
Present invention also offers a kind of examination and qualification BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 these 6 kinds non-common pattern of fusion method for distinguishing, it is characterized in that, said method comprising the steps of:
(1) RNA in blood sample is extracted;
(2) by the RNA reverse transcription cDNA in step (1);
(3) utilize the cDNA described in fluorescent PCR amplification step (2), carry out described other preliminary examination of non-common pattern of fusion;
(4) if pass through the preliminary examination of step (3), determine that described blood sample is not that described non-common pattern of fusion is other, then detect end; If by the preliminary examination of step (3), determine that described blood sample is that described non-common pattern of fusion is other, then carry out concrete type Quantitative measurement by fluorescent PCR amplification again.
Further, utilizing the preliminary examination of described step (3) to judge, described blood preparation belongs to a2 group, a3 group or be not that described non-common pattern of fusion is other.
Further, be classified as the amplimer of described step (3) and the nucleotides sequence of probe:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
E1F：5’-CTCGCAGAACTCGCAACAG-3’
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
Further, according to the judged result in step (3), if a2 group, then carry out the qualification of e6a2, e8a2, e19a2 type; If a3 group, then carry out the qualification of e1a3, e13a3, e14a3 type.
Further, in described step (4), for carrying out the concrete primer of type Quantitative measurement by the amplification of described fluorescent PCR and the nucleotides sequence of probe is classified as:
The primer of qualification e6a2 type and the nucleotide sequence of probe as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
The primer of qualification e8a2 type and the nucleotide sequence of probe as follows:
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
The primer of qualification e19a2 type and the nucleotide sequence of probe as follows:
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
The primer of qualification e1a3 type and the nucleotide sequence of probe as follows:
E1F：5’-CTCGCAGAACTCGCAACAG-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
The primer of qualification e13a3 type and the nucleotide sequence of probe as follows:
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
The primer of qualification e14a3 type and the nucleotide sequence of probe as follows:
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
Present invention also offers a kind of examination and qualification BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 these 6 kinds non-other test kits of common pattern of fusion, comprise blood rna extracting solution, reverse transcription reagents, pcr amplification reaction system, described pcr amplification reaction system comprises primer and probe, it is characterized in that, the nucleotide sequence of described primer and probe is as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
E1F：5’-CTCGCAGAACTCGCAACAG-3’
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
Because non-other incidence of common pattern of fusion of BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 these 6 kinds is low, therefore the present invention is by the design optimization of primed probe, two pipe PCR reactions are taked first to carry out blood sample primary dcreening operation by the method for multi-primers combined with fluorescent PCR, again the blood preparation belonging to a2 group or a3 group is carried out to the method for concrete type Quantitative measurement, which reduce the cost of examination, turn improve the flux of detection.
The design of primed probe of the present invention and being grouped as follows of examination system: according to BCR-ABL in fusion process, the relatively-stationary feature of fracture position on abl gene, examination system is divided into two groups: a2 group (fracture position is positioned at abl gene the 1st intron), by e6a2, e8a2, e19a2 type forms; A3 group (fracture position is positioned at abl gene intron 2), is made up of e1a3, e13a3, e14a3 type.Meanwhile, the exon 2 of abl gene designs downstream primer and the fluorescent probe of a2 group; 3rd exon designs downstream primer and the fluorescent probe of a3 group.Subsequently, according to downstream primer design be suitable for it carrying out PCR reaction upstream primer.The upstream primer of a2 group lays respectively at the 6th exon of BCR gene, the 8th exon and the 19th exon; Combine with downstream primer like this and just in time can detect e6a2, e8a2, e19a2 tri-kinds of types.In like manner, the upstream primer of a3 group lays respectively at the 1st exon of BCR gene, the 13rd exon and the 14th exon, with downstream primer combine detection e1a3, e13a3 and e14a3 tri-kinds of types.This design three upstream primers share the matching method of a downstream primer and probe, decrease in a tube reaction system situation mutually suppressing owing to there are many primed probe to disturb, both save sensitivity and specificity that cost turn increases detection system.No matter be in a2 group or in a3 group simultaneously, owing to being shared often organizing interior downstream primer, so the principle of first design with different other upstream primer of pattern of fusion in the same group of total downstream primer bamboo product of group should be followed, if not so design is in advance with other its downstream primer of upstream primer bamboo product of the pattern of fusion of in group, then other its downstream primer of upstream primer bamboo product of another pattern of fusion in same group is designed, the phenomenon that the downstream primer of very possible generation twice design is inconsistent, and cause downstream primer to redesign, thus bring great time waste.
According to statistics, these 6 kinds non-common pattern of fusion not in, the incidence of any one type is probably at 1-2%, thus easy to be out in the cold and undetected in clinical detection.According to utilizing multiple fluorescence PCR to carry out examination in single tube, the fluorophor that the probe for each type adopts is necessarily different, and this not only can increase testing cost, then sensitivity, specificity can not meet clinical requirement, and result reliability is also poor.If identify separately each type, then need to carry out six pipe detections, simultaneously also in view of these 6 kinds non-common pattern of fusion not in, the incidence of any one type is only about 1-2%, this just means in 1000 parts of blood samples, at least by 980 increment product do not belong to these 6 kinds non-common pattern of fusion not in any one, if all carry out six pipe detections to these at least 980 increment product, so not only to consume the more blood sample sample of patient, also greatly increase reagent and human cost simultaneously.The present invention utilizes the array mode of corresponding many upstream primers of a downstream primer probe, quantitative typing is carried out after first carrying out qualitatively screening, the reaction system of primary dcreening operation inspection is tapered to two pipes, so just in the primary dcreening operation stage, above-mentioned at least 980 increment product can be excluded, just need not carry out follow-up detection by quantitative and somatotype again, therefore this has not only saved cost, turn increases a batch of flux detected, for high-throughout blood sample screening provides convenient.Moreover the probe for each type can share identical fluorophor, also help and reduce reagent cost of development, for ease of the exploitation of product.In addition, this array mode is combined with fluorescent quantitative PCR technique, avoid the reaction of uncapping of nest-type PRC, improve the accuracy of result, avoid the generation of polluting; Also improve the easy judgement of result, make the more objective readability of detected result.Finally, ABL typical curve is introduced when concrete type qualification, both somatotype had been carried out, to carry out again quantitatively, be conducive to clinically in conjunction with generation development and the prognosis of the result comprehensive assessment diseases such as bone marrow examination, chromosome karyotype analysis, immunophenotyping, carry out MRD monitoring targetedly, predicting recurrence risk simultaneously.
Accompanying drawing explanation
Fig. 1 is primer of the present invention and probe design site plan
Fig. 2 uses primer of the present invention, probe and detection method to carry out the amplified fluorescence graphic representation of examination and type qualification to a blood sample to be checked
Fig. 3 uses primer of the present invention, probe and detection method to carry out the amplified fluorescence graphic representation of examination and type qualification to second blood sample to be checked
Fig. 4 uses primer of the present invention, probe and detection method to carry out the amplified fluorescence graphic representation of examination and type qualification to the 3rd blood sample to be checked
Fig. 5 uses primer of the present invention, probe and detection method to carry out the amplified fluorescence graphic representation of examination and type qualification to the 4th blood sample to be checked
Fig. 6 uses primer of the present invention, probe and detection method to carry out the amplified fluorescence graphic representation of examination and type qualification to the 5th blood sample to be checked
Fig. 7 uses primer of the present invention, probe and detection method to carry out the amplified fluorescence graphic representation of examination and type qualification to the 6th sample to be checked
Fig. 8 is the amplified fluorescence graphic representation using primer of the present invention, probe and detection method the 7th blood sample to be checked to be carried out to examination
Embodiment
Embodiment 1:
The extraction of blood rna: add 1ml1 × erythrocyte cracked liquid in 1.5ml centrifuge tube, gets blood sample 0.5ml to be checked, puts upside down mixing.Centrifugal 4000rpm, 3min, inhale and abandon supernatant, adds erythrocyte cracked liquid washing once, obtain required cell; Add l ml TotalRNA Isolation Reagent (Shanghai Pu Fei Bioisystech Co., Ltd), pressure-vaccum is until without obvious cell mass repeatedly, adds chloroform 200 μ l, and whirlpool mixing 30s, leaves standstill 10min on ice.Then, 4 DEG C centrifugal 14,000rpm, 10min.Be transferred in another centrifuge tube with pipettor Aspirate supernatant 450 μ l, add isopyknic pre-cold isopropanol, after putting upside down mixing, leaving standstill 10min on ice.4 DEG C centrifugal 14,000rpm, 10min.Then with 75% ethanol and dehydrated alcohol wash once centrifugal respectively.Drying at room temperature 5min, adds 50 μ l DEPC-H2O and dissolves, and obtains RNA extracting solution.
10 × erythrocyte splitting liquid formula is: NH
4cl82g, NaHCO
38.4g, EDTA-Na
23.72g, adds ddH
2o is settled to 1000ml.
Embodiment 2:
Reverse transcription: the RNA extracting solution 4 μ l(concentration in Example 1 is about 200ng/ μ l) add 1 μ l Primer mix(ReverTra AceqPCR RT Kit, spin (Shanghai) bio tech ltd purchased from Japan) and 3 μ l DEPC-H2O mix, 70 DEG C of denaturation 5min; 4 μ l5*RT buffer(ReverTra Ace qPCR RT Kit are added on ice after quenching 1min), 1 μ l Enzyme Mix(ReverTra Ace qPCR RT Kit), and add 7 μ l DEPC-H
20 to cumulative volume is 20 μ l.37 DEG C of 60min react rear 98 DEG C of 5min deactivations, and gained is the cDNA of blood sample to be checked.
Embodiment 3:
Fluorescent PCR primary dcreening operation: according to each material as shown in table 1 and consumption configuration preliminary screening agent.Preliminary screening agent is altogether containing 3 reaction tubess: each pipe of a2 group, a3 group and ABL; Wherein ABL is internal reference detector tube, for judging that RNA extracts quality and whether meets the requirements.Add each 2 μ l of gained cDNA in embodiment 2.Detect by following program: 95 DEG C of denaturation 30s; 95 DEG C of 10s, 58 DEG C of 35s, totally 40 circulations; Fluorescence is gathered when 58 DEG C.HUNDERBIRD Probe qPCR Mix spins (Shanghai) bio tech ltd purchased from Japan.
Table 1
Embodiment 4:
First blood sample to be checked is carried out RNA extraction by the method described in embodiment 1-3, and obtain cDNA and fluorescent PCR primary dcreening operation, primary dcreening operation shows first blood sample to be checked and belongs to a2 group.Acquired results is as shown in Fig. 2-A.Then each type of a2 group to be identified respectively and quantitatively.Reagent configuration is as shown in table 2, and adds each 2 μ l of obtained cDNA.Trace routine is with embodiment 3.Through identifying that first blood sample to be checked is BCR-ABL fusion gene e8a2 type, as shown in fig. 2-b, calculate its relative quantification result (BCR-ABL e8a2/ABL) according to inspection software is 161.81% to acquired results.Finally this sample is carried out sequence verification, be defined as BCR-ABL fusion gene e8a2 type.
Table 2
Embodiment 5:
Carry out RNA extraction to second blood sample to be checked by described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Fig. 3-A: primary dcreening operation shows second blood sample to be checked and belongs to a3 group.Again each type of a3 group to be identified respectively and quantitatively.Reagent configuration is as shown in table 3, and adds each 2 μ l of obtained cDNA.Trace routine is with embodiment 3.Acquired results is as shown in Fig. 3-B: through identifying that first blood sample to be checked is BCR-ABL fusion gene e1a3 type, calculating its relative quantification result (BCR-ABL e1a3/ABL) according to inspection software is 50.97%.This sample is carried out order-checking detection validation, is defined as BCR-ABL fusion gene e1a3 type.
Table 3
Embodiment 6:
Carry out RNA extraction to the 3rd blood sample to be checked by described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Fig. 4-A: primary dcreening operation shows the 3rd blood sample to be checked and belongs to a3 group.Again each type of a3 group to be identified respectively and quantitatively.Reagent configuration and trace routine are with embodiment 5.Acquired results is as shown in Fig. 4-B: through identifying that the 3rd blood sample to be checked is BCR-ABL fusion gene e13a3 type, calculating its relative quantification result (BCR-ABL e13a3/ABL) according to inspection software is 384.62%.This sample is carried out order-checking detection validation, is defined as BCR-ABL fusion gene e13a3 type.
Embodiment 7:
By described in embodiment 1-3, RNA extraction is carried out to the 4th blood sample to be checked, obtains cDNA and fluorescent PCR primary dcreening operation, acquired results as shown in fig. 5-A: primary dcreening operation shows the 4th blood sample to be checked and belongs to a2 group.Again each type of a2 group to be identified respectively and quantitatively.Reagent configuration and trace routine are with embodiment 4.Acquired results is as shown in fig. 5-b: through identifying that the 4th blood sample to be checked is BCR-ABL fusion gene e19a2 type, calculating its relative quantification result (BCR-ABL e19a2/ABL) according to inspection software is 70.88%.This sample is carried out order-checking detection validation, is defined as BCR-ABL fusion gene e19a2 type.
Embodiment 8:
Carry out RNA extraction to the 5th blood sample to be checked by described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Fig. 6-A: primary dcreening operation shows the 5th blood sample to be checked and belongs to a2 group.Again each type of a2 group to be identified respectively and quantitatively.Reagent configuration and trace routine are with embodiment 4.Acquired results is as shown in figure 6-b: through identifying that the 5th blood sample to be checked is BCR-ABL fusion gene e6a2 type, calculating its relative quantification result (BCR-ABL e6a2/ABL) according to inspection software is 70.95%.This sample is carried out order-checking detection validation, is defined as BCR-ABL fusion gene e6a2 type.
Embodiment 9:
Carry out RNA extraction to the 6th blood sample to be checked by described in embodiment 1-3, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Fig. 7-A: primary dcreening operation shows the 6th blood sample to be checked and belongs to a3 group.Again each type of a3 group to be identified respectively and quantitatively.Reagent configuration and trace routine are with embodiment 5.Acquired results is as shown in Fig. 7-B: through identifying that the 6th blood sample to be checked is BCR-ABL fusion gene e14a3 type, calculating its relative quantification result (BCR-ABL e14a3/ABL) according to inspection software is 96.29%.This sample is carried out order-checking detection validation, is defined as BCR-ABL fusion gene e14a3 type.
Embodiment 10:
By described in embodiment 1-3, RNA extraction is carried out to the 7th blood sample to be checked, obtain cDNA and fluorescent PCR primary dcreening operation, acquired results is as shown in Figure 8: the 7th blood sample to be checked does not belong to a2 group, do not belong to a3 group yet, product everyone to get rid of the 7th blood sample to be checked be e6a2, e8a2, e19a2, the possibility of e1a3, e13a3, e14a3 type.Dyed body karyotyping results verification is that caryogram is normal.
Embodiment 11:
According to the BCR-ABL fusion gene type result of embodiment 4 to embodiment 10 gained, summarize.Result is as shown in table 4.
Table 4
As shown in Table 4, the present invention designs for detecting the non-common pattern of fusion of BCR-ABL fusion gene other e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 method, primer, probe and test kit can detect this six kinds of types.The result detected is all consistent with the result that order-checking detects or chromosome karyotype analysis obtains.
SEQUENCE LISTING
<110> Shenyang company limited of Ai Dikang medical test institute
<120> examination and the non-common pattern of fusion method for distinguishing of qualification BCR-ABL, primer, probe and test kit
<130>
<160> 13
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> artificial sequence
<400> 1
gagccagaag caacaaagat g 21
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
ttcctgtcca gcatcaatga g 21
<210> 3
<211> 19
<212> DNA
<213> artificial sequence
<400> 3
tggaggaggt gggcatcta 19
<210> 4
<211> 18
<212> DNA
<213> artificial sequence
<400> 4
agttccaacg agcggctt 18
<210> 5
<211> 26
<212> DNA
<213> artificial sequence
<400> 5
cccttcagcg gccagtagca tctgac 26
<210> 6
<211> 19
<212> DNA
<213> artificial sequence
<400> 6
ctcgcagaac tcgcaacag 19
<210> 7
<211> 21
<212> DNA
<213> artificial sequence
<400> 7
gatgctgacc aactcgtgtg t 21
<210> 8
<211> 20
<212> DNA
<213> artificial sequence
<400> 8
gtcatcgtcc actcagccac 20
<210> 9
<211> 20
<212> DNA
<213> artificial sequence
<400> 9
ttttggtttg ggcttcacac 20
<210> 10
<211> 24
<212> DNA
<213> artificial sequence
<400> 10
ctccgggtct taggctataa tcac 24
<210> 11
<211> 22
<212> DNA
<213> artificial sequence
<400> 11
gatacgaagg gagggtgtac ca 22
<210> 12
<211> 18
<212> DNA
<213> artificial sequence
<400> 12
ctcggccagg gtgttgaa 18
<210> 13
<211> 29
<212> DNA
<213> artificial sequence
<400> 13
tgcttctgat ggcaagctct acgtctcct 29
Claims (6)
1., for non-other primer of common pattern of fusion of examination BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 these 6 kinds and probe, it is characterized in that, the nucleotide sequence of described primer and probe is as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
E1F：5’-CTCGCAGAACTCGCAACAG-3’
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
2. primer as claimed in claim 1 and probe, is characterized in that, described primer and probe also comprise for increasing as the primer of the house-keeping gene ABL of internal reference and probe, and its nucleotide sequence is as follows:
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
3., for the identification of non-other primer of common pattern of fusion of BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 these 6 kinds and probe, it is characterized in that,
The primer of qualification e6a2 type and the nucleotide sequence of probe as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
The primer of qualification e8a2 type and the nucleotide sequence of probe as follows:
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
The primer of qualification e19a2 type and the nucleotide sequence of probe as follows:
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
The primer of qualification e1a3 type and the nucleotide sequence of probe as follows:
E1F：5’-CTCGCAGAACTCGCAACAG-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
The primer of qualification e13a3 type and the nucleotide sequence of probe as follows:
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
The primer of qualification e14a3 type and the nucleotide sequence of probe as follows:
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
4. primer as claimed in claim 3 and probe, is characterized in that, described primer and probe also comprise amplification as the primer of the house-keeping gene ABL of internal reference and probe, and its nucleotide sequence is as follows:
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
5. prepare non-other primer of common pattern of fusion of examination BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 and detecting probe method for one kind: according to BCR-ABL in fusion process, the relatively-stationary feature of fracture position on abl gene, examination system is divided into two groups: a2 group, fracture position is positioned at abl gene the 1st intron, by e6a2, e8a2, e19a2 type forms; A3 group, fracture position is positioned at abl gene intron 2, is made up of e1a3, e13a3, e14a3 type; Meanwhile, the exon 2 of abl gene designs downstream primer and the fluorescent probe of a2 group; 3rd exon designs downstream primer and the fluorescent probe of a3 group; Subsequently, according to downstream primer design be suitable for it carrying out PCR reaction upstream primer; The upstream primer of a2 group lays respectively at the 6th exon of BCR gene, the 8th exon and the 19th exon; Combine with downstream primer like this and just in time can detect e6a2, e8a2, e19a2 tri-kinds of types; In like manner, the upstream primer of a3 group lays respectively at the 1st exon of BCR gene, the 13rd exon and the 14th exon, with downstream primer combine detection e1a3, e13a3 and e14a3 tri-kinds of types; The nucleotide sequence that described method obtains primer and probe is as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
E1F：5’-CTCGCAGAACTCGCAACAG-3’
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’。
6. an examination and qualification BCR-ABL fusion gene e6a2, e8a2, e19a2, e1a3, e13a3 and e14a3 these 6 kinds non-other test kits of common pattern of fusion, comprise blood rna extracting solution, reverse transcription reagents, pcr amplification reaction system, described pcr amplification reaction system comprises primer and probe, it is characterized in that, the nucleotide sequence of described primer and probe is as follows:
E6F：5’-GAGCCAGAAGCAACAAAGATG-3’
E8F：5’-TTCCTGTCCAGCATCAATGAG-3’
E19F：5’-TGGAGGAGGTGGGCATCTA-3’
A2R：5’-AGTTCCAACGAGCGGCTT-3’
A2P：5’-FAM-CCCTTCAGCGGCCAGTAGCATCTGAC-TAMRA-3’
E1F：5’-CTCGCAGAACTCGCAACAG-3’
E13F：5’-GATGCTGACCAACTCGTGTGT-3’
E14F：5’-GTCATCGTCCACTCAGCCAC-3’
A3R：5’-TTTTGGTTTGGGCTTCACAC-3’
A3P：5’-FAM-CTCCGGGTCTTAGGCTATAATCAC-TAMRA-3’
ABL-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’。
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| CN105838792B (en) * | 2016-04-22 | 2019-07-12 | 上海荻硕贝肯生物科技有限公司 | For the primer of qualitative detection leukemia fusion gene, probe, kit and method |
| CN107674914A (en) * | 2016-08-02 | 2018-02-09 | 熊术道 | One step single tube standard measure RT PCR detect the expression quantity of chronic myelogenous leukemia BCR ABL1 genes |
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| CN102533740A (en) * | 2011-09-07 | 2012-07-04 | 杭州艾迪康医学检验中心有限公司 | Reverse transcription-polymerase chain reaction (RT-PCR) primer of human leukemia fusion gene BCR-ABL and application method thereof |
| CN102758006A (en) * | 2012-04-25 | 2012-10-31 | 武汉艾迪康医学检验所有限公司 | Kit for detecting relative expression of leukemia BCR/ABL (b3a2, b2a2) fusion gene |
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| CN102533740A (en) * | 2011-09-07 | 2012-07-04 | 杭州艾迪康医学检验中心有限公司 | Reverse transcription-polymerase chain reaction (RT-PCR) primer of human leukemia fusion gene BCR-ABL and application method thereof |
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