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CN-122012710-A - IGH rearrangement detection reverse transcription primer group, quantitative screening detection kit and application

CN122012710ACN 122012710 ACN122012710 ACN 122012710ACN-122012710-A

Abstract

The invention provides an IGH rearrangement detection reverse transcription primer set, a quantitative screening detection kit and application, wherein the reverse transcription primer set is specifically designed for common cleavage sites of IGH, T base close to the 3 'end in the reverse transcription primer is not modified so as not to influence the normal function of reverse transcriptase, the T base at the 5' end of the reverse transcription primer is replaced by U base so as to be digested by uracil-N-glycosylase (UNG), the residual reverse transcription primer after the reverse transcription is partially digested by UDG, reverse transcription is carried out through the reverse transcription primer set to obtain IGH gene cDNA and fusion gene cDNA of different types, and the specific primers and probes respectively designed at the upstream of DUX4, EPOR, CRLF2 and ABL1 genes are combined, so that the quantitative detection of any one of three types of rearrangements can be realized, and the kit has remarkable application value for clinical auxiliary diagnosis.

Inventors

  • LIU HUICHAO
  • WANG LI
  • PENG LING

Assignees

  • 武汉希诺医学检验实验室有限公司

Dates

Publication Date
20260512
Application Date
20260205

Claims (10)

  1. An IGH rearrangement detecting reverse transcription primer set comprising a reverse transcription primer set for detecting an IGH rearrangement, the rearrangement type comprising at least one of IGH-DUX4, IGH-EPOR, and IGH-CRLF2, wherein: the IGH-DUX4 reverse transcription primer group comprises a nucleotide sequence shown as SEQ ID NO. 1-34; and/or, the reverse transcription primer group of IGH-EPOR comprises a nucleotide sequence shown as SEQ ID NO. 35-44; and/or, the reverse transcription primer group of IGH-CRLF2 comprises a nucleotide sequence shown as SEQ ID NO. 45-55.
  2. 2. The use of the reverse transcription primer set of claim 1, wherein said use comprises: a. preparing an IGH rearrangement type detection product; b. And (5) preparing an IGH fusion proportion quantitative detection product.
  3. An IGH quantitative screening kit comprising the reverse transcription primer set of claim 1, and a PCR primer probe set for detecting at least one rearrangement of IGH-DUX4, IGH-EPOR, and IGH-CRLF 2.
  4. 4. The kit of claim 3, wherein the set of PCR primers for detecting IGH-DUX4 rearrangement comprises a probe having a nucleotide sequence as set forth in SEQ ID NO. 57 and primers as set forth in SEQ ID NO. 58-59; And/or the PCR primer probe group for detecting IGH-EPOR rearrangement comprises a probe with a nucleotide sequence shown as SEQ ID NO. 60 and primers shown as SEQ ID NO. 61-62; And/or the PCR primer probe group for detecting the IGH-EPORIGH-CRLF2 rearrangement comprises a probe with a nucleotide sequence shown as SEQ ID NO. 63 and a primer shown as SEQ ID NO. 64-65.
  5. 5. The kit of claim 3, further comprising a reference gene reverse transcription primer, and a primer set and a probe for PCR amplification.
  6. 6. The kit according to claim 5, wherein the nucleotide sequence of the reference gene reverse transcription primer is shown as SEQ ID NO. 56; and/or the primer group and the probe for PCR amplification of the reference gene comprise a probe with a nucleotide sequence shown as SEQ ID NO. 66 and a primer group with a nucleotide sequence shown as SEQ ID NO. 67-68.
  7. 7. The kit of claim 5 or 6, wherein any of the probes in the kit are modified with a different fluorescent label.
  8. 8. The kit of claim 3, wherein the concentration of any of the reverse transcription primers in the reverse transcription primer set is 0.1 to 1uM.
  9. 9. The kit of claim 3, further comprising at least one of a reverse transcription reaction reagent, a PCR reaction reagent, an RNA digestive enzyme, and a UNG enzyme.
  10. 10. A method for quantitatively screening IGH rearrangements for non-diagnostic purposes comprising the use of a kit according to claim 3 and performing the steps of: 1) Extracting sample RNA; 2) Reverse transcription of the RNA using reverse transcription primers to generate cDNA; 3) Taking cDNA as a template, adding a PCR primer probe group and UNG enzyme, and carrying out PCR amplification; 4) Judging the detection type and fusion ratio according to the detection signal of the probe.

Description

IGH rearrangement detection reverse transcription primer group, quantitative screening detection kit and application Technical Field The invention relates to the field of detection kits, in particular to an IGH rearrangement detection reverse transcription primer group, a quantitative screening detection kit and application. Background B-cell acute lymphoblastic leukemia (B-ALL) is characterized by a variety of genomic alterations, with gene fusion involving immunoglobulin heavy chain (IGH) sites also being one of the most common genomic alterations. However, immunoglobulin heavy chains (IGH) are large and highly polymorphic in their region, making IGH-related fusion genes difficult to detect using conventional detection methods. There are more than ten reported IGH fusion partners, including DUX4, EPOR, CRLF2, CEBPD, SPIDR, CEBPB, MYC, BCL2, BCL3, BCL6, CCND1, MUM1, etc., with DUX4, EPOR, CRLF2 being more common. DUX4 is a dual homeobox embryo Transcription Factor (TF) that is normally expressed in germ line cells and silenced in somatic cells. However, under pathological conditions, such as malnutrition, leukemia and other types of cancer, DUX4 re-expression in somatic cells has been found to be responsible for its pathogenesis. DUX4 is a gene located on chromosome 4 in the D4Z4 large satellite repeat, and has a homologous polymorphic repeat on chromosome 10. DUX4 rearrangement is found in 4% -7% B-ALL, mainly in children and teenagers. Often with lower white blood cell counts than other types of B-ALL, line transformations, especially single-core transformations, tend to occur during treatment. The most common DUX4 partner gene found in current research is IGH, and the DUX4 gene can undergo insertion translocation with IGH in different forms, rather than equilibrium translocation, and the end result is overexpression of DUX 4. EPOR IGH fusion genes are formed by t (14; 19) (q 32; p 13) translocations. The EPOR gene is an EGFR gene (Erythropoietin Receptor gene). The EPOR gene is located at the 19p13 position in the human genome and encodes a membrane-bound receptor protein, known as the epidermal growth factor receptor (EPOR). The epidermal growth factor receptor (EPOR) is a receptor protein on the cell membrane that binds to erythropoietin (Erythropoietin, abbreviated as EPO) and mediates the transmission of EPO signals. EPO is a hormone secreted by the kidney and plays a key role in erythropoiesis and maturation. The IGH gene is Immunoglobulin heavy chain gene (Immunoglobulin HEAVY CHAIN GENE). The IGH gene is located at position 14q 32. Immunoglobulin genes, including immunoglobulin heavy chain genes (IGH), light chain (kappa) chain genes (IGK), and light chain (lambda) chain genes (IGL), frequently undergo a break rearrangement in B cell tumors. These rearrangements result in juxtaposition (fusion) of IG enhancers to oncogenes (such as MYC and BCL 2), resulting in their overexpression and activation. According to literature reports, the EPOR:: IGH gene is relatively common in young B-ALL and has highly invasive characteristics. Conventional examination formats such as clinical, morphological, immunological, cytogenetics have made it difficult to find EPOR:: the presence of IGH. Philadelphia chromosome-like (Ph-like) ALL is a newly established subtype of acute lymphoblastic leukemia. Although Ph-like ALL does not express BCR-ABL fusion genes, the behavior is similar to true BCR/ABL1 positive cases. This subtype carries different molecular variations, most commonly CRLF2 rearrangements. CRLF2 gene rearrangements were found in 50% of Ph-like ALL, with CRLF2:: IGH fusion and P2RY8:: CRLF2 being the most common. CRLF2 IGH fusion is more common in adults and is consistently associated with Ph-like features. The current common methods for detecting gene rearrangements include G-banding chromosomal karyotyping, FISH probes, RNA-Seq, whole genome, and the like. However, for detection of IGH rearrangements, methods based on second generation sequencing such as RNA-Seq, whole genome, etc. are generally costly and less sensitive, and conventional detection methods such as G-banding chromosomal karyotyping and FISH probes do not detect it well, for the following reasons: 1. For karyotyping analysis, IGH rearrangements are cytogenetically cryptic and difficult to detect because of the small repetitive sequence inserted into the IGH site or, in the case of reverse translocation, the fusion partners IGH and DUX4 are both located in the subtelomere region; The fish detection difficulty is mainly that the sequence and position of the IGH rearrangement breakpoint change greatly, so that the design of the separated fluorescent probe becomes difficult, and fusion partners such as DUX4 and the like have larger homologous interference to cause cross reaction. Disclosure of Invention The invention provides an IGH rearrangement detection reverse transcription primer group and a quantitative screening detection kit, which are