CN117757910A

CN117757910A - Leukemia fusion gene single tube multiplex PCR detection method and kit thereof

Info

Publication number: CN117757910A
Application number: CN202311844553.0A
Authority: CN
Inventors: 郎兴萍; 肖晟; 金丽琴; 王祎婷; 严晓鑫
Original assignee: Suzhou Jingzhun Medical Technology Co ltd
Current assignee: Suzhou Jingzhun Medical Technology Co ltd
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2024-03-26

Abstract

The invention relates to a leukemia fusion gene single-tube multiplex PCR detection method and a kit thereof, which specifically adopt GSP1 primer to carry out a first PCR reaction, then GSP2 primer to carry out a second PCR reaction, and final PCR products respectively adopt GSP1MF and GSP2MR to carry out a first generation sequencing.

Description

Leukemia fusion gene single tube multiplex PCR detection method and kit thereof

Technical Field

The invention relates to the field of biotechnology and gene detection, in particular to a leukemia fusion gene single-tube multiplex PCR detection method and a kit thereof.

Background

Leukemia (leukemia), a malignant tumor of the hematopoietic system, is a group of highly heterogeneous hematological malignancies characterized by abnormal proliferation of leukemia cells with a differentiation maturation disorder. Clinically, anemia, hemorrhage, fever, hepatosplenomegaly and lymphadenopathy of different degrees appear, which can endanger life. Leukemia fusion gene (fusion gene) is a molecular biology specific marker of leukemia. In recent years, due to the development of molecular biology techniques, the understanding of the molecular genetic changes of leukemia cells has been in progress. Leukemia has been reported to date to involve at least tens of fusion genes. It has been recognized that structural chromosomal aberrations, including deletions, duplications, inversions, translocations, etc., exist in most leukemias, resulting in structural variation of protooncogenes and cancer suppressor genes, which activate or deactivate, resulting in new fusion genes encoding fusion proteins. Some genes are transcription factors for regulating cell proliferation, differentiation and apoptosis, and when the genes are mutated, downstream signal transmission paths are directly influenced, so that the cell proliferation capacity is enhanced, apoptosis disorder, differentiation disorder and the like are caused, and leukemia phenotype is generated. Some typical leukemia fusion genes are specific molecular diagnostic markers for certain leukemias, such as the BCR-ABL fusion gene, which can occur in more than 95% of Chronic Myelogenous Leukemia (CML). The prognosis effect of patients has a certain relation with the type of fusion genes, such as PML-RARa fusion genes specific to Acute Promyelocytic Leukemia (APL), and has very good prognosis and low recurrence rate when the APL patients are subjected to all-trans retinoic acid (ATRA) induced remission treatment. While some genes, such as MLL related fusion genes, have poor prognosis and high mortality.

Sense of fusion gene detection for leukemia diagnosis: clinical practice shows that the subjective components of a tester are relatively large, the mutual coincidence rate and the correct rate are limited to a certain extent, and along with the rapid development of cell and molecular biology technology and the continuous deep research on leukemia pathogenesis, the gene and phenotype changes in the leukemia pathogenesis process are recognized to have important significance for the diagnosis and treatment of various leukemia, so that the leukemia MICM typing is proposed. Recent years of research on the molecular characteristics of leukemia have significantly progressed, and in particular, chromosomal translocation to form fusion genes, have been used as molecular biological specific markers for diagnosis of different types of leukemia and the only basis for determining diagnosis, such as acute promyelocytic leukemia APL: PML/RARA, t (15; 17) (q 21; q 22); acute myeloid leukemia AML-M4Eo: CBFB/MYH11, inv (16) (p 13; q 22); slow downMyeloid leukemia CML or partially acute lymphoblastic leukemia ALL: BCR/ABL, t (9; 22) (q 34; q 11); AML-M2: AML1/ETO, t (8; 21) (q 22; q 22); ALL-L3: MYC/IgH, t (8; 14) (q 24; q 32); AML-M4/M5:11q23MLL anomalies, etc. The leukemia fusion gene can be detected by reverse transcription PCR (RT-PCR) technology, which is helpful for evaluating the acute degree, cloning property and typing of leukemia, and makes the diagnosis and typing of leukemia more scientific and standardized. In the catalogue of clinical examination projects of medical institutions issued by the Ministry of health of 2007, leukemia fusion gene examination using RT-PCR or real-time PCR technology is required, and mainly 6 fusion genes are involved in the examination, including BCR/ABL, PML/RARA, AML1/MPSI/EVI1, DEK/CAN, AML1/MTG8 and E2A/PBX1.RT-PCR can be performed 5-8 months earlier than the traditional cytology method and clinical symptoms, and 1X 10 can be detected ⁶ One of the nucleated cells has incomparable specificity and sensitivity to other methods in early diagnosis of leukemia.

Meaning of fusion gene detection on leukemia treatment and prognosis judgment: cytogenetic typing has close relation with disease prognosis, and has great significance for guiding the selection of clinical personalized treatment schemes and judging prognosis. The acute leukemia has PML/RARA, CBFB/MYH11 and AML1/ETO fusion genes, has better prognosis, complete chemotherapy and high remission rate, can be relieved or cured for a long time, and does not claim to be transplanted for hematopoietic stem cells in early stage; in contrast, ALL of BCR/ABL fusion gene has poor response to chemotherapy and high recurrence rate for AML with MLL abnormality and MYC/IgH fusion gene, and it is suggested that it is conditionally active for hematopoietic stem cell transplantation. With the detection of the fusion gene, the method can guide scientific and reasonable selection of a long-term treatment scheme during primary treatment, and avoid unnecessary under-treatment or over-treatment.

From the trend, chromosome analysis and related fusion gene molecule detection technology play an increasingly important role in blood system tumor diagnosis, typing and treatment guidance. In WHO disease typing, each new modification increases the specific gravity of the relevant genetic test results. With the clinical application of new target drugs, the detection of related target genes is increasing. In addition, fusion gene alterations are one of the important bases for selection of therapeutic regimens in tumor clinical treatment guidelines promulgated by the national cancer complex (The National Comprehensive Cancer Network, NCCN) and the british committee for hematology standards (The British Committee for Standards in Haematology, BCSH).

Disclosure of Invention

The invention aims to provide a leukemia fusion gene single-tube multiplex PCR detection method, which targets a fusion gene:

CBFB::MYH11，KMT2A::MLLT7，KMT2A::MLLT4，KMT2A::ELL，KMT2A::EPS15，KMT2A::MLLT6，KMT2A::MLLT10，KMT2A::PTD，TCF3::PBX1，TCF3::HLF，STIL::TAL1，ETV6::RUNX1，RUNX1::RUNX1T1，RUNX1::MECOM，RUNX1::EAP，FUS::ERG，RPN1::MECOM，KMT2A::AFF1，KMT2A::MLLT3，KMT2A::MLLT11，KMT2A::MLLT1，BCR::ABL1，ETV6::ABL1，ETV6::PDGFRB，ZMYM2::FGFR1，BCR::FGFR1，DEK::NUP214，SET::NUP214，FIP1L1::PDGFRA，NUP98::HOXA9，NUP98::HOXD13，ZBTB16::RARA，PML::RARA，NPM1::RARA，NPM1::ALK，NPM1::MLF1，AML1::MTG16，BCR::JAK2，CALM::AF10，CBFA2T3::GLIS2，E2A::ZNF384，EBF1::PDGFRB，MLL::SEPT6，NUP98::HOXA11，NUP98::HOXA13，NUP98::HOXC11，NUP98::NSD1，PCM1::JAK2，PRKAR1A::RARA，STAT5B::RARA，TEL::JAK2，TEL::PDGFRA，NCOR1::LYN，ETV6::LYN，NUP98::KDM5A，NUP98::DDX10。

the technical method comprises the steps of performing a first PCR reaction by using a GSP1 primer, then performing a second PCR reaction by using a GSP2 primer, and performing first-generation sequencing on a final PCR product by using GSP1MF and GSP2MR respectively;

wherein, the GSP1 primer sequence is shown in SEQ ID NO. 1-83; the GSP2 primer sequence is shown as SEQ ID NO. 84-171;

the GSP2MF primer sequence is shown as SEQ ID NO. 84-117, and the GSP2MR primer sequence is shown as SEQ ID NO. 118-171.

The technical method comprises the following steps:

1) Extracting RNA of a tissue sample to be detected, and performing reverse transcription to obtain cDNA;

2) Performing a first round of PCR reaction;

3) Performing a second round of PCR reaction;

4) Electrophoresis detection and magnetic bead purification for sequencing.

The invention also relates to a leukemia fusion gene single-tube multiplex PCR detection kit, which targets the fusion gene:

CBFB::MYH11，KMT2A::MLLT7，KMT2A::MLLT4，KMT2A::ELL，KMT2A::EPS15，KMT2A::MLLT6，KMT2A::MLLT10，KMT2A::PTD，TCF3::PBX1，TCF3::HLF，STIL::TAL1，ETV6::RUNX1，RUNX1::RUNX1T1，RUNX1::MECOM，RUNX1::EAP，FUS::ERG，RPN1::MECOM，KMT2A::AFF1，KMT2A::MLLT3，KMT2A::MLLT11，KMT2A::MLLT1，BCR::ABL1，ETV6::ABL1，ETV6::PDGFRB，ZMYM2::FGFR1，BCR::FGFR1，DEK::NUP214，SET::NUP214，FIP1L1::PDGFRA，NUP98::HOXA9，NUP98::HOXD13，ZBTB16::RARA，PML::RARA，NPM1::RARA，NPM1::ALK，NPM1::MLF1，AML1::MTG16，BCR::JAK2，CALM::AF10，CBFA2T3::GLIS2，E2A::ZNF384，EBF1::PDGFRB，MLL::SEPT6，NUP98::HOXA11，NUP98::HOXA13，NUP98::HOXC11，NUP98::NSD1，PCM1::JAK2，PRKAR1A::RARA，STAT5B::RARA，TEL::JAK2，TEL::PDGFRA，NCOR1::LYN，ETV6::LYN，NUP98::KDM5A，NUP98::DDX10；

it includes GSP1 primer and GSP2 primer, GSP2MF and GSP2MR primer;

The current common techniques for fusion gene detection are as follows:

fluorescent In Situ Hybridization (FISH) 1: FISH is a traditional gold standard method for detecting fusion genes, which can observe rearrangement of specific genes on chromosomes directly at the cellular level. The technology uses a fluorescent-labeled probe to combine with a target chromosome region, and adopts a separation probe or a fusion probe to detect the occurrence of gene fusion.

2 nd, reverse transcription polymerase chain reaction (RT-PCR): RT-PCR is a common technique for detecting fusion genes in leukemia. It involves transcription of RNA into the corresponding DNA and PCR amplification using specific primers. This technique allows the detection of the presence and quantity of a particular fusion gene,

3, high throughput nucleic acid sequencing techniques, such as whole genome sequencing or targeted sequencing, can help identify fusion genes and other pathogenic variations in leukemia. Such a method enables comprehensive analysis of the sequence of a genome or a specific gene region, including the structure and variation of the fusion gene.

4, PCR amplification and Sanger sequencing: primers were designed for specific fusion gene sequences for PCR amplification and then sequence analysis using Sanger sequencing technology. This method allows to confirm the presence of the fusion gene and to determine its specific sequence structure.

5 th, conventional multiplex PCR (Multiplex PCR): this technique allows the simultaneous detection of fusion of multiple genes, which are detected by one PCR reaction. The method generally judges the existence of fusion genes by agarose gel electrophoresis.

Disadvantages/shortcomings of conventional techniques:

FISH does not provide detailed sequence information of the fusion gene, and the range of gene rearrangements detected by the separation probe is large, and false positive results may occur.

RT-PCR can only detect a single fusion gene.

High throughput nucleic acid sequencing techniques are costly, requiring highly specialized laboratory and analytical skills; deeper sequencing may be required for rare or low frequency fusion events and data analysis requires some time and expertise.

PCR amplification and Sanger sequencing only detect a single fusion gene.

The traditional multiplex PCR needs to divide multiple tubes to carry out PCR reaction, respectively carries out electrophoresis preliminary identification after one round of PCR, then selects corresponding primers to carry out a second round of PCR reaction according to the result, carries out electrophoresis identification again, has more complicated process, and can generate false positive results caused by non-specific bands only according to the result of electrophoresis band interpretation.

Drawings

FIG. 1 is a graph showing agarose assay results according to an embodiment of the invention;

FIG. 2 shows the sequencing results of BCR-ABL1p 190;

FIG. 3 shows the sequencing results of BCR-ABL1p 210;

FIG. 4 shows the result of RUNX1-RUNX1T1 sequencing;

FIG. 5 shows NCOR1-LYN sequencing results;

FIG. 6 shows the sequencing results of KMT2A-AFF1.

Detailed Description

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Unless defined otherwise, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used in this specification is well known and commonly employed in the art.

The invention relates to a single-tube multiplex PCR detection technical method for leukemia fusion genes, which targets the fusion genes:

compared with 36 groups of common fusion genes of leukemia in the literature, 20 newly reported leukemia related fusion genes are added, and the genes are respectively: AML 1:MTG 16, BCR:JAK 2, CALM:AF 10, CBFA2T 3:GLIS 2, E2 A:ZNF 384, EBF 1:PDGFRB, MLL:SEPT 6, NUP 98:HOXA 11, NUP 98:HOXA 13, NUP 98:HOXC 11, NUP 98:NSD 1, PCM 1:JAK 2, PRKAR1 A:RARA, STAT5 B:RARA, TEL:JAK 2, TEL:PDGFRA, NCOR 1:LYN, ETV 6:LYN, NUP 98:KDM 5A, NUP 98:DDX 10, the incidence of these genes in leukemia being up to increasing specific gravity. For example, 15 cases of AML have been found in AML and MDS: MTG16 fusion; 11 cases of BCR were found in myeloid tumors and ALL:: JAK2 fusion; CBFA2T3 is found in 20 cases of normal karyotype children AML, GLIS2 fusion gene; of 193 cases of B-ALL with ZNF384 rearrangement, 60 cases (31%) detected ZNF384 fused with E2A; of 164 pediatric AML patients, 76 (46%) found known fusions involving MLL, with MLL-SEPT6 fusions found in 6 patients (8%), 1421 AML patients, 15% with FLT3-ITD and 7% cytogenetically normal AML patients detected NUP 98:NSD1 fusions; PCM1 in which JAK2 fusion has been reported for 16 or more, and in myelogenous/gonomic tumors with JAK2 rearrangement in the 2022 5 th edition WHO, PCM1 is the most common rearrangement partner gene for JAK 2; LYN fusion has been reported in 3 cases at present, and patients found by the company are successfully treated by dasatinib for the first time; in 2393 children AML, 47 (2.0%) detected NUP98: KDM5A fusion; NUP98-DDX10 fusion 14 have been reported, including 8 new (6 AML,1 MDS and 1 CML acceleration phase) myeloid tumors, 6 treatment-related (3 AML,2 MDS,1 CML) myeloid tumors.

The technical method of the invention comprises the steps of carrying out a first PCR reaction by using a GSP1 primer and then carrying out a second PCR reaction by using a GSP2 primer. The method comprises the following steps:

2) Performing a first round of PCR reaction;

3) Performing a second round of PCR reaction;

4) Electrophoresis detection and magnetic bead purification for sequencing.

In the prior art, the multiplex PCR fusion gene technology is used for detecting 36 groups of leukemia common fusion genes, 8 tubes are needed for carrying out the first round of PCR, if the result is positive, single-tube primers (generally 5-7 pairs) are respectively adopted for carrying out the 2 nd round of PCR to obtain a specific fusion gene, the result of one positive of a single sample at least needs 13-15 tubes of PCR reaction, the process is very complicated, and the final result is judged by only electrophoresis strips, so that the false positive result is very easy.

The multiple PCR technology provided by the invention detects 56 groups of leukemia common fusion genes, all primers are mixed in the same PCR tube to perform two rounds of PCR experiments, the experiments can be completed by only 2 PCR tubes, 83% (10/12) experiment cost and detection time can be saved, the final result adopts mixed primers to perform first-generation sequencing, the result is more objective according to the sequence interpretation result, and the false positive result caused by electrophoresis impurity bands does not exist.

Compared with the conventional generation sequencing which only adopts a single forward primer and a single reverse primer for sequencing, the invention has higher sequencing efficiency. Because 56 pairs of fusion genes have multiple target sequences with similar lengths, inaccurate results can occur by interpretation of the length of the bands after electrophoresis alone. The invention can directly sequence by mixing the primers, and the missed detection or false negative result caused by the wrong selection of the sequencing primers is not needed. This also allows multiple PCR result interpretation to be more objective and accurate to obtain fusion gene sequences without relying on large-scale PCR reactions and agarose gel electrophoresis. The method can be popularized to the detection of similar fusion genes by multiplex PCR, such as the detection of fusion genes of other solid tumors such as lung cancer, sarcoma and the like.

The primer sequences related to the invention are shown in the following tables 1-4;

TABLE 1 GSP1 primer sequences

Table 2. Forward mix primer GSP2MF:

TABLE 3 reverse mix primer GSP2MR:

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TABLE 4 internal reference primer sequences (4 strips total)

1. Materials and methods

1.1RNA extraction

The operation is carried out according to the instruction of the selected extraction kit, and the extraction kit is preserved at the temperature of minus 80 ℃ for standby.

1.2 reverse transcription

cDNA synthesis was performed according to the selected reverse transcription kit instructions and stored at-20℃until use.

1.3 primer design and Synthesis

Primer design: specific primers GSP1 and GSP2 are designed aiming at known common fusion genes, and more newly discovered genes or genes reported recently in literature are added, so that the coverage is more complete. The invention comprises leukemia fusion genes as follows, 56 groups of fusion genes in total: CBFB: MYH11, KMT2A: MLLT7, KMT2A: A, a, a, the.

1.4 round 1 and round 2 PCR primers were mixed into a single tube, GSP1 and GSP2, respectively. Meanwhile, forward and reverse primers in GSP2 are selected and respectively mixed and marked as GSP2MF and GSP2MR.

First round PCR reaction

20. Mu.l of reaction system, 2x QIAGEN Multiplex PCR Master Mix 10. Mu.l, 2. Mu.l of Q-Solution (5X), 1.34. Mu.l of GSP1 Primers (0.04. Mu.M final concentration), 10-100ng of cDNA, ddH ₂ O is made up to 20ul. The sample was subjected to pre-denaturation at 95℃for 15min, denaturation at 95℃for 30s, annealing at 62℃for 30s and extension at 72℃for 60s in a PCR apparatus, followed by 25 cycles of extension at 72℃for 10min and standing at 4 ℃.

Second round PCR reaction

20 μl of the reaction system, 2x QIAGEN Multiplex PCR Master Mix 10 μl,2 μl of Q-Solution (5×), 3.6 μl of GSP2 Primers (0.1 μM final concentration), and 0.5ul,ddH2O 3.9ul of the first round PCR reaction. The sample was subjected to a PCR reaction at 95℃for 15min,95℃for 30s,62℃for 30s, and 72℃for 60s, followed by 34 cycles of 10min and 4℃for 10 min.

Wherein, GSP1 primer is adopted to carry out single-tube first round PCR, and then 0.5ul of the first round PCR product is directly taken to carry out second round nested PCR reaction. At the same time, the primers corresponding to the 56 groups of fusion genes are verified again and ensured to accurately detect the fusion genes.

1.5 agarose gel electrophoresis

And (3) judging the result of agarose gel electrophoresis, wherein at least 118bp of B2M gene target band (reference gene) is observed according to the result of the agarose gel electrophoresis, if the band exists, the first generation sequencing is carried out after the magnetic bead is purified, and if the band does not exist, the PCR is carried out again.

1.6 magnetic bead purification

22.5 mu L of AMPure XP magnetic beads (1.5X) are added into the rest 15 mu L of the PCR tube product of the second round, and the mixture is sucked and stirred uniformly and kept stand for 5min at room temperature; standing on a magnetic rack for 1min, sucking out supernatant with a pipette after the solution is clarified, and discarding; adding 200 mu L of 80% ethanol for cleaning for 2 times; sucking the residual liquid with a 10 mu l pipettor, opening the cover, and standing and drying on a magnetic rack for 2min; add 22 μl ddH2O for resuspension; aspirate about 20 μl with a pipette;

1.7 Generation sequencing

10. Mu.L of purified PCR product, 10. Mu.L of forward mixed primer GSP2MF (100. Mu.M, 34 forward primer 1:1 mix), 10. Mu.L of reverse mixed primer GSP2MR (100. Mu.M, 54 reverse primer 1:1 mix) were added to each new EP tube, and a generation sequencing was performed using the mixed primers.

The sequencing of the generation is performed by adopting mixed GSP2MF and GSP2MR primers. The original primary sequencing result is generally sequenced by adopting single forward and reverse primers, the false negative result can be generated by selecting the corresponding primers according to the length by multiple PCR, and the forward and reverse sequencing is respectively performed by adopting the forward mixed primers and the reverse mixed primers by the improved method, so that the false negative caused by the error of the selection of the primers can not be generated.

And comparing the sequencing result with a human reference genome to judge whether the fusion gene exists or not. The original multiplex PCR method is only interpreted according to agarose gel electrophoresis results, and the sequence cannot be determined. The improved method is convenient and rapid, is more objective, and can directly interpret specific subtypes of fusion genes.

As a result, as shown in FIG. 1, after the PCR2 product was purified, the expected length of the internal reference was 118bp (in red box), and if the internal reference was negative, the experiment was repeated. If the product electrophoresis image is shown in a green frame, the band is single and the yield is enough, and the sequencing can be directly carried out; if the product is shown in the blue box, the amount of the mixed bands is more and less, the separate gel cutting recovery and sequencing are considered.

Sanger sequencing results

Performing Blast on the forward and reverse sequencing result at NCBI, and judging that the fusion gene result is positive if the sequence result can be simultaneously compared with different genes; if the sequence result is aligned to the same gene, the judgment is negative.

Wherein 41 groups of fusion genes have been validated, CBFB: MYH11, KMT2A: MLLT7, KMT2A: MLLT4, KMT2A, ELL, KMT2A, EPS15, KMT2A, MLLT6, KMT2A, MLLT10, KMT2A, PTD, TCF3, MLLT1, TCF3, HLF, STIL 1, ETV6, RUNX1T1, RUX 1, MECOM, EAP, FUS, ERG, RPN1, MECOM, KMT2A, AFF1, KMT2A, MLLT3, KMT2A, MLLT11, KMT2A, MLLT1, BCR, ABL1, ETV6, PDRB, ZM 2, GFR1, GFR 214, RARA 1, RAP 98, RAP 1, RAP 98, and the like. Wherein, as shown in FIG. 2, BCR-ABL1p190, FIG. 3, BCR-ABL1p 210, FIG. 4, RUNX1-RUNX1T1, FIG. 5, NCOR1-LYN, and FIG. 6, KMT2A-AFF1.

Claims

1. A leukemia fusion gene single tube multiplex PCR detection method is characterized in that the method targets the fusion gene:

2. the leukemia fusion gene single tube multiplex PCR detection method according to claim 1, wherein,

3. The single tube multiplex PCR detection method for leukemia fusion genes according to claim 1 or 2, comprising the steps of:

2) Performing a first round of PCR reaction;

3) Performing a second round of PCR reaction;

4) Electrophoresis detection and magnetic bead purification for sequencing.

4. A leukemia fusion gene single tube multiplex PCR detection kit is characterized in that the kit targets a fusion gene:

it includes GSP1 primer and GSP2 primer, GSP2MF and GSP2MR primer;