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CN-121992085-A - Primer probe group for detecting three leukemia fusion genes of BCR-ABL1, ETV6-RUNX1 and TCF3-PBX1 and application thereof

CN121992085ACN 121992085 ACN121992085 ACN 121992085ACN-121992085-A

Abstract

The invention provides a primer probe set for detecting three leukemia fusion genes of BCR-ABL1, ETV6-RUNX1 and TCF3-PBX1 and application thereof, and establishes a method for simultaneously detecting three fusion gene RNAs based on capture and ligation reaction combined with multiplex fluorescence quantitative PCR. The primer probe group comprises a capture probe, a connection probe, an amplification primer and a Taq-Man probe, wherein the capture probe, the connection probe, the amplification primer and the Taq-Man probe are respectively used for detecting various types of three fusion genes and internal reference genes. The invention can directly crack and capture target RNA in whole blood, dried blood slices or bone marrow samples without nucleic acid extraction and reverse transcription, and simultaneously detect three fusion genes in the same fluorescent quantitative PCR reaction system. The detection sensitivity was 75 copies/reaction for BCR-ABL1, 6 copies/reaction for ETV6-RUNX1, and 29 copies/reaction for TCF3-PBX1, respectively.

Inventors

  • NI XIN
  • LI JINZHI
  • Han dai

Assignees

  • 首都医科大学附属北京儿童医院

Dates

Publication Date
20260508
Application Date
20251016

Claims (10)

  1. 1. A primer probe composition, wherein the primer probe composition comprises: A first ligation probe set according to SEQ ID NO.3-4, a second ligation probe set according to SEQ ID NO.14-15, and a third ligation probe set according to SEQ ID NO. 27-28; a first set of capture probes shown in SEQ ID NOS.7-13, a second set of capture probes shown in SEQ ID NOS.20-24, and a third set of capture probes shown in SEQ ID NOS.31-35; Taq-Man probe set shown in SEQ ID NO.45, SEQ ID NO.47 and SEQ ID NO. 48; Primer sets for amplifying fusion genes BCR-ABL1, ETV6-RUNX1 and TCF3-PBX 1.
  2. 2. The primer probe composition according to claim 1, wherein the primer set for amplifying the fusion genes BCR-ABL1, ETV6-RUNX1 and TCF3-PBX1 is shown as SEQ ID NO. 43-44.
  3. 3. The primer probe composition according to claim 1 or 2, wherein the primer probe composition further comprises a probe and a primer for detecting an internal reference gene, wherein the internal reference gene comprises an ABL1 gene, and/or the probe for detecting an internal reference gene comprises a ligation probe and a Taq-Man probe.
  4. 4. The primer probe composition of claim 3, comprising at least one of the following features: (1) The connection probe for detecting the reference gene is shown as SEQ ID NO. 36-41; (2) The Taq-Man probe for detecting the reference gene is shown as SEQ ID NO. 46; (3) The primer for detecting the reference gene ABL1 is shown as SEQ ID NO. 42-43; (4) The Taq-Man probe is modified with a detectable label; (5) The Taq-Man probe is provided with a locked nucleic acid modification or an MGB modification.
  5. 5. A test kit comprising the primer probe composition of any one of claims 1-4.
  6. 6. Use of the primer probe composition of any one of claims 1 to 4 or the detection kit of claim 5 for preparing a fusion gene detection product of at least one of BCR-ABL1, ETV6-RUNX1 and TCF3-PBX 1.
  7. 7. Use of the primer probe composition of any one of claims 1 to 4 or the detection kit of claim 5 in the preparation of a leukemia detection product.
  8. 8. The use according to claim 7, wherein the detecting comprises the steps of: (1) Obtaining a sample to be detected; (2) Mixing and incubating the connection probe and the capture probe with a sample to be detected for capture reaction, washing after the reaction is finished to remove the unhybridized probe, adding a connection system for connection reaction, mixing the product with the primer and the Taq-Man probe after the reaction is finished, carrying out PCR, and judging the detection result according to the PCR result.
  9. 9. Use according to claim 7 or 8, characterized in that the detection is a single tube detection.
  10. 10. The use according to claim 8, wherein the sample to be tested is a whole blood sample, a dried blood sample or a bone marrow sample.

Description

Primer probe group for detecting three leukemia fusion genes of BCR-ABL1, ETV6-RUNX1 and TCF3-PBX1 and application thereof Technical Field The invention relates to the technical field of gene detection, in particular to a primer probe group for detecting three leukemia fusion genes of BCR-ABL1, ETV6-RUNX1 and TCF3-PBX1 and application thereof. Background Acute lymphoblastic leukemia (acute lymphoblastic leukemia, ALL) is the most common type of cancer in children, accounting for 70% -80% of acute leukemia in children, and approximately 27.3% of acute leukemia in children, and is the most common cause of cancer death before 20 years of age. Fusion genes are common and important molecular biological abnormalities in leukemia. BCR-ABL1, ETV6-RUNX1 (also known as TEL-AML 1) and TCF3-PBX1 (also known as E2A-PBX 1) are the three most common fusion gene types for ALL patients. Fusion genes have heterogeneity and can produce multiple isoforms due to differences in gene cleavage sites and fusion sites. For example, the BCR-ABL1 fusion gene is formed by translocation of t (9; 22) (q 34; q 11), and depending on the cleavage site of the BCR gene, the major types of isoforms include p210 (e 13a2 or e14a 2), p190 (e 1a 2) and p230 (e 19a 2), and other fusion variants such as e1a3, e6a2 and e8a2 occur less frequently than these three typical fusions. Current studies have also found that there are multiple isomers of ETV6-RUNX1 and TCF3-PBX 1. The accurate detection of the fusion gene can provide important molecular basis for leukemia diagnosis and typing, prognosis evaluation, treatment scheme selection and monitoring of minimal residual disease. The existing detection method of the fusion gene mainly comprises chromosome karyotyping analysis, fluorescence In Situ Hybridization (FISH), immunohistochemical staining, next Generation Sequencing (NGS), reverse transcription fluorescence quantitative PCR and the like. The FISH method is a gold standard for detecting fusion genes, has strong specificity, is complex to operate, has high requirement on sheet production, has limited number of sites to be detected at one time, has low sample requirement on an immunohistochemical mode, has simple technology, has complex operation, has few fusion protein antibody types and high requirement on sample integrity, can realize comprehensive unbiased detection, has the opportunity to detect unknown fusion genes, has large data volume, high cost and long complex reporting period of data analysis. The fluorescent quantitative RT-PCR method has strong operability and high sensitivity, but is limited by a fluorescent channel and a probe, different isomers of fusion genes cannot be covered in a single reaction system, and if the isomers possibly existing in a plurality of fusion genes need to be comprehensively detected, more reaction holes need to be added, so that the cost is greatly increased, and the workload is increased. At present, no method for simultaneously detecting multiple isomers of three leukemia fusion genes in the same reaction system exists, so that a novel technology with high sensitivity and specificity, simplicity and rapidness is urgently needed to be established. Disclosure of Invention In order to solve the technical problems, the invention provides a scheme for detecting different isomers of three fusion genes based on capture and ligation combined fluorescence quantitative PCR, and the method can detect multiple transcripts of the fusion genes by using a set of primer probes without nucleic acid extraction and reverse transcription operation, so that whether three fusion genes of BCR-ABL1, ETV6-RUNX1 and TCF3-PBX1 exist in a sample can be detected simultaneously in a single tube. A first object of the present invention is to provide a primer probe composition comprising: A first ligation probe set according to SEQ ID NO.3-4, a second ligation probe set according to SEQ ID NO.14-15, and a third ligation probe set according to SEQ ID NO. 27-28; a first set of capture probes shown in SEQ ID NOS.7-13, a second set of capture probes shown in SEQ ID NOS.20-24, and a third set of capture probes shown in SEQ ID NOS.31-35; Taq-Man probe set shown in SEQ ID NO.45, SEQ ID NO.47 and SEQ ID NO. 48; Primer sets for amplifying fusion genes BCR-ABL1, ETV6-RUNX1 and TCF3-PBX 1. The invention designs a method for detecting leukemia fusion genes based on a primer probe composition and multiple fluorescence quantitative PCR (polymerase chain reaction), and particularly relates to screening and optimizing a connection probe, a capture probe and a Taq-Man probe. Specifically, the design of a connection probe and a capture probe is carried out through the conserved regions of different fusion gene variants, a series of connection probes and capture probes are screened, and a probe set with highest combination efficiency and highest detection sensitivity is selected. As for the Taq-Man probe, unlike the existing quantitative PCR, the Taq-Man