CN116240288A - Primers and probes for screening and identifying MYB rearrangement related genes, and combined detection method and kit - Google Patents
Primers and probes for screening and identifying MYB rearrangement related genes, and combined detection method and kit Download PDFInfo
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Abstract
The invention relates to a primer probe for screening and identifying MYB rearrangement related genes by using a fluorescent PCR technology, a combined detection method and a kit, wherein 10 genes related to MYB gene rearrangement are screened and identified, namely MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1 and MYB-FAM111A, MYB-MCF2. The method can be used for rapidly detecting the expression quantity of the MYB rearrangement related genes, has good specificity, high sensitivity, high degree of automation, simple operation and larger linear range. The method can be widely applied to early diagnosis and targeted therapy of related diseases related to 10 genes related to MYB rearrangement in clinic, provides reference basis for guiding scheme selection and prognosis judgment of personalized therapy, has important reference significance for tumor research, and can be used for monitoring tiny residual focus targets in a targeted manner and predicting recurrence risk.
Description
Technical Field
The invention belongs to the field of detection of rearranged genes in disease gene detection, and particularly relates to a primer, a probe, a combined detection method and a kit for screening 10 genes related to MYB rearrangement by adopting a multiple fluorescence PCR technology.
Technical Field
Chromosomal rearrangements are common in abnormal transcripts and corresponding fusion proteins produced by transgene expression in malignant tumors in the blood system. Most of malignant hematopathy and tumor are accompanied with mutation or fusion of genes in the occurrence and development processes, so that the method for monitoring the mutation and fusion of the genes of patients suffering from malignant hematopathy and tumor has important application values for diagnosis and typing, prognosis judgment, treatment guidance and the like of diseases.
MYB is the earliest one of the protooncogenes found, and has potent carcinogenesis. The chaperones of cleavage sites involved in MYB rearrangement are numerous, and thus the variety of fusion and rearrangement genes produced is also numerous. It is found that the gene and other related genes of the disease can form more than ten kinds of gene fusion, most of fusion occurs in blood diseases and adenoid diseases, and common are MYB-NFIB fusion genes in adenoid cystic cancers (including salivary gland adenoid cystic cancers and breast adenoid cystic cancers): MYB-AHL1 fusion gene in acute lymphoblastic leukemia, MYB-GATA1 fusion gene in Acute Myelogenous Leukemia (AML) and Acute Basophilic Leukemia (ABL), MYB-BDP1, MYB-PLAGL1, SLC12A9-MYB, FAM111A-MYB fusion gene in acute lymphoblastic leukemia and lymphoblastic lymphoma, and the like.
In current studies of gene rearrangement, methods for detecting rearranged genes mainly include chromosome karyotyping, fluorescence In Situ Hybridization (FISH), real-time fluorescence PCR detection (RQ-PCR), and second generation sequencing (NGS). Compared with chromosome karyotype analysis and fluorescence in situ hybridization technology, the sensitivity of the real-time fluorescence PCR detection reaches 10 -5 -10 -6 The detection of rearranged genes can be performed more rapidly and more accurately, which is also an effective method recommended in NCCN guidelines, ELN guidelines and expert consensus for detecting gene rearrangement. The sensitivity of the second generation sequencing technology can reach 10 -5 -10 -6 But its use in detection of rearranged genes is limited due to high cost and long detection period. Multiple fluorescent RT-PCR can amplify multiple target genes simultaneously, has the advantages of saving time, reducing cost and improving efficiency, and is especially suitable for projects with less sample size and more detection targets. Therefore, the multiplex fluorescence RT-PCR method has greater application value in the aspect of rearranged gene screening. The invention adopts fluorescent PCR technology to obtain a rapid, simple and accurate method for screening and identifying MYB rearrangement related genes. The method is suitable for comprehensively evaluating the occurrence and development of diseases by combining the results of bone marrow examination, chromosome karyotyping analysis, immune typing and the like of a large number of samples; meanwhile, the target of the tiny residual focus is monitored in a targeted way, and the recurrence risk is predicted. Provides basis for understanding the disease progress and the treatment effect, and has important reference significance for the research of tumors.
Disclosure of Invention
In view of the fact that the current method for screening MYB rearrangement related genes cannot simultaneously meet the requirements of economy, high efficiency, accurate results, monitoring and the like, the invention designs a set of primers and probes for detecting MYB rearrangement common genes by using a multiplex fluorescence PCR technology, and designs a screening and type identification scheme which has the advantages of high sensitivity, good specificity, large detection flux, rapidness and accuracy. Meanwhile, the PCR reaction condition and the reaction system are optimized by adjusting the concentration ratio of the primer and the probe, so that the amplification efficiency is optimal. Aims to assist clinical diagnosis of the existence of rearranged genes of patients, and provides assistance for diagnosis of related diseases caused by MYB gene rearrangement, prognosis judgment, targeted therapy at a gene level, diagnosis markers, pathogenesis and other researches.
The invention provides a method for screening and identifying 10 MYB rearrangement related genes of MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1, MYB-FAM111A, MYB-MCF2 by using a real-time fluorescence PCR technology, and provides 14 pairs of primer pairs and probes for detecting different rearrangement modes aiming at 10 different rearranged genes. The nucleotide sequence is as follows:
MYB-E8-F：5′-GAATTGCTCCTAATGTCAACCG-3′
MYB-PROBE：5′-FAM-AATGAGCTAAAAGGACAGCAGGTGCTACC-TAMRA-3′
(MYB Gene reference sequence: NG_ 012330.1)
GATA1-5R：5′-GCACTGAGTACCTGCCCGTT-3′
GATA1-6R：5′-TCTGAATACCATCCTTCCGCA-3′
(GATA 1 Gene reference sequence: NG_ 008846.2)
NFIB-E8-R：5'-GGCTGGTTTGTGGACTGGAT-3'
NFIB-E9-R：5′-GCCTCACCTTCAGTGGATGTAGT-3′
NFIB-E11-R：5′-TGTATCCTCACTGGTACTGGGG-3′
NFIB-E12-R：5′-CGGTAAGATGGGTGTCCTATTTG-3′
(NFIB Gene reference sequence: NG_ 008846.2)
AHI1-E14-R：5′-TGAGAAGAGGTGTTTGTTTGGG-3′
(AHI 1 Gene reference sequence: NG_ 008643.2)
QKI-E5-R：5'-GCATCTCTGTAGGTGCCATTCA-3′
(QKI Gene reference sequence: NG_ 029604.2)
MNX1-E2-R：5′-ACTTCCCCAGGAGGTTCGA-3′
(MNX 1 Gene reference sequence: NG_ 013212.1)
ESR1-E5-R：5′-CAGACGAGACCAATCATCAGGA-3′
(ESR 1 Gene reference sequence: NG_ 008493.2)
SLC45A2-E5-R：5′-CAGAAGCCCCAACATCCAAC-3′
(SLC 45A2 Gene reference sequence: NG_ 011691.2)
ACTG1-E5-R：5′-GAGTTGAAGGTGGTCTCGTGG-3′
(ACTG 1 Gene reference sequence: NG_ 011433.1)
FAM111A-E5-R：5′-GTGCTTCCGTGACCTCTGCT-3′
(FAM 111A Gene reference sequence: NG_ 042835.1)
MCF2-E8-R：5'-CATTGCACCTCTGGCAGATTAA-3′
(MCF 2 Gene reference sequence: NG_ 016439.1)
The primer probe for screening the rearranged genes also comprises a primer and a probe for amplifying a housekeeping gene ABL serving as an internal reference, and the nucleotide sequence of the primer probe is as follows:
ABL1-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL1-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL1-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’
the invention also provides a kit for detecting 10 MYB rearrangement-related genes, which comprises: the PCR reaction solution comprises primers and probes for amplifying 10 MYB rearrangement related genes and internal reference housekeeping genes ABL.
Further, the nucleotide sequences of the 10 MYB rearrangement related gene primers and probes are as follows:
MYB-E8-F：5′-GAATTGCTCCTAATGTCAACCG-3′
MYB-PROBE：5′-FAM-AATGAGCTAAAAGGACAGCAGGTGCTACC-TAMRA-3′
GATA1-5R：5′-GCACTGAGTACCTGCCCGTT-3′
GATA1-6R：5′-TCTGAATACCATCCTTCCGCA-3′
NFIB-E8-R：5′-GGCTGGTTTGTGGACTGGAT-3′
NFIB-E9-R：5′-GCCTCACCTTCAGTGGATGTAGT-3′
NFIB-E11-R：5′-TGTATCCTCACTGGTACTGGGG-3′
NFIB-E12-R：5′-CGGTAAGATGGGTGTCCTATTTG-3′
AHI1-E14-R：5′-TGAGAAGAGGTGTTTGTTTGGG-3'
QKI-E5-R：5'-GCATCTCTGTAGGTGCCATTCA-3′
MNX1-E2-R：5′-ACTTCCCCAGGAGGTTCGA-3′
ESR1-E5-R：5′-CAGACGAGACCAATCATCAGGA-3′
SLC45A2-E5-R：5′-CAGAAGCCCCAACATCCAAC-3′
ACTG1-E5-R：5′-GAGTTGAAGGTGGTCTCGTGG-3′
FAM111A-E5-R：5′-GTGCTTCCGTGACCTCTGCT-3'
MCF2-E8-R：5′-CATTGCACCTCTGGCAGATTAA-3′
further, the method also comprises the steps of providing upstream and downstream primers and probes for identifying 10 MYB rearrangement-related genes of MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1, MYB-FAM111A and MYB-MCF2, sharing one upstream primer and a corresponding probe in 8 th exons of the MYB genes, and pairing different downstream primers for detection. Finally, 14 pairs of upstream and downstream primers were prepared. The primer and probe sequences are as follows:
the upstream and downstream primers of 2 spliceosomes of MYB-GATA1 fusion gene are respectively as follows: MYB-E8-F and GATA1-5R, MYB-E8-F and GATA1-6R;
the upstream and downstream primers of the 4 spliceosomes of the MYB-NFIB fusion gene are respectively as follows: MYB-E8-F and NFIB-E8-R, MYB-E8-F and NFIB-E9-R, MYB-E8-F and NFIB-E11-R, MYB-E8-F and NFIB-E12-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-AHI1 rearranged gene detection are: MYB-E8-F, AHI1-E14-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-KQI rearranged gene detection are: MYB-E8-F, KQI-5R;
the nucleotide sequences of the upstream and downstream primers used for MYB-MNX1 rearranged gene detection are: MYB-E8-F, MNX1-E2-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-ESR1 rearranged gene detection are: MYB-E8-F, ESR1-E5-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-SLC45A2 rearranged gene detection are: MYB-E8-F, SLC45A2-E5-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-ACTG1 rearrangement gene detection are: MYB-E8-F, ACTG1-E5-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-FAM111A rearranged gene detection are: MYB-E8-F, FAM111A-E5-R;
the nucleotide sequence of the primer for detecting MYB-MCF2 rearranged genes is as follows: MYB-E8-F, MCF2-E8-R;
further, 10 MYB rearrangement-related genes including MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1 and MYB-FAM111A, MYB-MCF2 are the same as one PROBE MYB-PROBE.
The nucleotide sequence of the probe is as follows:
MYB-PROBE：5′-FAM-AATGAGCTAAAAGGACAGCAGGTGCTACC-TAMRA-3′
further, primers and probes for amplifying housekeeping gene ABL as an internal reference are also included, and the nucleotide sequences thereof are as follows:
ABL1-F：5’-GATACGAAGGGAGGGTGTACCA-3’
ABL1-R：5’-CTCGGCCAGGGTGTTGAA-3’
ABL1-P：5’-FAM-TGCTTCTGATGGCAAGCTCTACGTCTCCT-TAMRA-3’
further, the method also comprises the steps of screening and identifying 10 MYB rearrangement related genes by using a multiplex fluorescence PCR technology, and pairing the same upstream primer of the MYB rearrangement related genes, probes corresponding to the same and downstream primers on different rearrangement genes, wherein the MYB-GATA1, the MYB-NFIB, the MYB-AHI1, the MYB-QKI, the MYB-MNX1, the MYB-ESR1, the MYB-SLC45A2, the MYB-ACTG1 and the MYB-FAM111A, MYB-MCF2 are combined and detected. Screening of MYB rearrangement related genes into two groups is carried out in two reaction tubes, and the combined detection mode of the rearrangement genes comprises a 2-tube combination, wherein:
tube 1: screening for MYB-GATA1, MYB-AHI1, MYB-QKI, MYB-MNX1 and
MYB-ESR1 rearranged genes. The primer probe sequence is as follows:
MYB-E8-F：5'-GAATTGCTCCTAATGTCAACCG-3′
MYB-PROBE：5′-FAM-AATGAGCTAAAAGGACAGCAGGTGCTACC-TAMRA-3′
GATA1-5R：5′-GCACTGAGTACCTGCCCGTT-3′
GATA1-6R：5′-TCTGAATACCATCCTTCCGCA-3′
AHI1-E14-R：5′-TGAGAAGAGGTGTTTGTTTGGG-3′
QKI-E5-R：5′-GCATCTCTGTAGGTGCCATTCA-3′
MNX1-E2-R：5′-ACTTCCCCAGGAGGTTCGA-3′
ESR1-E5-R：5′-CAGACGAGACCAATCATCAGGA-3′
the upstream and downstream primers of the rearranged gene of tube 1 were paired as follows:
the nucleotide sequences for the 2 spliceosome detection primers and probes of the MYB-GATA1 fusion gene are: MYB-E8-F and GATA1-5R, MYB-E8-F and GATA1-6R;
the nucleotide sequences for MYB-AHI1 rearranged gene detection primers and probes are: MYB-E8-F, AHI1-E14-R;
the nucleotide sequences for the MYB-KQI rearranged gene detection primers and probes are: MYB-E8-F, KQI-5E-R;
the nucleotide sequences for MYB-MNX1 rearranged gene detection primers and probes are: MYB-E8-F, MNX1-E2-R;
the nucleotide sequences for the MYB-ESR1 rearranged gene detection primers and probes are: MYB-E8-F, ESR1-E5-R;
tube 2: screening for MYB-NFIB, MYB-SLC45A2, MYB-ACTG1, MYB-FAM111A and MYB-MCF2. The primer probe sequence is as follows:
MYB-E8-F：5′-GAATTGCTCCTAATGTCAACCG-3′
MYB-PROBE：5′-FAM-AATGAGCTAAAAGGACAGCAGGTGCTACC-TAMRA-3′
NFIB-E8-R：5'-GGCTGGTTTGTGGACTGGAT-3'
NFIB-E9-R：5′-GCCTCACCTTCAGTGGATGTAGT-3′
NFIB-E11-R：5′-TGTATCCTCACTGGTACTGGGG-3′
NFIB-E12-R：5′-CGGTAAGATGGGTGTCCTATTTG-3′
SLC45A2-E5-R：5′-CAGAAGCCCCAACATCCAAC-3′
ACTG1-E5-R：5′-GAGTTGAAGGTGGTCTCGTGG-3′
FAM111A-E5-R：5′-GTGCTTCCGTGACCTCTGCT-3′
MCF2-E8-R：5′-CATTGCACCTCTGGCAGATTAA-3′
the pairing mode of the upstream primer and the downstream primer of the rearranged gene of the 2 nd tube is as follows:
the upstream and downstream primers of the 4 splicing primers of the MYB-NFIB fusion gene are respectively as follows: MYB-E8-F and NFIB-E8-R, MYB-E8-F and NFIB-E9-R, MYB-E8-F and NFIB-E11-R, MYB-E8-F and NFIB-E12-R;
the nucleotide sequences for MYB-SLC45A2 rearranged gene detection primers and probes are as follows: MYB-E8-F, SLC45A2-E5-R;
the nucleotide sequences for the MYB-ACTG1 rearrangement gene detection primers and probes are: MYB-E8-F, ACTG1-E5-R;
the nucleotide sequences for MYB-FAM111A rearranged gene detection primers and probes are: MYB-E8-F, FAM111A-E5-R;
the nucleotide sequences for MYB-MCF2 rearranged gene detection primers and probes are: MYB-E8-F, MCF2-E8-R;
further, reagents such as 2×t5fast qPCR Mix, ROX Reference Dye ii (50×), ddH2O, etc. are included;
further, the kit further comprises a negative control and a positive control.
Further, the method for screening and identifying 10 MYB rearrangement modes of MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1, MYB-FAM111A and MYB-MCF2 comprises the following steps:
(1) Extracting fresh blood sample RNA;
(2) Reverse transcription of the RNA extracted in (1) into cDNA;
(3) And (5) carrying out preliminary screening on MYB rearrangement related genes. Screening reagents for each sample were divided into 3 reaction tubes: the first tube and the second tube are screening detection groups, and the third tube is an ABL reference gene detection group;
(4) According to the optimized PCR system, reagent system reagents such as 2×T5Fast qPCR Mix, ROX Reference Dye II (50), ddH2O and the like are added.
(5) Adding an upstream primer and a downstream primer and a probe for detecting MYB-GATA1, MYB-AHI1, MYB-QKI, MYB-MNX1 and MYB-ESR1 rearranged genes into a reaction tube 1;
(6) Adding an upstream primer and a downstream primer and a probe for detecting MYB-NFIB, MYB-SLC45A2, MYB-ACTG1, MYB-FAM111A and MYB-MCF2 rearranged genes into a reaction tube 2;
(7) The reaction tube 3 is added with an upstream primer ABL-F, a downstream primer ABL-R and a probe ABL-P for detecting the reference gene
(8) And (3) respectively adding the cDNA in the step (2) serving as a template into a reaction tube of the prepared target gene and an internal reference ABL reaction system.
(9) The prepared reaction system (total 20ul system) was subjected to qPCR amplification reaction.
(10) Analysis and further processing of detection results of genes related to MYB rearrangement:
amplification signal for reference gene ABL: if the CT value is less than 30, the whole detection process is effective; if its CT value is greater than 30, then the test needs to be re-validated.
Amplification signal for the gene of interest:
(1) When the internal reference is positive, the detection result is considered to be effective;
(2) Judging which of the first and second groups is positive according to the detection result in the step (1); positive judgment criteria: ct <36, positive; ct is more than or equal to 36 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative. If the first group of internal references are positive and the target genes are on line, identifying which of MYB-GATA1, MYB-AHI1, MYB-QKI, MYB-MNX1 and MYB-ESR1 rearranged genes is positive; if the second group of internal references are positive and the target gene is on line, it is necessary to identify which of MYB-NFIB, MYB-SLC45A2, MYB-ACTG1, MYB-FAM111A and MYB-MCF2 rearranged genes is positive. The invention has the beneficial effects that:
when MYB genes are rearranged, the cleavage sites are mainly concentrated on the 8-16 exons and introns, so that the MYB exons 8-16 are rearranged or fused with other genes; therefore, in the design of the primer, the upstream primer is designed aiming at the 8 th exon of MYB genes, and 14 pairs of primers of MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1 and MYB-FAM111A, MYB-MCF2 of 10 MYB rearrangement related genes are designed by using the same upstream sequence, and the downstream primer is designed aiming at different splicing modes on the exons of different ligand genes. In the primer design, the primer 3' end is avoided from using A base and repeating more than 3 bases as much as possible, the mismatch probability of the primer is reduced, and meanwhile, the length of a PCR amplification product is controlled within 150bp as much as possible, so that the detection range requirement of the fluorescent PCR technology is met. The invention uses the same upstream primer and probe and different downstream primers, so that the operation is simpler and faster than that of a plurality of pairs of different upstream and downstream primers.
2. In the design of the probe, a section of nucleic acid sequence of the probe on the 8 th exon of MYB, namely, 14 pairs of primers of 10 MYB rearrangement modes of MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1 and MYB-FAM111A, MYB-MCF2 are detected by using the same probe, so that the design can reduce the synthetic cost of the primers and the probes, and effectively, quickly and conveniently detect the heavy-row genes. In the design of the probe, the temperature is 10 ℃ higher than the Tm of the primer, the complementary sequence of more than 3 bases is avoided in the probe, and the probe is positioned as close to the upstream and downstream as possible. Meanwhile, when the primers and the probes are screened, complementary pairing of more than 3 bases between the two primers and between the primers and the probes is avoided as much as possible, so that the specificity of the primers and the probes can be improved, and the amplification efficiency of the primers can be improved.
On a combined scheme of screening MYB rearrangement related genes, the invention considers interference factors such as primer dimers, hairpin structures and the like and the consistency of MYB gene breakage and rearrangement positions, and screens MYB rearrangement related genes into two groups to be carried out in two tubes.
4. According to the invention, a real-time fluorescence PCR probe method is used for respectively constructing detection systems of related rearranged genes of internal reference genes ABL and MYB rearrangement, and the expression levels of 10 MYB rearrangement related genes of MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1 and MYB-FAM111A, MYB-MCF2 in a subject are detected. Compared with the prior FISH and delta CT methods, the kit has the advantages of good test specificity, high sensitivity, simple and convenient operation, high degree of automation, pollution prevention, larger linear range and the like, and can quantitatively detect tiny residual focus of genes related to MYB rearrangement. Meanwhile, the invention optimizes the reaction conditions such as primer concentration, probe ratio, the lowest lower limit of template concentration and the like by fumbling the amplification time and temperature, so that the amplification efficiency can be optimized, the target gene can be rapidly, effectively and accurately amplified, and the expression condition of MYB rearrangement related genes can be judged.
Drawings
FIG. 1 is an amplification plot of clinical sample screening with normal priming of internal controls to indicate that sample extraction is normal and no priming of both combinations 1 and 2 to indicate that the sample is free of MYB fusion genes.
FIG. 2 shows a sample screening amplification curve for a MYB-E8-GATA1-E5 positive quality control, set 1, indicating the presence of a MYB fusion gene in the reaction tube of set 1.
FIG. 3 to identify which fusion genes are present in screening positive samples, reactions were performed separately, and the reaction tubes for the MYB-E8-F, GATA1-E5-R combinations were found to be lined, and none of the other reaction tubes were lined, indicating that the screening positive samples contained MYB-E8-GATA1-E5 fusion genes.
FIG. 4 is an electropherogram showing that positive samples contained MYB-E8-GATA1-E5 fusion genes, without the presence of other fusion genes.
FIG. 5Sanger sequencing shows that the MYB-E8-GATA1-E5 fusion gene is present and that fusion points of both genes can be intuitively found.
Detailed Description
Example 1
The invention is used for assisting in clinical screening and identification of 10 MYB rearrangement related genes including MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1, MYB-FAM111A and MYB-MCF2. The method mainly comprises the following reagents: RNA extraction reagent, RNA reverse transcription reagent and detection system PCR reaction liquid.
And synthesizing a Taqman probe sequence with specificity of the QRT-PCR primer, wherein the 5 '-end of the Taqman probe sequence is marked with a corresponding FAM, and the 3' -end of the Taqman probe sequence is uniformly marked with a fluorescence quenching group TAM.
Wherein the detection system PCR reaction liquid comprises: reverTra Ace qPCR RT Kit;2×T5 Fast qPCR Mix, ROX Reference Dye II (50 x), ddH 2 The primer and probe of O, ACTIN reference gene and MYB-GATA1 target gene are 10 mu M, and the ratio of the primer to the probe is 1:1.
the primer and the probe for detecting the reference gene ABL and the target gene MYB-GATA1 have the following base sequences:
example 2
The operation flow of the invention is as follows:
(1) Extracting total RNA in blood: fresh blood was added to a clean 1.5ml centrifuge tube, 3 volumes of RZ (0.25 ml blood+075 ml RZ recommended) were added, and mixed well with shaking. The homogenized sample was left at 15-30℃for 5min to allow complete separation of the nucleic acid protein complexes. 200 μl of chloroform was added, the tube cap was capped, vigorously shaken for 15sec, and left at room temperature for 3min at 12000rpm for 10min, the sample was separated into three layers, yellow organic phase, middle layer and colorless aqueous phase, RNA was mainly in the aqueous phase, and the volume of the aqueous phase was about 50% of the lysate RZ reagent used. The aqueous phase was transferred to a new tube for the next operation. Slowly add 0.5 volume of absolute ethanol and mix well (precipitation may occur at this time). The resulting solution was transferred to the adsorption column CR3 together with the precipitate, and if the whole solution and the mixture could not be added to the adsorption column CR3 at one time at 12000rpm at 4 ℃, the solution was transferred to the adsorption column CR3 twice, centrifuged at 12000pm at 4 ℃ for 30sec, and the waste liquid in the collection tube was discarded. To the adsorption column CR3, 500. Mu.l of deproteinized solution RD was added, centrifuged at 12,000rpm at 4℃for 30sec, and the waste liquid was discarded, and CR3 was placed in a collection tube. To the adsorption column CR3, 500. Mu.l of a rinse solution RW was added, and the mixture was allowed to stand at room temperature for 2min, centrifuged at 12,000rpm at 4℃for 30sec, and the waste liquid was discarded. The previous step is repeated once. The column was placed in a 2ml collection tube and centrifuged at 2000rpm at 4℃for 2min to remove residual liquid. The column CR3 was transferred to a new 1.5ml centrifuge tube, 30-100. Mu.l RNase-Free ddHO was added, and the mixture was left at room temperature for 2min and centrifuged at 12000rpm at 4℃for 2min. The volume of the elution buffer should not be less than 30. Mu.l, and too small a volume affects recovery efficiency. And the RNA should be stored at-70℃to prevent degradation.
(2) RNA is inverted into cDNA. With reference to Rever Tra Ace qPCR RT Kit kit instruction configuration system, the reverse transcription PCR amplification reaction procedure was a two-step method.
(3) Reagent configuration: the primers and probe working solution for the target rearranged gene and internal reference were diluted to 10. Mu.M.
Preparing detection system PCR reaction liquid according to the parts of detection people, and subpackaging 20 mu L of each part:
x=20 μl of reaction solution X (n internal reference samples+n specimens of the gene of interest+1 positive control+1 negative control);
n is the number of detection samples.
(4) Sample adding: adding 2 μl of cDNA into the PCR reaction solution of the detection system; positive control and negative control 2 μl of positive control and negative control are directly added; the blank was added with 2. Mu.l of physiological saline or without any substance.
(5) Amplification: detection was performed on a real-time fluorescent PCR instrument (e.g., 7500 from us Applied Biosystems).
(6) And (3) judging results: and adjusting the threshold line to be above a background signal and a negative amplification line, and automatically calculating the copy number according to a standard curve and a CT value by the system.
Amplification signal for reference gene ABL:
CT value <30, it is successful to demonstrate that the sample is extracted, and the content of RNA accords with the detection requirement of the target gene; CT value >30, which may lead to inaccurate results, should be re-extracted.
Amplification signal for the gene of interest:
1) When the internal reference is positive, the detection result is considered to be effective;
2) Positive judgment criteria: ct <36, positive; ct is more than or equal to 35 and less than or equal to 38, is suspected positive and needs to be verified again; ct > 38, negative. If the first group is positive, identifying which of MYB-GATA1, MYB-AHI1, MYB-QKI, MYB-MNX1 and MYB-ESR1 rearranged genes is positive; if the second group is positive, it is necessary to identify which of MYB-NFIB, MYB-SLC45A2, MYB-ACTG1, MYB-FAM111A and MYB-MCF2 rearranged genes is positive.
Example 3
Clinical samples were taken for screening, and screening reagents were prepared according to the materials and amounts shown in Table 1 below for detection of rearranged genes according to examples 1 and 2. Screening reagents for each sample were divided into 3 reaction tubes: the first tube and the second tube are screening detection groups (namely, respectively corresponding to the combination 1 and the combination 2 of FIG. 1), and the third tube is a screening detection group ABL reference gene detection group for judging whether the quality of RNA extraction meets the requirement; each tube was charged with 2ul of cDNA. The results obtained are shown in FIG. 1: the reference gene ABL of the sample is all hatched, the RNA extraction of the sample is proved to be effective, but the reaction tubes 1 and 2 in the screening detection group are not all hatched, and the sample is determined to have no MYB rearrangement.
TABLE 1 reaction System, fluorescent PCR Material and dose configuration thereof
Example 4:
since there are very few samples positive for MYB rearrangement, a positive quality control sample containing MYB-E8-GATA1-E5 fusion gene was taken and subjected to multiplex fluorescence PCR screening as described in example 3, and the results are shown in FIG. 2: the first group was shown to be positive. Each rearranged gene of the first group was identified separately, the reagent configuration is shown in Table 2, and the detection procedure is the same as in example 3. The results obtained are shown in FIG. 3: qPCR detection identified as MYB-E8-GATA1-E5 fusion positive, demonstrating that our designed screening primers and methods can be used as large-scale screening for MYB rearrangement of clinical samples.
TABLE 2 reaction System, fluorescent PCR Material and dose configuration thereof
Example 5:
to verify the accuracy of the results of example 4, PCR amplification, gel electrophoresis and sequencing were performed on each fusion set of group 1, and the effectiveness and specificity of the designed primers were determined. The reagent configuration is shown in Table 3, and the template is 1ul of positive quality control sample containing MYB-E8-GATA1-E5 fusion genes. The results of gel electrophoresis are shown in FIG. 4: only the MYB-E8-GATA1-E5 fusion gene can successfully amplify the band, and the band is clear and bright. The primer specificity is good. After Sanger sequencing of the amplified product, the sequence is compared with a MYB-E8-GATA1-E5 standard fusion sequence, and the detected fusion gene of the MYB-E8-GATA1-E5 is confirmed, wherein the fusion positions of the two genes are shown in a sequencing figure 5.
TABLE 3 reaction System and PCR Material and dose configuration thereof
Claims (10)
1. The primers and probes for 10 MYB rearrangement-related genes, namely MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1 and MYB-FAM111A, MYB-MCF2, are screened and identified by using a real-time fluorescent PCR technology, and are characterized in that 14 pairs of detected primers and probes are provided for different rearranged genes.
2. The primer and probe of claim 1, comprising 10 MYB rearrangement-related genes of the total of MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1, MYB-FAM111A, MYB-MCF2, the nucleotide sequences of the primer and probe are as follows:
3. the primer and probe according to claim 2, wherein,
the nucleotide sequences of the primers used for detection of 2 spliceosomes of the MYB-GATA1 fusion gene are: the upstream and downstream primers of MYB-E8-F, GATA1-5R, GATA1-6R,2 pairs are respectively: MYB-E8-F and GATA1-5R, MYB-E8-F and GATA1-6R.
4. The primer and probe according to claim 2, wherein,
the nucleotide sequences of the primers used for detection of the 4 spliceosomes of the MYB-NFIB fusion gene are: MYB-E8-F, NFIB-E8-R, NFIB-E9-R, NFIB-E11-R, NFIB-E12-R,4 pairs of upstream and downstream primers are respectively: MYB-E8-F and NFIB-E8-R, MYB-E8-F and NFIB-E9-R, MYB-E8-F and NFIB-E11-R, MYB-E8-F and NFIB-E12-R.
5. The primer and probe according to claim 2, wherein,
the nucleotide sequences of the upstream and downstream primers used for MYB-AHI1 rearranged gene detection are: MYB-E8-F, AHI1-E14-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-KQI rearranged gene detection are: MYB-E8-F, KQI-E5-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-MNX1 rearranged gene detection are: MYB-E8-F, MNX1-E2-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-ESR1 rearranged gene detection are: MYB-E8-F, ESR1-E5-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-SLC45A2 rearranged gene detection are: MYB-E8-F, SLC45A2-E5-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-ACTG1 rearrangement gene detection are: MYB-E8-F, ACTG1-E5-R;
the nucleotide sequences of the upstream and downstream primers used for MYB-FAM111A rearranged gene detection are: MYB-E8-F, FAM111A-E5-R;
the nucleotide sequence of the primer for detecting MYB-MCF2 rearranged genes is as follows: MYB-E8-F, MCF2-E8-R.
6. The primer and probe according to any one of claims 1 to 5, further comprising a primer and probe for amplifying housekeeping gene ABL as an internal reference, the nucleotide sequence of which is as follows:
7. a combined detection method for screening and identifying 10 MYB rearrangement related genes of MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI, MYB-MNX1, MYB-ESR1, MYB-SLC45A2, MYB-ACTG1 and MYB-FAM111A, MYB-MCF2 by using a fluorescent PCR technology is characterized in that 5 kinds of the 10 rearrangement genes are combined together and divided into 2 pipes for screening and detection, wherein:
the combined tube 1 comprises primers and probes for screening 5 rearranged genes of MYB-GATA1, MYB-AHI1, MYB-QKI, MYB-MNX1 and MYB-ESR1, and comprises 6 pairs of primers, wherein the nucleotide sequences of the primers and the probes are as follows: MYB-E8-F, MYB-PROBE, GATA1-5R, GATA1-6R,
AHI1-R，KQI-5R，MNX1-E2-R，ESR1-E5-R；
the combined tube 2 comprises the screening of MYB-NFIB, MYB-SLC45A2, MYB-ACTG1,
Primers and probes for 5 rearranged genes of MYB-FAM111A, MYB-MCF21 comprise 8 pairs of primers, and the nucleotide sequences of the primers and probes are as follows: MYB-E8-F, MYB-PROBE, NFIB-E8-R, NFIB-E9-R, NFIB-E11-R, NFIB-E12-R, SLC45A2-E5-R, ACTG1-E5-R, FAM111A-E5-R, MCF2-E8-R.
8. The combination detection method according to claim 7, further comprising a primer and a probe for amplifying housekeeping gene ABL as an internal reference, the nucleotide sequence of which is as follows:
9. kit for detecting MYB rearrangement related genes with primers and probes according to any of claims 1-6, characterized in that the kit comprises:
(i) An RNA extraction reagent;
(ii) A reverse transcription reagent;
(iii) Detection system PCR amplification reaction solution: the primer and the probe for amplifying 10 genes related to MYB rearrangement are respectively provided with the following base sequences:
(iv) Detection system PCR amplification reaction solution: the primer and the probe of the reference gene are also included, and the base sequences of the primer and the probe are respectively as follows:
(v) Positive control and negative control.
10. Screening and identifying MYB-GATA1, MYB-NFIB, MYB-AHI1, MYB-QKI,
MYB-MNX1、MYB-ESR1、MYB-SLC45A2、MYB-ACTG1、
The method for 10 MYB rearrangement-related genes of MYB-FAM111A and MYB-MCF2 is characterized by comprising the following steps:
(1) Extracting RNA of a fresh blood sample;
(2) Reverse transcription of the RNA extracted in (1) into cDNA;
(3) And (5) carrying out preliminary screening on MYB rearrangement related genes. Screening reagents for each sample were divided into 3 reaction tubes: the first tube is a screening detection group, the second tube is an ABL reference gene detection group;
(4) Adding reagent system reagents such as 2×T5Fast qPCR Mix, ROX Reference Dye II (50), ddH2O and the like according to the optimized PCR system;
(5) Adding an upstream primer and a downstream primer and a probe for detecting MYB-GATA1, MYB-AHI1, MYB-QKI, MYB-MNX1 and MYB-ESR1 rearranged genes into a reaction tube 1;
(6) Adding an upstream primer and a downstream primer and a probe for detecting MYB-NFIB, MYB-SLC45A2, MYB-ACTG1, MYB-FAM111A and MYB-MCF2 rearranged genes into a reaction tube 2;
(7) Adding an upstream primer ABL-F, a downstream primer ABL-R and a probe ABL-P for detecting the reference gene into the reaction tube 3;
(8) Respectively adding the cDNA in the step (2) serving as a template into a reaction tube of the prepared target rearrangement gene and an internal reference ABL reaction system;
(9) A prepared reaction system (20 ul system in total) is used for carrying out qPCR amplification reaction;
(10) Analysis and further processing of detection results of MYB rearranged genes.
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